JP4965022B2 - Stabilized protein crystals, formulations containing the same, and methods of making the same - Google Patents

Description

ハロアミン（ｈａｌｏａｍｉｎｅ）Ｒ₂Ｎ−Ｈａｌ、アルケンおよびアルキン。
【０１８１】
ＩＶ．硫黄含有基は、以下により例示される：ジスルフィドＲＳＳＲ’、スルフヒドリルＲＳＨ、エポキシド
【０１８２】
【化２】

Ｖ．塩は以下により例示される；アルキルアンモニウム塩またはアリールアンモニウム塩Ｒ₄Ｎ＋、カルボキシレートＲＣＯＯ−、スルフェートＲＯＳＯ₃−、ホスフェートＲＯＰＯ₃”およびアミンＲ₃Ｎ。
【０１８３】
以下の表１は、放出機構により体系付けられるトリガーの例を挙げる。表１において、Ｒ＝は、多機能架橋剤であり、これは、アルキル、アリール、または架橋すべきタンパク質と反応し得る活性基を有する他の鎖であり得る。これらの反応基は、任意の種々の基（例えば、求核置換、フリーラジカル置換、または求電子置換に感受性の基（特に、ハロゲン化物、アルデヒド、カルボネート、ウレタン、キサンタン、エポキシドを含む））であり得る。
【０１８４】
【表１】

可逆性クロスリンカーのさらなる例は、Ｔ．Ｗ．Ｇｒｅｅｎ、Ｐｒｏｔｅｃｔｉｖｅ Ｇｒｏｕｐｓ ｉｎ Ｏｒｇａｎｉｃ Ｓｙｎｔｈｅｓｉｓ、Ｊｏｈｎ ＷｉｌｅｙおよびＳｏｎｓ（編）（１９８１）に記載される。可逆性保護基に使用するための任意の種々のストラテジーは、可逆性の制御された可溶化が可能な、架橋されたタンパク質結晶の生成に好適なクロスリンカー中に導入され得る。種々のアプローチが、この主題のＷａｌｄｍａｎｎの総説（Ａｎｇｅｗａｎｔｅ Ｃｈｅｍｉｅ Ｉｎｌ．Ｅｄ．Ｅｎｇｌ．３５、２０５６頁（１９９６））に列挙される。
【０１８５】
可逆性クロスリンカーの他の型は、ジスフィルド結合含有クロスリンカーである。このようなクロスリンカーにより形成されたトリガー破損架橋は、還元剤（例えば、システイン）の架橋したタンパク質結晶環境への添加である。
【０１８６】
ジスフィルドクロスリンカーは、Ｐｉｅｒｃｅ Ｃａｔａｌｏｇ ａｎｄ Ｈａｎｄｂｏｏｋ（１９９４−１９９５）に記載され、そしてより最近には、Ｇ．Ｔ．Ｈｅｒｍａｎｓｏｎによる「Ｂｉｏｃｏｎｊｕｇａｔｅ Ｔｅｃｈｎｉｑｕｅｓ」、（１９９６）、Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ、Ｄｉｖｉｓｉｏｎ ｏｆ Ｈａｒｃｏｕｒｔ Ｂｒａｃｅ ａｎｄ Ｃｏｍｐａｎｙ、５２５ Ｂ Ｓｔｒｅｅｔ、Ｓｕｉｔｅ １９９０、Ｓａｎ Ｄｉｅｇｏ、ＣＡ ９２１０１−４４９５に記載される。
このようなクロスリンカーの例は、以下を含む：
（同種二官能性（対称性））
（ＤＳＳ）− ジチオビス（スクシンイミジルプロピオネート）、Ｌｏｍａｎｔ剤としても知られる
（ＤＴＳＳＰ）−３−３’−ジチオビス（スルホスクシンイミジルプロピオネート）、ＤＳＰの水溶性版
（ＤＴＢＰ）− ジメチル３，３’−ジチオビスプロピオンイミデート・ＨＣｌ。
（ＢＡＳＥＤ）− ビス−（β−［４−アジドサリチルアミノ］エチル）ジスルフィド
（ＤＰＤＰＢ）− １，４−ジ−（３’−［２’−ピリジルジチオ］−プロピオンアミド）ブタン
（異種二官能性（非対称性））
（ＳＰＤＰ）− Ｎ−スクシンイミジル−３−（２−ピリジルジチオ）プロピオネート
（ＬＣ−ＳＰＤＰ）− スクシンイミジル−６−（３−［２−ピリジルジチオ］プロピオネート）ヘキサノエート
（スルホ−ＬＣ−ＳＰＤＰ）− スルホスクシンイミジル−６−（３−［２−ピリジルジチオ］プロピオネート）ヘキサノエート、ＬＣ−ＳＰＤＰの水溶性版
（ＡＰＤＰ）− Ｎ−（４−［ｐ−アジドサリチルアミド］ブチル）−３’−（２’−ピリジルジチオ）プロピオンアミド
（ＳＡＤＰ）− Ｎ−スクシンイミジル（４−アジドフェニル）１，３’−ジチオプロピオネート
（スルホ−ＳＡＤＰ）− スルホスクシンイミジル（４−アジドフェニル）１，３’−ジチオプロピオネート、ＳＡＤＰの水溶性版
（ＳＡＥＤ）− スルホスクシンイミジル−２−（７−アジド−４−メチルクマリン（ｍｅｔｈｙｃｏｕｍａｒｉｎ）−３−アセトアミド）エチル−１，３’ジチオプロピオネート
（ＳＡＮＤ）− スルホスクシンイミジル−２−（ｍ−アジド−ｏ−ニトロベンズアミド）エチル−１，３’−ジチオプロピオネート
（ＳＡＳＤ）− スルホスクシンイミジル−２−（ｐ−アジドサリチルアミド）エチル−１，３’−ジチオプロピオネート
（ＳＭＰＲ）− スクシンイミジル−４−（ｐ−マレイミドフェニル）ブチレート
（スルホ−ＳＭＰＲ）− スルホスクシンイミジル−４−（ｐ−マレイミドフェニル）ブチレート
（ＳＭＰＴ）− ４−スクシンイミジルオキシカルボニル−メチル−α−（２−ピリジルチオ）トルエン
（スルホ−ＬＣ−ＳＭＰＴ）− スルホスクシンイミジル−６−（α−メチル−α−（２−ピリジルチオ）トルアミド）ヘキサノエート。
【０１８７】
詳細には、Ｇ．Ｔ．Ｈｅｒｍａｎｓｏｎ、（１９９６）、Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ、Ｄｉｖｉｓｉｏｎ ｏｆ Ｈａｒｃｏｕｒｔ Ｂｒａｃｅ ＆ Ｃｏｍｐａｎｙ、５２５ Ｂ Ｓｔｒｅｅｔ、Ｓｕｉｔｅ １９００、Ｓａｎ Ｄｉｅｇｏ、ＣＡ ９２１０１−４４９５による「Ｂｉｏｃｏｎｊｕｇａｔｅ Ｔｅｃｈｎｉｑｕｅｓ」の、Ｚｅｒｏ−ｌｅｎｇｔｈ Ｃｒｏｓｓ−ｌｉｎｋｅｒｓ、Ｈｏｍｏｂｉｆｕｎｃｔｉｏｎａｌ Ｃｒｏｓｓ−ｌｉｎｋｅｒｓ ａｎｄ Ｈｅｔｅｒｏｂｉｆｕｎｃｔｉｏｎａｌ Ｃｒｏｓｓ−ｌｉｎｋｅｒｓのＩＩ部、３−５章を参照のこと。
【０１８８】
本発明のタンパク質の形成に有用な架橋タンパク質結晶はまた、ＰＣＴ特許出願ＰＣＴ／ＵＳ９１／０５４１５に示される方法に従って調製され得る。
【０１８９】
（ポリマーキャリアにおけるタンパク質結晶のカプセル化）
本発明の１つの実施態様によると、組成物は、タンパク質結晶が少なくとも１つのポリマーキャリアにおいて、カプセル化によりマイクロスフェア形態になるように、ポリマーキャリアのマトリックスとともにカプセル化される場合、それらのネイティブな三次構造および生物学的に活性な三次構造を保存するように生成される。結晶は、異なる生物学的環境への送達または特定の機能に効果を及ぼすために好適な独特の特性を有する、種々の生体適合性および／または生分解性ポリマーを使用してカプセル化され得る。従って、活性なタンパク質の溶解速度および送達速度は、特定のカプセル化技術、ポリマー組成物、ポリマー架橋、ポリマーの厚さ、ポリマー溶解度、タンパク質結晶の相対位置および程度、ならびにもしあれば、タンパク質結晶架橋の相対位置および程度により決定される。
【０１９０】
カプセル化されるタンパク質の結晶または処方物は、有機溶媒に溶解したポリマーキャリアに懸濁される。ポリマー溶液は、溶液に添加された後、タンパク質の結晶または処方物を完全に被膜するに十分な濃度に濃縮されなければならない。このような量は、約０．０２と約２０との間、好ましくは、約０．１と約２との間のポリマーに対するタンパク質結晶の重量比を提供する量である。タンパク質結晶は、約０．５分と約３０分の間、好ましくは約１分と約３分との間の時間の間、溶液中のポリマーと接触される。結晶は、懸濁されたままであるべきであるべきであり、そしてポリマーと接触させることにより結晶は被膜されるので、凝集し得ない。
【０１９１】
その接触後、結晶は被膜され、そして新生マイクロスフェアと呼ばれる。新生マイクロスフェアは、被膜化が起こっている間大きさが増加する。本発明の好ましい実施態様において、ポリマーキャリアおよび有機溶媒とともに懸濁された被膜化結晶または新生ミクロスエフェアは、乳化剤として公知の界面活性剤を含む大量の水溶液に移される。この水溶液において、懸濁された新生マイクロスフェアは、水相に浸漬され、ここで、有機溶媒は、エバポレートするか、またはポリマーから拡散させる。最終的に、ポリマーは、もはや可溶性ではなく、そしてタンパク質結晶または処方物をカプセル化して組成物を形成した沈殿相を形成する点に達する。このプロセスのこの局面は、ポリマーキャリアまたはポリマーの硬化といわれる。乳化剤は、硬化相プロセスの間の系での物質の種々の相間の界面の表面張力を低減するのを助ける。あるいは、被膜ポリマーが、いくらかの固有の界面活性を有する場合、別の界面活性剤を添加する必要はない。
【０１９２】
カプセル化タンパク質結晶を本発明に従って調製するために有用な乳化剤としては、ポリマー被覆タンパク質結晶もしくはポリマー被覆結晶処方物と溶液との間の表面張力を減少し得る、本明細書に例示されるようなポリ（ビニルアルコール）、界面活性剤および他の表面活性剤が挙げられる。
【０１９３】
本発明のマイクロスフェアを調製するために有用な有機溶媒としては、塩化メチレン、酢酸エチル、クロロホルムおよび他の非毒性溶媒が挙げられ、これらはポリマーの特性に依存する。ポリマーを溶解しそして究極的には非毒性である溶媒が、選択されるべきである。
【０１９４】
本発明の好ましい実施態様は、タンパク質結晶の結晶性が、カプセル化過程の間維持されることである。結晶性は、被覆過程の間、有機溶媒を使用して維持される（ここにおいては、結晶は可溶性ではない）。続いて、一旦被覆された結晶が水性溶媒に移されると、先の工程でのポリマーキャリアの迅速な硬化および結晶の十分な被覆が、溶解から結晶性材料を保護する。他の実施態様において、架橋されたタンパク質結晶の使用は、水性溶媒および有機溶媒の両方において、結晶性の維持を容易にする。
【０１９５】
タンパク質結晶を被覆するために、ポリマーキャリアとして使用されるポリマーは、ホモポリマーまたはコポリマーのいずれかであり得る。マイクロスフェアの加水分解速度は、大部分は、個々のポリマー種の加水分解速度によって決定される。一般的に、加水分解速度は、以下のように減少する：ポリカーボネート＞ポリエステル＞ポリウレタン＞ポリオルトエステル＞ポリアミド。生分解性ポリマーおよび生体適合性ポリマーの総説については、Ｗ．Ｒ．ＧｏｍｂｏｔｚおよびＤ．Ｋ．Ｐｅｔｔｉｔ、「Ｂｉｏｄｅｇｒａｄａｂｌｅ ｐｏｌｙｍｅｒｓ ｆｏｒ ｐｒｏｔｅｉｎ ａｎｄ ｐｅｐｔｉｄｅ ｄｒｕｇ ｄｅｌｉｖｅｒｙ」、Ｂｉｏｃｏｎｊｕｇａｔｅ Ｃｈｅｍｉｓｔｒｙ、第６巻、３３２〜３５１頁（１９９５）を参照のこと。
【０１９６】
好ましい実施態様において、ポリマーキャリアは、ＰＬＧＡのような単一のポリマー型からなる。次の好ましい実施態様において、ポリマーキャリアは、５０％ ＰＬＧＡおよび５０％アルブミンのようなポリマーの混合物であり得る。
【０１９７】
本発明に従うカプセル化タンパク質結晶を調製するための、ポリマーキャリアとして有用な他のポリマーは、以下からなる群から選択される、生体適合性／生分解性ポリマーを含む：ポリ（アクリル酸）、ポリ（シアノアクリレート）、ポリ（アミノ酸）、ポリ（無水物）、ポリ（デプシペプチド）、ポリ（エステル）（例えば、ポリ（乳酸）もしくはＰＬＡ）、ポリ（ｂ−ヒドロキシ酪酸）、ポリ（カプロラクトン）およびポリ（ジオキサノン）；ポリ（エチレングリコール）、ポリ（ヒドロキシプロピル）メタクリルアミド、ポリ［（オルガノ）ホスファゼン（ｐｈｏｓｐｈａｚｅｎｅ）］、ポリ（オルトエステル）、ポリ（ビニルアルコール）、ポリ（ビニルピロリドン）、無水マレイン酸アルキルビニルエーテルコポリマー、ｐｌｕｒｏｎｉｃポリオール、アルブミン、アルギン酸、セルロースおよびセルロース誘導体、コラーゲン、フィブリン、ゼラチン、ヒアルロン酸、オリゴサッカリド、グリカミノグリカン（ｇｌｙｃａｍｉｎｏｇｌｙｃａｎ）、硫酸化ポリサッカリド、それらの混合物ならびにコポリマー。他の有用なポリマーは、Ｊ．ＨｅｌｌｅｒおよびＲ．Ｗ．Ｂａｌａｒ、「Ｔｈｅｏｒｙ ａｎｄ Ｐｒａｃｔｉｃｅ ｏｆ Ｃｏｎｔｒｏｌｌｅｄ Ｄｒｕｇ Ｄｅｌｉｖｅｒｙ ｆｒｏｍ Ｂｉｏｄｅｇｒａｄａｂｌｅ Ｐｏｌｙｍｅｒｓ」Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ，Ｎｅｗ Ｙｏｒｋ、ＮＹ、（１９８０）；Ｋ．Ｃ．Ｒ．ＬｅｈｍａｎおよびＤ．Ｋ．Ｄｒｅｈｅｒ，Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｔｅｃｈｎｏｌｏｇｙ 第３巻、５− 頁（１９７９）；Ｅ．Ｍ．Ｒａｍａｄａｎ，Ａ．Ｅｌ−ＨｅｌｗおよびＹ．Ｅｌ−Ｓａｉｄ、Ｊｏｕｒｎａｌ ｏｆ Ｍｉｃｒｏｅｎｃａｐｓｕｌａｔｅｉｏｎ、第５巻、１２５頁（１９８８）に記載される。好ましいポリマーは、使用されるタンパク質結晶の特定のタンパク質成分、およびカプセル化結晶（処方物および組成物）の意図される使用に依存する。あるいは、タンパク質結晶をカプセル化するために、溶媒エバポレーション技術が使用され得る（Ｄ．Ｂａｂａｙ，Ａ．ＨｏｆｆｍａｎｎおよびＳ．Ｂｅｎｉｔａ，Ｂｉｏｍａｔｅｒｉａｌｓ 第９巻、４８２−４８８頁（１９８８）を参照のこと）。
【０１９８】
本発明の好ましい実施態様において、タンパク質結晶は、二重エマルジョン法（ｄｏｕｂｌｅ ｅｍｕｌｓｉｏｎ ｍｅｔｈｏｄ）（本明細書中に例証される）を使用して、ポリ乳酸−ｃｏ−グリコール酸のようなポリマーを用いて、少なくとも１つのポリマーキャリア中にカプセル化される。本発明の最も好ましい実施態様において、ポリマーは、ポリ乳酸−ｃｏ−グリコール酸（「ＰＬＧＡ」）である。ＰＬＧＡは、乳酸（「Ｌ」）とグリコール酸（「Ｇ」）とでの重縮合反応により調製されるコポリマーである。ＰＬＧＡポリマーの結晶性および疎水性を調節するために、種々の割合のＬおよびＧが使用され得る。ポリマーのより高度な結晶性は、結果としてより緩徐な溶解をもたらす。２０〜７０％のＧ含有量を有するＰＬＧＡポリマーは、非結晶性固体である傾向があるが、ＧまたはＬいずれかの高いレベルは、優れたポリマー結晶性をもたらす。ＰＬＧＡの調製についてのさらなる情報は、Ｄ．Ｋ．ＧｉｌｄｉｎｇおよびＡ．Ｍ．Ｒｅｅｄ、「Ｂｉｏｄｅｇｒａｄａｂｌｅ ｐｏｌｙｍｅｒｓ ｆｏｒ ｕｓｅ ｉｎ ｓｕｒｇｅｒｙ−ｐｏｌｙ（ｇｌｙｃｏｌｉｃ）／ｐｏｌｙ（ｌａｃｔｉｃ ａｃｉｄ） ｈｏｍｏ ａｎｄ ｃｏｐｏｌｙｍｅｒｓ：１」Ｐｏｌｙｍｅｒ 第２０巻、１４５９−１４６４頁（１９８１）を参照のこと。ＰＬＧＡは、水への曝露後、エステル結合の加水分解により分解されて、非毒性の乳酸およびグリコール酸のモノマーを生成する。
【０１９９】
別の実施態様において、二重層（ｄｏｕｂｌｅ−ｗａｌｌｅｄ）ポリマー被覆されたマイクロスフェアは、有益であり得る。二重層ポリマー被覆されたマイクロスフェアは、２つの別々のポリマー溶液を、塩化メチレンまたはポリマーを溶解し得る他の溶媒中に調製することによって生成され得る。タンパク質結晶をこの溶液の一方に添加し、そして分散させる。ここで、このタンパク質結晶は、第１のポリマーで被覆される。次いで、この第１のポリマーで被覆されたタンパク質結晶を含む溶液を、第２のポリマー溶液と混合する。（Ｐｅｋａｒｅｋ，Ｋ．Ｊ．；Ｊａｃｏｂ，Ｊ．Ｓ．およびＭａｔｈｉｏｗｉｔｚ，Ｅ． Ｄｏｕｂｌｅ−ｗａｌｌｅｄ ｐｏｌｙｍｅｒ ｍｉｃｒｏｓｐｈｅｒｅｓ ｆｏｒ ｃｏｎｔｒｏｌｌｅｄ ｄｒｕｇ ｒｅｌｅａｓｅ，Ｎａｔｕｒｅ，３６７，２５８−２６０を参照のこと）。今度は、第２のポリマーが、タンパク質結晶をカプセル化している第１のポリマーをカプセル化する。理想的には、次いで、この溶液を、表面活性剤もしくは乳化剤を含む、より大きな容積の水溶液中に滴下する。水溶液において、この溶媒は、２つのポリマー溶液からエバポレートされそしてポリマーを沈殿させる。
【０２００】
上記の工程は、タンパク質結晶、ＤＮＡ結晶またはＲＮＡ結晶のいずれかをこれらのいずれかの処方物中から使用して行われ、組成物を生成し得る。
【０２０１】
本発明に従う処方物は、タンパク質結晶および少なくとも１つの成分を含む。このような処方物は、５０℃で貯蔵された場合、５０℃の溶液中でのこのタンパク質の可溶性形態よりも、少なくとも６０倍のより長い有効期限によって特徴付けられる（Ｔ_1/2によって測定した場合）。あるいは、このような処方物は、４０℃および７５％の湿度で貯蔵された場合、４０℃および７５％の湿度で貯蔵されたこのタンパク質結晶の非処方物形態よりも、少なくとも５９倍のより長い有効期限によって特徴付けられる（Ｔ_1/2によって測定した場合）。あるいは、このような処方物は、５０℃で貯蔵された場合、５０℃で貯蔵されたこのタンパク質結晶の非処方物形態よりも、少なくとも６０％のより長い有効期限によって特徴付けられる（Ｔ_1/2によって測定した場合）。あるいは、このような処方物は、５０℃で４日間貯蔵された後のタンパク質のα−へリックス構造含有量の２０％未満の損失によって特徴付けられ、ここで、このタンパク質の可溶性形態は、５０℃で６時間貯蔵された後に、このタンパク質のα−へリックス構造含有量を５０％を超えて損失する（ＦＴＩＲによって測定した場合）。あるいは、このような処方物は、５０℃で４日間貯蔵された後のこのタンパク質のα−へリックス構造含有量の２０％未満の損失によって特徴付けれら、ここで、このタンパク質の可溶性形態は、５０℃で６時間貯蔵された後に、このタンパク質のα−へリックス構造含有量を５０％を超えて損失し（ＦＴＩＲによって測定した場合）、そしてここで、このような処方物は、５０℃で貯蔵される場合に、５０℃の溶液中でのこのタンパク質の可溶性形態よりも、少なくとも６０倍のより長い有効期限によって特徴付けられる（Ｔ_1/2によって測定した場合）。
【０２０２】
本発明に従う組成物は、上記のタンパク質結晶処方物のうちの１つ、および少なくとも１つのポリマーキャリアを含み、ここで、この処方物は、このポリマーキャリアのマトリックス中にカプセル化されている。
【０２０３】
あるいは、本発明に従う組成物は、タンパク質結晶および少なくとも１つの成分の処方物を含む。このような組成物は、５０℃で貯蔵された場合、５０℃の溶液中でのこのタンパク質の可溶性形態よりも、少なくとも６０倍のより長い有効期限によって特徴付けられ得る（Ｔ_1/2によって測定した場合）。あるいは、このような組成物は、４０℃および７５％の湿度で貯蔵された場合、４０℃および７５％の湿度で貯蔵されたこのタンパク質結晶の非処方物形態よりも、少なくとも５９倍のより長い有効期限によって特徴付けられる（Ｔ_1/2によって測定した場合）。あるいは、このような組成物は、５０℃で貯蔵された場合、５０℃で貯蔵されたこのタンパク質結晶の非処方物形態よりも、少なくとも６０％のより長い有効期限によって特徴付けられる（Ｔ_1/2によって測定した場合）。あるいは、このような組成物は、５０℃で４日間貯蔵された後のタンパク質のα−へリックス構造含有量の２０％未満の損失によって特徴付けられ、ここで、このタンパク質の可溶性形態は、５０℃で６時間貯蔵された後に、このタンパク質のα−へリックス構造含有量を５０％を超えて損失する（ＦＴＩＲによって測定した場合）。あるいは、このような組成物は、５０℃で４日間貯蔵された後のこのタンパク質のα−へリックス構造含有量の２０％未満の損失によって特徴付けれら、ここで、このタンパク質の可溶性形態は、５０℃で６時間貯蔵された後に、このタンパク質のα−へリックス構造含有量を５０％を超えて損失し（ＦＴＩＲによって測定した場合）、そしてここで、この処方物は、５０℃で貯蔵される場合に、５０℃の溶液中でのこのタンパク質の可溶性形態よりも、少なくとも６０倍のより長い有効期限によって特徴付けられる（Ｔ_1/2によって測定した場合）。
【０２０４】
本発明がより理解され得るように、以下に実施例を示す。これらの実施例は、例示のみの目的のためであり、いかなる様式によっても本発明の範囲を限定するように解釈され得ない。
【０２０５】
（実施例）
（実施例１ リパーゼ）
（Ｃａｎｄｉｄａ ｒｕｇｏｓａのリパーゼの結晶化）
材料：
Ａ−Ｃａｎｄｉｄａ ｒｕｇｏｓａのリパーゼの粉末
Ｂ−セライト（Ｃｅｌｉｔｅ）粉末（珪藻土）
Ｃ−ＭＰＤ（２−メチル−２，４−ペンタジオール）
Ｄ−５ｍＭ Ｃａアセテート緩衝液ｐＨ４．６
Ｅ−脱イオン水。
【０２０６】
（手順）
１ｋｇのリパーゼの粉末のアリコートを１ｋｇのセライトとよく混合し、次いで、２２Ｌの蒸留水を加えた。この混合物を攪拌してリパーゼの粉末を溶解させた。溶解が完了した後、酢酸を使用してｐＨを４．８に調整した。次に、この溶液を濾過して、セライトおよび溶解していない物質を除去した。次いで、この濾過物を３０Ｋカットオフ（ｃｕｔ−ｏｆｆ）中空繊維を使用してポンピング（ｐｕｍｐｉｎｇ）し、３０ｋＤ未満の分子量のタンパク質を全て除いた。蒸留水を加え、そしてリパーゼ濾過物を、残留物（ｒｅｔｅｎｔａｔｅ）の伝導率が蒸留水の伝導率と等しくなるまで中空繊維によりポンピングした。この時点で、蒸留水の添加を止め、そして５ｍＭのＣａ−アセテート緩衝液を加えた。次に、Ｃａ−アセテート緩衝液を、残留物の伝導率がＣａ−アセテート緩衝液の伝導率と等しくなるまで中空繊維によるポンピングにより送達した。その時点で、緩衝液の添加を止めた。このリパーゼ溶液を、３０ｍｇ／ｍｌ溶液にまで濃縮した。この結晶化を、ＭＰＤを攪拌しながらこのリパーゼ溶液に緩やかにポンピングすることによって開始させた。ＭＰＤが２０％容積／容積に達するまで、ＭＰＤの添加を続けた。この混合液を２４時間またはタンパク質の９０％が結晶化するまで攪拌した。得られた結晶を結晶化緩衝液で洗浄して、この結晶から可溶性物質を全て除去した。次いで、この結晶を新鮮な結晶化緩衝液中に懸濁して、４２ｍｇ／ｍｌのタンパク質濃度を達成した。
【０２０７】
（実施例２）
（賦形剤としてショ糖を使用するリパーゼ結晶の処方）
リパーゼ結晶の乾燥工程および貯蔵の間の安定性を増強するために、この結晶を賦形剤と共に処方した。この実施例において、リパーゼ結晶は、乾燥工程の前に、母液の存在下でスラリー形態で処方された。ショ糖（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）を、母液中のリパーゼ結晶に賦形剤として加えた。十分なショ糖を、母液（１０ｍＭ塩化カルシウムおよび２０％ ＭＰＤを含む１０ｍＭ酢酸ナトリウム緩衝液、ｐＨ４．８）中２０ｍｇ／ｍｌタンパク質濃度のリパーゼ結晶に、最終濃度が１０％に達するまで加えた。得られた懸濁物を室温で３時間反転させた。ショ糖での処置の後、この結晶を、この液体から、遠心分離（実施例６、方法４または５に記載される）により分離した。
【０２０８】
（実施例３）
（トレハロースを賦形剤として使用するリパーゼ結晶の処方）
リパーゼ結晶を、実施例２において、スクロースの代わりにトレハロース（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．、Ｓｔ．Ｌｏｕｉｓ、ＭＯ）を添加することにより処方し、母液中の最終濃度１０％にした。得られた懸濁物を、室温で３時間転倒混和（ｔｕｍｂｌｅ）し、そしてその結晶を、実施例６の方法４または５に記載のように、遠心分離により液体から分離した。
【０２０９】
（実施例４）
ポリエチレンオキシド（ＰＥＯ）を賦形剤として使用するリパーゼ結晶の処方）
リパーゼ結晶を、以下のように水中０．１％のポリエチレンオキシドを使用して処方した。この結晶（母液中２０ｍｇ／ｍｌ）を、スイングバケットローターを備えたＢｅｃｋｍａｎ ＧＳ−６Ｒ卓上遠心機において１０００ｒｐｍの遠心分離により母液から分離した。次いで、この結晶を、０．１％ポリエチレンオキシド（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．、Ｓｔ．Ｌｏｕｉｓ、ＭＯ）中で３時間懸濁し、次いで、実施例６の方法４または５に記載のように、遠心分離により分離した。
【０２１０】
（実施例５）
（メトキシポリエチレングリコール（ＭＯＰＥＧ）を賦形剤として使用するリパーゼ結晶の処方）
リパーゼ結晶を、実施例２にように、スクロースの代わりに母液中１０％のメトキシポリエチレングリコール（最終濃度）（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．、Ｓｔ．Ｌｏｕｉｓ、ＭＯ）を添加し、そして実施例６の方法４または５のように、３時間後に遠心分離により分離することにより処方した。
【０２１１】
（実施例６）
（結晶処方物を乾燥する方法）
（方法１．室温でのＮ₂ガス乾燥）
実施例１、１０、１４、および２１のように調製した結晶を、スイングバケットローターを備えたＢｅｃｋｍａｎ ＧＳ−６Ｒ卓上遠心機において１０００ｒｐｍの遠心分離により、５０ｍｌ ＦｉｓｃｈｅｒブランドＤｉｓｐｏｓａｂｌｅ遠心管（ポリプロピレン）中で、賦形剤を含む母液から分離した。次いで、この結晶を、約１０ｐｓｉ圧の窒素の気流をこの管に一晩通すことによって乾燥した。
【０２１２】
（方法２．真空オーブン乾燥）
実施例１、１０、１４、および２１のように調製した結晶を、まず、スイングバケットローターを備えたＢｅｃｋｍａｎ ＧＳ−６Ｒ卓上遠心機において１０００ｒｐｍの遠心分離を使用して、５０ｍｌ ＦｉｓｃｈｅｒブランドＤｉｓｐｏｓａｂｌｅポリプロピレン遠心管中で、母液／賦形剤溶液から分離した。次いで、湿った結晶を、室温にて２５ ｉｎ Ｈｇで真空オーブン（ＶＷＲ Ｓｃｉｅｎｔｉｆｉｃ Ｐｒｏｄｕｃｔｓ）中に配置し、そして少なくとも１２時間乾燥した。
【０２１３】
（方法３．凍結乾燥）
実施例１、１０、１４、および２１のように調製した結晶を、まず、スイングバケットローターを備えたＢｅｃｋｍａｎ ＧＳ−６Ｒ卓上遠心機において１０００ｒｐｍの遠心分離を使用して、５０ｍｌ ＦｉｓｃｈｅｒブランドＤｉｓｐｏｓａｂｌｅポリプロピレン遠心管中で、母液／賦形剤溶液から分離した。次いで、この湿った結晶を、Ｖｉｒｔｉｓ Ｌｙｏｐｈｉｌｉｚｅｒ Ｍｏｄｅｌ ２４を使用して、半分閉めたバイアル中で凍結乾燥した。貯蔵温度（ｓｈｅｌｆ ｔｅｍｐｅｒｔｕｒｅ）を、凍結工程の間ゆっくりと−４０℃へと低減した。この温度を１６時間保った。次いで、２次乾燥を、さらに８時間実行した。
【０２１４】
（方法４．有機溶媒および風乾）
実施例１、１０、１４、および２１のように調製した結晶を、まず、スイングバケットローターを備えたＢｅｃｋｍａｎ ＧＳ−６Ｒ卓上遠心機において１０００ｒｐｍの遠心分離を使用して、５０ｍｌ ＦｉｓｃｈｅｒブランドＤｉｓｐｏｓａｂｌｅポリプロピレン遠心管中で、母液／賦形剤溶液から分離した。次いで、この結晶を、有機溶媒（エタノールまたはイソプロパノールまたは酢酸エチルあるいは他の適切な溶媒のような）中に懸濁し、遠心分離し、その上清をデカントし、そして換気フード中で２日間室温で風乾した。
【０２１５】
（方法５．室温での風乾）
実施例１、１０、１４、および２１のように調製した結晶を、スイングバケットローターを備えたＢｅｃｋｍａｎ ＧＳ−６Ｒ卓上遠心機において１０００ｒｐｍの遠心分離により、５０ｍｌ ＦｉｓｃｈｅｒブランドＤｉｓｐｏｓａｂｌｅ遠心管（ポリプロピレン）中で、賦形剤を含む母液から分離した。続いて、結晶を、換気フード中で２日間風乾させた。
【０２１６】
（実施例７）
（可溶性リパーゼサンプル調製）
比較のために、可溶性リパーゼのサンプルを、リパーゼ結晶をリン酸緩衝化生理食塩水（ｐＨ７．４）に、２０ｍｇ／ｍｌに溶解することにより調製した。
【０２１７】
図２は、可溶性リパーゼについての安定性を示す。比活性は、時間とともに非常に迅速に減少する。２〜３時間以内、比活性は、約６６０マイクロモル／分／ｍｇタンパク質から約１００マイクロモル／分／ｍｇタンパク質に減少する（すなわち約８５％減少）。可溶性リパーゼについてのＴ_1/2を算出すると１．１２時間であった。
【０２１８】
（実施例８）
（リパーゼ活性を測定するためのオリーブオイルアッセイ）
実施例１〜７からのリパーゼ結晶を、ｐＨ７．７緩衝液中のオリーブオイルに対する活性について評価した。このアッセイを、Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｅｎｚｙｍｅｓ−Ｐｒｏｐｅｒｔｉｅｓ ａｎｄ Ａｓｓａｙ Ｍｅｔｈｏｄｓ、Ｒ．ＲｕｙｓｓｅｎおよびＡ．Ｌａｕｗｅｒｓ（編）、Ｓｃｉｅｎｔｉｆｉｃ Ｐｕｂｌｉｓｈｉｎｇ Ｃｏｍｐａｎｙ、Ｇｈｅｎｔ、Ｂｅｌｇｉｕｍ（１９７８）に記載される手順に対するわずかな改変形を使用して、滴定により実行した。
【０２１９】
（試薬）
１．オリーブオイルエマルジョン：
アラビアゴム（Ｓｉｇｍａ）１６．５ｇを、水１８０ｍｌ、オリーブオイル（Ｓｉｇｍａ）２０ｍｌに溶解し、そしてＱｕｉｃｋ Ｐｒｅｐミキサーを３分間使用して乳化した
２．滴定液：０．０５Ｍ ＮａＯＨ
３．溶液Ａ：３．０Ｍ ＮａＣｌ
４．溶液Ｂ：７５ｍＭ ＣａＣｌ₂・２Ｈ₂Ｏ
５．混合物：溶液Ａ４０ｍｌを、溶液Ｂ２０ｍｌおよび水１００ｍｌと混合した
６．０．５％アルブミン：
７．リパーゼ基質溶液（溶液７）を、オリーブオイルエマルジョン（溶液１）５０ｍｌを混合物（溶液５）４０ｍｌおよび０．５％アルブミン（溶液６）１０ｍｌに添加することにより調製した。
【０２２０】
（アッセイ手順）
リパーセ基質溶液（溶液７）を、水浴中で３７℃に温めた。まず、基質２０ｍｌを、反応容器に加え、そしてそのｐＨを、０．０５Ｍ ＮａＯＨ（溶液２）を使用して７．７に調整し、そしてかき混ぜながら３７℃に平衡化した。反応を、酵素を添加することにより開始した。反応の進行を、酵素と基質の混合物を０．０５Ｍ ＮａＯＨで滴定することによりそのｐＨを７．７に維持しながら、モニターした。
【０２２１】
比活性（マイクロモル／分／ｍｇタンパク質）は、（初速×１０００×滴定液の濃度）／（酵素の量）に等しかった。ゼロ点は、酵素を伴わない反応を実行すること（すなわち、反応混合物中で酵素の代わりに緩衝液を使用すること）により決定した。
【０２２２】
（実施例９）
（活性）
実施例１〜５からの乾燥結晶の貯蔵活性を、実施例８に記載のようなオリーブオイルアッセイを使用して測定した。乾燥結晶（５ｍｇ）を、リン酸緩衝化生理食塩水（「ＰＢＳ」）（ｐＨ７．４）１ｍｌに溶解し、そしてその活性を、オリーブオイルを基質として使用して測定した。
【０２２３】
（貯蔵安定性（ｓｈｅｌｆ ｓｔａｂｉｌｉｔｙ））
実施例２〜５からの乾燥結晶リパーゼ処方物の貯蔵安定性を、相対湿度７５％および温度４０℃に制御された湿潤チャンバー（ＨＯＴＰＡＣＫ）中で実行した。この結晶の活性を、乾燥サンプル５ｍｇをＰＢＳ緩衝液（ｐＨ７．４）に溶解し、オリーブオイルアッセイにおける活性を測定し、次いで最初の結果と比較することにより測定した。
【０２２４】
（方法５により乾燥した結晶処方物）
図３は、スクロース、トレハロース、およびＰＥＯを用いて処方されたリパーゼ結晶の貯蔵安定性プロフィールを示す。方法５により乾燥した場合、ＰＥＯが最も保護的な賦形剤であり、次にスクロース次いでトレハロースであった。
【０２２５】
（方法４により乾燥した結晶処方物）
図４は、スクロース、トレハロース、ＰＥＯ、およびＭＯＰＥＧを用いて処方されたリパーゼ結晶の貯蔵安定性プロフィールを示す。方法４により乾燥した場合、賦形剤ＰＥＯ、スクロースおよびＭＯＰＥＧは、Ｔ_1/2に対するそれらの効果により測定した場合に、酵素活性を保存するそれらの能力は同様であった。トレハロースは、他の賦形剤よりもリパーゼ活性の保護が劣った。
【０２２６】
（貯蔵安定性）
酵素の比活性が５０％減少するのに要する時間は、Ｔ_1/2として既知である。表２は、乾燥リパーゼ結晶の処方物についての比活性のＴ_1/2に対する効果を示す。リパーゼについて、スクロースが最も保護的な賦形剤であり、ポリエチレンオキシド（ＰＥＯ）、メトキシポリエチレングリコール（ＭＯＰＥＧ）、そして最後にトレハロースと続いた。スクロースは、比活性のＴ_1/2に対するその効果により測定した場合は、１０倍を超えてより保護的であった。
【０２２７】
【表２】

Ｔ_1/2を、Ｓｉｇｍａ Ｐｌｏｔプログラムを使用する非線形回帰分析により有効期限データから算出した。表２は、リパーゼ処方物が、ＰＥＯを賦形剤として使用した場合に可溶性よりも１，９２３倍安定であったことを示す。さらに、リパーゼ処方物は、スクロースを賦形剤として使用した場合に、可溶性リパーゼよりも２３，３００倍安定であった（表２）。ＭＯＰＥＧおよびＰＥＯを賦形剤として使用したリパーゼ結晶の処方物は、可溶性リパーゼよりも９，２７０倍および１７，８００倍安定であった（表２）。
【０２２８】
表２に示されるように、非処方結晶と比較して、処方された結晶の安定性は大きく増強されていた。表２に示されるように、例えば、トレハロースを用いて処方された結晶は、賦形剤を用いずに生成された非処方リパーゼ結晶よりも４０℃で５９倍安定であった。同様に、表２に示されるように、ＭＯＰＥＧを用いて処方された結晶は、非処方結晶よりも２８６倍安定であり、そしてＰＥＯを用いて処方された結晶は、賦形剤を用いずに生成された非処方リパーゼ結晶よりも４０℃で５４９倍安定であった。最後に、表２に示されるように、スクロースを用いて処方された結晶は、賦形剤を用いずに生成された非処方リパーゼ結晶よりも４０℃で７１８倍安定であった。
【０２２９】
（水分含量）
水分含量を、ＶＡ−０６ Ｖａｐｏｒｉｚｅｒを装備するＭｉｔｓｕｂｉｓｈｉ ＣＡ−０６ Ｍｏｉｓｔｕｒｅ Ｍｅｔｅｒ（Ｍｉｔｓｕｂｉｓｈｉ Ｃｈｅｍｉｃａｌ Ｃｏｒｐｏｒａｔｉｏｎ，Ｔｏｋｙｏ，Ｊａｐａｎ）を使用して、製造業者の指示に従って、Ｋａｒｌ Ｆｉｓｃｈｅｒ法によって決定した。
【０２３０】
【表３】

（結晶化度）
処方物の結晶強度を、定量的顕微鏡観察によって測定した。結晶が、乾燥後にその形状を維持するか否かを可視化するために、乾燥した結晶を、Ｉｍａｇｅ ＰｒｏＰｌｕｓソフトウェアとともにＣａｍｅｒａ ＡｄａｐｔｅｒとＤＸＣ−９７０ＭＤ ３ＣＣＤ Ｃｏｌｏｒ Ｖｉｄｅｏ Ｃａｍｅｒａを装着したＯｌｙｍｐｕｓ ＢＸ６０顕微鏡の下で検査した。乾燥させた結晶のサンプルを、ガラスカバーストリップで覆い、取り付け、そしてＯｌｙｍｐｕｓ ＵＰＬＡＮ Ｆ１対物レンズ １０×／０．３０ ＰＨ１を備えたＯｌｙｍｐｕｓ顕微鏡（位相差）使用して、１０倍の拡大率で検査した。
【０２３１】
この分析において、スクロース、トレハロース、ＰＥＯおよびＭＯＰＥＧでもともと処方された結晶は、４０℃および７５％湿度にて１２９日後に容易に可視化された。図５は、ＰＥＯで処方された結晶が、最初の時点で存在したことを示す。１２９日後に得られそして図６に示した類似の顕微鏡観察は、結晶化度が、期間全体にわたって維持されたことを実証する。類似のデータを、スクロース、ＭＯＰＥＧおよびトレハロースで処方した結晶について得た（データは示さず）。
【０２３２】
（ＦＴＩＲによる二次構造の特徴づけ）
フーリエ変換赤外分光法（「ＦＴＩＲ」）スペクトルを、Ｄｏｎｇらに記載されるように、Ｎｉｃｏｌｅｔモデル５５０Ｍａｇｎａシリーズ分光計で収集した（Ｄｏｎｇ，Ａ．，Ｃａｕｇｈｅｙ，Ｂ．，Ｃａｕｇｈｅｙ，Ｗ．Ｓ．，Ｂｈａｔ，Ｋ．Ｓ．およびＣｏｅ，Ｊ．Ｅ．Ｂｉｏｃｈｅｍｉｓｔｒｙ，１９９２；３１：９３６４−９３７０；Ｄｏｎｇ，Ａ．Ｐｒｅｓｔｒｅｌｓｋｉ，Ｓ．Ｊ．，Ａｌｌｉｓｏｎ，Ｓ．Ｄ．およびＣａｒｐｅｎｔｅｒ，Ｊ．Ｆ．Ｊ．Ｐｈａｒｍ．Ｓｃｉ．，１９９５；８４；４１５−４２４）。固体サンプルについて、１〜２ｍｇのタンパク質を、３５０ｍｇのＫＢｒ粉末で軽く施し、そして乱反射部品を用いて小さなカップを満たした。スペクトルを収集し、次いで、第２の誘導体を用いる二次構造の個々の成分の相対領域の決定のためのＧａｌａｃｔｉｃソフトウェアからのＧｒａｍｓ ３２、およびアミドＩ領域（１６００〜１７００ｃｍ^-1）の下でカーブフィッティングプログラムを用いて、処理した。
【０２３３】
比較のために、可溶性リパーゼサンプルを、リン酸緩衝化生理食塩水中にリパーゼ結晶を溶解することによって調製し、そして安定性についてＦＴＩＲによって分析した。
【０２３４】
二次構造を、以下のように決定した：ＦＴＩＲスペクトルを、Ｎｉｃｏｌｅｔモデル５５０ Ｍａｇｎａシリーズ分光計で収集した。可溶性リパーゼの１ｍｌのサンプルを、ＡＲＫ ＥＳＰのセレン化亜鉛結晶上に置いた。スペクトルを、最初の時点（０）およびほとんどの活性が損失したまたはほぼ０の活性になった後に収集した。次いで、獲得したデータを、第２の誘導体を用いる二次構造の個々の成分の相対領域の決定のためのＧａｌａｃｔｉｃソフトウェアからのＧｒａｍｓ ３２ソフトウェア、およびアミドＩ領域（１６００〜１７００ｃｍ^-1）の下でカーブフィッティングプログラムを用いて、処理した。
【０２３５】
【表４】

（結論）
表４は、可溶性リパーゼについて、約５０％および７５％のα−ヘリックスおよびβ−シート構造含量が、３日間にわたって損失したことを示す。ランダム構造の含量において対応する増加が存在した。
【０２３６】
対照的に、図４は、処方し乾燥した結晶が、可溶性酵素より非常に安定であることを示す。このような結晶は、４０℃および７５％湿度にて６６日後に、６．１％〜２１．１％の範囲のα−ヘリックス構造の損失を示した。この時点で、溶液中ランダムコイルは、３日間にわたって７〜５０％増加し、一方、結晶化度は、６６日後でさえ最小のランダムコイル含量を示した。
【０２３７】
ＦＴＩＲを用いて二次構造における変化をモニタリングすることで得られた表４のデータは、図４に示す活性データと相関した。詳細には、スクロース、ＰＥＯおよびＭＯＰＥＧで処方されたリパーゼは、トレハロースで処方した結晶（２１％のα−ヘリックス構造の損失を示し、そして最も低い活性プロフィールを有した）よりも、α−ヘリックス構造の有意により少ない損失を示し、かつ上昇した温度および湿度にて６６日間にわたって、より高い比活性を維持した。
【０２３８】
（実施例１０：ヒト血清アルブミン）
（ヒト血清アルブミンの結晶化）
１０グラムの粉末化したヒト血清アルブミンを、１００ｍＭリン酸緩衝液（ｐＨ５．５）（４℃）の７５ｍｌの攪拌した溶液に添加した。最終タンパク質濃度は１２０ｍｇ／ｍｌであった（溶液のＯＤ₂₈₀値から推定した）。最初に、硫酸アンモニウム溶液（７６７ｇ／ｌ）（脱イオン水で調製した）を、最終濃度が３５０ｇ／ｌまたは５０％飽和まで、このタンパク質溶液に添加した。次に、結晶化溶液を、５０％硫酸アンモニウム（ｐＨ５．５）中の５０ｍｇ／ｍｌアルブミン結晶１ｍｌを用いて「播種（ｓｅｅｄ）」した。種結晶（ｓｅｅｄ ｃｒｙｓｔａｌ）を、１００ｍＭリン酸緩衝液中５０％飽和の硫酸アンモニウム（ｐＨ５．５）の溶液との沈殿物を含まない結晶のサンプルを洗浄することによって調製した。播種した結晶化溶液を、４℃にて一晩、強力に回転するプラットフォーム上でインキュベートした。棒（ｒｏｄ）の形状の結晶（２０μｍ）は、約１６時間後の一晩の溶液中に出現した。
【０２３９】
（実施例１１）
（賦形剤としてゼラチンを用いるＨＳＡ結晶の処方）
ヒト血清アルブミン（ＨＳＡ）結晶の乾燥および保存の間の安定性を増強するために、この結晶を賦形剤とともに処方した。この実施例において、ＨＳＡ結晶を、乾燥前の母液の存在下でスラリー形態で処方した。ゼラチン（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）を、賦形剤としての母液中のリパーゼ結晶に添加した。十分なゼラチンを、母液（１００ｍＭリン酸緩衝液中２．５Ｍ硫酸アンモニウム（ｐＨ５．５））中のリパーゼ結晶に、タンパク質濃度２０ｍｇｓ／ｍｌにて、１０％の最終濃度に達するまで、添加した。得られた懸濁物を、室温にて３時間回転させた。ゼラチンでの処理の後、結晶を、実施例６の方法１に記載の遠心分離によって液体から分離した。
【０２４０】
（ＨＳＡ結晶の乾燥）
次いで、ＨＳＡ結晶を実施例６に記載の４つの方法によって乾燥させた。この結晶を、方法４の有機溶媒として冷エタノール（４℃）中に懸濁させた。
【０２４１】
（実施例１２）
（可溶性ヒト血清アルブミン調製）
比較のために、可溶性ＨＳＡサンプルを、水中２０ｍｇ／ｍｌでＨＳＡを溶解させることによって調製した。
【０２４２】
（実施例１３）
（貯蔵安定性（ｓｈｅｌｆ ｓｔａｂｉｌｉｔｙ））
ＨＳＡの乾燥した結晶処方物の貯蔵安定性を、５０℃の温度の水浴中で行った。この結晶の安定性を、ＦＴＩＲ分析により以下の構造崩壊によってモニタリングした。
【０２４３】
（水分含量）
水分含量を、ＶＡ−０６ Ｖａｐｏｒｉｚｅｒを装備するＭｉｔｓｕｂｉｓｈｉ ＣＡ−０６ Ｍｏｉｓｔｕｒｅ Ｍｅｔｅｒ（Ｍｉｔｓｕｂｉｓｈｉ Ｃｈｅｍｉｃａｌ Ｃｏｒｐｏｒａｔｉｏｎ，Ｔｏｋｙｏ，Ｊａｐａｎ）を使用して、製造業者の指示に従って、Ｋａｒｌ Ｆｉｓｃｈｅｒ法によって決定した。
【０２４４】
【表５】

（結晶化度）
処方物の結晶強度を、実施例９に記載のように、定量的顕微鏡観察によって測定した。図７は、ＨＳＡ結晶が、ゼラチンで処方物を調製した直後に、容易に可視化されたことを示す。図８は、結晶化度が、５０℃にて４日間後に維持されたことを示す。
【０２４５】
（ＦＴＩＲによる二次構造の特徴付け）
ＦＴＩＲスペクトルを、Ｄｏｎｇらに記載されるように、Ｎｉｃｏｌｅｔモデル５５０Ｍａｇｎａシリーズ分光計で収集した（Ｄｏｎｇ，Ａ．，Ｃａｕｇｈｅｙ，Ｂ．，Ｃａｕｇｈｅｙ，Ｗ．Ｓ．，Ｂｈａｔ，Ｋ．Ｓ．およびＣｏｅ，Ｊ．Ｅ．Ｂｉｏｃｈｅｍｉｓｔｒｙ，１９９２；３１：９３６４−９３７０；Ｄｏｎｇ，Ａ．Ｐｒｅｓｔｒｅｌｓｋｉ，Ｓ．Ｊ．，Ａｌｌｉｓｏｎ，Ｓ．Ｄ．およびＣａｒｐｅｎｔｅｒ，Ｊ．Ｆ．Ｊ．Ｐｈａｒｍ．Ｓｃｉ．，１９９５；８４；４１５−４２４）。固体サンプルについて、１〜２ｍｇのタンパク質を、３５０ｍｇのＫＢｒ粉末で軽く施し、そして乱反射部品を用いて小さなカップを満たした。スペクトルを収集し、次いで、第２の誘導体を用いる二次構造の個々の成分の相対領域の決定のためのＧａｌａｃｔｉｃソフトウェアからのＧｒａｍｓ ３２、およびアミドＩ領域（１６００〜１７００ｃｍ^-1）の下でカーブフィッティングプログラムを用いて、処理した。
【０２４６】
比較のために、可溶性ＨＳＡサンプルを、水中にＨＳＡ結晶を溶解することによって調製し、そして安定性についてＦＴＩＲによって試験した。二次構造を、実施例９に記載のように決定した。
【０２４７】
【表６】

（結論）
可溶性ＨＳＡは、溶液中で１時間後に７８％のα−ヘリックス含量の急速な減少、およびランダムコイル構造の対応する増加を示した。
【０２４８】
乾燥処方した結晶は、約１６％のα−ヘリックス含量の減少、β−シート含量の大幅な減少を示し、そしてランダム構造の含量の増加は示さなかった。
【０２４９】
（実施例１４：ペニシリンアシラーゼ）
（ペニシリンアシラーゼの結晶化）
Ｂｏｅｈｒｉｎｇｅｒ Ｍａｎｎｈｅｉｍからのペニシリンアシラーゼの硫酸アンモニウム懸濁物が、粗材料であった。ペニシリンアシラーゼの懸濁物を、脱イオン水を用いて１：３希釈した。この溶液を、３０Ｋメンブレンを用いるダイアフィルトレーションによって、Ａ_280nm／ｍｌで２００ＯＤの最終濃度まで濃縮した。次いで、この酵素溶液を、４Ｍ ＮａＨ₂ＰＯ₄・Ｈ₂Ｏを用いてＡ_280nm／ｍｌで１５０ＯＤｍまで希釈した。
【０２５０】
１．９Ｍ ＮａＰＯ₄の最終溶液濃度となる４Ｍおよび１Ｍの十分なＮａＨ₂ＰＯ₄・Ｈ₂Ｏの二相性溶液を、調製した。この場合、２８０ｍｌの１Ｍ ＮａＨ₂ＰＯ₄・Ｈ₂Ｏを、５４９ｍｌの４Ｍ ＮａＨ₂ＰＯ₄・Ｈ₂Ｏの頂部に注意深く重層した。この酵素溶液を、容器の側面に穏やかに注ぎ、１Ｍ層の上に層を形成した。船舶用インペラーと共にオーバーヘッド攪拌機を設定した。アジテーターを、その刃を４Ｍ／１Ｍ界面のすぐ下にして容器中に置いた。速度を、８．０の設定、すなわち６００ｒｐｍに調整した。このアジテーターのスイッチを入れ、１０分後に停止させた。インペラーを容器からはずした。種結晶の容積を、０．５容積％と０．１容積％との間で目盛り付きピペットを用いて測定し、そして２０Ｌの滅菌ポリカーボネート容器中に添加した。この種を、添加前に１０秒間よりも長く静置しなかった。この溶液を、フラットブレードインペラーを用いて約１分間、手動で攪拌した。この結晶化混合物を、２４時間静置させた。
【０２５１】
２４時間後、この容器を開け、そして溶液を３０秒間、手動で混合した。この溶液を、さらに２４時間沈澱させた。計４８時間後、上清のうちの１０ｍｌのサンプルを採取した。このサンプルを、０．２ミクロンＡｃｒｏｄｉｓｃを用いて濾過した。上清のＡ_280nmを測定した。上清のＡ_280nmが１．０ｍｇ／ｍｌよりも大きい場合、結晶化をさらに２４時間継続させた。上清の濃度が１．０ｍｇ／ｍｌ未満になった場合、結晶スラリーおよび上清のＡ_280nmを直接決定した。
【０２５２】
（実施例１５：ＰＡ結晶についてのペニシリンＧアッセイ）
ペニシリンアシラーゼについての活性アッセイの基礎は、この酵素によるベンジルペニシリンの酵素的加水分解を測定する滴定アッセイを含む。この酵素は、ペニシリンＧからのフェニルアセチル基の切断を触媒し、これによりｐＨの減少を引き起こす。この活性は、反応１分間に（２８℃にてｐＨ８を維持するために）必要な５０ｍＭ ＮａＯＨの容積の測定による。このアッセイは、塩化カリウムおよびＴｒｉｓを用いて作製された基質緩衝液を使用する。そしてこのアッセイを、Ｕ／ｍｇで報告した。
【０２５３】
（ペニシリンアシラーゼアッセイ）：
（このアッセイにおいて用いられる化学物質および溶液）：
１． ペニシリン−Ｇ、Ｓｉｇｍａ、カリウム塩
２． ０．０５Ｎ 水酸化ナトリウム
３． １．０Ｍ ＫＣ１、２０ｍＭ Ｔｒｉｓ緩衝液、ｐＨ８．０
４． １０ｍＭ Ｔｒｉｓ、１０ｍＭ ＣａＣｌ₂緩衝液、ｐＨ８．０
５． ０．０１Ｍ ＰＢＳ緩衝液、ｐＨ７．５
（基質溶液の調製）
１ｇのペニシリン−Ｇを、１０ｍｌの１．０Ｍ ＫＣ１、２０ｍＭ Ｔｒｉｓ緩衝液、ｐＨ８．０および約７０ｍｌのＤＩ水、１ｍｌの１０ｍＭ Ｔｒｉｓ、１０ｍＭ ＣａＣｌ₂溶液中に添加した。次いで、この溶液のｐＨを、０．０５Ｎ ＮａＯＨを用いて８．０に調整した。次いで、最終溶液容積を、１００ｍｌに調整した。この溶液を、各使用の前に新たに調製し、そして調製から５時間以内に使用した。
【０２５４】
（ＰＡサンプル溶液の調製）
ペニシリンアシラーゼのサンプルを、ＰＢＳ緩衝液、ｐＨ７．４中に溶解させた。代表的には、２ｍｇ〜４ｍｇ（乾燥重量）のタンパク質を、各アッセイに用いた。
【０２５５】
（ペニシリンＧの加水分解についてのアッセイ）
２０ｍｌのペニシリンアシラーゼ基質溶液を、滴定容器中に添加し、そして２８℃に平衡化させた。この酵素溶液をこの反応容器に添加した後、ｐＨを８．０に維持するために０．０５Ｎ ＮａＯＨを用いて反応混合物を滴定することにより、ペニシリン−Ｇの加水分解をモニタリングした。
【０２５６】
（実施例１６：ヒドロキシプロピル−β−シクロデキストリン（ＨＰＣＤ）を賦形剤として用いるＰＡ結晶の処方）：
乾燥および保存の間のＰＡ結晶の安定性を増強するために、この結晶を、賦形剤と共に処方した。本実施例では、ＰＡ結晶を、乾燥前に母液の存在下でスラリー形態で処方した。ヒドロキシプロピル−β−シクロデキストリン（ＨＰＣＤ）（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）を、賦形剤として母液中のＰＡ結晶に添加した。十分なＨＰＣＤを、母液（１．９Ｍ ＮａＨ₂ＰＯ₄、ｐＨ６．６）中２０ｍｇ／ｍのタンパク質濃度でリパーゼ結晶に添加して、１０％という最終濃度に到達させた。得られた懸濁物を、室温にて３時間転動（ｔｕｍｂｌｅｄ）させた。ＨＰＣＤを用いる処理後、この結晶を、実施例６の方法１に記載のように遠心分離によって液体から分離した。
【０２５７】
（実施例１７：母液自体を賦形剤として用いるＰＡ結晶の処方）：
ＰＡ結晶を、母液（１．９Ｍ ＮａＨ₂ＰＯ₄、ｐＨ６．６）を用いて処方した。この結晶を、実施例１６においてのように、遠心分離によって分離した。
【０２５８】
（実施例１８：ＰＡの乾燥）
次いで、実施例１５〜１７からのＰＡ結晶を、実施例６の方法１、３または４に従って乾燥させた。実施例６の方法４について用いた有機溶媒は、酢酸エチルであった。
【０２５９】
（実施例１９：可溶性ＰＡ調製物）：
比較標準として、可溶性ＰＡのサンプルを、２０ｍｇ／ｍｌで水中にＰＡ結晶を溶解させることによって調製した。
【０２６０】
可溶性ＰＡの安定性を、経時的に測定した。そして時間に対する５５℃でのプロット比活性を図９に示す。溶液中のＰＡ酵素活性は、迅速に減衰し、そして約２０時間後に検出できなかった。
【０２６１】
（実施例２０：乾燥結晶の活性）：
実施例１８からの乾燥結晶の活性を、実施例１５に記載される通りにＰｅｎ Ｇアッセイにおいて試験した。乾燥した結晶（５ｍｇ）を、１ｍｌの水中に溶解させ、そしてその活性を、基質としてＰｅｎ Ｇを用いて測定した。
【０２６２】
（貯蔵安定性）：
ＰＡ乾燥結晶処方物の貯蔵安定性を、ＰｉｅｒｃｅによるＲｅａｃｔｉｖｅ−Ｔｈｅｒｍ ＩＩＩ−Ｈｅａｔｉｎｇ／Ｓｔｉｒｒｉｎｇモジュールを５５℃の温度で用いて決定した。活性を、５ｍｇの乾燥サンプルを水中に溶解し、そして実施例１５に記載のＰｅｎ Ｇアッセイにおいて酵素活性を測定することにより測定し、そして最初の結果と比較した。図１０は、賦形剤を含むＰＡ結晶および賦形剤を含まないＰＡ結晶の貯蔵安定性プロフィールを示す。図１０はまた、窒素（方法１）または風乾（方法４）を用いて乾燥したＰＡ結晶処方物の貯蔵安定性プロフィールを示す。
【０２６３】
【表７】

Ｔ_1/2を、Ｓｉｇｍａ Ｐｌｏｔプログラムを用いる非線形回帰分析による有効期限データから算出した。処方された結晶の安定性は、処方されていない結晶に比較して、表７に示すように、増強された。例えば、ＨＰＣＤを用いて処方された結晶は、５５℃にて可溶性ＰＡより４１８倍安定であった（表７）。ＨＰＣＤを用いて処方された結晶は表７に示すように、賦形剤なしで作製された、処方されていないＰＡ結晶よりも、５５℃にて３．５倍安定であった。
【０２６４】
（水分含量）：
水分含量を、ＶＡ−０６ Ｖａｐｏｒｉｚｅｒ（Ｍｉｔｓｕｂｉｓｈｉ Ｃｈｅｍｉｃａｌ Ｃｏｒｐｏｒａｔｉｏｎ，Ｔｏｋｙｏ，Ｊａｐａｎ）を備えたＭｉｔｓｕｂｉｓｈｉ ＣＡ−０６ Ｍｏｉｓｔｕｒｅ Ｍｅｔｅｒを用いて製造業者の使用説明書に従ってＫａｒｌ Ｆｉｓｃｈｅｒ方法によって決定した。
【０２６５】
【表８】

（結晶性）：
処方物の結晶の完全性を、実施例９に記載される通りに定量的顕微鏡観察によって測定した。結晶によって示される通り、結晶性は、プロセスの間中維持され（データは示さず）、これは容易に可視可能であった。
【０２６６】
（ＦＴＩＲによる二次構造の特徴付け）：
安定性を、乾燥され、そして処方されたＰＡ結晶の二次構造含量を、実施例９に記載される通りにＦＴＩＲによって定量することによって評価した。可溶性ＰＡを、比較の目的のために用いた。
【０２６７】
【表９】

（結論）：
α−ヘリックス含量の減少は、可溶性ペニシリンアシラーゼについては１日後に７６％であった。対照的に、ＨＰＣＤを賦形剤として用い、そして処方物を実施例６の方法１によって乾燥した場合、５５℃の温度で４３日後にほんの３１％のα−ヘリックス含量が失われただけであった。
【０２６８】
（実施例２１：グルコースオキシダーゼ）
（グルコースオキシダーゼ結晶の調製）
本発明者らは、グルコースオキシダーゼの結晶を以下の通りに調製した。最初に、糖タンパク質を、アニオン交換クロマトグラフィーによって精製し、次いで結晶化パラメーターを最適化した（データは示さず）。
【０２６９】
これらの研究の結果として、本発明者らは、グルコースオキシダーゼの結晶化条件が、一般に、７％〜１７％のＰＥＧ ４０００またはＰＥＧ ６０００、８％〜２０％の２−プロパノールまたはエタノール、および３と６との間のｐＨに調整する緩衝液の範囲に入ることを見出した。しかし、この範囲内およびこの範囲付近の多くのセットの実験条件が、グルコースオキシダーゼおよび他の糖タンパク質の結晶化について満足な結果を生じ得ることを理解すべきである。当業者は、所望の大きさおよび品質の結晶を効率的に生じる正確な条件が、実験条件の差異（例えば、タンパク質および試薬の純度、攪拌速度、剪断力の効果、ならびに炭水化物含量）に起因して変化することを理解する。
【０２７０】
（グルコースオキシダーゼの大規模結晶化）
本発明者らは、グルコースオキシダーゼの予備的規模の結晶化に好ましい条件を決定した。予備的規模の結晶化は一般に、１００ｍｌ〜９００ｍｌの糖タンパク質を含む。
【０２７１】
（Ａ．種を用いない、一定ｐＨでの結晶化）
グルコースオキシダーゼを、水中でダイアフィルトレーションし、そして５と１５との間のＡ₂₈₀まで濃縮した。グルコースオキシダーゼ濃縮物を、ｐＨ５．０にて０．２Ｍ酢酸Ｎａ中に１８％ ＰＥＧ ６０００、３２％ ２−プロパノールを含有する１容積の結晶化試薬と混合した（１：１）。混合後、この溶液を６℃まで冷却した。グルコースオキシダーゼ結晶化溶液を、プロペラスターラーを用いて１００ｒｐｍにて２４時間攪拌した。この時間の間に、この結晶が徐々に形成された。
【０２７２】
（実施例２２：賦形剤としてトレハロースを使用するグルコースオキシダーゼの処方）
乾燥および貯蔵の間のグルコースオキシダーゼ（ＧＯＤ）結晶の安定性を増強するために、この結晶を、賦形剤とともに処方した。この実施例において、ＧＯＤ結晶を、乾燥の前に、母液の存在下でスラリー形態に処方した。トレハロース（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）を、賦形剤として母液中のＧＯＤ結晶に添加した。十分なトレハロースを、母液（３２％ イソプロパノールおよび９％ ＰＥＧ ６０００を含む１００ｍＭ 酢酸ナトリウム緩衝液（ｐＨ５．５））中の、タンパク質濃度が２０ｍｇ／ｍｌのＧＯＤ結晶に、終濃度が１０％に達するまで添加した。得られた懸濁物を、３時間室温で転倒混和した。トレハロースでの処理後、実施例６の方法１に記載のように遠心分離によって、結晶と液体とを分離した。
【０２７３】
（実施例２３：賦形剤としてラクチトール（ｌａｃｔｉｔｏｌ）を使用するグルコースオキシダーゼ結晶の処方）
母液に対して終濃度１０％になるまで（トレハロースの代わりに）ラクチトール（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）を添加することによって、グルコースオキシダーゼ結晶を実施例２２と同様に処方した。３時間の遠心分離後に、この結晶と母液／ラクチトール溶液とを分離した。
【０２７４】
（実施例２４：賦形剤としてヒドロキシプロピル−β−シクロデキストリン（ＨＰＣＤ）を使用するグルコースオキシダーゼ結晶の処方）
母液に対して終濃度１０％になるまで（トレハロースの代わりに）ヒドロキシプロピル−β−シクロデキストリン（ＨＰＣＤ）（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）を添加することによって、グルコースオキシダーゼ結晶をＨＰＣＤ使用して、実施例２２と同様に処方し、そして３時間インキュベートした。次いで、実施例６の方法１に記載のように、３時間の遠心分離後に、この結晶と母液／ＨＰＣＤ溶液とを分離した。
【０２７５】
（実施例２５：賦形剤としてゼラチンを使用するグルコースオキシダーゼ結晶の処方）
母液に対して終濃度１０％になるまで（トレハロースの代わりに）ゼラチン（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）を添加することによって、グルコースオキシダーゼ結晶を実施例２２と同様に処方した。３時間の遠心分離後に、この結晶と母液／ゼラチン溶液とを分離した。
【０２７６】
（実施例２６：賦形剤としてメトキシポリエチレングリコールを使用するグルコースオキシダーゼ結晶の処方）
母液に対して終濃度１０％になるまで、メトキシポリエチレングリコール（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）を添加することによって、グルコースオキシダーゼ結晶を実施例２２と同様に処方した。３時間の遠心分離後に、実施例６の方法１に記載のように、この結晶と母液／メトキシポリエチレングリコール溶液とを分離した。
【０２７７】
（実施例２７：賦形剤としてスクロースを使用するグルコースオキシダーゼ結晶の処方）
母液に対して終濃度１０％になるまでスクロース（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．，Ｓｔ．Ｌｏｕｉｓ，ＭＯ）を添加することによって、グルコースオキシダーゼ結晶を実施例２２と同様に処方した。３時間の遠心分離後に、この結晶と母液／スクロース溶液とを分離した。
【０２７８】
（実施例２８：グルコースオキシダーゼ処方物の乾燥）
上記のグルコースオキシダーゼ結晶処方物を、実施例６に記載の方法に従って乾燥させた。方法４は、有機溶媒として冷イソプロパノール（４℃）を使用した。
【０２７９】
（実施例２９：可溶性グルコースオキシダーゼの調製）
比較のために、可溶性グルコースオキシダーゼサンプルを、５０ｍＭ クエン酸緩衝液（ｐＨ６．０）中、２０ｍｇ／ｍｌでグルコースオキシダーゼ結晶を溶解することによって調製した。
【０２８０】
図１１は、５０℃での可溶性グルコースオキシダーゼの安定性を経時的に示す。比活性は時間がたつにつれて急速に減少する。２４時間後、比活性は、９０％を超えて減少する。可溶性グルコースオキシダーゼのＴ_1/2は、０．９１時間と算出された。
【０２８１】
（実施例３０：グルコースオキシダーゼ活性アッセイ）
以下のプロトコルを使用して、乾燥したグルコースオキシダーゼ結晶および結晶処方物の活性を決定した。
【０２８２】
化学薬品および溶液：
１．リン酸緩衝液（２０ｍＭ、ｐＨ７．３）、ＮａＣｌ（０．１Ｍ）溶液
２．２１ｍＭ Ｏ−ジアニシジンジヒドロクロリドストック溶液。使用前に、作業溶液として０．２１ｍＭまで希釈
３．２Ｍ グルコース溶液
４．ペルオキシド溶液（２ｍｇ／ｍｌ）
５．５０ｍＭ クエン酸緩衝液（ｐＨ６．０）。
【０２８３】
サンプル調製：
１．２ｍｇのグルコースオキシダーゼを１ｍｌの５０ｍＭ クエン酸緩衝液（ｐＨ６．０）に添加し、そして約２分間、十分にボルテックスする。この溶液を、室温で１時間転倒混和して、再構成した
２．上記の酵素溶液０．１ｍｌと４．９ｍｌの同じクエン酸緩衝液とを混合することによって希釈酵素溶液を調製する。
【０２８４】
酵素活性測定のためのアッセイ手順：
１．ＵＶ−Ｖｉｓ分光光度計によってこのアッセイをモニターした。速度モードを使用し、そして４６０ｎｍの波長および２５℃の温度を設定する
２．２５℃の水浴中でＯ−ジアニシジン／ホスフェート作業溶液を温め、そして使用の少なくとも２０分前に酸素でこの溶液を泡立てる
３．酵素溶液を添加せずに試薬溶液を使用してブランクを測定する
４．２．４ｍｌの酸素化したＯ−ジアニシジン／ホスフェート作業溶液、０．４ｍｌの２Ｍ グルコース溶液、および０．１ｍｌのペルオキシダーゼを、使い捨てキュベットにピペットで入れる
５．キュベット壁上に１０ｍｌの酵素サンプルを添加し（この工程でサンプルと試薬とが混合しないように、キュベットを傾ける）、そしてパラフィルム片で覆う。キュベットを２回反転させて素早く混合し、分光光度計のセル区画にキュベットを挿入し、そしてデータを収集し始める。
酵素比活性を以下の式を使用して算出する：
比活性＝Ａ＊Ｂ＊Ｃ／Ｄ＊Ｅ＊Ｆ
ここで、Ａ＝１分間あたりの４６０ｎｍでの吸光度単位の変化
Ｂ＝反応混合物容積（ｍｌ）
Ｃ＝希釈倍率
Ｄ＝１１．３（定数）
Ｅ＝使用した酵素の重量（ｍｇ）
Ｆ＝サンプル容積（ｍｌ）。
【０２８５】
（実施例３１：乾燥した結晶の活性）
グルコースオキシダーゼの乾燥した結晶処方物の活性を、実施例３０に記載のように測定した。
【０２８６】
（貯蔵（ｓｈｅｌｆ）安定性）
グルコースオキシダーゼ結晶処方物の貯蔵安定性の研究を行った。この場合、この処方物を、実施例６の方法４を用いて乾燥させ、そして２ｍｌのスクリューキャップ付きエッペンドルフチューブ中で１３日間、５０℃の温度にて水浴中に貯蔵した。特定の時点での活性を、５０ｍＭ クエン酸緩衝液（ｐＨ６．０）中に２ｍｇの乾燥したサンプルを溶解して、次いで、実施例３０に従って活性を測定することにより得た。
【０２８７】
５０℃で貯蔵した種々のグルコースオキシダーゼ処方物の貯蔵安定性を決定した。このデータを図１２に示す。ラクチトールは、上昇した温度において期間にわたり、グルコースオキシダーゼ比活性を保存するに最も有効な賦形剤であった。
【０２８８】
【表１０】

Ｔ_1/2を、Ｓｉｇｍａプロットプログラムを使用する非線形回帰分析により有効期限データから算出した。表１０は、トレハロースが賦形剤として使用された場合、グルコースオキシダーゼ処方物が可溶性グルコースオキシダーゼより、９５倍より安定であったことを示す。さらに、グルコースオキシダーゼ処方物は、賦形剤としてラクチトールが使用された場合、可溶性グルコースオキシダーゼより１２１倍より安定であった（表１０）。ＨＰＣＤまたはＭＯＰＥＧを賦形剤として使用するグルコースオキシダーゼ結晶処方物は、可溶性グルコースオキシダーゼより、それぞれ、６０倍および３２５倍より安定であった（表１０）。最後に、ゼラチンまたはスクロースを賦形剤として使用するグルコースオキシダーゼ結晶処方物は、可溶性グルコースオキシダーゼより、それぞれ、９９倍および８２倍より安定であった（表１０）。
【０２８９】
賦形剤としてトレハロース、ラクチトール、ＨＰＣＤ、ＭＯＰＥＧ、ゼラチンまたはスクロースのいずれかを用いて作製されたグルコースオキシダーゼ結晶処方物は、表１０に示されるように、Ｔ_1/2により測定した場合、賦形剤を用いずに作製されたグルコースオキシダーゼ結晶より、５０℃において２．５倍、３．２倍、１．６倍、８．６倍、２．６倍、または２．２倍、より安定であった。
【０２９０】
（水分含量）
水分含量を、ＶＡ−０６ Ｖａｐｏｒｉｚｅｒ（Ｍｉｔｓｕｂｉｓｈｉ Ｃｈｅｍｉｃａｌ Ｃｏｒｐｏｒａｔｉｏｎ，Ｔｏｋｙｏ，Ｊａｐａｎ）を備えたＭｉｔｓｕｂｉｓｈｉ ＣＡ−０６ Ｍｏｉｓｔｕｒｅ Ｍｅｔｅｒを使用して、製造業者の説明書に従ってＫａｒｌ Ｆｉｓｃｈｅｒ法により決定した。
【０２９１】
【表１１】

（結晶化度）
ＧＯＤ処方物の結晶完全性を、実施例９に記載のように定量的顕微鏡観察によって測定した。この実施例において、結晶は容易に結晶化された。このことにより、結晶化度は、プロセスの間中維持されたことを示す。
【０２９２】
図１３は、グルコースオキシダーゼ結晶がラクチトール処方物の調製直後に容易に視覚化されたことを示す。図１４は、結晶化度が５０℃で１３日間の後に維持されたことを実証する。結晶材料もまた、図１５に示すように、トレハロースでのグルコースオキシダーゼの処方後に容易に視覚化された。同様に、残った結晶材料も、図１６に示されるように、５０℃で１３日間の後に容易に視覚化された。
【０２９３】
（ＦＴＩＲによる二次構造の特徴）
実施例９に記載のように、乾燥したＧＯＤ結晶および処方したＧＯＤ結晶の二次構造量をＦＴＩＲにより定量することにより、安定性を評価した。比較のために、可溶性グルコースオキシダーゼサンプルを、５０ｍＭ クエン酸緩衝液（ｐＨ６．０）中にグルコースオキシダーゼ結晶を溶解し、そしてＡＲＫ ＥＳＰのセレン化亜鉛結晶上で約１ｍｌを配置することにより調製し、次いで、これを、ＦＴＩＲにより安定性について分析した。
【０２９４】
【表１２】

（結論）
可溶性グルコースオキシダーゼは、５０℃でわずか６時間以内で、そのα−ヘリックス含量の７５％を損失した。
【０２９５】
糖ラクチトール、スクロース、およびトレハロースは、高温での貯蔵の際のα−ヘリックス含量の損失を防止するのに、最も有効な賦形剤であった。ラクチトール、スクロース、およびトレハロース中で処方され、そして方法４によって乾燥されたグルコースオキシダーゼ結晶は、５０℃で４日後に、α−ヘリックス構造含量の１８．１〜１９．４％のみの損失を示した。
【０２９６】
（実施例３２：Ｃａｎｄｉｄａ ｒｕｇｏｓａリパーゼ結晶の乾燥）
材料：
Ａ−Ｃａｎｄｉｄａ ｒｕｇｏｓａリパーゼ（実施例１）
Ｂ−ポリ（エチレングリコール）、１００％、ＰＥＧ２００、３００、４００、または６００
Ｃ−アセトン
手順：
結晶懸濁液（１４０ｍｇ）の４ｍｌアリコートを、４つの１５ｍｌ管に添加する。次に、この懸濁液を、１０００ＲＰＭ〜３０００ＲＰＭの間で、１〜５分間の間、または結晶化緩衝液が除去されるまで遠心分離する。次いで、４ｍｌの液体ポリマー（２００と６００との間のいかなるＰＥＧも適切である）を各管に添加し、そしてこの内容物を均一になるまで混合する。この懸濁液を１０００ＲＰＭ〜３０００ＲＰＭの間で、１〜５分間の間、または液体ポリマーが除去されるまで、遠心分離する。次に、４ｍｌのアセトン（イソプロパノール、ブタノール、および他の溶媒もまた適切である）を各管に入れ、そして十分に混合する。結晶／有機溶媒懸濁液を、０．８ｃｍ×４ｃｍ ＢＩＯ−ＲＡＤポリ分取クロマトグラフィーカラム（スピンカラム）に移す。カラムを、有機溶媒を除去するために、１０００ＲＰＭで、１〜５分間の間、遠心分離する。最後に、窒素ガスをカラムに通し、自由流動粉末が生じるまで、結晶を乾燥させる。
【０２９７】
（実施例３３：Ｐｕｒａｆｅｃｔ（プロテアーゼ）４０００Ｌ結晶化）
材料：
Ａ−粗製ｐｕｒａｆｅｃｔ ４０００Ｌ
Ｂ−１５％ ＮａＳＯ₄溶液
手順：
１容量の粗製ｐｕｒａｆｅｃｔ酵素溶液を、２容量の１５％Ｎａ₂ＳＯ₄溶液と混合する。混合物を、室温で２４時間、または結晶化が完了するまで、攪拌する。結晶を１５％ Ｎａ₂ＳＯ₄溶液で洗浄し、可溶性酵素を除去する。結晶を新鮮な１５％ Ｎａ₂ＳＯ₄溶液中に懸濁し、２７ｍｇ／ｍｌのタンパク質濃度を得る。
【０２９８】
（実施例３４：Ｐｕｒａｆｅｃｔ結晶の乾燥）
材料：
Ａ−ｐｕｒａｆｅｃｔ結晶懸濁液
Ｂ−ポリ（エチレングリコール）、１００％ ＰＥＧ
２００、３００、４００、または６００
Ｃ−有機溶媒
手順：
結晶懸濁液（１４０ｍｇ）の４ｍｌアリコートを、４つの１５ｍｌ管に添加する。次に、この懸濁液を、１０００ＲＰＭ〜３０００ＲＰＭの間で、１〜５分間の間、または結晶化緩衝液が除去されるまで、遠心分離する。次いで、４ｍｌの液体ポリマー（２００と６００との間のいかなるＰＥＧも適切である）を各管に添加し、そしてこの内容物を均一になるまで混合する。この懸濁液を１０００ＲＰＭ〜３０００ＲＰＭの間で、１〜５分間の間、または液体ポリマーが除去されるまで、遠心分離する。次に、４ｍｌのアセトン（イソプロパノール、ブタノール、および他の溶媒もまた適切である）を各管に入れ、そして十分に混合する。結晶／有機溶媒懸濁液を、０．８ｃｍ×４ｃｍ ＢＩＯ−ＲＡＤポリ分取クロマトグラフィーカラム（スピンカラム）に移す。カラムを、有機溶媒を除去するために、１０００ＲＰＭで１〜５分間、遠心分離する。最後に、窒素ガスをカラムに通し、自由流動粉末が生じるまで、結晶を乾燥させる。
【０２９９】
（実施例３５：結晶化のためのＤＮＡの生成）
ｐＵＣプラスミド由来のプラスミド（例えば、ｐＳＰ６４）は、結晶化用のＤＮＡまたはｍＲＮＡのいずれかを産生するために使用され得る。本実施例において、プラスミドｐＳＰ６４（Ｐｒｏｍｅｇａ Ｂｉｏｌｏｇｉｃａｌ Ｒｅｓｅａｒｃｈ Ｐｒｏｄｕｃｔｓから入手可能）を用いて結晶化用のＤＮＡを生成させる。目的のタンパク質をコードするｃＤＮＡを、マルチプルクローニング部位で利用可能な多数の制限部位のいずれかに挿入する。次いで、組換えプラスミドを、Ｅ．ｃｏｌｉ細菌を形質転換するために使用する。次に、大量のプラスミドを、アンピシリン含有培地中の細菌の増殖により得る。組換えプラスミドを生産し、Ｅ．ｃｏｌｉ細胞を形質転換し、そして細菌増殖およびプラスミドＤＮＡ調製のための技術は「Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ、第２版」（１９８９）Ｓａｍｂｒｏｏｋ、Ｊ．、Ｆｒｉｔｓｃｈ、Ｅ．Ｆ．およびＴ．Ｍａｎｉａｔｉｓにおいて詳細に記載される。
【０３００】
続いて、プラスミドＤＮＡを、細胞をまず溶解し、次いでゲノムＤＮＡ、ＲＮＡ、および他の細胞材料からプラスミドＤＮＡをＣｓＣｌ勾配を用いて分離することにより、細菌培養物から精製する。これらの技術は、当該分野で周知であり、Ｓａｍｂｒｏｏｋらで詳細に議論されている。勾配精製ＤＮＡを、Ｔｒｉｓ−ＥＤＴＡ緩衝液飽和Ｎ−ブタノールで抽出し、そして最後にエタノールで抽出する。次に、プラスミドＤＮＡを、ＲＮＡ結晶化のためにｍＲＮＡの生成（実施例３６）、またはＤＮＡ結晶化のために目的の遺伝子の切り出し（実施例３７）に使用される、プラスミドのいずれかの線状化に供する。
【０３０１】
（実施例３６：結晶化のためのｍＲＮＡの生成）
ＳＰ６ ＲＮＡポリメラーゼと組み合わせたＳＰ６４プラスミド（Ｐｒｏｍｅｇａ Ｂｉｏｌｏｇｉｃａｌ Ｒｅｓｅａｒｃｈ Ｐｒｏｄｕｃｔｓから入手可能）を、５’キャップ化ＲＮＡ転写物のミリグラム量の生成のために使用する。実施例３５で調製したプラスミドを、遺伝子の切り出しの前に、ポリＡテールの下流の制限酵素で線状化する。この線状化したプラスミドを、２回のフェノール／クロロホルム抽出および２回のクロロホルム抽出によって精製する。次に、ＤＮＡを、ＮａＯＡｃ（０．３Ｍ）および２容量のＥｔＯＨで沈殿させる。次に、ペレットを、ＤＥＰＣ処理した蒸留した脱イオン水中におよそ１ｍｇ／ｍｌで再懸濁する。
【０３０２】
転写を、４００ｍＭ Ｔｒｉｓ ＨＣｌ（ｐＨ８．０）、８０ｍＭ ＭｇＣｌ₂、５０ｍＭ ＤＴＴ、および４０ｍＭスペルミジンで構成される緩衝液中で実施する。続く試薬を、室温で１容量のＤＥＰＣ処理水に対して以下の順に添加する：１容量のＳＰ６ ＲＮＡポリメラーゼ転写緩衝液；ｒＡＴＰ、ｒＣＴＰ、およびｒＵＴＰ（１ｍＭ濃度まで）；ｒＧＴＰ（０．５ｍＭ濃度まで）；７ｍｅＣ（５’）ｐｐｐ（５’）Ｇキャップアナログ（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ、Ｂｅｖｅｒｌｙ、Ｍａｓｓ．、０１９５１）（０．５ｍＭ濃度まで）；上記で調製した線状化ＤＮＡテンプレート（０．５ｍｇ／ｍｌ濃度まで）；ＲＮＡ（Ｐｒｏｍｅｇａ、Ｍａｄｉｓｏｎ、Ｗｉｓ．）（２０００Ｕ／ｍｌ濃度まで）；およびＳＰ６ ＲＮＡポリメラーゼ（Ｐｒｏｍｅｇａ、Ｍａｄｉｓｏｎ、Ｗｉｓ．）（３０００Ｕ／ｍｌ濃度まで）。転写混合物を、３７℃で１時間インキュベートする。
【０３０３】
次いで、ＤＮＡテンプレートを、使用した１μｇのＤＮＡテンプレート当たり２ＵのＲＱ１ ＤＮＡｓｅ（Ｐｒｏｍｅｇａ）を添加することにより消化する。消化反応を、１５分間実施する。転写ＲＮＡを、クロロホルム／フェノールで２回、およびクロロホルムで２回抽出する。上清溶液を、２容量のＥｔＯＨ中０．３Ｍ ＮａＯＡｃで沈殿させ、そしてこのペレットを、５００ｍｌ転写産物当たり１００ｍｌ ＤＥＰＣ処理脱イオン水で再懸濁する。最後に、上清溶液を、ＲＮＡｓｅ非含有Ｓｅｐｈａｄｅｘ Ｇ５０カラム（Ｂｏｅｈｒｉｎｇｅｒ Ｍａｎｎｈｅｉｍ ＃ １００ ４１１）に通す。得られるｍＲＮＡは、結晶化に用いられるのに十分に純粋である。
【０３０４】
（実施例３７：ＤＮＡ結晶化）
材料：
Ａ−精製プラスミドＤＮＡ（実施例３５）
Ｂ−スペルミン
Ｃ−ＭＰＤ（２−メチル−２，４−ペンタンジオール）
Ｄ−５ｍＭ酢酸カルシウム緩衝液（ｐＨ７．０）
Ｅ−脱イオン水
手順：
増幅されたＤＮＡ（実施例３５）を、制限消化によってプラスミドから除去する。挿入された遺伝子を、適切な分子量のゲルバンドからの目的遺伝子のアガロースゲル電気泳動および抽出によって、プラスミドビヒクルから精製する。
【０３０５】
ハンギングドロップ（ｈａｎｇｉｎｇ ｄｒｏｐ）技術を使用して、５ｍＭ酢酸ナトリウム／２０％ ＭＰＤ／１ｍＭスペリミン緩衝液（ｐＨ７．０）中５ｍｇ／ｍｌのＤＮＡを、９０％のＤＮＡが結晶化されるまで、室温でインキュベートする。得られる結晶を、結晶化緩衝液で洗浄し、全ての可溶性材料を結晶から除去する。次いで、５ｍｇ／ｍｌのＤＮＡ濃度を達成するまで、結晶を新鮮な結晶化緩衝液中に再懸濁する。
【０３０６】
（実施例３８：ＲＮＡ結晶化）
材料：
Ａ−精製ｍＲＮＡ（実施例３６）
Ｂ−スペルミン
Ｃ−ＭＰＤ（２−メチル−２，４−ペンタンジオール）
Ｄ−５ｍＭ酢酸カルシウム緩衝液（ｐＨ７．０）
Ｅ−脱イオン水
手順：
ハンギングドロップ技術を使用して、５ｍＭ酢酸カルシウム／２０％ ＭＰＤ／１ｍＭスペリミン緩衝液（ｐＨ７．０）中５ｍｇ／ｍｌのＲＮＡ（実施例３６）を、９０％のＲＮＡが結晶化されるまで、室温でインキュベートする。得られる結晶を、結晶化緩衝液で洗浄し、全ての可溶性材料を結晶から除去する。次いで、５ｍｇ／ｍｌのＲＮＡ濃度を達成するまで、結晶を新鮮な結晶化緩衝液中に再懸濁する。
【０３０７】
（実施例３９：ＤＮＡ結晶を用いるＨＩＶ ｇｐ１２０に対する免疫応答の誘発）
ＨＩＶ ｇｐ１６０をコードするＤＮＡを、実施例３６および３７の方法に従って調製する。次いで、ＨＩＶ ｇｐ１６０の結晶を、マウスの免疫化のために使用する。広範な中和免疫を誘発するワクチンを設計するために、ＨＩＶの初代分離株および実験分離株の両方の多くの遺伝的クローンは、Ａｉｄｓ Ｒｅｓｅａｒｃｈ ａｎｄ Ｒｅａｇｅｎｔ Ｐｒｏｇｒａｍ、Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅｓ ｏｆ Ａｌｌｅｒｇｙ ａｎｄ Ｉｎｆｅｃｔｉｏｕｓ Ｄｉｓｅａｓｅｓ、Ｒｏｃｋｖｉｌｌｅ ＭＤ ２０８５２から入手可能である。
【０３０８】
ＤＮＡ結晶を、結晶化緩衝液中で維持し、そして２００μｌ／マウスを後脚に注射する。ｇｐ１２０に対する免疫応答の発達を、１ヶ月の基準でＥＬＩＳＡにおいて対応するＶ３ループペプチドに対する血清抗体を測定することにより決定する。
【０３０９】
（実施例４０：ｍＲＮＡ結晶を用いるＨＩＶ ｇｐ１２０に対する免疫応答の誘発）
ＨＩＶ ｇｐ１６０をコードするＲＮＡを、実施例３５、３６および３８の方法に従って調製する。次いで、ＨＩＶ ｇｐ１６０ ｍＲＮＡの結晶を、マウスの免疫化のために使用する。広範な中和免疫を誘発するワクチンを設計するために、ＨＩＶの種々の初代分離株および実験分離株は、Ａｉｄｓ Ｒｅｓｅａｒｃｈ ａｎｄ Ｒｅａｇｅｎｔ Ｐｒｏｇｒａｍ、Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅｓ ｏｆ Ａｌｌｅｒｇｙ ａｎｄ Ｉｎｆｅｃｔｉｏｕｓ Ｄｉｓｅａｓｅｓ、Ｒｏｃｋｖｉｌｌｅ ＭＤ ２０８５２から入手可能である。
【０３１０】
ＲＮＡ結晶を、結晶化緩衝液中で維持し、そして２００μｌ／マウスを後脚に注射する。ｇｐ１２０に対する免疫応答の発達を、１ヶ月の基準でＥＬＩＳＡにおいて対応するＶ３ループペプチドに対する血清抗体を測定することにより決定する。
【０３１１】
（実施例４１ オリゴＤＮＡ結晶化）
材料：
Ａ−合成オリゴＤＮＡ
Ｂ−スペルミン
Ｃ−ＭＰＤ（２−メチル−２，４−ペンタンジオール）
Ｄ−５ｍＭ 酢酸Ｃａ緩衝液 ｐＨ７．０
Ｅ−脱イオン水
手順：
ハンギングドロップ（ｈａｎｇｉｎｇ ｄｒｏｐ）技術を用いて、５ｍＭ酢酸Ｍｇ／３０％ ＭＰＤ／１ｍＭスペルミン緩衝液（ｐＨ７．０）中５ｍｇ／ｍｌの合成オリゴＤＮＡを、このＤＮＡの９０％が結晶化するまで室温にてインキュベートする。得られた結晶を結晶化緩衝液で洗浄し、この結晶からすべての可溶性物質を除去する。次いで、この結晶を、新しい結晶化緩衝液中に再懸濁し、５ｍｇ／ｍｌのＤＮＡ濃度を達成する。
【０３１２】
（実施例４２ 遺伝子発現の阻害のためのアンチセンスＤＮＡ投与）
抑制されるべきｍＲＮＡ種のセンス鎖に相補的であるＤＮＡ配列をコードするオリゴＤＮＡ結晶を、実施例４１に記載するように生成する。次に、この結晶またはこの結晶を含む処方物を、遺伝子発現の阻害が意図される部位に投与する。引き続いて、細胞が、このＤＮＡ結晶または溶解されたＤＮＡを取り込み、そしてこのオリゴＤＮＡおよび宿主ｍＲＮＡが、相補的な塩基対を形成し、そして遺伝子発現が一定の時間阻害される。
【０３１３】
（カプセル化されたタンパク質結晶）
（実施例４３ Ｐｓｅｕｄｏｍｏｎａｓ ｃｅｐａｃｉａリパーゼの大規模結晶化）
１５ｋｇの粗製Ｐｓｅｕｄｏｍｏｎａｓ ｃｅｐａｃｉａリパーゼ（ＰＳ３０リパーゼ−Ａｍａｎｏ）（「ＬＰＳ」）のスラリーを、１００Ｌの蒸留した脱イオン水中に溶解し、そしてさらなる蒸留した脱イオン水を用いて容量を２００Ｌにした。この懸濁液を、Ａｉｒ Ｄｒｉｖｅ Ｌｉｇｈｔｎｉｎｇミキサー中で室温にて２時間混合し、次いで０．５μｍフィルターを通して濾過し、セリット（ｃｅｌｉｔｅ）を除去した。次いで、この混合物を、３Ｋ中空ファイバーフィルター膜カートリッジを使用して限外濾過し、そして１０Ｌ（１２１．４ｇ）に濃縮した。固体の酢酸カルシウムを、２０ｍＭ Ｃａ（ＣＨ₃ＣＯＯ）₂の濃度にまで添加した。必要に応じて、ｐＨを、濃酢酸を用いて５．５に調整した。この混合物を加熱し、そして３０℃の温度に維持した。硫酸マグネシウムを、０．２Ｍの濃度にまで添加し、続いてグルコポン（ｇｌｕｃｏｐｏｎ）を１％の濃度にまで添加した。次いで、イソプロパノールを２３％の最終濃度にまで添加した。得られた溶液を、３０℃にて３０分間混合し、次いで３０℃から１２℃にまで２時間にわたって冷却した。次いで、結晶化を１６時間進行させた。
【０３１４】
この結晶を沈殿させ、そして可溶性タンパク質を、末端に１０ｍｌのピペットを有するタイゴン管を備えた蠕動ポンプを用いて除去した。新しい結晶化溶液（２３％イソプロピルアルコール、０．２Ｍ ＭｇＳＯ₄、１％グルコポン、２０ｍＭ Ｃａ（ＣＨ₃ＣＯＯ）₂、ｐＨ５．５）を添加し、タンパク質の濃度を３０ｍｇ／ｍｌにした（１ｍｇ／ｍｌ溶液のＯ．Ｄ．２８０＝１．０、分光光度計を波長２８０で用いて測定した場合）。結晶の収量は、約１２０グラムであった。
【０３１５】
（架橋されたＬＰＳ結晶）
架橋されたＰｓｅｕｄｏｍｏｎａｓ ｃｅｐａｃｉａリパーゼ結晶（ＣｈｉｒｏＣＬＥＣ−ＰＣ^TMという名称で販売されており、Ａｌｔｕｓ Ｂｉｏｌｏｇｉｃｓ，Ｉｎｃ．（Ｃａｍｂｒｉｄｇｅ，Ｍａｓｓａｃｈｕｓｅｔｔｓ）から入手可能である）を用いて、実施例４８によって処方物を生成した。あるいは、上記のように調製したリパーゼ結晶は、任意の従来方法を用いて架橋され得る。
【０３１６】
（実施例４４ グルコースオキシダーゼ結晶の架橋）
次いで、本発明者らは、実施例２１において調製されたグルコースオキシダーゼ結晶を下記のように架橋した。この架橋手順は、グルタルアルデヒド、またはＴｒｉｓ緩衝液（２−アミノ−２−ヒドロキシメチル−１，３−プロパンジオール）、リジン、もしくはジアミノオクタンのいずれかを用いて前処理されたグルタルアルデヒドを含んだ。
【０３１７】
この架橋を、６０ｍｇのタンパク質を用いて行った。Ｔｒｉｓ前処理グルタルアルデヒド（４８ｍｇ Ｔｒｉｓ塩／１ｇグルタルアルデヒド）を、０．２Ｍリン酸ナトリウム、ｐＨ７．０中に懸濁した１ｇのＧＯ結晶あたり０．２ｇおよび０．６ｇの濃度にまで添加した。反応を、室温にて２時間進行させた。２時間後、この結晶をガラスファイバー紙を通して濾過および洗浄した。
【０３１８】
この架橋した結晶を、実施例４８に記載のようにカプセル化した。様々に架橋された結晶の間で、カプセル化プロセスにおいて差異はなかった。代表的なサンプルを、図２１に示す。
【０３１９】
（実施例４５ 架橋されたＣａｎｄｉｄａ ｒｕｇｏｓａリパーゼ結晶）
架橋したＣａｎｄｉｄａ ｒｕｇｏｓａリパーゼ結晶（ＣｈｉｒｏＣＬＥＣ−ＣＲ^TMという名称で販売されており、Ａｌｔｕｓ Ｂｉｏｌｏｇｉｃｓ，Ｉｎｃ．（Ｃａｍｂｒｉｄｇｅ，Ｍａｓｓａｃｈｕｓｅｔｔｓ）から入手可能である）を用いて、実施例４８によって処方物を生成した。あるいは、上記で調製したリパーゼ結晶は、任意の従来方法を用いて架橋され得る。
【０３２０】
（実施例４６ ヒト血清アルブミン結晶の架橋）
実施例１０において調製されたヒト血清アルブミン結晶に対して架橋を行った。この架橋反応を、５０％の飽和硫酸アンモニウムを含む母液中の攪拌された結晶溶液中で、４℃にて行った。ホウ酸塩で前処理したグルタルアルデヒドでの架橋の前には、この結晶を洗浄しなかった。
【０３２１】
前処理したグルタルアルデヒドを、１容量の５０％グルタルアルデヒド（「ＧＡ」）を、等量の３００ｍＭホウ酸ナトリウム（ｐＨ９）に添加することによって調製した。次いで、このグルタルアルデヒド溶液を、６０℃にて１時間インキュベートした。この溶液を、室温まで冷却し、そして濃ＨＣｌを用いてｐＨを５．５に調整した。次に、この溶液を、氷上で迅速に４℃にまで冷却した。
【０３２２】
前処理したグルタルアルデヒド（２５％）を、段階様式で、１５分間隔で０．０５％の増分（総濃度）を用いて、２％の濃度にまで結晶化溶液に添加した。使用された結晶化溶液のアリコートは、１ｍｌ〜５００ｍｌの容量の範囲に及んだ。次いで、この結晶を、５％ＧＡにし、そして４℃にて４時間インキュベートして架橋させた。最終的に、アルブミン架橋した結晶を、低速の遠心分離によって回収し、そしてｐＨ７．５、１００ｍＭ Ｔｒｉｓ ＨＣｌを用いて繰り返し洗浄した。この架橋された結晶が、凝集することなく高速で遠心分離され得るときに、洗浄を停止した。
【０３２３】
（実施例４７ ペニシリンアシラーゼの架橋）
前処理したグルタルアルデヒドを、実施例４６の方法によって調製した。
【０３２４】
前処理したグルタルアルデヒド（２５％）を、段階様式で、１５分間隔で０．０５％の増分（総濃度）を用いて、１．５％の濃度にまで結晶化溶液に添加した。使用された結晶化溶液のアリコートは、１ｍｌ〜５００ｍｌの容量の範囲に及んだ。最終的に、架橋した結晶を、低速の遠心分離によって回収し、そしてｐＨ７．５、１００ｍＭ Ｔｒｉｓ ＨＣｌを用いて繰り返し洗浄した。この架橋された結晶が、凝集することなく高速で遠心分離され得るときに、洗浄を停止した。
【０３２５】
（実施例４８ ポリ乳酸−コ−グリコール酸（ＰＬＧＡ）中のタンパク質結晶のマイクロカプセル化）
Ａ．糖タンパク質、タンパク質、酵素、ホルモン、抗体およびペプチド
Ｃａｎｄｉｄａ ｒｕｇｏｓａおよびＰｓｅｕｄｏｍｏｎａｓ ｃｅｐａｃｉａ由来のリパーゼ、Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ由来のグルコースオキシダーゼ、ならびにＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ由来のペニシリンアシラーゼの非架橋結晶を用いて、マイクロカプセル化を行った。さらに、Ｃａｎｄｉｄａ ｒｕｇｏｓａ由来のリパーゼ、Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ由来のグルコースオキシダーゼ、およびＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ由来のペニシリンアシラーゼの架橋された酵素結晶を用いて、マイクロカプセル化を行った。表１３は、この実施例によって生成されたマイクロスフェアのサンプルのおよその平均直径を示す。さらに、生成されたヒト血清アルブミンまたは任意の他のタンパク質結晶またはタンパク質結晶処方物は、この技術によってカプセル化され得る。
【０３２６】
【表１３】

Ｂ．乾燥結晶の調製：
実施例６によって乾燥された結晶または結晶処方物を、各々用いて、本発明のマイクロスフェアを生成し得る。本発明における使用のためのタンパク質結晶を乾燥させるための１つのプロセスは、空気乾燥を含む。
【０３２７】
実施例１からのＣａｎｄｉｄａ ｒｕｇｏｓａリパーゼ結晶（非架橋および架橋）、実施例２１および４４からのグルコースオキシダーゼ（非架橋および架橋）、ならびに実施例１４および４７からのペニシリンアシラーゼ（非架橋および架橋）の各々約５００ｍｇを、空気乾燥させた。最初に、母液を、３０００ｒｐｍでの５分間の遠心分離によって除去した。次に、結晶を、２５℃にて２日間、換気フード中に置いた。
【０３２８】
（Ｃ．ポリマーおよび溶媒）
タンパク質結晶をカプセル化するのに使用したポリマーは、ＰＬＧＡであった。ＰＬＧＡは、５０／５０ポリ（ＤＬ−ラクチド−ｃｏ−グリコリド）として、Ｂｉｒｍｉｎｇｈａｍ Ｐｏｌｙｍｅｒｓ，Ｉｎｃ．からロット番号Ｄ９７１８８から購入した。このロットは、ＨＦＩＰ中３０℃で、０．４４ｄｌ／ｇの固有の粘度を有していた。
【０３２９】
塩化メチレンは、分光学的グレードであり、そしてＡｌｄｒｉｃｈ Ｃｈｅｍｉｃａｌ Ｃｏ．Ｍｉｌｗａｕｋｅｅ，ＷＩから購入した。ポリビニルアルコールを、Ａｌｄｒｉｃｈ Ｃｈｅｍｉｃａｌ Ｃｏ．Ｍｉｌｗａｕｋｅｅ，ＷＩから購入した。
【０３３０】
（Ｄ．ＰＬＧＡにおける結晶のカプセル化）
結晶を、二重エマルジョン法を使用してＰＬＧＡ中にカプセル化した。一般的なプロセスは、以下の通りであった。乾燥タンパク質結晶またはタンパク質結晶のスラリーのいずれかを、まず、塩化メチレン中のポリマー溶液に添加した。その結晶をポリマーでコートし、そして新生マイクロスフェアとなった。次いで、有機溶媒溶液中のそのポリマーを、界面活性剤を含有するより大きな容量の水溶液へと移した。結果として、その有機溶媒は、蒸発し始め、そしてそのポリマーは硬化した。この実施例では、漸減濃度の乳化剤の２つの連続的な水溶液を、そのポリマーコートを硬化するために使用し、マイクロスフェアを形成した。以下の手順は、この一般的なプロセスの１つの例示であった。ポリマーサイエンスの当業者は、その手順の多くの改変が利用され得ること、および以下の実施例は本発明を限定することを意図されたものではないことを理解する。
【０３３１】
（１．０ 乾燥タンパク質結晶の使用）
実施例１に従って製造された架橋された、および架橋されていないＣａｎｄｉｄａ ｒｕｇｏｓａリパーゼの乾燥結晶、実施例２１および４５に従って製造された架橋された、および架橋されていないグルコースオキシダーゼ、実施例１４および４７に従って製造された架橋された、および架橋されていないペニシリンアシラーゼを、１５０ｍｇサンプルへと計量した。次いで、計量したタンパク質結晶を、２ｍｌの塩化メチレンおよび０．６ｇＰＬＧＡ／１ｍｌ溶媒のＰＬＧＡを含有する１５ｍｌのポリプロピレン遠心分離チューブ（Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）に直接添加した。その結晶を、溶媒の表面へ直接添加した。次いで、そのチューブを、２分間室温でボルテックスすることによって十分に混合し、ＰＬＧＡを含む溶媒中でタンパク質結晶を完全に分散させた。その結晶を、ポリマーで完全にコートした。新生マイクロスフェアを懸濁された状態に維持するために、さらにボルテックスまたは攪拌をして、さらなるコーティングを許容し得る。そのポリマーは、３．０節に記載されるように硬化され得る。
【０３３２】
（２．０ タンパク質結晶スラリーの使用）
Ｐｓｅｕｄｏｍｏｎａｓ ｃｅｐａｃｉａリパーゼの結晶スラリーを、２００μｌの母液当たり約５０ｍｇの結晶を使用して作製した。結晶スラリーを、塩化メチレンおよび０．６ｇのＰＬＧＡ／１ｍｌ溶媒のポリ（乳酸−ｃｏ−グリコール酸）の２ｍｌの溶液とともに、１５ｍｌポリプロピレン遠心分離チューブ（Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）へ迅速に注入した。そのニードルを溶媒の表面下に挿入し、そして溶液中に注入した。この場合、１５０ｍｇの総タンパク質、または６００μｌの水溶液を注入した。プラスチックシリンジＬｅｕｒ−ｌｏｋ（Ｂｅｃｔｏｎ−Ｄｉｃｋｉｎｓｏｎ＆Ｃｏｍｐａｎｙ）を使用して、２２ゲージ（Ｂｅｃｔｏｎ−Ｄｏｃｋｉｎｓｏｎ＆Ｃｏｍｐａｎｙ）のステンレススチールニードルを介して注入した。次いで、タンパク質結晶ＰＬＧＡスラリーを、２分間室温でボルテックスすることによって完全に混合した。その結晶をポリマーで完全にコートした。新生マイクロスフェアを懸濁した状態で維持し、さらにコーティングするために、必要に応じて、さらにボルテックスまたは攪拌した。
【０３３３】
（３．０ ポリマーコーティングの硬化）
液体ポリマーコートから塩化メチレンを除去することを容易にし、そしてそのポリマーをタンパク質結晶上で硬化させるために、２工程プロセスを利用した。工程間の差異は、乳化剤の濃度が第１の溶液においてずっと高く、そして第１の溶液の容量が、第２の溶液よりもずっと小さいことである。
【０３３４】
第１の工程において、ポリマーでコートされた結晶および塩化メチレン懸濁液を、室温で、０．５％塩化メチレンを含む水中１８０ｍｌの６％ポリビニルアルコール（本明細書中以下で「ＰＶＡ」）の攪拌したフラスコに滴下して加えた。この溶液を、１分間迅速に混合した。
【０３３５】
工程２において、新生マイクロスフェアを含有する第１のＰＶＡ溶液を、２．４リットルの冷（４℃）蒸留水中に迅速に注いだ。この最終浴を、４℃で１時間、溶液の表面を窒素下でおいて、穏やかに混合した。１時間後、マイクロスフェアを、０．２２μｍのフィルターを使用して濾過し、そして凝集を減少させるために０．１％Ｔｗｅｅｎ２０を含有する３リットルの蒸留水で洗浄した。
【０３３６】
（実施例４９）
（カプセル化した結晶の作製）
Ｐｓｅｕｄｏｍｏｎａｓ ｃｅｐａｃｉａリパーゼのカプセル化マイクロスフェアを、相分離技術によって調製する。実施例４３において調製された結晶ＬＰＳを、二重エマルジョン法を使用して、ポリ乳酸−ｃｏ−グリコール酸（「ＰＬＧＡ」）中にカプセル化する。タンパク質結晶の７００ｍｇのアリコートを、ＰＬＧＡを含有する塩化メチレン（０．６ｇ ＰＬＧＡ／１ｍｌ溶媒；１０ｍｌ）中に注入する。その混合物を、マイクロファインチップを供えたホモジナイザーを使用して、３０秒間、３，０００ｒｐｍでホモジナイスする。得られる懸濁液を、６％ポリ（ビニルアルコール）（「ＰＶＡ」）および塩化メチレン（４．５ｍｌ）を含有する攪拌しているタンク（９００ｍｌ）に移す。その溶液を、１，０００ｒｐｍで１分間混合する。ＰＶＡ溶液中のマイクロスフェアを、蒸留水中に浸漬させることによって沈殿させ、洗浄し、そして濾過する。次いで、そのマイクロスフェアを、凝集を減少させるために０．１％のＴｗｅｅｎを含有する蒸留水で洗浄し、そして２日間、４℃で窒素で乾燥させる。
【０３３７】
（実施例５０）
（Ａ．マイクロスフェアのタンパク質濃度）
実施例４８で調製したマイクロスフェアの総タンパク質含量を測定した。２５ｍｇのＰＬＧＡ／ＰＶＡマイクロスフェアの３連のサンプルを、１Ｎの水酸化ナトリウム中で４８時間混合させながらインキュベートした。次いで、タンパク質含量を、Ｂｒａｄｆｏｒｄ法（Ｍ．Ｍ．Ｂｒａｄｆｏｒｄ，Ａｎａｌｙｔｉｃａｌ Ｂｉｏｃｈｅｍｉｓｔｒｙ、ｖｏｌ．７２、２４８〜２５４頁（１９７６））およびＢｉｏＲａｄ Ｌａｂｏｒａｔｏｒｉｅｓ（Ｈｅｒｃｕｌｅｓ，ＣＡ）からの市販のキットを使用して見積もった。タンパク質を含有するマイクロスフェアを、結晶を含まないＰＬＧＡマイクロスフェアと比較した。これを表１４に示す。
【０３３８】
【表１４】

表１４から選択されたサンプルのミリグラム当たりの活性または比活性を決定した。これを表１５に示す。
【０３３９】
【表１５】

（実施例５１）
（マイクロスフェアからのタンパク質の放出）
実施例４８で調製されたＰＬＧＡマイクロスフェアからのタンパク質の放出を、０．２２μｍのフィルターを含有する微少遠心分離濾過チューブ中に５０ｍｇのタンパク質カプセル化ＰＬＧＡマイクロスフェアを配置することによって測定した。次に、６００μｌの放出緩衝液（０．２％Ｔｗｅｅｎ２０を含むリン酸緩衝化生理食塩水、ｐＨ７．４）を、フィルターの保持側のマイクロスフェアに添加した。そのチューブを３７℃でインキュベートし、溶解させる。経時的に放出させたタンパク質の量を測定するために、サンプルを異なる時間間隔で採取した。そのチューブを３０００ｒｐｍで１分間遠心分離し、そしてその濾過物をタンパク質活性および総タンパク質測定について取り出した。次いで、マイクロスフェアをさらなる６００μｌの放出緩衝液で再懸濁した。
【０３４０】
（Ａ．マイクロスフェアから放出された総タンパク質）
表１６は、ほぼ９０％のインプットしたタンパク質が全ての場合において回収されたことを示す。さらに、マイクロカプセル化したタンパク質結晶から放出された総Ｐｓｅｕｄｏｍｏｎａｓ ｃｅｐａｃｉａリパーゼのパーセントは、約５．７日間、またはインプットしたタンパク質の８０％を超えるものが、１５．８％／日の速度で放出されるまで、比較的定常であった。この長い迅速放出の後は、１日当たりわずか０．６％の放出のみが８日間続いた
対照的に、表１６は、Ｃａｎｄｉｄａ ｒｕｇｏｓａリパーゼ結晶が、まず最初ゆっくりとした放出を示し、続いて迅速な放出相が続いた反対のプロフィールを示したことをさらに示す。最初の３日間において、約１０％のタンパク質が２．４％／日の緩やかな勾配を伴って放出された。４日から１４日まで、さらにタンパク質の８０％が、直線的に、７．５％／日の勾配で、放出された。表１６に示した放出したプロフィールは、３７℃、ｐＨ７．４で得られた。
【０３４１】
これらのデータは、本発明のカプセル化したタンパク質が治療用タンパク質の生物学的送達に適していることを示す。送達の種々の速度は、タンパク質結晶、結晶の選択、結晶の架橋、カプセル化するポリマーの疎水性および親水性の特徴、カプセル化の数、マイクロスフェアの用量、および他の簡単に制御可能な変数の選択を操作することによって選択され得る。
【０３４２】
【表１６】

（Ｂ．マイクロスフェアから放出されたタンパク質の活性）
経時的に放出されたタンパク質の生物学的活性を、リパーゼマイクロスフェアについてのオリーブ油アッセイを用いて測定した。これらの結果を表１７に示す。
【０３４３】
表１７に示すように、放出されたタンパク質の生物学的活性は、マイクロスフェアが、活性タンパク質を保護および放出することを実証する。タンパク質の投入量に基づいて計算した放出された活性の累積率は、放出されたタンパク質の総量と密接な相関関係にあった（表１６と表１７とを比較のこと）。二つの異なる結晶リパーゼは、本質的に１００％活性なタンパク質を放出した。３７℃での液浸の７日後でさえ、マイクロスフェアから放出されたタンパク質は、完全に活性であった。
【０３４４】
【表１７】

上記に示す活性測定を、実施例８に記載したオリーブオイルアッセイを用いて行った。
【０３４５】
（実施例５２）
（ＰＬＧＡマイクロスフェアの顕微鏡検査）
結晶が、カプセル化後にインタクトであったか否かを可視化するために、実施例４８に従って調製したＰＬＧＡマイクロスフェアを、ＤＸＣ−９７０ＭＤ ３ＣＣＤカラービデオカメラ、カメラアダプター（ＣＭＡ Ｄ２）、Ｉｍａｇｅ ＰｒｏＰｌｕｓソフトウェアを備えたＯｌｙｍｐｕｓ ＢＸ６０マイクロスコープの下で検査した。乾燥マイクロスフェアのサンプルを、ガラスカバーガラスで覆い、マウントし、そしてＯｌｙｍｐｕｓ ＵＰＬＡＮ Ｅ１対物レンズ１０×０．３０ ＰＨ１（位相差）を備えるＯｌｙｍｐｕｓ顕微鏡を用いて１０×の拡大率の下で検査して、結晶を容易に可視化し、そして結晶サイズを決定した。マイクロスフェアサイズおよび結晶サイズを、ＯｌｙｍｐｕｓからのＩｍａｇｅ Ｐｒｏソフトウェアおよび製造業者によって提供される０．５〜１５０μｍサイジングビーズを用いて決定した。ＰＬＧＡマイクロスフェアの外側のサイズを決定し、結晶についても同様に決定した。
【０３４６】
図１７は、実施例４８の方法によってカプセル化されたＣａｎｄｉｄａ ｒｕｇｏｓａ由来のリパーゼの架橋酵素結晶を示す。結晶サイズは、約２５μｍであり、そしてマイクロスフェアは、約９０μｍであった。拡大率は、２５０×であった。
【０３４７】
図１８は、実施例４８の方法によってカプセル化されたＣａｎｄｉｄａ ｒｕｇｏｓａ由来のリパーゼの非架橋酵素結晶を示す。結晶サイズは、約２５μｍであり、そしてマイクロスフェアは、約１２０μｍであった。拡大率は、２５０×であった。
【０３４８】
図１９は、実施例４８の方法によってカプセル化されたＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ由来のペニシリンアシラーゼの架橋酵素結晶を示す。結晶サイズは、約２５μｍであり、そしてマイクロスフェアは、約７０μｍであった。拡大率は、２５０×であった。
【０３４９】
図２０は、実施例４８の方法によってカプセル化されたＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ由来のペニシリンアシラーゼの非架橋酵素結晶を示す。結晶サイズは、約５０μｍであり、そしてマイクロスフェアは、約９０μｍであった。拡大率は、２５０×であった。
【０３５０】
図２１は、実施例４８の方法によってカプセル化されたＡｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ由来のグルコースオキシダーゼの架橋酵素結晶を示す。結晶サイズは、０．５〜１μｍの範囲であり、そしてマイクロスフェアは、約５０μｍであった。拡大率は、５００×であった。
【０３５１】
図２２は、実施例４８の方法によってカプセル化されたＡｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ由来のグルコースオキシダーゼの非架橋酵素結晶を示す。結晶サイズは、０．５〜１μｍの範囲であり、そしてマイクロスフェアは、約５０μｍであった。拡大率は、５００×であった。
【０３５２】
図２３は、実施例４８の方法によって母液中にスラリーとしてカプセル化されたＰｓｅｕｄｏｍｏｎａｓ ｃｅｐａｃｉａ由来のリパーゼの非架橋酵素結晶を示す。結晶サイズは、約２．５μｍであり、そしてマイクロスフェアは、約６０μｍであった。拡大率は、５００×であった。
【０３５３】
図２４は、Ｐｓｅｕｄｏｍｏｎａｓ ｃｅｐａｃｉａ由来のリパーゼの非架橋酵素結晶を示す。結晶サイズは、約２．５μｍであった。拡大率は、１０００×であった。
【０３５４】
（実施例５３）
（タンパク質放出）
ＰＬＧＡマイクロスフェアからのタンパク質の放出を、０．２２μｍフィルターを含むマイクロ遠心濾過チューブ中に５０ｍｇのＰＬＧＡマイクロスフェアを置くことによって測定する。放出緩衝液（１０ｍＭ ＨＥＰＥＳ、ｐＨ７．４、１００ｍＭ ＮａＣｌ、０．０２％ Ｔｗｅｅｎ、０．０２％アジド）の６００μｌのアリコートを、フィルターの保持側面（ｒｅｔｅｎｔａｔｅ ｓｉｄｅ）上のマイクロスフェアへ添加して懸濁する。チューブを、３ｃｃバイアル栓で密閉し、パラフィルムを覆う。次いで、マイクロスフェアを、３７℃でインキュベートする。サンプルを、チューブの遠心分離（１３，０００ｒｐｍ、１分）によって経時的に得る。濾液を取り出し、そしてマイクロスフェアを、６００μｌの放出緩衝液を用いて再懸濁する。放出されたタンパク質の品質を、ＳＥＣ−ＨＰＬＣおよび酵素活性によってアッセイする。
【０３５５】
タンパク質結晶の形およびサイズを、本発明のタンパク質結晶処方物の溶解の割合か、または他の特性を調整するために選択し得る。
【０３５６】
（実施例５４）
（生物学的ポリマーを用いるリパーゼ結晶のカプセル化）
生物学的ポリマーはまた、タンパク質結晶のカプセル化に有用である。本実施例は、Ｃａｎｄｉｄａ ｒｕｇｏｓａリパーゼ結晶の架橋結晶および非架橋結晶のカプセル化を実証する。非架橋結晶および架橋結晶を、実施例１および実施例４５に記載のように調製した。抗体および化学薬品を、Ｓｉｇｍａから購入した。
【０３５７】
（１．０ 被覆結晶の調製）
１０ｍｇ／ｍｌにて１．５ｍｌのウシ血清アルブミン（「ＢＳＡ」）の溶液を、ｐＨ７に調整した５ｍＭリン酸緩衝液中で調製した。次いで、１５ｍｌのＣａｎｄｉｄａ ｒｕｇｏｓａリパーゼ結晶の１０ｍｇ／ｍｌ懸濁液を、５ｍＭ Ｋ／Ｎａリン酸緩衝液、１Ｍ ＮａＣｌ、ｐＨ７（「緩衝液」）中で調製した。ＢＳＡ溶液を結晶溶液へ添加し、そして二つの溶液を、十分に混合した。結晶を、オービタルシェーカー（ｏｒｂｉｔａｌ ｓｈａｋｅｒ）を使用してＢＳＡ中で３０分間ゆっくりと混合しながらインキュベートした。ＢＳＡとのインキュベーションに続いて、結晶を、真空ろ過によって一晩乾燥した。乾燥させた結晶をアルブミンを有さない緩衝液中に再懸濁した。結晶を、２８０ｎｍの吸光度で測定した洗浄液中にタンパク質を検出出来なくなるまでか、またはＡ_280nmが０．０１未満になるまで、緩衝液を用いて洗浄した。この結晶を、低速遠心分離によって回収した。
【０３５８】
（２．０ アルブミン被膜の検出）
被覆結晶を、ウェスタンブロットによって評価して、アルブミン層の存在を確認した。洗浄に続いて、被覆タンパク質結晶を、１００ｍＭ ＮａＯＨ中で一晩インキュベートして、マイクロスフェアを成分タンパク質へと溶解した。サンプルを、中和し、ろ過し、そしてＳａｍｂｒｏｏｋら、「Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ」，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｌａｂｏｒａｔｏｒｙ Ｐｒｅｓｓ，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ，Ｎｅｗ Ｙｏｒｋ（１９８９）に従ってＳＤＳ−ＰＡＧＥイムノブロットによって分析した。
【０３５９】
Ｃａｎｄｉｄａ ｒａｇｏｓａリパーゼのアルブミン被覆架橋結晶マイクロスフェアおよびアルブミン被覆非架橋結晶マイクロスフェアの両方のＳＤＳ−ＰＡＧＥイムノブロットの結果は、アルブミンと同じ分子量を有する単一の免疫反応種を示した。
【０３６０】
Ｃａｎｄｉｄａ ｒａｇｏｓａリパーゼのアルブミン被覆架橋結晶マイクロスフェアおよびアルブミン被覆非架橋結晶マイクロスフェアのサンプルを、ウシ血清アルブミンを特異的に認識しかつ結合する蛍光標識抗ＢＳＡ抗体と共にインキュベートした。次いで、過剰な抗体を、リン酸緩衝液を用いて洗浄することで取り除いた。蛍光顕微鏡下での、これらの蛍光性標識アルブミン被覆結晶マイクロスフェアの顕微鏡検査は、マイクロスフェアの特異的蛍光標識を示した。非被覆リパーゼ結晶をコントロールとして用い、そしてこれらは抗体の特異的結合を示さなかった。
【０３６１】
本発明者らは、上に本発明の多くの実施態様を記載したが、本発明者らの基礎的な構築物を変化させて、本発明のプロセスおよび組成物を利用する他の実施態様を提供し得ることは明白である。従って、本発明の範囲は、例示の目的で上に示した特定の実施態様によってよりはむしろ、ここに添付される特許請求の範囲によって定義されるべきであることが理解される。
【図面の簡単な説明】
【図１】 図１は、Ｃａｎｄｉｄａ ｒｕｇｏｓａリパーゼの以下の分子状態の相対的な安定性を示す：架橋アモルファス、液体、２０％有機溶媒における結晶、２０％有機溶媒における架橋結晶、有機溶媒を伴わない架橋結晶（「Ｘｌｉｎｋｅ ｐｐｔ」は、架橋沈殿物を示す）。
【図２】 図２は、４０℃での可溶性リパーゼの比活性を経時的に示す。
【図３】 図３は、４０℃および湿度７５％で方法１によって乾燥させたリパーゼ結晶処方物の貯蔵安定性を示す。
【図４】 図４は、４０℃および湿度７５％で方法４によって乾燥させたリパーゼ結晶処方物の貯蔵安定性を示す。
【図５】 図５は、初期時間０においてポリエチレンオキシドを用いて処方したリパーゼ結晶を示す。
【図６】 図６は、４０℃および湿度７５％で、１２９日間インキュベーションした後に、ポリエチレンオキシドを用いて処方したリパーゼ結晶を示す。
【図７】 図７は、初期時間０においてゼラチンを用いて処方したヒト血清アルブミン結晶を示す。
【図８】 図８は、５０℃にて４日間インキュベーションした後にゼラチンを用いて処方したヒト血清アルブミン結晶を示す。
【図９】 図９は、５５℃での可溶性ペニシニンアシラーゼの比活性を経時的に示す。
【図１０】 図１０は、５５℃での種々の乾燥ペニシリンアシラーゼ結晶処方物の貯蔵安定性を示す。
【図１１】 図１１は、可溶性グルコースオキシダーゼの比活性を経時的に示す。
【図１２】 図１２は、５０℃での種々の乾燥グルコースオキシダーゼ結晶処方物の貯蔵安定性を示す。
【図１３】 図１３は、初期時間０にてラクチトールを用いて処方したグルコースオキシダーゼ結晶を示す。
【図１４】 図１４は、５０℃にて１３日間インキュベーションした後にラクチトールを用いて処方したグルコースオキシダーゼ結晶を示す。
【図１５】 図１５は、初期時間０にてトレハロースを用いて処方したグルコースオキシダーゼ結晶を示す。
【図１６】 図１６は、５０℃にて１３日間のインキュベーション後にトレハロースを用いて処方したグルコースオキシダーゼ結晶を示す。
【図１７】 図１７は、Ｃａｎｄｉｄａ ｒｕｇｏｓａ由来のリパーゼのカプセル化し架橋した酵素結晶を示す。
【図１８】 図１８は、Ｃａｎｄｉｄａ ｒｕｇｏｓａ由来のリパーゼのカプセル化し架橋していない酵素結晶を示す。
【図１９】 図１９は、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ由来のペニシリンアシラーゼのカプセル化し架橋した酵素結晶を示す。
【図２０】 図２０は、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ由来のペニシリンアシラーゼのカプセル化し架橋していない酵素結晶を示す。
【図２１】 図２１は、Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ由来のグルコースオキシダーゼのカプセル化し架橋した酵素結晶を示す。
【図２２】 図２２は、Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ由来のグルコースオキシダーゼのカプセル化し架橋していない酵素結晶を示す。
【図２３】 図２３は、Ｐｓｅｕｄｏｍｏｎａｓ ｃｅｐａｃｉａ由来のリパーゼの架橋されていない酵素結晶のカプセル化した水性スラリーを示す。
【図２４】 図２４は、Ｐｓｅｕｄｏｍｏｎａｓ ｃｅｐａｃｉａ由来のリパーゼの架橋されていない酵素結晶を示す。[0001]
(Technical field of the invention)
The present invention relates to methods for the stabilization, storage, and delivery of biologically active macromolecules such as proteins, peptides, and nucleic acids. In particular, the present invention relates to protein or nucleic acid crystals, formulations containing them, and compositions. Provided are methods for crystallization of proteins and nucleic acids, and methods for the preparation of stabilized protein crystals or nucleic acid crystals for use in dry or slurry formulations. The crystals, crystal formulations, and crystal compositions of the present invention can be reconstituted with diluents for parenteral administration of biologically active polymer components.
[0002]
The methods of the invention are useful for preparing crystals of “naked” DNA and RNA sequences that encode therapeutic or immunogenic proteins and that can be administered parenterally. Dissolved DNA and RNA molecules that are subsequently taken up by cells and used to express proteins with the appropriate glycosylation pattern can be either therapeutic or immunogenic. Alternatively, the present invention is useful for preparing crystals, crystal formulations, and crystal compositions of RNA or DNA sense and antisense polynucleotides.
[0003]
The present invention further relates to encapsulating crystals, or crystal formulations of proteins, glycoproteins, enzymes, antibodies, hormones, and peptides in compositions for biological delivery to humans and animals. In accordance with the present invention, protein crystals or crystal formulations are encapsulated in a matrix containing a polymeric carrier to form a composition. The formulations and compositions enhance the retention of the protein's native biologically active tertiary structure and create a reservoir that can slowly release the active protein where and when needed. Such polymeric carriers include biocompatible polymers and biodegradable polymers. Biologically active proteins are subsequently controlled in a controlled manner over time, as determined by the particular encapsulation technique, polymer formation, crystal shape, crystal solubility, crystal crosslinking, and formulation conditions used. Released. For preparing stable formulations using methods, pharmaceutical ingredients or excipients for crystallizing proteins, and optionally encapsulating them in a polymeric carrier to produce a composition Methods and methods for using such protein crystal formulations and compositions for biomedical applications, including delivery of therapeutic proteins and vaccines, are provided. Additional uses for this protein crystal formulation and composition of the present invention include protein delivery in human food, agricultural feed, veterinary compositions, diagnostics, cosmetics, and personal care compositions.
[0004]
(Background of the Invention)
Proteins are used in a wide range of applications in the fields of pharmacy, veterinary products, cosmetics, and other consumer goods, food, feed, diagnostics, industrial chemistry and purification. Sometimes such uses have been limited by the inherent constraints of the proteins themselves, or have been constrained by the environment or medium in which they are used. Such constraints can result in poor protein stability, performance fluctuations, or high costs.
[0005]
It is inevitable that the higher order tertiary or quaternary structure of the protein should be preserved until the point where individual protein molecules are required to perform their unique functions. To date, a limiting factor for the use of proteins (especially in therapeutic regimes) is the sensitivity of the protein structure to chemical and physical modifications encountered during delivery.
[0006]
Various approaches have been utilized to overcome these barriers. However, these approaches often incur either loss of protein activity or additional expense of protein stabilizing carriers or formulations.
[0007]
One approach to overcoming barriers to the widespread use of proteins is the cross-linked enzyme crystal (“CLEC®”) technology (NL St. Clair and MA Navia, J. Am. Chem. Soc., 114, pages 4314-16 (1992)). See also PCT patent application PCT / US91 / 05415. Cross-linked enzyme crystals usually retain their activity in environments that are not compatible with enzyme function. Such environments include prolonged exposure to proteases, organic solvents, elevated temperatures, or extreme pH. In such an environment, the crosslinked enzyme crystals remain insoluble, stable and active.
[0008]
In general, despite recent advances in protein technology, the two issues discussed below continue to limit the use of biological macromolecules in industry and medicine. The first concerns the molecular stability and sensitivity of higher order tertiary structures to chemical and physical denaturation during manufacturing and storage. The second problem is that the field of biological delivery of therapeutic proteins is based on native proteins (eg, proteins, glycoproteins, etc.) at a rate where the provided vehicle is consistent with the needs of a particular patient or disease process. Enzymes, antibodies, hormones, nucleic acids, and peptides).
[0009]
(Polymer stability)
A number of factors distinguish biological macromolecules from traditional chemical entities such as their size, conformation, and amphiphilic properties. Macromolecules are not only sensitive to chemical degradation, but are also sensitive to physical degradation. They are sensitive to various environmental factors such as temperature, oxidant, pH, freezing, shaking, and shear stress (Cholwinski, M., Luckel, B. and Horn, H., Acta Helv. 71, 405 (1996)). In considering macromolecules for drug development, stability factors must be considered when choosing a production process.
[0010]
Maintenance of biological activity during the development and manufacture of a pharmaceutical product depends on the inherent stability of the polymer and the stabilization technology utilized. There are various protein stabilization techniques including:
a) Addition of chemical “stabilizers” to aqueous solutions or suspensions of proteins. For example, US Pat. No. 4,297,344 discloses heat stabilization of coagulation factor II and coagulation factor VIII, antithrombin III and plasminogen by the addition of selected amino acids. U.S. Pat. No. 4,783,441 discloses a method for stabilizing proteins by the addition of surfactants. US Pat. No. 4,812,557 discloses a method for stabilizing interleukin-2 using human serum albumin. The disadvantage of such a method is that each formulation is specific for the protein of interest and requires significant development effort.
[0011]
b) A freeze / thaw method in which the preparation is mixed with a cryoprotectant and stored at cryogenic temperatures. However, not all proteins survive a freeze / thaw cycle.
[0012]
c) Cold storage with cryoprotective adduct (usually glycerol).
[0013]
d) Storage in glass form as described in US Pat. No. 5,098,893. In this case, the protein is dissolved in a water-soluble substance or a water-swellable substance in an amorphous or glassy state.
[0014]
e) The most widely used method for protein stabilization is freeze-drying or lyophilization (Carpenter, JF, Pical, MJ, Chang, B.S. And Randolph, T.W., Pharm.Res., 14: (8) 969 (1997)). Lyophilization provides the most viable alternative whenever sufficient protein stability cannot be achieved in aqueous solution. One disadvantage of lyophilization is that it requires sophisticated processing, is time consuming, and expensive (Carpenter, JF, Pical, MJ, Chang, B. et al. S. and Randolph, TW, Pharm. Res., 14: (8) 969 (1997) and references cited therein). Furthermore, if the lyophilization is not performed carefully, the freezing and dehydration steps of this technique will at least partially denature most preparations. The result is frequently an irreversible aggregation of portions of the protein molecule, which makes the formulation unacceptable for parenteral administration.
[0015]
Most of the protein formulations produced by the above techniques require cryogenic storage (sometimes as low as -20 ° C). Exposure to elevated temperatures during shipping or storage can result in significant loss of activity. Thus, storage is not possible for many proteins, even at elevated temperatures or even at ambient temperatures.
[0016]
Proteins, peptides, and nucleic acids are increasingly used in the pharmaceutical, diagnostic, food, cosmetic, detergent, and research industries. There is a great need for alternative stabilization procedures that are fast, inexpensive and applicable to a wide range of biological macromolecules. In particular, there is a need for a stabilization procedure that does not rely on the use of excess excipients that can interfere with the function of these biological macromolecules.
[0017]
The stability of the small molecule drug is such that it can be resistant to extreme forces during the formulation process (see US Pat. No. 5,510,118). The forces associated with nanoparticle milling of relatively insoluble drug crystalline materials include: shear stress, turbulence, high impact impact, cavitation, and crushing. Small molecule crystalline compounds are understood to be much more stable to chemical degradation than the corresponding amosphas solids (Pical, MJ, Lukes, AL, Lang, JE, and Gaines, J. Pharm. Sci., 67, 767 (1978)). Unfortunately, macromolecule crystals such as proteins and nucleic acids present additional problems and difficulties not associated with small molecules.
[0018]
For the majority of this century, science and medicine have attempted to solve the problem of providing insulin in a useful form for diabetes. Attempts have been made to solve some of this protein stability and biological delivery problems. For example, US Pat. No. 5,506,203 describes the use of amorphous insulin in combination with an absorption enhancer. Solid state insulin was exclusively an amorphous material as shown by polarized light microscopy.
[0019]
Jensen et al. Co-precipitated insulin with an absorption enhancer for use in airway delivery of insulin (see PCT patent application WO 98/42368). Here, absorption enhancers have been described as surfactants (eg, fatty acid salts or bile salts). Insulin crystals less than 10 micrometers in diameter and lacking zinc are Made by Havelund (see PCT patent application WO 98/42749). Similarly, crystals were also made in the presence of surfactants to enhance pulmonary administration.
CA-A-1 196 864 relates to a composition comprising a crystal of insulin and a biodegradable matrix of a biodegradable polymer. The biodegradable polymer is further defined as any convenient biodegradable polymer that dissolves in an organic solvent and allows formation of a matrix therewith.
[0020]
To date, those skilled in the art have recognized that the great enhancement of crystalline state stability observed for small molecules is not converted to biological macromolecules (Pical, MJ and Rigsbee, D R., Pharm. Res., 14: 1379 (1997)). For example, an aqueous suspension of crystalline insulin is only slightly more stable (up to a factor of 2) compared to the corresponding amorphous phase suspension (Brange, J., Langkjaer, L., Havelund). S., and Volund, A., Pharm. Res., 9: 715 (1992)). In the solid state, lyophilized amorphous insulin is much more stable than lyophilized crystalline insulin under all conditions investigated (Pical, MJ and Rigsbee, DR, Pharm. Res., 14: 1379 (1997)).
[0021]
Molecular weight has a significant effect on all properties of a polymer, including volume, hydration, viscosity, diffusion, mobility, and stability of the polymer. (Cantor, CR and Schimmel, PR, Biophysical Chemistry, WH Freeman and Co., New York, 1980).
WO 96/18417 relates to a pharmaceutical composition comprising crystalline protein and polyethylene glycol or a pharmaceutically acceptable vegetable oil. This invention has been described by the authors as the unexpected high controlled release properties obtained when crystalline proteins are suspended in PEG solutions or gels, or vegetable oils. The authors specifically claim a composition comprising a crystalline protein selected from the group consisting of: crystalline erythropoietin (EPO), crystalline granulocyte colony stimulating factor (G-CSF). , Crystalline insulin, and crystalline granulocyte-macrophage colony stimulating factor (GM-CSF).
WO-A-9 641 873 relates to a formulation of a polynucleotide complex that is stabilized with a cryoprotective compound and lyophilized. The lyophilized formulation is then powdered or sieved into a dry powder formulation that can be used to deliver the polynucleotide complex.
[0022]
(Summary of the Invention)
The inventors have surprisingly found that biological macromolecules that are not stable when held in solution at ambient or elevated temperatures are nevertheless long-term at such temperatures in crystalline form. It has been discovered that it can be successfully stored in dry form. In practice, the five aspects of this discovery are particularly advantageous.
[0023]
First, the crystallinity of the material being stored is very important. This is because large-scale crystallization can be introduced as a final purification step and / or concentration step in a clinical manufacturing process (eg, a process for manufacturing a therapeutic agent or vaccine). Furthermore, large scale crystallization can replace some of the purification steps in the manufacturing process. For example, protein crystallization can streamline the production of a protein formulation, making it more convenient.
[0024]
Second, the polymer interactions that occur in solution are prevented or severely reduced in the crystalline state due to a substantial decrease in the overall reaction rate. This crystalline state is therefore uniquely suitable for the preservation of mixtures of biological macromolecules.
[0025]
Third, solid crystalline preparations can be easily reconstituted to produce easily used parenteral formulations with very high protein concentrations. Such protein concentrations are considered particularly useful when the formulation is intended for subcutaneous administration (see PCT patent application WO 97/04801). For subcutaneous administration, an injection volume of 1.5 ml or less is well tolerated. Thus, for proteins administered at 1 mg / ml on a weekly basis, a protein concentration of at least 50 mg / ml is required and 100-200 mg / ml is preferred. These concentrations are difficult to achieve in liquid formulations due to aggregation problems. These concentrations can be easily achieved in the crystal formulations of the present invention.
[0026]
Fourth, protein crystals also constitute a particularly advantageous form for pharmaceutical dosage preparations. The crystals can be used as a basis for sustained release formulations in vivo. As those skilled in the art will appreciate, particle size is important for crystal dissolution and release of activity. It is also known that the release rate is more predictable if the crystals have a substantially uniform particle size and do not contain amorphous precipitates (see EP 0 265 214). ). Thus, protein crystals can be advantageously used in implantable devices (see PCT patent application WO 96/40049). The implant reservoir is generally approximately 25-250 μl. Due to this volume limitation, high concentration formulations (greater than 10%) and minimal amounts of suspension vehicle are preferred. The protein crystals of the present invention can be readily formulated in non-aqueous suspensions at such high concentrations.
[0027]
Fifth, another advantage of crystals is that certain variables can be manipulated to regulate the release of macromolecules over time. For example, the crystal size, shape, formulation with excipients that result in dissolution, cross-linking, cross-linking to the polymer matrix and the level of encapsulation can all be manipulated to produce a delivery vehicle for the biological molecule. .
[0028]
The present invention uses the most stable form, crystalline form of the active protein, and (1) adding ingredients or excipients when it is necessary to stabilize the dried crystals, or (2) The protein crystal or crystal formulation is encapsulated in a polymeric carrier to produce a composition containing each crystal and subsequently allow the release of active protein molecules to overcome the above obstacles. . Any form of protein (including glycoproteins, antibodies, enzymes, hormones or peptides) can be crystallized and stabilized or encapsulated in the composition according to the methods of the invention. In addition, nucleic acids encoding such proteins can be similarly processed.
[0029]
Crystals can be encapsulated with a variety of polymeric carriers with unique properties suitable for delivery to different or specific environments or to provide specific functions. The rate of dissolution of the composition and hence the delivery of the active protein is dependent on crystal size, polymer composition, polymer cross-linking, crystal cross-linking, polymer thickness, polymer hydrophobicity, polymer crystallinity or polymer solubility. It can be adjusted by varying.
[0030]
Addition of ingredients or excipients to the crystals of the present invention or encapsulation of protein crystals or crystal formulations results in further stabilization of the protein ingredients.
[0031]
(Detailed description of the invention)
In order that the invention described herein may be more fully understood, the following detailed description is set forth. In this description, the following terms are used.
[0032]
Amorphous solid-an amorphous solid form of a protein. Sometimes called amorphous precipitation. It does not have a molecular lattice structure that is characteristic of the solid state of the crystal.
[0033]
Antisense polynucleotide—RNA or DNA encoding RNA that is complementary to the mRNA of a gene whose inhibition of expression is intended.
[0034]
Aqueous organic solvent mixture—a mixture comprising n% organic solvent (where n is between 1 and 99) and m% aqueous solvent (where m is 100-n).
[0035]
Biocompatible polymer—A polymer that is non-antigenic (when not used as an adjuvant), non-carcinogenic, non-toxic, and otherwise not inherently incompatible with living organisms. Examples include: poly (acrylic acid), poly (cyanoacrylate), poly (amino acid), poly (anhydride), poly (depsipeptide), poly (ester) (eg, poly (lactic acid) or PLA) , Poly (lactic acid coglycolic acid) or PLGA, poly (β-hydroxybutyrate), poly (caprolactone) and poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide), poly [( Organo) phosphazene], poly (orthoester), poly (vinyl alcohol), poly (vinyl pyrrolidone), maleic anhydride-alkyl vinyl ether copolymer, pluronic polyol, albumin, alginic acid, cellulose and cellulose derivatives, co Gen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycidyl Camino glycans, sulfated polysaccharide, their blends and copolymers.
[0036]
Biodegradable polymer—A polymer that degrades by hydrolysis or solubilization. Degradation can be non-uniform (primarily occurring at the particle surface) or uniform (degrading uniformly throughout the polymer matrix) or a combination of such processes.
[0037]
Biological macromolecules-biological polymers such as proteins, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). For purposes of this application, biological macromolecules are also referred to as macromolecules.
[0038]
Change in chemical composition-any change in the chemical component of the environment surrounding a protein or nucleic acid crystal or crystal formulation that affects the stability or dissolution rate of the crystal component.
[0039]
Change in shear force—under conditions of use, any change in environmental factors surrounding the protein or nucleic acid crystal or crystal formulation (eg, change in mechanical pressure (which may be positive or negative) Good), rotary stirring, centrifugation, tumbling, mechanical shaking and filtration pump).
[0040]
Composition—any of non-crosslinked protein crystals, cross-linked crystals, nucleic acid crystals or formulations containing them. These are encapsulated in a polymeric carrier to form coated particles. As used herein, a composition always refers to an encapsulated crystal or formulation.
[0041]
Controlled dissolution-dissolution of a protein or nucleic acid crystal or crystal formulation or release of the crystal components of the formulation, controlled by a factor selected from the group consisting of: the surface area of the crystal; the size of the crystal; The shape of the crystals; the concentration of the excipient component; the number and nature of the excipient component; the molecular weight of the excipient component and combinations thereof.
[0042]
Polymer made with monomer species greater than Copolymer-1.
[0043]
Crystal—One form of a solid state of a substance that is different from the amorphous solid state that is the second form. Crystals exhibit characteristic properties including lattice structures, characteristic shapes and optical properties (eg, refractive index). Crystals consist of atoms arranged in a three-dimensional, periodically repeating pattern (CS Barrett, Structure of Metals, 2nd edition, McGraw-Hill, New York, 1952, page 1). The crystals of the present invention can be proteins, glycoproteins, peptides, antibodies, therapeutic proteins, or DNA or RNA encoding such proteins.
[0044]
(Drying protein crystals or nucleic acid crystals) N₂Water by means including gas or inert gas drying, vacuum oven drying, lyophilization, washing with a volatile organic solvent followed by evaporation of this solvent, or evaporation of this solvent in a fume hood, Removal of organic solvent or liquid polymer. Typically, drying is achieved when the crystals become a free flowing powder. Drying can be performed by passing a gas stream through the wet crystals. The gas may be selected from the group consisting of nitrogen, argon, helium, carbon dioxide, air or combinations thereof.
[0045]
(Effective amount) a protein crystal or nucleic acid crystal of the invention or effective in treating, immunizing, boosting, protecting, repairing or detoxifying a subject or region to which they are administered for some period of time or The amount of crystal formulation or crystal composition.
[0046]
(Emulsifier) A surfactant that reduces the interfacial tension between the polymer-coated protein crystals and the solution.
[0047]
(Formulation or (protein crystal composition or nucleic acid crystal composition)) A combination of a protein crystal or nucleic acid crystal of the present invention and one or more ingredients or excipients (including sugar and biocompatible polymer). Examples of excipients are described in Handbook of Pharmaceutical Excipients, co-published by American Pharmaceutical Association and the Pharmaceutical Society of Great Britian. For the purposes of this application, “formulation” includes “crystal formulation”. Furthermore, “formulation” includes “protein crystal formulation or nucleic acid crystal formulation”.
[0048]
(Formulations for decontamination) Formulations selected from the following groups: Chemical wastes, herbicides, insecticides, pesticides and formulations for decontamination of environmental hazards.
[0049]
(Gene therapy) Treatment using a DNA formulation and / or composition encoding a protein that is deficient, deficient or poorly expressed in an individual. The crystals are injected into non-proliferating tissue where DNA is taken up into cells and expressed over a period of 1-6 months. The expressed protein serves to temporarily replace or supplement the endogenous protein. Gene therapy also provides a transgene in the opposite gene orientation as compared to the promoter, so that antisense mRNA serves to inhibit gene expression by being produced in vivo.
[0050]
(Glycoprotein) A protein or peptide covalently bound to a carbohydrate. The carbohydrate can be monomeric or can be composed of oligosaccharides.
[0051]
(Homopolymer) A polymer made of a single monomer species.
[0052]
(Immunotherapeutic agent) A protein derived from the above-mentioned tumor cell, virus or bacterium having a protein activity that induces protective immunity against the tumor cell, virus or bacterium. Immunotherapeutic agents can be administered directly (as a protein) or by injecting DNA or RNA encoding the protein (indirectly).
[0053]
The immunotherapeutic agent can also be a protein or glycoprotein cytokine or an immune cell costimulatory molecule that stimulates the immune system to reduce or eliminate the tumor cells, viruses or bacteria described above.
[0054]
(Liquid polymer) A pure liquid phase synthetic polymer, such as polyethylene glycol (PEG), in the absence of aqueous or organic solvents.
[0055]
(Polymer) protein, glycoprotein, peptide, therapeutic protein, DNA molecule or RNA molecule.
[0056]
Methods of Administration Protein crystals or nucleic acid crystals or crystal formulations or crystal compositions can be suitable for various modes or administration. These may include oral administration, parenteral administration, subcutaneous administration, intravenous administration, pulmonary administration, intralesional administration, or topical administration. Alternatively, the nucleic acid crystals can be covalently attached to gold particles, or other carrier beads, for delivery to non-proliferating tissue (eg, muscle) using a “DNA gun”.
[0057]
A non-replicating, non-integrating polynucleotide encoding a vaccine antigen, therapeutic protein, or immunotherapy protein that can be operably linked to a (naked DNA) promoter and inserted into a replication-competent plasmid. This DNA is free from association with proteins, virus particles, liposomal formulations, lipids and calcium phosphate precipitants that facilitate transfection.
[0058]
(Naked DNA vaccine) DNA encoding vaccine antigen or crystal of vaccine antigen and immunotherapeutic agent. The vaccine is injected into non-proliferating tissue where the DNA is taken up into cells and expressed over a period of 1-6 months. The nucleic acid crystal can be covalently bound to the gold particles to aid delivery to the site of administration.
[0059]
(Organic solvent) Any solvent of non-aqueous origin, including liquid polymers and mixtures thereof. Suitable organic solvents for the present invention are acetone, methyl alcohol, methyl isobutyl ketone, chloroform, 1-propanol, isopropanol, 2-propanol, acetonitrile, 1-butanol, 2-butanol, ethyl alcohol, cyclohexane, dioxane, ethyl acetate, Including dimethylformamide, dichloroethane, hexane, isooctane, methylene chloride, tert-butyl alcohol, toluene, carbon tetrachloride, or combinations thereof.
[0060]
Peptide Usually 3 to 35 amino acid residues, and frequently (but not necessarily showing larger protein fragments) small to medium molecular weight polypeptides.
[0061]
(Pharmaceutically effective amount) The amount of a protein crystal or nucleic acid crystal or crystal formulation or crystal composition that is administered over a period of time and is effective to treat a condition in a living organism.
[0062]
(Component) Any excipient (s), including pharmaceutical ingredients or pharmaceutical excipients. Excipients include, for example:
(Acidizing agent)
Acetic acid, glacial acetic acid, citric acid, fumaric acid, hydrochloric acid, dilute hydrochloric acid, malic acid, nitric acid, phosphoric acid, dilute phosphoric acid, sulfuric acid, tartaric acid.
[0063]
(Aerosol spray)
Butane, dichlorodifluoromethane, dichlorotetrafluoroethane, isobutane, propane, trichloromonofluoromethane.
[0064]
(Air replacement)
Carbon dioxide, nitrogen.
[0065]
(Alcohol denaturant)
Denatonium benzoate, methyl isobutyl ketone, sucrose octaacetate.
[0066]
(Alkalizing agent)
Strong ammonia solution, ammonium carbonate, diethanolamine, diisopropanolamine, potassium hydroxide, sodium bicarbonate, sodium borate, sodium carbonate, sodium hydroxide, trolamine.
[0067]
(See anti-caking agent: glidant section)
(Antifoaming agent)
Dimethicone, simethicone.
[0068]
(Preservative)
Benzalkonium chloride, benzalkonium chloride solution, benzethonium chloride, benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium chloride, chlorobutanol, chlorocresol, cresol, dehydroacetic acid, ethylparaben, methylparaben, Sodium methylparaben, phenol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric nitrate, potassium benzoate, potassium sorbate, propylparaben, sodium propylparaben, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid, thimerosal, thymol .
[0069]
(Antioxidant)
Ascorbic acid, accorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium formaldehydesulfoxylate, sodium metabisulfite, sodium thiosulfate, sulfur dioxide, tocopherol, Tocopherol excipient.
[0070]
(Buffering agent)
Acetic acid, ammonium carbonate, ammonium phosphate, boric acid, citric acid, butyric acid, phosphorous acid, potassium citrate, potassium metaphosphate, potassium phosphate monobasic, sodium acetate, sodium citrate, sodium butyrate solution, dibasic phosphorus Sodium phosphate, monobasic sodium phosphate.
[0071]
(Capsule lubricant)
See tablets and capsule lubricants.
[0072]
(Chelating agent)
Edetic acid disodium, ethylenediaminetetraacetic acid and its salts, edetic acid. (Coating agent)
Sodium carboxymethylcellulose, cellulose acetate, cellulose acetate phthalate, ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, methacrylic acid copolymer, methylcellulose, polyethylene glycol, polyvinyl acetate phthalate, shellac, Sucrose, titanium dioxide, carnauba wax, microcrystalline wax, zein.
[0073]
(Coloring agent)
Caramel, red, yellow, black or a blend of these, iron oxide.
[0074]
(Complexing agent)
Ethylenediaminetetraacetic acid and its salt (EDTA), edetic acid, ethanolic gentisate, oxyquinoline sulfate.
[0075]
(desiccant)
Calcium chloride, calcium sulfate, silicon dioxide.
[0076]
(Emulsifier and / or solubilizer)
Acacia, cholesterol, diethanolamine (adjunct), glyceryl monostearate, lanolin alcohol, lecithin, mono- and diglycerides, monoethanolamine (adjacent), oleic acid (adjacent), oleyl alcohol (stabilized) Agent), poloxamer, polyoxyethylene 50 stearate, polyoxy 35 castor oil, polyoxy 40 hydrogenated castor oil, polyoxy 10 oleyl ether, polyoxy 20 cetostearyl ester, polyoxy 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, propylene glycol diacetate, propylene glycol monostearate, sodium lauryl sulfate, sodium stearate, Sorbitan laurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, stearic acid, trolamine, emulsifying wax.
[0077]
(Filter aid)
Powdered cellulose, refined siliceous soil.
[0078]
(Odorant and fragrance)
Anethole, benzaldehyde, ethyl vanillin, menthol, methyl salicylate, monosodium glutamate, orange flower oil, peppermint, peppermint oil, peppermint essential oil, rose oil, stronger rose water, thymol, tolbalsum tincture, vanilla, vanilla tincture, Vanillin.
[0079]
(Lubricant and / or anti-caking agent)
Calcium silicate, magnesium silicate, colloidal silicon dioxide, talc.
[0080]
(Wetting agent)
Glycerin, hexylene glycol, propylene glycol, sorbitol.
[0081]
(Ointment base)
Lanolin, anhydrous lanolin, hydrophilic ointment, white ointment, yellow ointment, polyethylene glycol ointment, petrolatum, hydrophilic petrolatum, white petrolatum, rose water ointment, squalane.
[0082]
(Plasticizer)
Castor oil, diacetylated monoglyceride, diethyl phthalate, glycerin, mono- and di-acetylated monoglyceride, polyethylene glycol, propylene glycol, triacetin, triethyl citrate.
[0083]
(Polymer film)
Cellulose acetate.
[0084]
(solvent)
Acetone, alcohol, diluted alcohol, amylene hydrate, benzyl benzoate, butyl alcohol, carbon tetrachloride, chloroform, corn oil, cottonseed oil, ethyl acetate, glycerin, hexylene glycol, isopropyl alcohol, methyl alcohol, methyl chloride, methyleneisobutyl Ketone, mineral oil, peanut oil, polyethylene glycol, propylene carbonate, propylene glycol, sesame oil, water for injection, sterile water for injection, sterile water for washing, purified water.
[0085]
(Absorbent)
Powdered cellulose, charcoal, refined siliceous soil.
[0086]
(Carbon dioxide absorbent)
Barium hydroxide lime, soda lime.
[0087]
(Curing agent)
Hardened castor oil. Cetostearyl alcohol, cetyl alcohol, cetyl ester wax, hard fat, paraffin, polyethylene excipient, stearyl alcohol, emulsifying wax, white wax, yellow wax.
[0088]
(Suppository base)
Cocoa butter, hardened fat, polyethylene glycol.
[0089]
(Suspension and / or thickener)
Gum arabic, agar, alginic acid, aluminum monostearate, bentonite, purified bentonite, magma bentonite, carbomer 934p, carboxymethylcellulose calcium, carboxymethylcellulose sodium, carboxymethylcellulose sodium 12, carrageenan, microcrystalline and carboxymethylcellulose sodium cellulose, Dextrin, gelatin, guar gum, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, magnesium aluminum silicate, methylcellulose, pectin, polyethylene oxide, polyvinyl alcohol, povidone, propylene glycol alginate, silicon dioxide, silicon dioxide colloid, algin Sodium, tragacanth, xanthan gum.
[0090]
(Sweetener)
Aspartame, dextrate, dextrose, excipient dextrose, fructose, mannitol, saccharin, calcium saccharin, saccharin sodium, sorbitol, sorbitol solution, sucrose, compressed sugar, coated sugar, syrup.
[0091]
(Tablet binder)
Gum arabic, alginic acid, sodium carboxymethylcellulose, microcrystalline cellulose, dextrin, ethylcellulose, gelatin, liquid glucose, gar gum, hydroxypropylmethylcellulose, methylcellulose, polyethylene oxide, povidone, pregelatinized starch, syrup.
[0092]
(Tablet and / or capsule diluent)
Calcium carbonate, calcium phosphate (2 bases), calcium phosphate (3 bases), calcium sulfate, microcrystalline cellulose, powdered cellulose, dextrate, dextrin, dextrose excipient, fructose, kaolin, lactose, mannitol, sorbitol, starch, pregelatinized Starch, sucrose, compressed sugar, coated sugar.
[0093]
(Tablet disintegrant)
Alginic acid, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium glycolate starch, starch, pregelatinized starch.
[0094]
(Tablet and / or capsule lubricant)
Calcium stearate, glyceryl behenate, magnesium stearate, light mineral oil, polyethylene glycol, sodium stearyl fumarate, stearic acid, purified stearic acid, talc, hydrogenated vegetable oil, zinc stearate.
[0095]
(Tonic)
Dextrose, glycerin, mannitol, potassium chloride, sodium chloride.
[0096]
(Vehicle: Flavored and / or sweetened)
Aromatic elixir, compound benzaldehyde elixir, isoalcohol elixir, mint water, sorbitol solution, syrup, trough sum syrup.
[0097]
(Vehicle: oily)
Almond oil, corn oil, cottonseed oil, ethyl oleate, isopropyl myristate, isopropyl palmitate, mineral oil, light mineral oil, myristyl alcohol, octyldodecanol, olive oil, peanut oil, kyounin oil, sesame oil, soybean oil, squalin.
[0098]
(Vehicle: solid carrier)
Sugar sphere
(Vehicle: sterilization)
Bacteriostatic water for injection, bacteriostatic sodium chloride for injection.
[0099]
(Thickener) (See Suspension).
[0100]
(Water repellent)
Cyclomethicone, dimethicone, simethicone.
[0101]
(Wetting agent and / or solubilizer)
Benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, doxate sodium, nonoxynol 9, nonoxynol 10, octoxynol 9, poloxamer, polyoxyl 35, castor oil, polyoxyl 40, hydrogenated castor oil, polyoxyl 50 stearate, polyoxyl 10 oleyl Ether, polyoxyl 20, cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium lauryl sulfate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, tyloxapol .
[0102]
Preferred ingredients or excipients include the following salts: 1) amino acids (eg glycine, arginine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, proline), 2) carbohydrates (eg monosaccharides (eg glucose) , Fructose, galactose, mannose, arabinose, xylose, ribose), and 3) disaccharides (eg, lactose, trehalose, maltose, sucrose), and 4) polysaccharides (eg, maltodextrin, dextran, starch, glycogen), and 5) Alditol (eg, mannitol, xylitol, lactitol, sorbitol), 6) Glucuronic acid, galacturonic acid, 7) Cyclodextrin (eg, methylcyclodextrin, hydroxypropyl-β-cycl) Dextrin etc.), 8) inorganic salts (eg sodium chloride, potassium chloride, magnesium chloride, sodium and potassium phosphates, borates, ammonium carbonate and ammonium phosphate), and 9) organic salts (eg acetate) , Citrate, ascorbate, lactate), 10) emulsifiers or solubilizers (gum arabic, diethanolamine, glyceryl monostearate, lecithin, monoethanolamine, oleic acid, oleyl alcohol, poloxamer, Polysorbate, sodium lauryl sulfate, stearic acid, sorbitan monolaurate, sorbitan monostearate, and other sorbitan derivatives, polyoxyl derivatives, waxes, polyoxyethylene derivatives, sorbitan derivatives), 11) Thickening reagents (agar, alginic acid and its salts, gar gum, pectin, polyvinyl alcohol, polyethylene oxide, cellulose and its derivatives, propylene carbonate, polyethylene glycol, hexylene glycol, tyloxapol). A further preferred group of excipients or components comprises: sucrose, trehalose, lactose, sorbitol, lactitol, inositol, sodium and potassium salts (eg acetate, phosphate, citrate, borate) Glycine, arginine, polyethylene oxide, polyvinyl alcohol, polyethylene glycol, hexylene glycol, methoxypolyethylene glycol, gelatin, hydroxypropyl-β-cyclodextrin.
[0103]
(Polymer)-A macromolecule constructed by repeating simple chemical units of small molecules. The repeating units can be linearized or branched to form an interconnected network. The repeating unit is usually equivalent or nearly equivalent to the monomer.
[0104]
(Polymer carrier)-a polymer used to encapsulate protein crystals for protein delivery (including biological delivery). Such polymers include biocompatible polymers and biodegradable polymers. The polymer carrier can be a single polymer type or it can be composed of a mixture of polymer types. Polymers useful as polymer carriers include, for example: poly (acrylic acid), poly (cyanoacrylate), poly (amino acid), poly (anhydride), poly (depsipeptide), poly (ester) (eg Poly (lactic acid) or PLA, poly (lactic acid-co-glycolic acid) or PLGA, poly (B-hydroxybutyrate), poly (caprolactone), and poly (dioxanone)); poly (ethylene glycol); Poly ((hydroxypropyl) methacrylamide), poly ((organo) phosphazene), poly (orthoester), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymer, pluronic poly Any conventional material that encapsulates a protein, albumin, natural and synthetic polypeptides, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfurized polysaccharides, or protein crystals .
[0105]
(Protein)-A complex polymer composed of a chain of amino acids containing carbon, hydrogen, oxygen, nitrogen and usually sulfur and linked by peptide bonds. Proteins in this application refer to glycoproteins, antibodies, non-enzymatic proteins, enzymes, hormones and peptides. The molecular weight range of proteins includes 1000 dalton peptides to 600-1000 kilodalton glycoproteins. Small molecule proteins (less than 10,000 Daltons) may not be characterized by highly organized tertiary structures because they are so small. Here, this tertiary structure is organized around the hydrophobic core.
[0106]
In one embodiment of the invention, such a protein has a molecular weight of 10,000 daltons or greater. According to an alternative embodiment, the molecular weight is 20,000 daltons or more. According to another alternative embodiment, the molecular weight is 30,000 daltons or more. According to a further alternative embodiment, the molecular weight is greater than 40,000 daltons. According to another alternative embodiment, the molecular weight is 50,000 daltons or more.
[0107]
(Protein activity)-from binding, catalysis, signal transduction, transport, or other activity that induces a functional response within the environment in which the protein is used (eg, immune response, enzymatic activity, or combinations thereof) An activity selected from the group consisting of:
[0108]
Protein activity release rate--the amount of protein dissolved per unit time.
[0109]
(Protein crystal)-protein molecules aligned in a crystal lattice. Protein crystals contain a pattern of specific protein-protein interactions that repeat periodically in three dimensions. The protein crystals of the present invention do not include amorphous solid forms or protein precipitates (eg, those obtained by lyophilization of protein solutions).
[0110]
Protein crystal formulation--a combination of protein crystals encapsulated in a polymeric carrier to form coated particles. The coated particles of the protein crystal formulation can have a spherical morphology and can be microspheres up to 500 micrometers in diameter, or they can have other morphology and can be microparticles . For the purposes of this application, the “protein crystal formulation” is included in the term “composition”.
[0111]
Protein delivery system--One or more protein crystal formulations or compositions, a process for making a formulation, or a method or means for administering the formulation to the biological context.
[0112]
Protein loading—the protein content of the microspheres calculated as a percentage of the weight of the protein with respect to the weight of the dry formulation. A typical range of protein loading is 1-80%.
[0113]
Protein release--The release of active protein from the polymer carrier is controlled by one or more of the following factors: (1) degradation of the polymer matrix; (2) rate of crystal melting within the polymer matrix; (3) Diffusion of molten protein through the polymer matrix; (4) protein loading; and (5) diffusion of biological media into the protein crystal / polymer matrix.
[0114]
Prophylactically effective amount--a protein or nucleic acid crystal or protein or nucleic acid crystal formulation or protein or nucleic acid crystal effective to prevent a condition in a living organism that is administered for some time The amount of the composition.
[0115]
Reconstitution--melting of a protein or nucleic acid crystal or protein or nucleic acid crystal formulation or protein or nucleic acid crystal composition in an appropriate buffer or pharmaceutical formulation.
[0116]
Storage stability--loss of specific activity from native protein and / or change in secondary structure over time incubated under certain conditions.
[0117]
Stability--loss of specific activity from native protein and / or change in secondary structure over time in solution under certain conditions.
[0118]
Stabilization--A process that prevents loss of specific activity from native proteins and / or changes in secondary structure by preparing protein crystals or DNA or RNA crystal formulations containing excipients or components.
[0119]
Therapeutic protein--a protein as described above, which is administered to a formulation or composition, or a living organism in a pharmaceutical formulation or composition. Therapeutic proteins include all of the protein types described herein.
[0120]
Vaccine antigen--a protein from a pathogenic agent such as a virus, parasite, bacterium or tumor cell The protein activity of such vaccine antigens is an inducer of a protective immune response specific to pathogenic factors or tumors.
[0121]
(crystalline)
The crystallinity of macromolecules is of great importance for their storage and delivery in vivo. However, there are few techniques for preparing large quantities of such crystalline polymers that are stable outside the mother liquor. Since proteins or nucleic acid crystals are very brittle and contain a high proportion of solvent, they must be handled with considerable care. In X-ray crystallography, it is well known that the diffraction pattern from a polymer crystal is rapidly denatured upon dehydration in air. Usually, the crystal is carefully separated from its mother liquor and inserted into a capillary tube. The tube is sealed from the air along the inside of a small amount of mother liquor with dental wax or silicone grease to maintain hydration [McPherson, A. et al. , Preparation and Analysis of Protein Crystals, Robert E .; Kriequer Publishing, Malabar, page 214 (1969)]. Another technique is to collect data from polymer crystals at low temperatures. The crystals are prepared and then cooled rapidly to prevent ice lattice formation in the aqueous medium. Instead of ice, a hard glass form, place crystals with little damage in a container. The crystal is then maintained at 100 Kelvin to prevent crystal collapse [(Rodgers, DW Methods in Enzymology (Edited by Carter, CW and Sweet, RM) Academic Press, 276). Vol., Page 183 (1997)] This technique makes it possible to maintain the crystals outside these mother liquors, but cannot be used at temperatures above 100 Kelvin.
[0122]
In principle, dried crystals can be prepared by lyophilization. However, this technique involves rapid cooling of the material and can only be applied to freeze stable products. This aqueous solution is first frozen between −40 ° C. and −50 ° C. The ice is then removed under vacuum. This ice formation is usually destructive to the protein crystal lattice, resulting in a mixture of crystalline and amorphous precipitates.
[0123]
In the crystalline state, it is desirable to produce polymers that are pure and stable under storage conditions at ambient temperature. Such crystals constitute a particularly advantageous form of protein or nucleic acid for pharmaceutical and vaccine dosage preparations. The present invention provides formulations and compositions for storage of crystalline polymers, either as solid particles or dispersed in a non-aqueous solvent. Furthermore, the present invention can be applied to the storage of a single biological polymer or a mixture of polymers that do not interact with each other.
[0124]
In another embodiment, the present invention provides a method for making a biological macromolecule suitable for storage in suspension, comprising replacing the mother liquor with a non-aqueous solvent. In yet another embodiment, the crystalline slurry is used to spin out the first solvent and wash the remaining crystalline solid using a second organic solvent to remove water. And then solidified by evaporation of a non-aqueous solvent.
[0125]
Non-aqueous slurries of crystalline therapeutic proteins are particularly useful for subcutaneous delivery, while solid formulations are ideally suited for pulmonary administration. Pulmonary delivery is particularly useful for biological macromolecules that are difficult to deliver by other routes or administration (see, eg, PCT patent applications WO 96/32152, WO 95/24183, and WO 97/41833). ).
[0126]
The proteins referred to below include their own protein crystals or nucleic acid crystals comprising DNA or RNA encoding the proteins upon cellular uptake.
[0127]
The present invention advantageously provides protein or nucleic acid crystal compositions and formulations.
[0128]
(Stability of encapsulated crystals)
Those skilled in the art understand that protein stability is one of the most important obstacles to successful formulations of polymer microparticle delivery systems that control protein release. The stability of the protein encapsulated in a polymer carrier is investigated in three distinct stages: production of the protein crystal composition, release of the protein from the resulting composition, and in vivo stability after release of the protein. obtain. During the preparation of microparticles or microspheres containing soluble or amorphous proteins, the use of organic solvents and lyophilization is particularly detrimental to protein stability. Subsequently, the released protein is susceptible to moisture-induced aggregation, thus resulting in permanent inactivation.
[0129]
In order to achieve high protein stability during the preparation of protein formulations and protein compositions according to the invention, it is necessary to limit the mobility of individual protein molecules (best achieved in the crystalline solid state). Results). For the purposes of this application, the solid state is divided into two categories (amorphous and crystalline). The three-dimensional long-range order normally present in crystalline materials does not exist in the amorphous state. Furthermore, the position of the molecules relative to each other is more random in the amorphous or liquid state with respect to a highly ordered crystalline state. Thus, amorphous proteins can be less stable than their crystalline counterparts.
[0130]
FIG. 1 shows the relative stability of the following molecular states of Candida rugosa lipase (“CRL”): crosslinked amorphous, liquid, crystalline, 20% organic solvent in 20% organic solvent. Cross-linked crystallinity, cross-linked crystallinity without organic solvent. FIG. 1 shows that crystalline CRL retains 80% activity in over 20% organic solvent over 175 hours. In contrast, both the amorphous and soluble forms of the enzyme are completely inactivated within a few hours. The present invention advantageously utilizes the crystalline form of the protein due to their excellent stability characteristics.
[0131]
(Maintaining crystallinity)
In order to use protein crystals as a protein source for preparing protein formulations and protein compositions according to the present invention, the problem of melting protein crystals outside the crystallization solution (mother liquor) must be overcome. In order to maintain protein crystallinity, and hence stability, several approaches can be used in the production of protein crystal formulations and protein crystal compositions of the present invention:
1. The crystals remain in the mother liquor in the process of producing protein crystals encapsulated with the polymer carrier. Many compounds used in protein crystallization (eg, salts, PEG, and organic solvents) are compatible with the conditions under which the polymer is processed.
2. Melting kinetics. The rate of crystal melting outside the mother liquor depends on conditions such as pH, temperature, presence of metal ions (eg, Zn, Cu and Ca), and concentration of precipitant. By changing these conditions, the melting of the crystals can be slowed for several hours. At the same time, the process of microparticle formation is very fast and usually requires seconds to minutes to complete.
3. Dry protein crystals. The mother liquor can be removed by filtration and the remaining crystalline paste can be dried by air drying, under vacuum, by washing with a water miscible organic solvent, and / or by lyophilization.
4). Protein crystals can be chemically cross-linked to form non-melting crystals or slow melting crystals.
5). Crystal size and shape can be manipulated and controlled during the crystallization process. Thus, a range of crystalline forms are available that each have a different melting rate and subsequent different sustained release profile compared to the amorphous protein.
[0132]
(Protein composition)
The protein composition of the formulations and compositions of the present invention may be a composition that is naturally or synthetically modified. They can be glycoproteins, phosphoproteins, sulfur proteins, iodine proteins, methylated proteins, unmodified proteins or proteins containing other modifications. Such protein configurations include, for example, therapeutic proteins, prophylactic proteins (including antibodies), detergent proteins (including surfactant proteins), personal medical proteins (including cosmetic proteins), veterinary proteins, It can be any protein, including food protein, feed protein, diagnostic protein and purification protein.
[0133]
In one embodiment of the invention, such a protein has a molecular weight of 10,000 Daltons or greater. According to an alternative embodiment, the molecular weight is greater than 20,000 daltons. According to another alternative embodiment, the molecular weight is 30,000 daltons or more. According to a further alternative embodiment, the molecular weight may be 40,000 daltons or more. According to another alternative embodiment, the molecular weight is 50,000 daltons or more.
[0134]
Included in such proteins are enzymes such as hydrolases, isomerases, lyases, ligases, adenylate cyclases, transferases and oxidoreductases. Examples of hydrolases include elastase, esterase, lipase, nitrilase, amylase, pectinase, hydantoinase, asparaginase, urease, subtilisin, thermolysin and other proteases and lysozyme. Examples of lyases include aldolase and hydroxynitrile lyase. Examples of oxidoreductases include peroxidase, laccase, glucose oxidase, alcohol dehydrogenase and other dehydrogenases. Other enzymes include cellulase and oxidase.
[0135]
Examples of therapeutic or prophylactic proteins include hormones (eg, insulin, glucagon-like peptide 1 and parathyroid hormone), antibodies, inhibitors, growth factors, post-hormonal hormones, nerve growth hormones, blood clotting ) Factors, adhesion molecules, bone morphogenetic proteins and lectin trophic factors), cytokines (eg TGF-β, IL-2, IL-4, α-IFN, β-IFN, γ-IFN, TNF) IL-6, IL-8, lymphotoxin, IL-5, migration inhibitory factor, GMCSF, IL-7, IL-3, monocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, multidrug resistance protein, other Rinhokai , Toxoid, erythropoietin, factor VIII, amylin, TPA, dornase-α, α-1-antitrypsin, human growth hormone, nerve growth hormone, bone morphogenetic protein, urease, toxoid, fertility hormone (FSH and LSH ).
[0136]
The following therapeutic proteins are also included:
Leukocyte markers (eg, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD11a, CD11b, CD11c, CD13, CD14, CD18, CD19, CE20, CD22, CD23, CD27 and its ligand, CD28 and its ligand, B7 .1, B7.2, B7.3, CD29 and its ligand, CD30 and its ligand, CD40 and its ligand gp39, CD44, CD45 and isoform, Cdw52 (Campath antigen), CD56, CD58, CD69, CD72, CTLA- 4, LFA-1 and TCR),
Histocompatibility antigens (eg, MHC class I or MHC class II antigen, Lewis Y antigen, SLex, SLey, SLea and SLeb);
Integrins (eg, VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6 and LFA-1);
Adhesion molecules (eg, Mac-1 and p150, 95);
Selectins (eg L-selectin, P-selectin and E-selectin and their corresponding receptors VCAM-1, ICAM-1, ICAM-2 and LFA-3);
Interleukins (eg, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL -13, IL-14 and IL-15);
Interleukin receptors (eg, IL-1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-7R, IL-8R, IL-10R, IL-11R, IL-12R, IL-13R, IL-14R and IL-15R);
Chemokines (eg, PF4, RANTES, MIP1α, MCP1, NAP-2, Groα, Groβ and IL-8);
Growth factors (eg, TNFα, TGFβ, TSH, VEGF / VPF, PTHrP, EGF family, EGF, PDGF family, endothelin and gastrin releasing peptide (GRP));
Growth factor receptors (eg, TNFαR, RGFβR, TSHR, VEGFR / VPFR, FGFR, EGFR, PTHrPR, PDGFR family, EPO-R; GCSF-R and other hematopoietic receptors;
Interferon receptors (eg, IFNαR, IFNβR and IFNγR);
Ig and their receptors (eg, IgE, FceRI and FceRII);
Blood factors (eg complement C3b, complement C5a, complement C5b-9, Rh factor, fibronectin, fibrin and myelin related growth inhibitors).
[0137]
The protein component of the formulations and compositions of the present invention can be any natural protein antigen, synthetic protein antigen or recombinant protein antigen, eg, tetanus toxin, diphtheria toxin, viral surface protein (eg, CMV sugar) Protein B, H and gCIII, HTV-1 envelope glycoprotein, RSV envelope glycoprotein, HSV envelope glycoprotein, EBV envelope glycoprotein, VZV envelope glycoprotein, HPV envelope glycoprotein, influenza virus glycoprotein, hepatitis family surface antigen), Includes viral structural proteins, viral enzymes, parasitic proteins, parasitic glycoproteins, parasitic enzymes as well as bacterial proteins.
[0138]
Also included are tumor antigens such as her2-neu, mucin, CEA and endosialin. Allergens such as house dust mite antigen, lol p1 (grass) antigen and urushiol are included.
[0139]
Toxins such as Pseudomonas endotoxin and osteopontin / uropontin, snake venom and bee venom.
[0140]
Glycoprotein tumor associated antigens (eg carcinoembryonic antigen (CEA), human mucin, her-2 / neu and prostate specific antigen (PSA) [RA Henderson and OJ Finn, Advances in Immunology, 62, 217 -56 (1996)]).
[0141]
(Administration and biological delivery)
To date, therapeutic proteins have generally been administered by frequent injections due to their negligible oral bioavailability and short survival in plasma. The protein crystal formulations and compositions of the present invention include a microparticle-based sustained release system for protein drugs, which advantageously allows for improved patient compliance and convenience, more stable blood levels and a reduced potential dose. The slow and consistent release capability of the present invention can advantageously reduce dosage. This is due to more efficient delivery of the active protein. Significant cost savings can be achieved using the protein formulations and compositions described herein.
[0142]
Formulations and compositions comprising protein crystals in a polymer delivery carrier according to the present invention are also any convention used in vaccines, pharmaceuticals, personal medical formulations and compositions, veterinary formulations, or oral enzyme supplements. A typical carrier or adjuvant may be included. These carriers and adjuvants include, for example, Freund's adjuvant, ion exchanger, alumina, aluminum stearate, lecithin, buffer (phosphate), glycine, sorbic acid, potassium sorbate, a partial glyceride mixture of saturated vegetable fatty acids, Examples include water, salts or electrolytes (eg, protamine sulfate), disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium, trisilicates, cellulose-based materials, and polyethylene glycols. Adjuvants for topical or gel-based forms include, for example, sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycols and tree wax alcohols.
[0143]
In accordance with one embodiment of the invention, the protein crystals can be combined with any conventional material used for controlled release administration (including pharmaceutical controlled release administration). Such materials include, for example, coatings, shells and films (eg, enteric coatings and polymer coatings and films).
[0144]
The protein formulation in the polymer delivery carrier and composition (composition) according to the present invention can be a device such as an implantable device and can be a particulate protein delivery system.
[0145]
In one embodiment of the invention, the polymer crystal has the longest dimension between about 0.01 μm and about 500 μm, or between about 0.1 μm and about 100 μm. The most preferred embodiment is that the protein crystals of the protein crystal formulation component are between about 50 μm and about 100 μm in their longest dimension. Such crystals may have a shape selected from the group consisting of spherical, needle-like, rod-like, plate-like (eg, pentagonal and square), rhombus, cubic, bipyramids and columnar.
[0146]
In accordance with the present invention, the encapsulation of a protein crystal or protein crystal formulation in a polymer carrier to make a composition can be performed on cross-linked or non-cross-linked protein crystals. Such protein crystals are either commercially available or can be generated as exemplified herein.
[0147]
Protein or nucleic acid crystals or crystal formulations and compositions according to the present invention are cosmetic (eg creams, lotions, emulsions, foams, detergents, compacts, gels, mousses, slurries, powders, sprays, pastes, ointments, It can be used as an ingredient in personal medical compositions including salves, salves, drops, shampoos and sunscreens. For example, in topical creams and lotions, they can be used as humectants or to protect, soften, decolorize, cleanse, deproteinize, remove lipids, moisten, bleach, color or detoxify the skin. They can also be used as antioxidants in cosmetics.
[0148]
In accordance with the present invention, any individual (including humans, animals and plants) may be pharmaceutically effective amounts of protein or nucleic acid crystals for a period sufficient to treat the condition in the individual administered for a period of time. Alternatively, it can be treated in a pharmaceutically acceptable manner with a crystalline formulation or crystalline composition. Alternatively, an individual can receive a prophylactically effective amount of a protein or nucleic acid crystal or crystal formulation or composition of the invention that is effective to prevent the condition in the individual being administered over a period of time.
[0149]
Protein or nucleic acid crystals or crystal formulations or crystal compositions may be used alone or as part of a pharmaceutical preparation, personal medical preparation or veterinary preparation with or without adjuvant, or prophylactic preparation Can be administered as part of a product (eg, a vaccine). They can be administered by parenteral or oral routes. For example, they can be administered by oral, pulmonary, nasal, otic, anal, cutaneous, ocular, intravenous, intramuscular, intraarterial, intraperitoneal, mucosal, sublingual, subcutaneous, or intracranial routes. In either pharmaceutical, personal therapeutic or veterinary applications, protein or nucleic acid crystals or crystal formulations or crystal compositions can be administered locally to any epithelial surface. Such epithelial surfaces include the mouth, eye, ear, anus and nasal surfaces, which can be treated, protected, repaired or detoxified by application of protein or nucleic acid crystals or crystal formulations or crystal compositions.
[0150]
Pharmaceutical, personal therapeutic, veterinary or prophylactic formulations and compositions comprising protein or nucleic acid crystals or crystal formulations or crystal compositions according to the invention are also tablets, liposomes, granules, spheres, microparticles, microspheres And can be selected from the group consisting of capsules.
[0151]
For such uses, and other uses according to the present invention, protein or nucleic acid crystals or crystal formulations and compositions can be formulated into tablets. Such tablets are composed in a liquid-free, dust-free form for storage of protein or nucleic acid crystals or crystal formulations or crystal compositions that are easy to handle and retain an acceptable level of activity or capacity. The
[0152]
Alternatively, the protein or nucleic acid crystal or crystal formulation or crystal composition can be in a variety of convenient storage forms used for administration to provide the reaction composition. These include, for example, solid, semi-solid and liquid forms (eg, liquid solutions or suspensions, chillers, gels, creams, balms, emulsions, lotions, powders, sprays, foams, pastes, ointments, ointments ( salve), balm and drop).
[0153]
A protein or nucleic acid crystal or crystal formulation or crystal composition according to the invention may also include any conventional carrier or adjuvant used in pharmaceutical, personal medical or veterinary formulations. These carriers and adjuvants include, for example, Freund's adjuvant, ion exchangers, alumina, aluminum stearate, lecithin, buffer (phosphate), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of vegetable saturated fatty acids Water, salts or electrolytes (eg, protamine sulfate), disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium, trisilicates, cellulose-based materials and polyethylene glycols. For topical or gel-based forms, adjuvants may include, for example, carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycols and tree wax alcohols.
[0154]
The most effective dosage forms and dosing regimes of the protein or nucleic acid crystals or crystal formulations or crystal compositions of the present invention are the desired effect, previous treatment (if any), the health condition of the individual or the condition of the symptoms themselves, and Depends on the response to the protein or nucleic acid crystal or crystal formulation or composition and the judgment of the treating physician or clinician. The protein or nucleic acid crystal or crystal formulation or crystal composition is acceptable for pharmaceutical, vaccination, gene therapy, immunotherapy, personal medical composition or veterinary formulation at a point in time or over a series of treatments Can be formulated in any dosage form.
[0155]
The amount of protein or nucleic acid crystal or crystal formulation or crystal composition that provides a single dose will vary depending on the particular mode of administration, formulation, dosage level or frequency of administration. A typical preparation comprises between about 0.01% and about 99%, preferably between about 1% and about 50% protein or nucleic acid crystals (W / W). Alternatively, the preparation comprises between about 0.01% and about 80% protein crystals, preferably between about 1% and about 50% protein crystals (W / W). Alternatively, the preparation comprises between about 0.01% and about 80% protein crystal formulation, preferably between about 1% and about 50% protein crystal formulation (W / W).
[0156]
In improving an individual's condition, a maintenance dose of a protein or nucleic acid crystal or crystal formulation or crystal composition can be administered if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced as a function of symptoms to a level at which an improved condition is maintained. If symptoms are relieved to the desired level, treatment should be stopped. However, individuals may require intermittent treatment on a long-term basis based on any recurrence of their condition or symptoms.
[0157]
(Production of crystals, crystal formulations, and crystal compositions)
In accordance with one embodiment of the present invention, crystals, crystal formulations, and crystal compositions are prepared by the following steps: First, a protein or nucleic acid is crystallized. Next, excipients or ingredients selected from sugars, sugar alcohols, viscosity increasing agents, wetting or solubilizing agents, buffer salts, emulsifiers, antibacterial agents, antioxidants, and coatings are directly applied to the mother liquor. Added. Alternatively, the crystals are suspended in the excipient solution for a minimum of 1 hour to a maximum of 24 hours after the mother liquor is removed. The excipient concentration is typically about 0.01-30% weight / weight. Most preferably, it is about 0.1 to 10%. The component concentration is about 0.01 to 90%. The crystal concentration is about 0.01 to 95%. The mother liquor is then removed from the crystal slurry, either by filtration or by centrifugation. Subsequently, using a solution of 50-100% of one or more organic solvents (such as ethanol, methanol, isopropanol, or ethyl acetate), optionally at room temperature or a temperature of −20 ° C. to 25 ° C. Wash the crystals with either The crystals are dried by passing either a stream of nitrogen, air or inert gas over the crystals. Alternatively, the crystals are dried by air drying, by freeze drying, or by vacuum drying. This drying is carried out for a minimum of 1 hour up to a maximum of 72 hours after washing until the moisture content of the final product is less than 10% weight, most preferably less than 5%. Finally, if necessary, crystal micronization can be performed.
[0158]
When preparing protein crystals, protein crystal formulations or protein crystal compositions according to one embodiment of the invention, enhancers (eg, surfactants) are not added during crystallization. The excipient or component is crystallized at a concentration of about 1-10% weight / weight, alternatively about 0.1-25% weight / weight, alternatively about 0.1-50% weight / weight. It is added to the mother liquor after conversion. The excipient or component is incubated with the crystals in the mother liquor for about 0.1 to 3 hours. Alternatively, this incubation is performed for 0.1-12 hours. Alternatively, this incubation is performed for 0.1 to 24 hours.
[0159]
In another embodiment of the invention, the component or excipient is dissolved in a solution other than the mother liquor, and the protein crystals are removed from the mother liquor and suspended in the excipient solution or component solution. Ingredient or excipient concentrations and incubation times are the same as those described above.
[0160]
(Slow release form and sustained release vaccine)
In another embodiment of the invention, encapsulation of the lipase in a polymer carrier provides a composition useful for treating patients suffering from intestinal lipase deficiency. Such patients include patients with pancreatic fatty stool. This is because progressive pancreatic dysfunction requires oral lipase supplementation. Unfortunately, current therapies protect active lipase while moving through the gastrointestinal tract and release it in the small intestine where it is clinically required. It cannot be sufficiently adaptive (see L. Guarner et al., “Fate of oral enzymes in pancreatic insufficiency”, Gut, 34, 708-712 (1993)). The adaptability of the present invention in preparing slowly effective active lipases solves the current problems often associated with lipase supplementation. According to one embodiment of the present invention, the combination of encapsulated lipase crystals (composition) with non-encapsulated cross-linked lipase crystals or non-encapsulated cross-linked lipase formulations provides a drug therapy regimen. In this pharmacotherapy regimen, enzyme activity is available early from unencapsulated cross-linked lipase. As this material undergoes proteolytic degradation, the encapsulated enzyme (composition) begins to release enzyme activity into the more distal gut. Similar strategies can be used to solve the problem of supplementing other enzymes or therapeutic proteins.
[0161]
The present invention also includes other sustained release methodologies, such as silicon-based rings or rods preloaded with encapsulated protein crystals comprising hormones, antibodies or enzymes, or compositions containing them. )) Can be used. The purpose of the technology of the present invention is to provide a constant level of protein in the bloodstream over a period of weeks or months. Such implants can be inserted into the skin and can be safely replaced and removed when needed.
[0162]
Other formulations and compositions according to the invention include vaccine formulations and compositions comprising protein (antigen) crystals, adjuvants, and encapsulating polymers. The protein antigen is a viral glycoprotein, viral structural protein, viral enzyme, bacterial protein, or some engineered homologue of a viral or bacterial protein, or any immune enhancing protein (eg, cytokine) obtain. One embodiment of such a formulation or composition includes a single vaccine injection containing microspheres having three or more different release profiles. In this manner, the antigen formulation or antigen composition can be released for a duration sufficient to generate lasting immunity. Depending on the formulation or composition, multiple antigen boosts may be in a single unit form. Faster degradation preparations (compositions) can include immunogenic adjuvants to enhance the immune response. One advantage of such a system is that by using protein crystals, the native three-dimensional structure of this epitope is maintained and presented to the immune system in their native form.
[0163]
Once the immune system is primed, the need for an adjuvant effect can be reduced. Thus, less slowly antigenic adjuvants may be included in more slowly degrading inoculations, and perhaps no adjuvant may be needed in the most slowly degrading microspheres of this formulation and composition. . In this manner, the remote patient population need not be treated multiple times to provide protection against infectious diseases. Those skilled in the art of biological delivery of proteins recognize that many modifications to this theme are possible. Accordingly, the examples provided herein are not intended to limit the invention.
[0164]
In another embodiment of the invention, a combination vaccine can be produced, which induces immunity against multiple diseases with a single injection. As noted above, microspheres with different release profiles can be combined alone or in formulations and compositions. And this can include microspheres containing antigens from multiple pathogens to produce a combination vaccine (formulation and composition). For example, microspheres having multiple release profiles and containing antigenic crystals of measles, mumps, rubella, polio, and hepatitis B factors can be combined and administered to children. Alternatively, microspheres having multiple release profiles and containing different isolates of HIV gp120 can be combined to produce a vaccine for HIV-1 or HIV-2.
[0165]
Another advantage of the present invention is that protein crystals encapsulated in a polymer carrier and forming a composition containing microspheres can be dried by a lyophilization method. Lyophilization (ie, freeze-drying) separates moisture from the composition. The protein crystal composition is first frozen and then placed under high vacuum. Under vacuum, the crystalline H₂O sublimes, leaving a protein crystal composition containing only a small amount of bound water. Such a process further stabilizes the composition and allows for easier storage and transport at ambient temperatures typically encountered.
[0166]
This feature is particularly desirable for therapeutic proteins and vaccines that can be dispensed into any desired increment of a single dose, such as a single dose sterile container ("ampoule"), or slurry, in a formulation or composition. The ampule containing the dispensed slurry, formulation or composition can then be capped, batch frozen, and lyophilized under sterile conditions. Such sterile containers can be transported throughout the world and stored at ambient temperature. Such systems are useful for providing sterile vaccines and therapeutic proteins to remote locations and undeveloped areas of the world. At the time of use, the ampoule is rehydrated and dosed with a selected sterile solvent or sterile buffer. Under this scenario, minimal refrigeration or no refrigeration is required.
[0167]
(Protein crystallization)
Protein crystals are generated by controlled protein crystallization from aqueous solutions or aqueous solutions containing organic solvents. Solution conditions that can be controlled include, for example, solvent, organic solvent evaporation rates, the presence of appropriate co-solutes and buffers, pH and temperature. A comprehensive review of various factors affecting protein crystallization can be found in McPherson, Methods Enzymol. 114, pages 112-20 (1985).
[0168]
McPherson and Gilland, J.M. Crystal Growth, 90, pages 51-59 (1988) compiles a comprehensive list of crystallized proteins and nucleic acids and the conditions under which they were crystallized. An overview of crystals and crystallization formulations, as well as a collection of elucidated protein and nucleic acid structure coordinates, can be found at Brookhaven National Laboratory's Protein Data Bank (http://www.pdb.bnl.gov; Bernstein et al., J. Mol. Biol. 112, pp. 535-42 (1977)). These references can be used as a prelude to the formation of appropriate protein crystals to determine the conditions required for protein crystallization and can lead to crystallization strategies for other proteins. Alternatively, in many cases, appropriate crystallization conditions can be created by intelligent trial and error research strategies, provided that acceptable levels of purity can be achieved for many proteins (eg, C W. Carter, Jr. and C. W. Carter, J. Biol. Chem., 254, pages 12219-23 (1979)).
[0169]
In general, crystals are produced by combining the protein to be crystallized with an appropriate aqueous solvent or an aqueous solvent containing an appropriate crystallization factor (eg, a salt or organic solvent). The solvent is combined with the protein and subjected to stirring at a temperature that has been experimentally determined to be suitable for inducing crystallization and acceptable for maintaining the activity and stability of the protein. obtain. The solvent can optionally include coexisting solutes (eg, divalent cations, cofactors, or chaotropes), and buffer species to control pH. The need for coexisting solutes and their concentrations are determined experimentally to promote crystallization.
[0170]
It is important to distinguish between amorphous precipitates and crystalline materials. Crystalline material is the solid state form of the material, which is different from the amorphous solid state. Crystals exhibit characteristic properties including lattice structures, characteristic shapes, and optical characteristics (eg, refractive index and birefringence). A crystal consists of atoms arranged in a pattern that repeats periodically in three dimensions. In contrast, an amorphous material is an amorphous solid state of the material, sometimes referred to as an amorphous precipitate. Such precipitates do not have any molecular lattice structure characteristic of the crystalline solid state and do not exhibit birefringence or other spectroscopic features typical of the crystalline form of the material.
[0171]
In industrial scale processes, controlled precipitation leading to crystallization is best performed by simply combining proteins, precipitants, co-solutes, and optionally buffers in batch processes. obtain. As another option, the protein can be crystallized by using a protein precipitate as a starting material. In this case, the protein precipitate is added to the crystallization solution and incubated until crystal formation. Alternative laboratory crystallization methods (eg, dialysis or diffusion deposition) can also be employed. McPherson (supra) and Gilland (supra) include a comprehensive list of suitable conditions in their review of the crystallization literature.
[0172]
Occasionally, where the crystallized protein is to be cross-linked, incompatibility between the intended cross-linking agent and the crystallization medium may necessitate exchanging the crystals for a more suitable solvent system.
[0173]
Many proteins for which crystallization conditions have already been described can be used to prepare protein crystals according to the present invention. However, it is noted that the conditions reported in most of the references cited above are often optimized to produce slightly larger diffraction quality crystals. Should. Therefore, some adjustment of these conditions may be required to provide a high yield process for large scale production of fewer crystals used in making the protein crystals of the present invention. Will be recognized by those skilled in the art.
[0174]
(Crosslinking protein crystals)
According to one embodiment of the invention, for example, the rate of protein release from the polymer carrier (composition) can be slow and using a crosslinker (such as a biocompatible crosslinker). Can be controlled by the use of chemically cross-linked protein crystals. Thus, once protein crystals are grown in a suitable medium, they can be cross-linked.
[0175]
Crosslinking can be performed using a reversible crosslinker (in parallel or sequentially). The resulting cross-linked protein crystals are characterized by a reactive multi-functional linker. The trigger is taken as another group. Reactive functionalities are involved in binding reactive amino acid side chains of proteins together, and triggers can affect one or more conditions (eg, pH, temperature, or thermodynamics) of the surrounding environment. It consists of bonds that can be broken by changing the activity of the water. This is illustrated as follows:
XYZ + 2AA residue-> AA₁-X-Y-Z-AA₂
Environmental changes ---> AA₁-X + Y-Z-AA₂
-Where X and Z are groups having reactive functional groups;
-Where Y is the trigger,
-Where AA₁And AA₂Indicates reactive amino acid residues on the same protein or on two different proteins. The bond between the crosslinker and the protein can be a covalent bond, an ionic bond, or a hydrogen bond. Changes in the surrounding environment result in cleavage of the trigger bond and dissociation of the protein. Therefore, when the crosslink in the protein crystal crosslinked with such a reversible crosslinker breaks, the protein crystal dissociates and thus loses activity.
[0176]
Alternatively, the crosslinker and trigger reactive functional groups may be the same as follows:
X-Z + 2AA residue-> AA₁-XZ-AA₂
Environmental changes ---> AA₁  + X-Z-AA₂.
[0177]
The crosslinker can be a homologous functional group (X = Y) or a heterogeneous functional group (X is not the same as Y). The reactive functional groups X and Y can be, but are not limited to, the following functional groups (where R, R ′, R ″ and R ′ ″ can be alkyl groups, aryl groups or hydrogen groups): :
I. Reactive acyl donors are exemplified by: carboxylate ester RCOOR ', amide RCONHR', acyl azide RCON_Three, Carbodiimide R—N═C═N—R ′, N-hydroxyimide ester RCO—O—NR ′, imido ester R—C═NH 2 ′ (OR ′), anhydride RCO—O—COR ′, carbonate RO— CO—O—R ′, urethane RNHCONHR ′, acidic halide RCOHal (where Hal is one halogen), acyl hydrazide RCONNR′R ″, O-acyscure RCO—O—C═NR '(-NR "R'").
[0178]
II. Reactive carbonyl groups are exemplified by: aldehyde RCHO, and ketone RCOR ', acetal RCO (H₂) R ', Ketal RR'CO₂R'R ". Reactive carbonyls containing functional groups known to those skilled in the art of protein immobilization and cross-linking are described in the following literature [Pierce Catalog and Handbook, Pierce Chemical Company, Rockford, Illinois (1994) SS Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press, Boca Raton, Florida (1991)].
[0179]
III. Alkyl or aryl donors are exemplified by: alkyl halides or aryl halides R-Hal, azide RN_Three, Sulfate ester RSO_ThreeR ', phosphate ester RPO (OR'_Three), Alkyloxonium salts R_ThreeO +, sulfonium R_ThreeS +, nitrate ester RONO₂Michael acceptor RCR '= CR' "COR", fluorinated aryl ArF, isonitrile
[0180]
[Chemical 1]

Haloamine R₂N-Hal, alkene and alkyne.
[0181]
IV. Sulfur-containing groups are exemplified by: disulfide RSSR ', sulfhydryl RSH, epoxide
[0182]
[Chemical 2]

V. Salts are exemplified by: alkyl ammonium salts or aryl ammonium salts R_FourN +, carboxylate RCOO-, sulfate ROSO_Three-, Phosphate ROPO_ThreeAnd Amine R_ThreeN.
[0183]
Table 1 below gives examples of triggers organized by the release mechanism. In Table 1, R = is a multifunctional cross-linking agent, which can be alkyl, aryl, or other chain with an active group that can react with the protein to be cross-linked. These reactive groups can be any of a variety of groups (eg, groups that are sensitive to nucleophilic substitution, free radical substitution, or electrophilic substitution, particularly including halides, aldehydes, carbonates, urethanes, xanthan, epoxides). possible.
[0184]
[Table 1]

Further examples of reversible crosslinkers are described in T.W. W. Green, Protective Groups in Organic Synthesis, John Wiley and Sons (ed.) (Ed.) (1981). Any of a variety of strategies for use with reversible protecting groups can be introduced into a crosslinker suitable for the production of cross-linked protein crystals capable of reversible controlled solubilization. Various approaches are listed in a review by Waldmann on this subject (Angewante Chemie Inl. Ed. Engl. 35, 2056 (1996)).
[0185]
Another type of reversible crosslinker is a disulfide bond-containing crosslinker. The trigger break bridge formed by such a crosslinker is the addition of a reducing agent (eg, cysteine) to the crosslinked protein crystal environment.
[0186]
Disulfide crosslinkers are described in Pierce Catalog and Handbook (1994-1995), and more recently, G. T.A. “Bioconjugate Techniques” by Hermanson, (1996), Academic Press, Division of Harcourt Brace and Company, 525 B Street, Suite 1990, San Diego, CA 9210144.
Examples of such crosslinkers include the following:
(Same bifunctionality (symmetry))
(DSS) -dithiobis (succinimidyl propionate), also known as Lomant agent
(DTSSP) -3-3'-dithiobis (sulfosuccinimidylpropionate), a water soluble version of DSP
(DTBP) -dimethyl 3,3'-dithiobispropionimidate.HCl.
(BASED) -bis- (β- [4-azidosalicylamino] ethyl) disulfide
(DPDPB)-1,4-di- (3 '-[2'-pyridyldithio] -propionamido) butane
(Heterogeneous bifunctional (asymmetric))
(SPDP) -N-succinimidyl-3- (2-pyridyldithio) propionate
(LC-SPDP) -succinimidyl-6- (3- [2-pyridyldithio] propionate) hexanoate
(Sulfo-LC-SPDP) -sulfosuccinimidyl-6- (3- [2-pyridyldithio] propionate) hexanoate, a water-soluble version of LC-SPDP
(APDP) -N- (4- [p-azidosalicylamido] butyl) -3 '-(2'-pyridyldithio) propionamide
(SADP) -N-succinimidyl (4-azidophenyl) 1,3'-dithiopropionate
(Sulfo-SADP) -sulfosuccinimidyl (4-azidophenyl) 1,3'-dithiopropionate, a water-soluble version of SADP
(SAED) -sulfosuccinimidyl-2- (7-azido-4-methylcoumarin-3-acetamido) ethyl-1,3 'dithiopropionate
(SAND) -sulfosuccinimidyl-2- (m-azido-o-nitrobenzamido) ethyl-1,3'-dithiopropionate
(SASD) -sulfosuccinimidyl-2- (p-azidosalicylamido) ethyl-1,3'-dithiopropionate
(SMPR) -succinimidyl-4- (p-maleimidophenyl) butyrate
(Sulfo-SMPR) -sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate
(SMPT) -4-succinimidyloxycarbonyl-methyl-α- (2-pyridylthio) toluene
(Sulfo-LC-SMPT) -sulfosuccinimidyl-6- (α-methyl-α- (2-pyridylthio) toluamide) hexanoate.
[0187]
For details, see G. T.A. Hermanson, (1996), Academic Press, Division of Harcourt Brace & Company, 525 B Street, Suite 1900, San Diego, according to the CA 92101-4495 of "Bioconjugate Techniques", Zero-length Cross-linkers, Homobifunctional Cross-linkers and Heterobifunctional See Section II, Section 3-5 of Cross-linkers.
[0188]
Crosslinked protein crystals useful for the formation of the proteins of the invention can also be prepared according to the methods set forth in PCT patent application PCT / US91 / 05415.
[0189]
(Encapsulation of protein crystals in polymer carrier)
According to one embodiment of the invention, the compositions are those native when they are encapsulated with a matrix of polymer carriers such that the protein crystals are in microsphere form upon encapsulation in at least one polymer carrier. Generated to preserve tertiary structure and biologically active tertiary structure. Crystals can be encapsulated using a variety of biocompatible and / or biodegradable polymers with unique properties suitable for delivery to different biological environments or for effecting specific functions. Thus, the dissolution rate and delivery rate of the active protein depends on the specific encapsulation technique, polymer composition, polymer cross-linking, polymer thickness, polymer solubility, relative position and extent of protein crystals, and protein crystal cross-linking, if any. Is determined by the relative position and degree.
[0190]
The protein crystal or formulation to be encapsulated is suspended in a polymer carrier dissolved in an organic solvent. After the polymer solution is added to the solution, it must be concentrated to a concentration sufficient to completely coat the protein crystals or formulation. Such an amount is an amount that provides a weight ratio of protein crystals to polymer between about 0.02 and about 20, preferably between about 0.1 and about 2. The protein crystals are contacted with the polymer in solution for a time between about 0.5 minutes and about 30 minutes, preferably between about 1 minute and about 3 minutes. The crystals should remain suspended and cannot be agglomerated because the crystals are coated by contact with the polymer.
[0191]
After that contact, the crystals are coated and are called nascent microspheres. New born microspheres increase in size while filming occurs. In a preferred embodiment of the invention, the coated crystals or nascent microspheres suspended with a polymer carrier and an organic solvent are transferred to a large volume of aqueous solution containing a surfactant known as an emulsifier. In this aqueous solution, the suspended nascent microspheres are immersed in the aqueous phase, where the organic solvent is allowed to evaporate or diffuse out of the polymer. Eventually, the polymer is no longer soluble and reaches a point where it forms a precipitated phase that encapsulates the protein crystals or formulation to form the composition. This aspect of the process is referred to as polymer carrier or polymer curing. The emulsifier helps to reduce the surface tension of the interface between the various phases of the material in the system during the curing phase process. Alternatively, if the coating polymer has some inherent surface activity, it is not necessary to add another surfactant.
[0192]
Emulsifiers useful for preparing encapsulated protein crystals in accordance with the present invention include those exemplified herein that can reduce the surface tension between the polymer-coated protein crystals or polymer-coated crystal formulation and the solution. Poly (vinyl alcohol), surfactants and other surfactants.
[0193]
Organic solvents useful for preparing the microspheres of the present invention include methylene chloride, ethyl acetate, chloroform and other non-toxic solvents, depending on the properties of the polymer. A solvent that dissolves the polymer and is ultimately non-toxic should be selected.
[0194]
A preferred embodiment of the present invention is that the crystallinity of the protein crystals is maintained during the encapsulation process. Crystallinity is maintained using an organic solvent during the coating process (where the crystals are not soluble). Subsequently, once the coated crystals are transferred to an aqueous solvent, rapid curing of the polymer carrier in the previous step and sufficient coating of the crystals protects the crystalline material from dissolution. In other embodiments, the use of cross-linked protein crystals facilitates the maintenance of crystallinity in both aqueous and organic solvents.
[0195]
The polymer used as a polymer carrier to coat protein crystals can be either a homopolymer or a copolymer. The hydrolysis rate of microspheres is largely determined by the hydrolysis rate of the individual polymer species. In general, the hydrolysis rate decreases as follows: polycarbonate> polyester> polyurethane> polyorthoester> polyamide. For a review of biodegradable and biocompatible polymers, see W.W. R. Gombotz and D.W. K. See Pettit, “Biodegradable polymers for protein and peptide drug delivery”, Bioconjugate Chemistry, Vol. 6, pages 332-351 (1995).
[0196]
In a preferred embodiment, the polymer carrier consists of a single polymer type such as PLGA. In the next preferred embodiment, the polymer carrier can be a mixture of polymers such as 50% PLGA and 50% albumin.
[0197]
Other polymers useful as polymer carriers for preparing encapsulated protein crystals according to the present invention include biocompatible / biodegradable polymers selected from the group consisting of: poly (acrylic acid), poly (Cyanoacrylate), poly (amino acid), poly (anhydride), poly (depsipeptide), poly (ester) (eg, poly (lactic acid) or PLA), poly (b-hydroxybutyric acid), poly (caprolactone) and poly (Dioxanone); poly (ethylene glycol), poly (hydroxypropyl) methacrylamide, poly [(organo) phosphazene], poly (orthoester), poly (vinyl alcohol), poly (vinyl pyrrolidone), maleic anhydride Alkyl vinyl ether copolymer, pl onic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glyceryl Camino glycans (glycaminoglycan), sulfated polysaccharide, mixtures thereof and copolymers. Other useful polymers are described in J. Org. Heller and R.W. W. Baller, “Theory and Practice of Controlled Drug Delivery from Biodegradable Polymers” Academic Press, New York, NY, (1980); C. R. Lehman and D.M. K. Dreher, Pharmaceutical Technology Volume 3, page 5- (1979); M.M. Ramadan, A .; El-Helw and Y.W. El-Said, Journal of Microencapsulation, Volume 5, page 125 (1988). Preferred polymers depend on the specific protein component of the protein crystals used and the intended use of the encapsulated crystals (formulations and compositions). Alternatively, solvent evaporation techniques can be used to encapsulate protein crystals (see D. Babay, A. Hoffmann and S. Benita, Biomaterials 9, 482-488 (1988)).
[0198]
In a preferred embodiment of the present invention, protein crystals are prepared using a polymer such as polylactic acid-co-glycolic acid using a double emulsion method (exemplified herein). Encapsulated in at least one polymer carrier. In the most preferred embodiment of the invention, the polymer is polylactic-co-glycolic acid (“PLGA”). PLGA is a copolymer prepared by a polycondensation reaction of lactic acid (“L”) and glycolic acid (“G”). Various ratios of L and G can be used to adjust the crystallinity and hydrophobicity of the PLGA polymer. The higher crystallinity of the polymer results in slower dissolution. PLGA polymers with 20-70% G content tend to be amorphous solids, but high levels of either G or L result in excellent polymer crystallinity. More information on the preparation of PLGA can be found in K. Gilding and A.M. M.M. See Reed, “Biodegradable polymers for use in surgery-poly (glycic) / poly (lactic acid) homo and copolymers: 1”, Polymer 20, pages 1459-1464 (1981). PLGA is degraded by hydrolysis of ester bonds after exposure to water to produce non-toxic lactic and glycolic acid monomers.
[0199]
In another embodiment, double-walled polymer-coated microspheres can be beneficial. Double layer polymer-coated microspheres can be produced by preparing two separate polymer solutions in methylene chloride or other solvent that can dissolve the polymer. Protein crystals are added to one side of this solution and dispersed. Here, the protein crystal is coated with a first polymer. The solution containing protein crystals coated with the first polymer is then mixed with the second polymer solution. (See Pekarek, KJ; Jacob, JS, and Mathiowitz, E. Double-walled polymer microspheres for controlled drug release, Nature, 367, 258-260). This time, the second polymer encapsulates the first polymer encapsulating the protein crystals. Ideally, this solution is then dropped into a larger volume of aqueous solution containing a surfactant or emulsifier. In aqueous solution, the solvent is evaporated from the two polymer solutions and precipitates the polymer.
[0200]
The above steps may be performed using either protein crystals, DNA crystals or RNA crystals from any of these formulations to produce a composition.
[0201]
The formulation according to the invention comprises protein crystals and at least one component. Such formulations are characterized by an expiration date that is at least 60 times longer than the soluble form of this protein in solution at 50 ° C. when stored at 50 ° C. (T_1/2When measured by). Alternatively, such a formulation, when stored at 40 ° C. and 75% humidity, is at least 59 times longer than the non-formulated form of this protein crystal stored at 40 ° C. and 75% humidity. Characterized by an expiration date (T_1/2When measured by). Alternatively, such a formulation is characterized by a shelf life of at least 60% longer when stored at 50 ° C. than the non-formulated form of the protein crystals stored at 50 ° C. (T_1/2When measured by). Alternatively, such formulations are characterized by a loss of less than 20% of the α-helix structure content of the protein after storage at 50 ° C. for 4 days, wherein the soluble form of the protein is 50 After storage at 6 ° C. for 6 hours, the protein loses the α-helix structure content by more than 50% (as measured by FTIR). Alternatively, such formulations may be characterized by less than 20% loss of the protein's α-helical structure content after storage at 50 ° C. for 4 days, wherein the soluble form of the protein is After storage for 6 hours at 50 ° C., the α-helix structure content of the protein is lost by more than 50% (as measured by FTIR), and such formulations are now at 50 ° C. When stored, it is characterized by an expiration date that is at least 60 times longer than the soluble form of this protein in solution at 50 ° C. (T_1/2When measured by).
[0202]
The composition according to the invention comprises one of the above protein crystal formulations and at least one polymer carrier, wherein the formulation is encapsulated in a matrix of the polymer carrier.
[0203]
Alternatively, the composition according to the invention comprises a formulation of protein crystals and at least one component. Such a composition, when stored at 50 ° C., can be characterized by an expiration date that is at least 60 times longer than the soluble form of this protein in a 50 ° C. solution (T_1/2When measured by). Alternatively, such a composition, when stored at 40 ° C. and 75% humidity, is at least 59 times longer than the non-formulated form of this protein crystal stored at 40 ° C. and 75% humidity. Characterized by an expiration date (T_1/2When measured by). Alternatively, such compositions are characterized by an expiration date that is at least 60% longer when stored at 50 ° C. than non-formulated forms of the protein crystals stored at 50 ° C. (T_1/2When measured by). Alternatively, such compositions are characterized by a loss of less than 20% of the α-helical structure content of the protein after storage at 50 ° C. for 4 days, wherein the soluble form of the protein is 50 After storage at 6 ° C. for 6 hours, the protein loses the α-helix structure content by more than 50% (as measured by FTIR). Alternatively, such a composition may be characterized by less than 20% loss of the protein's α-helical structure content after storage at 50 ° C. for 4 days, wherein the soluble form of the protein is After being stored at 50 ° C. for 6 hours, the α-helix structure content of the protein was lost by more than 50% (as measured by FTIR), and now the formulation was stored at 50 ° C. When it is characterized by a shelf-life of at least 60 times longer than the soluble form of this protein in solution at 50 ° C. (T_1/2When measured by).
[0204]
In order that the present invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and cannot be construed to limit the scope of the invention in any manner.
[0205]
(Example)
(Example 1 Lipase)
(Candida rugosa lipase crystallization)
material:
A-Candida rugosa lipase powder
B-Celite powder (diatomaceous earth)
C-MPD (2-methyl-2,4-pentadiol)
D-5 mM Ca acetate buffer pH 4.6
E-deionized water.
[0206]
(procedure)
An aliquot of 1 kg of lipase powder was mixed well with 1 kg of celite, then 22 L of distilled water was added. The mixture was stirred to dissolve the lipase powder. After dissolution was complete, the pH was adjusted to 4.8 using acetic acid. The solution was then filtered to remove celite and undissolved material. The filtrate was then pumped using a 30K cut-off hollow fiber to remove all proteins with a molecular weight of less than 30 kD. Distilled water was added and the lipase filtrate was pumped with hollow fibers until the conductivity of the retentate was equal to the conductivity of the distilled water. At this point, the addition of distilled water was stopped and 5 mM Ca-acetate buffer was added. The Ca-acetate buffer was then delivered by hollow fiber pumping until the residue conductivity was equal to the Ca-acetate buffer conductivity. At that point, buffer addition was stopped. The lipase solution was concentrated to a 30 mg / ml solution. The crystallization was initiated by gently pumping the lipase solution into the lipase solution while stirring the MPD. The addition of MPD was continued until MPD reached 20% volume / volume. The mixture was stirred for 24 hours or until 90% of the protein crystallized. The resulting crystals were washed with a crystallization buffer to remove any soluble material from the crystals. The crystals were then suspended in fresh crystallization buffer to achieve a protein concentration of 42 mg / ml.
[0207]
(Example 2)
(Formulation of lipase crystals using sucrose as an excipient)
In order to enhance the stability of the lipase crystals during the drying process and storage, the crystals were formulated with excipients. In this example, the lipase crystals were formulated in slurry form in the presence of mother liquor prior to the drying step. Sucrose (Sigma Chemical Co., St. Louis, MO) was added as an excipient to the lipase crystals in the mother liquor. Sufficient sucrose was added to lipase crystals at 20 mg / ml protein concentration in mother liquor (10 mM sodium acetate buffer, pH 4.8 containing 10 mM calcium chloride and 20% MPD) until the final concentration reached 10%. The resulting suspension was inverted for 3 hours at room temperature. After treatment with sucrose, the crystals were separated from the liquid by centrifugation (described in Example 6, Method 4 or 5).
[0208]
(Example 3)
(Formulation of lipase crystals using trehalose as an excipient)
Lipase crystals were formulated in Example 2 by adding trehalose (Sigma Chemical Co., St. Louis, MO) instead of sucrose to a final concentration of 10% in the mother liquor. The resulting suspension was tumbled for 3 hours at room temperature and the crystals were separated from the liquid by centrifugation as described in Method 4 or 5 of Example 6.
[0209]
Example 4
(Formulation of lipase crystals using polyethylene oxide (PEO) as an excipient)
Lipase crystals were formulated using 0.1% polyethylene oxide in water as follows. The crystals (20 mg / ml in the mother liquor) were separated from the mother liquor by centrifugation at 1000 rpm in a Beckman GS-6R tabletop centrifuge equipped with a swing bucket rotor. The crystals are then suspended in 0.1% polyethylene oxide (Sigma Chemical Co., St. Louis, Mo.) for 3 hours and then centrifuged as described in Method 6 or 5 of Example 6. separated.
[0210]
(Example 5)
(Formulation of lipase crystals using methoxypolyethylene glycol (MOPEG) as an excipient)
Lipase crystals were added to 10% methoxypolyethylene glycol (final concentration) (Sigma Chemical Co., St. Louis, MO) in mother liquor instead of sucrose as in Example 2, and Method 4 of Example 6 Alternatively, as in 5, it was formulated by separation by centrifugation after 3 hours.
[0211]
(Example 6)
(Method of drying crystal formulation)
(Method 1. N at room temperature₂Gas drying)
Crystals prepared as in Examples 1, 10, 14, and 21 were centrifuged in a Beckman GS-6R tabletop centrifuge equipped with a swinging bucket rotor at 1000 rpm in a 50 ml Fischer brand Disposable centrifuge tube (polypropylene). Separated from mother liquor containing excipients. The crystals were then dried by passing a stream of nitrogen at about 10 psi pressure through the tube overnight.
[0212]
(Method 2. Vacuum oven drying)
Crystals prepared as in Examples 1, 10, 14, and 21 were first prepared using a 50 ml Fischer brand Disposable polypropylene centrifuge tube using a 1000 rpm centrifuge in a Beckman GS-6R tabletop centrifuge equipped with a swinging bucket rotor. In, separated from the mother liquor / excipient solution. The wet crystals were then placed in a vacuum oven (VWR Scientific Products) at room temperature at 25 in Hg and dried for at least 12 hours.
[0213]
(Method 3. Freeze drying)
Crystals prepared as in Examples 1, 10, 14, and 21 were first prepared using a 50 ml Fischer brand Disposable polypropylene centrifuge tube using a 1000 rpm centrifuge in a Beckman GS-6R tabletop centrifuge equipped with a swinging bucket rotor. In, separated from the mother liquor / excipient solution. The wet crystals were then lyophilized in a half-closed vial using a Virtis Lyophilizer Model 24. The shelf temperature was slowly reduced to −40 ° C. during the freezing process. This temperature was maintained for 16 hours. Secondary drying was then performed for an additional 8 hours.
[0214]
(Method 4. Organic solvent and air drying)
Crystals prepared as in Examples 1, 10, 14, and 21 were first prepared using a 50 ml Fischer brand Disposable polypropylene centrifuge tube using a 1000 rpm centrifuge in a Beckman GS-6R tabletop centrifuge equipped with a swinging bucket rotor. In, separated from the mother liquor / excipient solution. The crystals are then suspended in an organic solvent (such as ethanol or isopropanol or ethyl acetate or other suitable solvent), centrifuged, the supernatant decanted, and left in a fume hood for 2 days at room temperature. Air dried.
[0215]
(Method 5. Air drying at room temperature)
Crystals prepared as in Examples 1, 10, 14, and 21 were centrifuged in a Beckman GS-6R tabletop centrifuge equipped with a swing bucket rotor at 1000 rpm in a 50 ml Fischer brand Disposable centrifuge tube (polypropylene). Separated from mother liquor containing excipients. Subsequently, the crystals were air-dried in a fume hood for 2 days.
[0216]
(Example 7)
(Preparation of soluble lipase sample)
For comparison, a sample of soluble lipase was prepared by dissolving lipase crystals in phosphate buffered saline (pH 7.4) at 20 mg / ml.
[0217]
FIG. 2 shows the stability for soluble lipase. Specific activity decreases very rapidly with time. Within 2-3 hours, the specific activity decreases from about 660 micromole / minute / mg protein to about 100 micromole / minute / mg protein (ie, about 85% reduction). T for soluble lipase_1/2Was calculated to be 1.12 hours.
[0218]
(Example 8)
(Olive oil assay to measure lipase activity)
The lipase crystals from Examples 1-7 were evaluated for activity against olive oil in pH 7.7 buffer. This assay was performed as described in Pharmaceutical Enzymes-Properties and Assay Methods, R.A. Ruyssen and A.M. Titration was performed using a slight modification to the procedure described in Lauwers (ed.), Scientific Publishing Company, Ghent, Belgium (1978).
[0219]
(reagent)
1. Olive oil emulsion:
16.5 g of gum arabic (Sigma) was dissolved in 180 ml of water, 20 ml of olive oil (Sigma) and emulsified using a Quick Prep mixer for 3 minutes.
2. Titration solution: 0.05M NaOH
3. Solution A: 3.0M NaCl
4). Solution B: 75 mM CaCl₂・ 2H₂O
5). Mixture: 40 ml of solution A was mixed with 20 ml of solution B and 100 ml of water
6. 0.5% albumin:
7). A lipase substrate solution (Solution 7) was prepared by adding 50 ml of olive oil emulsion (Solution 1) to 40 ml of the mixture (Solution 5) and 10 ml of 0.5% albumin (Solution 6).
[0220]
(Assay procedure)
The lipase substrate solution (Solution 7) was warmed to 37 ° C. in a water bath. First, 20 ml of substrate was added to the reaction vessel and the pH was adjusted to 7.7 using 0.05 M NaOH (solution 2) and equilibrated to 37 ° C. with agitation. The reaction was started by adding the enzyme. The progress of the reaction was monitored while maintaining the pH at 7.7 by titrating the enzyme and substrate mixture with 0.05M NaOH.
[0221]
The specific activity (micromole / min / mg protein) was equal to (initial velocity × 1000 × titrant concentration) / (enzyme amount). The zero point was determined by performing the reaction without enzyme (ie, using a buffer instead of enzyme in the reaction mixture).
[0222]
Example 9
(Activity)
The storage activity of the dried crystals from Examples 1-5 was measured using an olive oil assay as described in Example 8. Dry crystals (5 mg) were dissolved in 1 ml of phosphate buffered saline (“PBS”) (pH 7.4) and the activity was measured using olive oil as a substrate.
[0223]
(Shelf stability)
The storage stability of the dry crystalline lipase formulations from Examples 2-5 was carried out in a humid chamber (HOTPACK) controlled at a relative humidity of 75% and a temperature of 40 ° C. The activity of the crystals was measured by dissolving a 5 mg dry sample in PBS buffer (pH 7.4), measuring the activity in an olive oil assay and then comparing it to the initial results.
[0224]
(Crystal formulation dried by Method 5)
FIG. 3 shows the storage stability profile of lipase crystals formulated with sucrose, trehalose, and PEO. When dried by Method 5, PEO was the most protective excipient, followed by sucrose and then trehalose.
[0225]
(Crystal formulation dried by Method 4)
FIG. 4 shows the storage stability profile of lipase crystals formulated with sucrose, trehalose, PEO, and MOPEG. When dried by Method 4, the excipients PEO, sucrose and MOPEG are T_1/2Their ability to preserve enzyme activity was similar as measured by their effect on. Trehalose was less protected for lipase activity than other excipients.
[0226]
(Storage stability)
The time required for a 50% decrease in the specific activity of the enzyme is T_1/2Is known as Table 2 shows the specific activity T for dry lipase crystal formulations._1/2The effect on is shown. For lipase, sucrose was the most protective excipient, followed by polyethylene oxide (PEO), methoxypolyethylene glycol (MOPEG), and finally trehalose. Sucrose has a specific activity of T_1/2It was more than 10 times more protective when measured by its effect on.
[0227]
[Table 2]

T_1/2Was calculated from the expiry date data by non-linear regression analysis using the Sigma Plot program. Table 2 shows that the lipase formulation was 1,923 times more stable than soluble when PEO was used as an excipient. Furthermore, the lipase formulation was 23,300 times more stable than soluble lipase when sucrose was used as an excipient (Table 2). The formulation of lipase crystals using MOPEG and PEO as excipients was 9,270 and 17,800 times more stable than soluble lipase (Table 2).
[0228]
As shown in Table 2, the stability of the formulated crystals was greatly enhanced compared to the non-formulated crystals. As shown in Table 2, for example, crystals formulated with trehalose were 59 times more stable at 40 ° C. than non-formulated lipase crystals produced without excipients. Similarly, as shown in Table 2, crystals formulated with MOPEG are 286 times more stable than non-formulated crystals, and crystals formulated with PEO have no excipients. It was 549 times more stable at 40 ° C. than the produced non-formulated lipase crystals. Finally, as shown in Table 2, crystals formulated with sucrose were 718 times more stable at 40 ° C. than unformulated lipase crystals produced without excipients.
[0229]
(Water content)
The moisture content was determined by the Karl Fischer method using a Mitsubishi CA-06 Moisture Meter (Mitsubishi Chemical Corporation, Tokyo, Japan) equipped with a VA-06 Vaporizer according to the manufacturer's instructions.
[0230]
[Table 3]

(Crystallinity)
The crystal strength of the formulation was measured by quantitative microscopy. To visualize whether the crystals maintain their shape after drying, the dried crystals were examined under an Olympus BX60 microscope equipped with a Camera Adapter and DXC-970MD 3CCD Color Video Camera with Image ProPlus software. Samples of the dried crystals were covered with a glass cover strip, mounted, and examined at 10 × magnification using an Olympus microscope (phase contrast) equipped with an Olympus UPLAN F1 objective 10 × / 0.30 PH1. .
[0231]
In this analysis, crystals originally formulated with sucrose, trehalose, PEO and MOPEG were easily visualized after 129 days at 40 ° C. and 75% humidity. FIG. 5 shows that crystals formulated with PEO were present at the first time point. Similar microscopic observations obtained after 129 days and shown in FIG. 6 demonstrate that crystallinity was maintained throughout the period. Similar data was obtained for crystals formulated with sucrose, MOPEG and trehalose (data not shown).
[0232]
(Characteristic of secondary structure by FTIR)
Fourier Transform Infrared Spectroscopy (“FTIR”) spectra were collected on a Nicolet model 550 Magna series spectrometer as described by Dong et al. Bhat, KS and Coe, JE Biochemistry, 1992; 31: 9364-9370; Dong, A. Prestrelski, SJ, Allison, SD and Carpenter, JFJ Pharm. Sci., 1995; 84; 415-424). For solid samples, 1-2 mg of protein was lightly applied with 350 mg of KBr powder and a small cup was filled with diffuse reflection components. The spectra were collected and then Grams 32 from Galactic software for the determination of the relative regions of the individual components of the secondary structure using the second derivative, and the amide I region (1600-1700 cm^-1) Using a curve fitting program.
[0233]
For comparison, soluble lipase samples were prepared by dissolving lipase crystals in phosphate buffered saline and analyzed by FTIR for stability.
[0234]
Secondary structure was determined as follows: FTIR spectra were collected on a Nicolet model 550 Magna series spectrometer. A 1 ml sample of soluble lipase was placed on ARK ESP zinc selenide crystals. Spectra were collected at the first time point (0) and after most activity was lost or nearly zero activity. The acquired data was then used to determine the relative regions of individual components of the secondary structure using the second derivative, Grams 32 software from Galactic software, and the amide I region (1600-1700 cm^-1) Using a curve fitting program.
[0235]
[Table 4]

(Conclusion)
Table 4 shows that about 50% and 75% α-helix and β-sheet structure content was lost over 3 days for soluble lipase. There was a corresponding increase in the content of random structures.
[0236]
In contrast, FIG. 4 shows that formulated and dried crystals are much more stable than soluble enzymes. Such crystals showed a loss of α-helix structure ranging from 6.1% to 21.1% after 66 days at 40 ° C. and 75% humidity. At this point, the random coil in solution increased by 7-50% over 3 days, while the crystallinity showed minimal random coil content even after 66 days.
[0237]
The data in Table 4 obtained by monitoring changes in secondary structure using FTIR correlated with the activity data shown in FIG. Specifically, the lipase formulated with sucrose, PEO and MOPEG has an α-helix structure rather than crystals formulated with trehalose (showing 21% loss of α-helix structure and having the lowest activity profile). And maintained higher specific activity over 66 days at elevated temperature and humidity.
[0238]
(Example 10: Human serum albumin)
(Crystallization of human serum albumin)
Ten grams of powdered human serum albumin was added to 75 ml of a stirred solution of 100 mM phosphate buffer (pH 5.5) (4 ° C.). Final protein concentration was 120 mg / ml (OD of solution₂₈₀Estimated from the value). First, ammonium sulfate solution (767 g / l) (prepared with deionized water) was added to the protein solution to a final concentration of 350 g / l or 50% saturation. The crystallization solution was then “seeded” with 1 ml of 50 mg / ml albumin crystals in 50% ammonium sulfate (pH 5.5). Seed crystals were prepared by washing a sample of crystals without a precipitate with a solution of 50% saturated ammonium sulfate (pH 5.5) in 100 mM phosphate buffer. The seeded crystallization solution was incubated on a powerful rotating platform at 4 ° C. overnight. Rod-shaped crystals (20 μm) appeared in the overnight solution about 16 hours later.
[0239]
(Example 11)
(Formulation of HSA crystals using gelatin as an excipient)
In order to enhance the stability of human serum albumin (HSA) crystals during drying and storage, the crystals were formulated with excipients. In this example, HSA crystals were formulated in slurry form in the presence of mother liquor prior to drying. Gelatin (Sigma Chemical Co., St. Louis, MO) was added to the lipase crystals in the mother liquor as an excipient. Sufficient gelatin was added to lipase crystals in mother liquor (2.5 M ammonium sulfate (pH 5.5) in 100 mM phosphate buffer) at a protein concentration of 20 mgs / ml until a final concentration of 10% was reached. The resulting suspension was rotated at room temperature for 3 hours. After treatment with gelatin, the crystals were separated from the liquid by centrifugation as described in Method 1 of Example 6.
[0240]
(Drying of HSA crystals)
The HSA crystals were then dried by the four methods described in Example 6. The crystals were suspended in cold ethanol (4 ° C.) as method 4 organic solvent.
[0241]
(Example 12)
(Soluble human serum albumin preparation)
For comparison, soluble HSA samples were prepared by dissolving HSA at 20 mg / ml in water.
[0242]
(Example 13)
(Shelf stability)
Storage stability of the dried crystalline formulation of HSA was performed in a water bath at a temperature of 50 ° C. The stability of the crystals was monitored by FTIR analysis by the following structural collapse.
[0243]
(Water content)
The moisture content was determined by the Karl Fischer method using a Mitsubishi CA-06 Moisture Meter (Mitsubishi Chemical Corporation, Tokyo, Japan) equipped with a VA-06 Vaporizer according to the manufacturer's instructions.
[0244]
[Table 5]

(Crystallinity)
The crystal strength of the formulation was measured by quantitative microscopy as described in Example 9. FIG. 7 shows that HSA crystals were easily visualized immediately after preparing the formulation with gelatin. FIG. 8 shows that the crystallinity was maintained after 4 days at 50 ° C.
[0245]
(Characteristic of secondary structure by FTIR)
FTIR spectra were collected on a Nicolet model 550 Magna series spectrometer as described by Dong et al. (Dong, A., Caughey, B., Caughey, WS, Bhat, KS, and Coe, J. E. Biochemistry, 1992; 31: 9364-9370; Dong, A. Prestrelski, SJ, Allison, SD and Carpenter, JFJ Pharm.Sci., 1995; 84; 424). For solid samples, 1-2 mg of protein was lightly applied with 350 mg of KBr powder and a small cup was filled with diffuse reflection components. The spectra were collected and then Grams 32 from Galactic software for the determination of the relative regions of the individual components of the secondary structure using the second derivative, and the amide I region (1600-1700 cm^-1) Using a curve fitting program.
[0246]
For comparison, soluble HSA samples were prepared by dissolving HSA crystals in water and tested for stability by FTIR. Secondary structure was determined as described in Example 9.
[0247]
[Table 6]

(Conclusion)
Soluble HSA showed a rapid decrease in α-helix content of 78% after 1 hour in solution, and a corresponding increase in random coil structure.
[0248]
The dry formulated crystals showed about a 16% decrease in α-helix content, a significant decrease in β-sheet content, and no increase in random structure content.
[0249]
(Example 14: Penicillin acylase)
(Crystallization of penicillin acylase)
The ammonium sulfate suspension of penicillin acylase from Boehringer Mannheim was the crude material. The penicillin acylase suspension was diluted 1: 3 with deionized water. This solution was diafiltered using a 30K membrane,_280nmConcentrated to a final concentration of 200 OD / ml. This enzyme solution was then added to 4M NaH.₂PO_Four・ H₂A with O_280nm/ Ml to 150 ODm.
[0250]
1.9M NaPO_Four4M and 1M sufficient NaH to give a final solution concentration of₂PO_Four・ H₂A biphasic solution of O was prepared. In this case, 280 ml of 1M NaH₂PO_Four・ H₂549 ml of 4M NaH₂PO_Four・ H₂Carefully layered on top of O. The enzyme solution was gently poured onto the side of the container to form a layer on top of the 1M layer. An overhead stirrer was set up along with the ship impeller. The agitator was placed in the container with its blade just below the 4M / 1M interface. The speed was adjusted to a setting of 8.0, ie 600 rpm. The agitator was switched on and stopped after 10 minutes. The impeller was removed from the container. The volume of seed crystals was measured with a graduated pipette between 0.5% and 0.1% by volume and added into a 20 L sterile polycarbonate container. This seed was not allowed to stand for more than 10 seconds before addition. This solution was manually stirred using a flat blade impeller for about 1 minute. The crystallization mixture was allowed to stand for 24 hours.
[0251]
After 24 hours, the container was opened and the solution was mixed manually for 30 seconds. This solution was allowed to settle for an additional 24 hours. After a total of 48 hours, a 10 ml sample of the supernatant was taken. The sample was filtered using a 0.2 micron Acrodisc. A of the supernatant_280nmWas measured. A of the supernatant_280nmIf is greater than 1.0 mg / ml, crystallization was continued for an additional 24 hours. When the concentration of the supernatant is less than 1.0 mg / ml, the crystal slurry and the supernatant A_280nmWas determined directly.
[0252]
Example 15: Penicillin G assay on PA crystals
The basis of the activity assay for penicillin acylase includes a titration assay that measures the enzymatic hydrolysis of benzylpenicillin by this enzyme. This enzyme catalyzes the cleavage of the phenylacetyl group from penicillin G, thereby causing a decrease in pH. This activity is by measuring the volume of 50 mM NaOH required per minute of the reaction (to maintain pH 8 at 28 ° C.). This assay uses a substrate buffer made with potassium chloride and Tris. The assay was then reported in U / mg.
[0253]
(Penicillin acylase assay):
(Chemicals and solutions used in this assay):
1. Penicillin-G, Sigma, potassium salt
2. 0.05N sodium hydroxide
3. 1.0 M KC1, 20 mM Tris buffer, pH 8.0
4). 10 mM Tris, 10 mM CaCl₂Buffer, pH 8.0
5). 0.01M PBS buffer, pH 7.5
(Preparation of substrate solution)
1 g of penicillin-G was added to 10 ml of 1.0 M KCl, 20 mM Tris buffer, pH 8.0 and about 70 ml DI water, 1 ml 10 mM Tris, 10 mM CaCl.₂Added in solution. The pH of this solution was then adjusted to 8.0 using 0.05N NaOH. The final solution volume was then adjusted to 100 ml. This solution was prepared fresh before each use and used within 5 hours of preparation.
[0254]
(Preparation of PA sample solution)
A sample of penicillin acylase was dissolved in PBS buffer, pH 7.4. Typically, 2 mg to 4 mg (dry weight) of protein was used for each assay.
[0255]
(Assay for Penicillin G Hydrolysis)
20 ml of penicillin acylase substrate solution was added into the titration vessel and equilibrated to 28 ° C. After the enzyme solution was added to the reaction vessel, the hydrolysis of penicillin-G was monitored by titrating the reaction mixture with 0.05N NaOH to maintain the pH at 8.0.
[0256]
Example 16: PA crystal formulation using hydroxypropyl-β-cyclodextrin (HPCD) as excipient.
In order to enhance the stability of PA crystals during drying and storage, the crystals were formulated with excipients. In this example, PA crystals were formulated in slurry form in the presence of mother liquor before drying. Hydroxypropyl-β-cyclodextrin (HPCD) (Sigma Chemical Co., St. Louis, MO) was added as an excipient to the PA crystals in the mother liquor. Enough HPCD was added to the mother liquor (1.9M NaH₂PO_Four, PH 6.6) at a protein concentration of 20 mg / m was added to the lipase crystals to reach a final concentration of 10%. The resulting suspension was tumbled for 3 hours at room temperature. After treatment with HPCD, the crystals were separated from the liquid by centrifugation as described in Method 1 of Example 6.
[0257]
(Example 17: PA crystal formulation using mother liquor itself as excipient):
PA crystals were dissolved in the mother liquor (1.9 M NaH₂PO_Four, PH 6.6). The crystals were separated by centrifugation as in Example 16.
[0258]
(Example 18: Drying of PA)
The PA crystals from Examples 15-17 were then dried according to Method 1, 3 or 4 of Example 6. The organic solvent used for Method 4 of Example 6 was ethyl acetate.
[0259]
Example 19: Soluble PA preparation:
As a comparative standard, a sample of soluble PA was prepared by dissolving PA crystals in water at 20 mg / ml.
[0260]
The stability of soluble PA was measured over time. The plot specific activity at 55 ° C. with respect to time is shown in FIG. PA enzyme activity in solution decayed rapidly and was not detectable after about 20 hours.
[0261]
(Example 20: Activity of dry crystals):
The activity of the dried crystals from Example 18 was tested in the Pen G assay as described in Example 15. The dried crystals (5 mg) were dissolved in 1 ml water and the activity was measured using Pen G as a substrate.
[0262]
(Storage stability):
The storage stability of the PA dry crystal formulation was determined using a Reactive-Therm III-Heating / Stirring module by Pierce at a temperature of 55 ° C. Activity was measured by dissolving a 5 mg dry sample in water and measuring enzyme activity in the Pen G assay described in Example 15, and compared to initial results. FIG. 10 shows the storage stability profiles of PA crystals with and without excipients. FIG. 10 also shows the storage stability profile of PA crystal formulations dried using nitrogen (Method 1) or air-dried (Method 4).
[0263]
[Table 7]

T_1/2Was calculated from expiration date data by non-linear regression analysis using the Sigma Plot program. The stability of formulated crystals was enhanced as shown in Table 7 compared to unformulated crystals. For example, crystals formulated with HPCD were 418 times more stable than soluble PA at 55 ° C. (Table 7). Crystals formulated with HPCD were 3.5 times more stable at 55 ° C. than unformulated PA crystals made without excipients, as shown in Table 7.
[0264]
(Moisture content):
The moisture content was determined by the Karl Fischer method using a Mitsubishi CA-06 Moisture Meter with a VA-06 Vaporizer (Mitsubishi Chemical Corporation, Tokyo, Japan) according to the manufacturer's instructions.
[0265]
[Table 8]

(crystalline):
The crystallinity of the formulation was measured by quantitative microscopy as described in Example 9. As shown by the crystals, crystallinity was maintained throughout the process (data not shown), which was readily visible.
[0266]
(Characterization of secondary structure by FTIR):
Stability was assessed by quantifying the secondary structure content of the dried and formulated PA crystals by FTIR as described in Example 9. Soluble PA was used for comparison purposes.
[0267]
[Table 9]

(Conclusion):
The decrease in α-helix content was 76% after 1 day for soluble penicillin acylase. In contrast, when HPCD was used as the excipient and the formulation was dried by Method 1 of Example 6, only a 31% α-helix content was lost after 43 days at a temperature of 55 ° C. It was.
[0268]
(Example 21: Glucose oxidase)
(Preparation of glucose oxidase crystals)
The present inventors prepared glucose oxidase crystals as follows. First, the glycoprotein was purified by anion exchange chromatography and then the crystallization parameters were optimized (data not shown).
[0269]
As a result of these studies, we have found that the crystallization conditions for glucose oxidase are generally 7% to 17% PEG 4000 or PEG 6000, 8% to 20% 2-propanol or ethanol, and 3, It was found to fall within the buffer range to adjust the pH to between 6. However, it should be understood that many sets of experimental conditions within and near this range can produce satisfactory results for crystallization of glucose oxidase and other glycoproteins. Those skilled in the art will appreciate that the exact conditions that efficiently produce crystals of the desired size and quality are due to differences in experimental conditions (eg, protein and reagent purity, stirring speed, shear force effects, and carbohydrate content). Understand that changes.
[0270]
(Large-scale crystallization of glucose oxidase)
We have determined favorable conditions for preliminary scale crystallization of glucose oxidase. Preliminary scale crystallization generally contains between 100 ml and 900 ml of glycoprotein.
[0271]
(A. Crystallization at constant pH without seed)
Glucose oxidase is diafiltered in water and A between 5 and 15₂₈₀Until concentrated. The glucose oxidase concentrate was mixed with 1: 1 volume of crystallization reagent containing 18% PEG 6000, 32% 2-propanol in 0.2 M Na acetate at pH 5.0 (1: 1). After mixing, the solution was cooled to 6 ° C. The glucose oxidase crystallization solution was stirred for 24 hours at 100 rpm using a propeller stirrer. During this time, the crystals gradually formed.
[0272]
Example 22: Formulation of glucose oxidase using trehalose as excipient
In order to enhance the stability of glucose oxidase (GOD) crystals during drying and storage, the crystals were formulated with excipients. In this example, GOD crystals were formulated in slurry form in the presence of mother liquor before drying. Trehalose (Sigma Chemical Co., St. Louis, MO) was added as an excipient to the GOD crystals in the mother liquor. Sufficient trehalose is added to GOD crystals with a protein concentration of 20 mg / ml in mother liquor (100 mM sodium acetate buffer (pH 5.5) containing 32% isopropanol and 9% PEG 6000) until the final concentration reaches 10%. Added. The obtained suspension was mixed by inversion for 3 hours at room temperature. After treatment with trehalose, crystals and liquid were separated by centrifugation as described in Method 1 of Example 6.
[0273]
Example 23: Formulation of glucose oxidase crystals using lactitol as excipient
Glucose oxidase crystals were formulated as in Example 22 by adding lactitol (Sigma Chemical Co., St. Louis, MO) to a final concentration of 10% with respect to the mother liquor (in place of trehalose). After centrifuging for 3 hours, the crystals were separated from the mother liquor / lactitol solution.
[0274]
Example 24: Formulation of glucose oxidase crystals using hydroxypropyl-β-cyclodextrin (HPCD) as excipient
By adding hydroxypropyl-β-cyclodextrin (HPCD) (Sigma Chemical Co., St. Louis, MO) to a final concentration of 10% with respect to the mother liquor, the glucose oxidase crystals were converted to HPCD. Used, formulated as in Example 22 and incubated for 3 hours. The crystals and the mother liquor / HPCD solution were then separated after 3 hours of centrifugation as described in Method 1 of Example 6.
[0275]
Example 25: Formulation of glucose oxidase crystals using gelatin as excipient
Glucose oxidase crystals were formulated as in Example 22 by adding gelatin (Sigma Chemical Co., St. Louis, MO) to a final concentration of 10% with respect to the mother liquor (in place of trehalose). After centrifuging for 3 hours, the crystals were separated from the mother liquor / gelatin solution.
[0276]
Example 26: Formulation of glucose oxidase crystals using methoxypolyethylene glycol as excipient
Glucose oxidase crystals were formulated as in Example 22 by adding methoxypolyethylene glycol (Sigma Chemical Co., St. Louis, MO) to a final concentration of 10% with respect to the mother liquor. After centrifuging for 3 hours, the crystals were separated from the mother liquor / methoxypolyethylene glycol solution as described in Method 1 of Example 6.
[0277]
Example 27: Formulation of glucose oxidase crystals using sucrose as excipient
Glucose oxidase crystals were formulated as in Example 22 by adding sucrose (Sigma Chemical Co., St. Louis, MO) to a final concentration of 10% with respect to the mother liquor. After centrifuging for 3 hours, the crystals were separated from the mother liquor / sucrose solution.
[0278]
Example 28: Drying of glucose oxidase formulation
The glucose oxidase crystal formulation was dried according to the method described in Example 6. Method 4 used cold isopropanol (4 ° C.) as the organic solvent.
[0279]
(Example 29: Preparation of soluble glucose oxidase)
For comparison, a soluble glucose oxidase sample was prepared by dissolving glucose oxidase crystals at 20 mg / ml in 50 mM citrate buffer (pH 6.0).
[0280]
FIG. 11 shows the stability of soluble glucose oxidase at 50 ° C. over time. Specific activity decreases rapidly over time. After 24 hours, the specific activity decreases by more than 90%. T of soluble glucose oxidase_1/2Was calculated to be 0.91 hours.
[0281]
(Example 30: Glucose oxidase activity assay)
The following protocol was used to determine the activity of dried glucose oxidase crystals and crystal formulations.
[0282]
Chemicals and solutions:
1. Phosphate buffer solution (20 mM, pH 7.3), NaCl (0.1 M) solution
2. 21 mM O-dianisidine dihydrochloride stock solution. Dilute to 0.21 mM as working solution before use
3.2M glucose solution
4). Peroxide solution (2mg / ml)
5. 50 mM citrate buffer (pH 6.0).
[0283]
Sample preparation:
1.2 mg glucose oxidase is added to 1 ml 50 mM citrate buffer (pH 6.0) and vortexed well for about 2 minutes. This solution was reconstituted by inversion for 1 hour at room temperature.
2. Prepare a diluted enzyme solution by mixing 0.1 ml of the above enzyme solution with 4.9 ml of the same citrate buffer.
[0284]
Assay procedure for measuring enzyme activity:
1. The assay was monitored by a UV-Vis spectrophotometer. Use speed mode and set wavelength of 460nm and temperature of 25 ° C
2. Warm the O-dianisidine / phosphate working solution in a 25 ° C. water bath and bubble this solution with oxygen at least 20 minutes before use.
3. Measure blanks using reagent solution without adding enzyme solution
4. Pipette 2.4 ml oxygenated O-dianisidine / phosphate working solution, 0.4 ml 2M glucose solution, and 0.1 ml peroxidase into a disposable cuvette.
5). Add 10 ml enzyme sample onto the cuvette wall (tilt the cuvette so that the sample and reagent do not mix in this step) and cover with a piece of parafilm. Invert the cuvette twice to mix quickly, insert the cuvette into the cell compartment of the spectrophotometer, and begin collecting data.
The enzyme specific activity is calculated using the following formula:
Specific activity = A * B * C / D * E * F
Where A = change in absorbance unit at 460 nm per minute
B = reaction mixture volume (ml)
C = dilution ratio
D = 11.3 (constant)
E = weight of enzyme used (mg)
F = sample volume (ml).
[0285]
Example 31: Activity of dried crystals
The activity of the dried crystal formulation of glucose oxidase was measured as described in Example 30.
[0286]
(Shelf stability)
The storage stability of glucose oxidase crystal formulation was studied. In this case, the formulation was dried using Method 4 of Example 6 and stored in a water bath at a temperature of 50 ° C. for 13 days in a 2 ml screw-cap Eppendorf tube. Activity at a specific time point was obtained by dissolving 2 mg of the dried sample in 50 mM citrate buffer (pH 6.0) and then measuring the activity according to Example 30.
[0287]
The storage stability of various glucose oxidase formulations stored at 50 ° C. was determined. This data is shown in FIG. Lactitol was the most effective excipient in preserving glucose oxidase specific activity over time at elevated temperatures.
[0288]
[Table 10]

T_1/2Was calculated from the expiry date data by non-linear regression analysis using the Sigma plot program. Table 10 shows that the glucose oxidase formulation was 95 times more stable than soluble glucose oxidase when trehalose was used as an excipient. Furthermore, the glucose oxidase formulation was 121 times more stable than soluble glucose oxidase when lactitol was used as an excipient (Table 10). Glucose oxidase crystal formulations using HPCD or MOPEG as excipients were 60 times and 325 times more stable than soluble glucose oxidase, respectively (Table 10). Finally, glucose oxidase crystal formulations using gelatin or sucrose as excipients were 99 and 82 times more stable than soluble glucose oxidase, respectively (Table 10).
[0289]
Glucose oxidase crystal formulations made using either trehalose, lactitol, HPCD, MOPEG, gelatin or sucrose as excipients, as shown in Table 10,_1/2From glucose oxidase crystals prepared without using excipients, 2.5 times, 3.2 times, 1.6 times, 8.6 times, 2.6 times, or 2 at 50 ° C. .2 times more stable.
[0290]
(Water content)
The moisture content was determined by the Karl Fischer method using a Mitsubishi CA-06 Moisture Meter with a VA-06 Vaporizer (Mitsubishi Chemical Corporation, Tokyo, Japan) according to the manufacturer's instructions.
[0291]
[Table 11]

(Crystallinity)
The crystal integrity of the GOD formulation was measured by quantitative microscopy as described in Example 9. In this example, the crystals were easily crystallized. This indicates that crystallinity was maintained throughout the process.
[0292]
FIG. 13 shows that glucose oxidase crystals were easily visualized immediately after preparation of the lactitol formulation. FIG. 14 demonstrates that the crystallinity was maintained after 13 days at 50 ° C. The crystalline material was also easily visualized after formulation of glucose oxidase with trehalose, as shown in FIG. Similarly, the remaining crystalline material was easily visualized after 13 days at 50 ° C., as shown in FIG.
[0293]
(Characteristics of secondary structure by FTIR)
As described in Example 9, the stability was evaluated by quantifying the secondary structure amount of the dried GOD crystal and the formulated GOD crystal by FTIR. For comparison, a soluble glucose oxidase sample was prepared by dissolving glucose oxidase crystals in 50 mM citrate buffer (pH 6.0) and placing about 1 ml on the zinc selenide crystals of ARK ESP, This was then analyzed for stability by FTIR.
[0294]
[Table 12]

(Conclusion)
Soluble glucose oxidase lost 75% of its α-helix content within only 6 hours at 50 ° C.
[0295]
The sugar lactitol, sucrose, and trehalose were the most effective excipients to prevent loss of α-helix content during storage at high temperatures. Glucose oxidase crystals formulated in lactitol, sucrose, and trehalose and dried by method 4 showed a loss of only 18.1-19.4% of α-helix structure content after 4 days at 50 ° C. .
[0296]
(Example 32: Drying of Candida rugosa lipase crystals)
material:
A-Candida rugosa lipase (Example 1)
B-poly (ethylene glycol), 100%, PEG 200, 300, 400, or 600
C-acetone
procedure:
A 4 ml aliquot of the crystal suspension (140 mg) is added to four 15 ml tubes. The suspension is then centrifuged between 1000 RPM and 3000 RPM for 1-5 minutes or until the crystallization buffer is removed. Then 4 ml of liquid polymer (any PEG between 200 and 600 is suitable) is added to each tube and the contents are mixed until uniform. The suspension is centrifuged between 1000 RPM and 3000 RPM for 1-5 minutes or until the liquid polymer is removed. Next, 4 ml of acetone (isopropanol, butanol, and other solvents are also suitable) is placed in each tube and mixed thoroughly. The crystal / organic solvent suspension is transferred to a 0.8 cm × 4 cm BIO-RAD poly-preparative chromatography column (spin column). The column is centrifuged at 1000 RPM for 1-5 minutes to remove organic solvent. Finally, nitrogen gas is passed through the column and the crystals are dried until free flowing powder is formed.
[0297]
(Example 33: Purafact (protease) 4000L crystallization)
material:
A-Crude purafect 4000L
B-15% NaSO_Foursolution
procedure:
1 volume of crude purefect enzyme solution was added to 2 volumes of 15% Na₂SO_FourMix with solution. The mixture is stirred at room temperature for 24 hours or until crystallization is complete. 15% Na₂SO_FourWash with solution to remove soluble enzyme. Crystals are fresh 15% Na₂SO_FourSuspend in solution to obtain a protein concentration of 27 mg / ml.
[0298]
(Example 34: Drying of Puraffect crystals)
material:
A-purafect crystal suspension
B-poly (ethylene glycol), 100% PEG
200, 300, 400, or 600
C-organic solvent
procedure:
A 4 ml aliquot of the crystal suspension (140 mg) is added to four 15 ml tubes. The suspension is then centrifuged between 1000 RPM and 3000 RPM for 1-5 minutes or until the crystallization buffer is removed. Then 4 ml of liquid polymer (any PEG between 200 and 600 is suitable) is added to each tube and the contents are mixed until uniform. The suspension is centrifuged between 1000 RPM and 3000 RPM for 1-5 minutes or until the liquid polymer is removed. Next, 4 ml of acetone (isopropanol, butanol, and other solvents are also suitable) is placed in each tube and mixed thoroughly. The crystal / organic solvent suspension is transferred to a 0.8 cm × 4 cm BIO-RAD poly-preparative chromatography column (spin column). The column is centrifuged at 1000 RPM for 1-5 minutes to remove the organic solvent. Finally, nitrogen gas is passed through the column and the crystals are dried until free flowing powder is formed.
[0299]
Example 35: Production of DNA for crystallization
A plasmid derived from the pUC plasmid (eg, pSP64) can be used to produce either DNA or mRNA for crystallization. In this example, plasmid pSP64 (available from Promega Biological Research Products) is used to generate DNA for crystallization. The cDNA encoding the protein of interest is inserted into any of a number of restriction sites available at the multiple cloning site. The recombinant plasmid was then transformed into E. coli. Used to transform E. coli bacteria. Large quantities of plasmid are then obtained by bacterial growth in ampicillin-containing media. Producing a recombinant plasmid; Techniques for transforming E. coli cells and bacterial growth and plasmid DNA preparation are described in “Molecular Cloning, 2nd Edition” (1989) Sambrook, J. et al. Fritsch, E .; F. And T. It is described in detail in Maniatis.
[0300]
Subsequently, plasmid DNA is purified from bacterial cultures by first lysing the cells and then separating the plasmid DNA from genomic DNA, RNA, and other cellular material using a CsCl gradient. These techniques are well known in the art and are discussed in detail in Sambrook et al. The gradient purified DNA is extracted with Tris-EDTA buffer saturated N-butanol and finally with ethanol. The plasmid DNA is then either of the plasmid lines used to generate mRNA for RNA crystallization (Example 36) or to excise the gene of interest for DNA crystallization (Example 37). We use for state.
[0301]
(Example 36: Production of mRNA for crystallization)
The SP64 plasmid (available from Promega Biological Research Products) in combination with SP6 RNA polymerase is used for the production of milligram quantities of 5 'capped RNA transcripts. The plasmid prepared in Example 35 is linearized with a restriction enzyme downstream of the poly A tail prior to gene excision. The linearized plasmid is purified by two phenol / chloroform extractions and two chloroform extractions. The DNA is then precipitated with NaOAc (0.3M) and 2 volumes of EtOH. The pellet is then resuspended at approximately 1 mg / ml in DEPC-treated distilled deionized water.
[0302]
Transcription was performed using 400 mM Tris HCl (pH 8.0), 80 mM MgCl.₂, 50 mM DTT, and 40 mM spermidine. Subsequent reagents are added to 1 volume of DEPC-treated water at room temperature in the following order: 1 volume of SP6 RNA polymerase transcription buffer; rATP, rCTP, and rUTP (up to 1 mM concentration); rGTP (up to 0.5 mM concentration) ); 7 meC (5 ′) ppp (5 ′) G cap analog (New England Biolabs, Beverly, Mass., 01951) (up to 0.5 mM concentration); linearized DNA template prepared above (0.5 mg / ml) RNA (Promega, Madison, Wis.) (Up to 2000 U / ml concentration); and SP6 RNA polymerase (Promega, Madison, Wis.) (Up to 3000 U / ml concentration). The transcription mixture is incubated at 37 ° C. for 1 hour.
[0303]
The DNA template is then digested by adding 2 U RQ1 DNAse (Promega) per 1 μg DNA template used. The digestion reaction is performed for 15 minutes. Transcribed RNA is extracted twice with chloroform / phenol and twice with chloroform. The supernatant solution is precipitated with 2 volumes of 0.3 M NaOAc in EtOH and the pellet is resuspended in 100 ml DEPC-treated deionized water per 500 ml transcript. Finally, the supernatant solution is passed through an RNAse-free Sephadex G50 column (Boehringer Mannheim # 100 411). The resulting mRNA is sufficiently pure to be used for crystallization.
[0304]
(Example 37: DNA crystallization)
material:
A-Purified plasmid DNA (Example 35)
B-spermine
C-MPD (2-methyl-2,4-pentanediol)
D-5 mM calcium acetate buffer (pH 7.0)
E-deionized water
procedure:
Amplified DNA (Example 35) is removed from the plasmid by restriction digestion. The inserted gene is purified from the plasmid vehicle by agarose gel electrophoresis and extraction of the gene of interest from an appropriate molecular weight gel band.
[0305]
Using a hanging drop technique, 5 mg / ml DNA in 5 mM sodium acetate / 20% MPD / 1 mM sperimine buffer (pH 7.0) was allowed to stand at room temperature until 90% DNA was crystallized. Incubate. The resulting crystals are washed with crystallization buffer to remove any soluble material from the crystals. The crystals are then resuspended in fresh crystallization buffer until a DNA concentration of 5 mg / ml is achieved.
[0306]
(Example 38: RNA crystallization)
material:
A-purified mRNA (Example 36)
B-spermine
C-MPD (2-methyl-2,4-pentanediol)
D-5 mM calcium acetate buffer (pH 7.0)
E-deionized water
procedure:
Using hanging drop technique, 5 mg / ml RNA (Example 36) in 5 mM calcium acetate / 20% MPD / 1 mM sperimine buffer, pH 7.0, was allowed to cool until 90% RNA was crystallized. Incubate with. The resulting crystals are washed with crystallization buffer to remove any soluble material from the crystals. The crystals are then resuspended in fresh crystallization buffer until an RNA concentration of 5 mg / ml is achieved.
[0307]
Example 39: Induction of immune response against HIV gp120 using DNA crystals
DNA encoding HIV gp160 is prepared according to the methods of Examples 36 and 37. The crystals of HIV gp160 are then used for immunization of mice. In order to design a vaccine that induces broadly neutralizing immunity, many genetic clones of both primary and experimental isolates of HIV have been identified by Aids Research and Reagent Program, National Institutes of Allergy and Infectious Diseases, Rockville MD. Available from 20852.
[0308]
DNA crystals are maintained in crystallization buffer and 200 μl / mouse is injected into the hind limb. Development of an immune response against gp120 is determined by measuring serum antibodies against the corresponding V3 loop peptide in an ELISA on a 1 month basis.
[0309]
Example 40: Induction of immune response against HIV gp120 using mRNA crystals
RNA encoding HIV gp160 is prepared according to the methods of Examples 35, 36 and 38. The crystal of HIV gp160 mRNA is then used for immunization of mice. To design a vaccine that induces extensive neutralizing immunity, various primary and experimental isolates of HIV are available from Aids Research and Reagent Program, National Institutes of Allergies and Infectious Diseases, Rockville MD 20852. .
[0310]
RNA crystals are maintained in crystallization buffer and 200 μl / mouse is injected into the hind limb. Development of an immune response against gp120 is determined by measuring serum antibodies against the corresponding V3 loop peptide in an ELISA on a 1 month basis.
[0311]
Example 41 Oligo DNA Crystallization
material:
A-Synthetic oligo DNA
B-spermine
C-MPD (2-methyl-2,4-pentanediol)
D-5 mM Ca acetate buffer pH 7.0
E-deionized water
procedure:
Using a hanging drop technique, 5 mg / ml synthetic oligo DNA in 5 mM Mg acetate / 30% MPD / 1 mM spermine buffer (pH 7.0) was brought to room temperature until 90% of this DNA crystallized. And incubate. The resulting crystals are washed with a crystallization buffer to remove any soluble material from the crystals. The crystals are then resuspended in fresh crystallization buffer to achieve a DNA concentration of 5 mg / ml.
[0312]
Example 42 Administration of antisense DNA for inhibition of gene expression
Oligo DNA crystals encoding DNA sequences that are complementary to the sense strand of the mRNA species to be suppressed are generated as described in Example 41. The crystal or formulation containing the crystal is then administered to the site where inhibition of gene expression is intended. Subsequently, cells take up the DNA crystals or lysed DNA, and the oligo DNA and host mRNA form complementary base pairs and gene expression is inhibited for a period of time.
[0313]
(Encapsulated protein crystal)
Example 43 Large-scale crystallization of Pseudomonas cepacia lipase
A slurry of 15 kg of crude Pseudomonas cepacia lipase (PS30 lipase-Amano) (“LPS”) was dissolved in 100 L of distilled deionized water and brought to a volume of 200 L with additional distilled deionized water. This suspension was mixed in an Air Drive Lightning mixer for 2 hours at room temperature and then filtered through a 0.5 μm filter to remove celite. The mixture was then ultrafiltered using a 3K hollow fiber filter membrane cartridge and concentrated to 10 L (121.4 g). Solid calcium acetate was added to 20 mM Ca (CH_ThreeCOO)₂To a concentration of. If necessary, the pH was adjusted to 5.5 using concentrated acetic acid. The mixture was heated and maintained at a temperature of 30 ° C. Magnesium sulfate was added to a concentration of 0.2M, followed by glucopon to a concentration of 1%. Isopropanol was then added to a final concentration of 23%. The resulting solution was mixed at 30 ° C. for 30 minutes and then cooled from 30 ° C. to 12 ° C. over 2 hours. Crystallization was then allowed to proceed for 16 hours.
[0314]
The crystals were precipitated and soluble protein was removed using a peristaltic pump equipped with a Tygon tube with a 10 ml pipette at the end. New crystallization solution (23% isopropyl alcohol, 0.2M MgSO_Four1% glucopon, 20 mM Ca (CH_ThreeCOO)₂, PH 5.5) was added to a protein concentration of 30 mg / ml (1 mg / ml solution OD 280 = 1.0, measured using spectrophotometer at wavelength 280). Crystal yield was about 120 grams.
[0315]
(Crosslinked LPS crystal)
Cross-linked Pseudomonas cepacia lipase crystals (ChiroCLEC-PC^TMSold under the name Altus Biology, Inc. A formulation was produced according to Example 48 using (available from Cambridge, Massachusetts). Alternatively, the lipase crystals prepared as described above can be cross-linked using any conventional method.
[0316]
Example 44 Crosslinking of glucose oxidase crystals
Next, the inventors cross-linked the glucose oxidase crystals prepared in Example 21 as follows. This cross-linking procedure included glutaraldehyde or glutaraldehyde pretreated with either Tris buffer (2-amino-2-hydroxymethyl-1,3-propanediol), lysine, or diaminooctane. .
[0317]
This crosslinking was performed with 60 mg of protein. Tris pretreated glutaraldehyde (48 mg Tris salt / 1 g glutaraldehyde) was added to a concentration of 0.2 g and 0.6 g per 1 g GO crystals suspended in 0.2 M sodium phosphate, pH 7.0. The reaction was allowed to proceed for 2 hours at room temperature. After 2 hours, the crystals were filtered and washed through glass fiber paper.
[0318]
The crosslinked crystals were encapsulated as described in Example 48. There was no difference in the encapsulation process between the variously crosslinked crystals. A representative sample is shown in FIG.
[0319]
Example 45 Cross-linked Candida rugosa lipase crystals
Cross-linked Candida rugosa lipase crystals (ChiroCLEC-CR^TMSold under the name Altus Biology, Inc. A formulation was produced according to Example 48 using (available from Cambridge, Massachusetts). Alternatively, the lipase crystals prepared above can be cross-linked using any conventional method.
[0320]
Example 46 Crosslinking of Human Serum Albumin Crystals
The human serum albumin crystals prepared in Example 10 were cross-linked. This crosslinking reaction was carried out at 4 ° C. in a stirred crystal solution in a mother liquor containing 50% saturated ammonium sulfate. The crystals were not washed prior to crosslinking with borate pretreated glutaraldehyde.
[0321]
Pretreated glutaraldehyde was prepared by adding 1 volume of 50% glutaraldehyde (“GA”) to an equal volume of 300 mM sodium borate (pH 9). The glutaraldehyde solution was then incubated at 60 ° C. for 1 hour. The solution was cooled to room temperature and the pH was adjusted to 5.5 using concentrated HCl. The solution was then quickly cooled to 4 ° C. on ice.
[0322]
Pretreated glutaraldehyde (25%) was added to the crystallization solution to a concentration of 2% in 0.05% increments (total concentration) at 15 minute intervals in a stepwise manner. Aliquots of the crystallization solution used ranged from 1 ml to 500 ml volumes. The crystals were then brought to 5% GA and incubated at 4 ° C. for 4 hours to crosslink. Finally, albumin cross-linked crystals were recovered by low speed centrifugation and washed repeatedly with pH 7.5, 100 mM Tris HCl. Washing was stopped when the crosslinked crystals could be centrifuged at high speed without agglomeration.
[0323]
Example 47 Crosslinking of penicillin acylase
Pretreated glutaraldehyde was prepared by the method of Example 46.
[0324]
Pretreated glutaraldehyde (25%) was added to the crystallization solution to a concentration of 1.5% using a 0.05% increment (total concentration) at 15 minute intervals in a stepwise fashion. Aliquots of the crystallization solution used ranged from 1 ml to 500 ml volumes. Finally, the cross-linked crystals were collected by low speed centrifugation and washed repeatedly with pH 7.5, 100 mM Tris HCl. Washing was stopped when the crosslinked crystals could be centrifuged at high speed without agglomeration.
[0325]
Example 48 Microencapsulation of protein crystals in polylactic acid-co-glycolic acid (PLGA)
A. Glycoproteins, proteins, enzymes, hormones, antibodies and peptides
Microencapsulation was performed using non-crosslinked crystals of lipase from Candida rugosa and Pseudomonas cepacia, glucose oxidase from Aspergillus niger, and penicillin acylase from Escherichia coli. Furthermore, microencapsulation was performed using a cross-linked enzyme crystal of lipase derived from Candida rugosa, glucose oxidase derived from Aspergillus niger, and penicillin acylase derived from Escherichia coli. Table 13 shows the approximate average diameter of the microsphere samples produced by this example. Furthermore, the produced human serum albumin or any other protein crystal or protein crystal formulation can be encapsulated by this technique.
[0326]
[Table 13]

B. Preparation of dry crystals:
Each of the crystals or crystal formulations dried according to Example 6 can be used to produce the microspheres of the present invention. One process for drying protein crystals for use in the present invention involves air drying.
[0327]
Candida rugosa lipase crystals from Example 1 (non-crosslinked and crosslinked), glucose oxidase from Examples 21 and 44 (non-crosslinked and crosslinked), and penicillin acylase from Examples 14 and 47 (non-crosslinked and crosslinked), respectively About 500 mg was air dried. First, the mother liquor was removed by centrifugation for 5 minutes at 3000 rpm. The crystals were then placed in a fume hood for 2 days at 25 ° C.
[0328]
(C. Polymer and solvent)
The polymer used to encapsulate the protein crystals was PLGA. PLGA is available from Birmingham Polymers, Inc. as 50/50 poly (DL-lactide-co-glycolide). From Lot No. D97188. This lot had an inherent viscosity of 0.44 dl / g at 30 ° C. in HFIP.
[0329]
Methylene chloride is spectroscopic grade and is available from Aldrich Chemical Co. Purchased from Milwaukee, WI. Polyvinyl alcohol was obtained from Aldrich Chemical Co. Purchased from Milwaukee, WI.
[0330]
(D. Crystal Encapsulation in PLGA)
The crystals were encapsulated in PLGA using the double emulsion method. The general process was as follows. Either a dry protein crystal or a slurry of protein crystals was first added to the polymer solution in methylene chloride. The crystals were coated with a polymer and became new microspheres. The polymer in organic solvent solution was then transferred to a larger volume of aqueous solution containing surfactant. As a result, the organic solvent began to evaporate and the polymer was cured. In this example, two successive aqueous solutions of decreasing concentrations of emulsifier were used to cure the polymer coat to form microspheres. The following procedure was one example of this general process. Those skilled in the art of polymer science will appreciate that many modifications of the procedure can be utilized and that the following examples are not intended to limit the invention.
[0331]
(1.0 Use of dried protein crystals)
Cross-linked and non-cross-linked Candida rugosa lipase dried crystals prepared according to Example 1, cross-linked and non-cross-linked glucose oxidase prepared according to Examples 21 and 45, according to Examples 14 and 47 The cross-linked and non-cross-linked penicillin acylase produced was weighed into a 150 mg sample. The weighed protein crystals were then added directly to a 15 ml polypropylene centrifuge tube (Fisher Scientific) containing 2 ml of methylene chloride and 0.6 g PLGA / 1 ml solvent PLGA. The crystals were added directly to the surface of the solvent. The tube was then mixed well by vortexing for 2 minutes at room temperature to completely disperse the protein crystals in a solvent containing PLGA. The crystals were completely coated with the polymer. In order to keep the nascent microspheres suspended, further vortexing or agitation may be used to allow further coating. The polymer can be cured as described in Section 3.0.
[0332]
(2.0 Use of protein crystal slurry)
A crystal slurry of Pseudomonas cepacia lipase was made using about 50 mg of crystals per 200 μl of mother liquor. The crystal slurry was rapidly injected into a 15 ml polypropylene centrifuge tube (Fisher Scientific) with 2 ml solution of methylene chloride and 0.6 g PLGA / 1 ml solvent poly (lactic acid-co-glycolic acid). The needle was inserted below the surface of the solvent and injected into the solution. In this case, 150 mg total protein, or 600 μl aqueous solution was injected. The plastic syringe Leur-lok (Becton-Dickinson & Company) was used to inject through a 22 gauge (Becton-Dockinson & Company) stainless steel needle. The protein crystal PLGA slurry was then thoroughly mixed by vortexing for 2 minutes at room temperature. The crystals were completely coated with the polymer. The nascent microspheres were maintained in suspension and further vortexed or agitated as necessary to further coat.
[0333]
(3.0 Curing of polymer coating)
A two-step process was utilized to facilitate removal of methylene chloride from the liquid polymer coat and to cure the polymer on protein crystals. The difference between the processes is that the concentration of the emulsifier is much higher in the first solution and the volume of the first solution is much smaller than in the second solution.
[0334]
In the first step, a polymer-coated crystal and methylene chloride suspension of 180 ml of 6% polyvinyl alcohol in water (hereinafter “PVA”) in water containing 0.5% methylene chloride at room temperature. Add dropwise to the stirred flask. This solution was mixed rapidly for 1 minute.
[0335]
In Step 2, the first PVA solution containing nascent microspheres was quickly poured into 2.4 liters of cold (4 ° C.) distilled water. The final bath was gently mixed with nitrogen at the surface of the solution for 1 hour at 4 ° C. After 1 hour, the microspheres were filtered using a 0.22 μm filter and washed with 3 liters of distilled water containing 0.1% Tween 20 to reduce aggregation.
[0336]
(Example 49)
(Preparation of encapsulated crystals)
Encapsulated microspheres of Pseudomonas cepacia lipase are prepared by a phase separation technique. Crystalline LPS prepared in Example 43 is encapsulated in polylactic-co-glycolic acid (“PLGA”) using a double emulsion method. A 700 mg aliquot of protein crystals is injected into methylene chloride containing PLGA (0.6 g PLGA / 1 ml solvent; 10 ml). The mixture is homogenized at 3,000 rpm for 30 seconds using a homogenizer equipped with a microfine chip. The resulting suspension is transferred to a stirred tank (900 ml) containing 6% poly (vinyl alcohol) (“PVA”) and methylene chloride (4.5 ml). The solution is mixed for 1 minute at 1,000 rpm. The microspheres in the PVA solution are precipitated by immersion in distilled water, washed and filtered. The microspheres are then washed with distilled water containing 0.1% Tween to reduce aggregation and dried with nitrogen at 4 ° C. for 2 days.
[0337]
(Example 50)
(A. Protein concentration of microspheres)
The total protein content of the microspheres prepared in Example 48 was measured. Triplicate samples of 25 mg PLGA / PVA microspheres were incubated in 1N sodium hydroxide for 48 hours with mixing. The protein content was then estimated using the Bradford method (MM Bradford, Analytical Biochemistry, vol. 72, pages 248-254 (1976)) and commercially available kits from BioRad Laboratories (Hercules, CA). Microspheres containing protein were compared to PLGA microspheres without crystals. This is shown in Table 14.
[0338]
[Table 14]

The activity per milligram or specific activity of the sample selected from Table 14 was determined. This is shown in Table 15.
[0339]
[Table 15]

(Example 51)
(Protein release from microspheres)
Protein release from the PLGA microspheres prepared in Example 48 was measured by placing 50 mg protein-encapsulated PLGA microspheres in a microcentrifuge filtration tube containing a 0.22 μm filter. Next, 600 μl of release buffer (phosphate buffered saline containing 0.2% Tween 20, pH 7.4) was added to the microspheres on the filter holding side. Incubate the tube at 37 ° C. to dissolve. Samples were taken at different time intervals to determine the amount of protein released over time. The tube was centrifuged at 3000 rpm for 1 minute and the filtrate was removed for protein activity and total protein measurement. The microspheres were then resuspended with an additional 600 μl of release buffer.
[0340]
(A. Total protein released from microspheres)
Table 16 shows that nearly 90% of the input protein was recovered in all cases. In addition, the percentage of total Pseudomonas cepacia lipase released from microencapsulated protein crystals is released at a rate of 15.8% / day for about 5.7 days, or over 80% of the input protein. Until then, it was relatively steady. After this long rapid release, only 0.6% release per day lasted for 8 days.
In contrast, Table 16 further shows that Candida rugosa lipase crystals initially showed slow release followed by a rapid release phase followed by the opposite profile. In the first 3 days, approximately 10% of the protein was released with a gentle slope of 2.4% / day. From day 4 to day 14, an additional 80% of the protein was released linearly with a slope of 7.5% / day. The released profiles shown in Table 16 were obtained at 37 ° C. and pH 7.4.
[0341]
These data indicate that the encapsulated proteins of the present invention are suitable for biological delivery of therapeutic proteins. Different rates of delivery depend on protein crystals, crystal selection, crystal cross-linking, hydrophobic and hydrophilic characteristics of the encapsulating polymer, number of encapsulations, microsphere dose, and other easily controllable variables Can be selected by manipulating the selection.
[0342]
[Table 16]

(B. Activity of proteins released from microspheres)
The biological activity of the protein released over time was measured using the olive oil assay for lipase microspheres. These results are shown in Table 17.
[0343]
As shown in Table 17, the biological activity of the released protein demonstrates that the microspheres protect and release the active protein. The cumulative rate of released activity calculated based on protein input was closely correlated with the total amount of protein released (compare Table 16 and Table 17). Two different crystalline lipases released essentially 100% active protein. Even after 7 days of immersion at 37 ° C., the protein released from the microspheres was fully active.
[0344]
[Table 17]

The activity measurements shown above were performed using the olive oil assay described in Example 8.
[0345]
(Example 52)
(Microscopic inspection of PLGA microspheres)
To visualize whether the crystals were intact after encapsulation, PLGA microspheres prepared according to Example 48 were prepared using Olympus equipped with a DXC-970MD 3CCD color video camera, camera adapter (CMA D2), and Image ProPlus software. Inspected under a BX60 microscope. Dry microsphere samples were covered with glass coverslips, mounted, and examined under an Olympus microscope with an Olympus UPLAN E1 objective 10 × 0.30 PH1 (phase difference) under a magnification of 10 ×. The crystals were easily visualized and the crystal size was determined. Microsphere size and crystal size were determined using Image Pro software from Olympus and 0.5-150 μm sizing beads provided by the manufacturer. The outer size of the PLGA microspheres was determined and the crystals were determined in the same way.
[0346]
FIG. 17 shows a Candida rugosa lipase cross-linked enzyme crystal encapsulated by the method of Example 48. The crystal size was about 25 μm and the microspheres were about 90 μm. The enlargement ratio was 250 ×.
[0347]
FIG. 18 shows non-crosslinked enzyme crystals of lipase from Candida rugosa encapsulated by the method of Example 48. The crystal size was about 25 μm and the microspheres were about 120 μm. The enlargement ratio was 250 ×.
[0348]
FIG. 19 shows a cross-linked enzyme crystal of penicillin acylase from Escherichia coli encapsulated by the method of Example 48. The crystal size was about 25 μm and the microspheres were about 70 μm. The enlargement ratio was 250 ×.
[0349]
FIG. 20 shows non-crosslinked enzyme crystals of penicillin acylase from Escherichia coli encapsulated by the method of Example 48. The crystal size was about 50 μm and the microspheres were about 90 μm. The enlargement ratio was 250 ×.
[0350]
FIG. 21 shows a cross-linked enzyme crystal of glucose oxidase from Aspergillus niger encapsulated by the method of Example 48. The crystal size ranged from 0.5-1 μm and the microspheres were about 50 μm. The enlargement ratio was 500 ×.
[0351]
FIG. 22 shows non-crosslinked enzyme crystals of glucose oxidase from Aspergillus niger encapsulated by the method of Example 48. The crystal size ranged from 0.5-1 μm and the microspheres were about 50 μm. The enlargement ratio was 500 ×.
[0352]
FIG. 23 shows non-crosslinked enzyme crystals of lipase from Pseudomonas cepacia encapsulated as a slurry in mother liquor by the method of Example 48. The crystal size was about 2.5 μm and the microspheres were about 60 μm. The enlargement ratio was 500 ×.
[0353]
FIG. 24 shows a non-crosslinked enzyme crystal of a lipase derived from Pseudomonas cepacia. The crystal size was about 2.5 μm. The enlargement ratio was 1000 ×.
[0354]
(Example 53)
(Protein release)
Protein release from PLGA microspheres is measured by placing 50 mg of PLGA microspheres in a microcentrifuge filter tube containing a 0.22 μm filter. A 600 μl aliquot of release buffer (10 mM HEPES, pH 7.4, 100 mM NaCl, 0.02% Tween, 0.02% azide) is added to the microspheres on the retention side of the filter and suspended. To do. The tube is sealed with a 3 cc vial stopper and the parafilm is covered. The microspheres are then incubated at 37 ° C. Samples are obtained over time by centrifugation of the tube (13,000 rpm, 1 minute). The filtrate is removed and the microspheres are resuspended with 600 μl of release buffer. The quality of the released protein is assayed by SEC-HPLC and enzyme activity.
[0355]
The shape and size of the protein crystals can be selected to adjust the rate of dissolution or other properties of the protein crystal formulation of the present invention.
[0356]
(Example 54)
(Encapsulation of lipase crystals using biological polymers)
Biological polymers are also useful for the encapsulation of protein crystals. This example demonstrates the encapsulation of cross-linked and non-cross-linked crystals of Candida rugosa lipase crystals. Non-crosslinked crystals and crosslinked crystals were prepared as described in Example 1 and Example 45. Antibodies and chemicals were purchased from Sigma.
[0357]
(1.0 Preparation of coated crystals)
A solution of 1.5 ml bovine serum albumin (“BSA”) at 10 mg / ml was prepared in 5 mM phosphate buffer adjusted to pH 7. Then, a 10 mg / ml suspension of 15 ml of Candida rugosa lipase crystals was prepared in 5 mM K / Na phosphate buffer, 1 M NaCl, pH 7 (“buffer”). BSA solution was added to the crystal solution and the two solutions were mixed well. The crystals were incubated with gentle mixing in BSA for 30 minutes using an orbital shaker. Following incubation with BSA, the crystals were dried overnight by vacuum filtration. The dried crystals were resuspended in a buffer without albumin. Crystals can be detected in washings measured at an absorbance of 280 nm until no protein can be detected or A_280nmWashed with buffer until was less than 0.01. The crystals were recovered by low speed centrifugation.
[0358]
(2.0 Detection of albumin coating)
The coated crystals were evaluated by Western blot to confirm the presence of the albumin layer. Following washing, the coated protein crystals were incubated overnight in 100 mM NaOH to dissolve the microspheres into the component proteins. Samples were neutralized, filtered and analyzed by SDS-Blot according to Sambrook et al., “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).
[0359]
SDS-PAGE immunoblot results of both albumin-coated cross-linked crystal microspheres and albumin-coated non-cross-linked crystal microspheres of Candida ragosa lipase showed a single immunoreactive species with the same molecular weight as albumin.
[0360]
Samples of Candida ragosa lipase albumin-coated crosslinked crystal microspheres and albumin-coated uncrosslinked crystal microspheres were incubated with a fluorescently labeled anti-BSA antibody that specifically recognizes and binds bovine serum albumin. Excess antibody was then removed by washing with phosphate buffer. Microscopic examination of these fluorescently labeled albumin coated crystal microspheres under a fluorescence microscope showed specific fluorescent labeling of the microspheres. Uncoated lipase crystals were used as controls and they did not show specific binding of the antibody.
[0361]
Although we have described many embodiments of the present invention above, we have altered our basic constructs to provide other embodiments that utilize the processes and compositions of the present invention. Obviously it can be done. Accordingly, it is understood that the scope of the invention should be defined by the claims appended hereto rather than by the specific embodiments shown above for purposes of illustration.
[Brief description of the drawings]
FIG. 1 shows the relative stability of the following molecular states of Candida rugosa lipase: cross-linked amorphous, liquid, crystal in 20% organic solvent, cross-linked crystal in 20% organic solvent, without organic solvent Cross-linked crystals (“Xlinke ppt” indicates cross-linked precipitate).
FIG. 2 shows the specific activity of soluble lipase over time at 40 ° C.
FIG. 3 shows the storage stability of a lipase crystal formulation dried by Method 1 at 40 ° C. and 75% humidity.
FIG. 4 shows the storage stability of a lipase crystal formulation dried by Method 4 at 40 ° C. and 75% humidity.
FIG. 5 shows lipase crystals formulated with polyethylene oxide at an initial time of 0.
FIG. 6 shows lipase crystals formulated with polyethylene oxide after incubation for 129 days at 40 ° C. and 75% humidity.
FIG. 7 shows human serum albumin crystals formulated with gelatin at an initial time of 0.
FIG. 8 shows human serum albumin crystals formulated with gelatin after incubation at 50 ° C. for 4 days.
FIG. 9 shows the specific activity of soluble penicinine acylase at 55 ° C. over time.
FIG. 10 shows the storage stability of various dry penicillin acylase crystal formulations at 55 ° C.
FIG. 11 shows the specific activity of soluble glucose oxidase over time.
FIG. 12 shows the storage stability of various dry glucose oxidase crystal formulations at 50 ° C.
FIG. 13 shows glucose oxidase crystals formulated with lactitol at an initial time of 0.
FIG. 14 shows glucose oxidase crystals formulated with lactitol after incubation at 50 ° C. for 13 days.
FIG. 15 shows glucose oxidase crystals formulated with trehalose at an initial time of 0.
FIG. 16 shows glucose oxidase crystals formulated with trehalose after 13 days incubation at 50 ° C.
FIG. 17 shows encapsulated and cross-linked enzyme crystals of lipase from Candida rugosa.
FIG. 18 shows encapsulated and uncrosslinked enzyme crystals of lipase from Candida rugosa.
FIG. 19 shows encapsulated and crosslinked enzyme crystals of penicillin acylase from Escherichia coli.
FIG. 20 shows encapsulated and uncrosslinked enzyme crystals of penicillin acylase from Escherichia coli.
FIG. 21 shows encapsulated and crosslinked enzyme crystals of glucose oxidase from Aspergillus niger.
FIG. 22 shows encapsulated and uncrosslinked enzyme crystals of glucose oxidase from Aspergillus niger.
FIG. 23 shows an encapsulated aqueous slurry of non-crosslinked enzyme crystals of lipase from Pseudomonas cepacia.
FIG. 24 shows an uncrosslinked enzyme crystal of lipase from Pseudomonas cepacia.

Claims (6)

Less than:
(A) enzyme crystals, and (b) at least one excipient selected from the group consisting of sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, and methoxypolyethylene glycol;
A formulation comprising, wherein said formulation, when measured by the T _1/2, than the soluble form of a crystal of the enzyme in a solution of 40 to 55 ° C., the formulation 40 to 55 ° C. A formulation having an expiration date that is at least 60 times longer when stored in. 2. The formulation of claim 1, wherein the enzyme is a lipase and the formulation is T _1/2 A formulation having an expiration date that is at least 60 times longer when stored at 40 ° C. than the soluble form of the enzyme crystals in solution at 40 ° C. as measured by 2. The formulation of claim 1, wherein the enzyme is penicillin acylase and the formulation is T _1/2 A formulation having an expiration date that is at least 60 times longer when stored at 55 ° C. than the soluble form of the enzyme crystals in a solution at 55 ° C. as measured by 2. The formulation of claim 1, wherein the enzyme is glucose oxidase and the formulation is T _1/2 A formulation having an expiration date that is at least 60 times longer when stored at 50 ° C. than the soluble form of the enzyme crystals in solution at 50 ° C. A composition for the release of an enzyme , the composition comprising:
(A) prescribing as claimed in any one of claims 1-4; and,
(B) at least one polymer carrier;
A composition comprising: wherein the formulation is encapsulated within a matrix of the polymer carrier. 6. The composition of claim 5 , wherein the polymer carrier is poly (acrylic acid), poly (cyanoacrylate), poly (amino acid), poly (anhydride), poly (depsipeptide), poly (ester). , Poly (lactic acid), poly (lactic acid-co-glycolic acid) ie PLGA, poly (β-hydroxybutyric acid), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide ), Poly [(organo) phosphazene], poly (orthoester), poly (vinyl alcohol), poly (vinyl pyrrolidone), maleic anhydride-alkyl vinyl ether copolymer, pluronic polyol, albumin, alginate, cellulose and cellulose derivatives, collagen, Fibrin, z Chin, hyaluronic acid, oligosaccharides, glycans amino glycans, is a polymer selected from one or more of the group consisting of sulfated polysaccharides, mixtures thereof and copolymers, compositions.

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