x DK 159274 Bx DK 159274 B

Den foreliggende opfindelse angår hidtil ukendte human insulinanaloger, der har den samme eller en større negativ ladning ved neutralt pH end human insulin, og med hurtigt indsættende virkning ved subkutane injektioner og inji-5 cerbare insulinopløsninger med hurtigt indsættende virkning, der indeholder sådanne insulinanaloger.The present invention relates to novel human insulin analogues having the same or a greater negative charge at neutral pH than human insulin, and having a fast insertion effect by subcutaneous injections and injectable fast-acting insulin solutions containing such insulin analogues.

Til behandling af diabetes mellitus er der foreslået og anvendt mange forskellige insulinpræparater. Nogle af disse præparater er hurtigtvirkende, og andre præparater 10 har mere eller mindre retarderet virkning.For the treatment of diabetes mellitus, many different insulin preparations have been proposed and used. Some of these compositions are fast acting and other compositions 10 have more or less retarded effect.

Hurtigtvirkende insulinpræparater kan anvendes i akutte situationer, såsom insulinchock, under operation, under graviditet og ved alvorlige infektioner. Desuden kan hyppige daglige injektioner af hurtigtvirkende insulinpræparater 15 forbedre kontrollen hos diabetikere, som har vist sig at være vanskelige at kontrollere med længerevirkende insulin.Fast-acting insulin preparations can be used in acute situations, such as insulin shock, during surgery, during pregnancy and in severe infections. In addition, frequent daily injections of fast-acting insulin preparations may improve the control of diabetics who have been found to be difficult to control with longer-acting insulin.

I de senere år har der været en stigende interesse for at tilnærme insulinbehandlingen til insulinudskillelsen fra betaceller i den sunde organisme, d.v.s. tilførsel af 20 insulin i forbindelse med måltider og opretholdelse af et basalt insulinniveau. Kliniske undersøgelser har vist, at diabetikere kan opnå tilnærmelsesvis normale insulin- og glu-cosekoncentrationer ved hjælp af en daglig injektion af insulin med retarderet virkning til at dække det basale behov 25 suppleret med injektioner med mindre mængder (bolus) af hurtigtvirkende insulin før hovedmåltiderne.In recent years, there has been a growing interest in approximating insulin therapy to insulin secretion from beta cells in the healthy organism, i.e. supply of 20 insulin in connection with meals and maintaining a basal insulin level. Clinical studies have shown that diabetics can achieve approximately normal insulin and glucose concentrations using a daily injection of retarded insulin to meet basic need supplemented with smaller bolus injections of fast-acting insulin before main meals.

Hurtigtvirkende insuliner anvendes også i blanding med middel- og længerevirkende insuliner til behandling af diabetikere, der kræver en stærkere initial virkning foruden 30 den forsinkede virkning af middel- og længerevirkende insuliner.Rapid-acting insulins are also used in admixture with medium- and longer-acting insulins to treat diabetics who require a stronger initial effect in addition to the delayed action of medium- and longer-acting insulins.

Endelig anvendes hurtigtvirkende insulin i kontinuerte insulinindgiftsystemer.Finally, fast-acting insulin is used in continuous insulin delivery systems.

Ved subkutan injektion af hurtigtvirkende insulin-35 opløsninger er der blevet observeret en initial forsinkelse i absorptionen (Binder, Diabetes Care ]_, nr. 2 (1984), 188- 199). En forsinkelse i absorptionen, som resulterer i en langsommere indsætning af virkningen, er imidlertid megetBy subcutaneous injection of fast-acting insulin solutions, an initial delay in absorption has been observed (Binder, Diabetes Care, No. 2 (1984), 188-199). However, a delay in absorption which results in a slower insertion of the effect is considerable

2 DK 159274 B2 DK 159274 B

uheldig, når der tilstræbes en stringent metabolisk kontrol. Blanding af hurtigtvirkende insulinopløsninger med længere-virkende insulinpræparater kan desuden resultere i formindsket absorptionshastighed af det hurtigtvirkende insulin.unfortunate when striving for rigorous metabolic control. In addition, mixing fast-acting insulin solutions with longer-acting insulin preparations may result in decreased absorption rate of the fast-acting insulin.

5 Der er derfor et behov for hurtigtvirkende insulin opløsninger med en hurtigere indsættende virkning ved subkutan injektion og en forbedret blandbarhed med protraherede insulinpræparater.Therefore, there is a need for fast-acting insulin solutions with a faster onset of action by subcutaneous injection and an improved miscibility with protracted insulin preparations.

En yderligere ulempe ved kendte hurtigtvirkende 10 insulinopløsninger er insulins tendens til at fibrillere og fælde ud fra insulinopløsninger, der anvendes til kontinuerlig insulinindgift, hvorved både mekaniske dele og infusionskateteret tilstoppes.A further disadvantage of known fast-acting insulin solutions is the tendency of insulin to fibrillate and precipitate from insulin solutions used for continuous insulin administration, thereby clogging both mechanical parts and the infusion catheter.

Der er endelig et behov for alternative insulinpræ-15 parater til behandling af patienter, der er blevet resistente overfor normal insulin.Finally, there is a need for alternative insulin preparations to treat patients who have become resistant to normal insulin.

Det er formålet med den foreliggende opfindelse at tilvejebringe hidtil ukendte hurtigtvirkende insulinopløsninger med hurtigre indsættende virkning ved subkutan injektion 20 eller andre administrationsmåder, og som desuden kan have en eller flere af følgende forbedrede egenskaber: 1) forbedret blandbarhed med protraherede insu- 1inpræparater, 2) formindsket tendens til fibrillering ved an- 25 vendelse i bl.a. implanterbare indgiftssystemer, og 3) anvendelighed til behandling af resistente patienter (lav affinitet til på forhånd eksisterende antistoffer).It is the object of the present invention to provide novel fast-acting insulin solutions with faster insertion by subcutaneous injection 20 or other modes of administration, and which may further have one or more of the following improved properties: 1) improved miscibility with protracted insulin preparations; 2) diminished tendency to fibrillation when used in i. implantable administration systems, and 3) utility in the treatment of resistant patients (low affinity for pre-existing antibodies).

Formålene med den foreliggende opfindelse opnås med 30 injicerbare vandige opløsninger af de i det efterfølgende beskrevne hidtil ukendte human insulin analoger.The objects of the present invention are achieved with 30 injectable aqueous solutions of the human insulin analogues described hereinafter.

Der er i årenes løb blevet beskrevet et stort antal af insulinanaloger. Mårke et al. (Hoppe-Seyler's Z.Physiol. Chem., 360. (1979), 1619-1632) beskriver syntese af analoger 35 til human insulin, der adskiller sig fra human insulin ved, at en enkelt aminosyre i positionerne 2, 5, 6, 7, 8 og 11 i A-kæden og 5, 7, 13 og 16 i B-kæden er blevet udskiftet medOver the years, a large number of insulin analogues have been described. Mårke et al. (Hoppe-Seyler's Z.Physiol. Chem., 360. (1979), 1619-1632) discloses synthesis of analogues of human insulin that differ from human insulin in that a single amino acid at positions 2, 5, 6, 7, 8 and 11 in the A chain and 5, 7, 13 and 16 in the B chain have been replaced by

3 DK 159274 B3 DK 159274 B

det formål at opnå ny indsigt i den interessante sammenhæng mellem struktur og aktivitet af insulin. Yderligere undersøgelser har til formål at modificere hovedreceptorbindingsare-alet i insulin (B(20)-B(26)) med det formål at undersøge ind-5 virkningen af sådanne mutationer på receptorbindingsaktiviteten. Sådanne kendte insulinanaloger vil imidlertid ikke have de ovenfor beskrevne forbedrede egenskaber.the purpose of gaining new insight into the interesting connection between the structure and activity of insulin. Further studies are aimed at modifying the main receptor binding site in insulin (B (20) -B (26)) with the aim of investigating the effect of such mutations on the receptor binding activity. However, such known insulin analogues will not have the improved properties described above.

Det er kendt, at sulfaterede insuliner har en væsentlig mindre tendens til fibrillering (Albisser et al., 10 Desired Characteristics of insulin to be used in infusion pumps. In: Gueriguian J.L. et al., eds. US Pharmacopeial Convention, Rockwille, Maryland, pp. 84-95) og udviser en lav antigenicitet. Sulfaterede insuliner er imidlertid en heterogen blanding af mindst ni forskellige insulinderivater, der 15 indeholder i middel 4,5 sulfatestergrupper pr. molekyle. Sulfaterede insuliner har desuden en formindsket insulinaktivitet, der andrager ca. 20% af aktiviteten af naturligt insulin. En yderligere ulempe ved sulfaterede insuliner i sammenligning med naturligt insulin er, at de indeholder aminosyre-20 rester, der er kemisk modificeret, d.v.s. aminosyrer, der ikke forekommer naturligt.Sulfated insulins are known to have a significantly lower tendency for fibrillation (Albisser et al., 10 Desired Characteristics of Insulin to be Used in Infusion Pumps. In: Gueriguian JL et al., Eds. US Pharmacopeial Convention, Rockwille, Maryland, pp. 84-95) and exhibit a low antigenicity. However, sulfated insulins are a heterogeneous mixture of at least nine different insulin derivatives containing an average of 4.5 sulfate ester groups per ml. molecule. In addition, sulfated insulins have a decreased insulin activity, which is approx. 20% of the activity of natural insulin. A further disadvantage of sulfated insulins as compared to natural insulin is that they contain amino acid residues that are chemically modified, i.e. amino acids that do not occur naturally.

Det er derfor et yderligere formål med den foreliggende opfindelse at tilvejebringe insulinanaloger, der er homogene, kun indeholder naturligt forekommende aminosyrer, 25 og fortrinsvis har en højere biologisk aktivitet end sulfaterede insuliner.Therefore, it is a further object of the present invention to provide insulin analogs that are homogeneous, contain only naturally occurring amino acids, and preferably have a higher biological activity than sulfated insulins.

Med "insulinanaloger" som anvendt her menes en forbindelse med en molekylær struktur, der svarer til strukturen af human insulin omfattende disulfidbroerne mellem A(7)Cys og 30 B(7)Cys og mellem A(20)Cys og B(19)Cys og en intern disulfidbro mellem A(6)Cys og A(ll)Cys, og som har insulinaktivitet.By "insulin analogues" as used herein is meant a compound having a molecular structure corresponding to the structure of human insulin comprising the disulfide bridges between A (7) Cys and 30 B (7) Cys and between A (20) Cys and B (19) Cys and an internal disulfide bridge between A (6) Cys and A (II) Cys, which has insulin activity.

Den foreliggende opfindelse er baseret på den overraskende erkendelse, at visse insulinanaloger, i hvilke mindst en af aminosyreresterne i human insulin er blevet sub-35 stitueret med en naturligt forekommende aminosyrerest, har den ønskede hurtigere virkning.The present invention is based on the surprising realization that certain insulin analogues in which at least one of the amino acid residues in human insulin has been substituted with a naturally occurring amino acid residue have the desired faster effect.

De hidtil ukendte, hurtigtvirkende human insulinanaloger ifølge opfindelsen fremkommer ved at erstatte enThe novel fast-acting human insulin analogues of the invention emerge by replacing one

4 DK 159274 B4 DK 159274 B

eller flere af aminosyreresterne i human insulin med naturligt forekommende aminosyrerester, der giver anledning til mindre selvassociering til dimere, tetramere, hexamere eller polymere, og har samme ladning eller en større negativ lad-5 ning ved neutralt pH end ladningen af human insulin.or more of the amino acid residues in human insulin with naturally occurring amino acid residues, giving rise to less self-association to dimers, tetramers, hexamers or polymers, and having the same charge or a greater negative charge at neutral pH than the loading of human insulin.

Til tilvejebringelse af en formindsket tendens til selvassociering til dimere, tetramere, hexamere eller polymere er visse rester i human insulin fortrinsvis erstattet med andre aminosyrerester, der er mere hydrophile end de na-10 turlige aminosyrerester på den pågældende position i moleky let. Ved visse positioner i insulinmolekylet vil substitutioner med en aminosyrerest med mere bulk også bevirke en formindsket tendens af insulinmolekylet til at associere til dimere, tetramere, hexamere eller polymere.To provide a diminished tendency for self-association to dimers, tetramers, hexamers or polymers, certain residues in human insulin are preferably replaced by other amino acid residues that are more hydrophilic than the natural amino acid residues at that particular position in the molecule. At certain positions in the insulin molecule, substitutions with a more bulk amino acid residue will also cause a decreased tendency of the insulin molecule to associate with dimers, tetramers, hexamers or polymers.

15 Insulinanalogerne ifølge opfindelsen er ejendomme lige ved, at de har den almene formel (I) A-kæde (e'OCM*XvaaGiuXG,nXc'«XCivLxAxAxIc»*Is*'Xx Xt,,agiXi,tXa,"at,,Ic,,X o 1 2 3 4 S β 7| * · 10 11 I! 11 14 15 16 17 18 19 201 21The insulin analogues of the invention are properties just in that they have the general formula (I) A chain (e'OCM * XvaaGiuXG, nXc '"XCivLxAxAxIc" * Is * "Xx Xt ,, agiXi, tXa" ,, X o 1 2 3 4 S β 7 | * · 10 11 I! 11 14 15 16 17 18 19 201 21

SS

VV

(* γx X*,nXGinXx X*Xr^X^XJLXv.xJvXx Χ°ΙυΧx XL,iXx Xx Xx X,*Xx 1 2 3 4 $ 8 7 3 * 10 11 12 13 14 15 16 17 18 19 2<j 21 22 23 24 25 28 27 28 29 30 2 0 B-kæde(* γx X *, nXGinXx X * Xr ^ X ^ XJLXv.xJvXx Χ ° ΙυΧx XL, iXx Xx Xx X, * Xx 1 2 3 4 $ 8 7 3 * 10 11 12 13 14 15 16 17 18 19 2 <j 21 22 23 24 25 28 27 28 29 30 2 0 B-chain

s DK 159274 Bs DK 159274 B

hvori X'erne er aminosyreresterne i human insulin eller ens eller forskellige aminosyrerestsubstitutioner valgt blandt naturligt forekommende aminosyrer, med den betingelse, at mindst et X og højst 4 af X'erne er forskellige fra amino-5 syreresten i human insulin på den tilsvarende position i insulinmolekylet, at mindst et X i B-kæden er forskellig fra aminosyreresten i human insulin på den tilsvarende position i human insulin molekylet og at når X i position B(5) er Ala, X i position B(9) er Leu, X i position B(10) er Asn eller Leu, 10 X i position B(12) er Asn, eller X i position B(26) er Ala, så er mindst en af de øvrige X'er forskellige fra aminosyreresten i human insulin på den tilsvarende position i insulinmolekylet, og med den yderligere betingelse, at op til 4 ami-nosyrerester kan være blevet fjernet fra den N-terminale ende 15 på B-kæden, og op til 5 aminosyrerester kan være fjernet ved den C-terminale ende af B-kæden.wherein the Xs are the amino acid residues of human insulin or the same or different amino acid residue substitutions selected from naturally occurring amino acids, provided that at least one X and at most 4 of the Xs are different from the amino acid residue in human insulin at the corresponding position in the insulin molecule, that at least one X in the B chain is different from the amino acid residue in human insulin at the corresponding position in the human insulin molecule and that when X at position B (5) is Ala, X at position B (9) is Leu, X is position B (10) is Asn or Leu, 10 X at position B (12) is Asn, or X at position B (26) is Ala, then at least one of the other Xs is different from the amino acid residue in human insulin on it. corresponding position in the insulin molecule, and with the additional condition that up to 4 amino acid residues may have been removed from the N-terminal end 15 of the B chain and up to 5 amino acid residues may be removed at the C-terminal end of B -the chain.

Fortrinsvis er i det mindste hovedparten af amino-syrerestsubstitutionerne mere hydrofile end aminosyreresten på det tilsvarende sted i human insulinmolekylet, og mere 20 fortrinsvis er alle aminosyrerestsubstitutionerne mere hydrofile end de tilsvarende human insulinaminosyrerester.Preferably, at least most of the amino acid residue substitutions are more hydrophilic than the amino acid residue at the corresponding site in the human insulin molecule, and more preferably all the amino acid residue substitutions are more hydrophilic than the corresponding human insulin amino acid residues.

Med hensyn til hydrofilicitet kan der henvises til C. Frommel, J.Theor.Biol. 111 (1984), 247-260, tabel 1.With regard to hydrophilicity, reference can be made to C. Frommel, J. Theor.Biol. 111 (1984), 247-260, Table 1.

Aminosyrerestsubstitutionerne vælges fortrinsvis 25 fra gruppen bestående af Asp, Glu, Ser, Thr, His og Ile og er mere foretrukket negativt ladede aminosyrer, d.v.s. Asp og/eller Glu.Preferably, the amino acid residue substitutions are selected from the group consisting of Asp, Glu, Ser, Thr, His and Ile and are more preferably negatively charged amino acids, i.e. Asp and / or Glu.

De hidtil ukendte human insulinanaloger kan fortrinsvis indeholde Asp og/eller Glu i stedet for en eller 30 flere hydroxyaminosyrer i human insulin, eller i stedet for en eller flere Gin og Asn i human insulin.The novel human insulin analogs may preferably contain Asp and / or Glu in place of one or more hydroxy amino acids in human insulin, or in place of one or more Gin and Asn in human insulin.

De hidtil ukendte human insulinanaloger kan yderligere fortrinsvis indeholde Ser og/eller Thr eller Asp og/eller Glu i stedet for en eller flere af aminosyreresterne 35 i human insulin med en aliphatisk og/eller aromatisk sidekæde.The novel human insulin analogues may further preferably contain Ser and / or Thr or Asp and / or Glu in place of one or more amino acid residues of human insulin with an aliphatic and / or aromatic side chain.

De hidtil ukendte human insulinanaloger kan også fortrinsvis indeholde His i stedet for en eller flere af ami-The novel human insulin analogs may also preferably contain His instead of one or more of the amine.

6 DK 159274B6 DK 159274B

nosyreresterne i human insulin med en aliphatisk og/eller aromatisk sidekæde eller i stedet for en eller flere af hy-droxyaminosyrerne i human insulin.the human acid residues in human insulin with an aliphatic and / or aromatic side chain or in place of one or more of the hydroxy amino acids in human insulin.

Foretrukne substitutionssteder er ved positionerne 5 B9, BIO, B12, B26, B27 og B28, fortrinsvis B9, B12, B27 og B28, i hvilke positioner én substitution kan være tilstrækkelig til opnåelse af en reduceret tendens til selvassociering og en hurtigere virkning ved indgift.Preferred substitution sites are at positions 5 B9, B10, B12, B26, B27 and B28, preferably B9, B12, B27 and B28, in which positions one substitution may be sufficient to achieve a reduced tendency for self-association and a faster effect on administration.

Aminosyrerestsubstitutionen i position B9 kan væl-10 ges fra gruppen bestående af Asp, Pro, Glu, Ile, Leu, Val,The amino acid residue substitution at position B9 may be selected from the group consisting of Asp, Pro, Glu, Ile, Leu, Val,

His, Thr, Gin, Asn, Met, Tyr, Trp og Phe, og mere foretrukket fra gruppen bestående af Asp, Glu, Gin, Asn og His.His, Thr, Gln, Asn, Met, Tyr, Trp and Phe, and more preferably from the group consisting of Asp, Glu, Gln, Asn and His.

Aminosyrerestsubstitutionen i position B12 kan vælges fra gruppen bestående af Ile og Tyr. Aminosyrerestsubsti-15 tutionen i position BIO kan vælges fra gruppen bestående af Asp, Arg, Glu, Asn og Gin og i position B26, B27 og B28 er aminosyrerestsubstitutionerne fortrinsvis Asp eller Glu.The amino acid residue substitution at position B12 can be selected from the group consisting of Ile and Tyr. The amino acid residue substitution at position BIO can be selected from the group consisting of Asp, Arg, Glu, Asn and Gln and at positions B26, B27 and B28, the amino acid residue substitutions are preferably Asp or Glu.

I de resterende positioner af insulinmolekylet menes mindst to substitutioner (fortrinsvis i kombination med 20 de ovenfor nævnte positioner) at være nødvendige for at opnå de forbedrede egenskaber. I disse positioner kan substitutioner foretages som følger:In the remaining positions of the insulin molecule, at least two substitutions (preferably in combination with the above-mentioned positions) are believed to be necessary to achieve the improved properties. In these positions, substitutions can be made as follows:

7 DK 159274 B7 DK 159274 B

Position Foretrukne aminosvrerestsubstitutioner_ A8 His, Gly, Gin, Glu, Ser, Asn, Asp, Pro A9 Gly, Asp, Glu, Thr, His, Gin, Asn, Ala, Pro A10 Leu, Pro, Val, His, Ala, Glu, Asp, Thr, Gin, Asn 5 A13 Pro, Val, Arg, His, Ala, Glu, Asp, Thr, Gly, Gin,Position Preferred amino acid residue substitutions_ A8 His, Gly, Gin, Glu, Ser, Asn, Asp, Pro A9 Gly, Asp, Glu, Thr, His, Gin, Asn, Ala, Pro A10 Leu, Pro, Val, His, Ala, Glu, Asp, Thr, Gln, Asn 5 A13 Pro, Val, Arg, His, Ala, Glu, Asp, Thr, Gly, Gln,

Asn, Asp A21 Asp, GluAsn, Asp A21 Asp, Glu

Bl Glu, Asp, Thr, Ser B2 Arg, His, Ala, Glu, Asp, Thr, Pro, Gly, Gin, Ser, 10 Asn B5 Glu, Asp, Thr, Ser, Gin, Asn B14 Glu, Asp, Asn, Gin, Ser, Thr, Gly B16 Asp, Glu, Gin, Asn, Ser, Thr, His, Arg B17 Ser, Thr, Asn, Gin, Glu, Asp, His 15 BIS Ser, Thr, Asn, Gin, His B20 Gin, Ser, Asn, Asp, Glu, ArgB1 Glu, Asp, Thr, Ser B2 Arg, His, Ala, Glu, Asp, Thr, Pro, Gly, Gin, Ser, Asn B5 Glu, Asp, Thr, Ser, Gin, Asn B14 Glu, Asp, Asn, Gin, Ser, Thr, Gly B16 Asp, Glu, Gin, Asn, Ser, Thr, His, Arg B17 Ser, Thr, Asn, Gin, Glu, Asp, His 15 BIS Ser, Thr, Asn, Gin, His B20 Gin , Ser, Asn, Asp, Glu, Arg

Yderligere foretrukne forbindelser ifølge den foreliggende opfindelse er insulinanaloger, i hvilke substitutioner er ved de følgende positioner: B27, B12, B9, (B27+B9), 20 (B27+A21) , (B27+B12), (B12+A21), (B27+B17), (B27+A13), (B27+B16) , (B27+A10), (B27+B28), (B27+B26), (B27+B10), (B27 + B1) , (B27 + B2), (B27+B5), (B27+B14), (B27+B18), (B27+B20), (B12+B17), (B12+A10), (B12+A13), (B12+B16) , (B12 + B1) , (B12 + B2) , (B12+B5) , (B12+B10), (B12+B26), 25 (B12+B28), (B9+B17), (B9+A13), (B9+B16), (B9+A8), (B9+A9), (B9+A10), (B9+B1), (B9+B2), (B9+B5), (B9+B10), (B9+B12), (B9+B14), (B9+B28), (B9+B18), (B9+B20), (B9+B26), (B27+B9+A21) , (B9+B27+A8) , (B27+B12+A21), (B27+B12+B9) , (B9+B12+B27+B17) , (B9+B12+B27+A13) , (B9+B12+B27+B16) og 30 (B12+B16+B17+B27+A10+A13).Further preferred compounds of the present invention are insulin analogues in which substitutions are at the following positions: B27, B12, B9, (B27 + B9), 20 (B27 + A21), (B27 + B12), (B12 + A21), (B27 + B17), (B27 + A13), (B27 + B16), (B27 + A10), (B27 + B28), (B27 + B26), (B27 + B10), (B27 + B1), (B27 + B2), (B27 + B5), (B27 + B14), (B27 + B18), (B27 + B20), (B12 + B17), (B12 + A10), (B12 + A13), (B12 + B16) ), (B12 + B1), (B12 + B2), (B12 + B5), (B12 + B10), (B12 + B26), 25 (B12 + B28), (B9 + B17), (B9 + A13) , (B9 + B16), (B9 + A8), (B9 + A9), (B9 + A10), (B9 + B1), (B9 + B2), (B9 + B5), (B9 + B10), ( (B9 + B12), (B9 + B14), (B9 + B28), (B9 + B18), (B9 + B20), (B9 + B26), (B27 + B9 + A21), (B9 + B27 + A8) , (B27 + B12 + A21), (B27 + B12 + B9), (B9 + B12 + B27 + B17), (B9 + B12 + B27 + A13), (B9 + B12 + B27 + B16), and 30 (B12 + B16 + B17 + B27 + A10 + A13).

Foretrukne udførelsesformer for formel I er som følger: 8Preferred embodiments of Formula I are as follows:

DK 159274 BDK 159274 B

A-kæde s 1 2 3 4 5 β 7| 8 B 10 11 12 13 14 15 18 17 18 19 20 | 21 ;; / @@@{q^©@@0©@0 j 2 3 4 6 8 7 8 B 10 11 12 13 14 16 18 17 18 1fl 20 21 22 23 24 25 26 27 28 29 30 B-kæde A-kæde r “s"i 1 2 3 4 6 8 7| 8 B 10 11 12 13 14 15 18 17 18 19 20| 21 li s / 1 2 3 4 6 8 7 8 B 10 11 12 13 14 16 16 17 18 18 20 21 22 23 24 25 28 27 28 29 30 B-kæde A-kædeA-chain s 1 2 3 4 5 β 7 | 8 B 10 11 12 13 14 15 18 17 18 19 20 | 21 ;; / @@@ {q ^ © @@ 0 © @ 0 j 2 3 4 6 8 7 8 B 10 11 12 13 14 16 18 17 18 1fl 20 21 22 23 24 25 26 27 28 29 30 B-chain A-chain r “s" i 1 2 3 4 6 8 7 | 8 B 10 11 12 13 14 15 18 17 18 19 20 | 21 li s / 1 2 3 4 6 8 7 8 B 10 11 12 13 14 16 16 17 18 18 20 21 22 23 24 25 28 27 28 29 30 B-chain A-chain

i s 1—8 I -COOHi s 1-8 I -COOH

^ly)^ii^(vaj)^i^^i^^y»)(cy*)^^^^^I^(cy»)^^^u)^r)(Qin)(Leu)(Qiu)^»^^i)(Cy»)^»n) 1 2 3 4 6 8 7j 8 B 10 11 12 13 14 15 16 17 18 19 2θ| 21 I 8 NH2- ί ί 1 2 3 4 6 8 7 8 B 10 11 12 13 14 15 18 17 18 1fl 20 21 22 23 24 25 28 27 28 29 30 B-kædeLy ^) ^ ii ^ (Vaj) ^ i ^^ ^^ in the y ') (cy *) ^^^^^ I ^ (cy') ^^^ u) ^ s) (Qin) (Leu) (Qiu ) ^ »^^ i) (Cy») ^ »n) 1 2 3 4 6 8 7j 8 B 10 11 12 13 14 15 16 17 18 19 2θ | 21 I 8 NH2- ί ί 1 2 3 4 6 8 7 8 B 10 11 12 13 14 15 18 17 18 1fl 20 21 22 23 24 25 28 27 28 29 30 B-chain

9 DK 159274B9 DK 159274B

i hvilke X'erne defineres som ovenfor.in which the Xs are defined as above.

Under henvisning til formel I er andre foretrukne insulinanaloger ifølge den foreliggende opfindelse sådanne, i hvilke X i position B27 er Glu, X i position B12 er Ile eller 5 Tyr, X i position A21 er Asp og i position B27 er Glu, X i position B9 er Asp, X i position A21 og i position B9 er Asp og i position B27 er Glu, X i position A8 er His, i position B9 er Asp og i position B27 er Glu, X i position BIO er Asp, X i position B28 er Asp, eller X i position B9 er Asp og i 10 position B27 er Glu.Referring to Formula I, other preferred insulin analogues of the present invention are those in which X at position B27 is Glu, X at position B12 is Ile or Tyr, X at position A21 is Asp and at position B27 is Glu, X at position B9 is Asp, X at position A21 and at position B9 is Asp and at position B27 is Glu, X at position A8 is His, at position B9 is Asp and at position B27 is Glu, X at position BIO is Asp, X at position B28 is Asp, or X at position B9 is Asp and at position B27 is Glu.

Ifølge et andet aspekt af den foreliggende opfindelse tilvejebringes der injicerbare opløsninger med hurtigt indsættende insulinaktivitet. De injicerbare insulinopløsninger ifølge opfindelsen er ejendommelige ved, at de indeholder 15 human insulinanaloger ifølge opfindelsen eller et farmaceutisk akceptabelt salt deraf i vandig opløsning, fortrinsvis ved neutral pH. Det vandige medium kan gøres isotonisk ved tilsætning af f.eks. natriumchlorid og glycerol. Også puffere, såsom acetat eller citrat og konserveringsmidler, såsom 20 m-cresol, fenol eller methyl 4-hydroxybenzoat kan tilsættes. Endvidere kan insulinopløsningerne indeholde zinkioner.According to another aspect of the present invention, injectable solutions are provided with rapidly inserting insulin activity. The injectable insulin solutions of the invention are characterized in that they contain 15 human insulin analogues of the invention or a pharmaceutically acceptable salt thereof in aqueous solution, preferably at neutral pH. The aqueous medium can be made isotonic by the addition of e.g. sodium chloride and glycerol. Also buffers such as acetate or citrate and preservatives such as 20 m-cresol, phenol or methyl 4-hydroxybenzoate may be added. Furthermore, the insulin solutions may contain zinc ions.

Human insulinanalogerne ifølge opfindelsen kan erstatte human eller svineinsulin i de hidtil kendte hurtigtvirkende insulinopløsninger.The human insulin analogues of the invention can replace human or porcine insulin in the known fast-acting insulin solutions.

25 Efter fremkomsten af rekombinant DNA-teknologien har mulighederne for protein-engineering vist sig at være enorme. Ved såkaldt "site specific mutageneseteknik" er det muligt at ændre et gen, der koder for et naturligt forekommende protein ved at substituere en eller flere af codonerne 30 i det naturlige gen med codoner for andre naturligt forekommende aminosyrer. Alternativt kan det modificerede gen fremstilles ved kemisk syntese af hele DNA-sekvensen på velkendt måde. Formålet med en sådan manipulation med et gen for et naturligt protein vil typisk være at ændre egenskaberne af 35 det naturlige protein på ønsket måde.25 Following the advent of recombinant DNA technology, the possibilities of protein engineering have proven enormous. By so-called "site specific mutagenesis technique" it is possible to alter a gene encoding a naturally occurring protein by substituting one or more of the codons 30 in the natural gene with codons for other naturally occurring amino acids. Alternatively, the modified gene can be prepared by chemical synthesis of the entire DNA sequence in a well-known manner. The purpose of such manipulation with a gene for a natural protein will typically be to alter the properties of the natural protein in the desired manner.

De hidtil ukendte insulinanaloger kan fremstilles ved at ændre proinsulingenet ved erstatning af codoner på det passende sted i det naturlige human proinsulingen med codo-The novel insulin analogs can be prepared by altering the proinsulin network by replacing codons at the appropriate site in the natural human proinsulin with the codon.

10 DK 159274 B10 DK 159274 B

ner, der koder for de ønskede aminosyrerestsubstitutioner, eller ved at syntetisere hele DNA-sekvensen, der koder for den ønskede insulinanalog. Det nye modificerede eller syntetiske gen, der koder for den ønskede insulinanalog, indsættes 5 derpå i en egnet ekspressionsvektor, der derpå overføres til en passende værtsorganisme, f.eks. E. coli. Bacillus eller gær, der er i stand til at udtrykke det ønskede produkt. Det udtrykte produkt isoleres derpå fra cellerne eller fra kulturvæsken, afhængigt af, om det udtrykte produkt udskilles 10 fra cellerne eller ej.coding for the desired amino acid residue substitutions, or by synthesizing the entire DNA sequence encoding the desired insulin analog. The new modified or synthetic gene encoding the desired insulin analog is then inserted into a suitable expression vector which is then transferred to a suitable host organism, e.g. E. coli. Bacillus or yeast capable of expressing the desired product. The expressed product is then isolated from the cells or from the culture fluid, depending on whether the expressed product is excreted from the cells or not.

De hidtil ukendte insulinanaloger kan også fremstilles ved kemisk syntese ifølge fremgangsmåder, der er analoge med den af Mårki et al. (Hoppe-Seyler's Z. Physiol. Chem., 360 (1979), 1619-1632) beskrevne metode. De kan også 15 dannes ud fra individuelt in vitro fremstillede A- og B-kæder indeholdende de pågældende aminosyrerestsubstitutioner, hvorefter de modificerede A- og B-kæder forbindes ved etablering af disulfidbroer ifølge kendte metoder (f.eks. chance et al.,The novel insulin analogs may also be prepared by chemical synthesis according to methods analogous to that of Mårki et al. (Hoppe-Seyler's Z. Physiol. Chem., 360 (1979), 1619-1632). They can also be formed from individually in vitro A and B chains containing the relevant amino acid residue substitutions, after which the modified A and B chains are joined by establishing disulfide bridges by known methods (e.g. Chance et al.,

In: Rick DH, Gross E (eds.) Peptides: Synthesis - Structure-20 Function. Proceedings of the Seventh American Peptid Symposium, Illinois, pp. 721-728).In: Rick DH, Gross E (eds.) Peptides: Synthesis - Structure-20 Function. Proceedings of the Seventh American Peptide Symposium, Illinois, pp. 721-728).

De hidtil ukendte insulinanaloger fremstilles fortrinsvis ved omsætning af en biosyntetisk precursor med den almene formel II: 11The novel insulin analogs are preferably prepared by reacting a biosynthetic precursor of the general formula II: 11

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A-kæde , 2 i 4 g « tT 8 9 10 11 H 13 14 1* 18 17 1β 1» *°| 21 q®©©ø©^^ VTTTYTVTYVTV 13 14 1» 13 17 1. 1» *> 21 12 ” 24 25 ** 27 “ " Π B-kæde hvor Qn er en peptidkæde med n naturligt forekommende amino-syrerester, R er Lys eller Arg, n er et helt tal fra 0 til 33, m er 0 eller 1, og X'erne defineres som ovenfor, med den betingelse, at peptidkæden -Qn“R" ikke indeholder to nabo-5 stillede basiske aminosyrerester, med en L-threoninester i nærvær af trypsin eller et trypsinderivat efterfulgt af konvertering af den fremkomne threoninester af humaninsulinana-logen til humaninsulinanalogen ifølge kendte metoder. Denne såkaldte "transpeptideringsreaktion" er beskrevet i US pa-10 tentskrift nr. 4.343.898.A chain, 2 in 4 g «tT 8 9 10 11 H 13 14 1 * 18 17 1β 1» * ° | 21 q® ©♦ ø © ^^ VTTTYTVTYVTV 13 14 1 »13 17 1. 1» *> 21 12 ”24 25 ** 27“ "Π B chain where Qn is a peptide chain with n naturally occurring amino acid residues, R is Lys or Arg, n is an integer from 0 to 33, m is 0 or 1 and the Xs are defined as above, provided that the peptide chain -Qn "R" does not contain two neighboring basic amino acid residues, with an L-threonine ester in the presence of trypsin or a trypsin derivative followed by conversion of the resulting threonine ester of the human insulin analog to the human insulin analogue by known methods. This so-called "transpeptidation reaction" is described in U.S. Patent No. 4,343,898.

Ved transpeptideringsreaktionen fraskæres broen -(Qn-R-)xn“ mellem aminosyre 29 i B-kæden og aminosyre 1 i A-kæden og en threoninestergruppe kobles på den C-terminale ende af B29Lys.In the transpeptidation reaction, the bridge - (Qn-R-) xn ′ is separated between amino acid 29 of the B chain and amino acid 1 of the A chain and a threonine ester group is coupled at the C-terminal end of B29Lys.

15 Precursorerne med formel II kan fremstilles ifølge en fremgangsmåde, der er analog med den i EP patentansøgning nr. 0163529A beskrevne fremgangsmåde. Ved denne fremgangsmåde indsættes en DNA-sekvens, der koder for den pågældende precursor, i en egnet udtrykkelsesvektor, som, når den overføres 20 til gær, er i stand til at udtrykke og secernere den ønskede forbindelse med korrekt anbragte disulfidbroer. Det udtrykte produkt isoleres derpå fra dyrkningsmediet.The precursors of formula II can be prepared according to a method analogous to the process described in EP patent application 0163529A. In this method, a DNA sequence encoding the particular precursor is inserted into a suitable expression vector which, when transferred to yeast, is able to express and secrete the desired compound with properly placed disulfide bridges. The expressed product is then isolated from the culture medium.

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!!

De foreliggende insulinanaloger kan også fremstilles ved omsætning af en biosyntetisk precursor med den generelle formel III: A-kædeThe present insulin analogs may also be prepared by reacting a biosynthetic precursor of the general formula III: A chain

fGlyY l,*X,olXB,0iG,nXc»0(c*»X x Y * Y x YcytYs«fY x YT»,YGtnYl«iJGtuYAt,YTrrTcy«Y X JfGlyY l, * X, olXB, 0iG, nXc »0 (c *» X x Y * Y x YcytYs «fY x YT», YGtnYl «iJGtuYAt, YTrrTcy« Y X J

1 2 2 4 S 6 7j 8 i IC II » 11 14 15 ti «7 18 18 20j 21 ^ 1 2 a 4 5 8 7 8 8 10 11 12 13 14 IS 18 17 18 18 20 21 22 23 24 2$ 26 27 28 29 30 m B-kæde hvor V og T er henholdsvis Lys eller Arg og X'erne defineres 5 som ovenfor, i vandig opløsning med trypsin og carboxypepti-dase B og udvinding af human insulinanalogen fra reaktionsopløsningen.1 2 2 4 S 6 7j 8 i IC II »11 14 15 ti« 7 18 18 20j 21 ^ 1 2 a 4 5 8 7 8 8 10 11 12 13 14 IS 18 17 18 18 20 21 22 23 24 2 $ 26 27 28 29 30 m B chain where V and T are Lys or Arg and the Xs are defined as above, in aqueous solution with trypsin and carboxypeptidase B and recovery of the human insulin analog from the reaction solution.

Precursorerne med den ovenfor anførte formel III IThe precursors of the above formula III I

kan fremstilles ifølge en fremgangsmåde, der er analog med 10 den i EP patentansøgning nr. 86302133.3 beskrevne fremgangs- j måde. Ifølge denne fremgangsmåde indsættes en DNA-sekvens, der koder for precursoren, i et egnet gærudtrykkelsesmedium, som, når det overføres til gær, er i stand til at udtrykke og secernere det udtrykte produkt med korrekt anbragte disulfid-15 broer i dyrkningsmediet.may be prepared according to a method analogous to the process described in EP Patent Application No. 86302133.3. According to this method, a DNA sequence encoding the precursor is inserted into a suitable yeast expression medium which, when transferred to yeast, is able to express and secrete the expressed product with properly placed disulfide bridges in the culture medium.

Den foreliggende opfindelse tænkes at omfatte visse deriveringer eller yderligere substitutioner i insulinanalo-gerne forudsat disse deriveringer eller yderligere substitutioner ikke har nogen væsentlig indflydelse på det ovenfor 20 beskrevne formål med opfindelsen. Det er således muligt at derivere en eller flere af de funktionelle grupper i aminosy-reresterne. Eksempler på en sådan derivering er en i og for sig kendt omdannelse af syregrupperne i insulinmolekylet tilThe present invention is contemplated to include certain derivations or additional substitutions in the insulin analogs, provided these derivatives or additional substitutions have no significant influence on the object of the invention described above. Thus, it is possible to derivate one or more of the functional groups in the amino acid residues. Examples of such derivation are a per se known conversion of the acid groups in the insulin molecule to

13 DK 159274 B13 DK 159274 B

ester- eller amidgrupper, omdannelse af alkoholgrupper til alkoxygrupper eller omvendt, og selektiv deamidering. Eksempelvis kan A21Asn deamideres til A21Asp ved hydrolyse i surt medium, eller B3Asn kan deamideres til B3Asp i neutralt medi-5 um.ester or amide groups, conversion of alcohol groups to alkoxy groups or vice versa, and selective deamidation. For example, A21Asn can be deamidated to A21Asp by hydrolysis in acidic medium, or B3Asn can be deamidated to B3Asp in neutral medium.

Endvidere er det muligt at modificere de foreliggende insulinanaloger enten ved tilsætning eller fjernelse af aminosyrerester ved de N- eller C-terminale ender. Insulin-analogerne ifølge opfindelsen kan mangle indtil fire aminosy-10 rerester ved den N-terminale ende på B-kæden og op til fem aminosyrerester ved den C-terminale ende på B-kæden uden væsentlig indflydelse på de almene egenskaber hos insulinanalo-gen. Eksempler på sådanne modificerede insulinanaloger er insulinanaloger, der mangler BIPhe eller B30Thr aminosyre-15 resten.Furthermore, it is possible to modify the present insulin analogs either by the addition or removal of amino acid residues at the N- or C-terminal ends. The insulin analogues of the invention may lack up to four amino acid residues at the N-terminal end of the B chain and up to five amino acid residues at the C-terminal end of the B chain without significantly affecting the general properties of the insulin analog. Examples of such modified insulin analogues are insulin analogs lacking the BIPhe or B30Thr amino acid residue.

Der kan også indføres naturligt forekommende aminosyrerester ved en eller flere ender af polypeptidkæderne under forudsætning af, at dette ikke har nogen væsentlig indflydelse på det ovenfor beskrevne formål.Naturally occurring amino acid residues may also be introduced at one or more ends of the polypeptide chains, provided that this has no significant influence on the purpose described above.

20 Sådanne deletioner eller additioner ved enderne af polypeptidkæden i de foreliggende insulinanaloger kan udføres in vitro på insulinanalogerne med aminosyresubstitutioner ifølge den foreliggende opfindelse; eller genet for de hidtil ukendte insulinanaloger ifølge den foreliggende opfindelse 25 kan modificeres enten ved tilsætning eller fjernelse af kodonerne, der svarer til de ekstra aminosyrerester henholdsvis de manglende aminosyrerester ved enderne af polypeptidkæden.Such deletions or additions at the ends of the polypeptide chain of the present insulin analogs may be performed in vitro on the insulin analogues with amino acid substitutions of the present invention; or the gene for the novel insulin analogues of the present invention 25 can be modified either by the addition or removal of the codons corresponding to the extra amino acid residues or the missing amino acid residues at the ends of the polypeptide chain, respectively.

De for aminosyrerne anvendte forkortelser er som anført i J.Biol.Chem. 243 (1968), 3558. Aminosyrerne er i L 30 konfigurationen.The abbreviations used for the amino acids are as set forth in J. Biol.Chem. 243 (1968), 3558. The amino acids are in the L 30 configuration.

Som anvendt i det følgende betyder B(l-29) en forkortet B-kæde af human insulin fra BlPh til B29Lys og A(1-21) betyder A-kæden i human insulin.As used below, B (1-29) means a shortened B chain of human insulin from BlPh to B29Lys and A (1-21) means the A chain of human insulin.

De substitutioner, der foretages i human insulin-35 molekylet ifølge den foreliggende opfindelse, er angivet med et præfix henførende til human insulin. Som et eksempel herpå betyder B27Glu human insulin en human insulinanalog, hvor Glu er blevet substitueret for Thr i position 27 i B-kæden.The substitutions made in the human insulin molecule of the present invention are indicated by a prefix relating to human insulin. As an example, B27Glu human insulin means a human insulin analog in which Glu has been substituted for Thr at position 27 in the B chain.

14 DK 159274 B14 DK 159274 B

B27G1U, B9Asp human insulin betyder en human insulinanalog, hvor Glu er blevet substitueret for Thr i position 27 i B-kæden, og Asp er blevet substitueret for Ser i position 9 i B-kæden. B27Glu, B(l-29)-Ala-Ala-Lys-A(l-21) human insulin 5 betyder en precursor for insulinanalogen (se formel II), hvor !B27G1U, B9Asp human insulin means a human insulin analog in which Glu has been substituted for Thr at position 27 in the B chain and Asp has been substituted for Ser at position 9 in the B chain. B27Glu, B (l-29) -Ala-Ala-Lys-A (l-21) human insulin 5 means a precursor for the insulin analog (see formula II), wherein!

Glu er blevet substitueret for Thr i position 27 i den for- i kortede B-kæde (se ovenfor), og hvor B(1-29)-kæden og A-kæden (A(l-21)) er forbundet med peptidsekvensen Ala-Ala-Lys. Med mindre andet anføres er det underforstået, at B(1-29)-kæden i 10 og A(l-21)-kæden er forbundet ved disulfidbroer mellem A(7)Cys og B(7)Cys henholdsvis mellem A(20)Cys og B(19)Cys som i human insulin, og at A-kæden indeholder den interne disulfidbro mellem A(6)Cys og A(ll)Cys.Glu has been substituted for Thr at position 27 of the short-chain B chain (see above), and wherein the B (1-29) chain and the A chain (A (1-21)) are linked to the peptide sequence Ala -Ala-Lys. Unless otherwise stated, it is understood that the B (1-29) chain in 10 and the A (1-21) chain is connected by disulfide bridges between A (7) Cys and B (7) Cys respectively between A (20) Cys and B (19) Cys as in human insulin and that the A chain contains the internal disulfide bridge between A (6) Cys and A (II) Cys.

Som det allerede er nævnt, er hensigten med den 15 foreliggende opfindelse at tilvejebringe hurtigtvirkende, injicerbare insulinopløsninger. Under bestræbelserne herpå blev det først og fremmest taget i betragtning, at der findes betydelige forskelle mellem insulin i et depot eller bolus og insulin i kredsløbet, herunder især en helt uundgåelig for-20 skel i insulinkoncentration. Specifikt er insulin i det cirkulerende blod særdeles fortyndet, nemlig 10-11 til 10”8 M, og er i monomer form med muligvis noget insulin i dimer form.As already mentioned, the object of the present invention is to provide fast-acting injectable insulin solutions. In this endeavor, it was first and foremost taken into account that there are significant differences between insulin in a repository or bolus and insulin in the circulation, including, in particular, a completely unavoidable difference in insulin concentration. Specifically, insulin in the circulating blood is highly diluted, namely 10-11 to 10 "8 M, and is in monomeric form with possibly some insulin in dimer form.

Det meget mere koncentrerede insulin lagret i betacellegranuler i pancreas og i den almindelige, administrerbare opløs-25 ning er stort set, om ikke hovedsageligt, i den ikke-aktive hexamere form for eskempel som den velkendte 2 zink hexamer.The much more concentrated insulin stored in beta cell granules in the pancreas and in the common, administerable solution is largely, if not mainly, in the non-active hexameric form of peel such as the well-known 2 zinc hexamer.

Det er kendt, at human insulin i opløsning eksisterer i mange molekylære former, nemlig den monomere, den dimere, den tetramere og den hexamere (Blundell et al., Advances 30 in Protein Chemistry, Academic Press, New York og London, vol. 26, (1972), 279 - 330), hvor de oligomere former er favoriseret ved høje insulinkoncentrationer, og de monomere er den aktive insul inform. Den tetramere og hexamere er ikke aktive former, og selv den dimere kan være inaktiv. Den for 35 den foreliggende opfindelse tilgrundliggende idé går ud på, at det erkendte og accepterede forsinkede absorptionsfænomen (Binder, Diabetes Care 7, nr. 2 (1984) , 188 - 199) for en stor dels vedkommende skyldes den tid, der kræves til at in-It is known that human insulin in solution exists in many molecular forms, namely the monomer, the dimer, the tetramer and the hexamer (Blundell et al., Advances 30 in Protein Chemistry, Academic Press, New York and London, vol. 26 , (1972), 279 - 330), wherein the oligomeric forms are favored at high insulin concentrations and the monomers are the active insulin inform. The tetramers and hexamers are not active forms, and even the dimers may be inactive. The idea underlying the present invention is that the recognized and accepted delayed absorption phenomenon (Binder, Diabetes Care 7, no. 2 (1984), 188-199) is largely due to the time required to in-

15 DK 159274 B15 DK 159274 B

sulinet disassocieres fra den hexamere, tetramere og dimere form til den (aktive) monomere form.the sulin is disassociated from the hexameric, tetrameric and dimeric form to the (active) monomeric form.

Human insulinanalogerne ifølge den foreliggende opfindelse opnår deres hurtige virkning gennem en molekylær 5 struktur, der ikke umiddelbart er i stand til at danne dimere, tetramere, hexamere eller polymere, d.v.s. med en formindsket tendens til selvassociering til dimere, tetramere, hexamere eller polymere med eller uden tilstedeværelse af zinkioner.The human insulin analogues of the present invention achieve their rapid action through a molecular structure which is not immediately capable of forming dimers, tetramers, hexamers or polymers, i.e. with a reduced tendency for self-association to dimers, tetramers, hexamers or polymers with or without the presence of zinc ions.

10 Det har i lang tid været kendt fra de betydelige artsforskelle i aminosyresekvensen, som findes i insulin, at ikke alle de aminosyrerester, der findes i insulinmolekylet er væsentlige for insulinaktiviteten, og at nogle af de aminosyrer, der er uvæsentlige for insulinaktiviteten, er vig-15 tige for de fysiske egenskaber af insulinmolekylet. Det er kendt, at marsvineinsulin er ude af stand til at dimerisere. Sulfateret insulin og tetranitrotyrosininsulin dimeriserer ikke. Således kan mange af aminosyreresterne i human insulinmolekylet ændres uden en væsentlig formindskelse af insulin-20 aktiviteten. Aminosyresubstitutionerne i human insulinmolekylet ifølge den foreliggende opfindelse er rettet mod, at dannelse af dimere, tetramere, hexamere eller polymere undgås uden at ødelægge insulinaktiviteten.10 It has long been known from the significant species differences in the amino acid sequence found in insulin that not all of the amino acid residues found in the insulin molecule are essential for insulin activity and that some of the amino acids that are insignificant to insulin activity are important. -15 for the physical properties of the insulin molecule. It is known that guinea pig insulin is unable to dimerize. Sulfated insulin and tetranitrotyrosine insulin do not dimerize. Thus, many of the amino acid residues in the human insulin molecule can be altered without a substantial decrease in insulin activity. The amino acid substitutions of the human insulin molecule of the present invention are directed to avoid formation of dimers, tetramers, hexamers or polymers without destroying insulin activity.

Aminosyreresterne i positionerne i A-kæden og B-25 kæden i formel I, hvor substitutionerne kan foretages, er ikke væsentlige for insulinaktiviteten, men de er vigtige for evnen af human insulin til at aggregere i dimere, tetramere, hexamere eller polymere eller for opløselighed af human insulin. De foreliggende aminosyrerestsubstitutioner interfere-30 rer med de atom-til-atom kontakter mellem tilstødende insulinmolekyler, som letter aggregationen til dimere, tetramere, hexamere eller polymere.The amino acid residues at the positions of the A chain and the B-25 chain of formula I, where the substitutions can be made, are not essential for insulin activity, but are important for the ability of human insulin to aggregate into dimers, tetramers, hexamers or polymers or for solubility. of human insulin. The present amino acid residue substitutions interfere with the atom-to-atom contacts between adjacent insulin molecules which facilitate aggregation into dimers, tetramers, hexamers or polymers.

Som det kunne forventes er ændringer i visse positioner i huamn insulinmolekylet mere effektive end andre.As might be expected, changes in certain positions in the huamn insulin molecule are more effective than others.

35 Stort set vil en enkelt substitution foretaget i B-kæden være tilstrækkelig til at mindske tendensen til selv-associering, hvorimod mindst to ændringer af andre rester kan være nødvendige. Substitutionerne i A-kæden tjener hovedsageligt til at 1635 Basically, a single substitution in the B chain will be sufficient to reduce the tendency for self-association, whereas at least two changes to other residues may be necessary. The A-chain substitutions mainly serve to 16

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forbedre opløseligheden af det dissocierede molekyle. Foretrukne positioner, i hvilke der foretages aminosyrerestsub-stitutioner er B9, B12, BIO, B26, B27 og B28 alene, i kombination med hinanden eller sammen med substitutioner andre 5 steder i insulinmolekylet som angivet i formel I.improve the solubility of the dissociated molecule. Preferred positions in which amino acid residue substitutions are made are B9, B12, B10, B26, B27 and B28 alone, in combination with each other or together with substitutions at other sites in the insulin molecule as set forth in Formula I.

Substitutionen af en eller flere negativt ladede aminosyrerester for en uladet eller positivt ladet aminosyre-rest gør ladningen af human insulinanalogen mere negativ ved neutralt pH og sænker det isolektriske punkt i forhold til 10 human insulin. Karakteristisk har human insulinanalogerne ifølge opfindelsen den samme eller en mere negativ ladning (ved neutralt pH) og et lavere isoelektrisk punkt end human insulin.The substitution of one or more negatively charged amino acid residues for an uncharged or positively charged amino acid residue makes the charge of the human insulin analog more negative at neutral pH and lowers the isolectric point relative to 10 human insulin. Typically, the human insulin analogues of the invention have the same or a more negative charge (at neutral pH) and a lower isoelectric point than human insulin.

Som oftest vil fra 1 til 3 substitutioner opfylde 15 de umiddelbare mål for opfindelsen, nemlig at tilvejebringe et mere hurtigtvirkende insulin, og sådanne repræsenterer foretrukne udførelsesformer for opfindelsen. Ved at anvende 2-3 substitutioner kan der desuden opnås en forbedret blandbarhed med protraherede insulinpræparater. Imidlertid anses 20 det for at være en fordel, at de umiddelbare mål for opfindelsen kan nås, også med et større antal substitutioner end tre, eftersom ønskelige sekundære mål herved kan opnås.Most often, from 1 to 3 substitutions, 15 will meet the immediate objectives of the invention, namely to provide a more rapid-acting insulin, and such represent preferred embodiments of the invention. Furthermore, by using 2-3 substitutions, an improved miscibility with protracted insulin preparations can be obtained. However, it is considered to be an advantage that the immediate objects of the invention can be achieved, even with a greater number of substitutions than three, since desirable secondary objectives can be achieved thereby.

Især kan et yderligere antal substitutioner, f.eks.In particular, a further number of substitutions, e.g.

4 eller 5 substitutioner af aminosyrerester, resultere i en 25 human insulinanalog, som også har mindre tendens til fibril-lering eller interfacepolymerisering, hvilket især er ønskeligt, når insulinopløsningen er beregnet til kontinuert tilførsel. Human insulinanalogerne ifølge opfindelsen vil dog ikke indeholde mere end 4 substitutioner i insulinmolekylet.4 or 5 substitutions of amino acid residues result in a human insulin analog, which also has less tendency to fibrillation or interface polymerization, which is particularly desirable when the insulin solution is intended for continuous administration. However, the human insulin analogues of the invention will contain no more than 4 substitutions in the insulin molecule.

30 Der foretrækkes 2-4 substitutioner.2-4 substitutions are preferred.

Gener, der koder for precursorerne for de foreliggende insulinanaloger kan fremstilles ved modificering af gener, der koder for de ovennævnte insulinprecursorer med formlen (II) eller (III), i hvilke alle X'er er aminosyreres-35 terne i human insulin, ved site specific mutagenese for at indføre eller substituere med codoner, der koder for den ønskede mutation. DNA-sekvenser, der koder for precursoren for insulinanalogen kan også fremstilles ved enzymatisk synteseGenes encoding the precursors of the present insulin analogues can be produced by modifying genes encoding the above insulin precursors of formula (II) or (III), in which all Xs are the amino acid residues of human insulin, at the site specific mutagenesis to introduce or substitute with codons encoding the desired mutation. DNA sequences encoding the precursor of the insulin analog can also be prepared by enzymatic synthesis

„ DK 159274 B"DK 159274 B

fra oligonucleotider svarende til hele insulinanalog-precursorgenet eller dele deraf.from oligonucleotides corresponding to the whole insulin analog precursor gene or portions thereof.

DNA-sekvenser, der indeholder et gen med den ønskede mutation af insulingenet kombineres derpå med fragmenter, 5 der koder for TPI-promoteren (TPIp) (T. Alber and G. Kawasaki. Nucleotide Sequence of the Triose Phosphate Isomerase Gene of Saccharomyces cerevisiae. J.Mol.Applied Genet. 1 (1982) 419-434), MFal leader-sekvensen (J. Kurjan and I.DNA sequences containing a gene with the desired mutation of the insulin gene are then combined with fragments encoding the TPI promoter (TPIp) (T. Alber and G. Kawasaki. Nucleotide Sequence of the Triose Phosphate Isomerase Gene of Saccharomyces cerevisiae). J. Mol Applied Genet. 1 (1982) 419-434), the MFal leader sequence (J. Kurjan and I.

Herskowitz, Structure of a Yeast Pheromone Gene (MFal): A 10 Putative α-Factor Precursor Contains four Tandem Copies of Mature α-Factor. Cell 30 (1982) 933-943) og transskriptions-terminatorsekvensen fra TPI fra S. cerevisiae (TPIrji). Disse fragmenter tilvejebringer sekvenser, der sikrer en høj transskriptionsgrad af genet for precursoren og tilvejebringer 15 også en præsekvens, der kan bevirke lokaliseringen af precursoren i sekretionsvejen og dens senere udskillelse i vækstmediet. Ekspressionsenhederne forsynes desuden med gær 2μ replikationsinitieringssitet og en selekterbar markør, LEU 2.Herskowitz, Structure of a Yeast Pheromone Gene (MFal): A 10 Putative α-Factor Precursor Contains Four Tandem Copies of Mature α-Factor. Cell 30 (1982) 933-943) and the transcription terminator sequence of TPI from S. cerevisiae (TPIrji). These fragments provide sequences which ensure a high degree of transcription of the precursor gene, and also provide a preconcept that can effect the localization of the precursor in the secretory pathway and its subsequent excretion into the growth medium. In addition, the expression units are provided with yeast 2μ replication initiation site and a selectable marker, LEU 2.

Ved in vivo modning af α-faktoren i gær, fjernes de 20 sidste (C-terminale) seks aminosyrer af MFal leaderpeptidet (Lys-Arg-Glu-Ala-Glu-Ala) fra α-faktor precursoren ved sekvensvis virkning af en endopeptidase, der genkender Lys-Arg-sekvensen, og en arainodipeptidase, der fjerner Glu-Ala-amino-syreresterne (Julius, D. et al. Cell 32 (1983), 839-852). For 25 at eliminere behovet for gæraminodipeptidasen blev den sekvens, der koder for den C-terminale Glu-Ala-Glu-Ala i MFal-leaderen, fjernet fra MFal-leaderen ved in vitro mutagenese.By in vivo maturation of the α-factor in yeast, the last 20 (C-terminal) six amino acids of the MFα1 leader peptide (Lys-Arg-Glu-Ala-Glu-Ala) are removed from the α-factor precursor by sequential action of an endopeptidase, recognizing the Lys-Arg sequence, and an arainodipeptidase that removes the Glu-Ala amino acid residues (Julius, D. et al. Cell 32 (1983), 839-852). To eliminate the need for the yeast aminodipeptidase, the sequence encoding the C-terminal Glu-Ala-Glu-Ala in the MFal leader was removed from the MFal leader by in vitro mutagenesis.

I den efterfølgende tekst betyder "MFal leaderen" hele leadersekvensen, mens MFal-leader (minus Glu-Ala-Glu-Ala) 30 betyder en leadersekvens, hvori den C-terminale Glu-Ala-Glu-Ala sekvens er blevet fjernet.In the following text, the "MFal leader" means the entire leader sequence, while the MFal leader (minus Glu-Ala-Glu-Ala) 30 means a leader sequence in which the C-terminal Glu-Ala-Glu-Ala sequence has been removed.

Eksempel 1Example 1

Fremstilling af B27Glu human insulin B27Glu human insulin blev fremstillet ved transpep-35 tidering af B27Glu, B(l-29)-Ala-Ala-Lys-A(l-21) human insulin 18Preparation of B27Glu human insulin B27Glu human insulin was prepared by transpidating B27Glu, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin 18

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med Thr-OBu*· og acidolyse af den opnåede theroninester med trifloureddikesyre. Fremstilling bestod af følgende trin: I. Konstruktion af et aen. der koder for B27Glu, (1-29)-Ala-Ala-Lvs-A(1-21) insulin 5 Et gærkodonoptimeret strukturelt gen for B(l-29)-with Thr-OBu * and acidolysis of the obtained theronine ester with trifluoroacetic acid. Preparation consisted of the following steps: I. Construction of a vein. encoding B27Glu, (1-29) -Ala-Ala-Lvs-A (1-21) insulin 5 A yeast codon-optimized structural gene for B (1-29) -

Ala-Ala-Lys-A(l-21) human insulin blev konstrueret som følger:Ala-Ala-Lys-A (1-21) human insulin was constructed as follows:

De følgende 10 oligonucleotider blev syntetiseret på en automatisk DNA-synthesizer under anvendelse af phos-10 phoramiditkemi på en glasporesupport (S.L. Beaucage og M.H. Caruthers (1981) Tetrahydron Letters 22., 1859-1869) :The following 10 oligonucleotides were synthesized on an automatic DNA synthesizer using phos-10 phoramidite chemistry on a glass pore support (S.L. Beaucage and M.H. Caruthers (1981) Tetrahydron Letters 22, 1859-1869):

I: AAAGATTCGTTAACCAACACTTGTGCGGTTCCCACIn: AAAGATTCGTTAACCAACACTTGTGCGGTTCCCAC

35- mer35- mer

II: AACCAAGTGGGAACCGCACAAGTGTTGGTTAACGAAII: AACCAAGTGGGAACCGCACAAGTGTTGGTTAACGAA

15 36-mer15 36-mer

III: TTGGTTGAAGCTTTGTACTTGGTTTGCGGTGAAAGAGGTTTCTIII: TTGGTTGAAGCTTTGTACTTGGTTTGCGGTGAAAGAGGTTTCT

43-mer43-mer

IV: GTAGAAGAAACCTCTTTCACCGCAAACCAAGTACAAAGCTTCIV: GTAGAAGAAACCTCTTTCACCGCAAACCAAGTACAAAGCTTC

42-mer42-mer

20 V: TCTACACTCCTAAGGCTGCTAAGGGTATTGTC20 V: TCTACACTCCTAAGGCTGCTAAGGGTATTGTC

32- mer32- mer

VI: ATTGTTCGACAATACCCTTAGCAGCCTTAGGAGTVI: ATTGTTCGACAATACCCTTAGCAGCCTTAGGAGT

34-mer34-mer

VII: GAACAATGCTGTACCTCCATCTGCTCCTTGTACCAATVII: GAACAATGCTGTACCTCCATCTGCTCCTTGTACCAAT

25 37-mer25 37-mer

VIII: TTTTCCAATTGGTACAAGGAGCAGATGGAGGTACAGCVIII: TTTTCCAATTGGTACAAGGAGCAGATGGAGGTACAGC

37-mer37-mer

IX: TGGAAAACTACTGCAACTAGACGCAGCCCGCAGGCTIX: TGGAAAACTACTGCAACTAGACGCAGCCCGCAGGCT

36- mer36- mer

30 X: CTAGAGCCTGCGGGCTGCGTCTAGTTGCAGTAG30 X: CTAGAGCCTGCGGGCTGCGTCTAGTTGCAGTAG

33- mer 5 duplekser A-E blev dannet fra de ovennævnte 10 oligonucleotider som vist på fig. 1.33 through 5 duplexes A-E were formed from the above 10 oligonucleotides as shown in FIG. First

20 pmol af hver af duplekserne A-E blev dannet fra 35 de tilsvarende par af 5'-phosphorylerede oligonucleotider I-X20 pmol of each of the duplexes A-E were formed from the corresponding pairs of 5'-phosphorylated oligonucleotides I-X

19 DK 159274 B19 DK 159274 B

ved opvarmning i 5 minutter ved 90“C efterfulgt af afkøling til stuetemperatur i løbet af 75 minutter. 33-meren (X) i dupleks E blev ikke 5'-phosphoryleret for at undgå dimerise-ring omkring de selvkomplementære Xbal-ender under ligerin-5 gen. De fem duplekser blev blandet og behandlet med T4 ligase. Det syntetiske gen blev isoleret som et 182/183 bp bånd efter elektroforese af ligeringsblandingen på en 2% agarose gel.by heating for 5 minutes at 90 ° C followed by cooling to room temperature over 75 minutes. The 33-mer (X) in duplex E was not 5'-phosphorylated to avoid dimerization around the self-complement Xba ends during ligation. The five duplexes were mixed and treated with T4 ligase. The synthetic gene was isolated as an 182/183 bp band after electrophoresis of the ligation mixture on a 2% agarose gel.

Det opnåede syntetiske gen er vist i fig. 1.The synthetic gene obtained is shown in FIG. First

10 Det syntetiske gen blev ligeret til et 4 kb Kpnl-The synthetic gene was ligated to a 4 kb KpnI gene.

EcoRl-fragment og et 8 kb Xabl-Kpnl-fragment fra pMT644 og en 0,3 kb EcoRl-Hgal-fragment fra pKFN9 til opnåelse af følgende struktur TPIp-MFal-leader-B(1-29)-Ala-Ala-Lys-A(1-21)-TPIT.EcoRl fragment and an 8 kb Xabl-Kpnl fragment from pMT644 and a 0.3 kb EcoRl-Hgal fragment from pKFN9 to obtain the following structure TPIp-MFal leader-B (1-29) -Ala-Ala-Lys -A (1-21) -TPIT.

Plasmid pMT644 indeholder DNA-sekvensen TPIp-MFal-15 leader-B(l-29)-A(l-21)-TPIT, og dets konstruktion er beskrevet i dansk patentansøgning nr. 1293/85. Konstruktionen af plasmid pKFN9 er beskrevet i det følgende.Plasmid pMT644 contains the DNA sequence TPIp-MFal-15 leader-B (l-29) -A (l-21) -TPIT, and its construction is described in Danish Patent Application No. 1293/85. The construction of plasmid pKFN9 is described below.

Ligeringsblandingen blev anvendt til at transformere kompetente E. coli stammer (r“, m+) (MT172). 30 ampicil-20 linresistente kolonier blev overført til plader indeholdende minimalmedium M9 (T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982, p. 68) resulterende i 8 Leu+ kolonier. Maxam-Gilbert sekventering af et 32P-Xbal-EcoRl fragment viste, at tre plasmider indeholdt et gen med den 25 ønskede sekvens. Et plasmid pKFN27 blev udvalgt til yderligere anvendelse.The ligation mixture was used to transform competent E. coli strains (r +, m +) (MT172). Thirty ampicillin-20 line resistant colonies were transferred to plates containing minimal medium M9 (T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982, p. 68) resulting in 8 Leu + colonies. Maxam-Gilbert sequencing of a 32P-XbaI-EcoR1 fragment showed that three plasmids contained a gene of the desired sequence. A plasmid pKFN27 was selected for further use.

Konstruktionen af pKFN27 er illustreret i fig. 2.The construction of pKFN27 is illustrated in FIG. 2nd

Konstruktion af plasmid pKFN9Construction of plasmid pKFN9

Formålet med konstruktionen af plasmid pKFN9 var at 30 opnå et plasmid, der indeholder et Hgal-site umiddelbart efter MFal-leadersekvensen. Plasmid pMT544 (hvis konstruktion er beskrevet i dansk patentansøgning nr. 278/85) blev skåret med Xbal, og ca. 250 baser blev fjernet fra 3'-enden ved ExoIII nucleasebehandling. En syntetisk 32-mer primer 35 GGATAAAAGAGAGGCGCGTCTGAAGCTCACTC indeholdende en Hgal-sekvens blev annealeret til det delvis enkeltstrengede DNA. Et dob-The purpose of constructing plasmid pKFN9 was to obtain a plasmid containing an Hgal site immediately after the MFα1 leader sequence. Plasmid pMT544 (the construction of which is described in Danish Patent Application No. 278/85) was cut with Xbal, and ca. 250 bases were removed from the 3 'end by ExoIII nuclease treatment. A 32-mer synthetic primer 35 GGATAAAAGAGAGGCGCGTCTGAAGCTCACTC containing an Hgal sequence was annealed to the partially single-stranded DNA. A double-

20 DK 159274 B20 DK 159274 B

beltstrenget cirkulært DNA blev fremstillet ved udfyldning med Klenow polymerase og ligering med T4 ligase. Efter transformering af E. coli (r“, m+) (MT172) blev der identificeret kolonier, der indeholdt muteret plasmid, ved kolonihybridise-5 ring med en 51-32P-mærket 32-mer primer. Tilstedeværelsen af et nyt Hgal site blev bekræftet ved restriktionsenzymskæring (EcoRl+Hgal, Hind3+Hgal). Efter retransformation blev der udvalgt en mutant pKFN9 til yderligere anvendelse. Konstruktionen af pKFN9 er illustreret i fig. 3.Belt stranded circular DNA was prepared by filling with Klenow polymerase and ligation with T4 ligase. After transformation of E. coli (r +, m +) (MT172), colonies containing mutated plasmid were identified by colony hybridization with a 51-32P-labeled 32-mer primer. The presence of a new Hgal site was confirmed by restriction enzyme cutting (EcoRl + Hgal, Hind3 + Hgal). After retransformation, a mutant pKFN9 was selected for further use. The construction of pKFN9 is illustrated in FIG. Third

10 Plasmid pKNF27 blev lineariseret i det unikke Xbal site lige efter det syntetiske insulinprecursorgen. For ikke at ødelægge Xabl sitet ved det nedenfor beskrevne udfyldningstrin, blev en 19-mer Hind3-Xbal dobbeltstrenget linker10 Plasmid pKNF27 was linearized in the unique Xbal site just after the synthetic insulin precursor. In order not to destroy the Xabl site at the fill step described below, a 19-mer Hind3-Xbal double-stranded linker

Xbal Hind3Xbal Hind3

15 CTAGAAGAGCCCAAGACTA15 CTAGAAGAGCCCAAGACTA

TTCTCGGGTTCTGATTCGATTCTCGGGTTCTGATTCGA

ligeret til begge ender af det lineariserede plasmid. Linke-ren blev 5'-phosphoryleret ved den enkeltstrengede Xbal-ende, men blev efterladt uphosphoryleret ved Hind3 enden, hvorved 20 polymeriseringen af linkeren under ligeringstrinnet og ringslutningen af DNA blev undgået, jfr. fig. 4.ligated to both ends of the linearized plasmid. The linker was 5 'phosphorylated at the single stranded XbaI end, but was left unphosphorylated at the Hind3 end, thereby avoiding the polymerization of the linker during the ligation step and DNA cycling, cf. FIG. 4th

5'mononucleotiderne blev fjernet fra 3'-enderne af det opnåede lineære, dobbeltstrengede DNA ved ExoIII-nucleasebehandling. ExoIII-nucleasebehandlingen blev udført 25 ved 23"C under betingelser, hvor ca. 250 nucleotider blev fjernet fra begge 3'-enderne af DNA-strengen (L. Guo og R. Wu (1983), Methods in Enzymology 100, 60-96).The 5'mononucleotides were removed from the 3 'ends of the linear double-stranded DNA obtained by ExoIII nuclease treatment. ExoIII nuclease treatment was performed at 23 ° C under conditions where approximately 250 nucleotides were removed from both 3 'ends of the DNA strand (L. Guo and R. Wu (1983), Methods in Enzymology 100, 60-96 ).

En 5'-phosphoryleret 25-mer mutageneseprimer d(GTTTCTTCTACGAACCTAAGGCTGC) blev annealeret til mutations-30 stedet. Efter udfyldning med Klenowpolymerase i nærværelse af T4 ligase blev dobbeltstrenget DNA fordøjet med Xbal. Hetero-dupleks cirkulært DNA med mutationen i den ene streng blev derpå dannet med T4 ligase.A 5 'phosphorylated 25-mer mutagenesis primer d (GTTTCTTCTACGAACCTAAGGCTGC) was annealed to the mutation site. After filling with Klenow polymerase in the presence of T4 ligase, double-stranded DNA was digested with XbaI. Hetero-duplex circular DNA with the mutation in one strand was then formed with T4 ligase.

Ligeringsblandingen blev transformeret i E. coli 35 (r“, m+) (MT172), idet der blev selekteret for ampicillin- resistens.The ligation mixture was transformed into E. coli 35 (r +, m +) (MT172), selecting for ampicillin resistance.

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Mutanter blev identificeret ved kolonihybridisering med den 51-32P-mærkede 25-mer mutageneseprimer. Efter re-transformering blev plasmid pKFN37 fra en af de fremkomne kolonier påvist at indeholde den ønskede mutation ved DNA-5 sekventering af et 0,5 kb Xbal-EcoRl-fregment (A. Maxam og W. Gilbert (1980) Methods in Enzymology 65, 499-560).Mutants were identified by colony hybridization with the 51-32P-labeled 25-mer mutagenesis primer. After re-transformation, plasmid pKFN37 from one of the resulting colonies was shown to contain the desired mutation by DNA sequencing of a 0.5 kb XbaI-EcoRl fragment (A. Maxam and W. Gilbert (1980) Methods in Enzymology 65 , 499-560).

II. TransformeringII. Transformation

S. cerevisiae stamme MT663 (E2-7B X E11-3CS. cerevisiae strain MT663 (E2-7B X E11-3C

a/a,Dtpi/otpi, pet 4-3/pep 4-3) blev dyrket på YPGaL (1% 10 Bacto gærekstrakt, 2% Bactopepton, 2% galactose, 1% lactat) til ODgoonm på 0,6.a / a, Dtpi / otpi, pet 4-3 / pep 4-3) were grown on YPGaL (1% 10 Bacto yeast extract, 2% Bactopeptone, 2% galactose, 1% lactate) to ODgoonm of 0.6.

100 ml kulturvæske blev høstet ved centrifugering, vasket med 10 ml vand, recentrifugeret og resuspenderet i 10 ml 1,2 M sorbitol, 25 mM Na2EDTA pH = 8,0 og 6,7 mg/ml di-15 thiotreitol. Suspensionen blev inkuberet ved 30°C i 15 minutter og centrifugeret, og cellerne blev resuspenderet i 10 ml 1,2 M sorbitol, 10 mM Na2EDTA, 0,1 M natriumcitrat pH = 5,8 og 2 mg Novozym, 234. Suspensionen blev inkuberet ved 30°C i 30 minutter, cellerne blev samlet ved centrifugering, vasket 20 i 10 ml 1,2 M sorbitol og i 10 ml CAS (1,2 M sorbitol, 10 mM CaCl2, 10 mM Tris (Tris = Tris(hydroxymethyl)-aminometan) pH = 7,5) og resuspenderet i 2 ml CAS. Til transformering blev 0,1 ml CAS-resuspenderede celler blandet med ca. 1 μG plasmid pKFN37 og henstillet ved stuetemperatur i 15 minutter. 1 ml 25 20% polyethylenglycol 4000, 10 mM CaCl2, 10 mM Tris pH = 7,5 blev tilsat, og blandingen blev henstillet i yderligere 30 minutter ved stuetemperatur. Blandingen blev centrifugeret, og pillerne blev resuspenderet i 0,1 ml SOS (1,2 M sorbitol, 33% v/v YPGaL, 6,7 mM CaCl2, 14 μg/ml leucin) og inkuberet 30 ved 30°C i 2 timer. Suspensionen blev derpå centrifugeret, og pillerne blev resuspenderet i 0,5 ml 1,2 M sorbitol. Der blev tilsat 6 ml topagar (SC mediet ifølge Sherman et al., (Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981) med leucin udeladt og indeholdende 1,2 M sorbitol plus 35 2,5% agar) ved 52“C, og suspensionen blev hældt ud på plader indeholdende det samme agarstørknede, sorbitolholdige medium.100 ml of culture fluid was harvested by centrifugation, washed with 10 ml of water, recentrifuged and resuspended in 10 ml of 1.2 M sorbitol, 25 mM Na 2 EDTA pH = 8.0 and 6.7 mg / ml di-thiotreitol. The suspension was incubated at 30 ° C for 15 minutes and centrifuged and the cells resuspended in 10 ml of 1.2 M sorbitol, 10 mM Na 2 EDTA, 0.1 M sodium citrate pH = 5.8 and 2 mg of Novozyme, 234. The suspension was incubated at 30 ° C for 30 minutes, cells were collected by centrifugation, washed 20 in 10 ml 1.2 M sorbitol and in 10 ml CAS (1.2 M sorbitol, 10 mM CaCl 2, 10 mM Tris (Tris = Tris (hydroxymethyl) -aminomethane) pH = 7.5) and resuspended in 2 ml of CAS. For transformation, 0.1 ml of CAS resuspended cells were mixed with ca. 1 μg of plasmid pKFN37 and left at room temperature for 15 minutes. 1 ml of 20% polyethylene glycol 4000, 10 mM CaCl 2, 10 mM Tris pH = 7.5 was added and the mixture was allowed to stand for an additional 30 minutes at room temperature. The mixture was centrifuged and the pills resuspended in 0.1 ml of SOS (1.2 M sorbitol, 33% v / v YPGaL, 6.7 mM CaCl 2, 14 μg / ml leucine) and incubated at 30 ° C for 2 hours. . The suspension was then centrifuged and the pills were resuspended in 0.5 ml of 1.2 M sorbitol. 6 ml of top agar (the SC medium of Sherman et al. (Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981) was added with leucine omitted and containing 1.2 M sorbitol plus 35 2.5% agar) at 52 ° C and the suspension was poured onto plates containing the same agar-dried sorbitol-containing medium.

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Transformantkolonier blev taget op efter 3 dage ved 30°C, reisoleret og anvendt til at starte flydende kulturer. En sådan transformant KFN40 (=MT663/pKFN37) blev valgt til yderligere karakterisering.Transformant colonies were taken up after 3 days at 30 ° C, reinsulated and used to start liquid cultures. Such a transformant KFN40 (= MT663 / pKFN37) was selected for further characterization.

5 III. Udtrykke Ise af B27Glu. Bf 1-29) -Ala-Ala-Lvs-AΓ1-2H insulinorecursor Gærstamme KFN40 blev dyrket på YPD medium (1% gærekstrakt, 2% pepton, (begge fra Difco Laboratories), og 2% glucose). En 10 ml kultur af stammen blev rystet ved 30°C til 10 et ODgoo 26. Efter centrifugering blev supernatanten analyseret ved reverse phase HPLC, og man fandt 13,5 mg/1 precursor.III. Express Ise by B27Glu. Bf 1-29) -Ala-Ala-Lvs-AΓ1-2H insulinorecursor Yeast strain KFN40 was grown on YPD medium (1% yeast extract, 2% peptone, (both from Difco Laboratories), and 2% glucose). A 10 ml culture of the strain was shaken at 30 ° C to an ODgoo 26. After centrifugation, the supernatant was analyzed by reverse phase HPLC and 13.5 mg / l precursor was found.

Analogen i supernatanten blev koncentreret på en kationbyttersøjle ved lavt pH efterfulgt af desorption med en 15 passende pufferopløsning. Krystallisering blev udført med en alkoholisk citratpuffer.The analog in the supernatant was concentrated on a low pH cation exchange column followed by desorption with a suitable buffer solution. Crystallization was performed with an alcoholic citrate buffer.

IV. Transpeptiderinq 0,2 mol (47,1 g) Thr-OBu*-, HOAC blev opløst i DMF resulterende i 100 ml opløsning, 50 ml 76,5% v/v DMF i vand 20 blev tilsat og 10 g rå B27Glu, B(l-29)-Ala-Ala-Lys-A(l-21) human insulin blev opløst i blandingen, som blev thermostate-ret ved 12“C. Derpå blev tilsat 1 g trypsin i 25 ml 0,05 M calciumacetat, og efter 24 timer ved 12“C blev blandingen sat til 2 liter acetone og de udfældede peptider blev isoleret 25 ved centrifugering og tørret in vacuo. B27Glu, B30Thr-0But human insulin blev renset på en HPLC søjle med silica-C18 som søjlemateriale.IV. Transpeptidation 0.2 mol (47.1 g) of Thr-OBu * -, HOAC was dissolved in DMF resulting in 100 ml of solution, 50 ml of 76.5% v / v DMF in water was added and 10 g of crude B27Glu, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin was dissolved in the mixture, which was thermostated at 12 ° C. Then 1 g of trypsin was added in 25 ml of 0.05 M calcium acetate and after 24 hours at 12 ° C the mixture was added to 2 liters of acetone and the precipitated peptides were isolated by centrifugation and dried in vacuo. B27Glu, B30Thr-0But human insulin was purified on an HPLC column with silica-C18 as a column material.

V. Konvertering til B27 human insulin B27Glu, B3OThr-OBu*· human insulin blev opløst i 100 30 ml trifloureddikesyre. Efter 2 timer ved stuetemperatur blev opløsningen lyophiliseret. Det lyophiliserede pulver blev opløst i 400 ml 47,5 mM natriumcitrat ved pH 7. Peptiderne blev udfældet ved pH 5,5 efter tilsætning af 2,4 ml 1 M ZnCl2/ isoleret ved centrifugering og tørret i vacuum. Pro-V. Conversion to B27 human insulin B27Glu, B3OThr-OBu * human insulin was dissolved in 100 30 ml of trifluoroacetic acid. After 2 hours at room temperature, the solution was lyophilized. The lyophilized powder was dissolved in 400 ml of 47.5 mM sodium citrate at pH 7. The peptides were precipitated at pH 5.5 after the addition of 2.4 ml of 1 M ZnCl 2 / isolated by centrifugation and dried in vacuo. pro-

23 DK 159274 B23 DK 159274 B

duktet blev renset ved anionbytterkromatografi og udsaltet ved gelfiltrering. Udbytte: 1,7 g B27Glu human insulin.the product was purified by anion exchange chromatography and salted by gel filtration. Yield: 1.7 g B27Glu human insulin.

Eksempel 2Example 2

Fremstilling af B9Asp human insulin 5 B9Asp human insulin blev fremstillet ved transpep- tidering af B9Asp, B(l-29)-Ala-Ala-Lys-A(l-21) human insulin med Thr-OBut og acidolyse af den opnåede threoninester med trifluoreddikesyre.Preparation of B9Asp human insulin B9Asp human insulin was prepared by transpeptidation of B9Asp, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin with Thr-OBut and acidolysis of the obtained threonine ester with trifluoroacetic acid.

I. Fremstilling af et gen, der koder for B9Asp, B(l-29)-Ala-10 Ala-Lvs-A(l-21) huamn insulinI. Preparation of a gene encoding B9Asp, B (l-29) -Ala-10 Ala-Lvs-A (l-21) human insulin

Dette gen blev fremstillet på samme måde som beskrevet for genet, der koder for B27Glu, B(l-29)-Ala-Ala-Lys-A(l-21) human insulin ved site specific mutagenese af pKFN27 styret af en 23-mer mutageneseprimer 15 d(CTTGTGCGGTGACCACTTGGTTG). Plasmid pKFN38 påvistes at indeholde den ønskede mutation.This gene was prepared in the same manner as described for the gene encoding B27Glu, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin by site specific mutagenesis of pKFN27 controlled by a 23 mer mutagenesis primer 15 d (CTTGTGCGGTGACCACTTGGTTG). Plasmid pKFN38 was found to contain the desired mutation.

II. TransformeringII. Transformation

Plasmid pKFN38 blev transformeret i S. cerevisiae stamme MT663 ved den samme fremgangsmåde som i eksempel 1, 20 II, og en transformant KFN41 blev isoleret.Plasmid pKFN38 was transformed into S. cerevisiae strain MT663 by the same procedure as in Examples 1, 20 II, and a transformant KFN41 was isolated.

III. Udtrykkelse af B9Asp, Bf1-29)-Ala-Ala-Lvs-A(1-21) human insulin Gærstamme KFN41 blev dyrket på YPD medium som beskrevet i eksempel 1, III. 2,5 mg/1 insulinanalogprecursor 25 blev fundet i supernatanten.III. Expression of B9Asp, Bf1-29) -Ala-Ala-Lvs-A (1-21) human insulin Yeast strain KFN41 was grown on YPD medium as described in Example 1, III. 2.5 mg / l insulin analog precursor 25 was found in the supernatant.

IV. Transpeptidering 7,4 g rå B9Asp, B(l-29)-Ala-Ala-Lys-A(l-21) human insulin blev transpeptideret som beskrevet i eksempel 1, IV resulterende i B9Asp, B3OThr-OBut human insulin.IV. Transpeptidation 7.4 g of crude B9Asp, B (l-29) -Ala-Ala-Lys-A (l-21) human insulin were transpeptided as described in Example 1, IV resulting in B9Asp, B3OThr-OBut human insulin.

24 DK 159274 B24 DK 159274 B

V. Konvertering B9Asp, B3 OThr-OBu"*- human insulin blev konverteret til B9Asp human insulin som beskrevet i eksempel 1, V. Udbytte: 0,83 g B9Asp human insulin.V. Conversion B9Asp, B3 OThr-OBu + - human insulin was converted to B9Asp human insulin as described in Example 1, V. Yield: 0.83 g B9Asp human insulin.

5 Eksempel 3Example 3

Fremstilling af B9Asp, B27Glu human insulin B9Asp, B27Glu human insulin blev fremstillet ved transpeptidering af B9Asp, B27Glu, B(l-29)-Ala-Ala-Lys-A(l- 21) human insulin med Thr-OBu*- og acidolyse af den opnåede 10 threoninester med trifloureddikesyre.Preparation of B9Asp, B27Glu human insulin B9Asp, B27Glu human insulin was prepared by transpeptidation of B9Asp, B27Glu, B (l-29) -Ala-Ala-Lys-A (l-21) human insulin with Thr-OBu * - and acidolysis of the obtained 10 threonine ester with trifluoroacetic acid.

I. Konstruktion af et gen, der koder for B9Asp. B27Glu. Bfl-29)-Ala-Ala-Lvs-Af1-21) human insulinI. Construction of a gene encoding B9Asp. B27Glu. Bfl-29) -Ala-Ala-Lvs-Af1-21) human insulin

Et 367 bp EcoRl-Hind3 fragment fra pKFN38 (se eksempel 2) og et 140 bp Hind3-Xbal-fragment fra pKFN37 (se 15 eksempel 1) blev ligeret til det store Xbal-EcoRl fragment fra plasmid pUC13 (dette plasmid er konstrueret som beskrevet for pUC8 og pUC9 af Vieira et al. (1982), Gene 19, 259-268). Ligeringsblandingen blev transformeret i E. coli (MT 172), idet der blev selekteret for ampicillinresistens. Plasmider 20 blev fremstillet fra et antal transformanter og analyseret ved fordøjelse med Pstl og med Hind3. 0,5 kb Xbal-EcoRl-frag-mentet fra et plasmid, der viste det korrekte restriktionsenzymmønster, blev ligeret til et 7,8 kb Xbal-Kpnl-fragment og et 4,3 kb Kpnl-EcoRl-fragment, begge fra pMT644 (beskrevet 25 i dansk patentansøgning nr. 1293/84). Ligeringsblandingen blev transformeret i E. coli (MT172), idet der blev selekteret for ampicillinresistens. Plasmid pKFN43 fra en af de fremkomne kolonier viste sig at indeholde genet for den ønskede insulinderivatprecursor ved DNA sekventering af et 0,5 30 kb Xbal-EcoRl-fragment. Konstruktionen af pKFN43 er illustreret i fig. 5.A 367 bp Eco RI-Hind3 fragment from pKFN38 (see Example 2) and a 140 bp Hind3-XbaI fragment from pKFN37 (see 15 Example 1) were ligated to the large XbaI-EcoR1 fragment from plasmid pUC13 (this plasmid is constructed as described for pUC8 and pUC9 by Vieira et al. (1982), Gene 19, 259-268). The ligation mixture was transformed into E. coli (MT 172), selecting for ampicillin resistance. Plasmids 20 were prepared from a number of transformants and analyzed by digestion with Pstl and with Hind3. The 0.5 kb XbaI-EcoRl fragment from a plasmid showing the correct restriction enzyme pattern was ligated to a 7.8 kb XbaI-Kpnl fragment and a 4.3 kb Kpnl-EcoR1 fragment, both from pMT644 ( described in Danish Patent Application No. 1293/84). The ligation mixture was transformed into E. coli (MT172), selecting for ampicillin resistance. Plasmid pKFN43 from one of the resulting colonies was found to contain the gene of the desired insulin derivative precursor by DNA sequencing of a 0.5 30 kb XbaI-EcoR1 fragment. The construction of pKFN43 is illustrated in FIG. 5th

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II. TransformeringII. Transformation

Plasmid pKFN40 blev transformeret i S. crevisiae stamme MT663 ifølge samme fremgangsmåde som i eksempel 1, II og en transformant KFN44 blev isoleret.Plasmid pKFN40 was transformed into S. crevisiae strain MT663 according to the same procedure as in Example 1, II and a transformant KFN44 was isolated.

5 III. Udtrvkkelse af B9Asp. B27Glu. B(l-29)-Ala-Ala-Lvs(A(l-21) human insulin Gærstamme KFN44 blev dyrket på YPD medium som beskrevet i eksempel 1, III. Der blev fundet 7,3 mg/1 insulin-analogprecursor i supernatanten.III. Expression of B9Asp. B27Glu. B (1-29) -Ala-Ala-Lvs (A (1-21) Human Insulin Yeast strain KFN44 was grown on YPD medium as described in Example 1, III. 7.3 mg / l insulin analog precursor was found in the supernatant. .

10 IV. Transpeptidering 12,7 g rå B9Asp, B27Glu, B(l-29)-Ala-Ala-Lys-A(l-21) human insulin blev transpeptideret som beskrevet i eksempel 1, IV og gav B9Asp, B27Glu, B30Thr-OBut human insulin.IV. Transpeptidation 12.7 g of crude B9Asp, B27Glu, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin were transpeptided as described in Example 1, IV to give B9Asp, B27Glu, B30Thr-OBut human insulin.

V. Konvertering 15 B9Asp, B27Glu, B30Thr-OBu*- human insulin blev kon verteret til B9Asp, B27Glu, B30Thr human insulin og renset som beskrevet i eksempel 1, V. Udbytte: 1,0 g B9Asp, B27Glu human insulin.V. Conversion 15 B9Asp, B27Glu, B30Thr-OBu * - human insulin was converted to B9Asp, B27Glu, B30Thr human insulin and purified as described in Example 1, V. Yield: 1.0 g B9Asp, B27Glu human insulin.

Eksempel 4 20 Fremstilling af A8His. B9Asp, B27Glu human insulin A8His, B9Asp, B27Glu human insulin blev fremstillet ved transpeptidering af A8His, B9Asp, B27Glu, B(l-29)-Ala-Ala-Lys-A(l-21) human insulin med Thr-OBu^ og acidolyse af den fremkomne threoninester med trifloureddikesyre som be-25 skrevet i eksempel l.Example 4 Preparation of A8His. B9Asp, B27Glu human insulin A8His, B9Asp, B27Glu human insulin was prepared by transpeptiding A8His, B9Asp, B27Glu, B (l-29) -Ala-Ala-Lys-A (l-21) human insulin with Thr-OBu acidolysis of the resulting threonine ester with trifluoroacetic acid as described in Example 1.

I. Fremstilling af et gen, der koder for A8His, B9Asp, B27Glu. B(1-2 9)-Ala—Ala-Lvs-A(1-21) human insulinI. Preparation of a gene encoding A8His, B9Asp, B27Glu. B (1-29) -Ala-Ala-Lvs-A (1-21) human insulin

Dette gen blev fremstillet ved oligonucleotidstyret mutagenese under anvendelse af en brudt duplex procedure (Y.This gene was prepared by oligonucleotide-guided mutagenesis using a broken duplex procedure (Y.

30 Morinaga, T. Franceschini, S. Inouye og M. Inouye (1984), Biotechnology 2, 636 - 639). Det pUC13 deriverede plasmid, 2630 Morinaga, T. Franceschini, S. Inouye, and M. Inouye (1984), Biotechnology 2, 636 - 639). The pUC13 derivative plasmid, 26

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der koder for MFal leadersekvensen og B9Asp, B27Glu human insulin precursor (fig. 5) blev skåret med Hpal og Xbal. Det store fragment blev blandet med det Ndel lineariserede plasmid. Efter varmedenaturering og afkøling indeholder blandin-5 gen brudte duplexer med et enkeltstrenget "vindue" i området svarende til insulinprecursorgenet (Hpal-Xbal). Den 37-mere mutagene umage primer d(GAACAATGCTGTCACTCCATCTGCTCCTTGTACCAAT) blev hybridiseret til den brudte duplex efterfulgt af udfyldning med Klenow 10 polymerase og ligering. Blandingen blev anvendt til transformering af E. coli (MT172) under selektering for ampicillin-resistens. Mutanter blev identificeret ved kolonihybridise-ring med en 18-mer 513^P-mærket probe d(AATGCTGTCACTCCATCT). Efter retransformering viste det sig, at et plasmid fra en af 15 de fremkomne kolonier indeholdt den ønskede mutation ved DNA sekventering af et 0,5 kb Xbal-EcoRl fragment. Dette plasmid blev anvendt til konstruktion af gærplasmidet pKFN102 som beskrevet i eksempel 3 for konstruktionen af pKFN43.encoding the MFal leader sequence and B9Asp, B27Glu human insulin precursor (Fig. 5) was cut with Hpal and Xbal. The large fragment was mixed with the Nde I linearized plasmid. After heat denaturation and cooling, the mixture contains broken duplexes with a single-stranded "window" in the region corresponding to the insulin precursor gene (Hpal-Xbal). The 37-mer mutagenic umage primer d (GAACAATGCTGTCACTCCATCTGCTCCTTGTACCAAT) was hybridized to the broken duplex followed by filling with Klenow 10 polymerase and ligation. The mixture was used to transform E. coli (MT172) while selecting for ampicillin resistance. Mutants were identified by colony hybridization with an 18-mer 513 µP-labeled probe d (AATGCTGTCACTCCATCT). After retransformation, it was found that a plasmid from one of the 15 colonies contained the desired mutation by DNA sequencing of a 0.5 kb XbaI-EcoR1 fragment. This plasmid was used to construct the yeast plasmid pKFN102 as described in Example 3 for the construction of pKFN43.

II. Transformering 20 Plasmid pKFN102 blev transformeret i S. cerevisiae stamme MT663 ved samme fremgangsmåde som i eksempel 1, II og en transformant KFN109 blev isoleret.II. Transformation Plasmid pKFN102 was transformed into S. cerevisiae strain MT663 by the same procedure as in Example 1, II and a transformant KFN109 was isolated.

III. Udtrvkkelse af A8His. B9Asp. B27Glu. Bfl-29)-Ala-Ala-Lvs-Af1-21^ human insulin 25 Gærstamme KFN109 blev dyrket på YPD medium som be skrevet i eksempel 1, III. Der fandtes 21,5 mg/1 insulinanalog precursor i supernatanten.III. Expression of A8His. B9Asp. B27Glu. Bfl-29) -Ala-Ala-Lvs-Af1-21 ^ human insulin 25 Yeast strain KFN109 was grown on YPD medium as described in Example 1, III. 21.5 mg / l insulin analogue precursor was found in the supernatant.

IV-V. Transpeotiderina og konvertering 22,0 g rå A8His, B9Asp, B27Glu, B(l-29)-Ala-Ala-30 Lys-A(1-21) human insulin blev transpeptideret, konverteret og renset som beskrevet i eksempel l, IV-V. Udbytte: 4,0 g A8HisB9AspB27Glu human insulin.IV-V. Transpeotiderin and Conversion 22.0 g of crude A8His, B9Asp, B27Glu, B (1-29) -Ala-Ala-30 Lys-A (1-21) human insulin were transpeptidized, converted and purified as described in Example 1, IV V. Yield: 4.0 g of A8HisB9AspB27Glu human insulin.

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Eksempel 5Example 5

Fremstilling af B12Ile human insulin B12Ile human insulin blev fremstillet ved transpep-tidering af 812116, B(l-29)-Ala-Ala-Lys-A(l-21) human insulin 5 med Thr-OBu*· og acidolyse af den fremkomne threoninester med trifloureddikesyre som beskrevet i eksempel 1.Preparation of B12Ile human insulin B12Ile human insulin was prepared by transpeptidation of 812116, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin 5 with Thr-OBu * and acidolysis of the resultant threonine ester with trifluoroacetic acid as described in Example 1.

I. Konstruktion af et gen, der koder for B12Ile. B(l-29^-Ala-Ala-Lvs-A(l-21) human insulinI. Construction of a gene coding for B12Ile. B (1-29) -Ala-Ala-Lvs-A (1-21) human insulin

Et 0,5 kb EcoRl-Xbal fragment af pMT598 (konstruk-10 tionen af et plasmid pMT598 er beskrevet i EP patentansøgning nr. 0163529A), der koder for MFal-leader-(minus Glu-Ala-Glu-Ala)-B(l-29)-Ala-Ala-Lys-A(l-21) blev indsat i M13 mplO RF fag skåret med Xbal-EcoRl, og det tilsvarende enkeltstrengede DNA blev renset fra den M13 mp 10 rekombinante fag. Det en-15 keltstrengede templat DNA blev hybridiseret til en 27-mer mutageneseprimer NOR-92 d(GTAGAGAGCTTCGATCAGGTGTGAGCC) og en M13 universel sekventeringsprimer d(TCCCAGTCACGACGT). Primerne blev forlænget med dNTP og Klenow polymerase og ligeret med T4 DNA ligase. Mutageneseprimeren KFN92 blev valgt med 20 henblik på ødelæggelse af et BstNl site (unikt i Xbal-EcoRl fragmentet). For at selektere mod umuteret EcoRl-Xbal fragment blev blandingen derfor skåret med BstNl og derpå med EcoRl og Xbal og ligret til EcoRl- og Xbal-skåret pUC13 vektor. Fra en af de fremkomne transformanter blev valgt et 25 plasmid pMT760, som manglede BstNl sitet i den sekvens, der koder for insulin. Den ønskede muterede sekvens blev verificeret ved Maxam-Gilbert DNA sekventering. Plasmid pMT760 indeholder en 0,5 kb EcoRl-Xbal sekvens svarende til det samme fragment fra pMT598 (se ovenfor) bortset fra en mutation ved 30 B12 (Val -*· Ile). Denne muterede sekvens blev derpå flyttet til et gærudtrykkelsesplasmid ved ligering af 0,5 kb EcoRl-Xabl fragmentet fra pMT760 til et 7,8 kb Xbal-Kpnl og et 4,3 Kpnl-EcoRl fragment fra pMT644 resulterende i pMTA.A 0.5 kb EcoR1-XbaI fragment of pMT598 (the construction of a plasmid pMT598 is described in EP Patent Application No. 0163529A) encoding MFal leader (minus Glu-Ala-Glu-Ala) -B ( 1-29) -Ala-Ala-Lys-A (1-21) was inserted into M13 mp10 RF phage cut with XbaI-EcoRl and the corresponding single-stranded DNA was purified from the M13 mp 10 recombinant phage. The single-stranded template DNA was hybridized to a 27-mer mutagenesis primer NOR-92 d (GTAGAGAGCTTCGATCAGGTGTGAGCC) and an M13 universal sequencing primer d (TCCCAGTCACGACGT). The primers were extended with dNTP and Klenow polymerase and ligated with T4 DNA ligase. The mutagenesis primer KFN92 was selected for destruction of a BstN1 site (unique in the XbaI-EcoR1 fragment). Therefore, to select against unmutated EcoRl-Xbal fragment, the mixture was cut with BstN1 and then with EcoRl and Xbal and ligated to EcoRl and Xbal cut pUC13 vector. From one of the resulting transformants, a plasmid pMT760 was selected which lacked the BstN1 site in the sequence encoding insulin. The desired mutated sequence was verified by Maxam-Gilbert DNA sequencing. Plasmid pMT760 contains a 0.5 kb EcoR1-XbaI sequence corresponding to the same fragment from pMT598 (see above) except for a mutation at 30 B12 (Val - * · Ile). This mutated sequence was then moved to a yeast expression plasmid by ligating the 0.5 kb EcoR1-Xabl fragment from pMT760 to a 7.8 kb XbaI-Kpnl and a 4.3 Kpnl-EcoR1 fragment from pMT644 resulting in pMTA.

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II-V. Transformering, udtrvkkelse. transpeptidering. konverteringII-V. Transformation, expression. transpeptidation. conversion

Plasmid pMTA blev transformeret i gærstamme MT663 som beskrevet i eksempel 1, II og den transformerede stamme 5 MTA blev dyrket som beskrevet i eksempel l, III. Der blev fundet 10,4 mg/1 insulinanalogprecursor i supernatanten. 10 g af den rå analogprecursor blev transpeptideret, konverteret og renset som beskrevet i eksempel 1, IV-V. Udbytte: 1,3 g B12Ile human insulin.Plasmid pMTA was transformed into yeast strain MT663 as described in Examples 1, II and the transformed strain 5 MTA was grown as described in Examples 1, III. 10.4 mg / l insulin analogue precursor was found in the supernatant. 10 g of the crude analog precursor was transpeptidized, converted and purified as described in Example 1, IV-V. Yield: 1.3 g of B12Ile human insulin.

10 Eksempel 6Example 6

Fremstilling af B12Tvr human insulin B12Tyr, human insulin kan fremstilles ved transpep-tidering af B12Tyr, B(1-29)-Ala-Ala-Lys-A(1-21) human insulin med Thr-OBu*· og acidolyse af den fremkomne threoninester med 15 trifloureddikesyre som beskrevet i eksempel 1.Preparation of B12Tru human insulin B12Tyr, human insulin can be prepared by transpeptidation of B12Tyr, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin with Thr-OBu * and acidolysis of the resultant threonine ester with trifluoroacetic acid as described in Example 1.

I. Konstruktion af et gen, der koder for B12Tvr. Bfl-29)-Ala-Ala-Lvs-Af1-211 human insulinI. Construction of a gene encoding B12Tvr. Bfl-29) -Ala-Ala-Lvs-Af1-211 human insulin

Genet blev fremstillet ved en fremgangsmåde analog med fremgangsmåden til fremstilling af genet, der koder for 20 B12Ile, B(l-29)-Ala-Ala-Lys-A(l-21) human insulin med undtagelse af, at primeren KFN93 d(GTAGAGAGCTTCGTACAGGTGTGAGCC) blev anvendt i stedet for KFN92.The gene was prepared by a method analogous to the method of producing the gene encoding 20 B12Ile, B (l-29) -Ala-Ala-Lys-A (l-21) human insulin except that the primer KFN93 d ( GTAGAGAGCTTCGTACAGGTGTGAGCC) was used in place of KFN92.

II-IV. Transformering, udtrvkkelse. transpeptidering. konvertering 25 Trin II-III blev udført som beskrevet i eksempel 1.II-IV. Transformation, expression. transpeptidation. conversion 25 Stages II-III were performed as described in Example 1.

Der blev fundet 1,7 mg/1 insulinanalogprecursor i supernatanten. Den rå analogprecursor kan transpeptideres, konverteres og renses som beskrevet i eksempel 1, VI-V resulterende i B12Tyr human insulin.1.7 mg / l insulin analogue precursor was found in the supernatant. The crude analog precursor can be transpeptidized, converted and purified as described in Example 1, VI-V resulting in B12Tyr human insulin.

30 Eksempel 7Example 7

Fremstilling af BlOAsp human insulinPreparation of BlOAsp human insulin

BlOAsp human insulin blev fremstillet ved transpep-tidering af BlOAsp, B(l-29)-Ala-Ala-Lys-A(l-21) human insulinBlOAsp human insulin was prepared by transpeptidation of BlOAsp, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin

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med Thr OBu1- og acidolyse af den fremkomne threoninester med trifloureddikesyre som beskrevet i eksempel 1.with Thr OBu1 and acidolysis of the resulting threonine ester with trifluoroacetic acid as described in Example 1.

I. Konstruktion af et gen, der koder for BIOAsp. B(l-29)-Ala-Ala-Lvs-A(l-21) human insulin 5 Genet blev konstrueret ifølge en fremgangsmåde, der er analog med fremgangsmåden til fremstilling af det gen, der koder for B12Ile, B(l-29)-Ala-Ala-Lys-A(l-21) human insulin med undtagelse af, at primeren KFN94 (d(AGCTTCCACCAGATCTGAGCCGCACAG) blev anvendt i stedet for 10 KFN92.I. Construction of a gene encoding BIOAsp. B (l-29) -Ala-Ala-Lvs-A (l-21) human insulin 5 The gene was constructed according to a method analogous to the method of producing the gene encoding B12Ile, B (l-29) ) -Ala-Ala-Lys-A (1-21) human insulin except that the primer KFN94 (d (AGCTTCCACCAGATCTGAGCCGCACAG) was used instead of 10 KFN92.

II-IV. Transformering, udtrykkelse. transpeptidering. konverteringII-IV. Transformation, expression. transpeptidation. conversion

Trin II-III blev udført som beskrevet i eksempel 1.Stages II-III were performed as described in Example 1.

Der blev fundet 36 mg/1 insulinanalogprecursor i supernatan-15 ten. Den rå analogprecursor blev transpeptideret, konverteret og renset som beskrevet i eksempel 1, IV-V. Udbytte 7,6 g BIOAsp human insulin.36 mg / l insulin analogue precursor was found in the supernatant. The crude analog precursor was transpeptidized, converted and purified as described in Example 1, IV-V. Yield 7.6 g BIOAsp human insulin.

Eksempel 8Example 8

Fremstilling af B28Asp human insulin 20 B28Asp human insulin blev fremstillet ved transpep- tidering af B28Asp, B(l-29)-Ala-Ala-Lys-A(l-21) human insulin med Thr-OMe og hydrolyse af den fremkomne threoninester ved pH på ca. 8 til 12.Preparation of B28Asp human insulin 20 B28Asp human insulin was prepared by transpeptiding B28Asp, B (1-29) -Ala-Ala-Lys-A (1-21) human insulin with Thr-OMe and hydrolysis of the resulting threonine ester at pH of approx. 8 to 12.

Konstruktion af et gen, der koder for B28Asp, B(l-29)-Ala-25 Ala-Lvs-Af1-21) human insulinConstruction of a gene encoding B28Asp, B (1-29) -Ala-25 Ala-Lvs-Af1-21) human insulin

Et 0,5 kb EcoRl-Xbal fragment af pMT 462 (konstruktionen af plasmid pMT 462 er beskrevet i dansk patentansøgning nr. 1257/86), der koder for Mfal leaderen (minus Glu-Ala-Glu-Ala)-B-C-A, dvs. det humane proinsulingen, anbragt 30 efter den modificreede MFal leader, blev indsat i M13 mplO RF fag skåret med Xbal-EcoRl, og det tilsvarende enkeltstrengede DNA blev renset fra M13 mplO rekombinantfagen. Det enkeltstrengede templat DNA blev hybridiseret til en mutagen 41-mer primer NOR205 d(TTCCACAATGCCCTTAGCGGCCTTGTCTGTGTAGAAGAAGC) og 30A 0.5 kb EcoR1-XbaI fragment of pMT 462 (the construction of plasmid pMT 462 is described in Danish Patent Application No. 1257/86) encoding the Mfal ladder (minus Glu-Ala-Glu-Ala) -B-C-A, i.e. the human proinsulin, placed 30 after the modified MFal leader, was inserted into the M13 mplO RF phage cut with Xbal-EcoR1, and the corresponding single-stranded DNA was purified from the M13 mplO recombinant phage. The single-stranded template DNA was hybridized to a mutagenic 41-mer primer NOR205 d (TTCCACAATGCCCTTAGCGGCCTTGTCTGTGTAGAAGAAGC) and 30

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en M13 universel sekventeringsprimer d(TCCCAGTCACGACGT). Primerne blev forlænget med dNTP og Klenow polymserase og ligeret med T4 DNA ligase.and M13 universal sequencing primer d (TCCCAGTCACGACGT). The primers were extended with dNTP and Klenow polymerase and ligated with T4 DNA ligase.

Efter phenolekstraktion, ethanoludfældning og re-5 suspension blev DNA skåret med restriktionsenzymerne Apal, Xbal og EcoRl. Efter yderligere en phenolekstraktion, ethanoludfældning og resuspension blev DNA ligeret til EcoRl-Xbal skåret pUC13. Ligationsblandingen blev transformeret i en E. coli (r”m+) stamme, og plasmider blev fremstillet fra 10 et antal transformanter. Plasmidpræparater blev skåret med EcoRl og Xbal, og de præparater, der viste bånd ved både 0,5 og 0,6 kb, blev retransformeret i E. coli. Fra retransforme-ringen blev valgt en transformant kun indeholdende pUC13 med et 0,5 indsat fragment.After phenol extraction, ethanol precipitation and re-suspension, the DNA was cut with the restriction enzymes Apal, Xbal and EcoRl. After a further phenol extraction, ethanol precipitation and resuspension, the DNA was ligated to EcoRl-Xbal cut pUC13. The ligation mixture was transformed into an E. coli (r + m +) strain, and plasmids were prepared from a number of transformants. Plasmid preparations were cut with Eco RI and XbaI, and the preparations showing bands at both 0.5 and 0.6 kb were retransformed into E. coli. From the retransformation, a transformant containing only pUC13 with a 0.5 inserted fragment was selected.

15 Fra en af transformanterne blev der valgt et plas mid pMT881 med den ønskede mutation ved Β2δ (Pro - Asp). Den muterede sekvens blev verificeret ved Maxam-Gilbert DNA-se-kventering. Den muterede sekvens blev derpå flyttet til et gærudtrykkelsesplasmid ved ligering af et 0,5 kb EcoRl-Xbal 20 fragment fra ρΜΤδδΙ til en 7,δ kb Xbal-Kpnl og et 4,3 Kpnl-EcoRl fragment fra pMT644 resulterende i pMTAl.From one of the transformants, a plasmid mid pMT881 with the desired mutation at ved2δ (Pro - Asp) was selected. The mutated sequence was verified by Maxam-Gilbert DNA Sequencing. The mutated sequence was then moved to a yeast expression plasmid by ligating a 0.5 kb EcoRl-Xbal 20 fragment from ρΜΤδδΙ to a 7, δ kb Xbal-KpnI and a 4.3 KpnI-EcoRl fragment from pMT644 resulting in pMTA1.

II. TransformeringII. Transformation

Plasmid pMTAl blev transformeret til S. cerevisiae stamme MT663 ved den samme procedure som i eksempel 1, II og 25 en transformant MTA1 blev isoleret.Plasmid pMTA1 was transformed into S. cerevisiae strain MT663 by the same procedure as in Examples 1, II and 25, a transformant MTA1 was isolated.

III. Udtrykkelse af B23Asp, B(l-29)-Ala-Ala-Lvs-Af1-21) human insulin Gærstamme MTA1 blev dyrket på YPD medium som beskrevet i eksempel 1, III. Der blev fundet 7,2 mg/1 insulin-30 analogprecursor i supernatanten.III. Expression of B23Asp, B (1-29) -Ala-Ala-Lvs-Af1-21) Human Insulin Yeast strain MTA1 was grown on YPD medium as described in Example 1, III. 7.2 mg / l insulin-30 analog precursor was found in the supernatant.

IV. TransoeotiderincrIV. Transoeotiderincr

Den rå B26Asp, B(l-29)-Ala-Ala-Lys-A(l-21) blev transpeptideret som beskrevet i eksempel I, IV ved at substiThe crude B26Asp, B (1-29) -Ala-Ala-Lys-A (1-21) was transpeptidated as described in Examples I, IV by substituting

31 DK 159274 B31 DK 159274 B

tuere Thr-OB^ med Thr-OMe resulterende i B28 Asp, B30Thr-OMe human insulin.culture with Thr-OMe resulting in B28 Asp, B30Thr-OMe human insulin.

V. Konvertering B28Asp, B30Thr-OMe human insulin blev dispergeret i 5 vand til 1% (w/v) og blev opløst ved tilsætning af IN natriumhydroxid til en pH-værdi på 10,0. pH-værdien blev holdt konstant ved 10,0 i 24 timer ved 25”C. Det dannede B28Asp human insulin blev udfældet ved tilsætning af natriumchlorid til ca. 8% (w/v), natriumacetattrihydrat til ca. 1,4% (w/v) 10 og zinkacetatdihydrat til ca. 0,01% (w/v) efterfulgt af tilsætning af IN saltsyre til pH 5,5. Bundfaldet blev isoleret ved centrifugering og renset ved anionbytterkromatografi og udsaltet ved gelfiltrering. Udbytte: 0,2 g B28Asp human insulin.V. Conversion B28Asp, B30Thr-OMe human insulin was dispersed in 5 water to 1% (w / v) and dissolved by the addition of 1N sodium hydroxide to a pH of 10.0. The pH was kept constant at 10.0 for 24 hours at 25 ° C. The resulting B28Asp human insulin was precipitated by the addition of sodium chloride to ca. 8% (w / v), sodium acetate trihydrate to approx. 1.4% (w / v) 10 and zinc acetate dihydrate to approx. 0.01% (w / v) followed by addition of 1N hydrochloric acid to pH 5.5. The precipitate was isolated by centrifugation and purified by anion exchange chromatography and salted by gel filtration. Yield: 0.2 g of B28Asp human insulin.

15 Eksempel 9Example 9

Fremstilling af A21Asp, B9Asp. B27Glu human insulin A2lAsp, B9Asp, B27Glu human insulin blev fremstillet fra B9Asp, B27Glu human insulin ved selektiv deamidering (hydrolyse af en 5% opløsning i 14 dage ved 37”C, pH 2,5).Preparation of A21Asp, B9Asp. B27Glu human insulin A2lAsp, B9Asp, B27Glu human insulin was prepared from B9Asp, B27Glu human insulin by selective deamidation (hydrolysis of a 5% solution for 14 days at 37 ° C, pH 2.5).

20 Det deamiderede produkt blev isoleret ved anionbytterkromatografi.The deamidated product was isolated by anion exchange chromatography.

Eksempel 10Example 10

Fremstilling af B27Glu. A21Asp human insulin B27Glu, A2lAsp human insulin blev fremstillet ved 25 transpeptidering af B27G1U, A21Asp, B(l-29)-Ala-Ala-Lys-A(l-21) med ThrOBu-*- og acidolyse af den fremkomne threoninester med trifloureddikesyre som beskrevet i eksempel 1.Preparation of B27Glu. A21Asp human insulin B27Glu, A2AAsp human insulin was prepared by transpeptidation of B27G1U, A21Asp, B (l-29) -Ala-Ala-Lys-A (l-21) with ThrOBu - * - and acidolysis of the resulting threonine ester with trifluoroacetic acid as described in Example 1.

B27GluA21AspB(1-21)-Ala-Ala-Lys-A(1-21) blev fremstillet fra B27G1U,B(1-29)-Ala-Ala-Lys-A(1-21) (se eksempel 30 1) ved deamidering som beskrevet i eksempel 9.B27GluA21AspB (1-21) -Ala-Ala-Lys-A (1-21) was prepared from B27G1U, B (1-29) -Ala-Ala-Lys-A (1-21) (see Example 30 1) at deamidation as described in Example 9.

Karakterisering af human insulinanaloger ifølge den foreliggende opfindelseCharacterization of human insulin analogues of the present invention

Bestemmelse af molekylvægte (Gutfreund H. Biochemical Journal 42 (544), 1948).Determination of molecular weights (Gutfreund H. Biochemical Journal 42 (544), 1948).

„ DK 159274 B"DK 159274 B

3232

Metode: Knauer Membran OsmometerMethod: Knauer Membrane Osmometer

Type: 1.00Type: 1.00

Membran. Schleicher and Schull Type: R52 5 Solvens: 0,05 M NaCl pH 7,5Membrane. Schleicher and Schull Type: R52 Solvent: 0.05 M NaCl pH 7.5

Temp.: 21°CTemp: 21 ° C

Resultater: Alle typer insulin blev målt ved en koncentra tion på 4 mg/ml.Results: All types of insulin were measured at a concentration of 4 mg / ml.

Tabel 1 10 Insulintvpe_Molekylvægt k DaltonTable 1 10 Insulin Intake_Molecular Weight k Dalton

Human 2 Zn insulin 36 ± 2Human 2 Zn insulin 36 ± 2

Human Zn fri insulin 29 ± 1Human Zn free insulin 29 ± 1

Zn fri B27Glu human insulin 22 ± 1 15 - - B12Ile human insulin 17 ± 1 - - B27Glu, A21Asp human insulin 8 ± 1 - - B9Asp, B27Glu human insulin 6 ± 1 - - B9Asp human insulin 6 ± 1 - - B9Asp, B27Glu, A21Asp human insulin 6 ± 1 20 - - B9Asp, B27Glu, A8His human insulin 9 ± 3 - - BlOAsp human insulin 12+1 - - B28Asp human insulin 9 ± 2Zn free B27Glu human insulin 22 ± 1 - - B12Ile human insulin 17 ± 1 - - B27Glu, A21Asp human insulin 8 ± 1 - - B9Asp, B27Glu human insulin 6 ± 1 - - B9Asp human insulin 6 ± 1 - - B9Asp, B27Glu , A21Asp human insulin 6 ± 1 20 - - B9Asp, B27Glu, A8His human insulin 9 ± 3 - - BlOAsp human insulin 12 + 1 - - B28Asp human insulin 9 ± 2

Det fremgår af ovenstående tabel 1, at human insu-linanalogerne har en væsentlig reduceret molekylvægt sammen-25 lignet med human insulin, hvilket betyder, at selvassocieringen til dimere, tetramere og hexamere er mindre udtalt eller endog mangler i adskillige tilfælde.It can be seen from the above Table 1 that the human insulin analogs have a substantially reduced molecular weight compared to human insulin, which means that the self-association to dimers, tetramers and hexamers is less pronounced or even lacking in several cases.

Tabel 2Table 2

33 DK 159274B33 DK 159274B

Halveringstid og biologisk styrkeHalf-life and biological potency

Human insulinanalog T 1/2* Biologisk styrke** (% af % af human insulin 5 human (95% conf.interval) _insulin_ B27Glu human insulin 78 101 (83-123) B9Asp, B27Glu human insulin 54 110 (90-139) B12Ile human insulin 78 91 (80-103) 10 B27Glu, A2lAsp human insulin 56 64 (58-71) B9Asp human insulin 52 80 (72-90) A21Asp, B9Asp, B27Glu human insulin 56 75 (66-85) A8His, B9Asp, B27Glu human insulin 68 116 (101-135)Human insulin analogue T 1/2 * Biological strength ** (% of% of human insulin 5 human (95% conf. Interval) _insulin_ B27Glu human insulin 78 101 (83-123) B9Asp, B27Glu human insulin 54 110 (90-139) B12Ile human insulin 78 91 (80-103) 10 B27Glu, A2lAsp human insulin 56 64 (58-71) B9Asp human insulin 52 80 (72-90) A21Asp, B9Asp, B27Glu human insulin 56 75 (66-85) A8His, B9Asp , B27Glu human insulin 68 116 (101-135)

BlOAsp human insulin 64 104 (92-18) 15 B28Asp human insulin 53 104 (95-114) * Tid til 50% forsvinden fra injektionsstedet (subkutant) hos grise. Fremgangsmåde ifølge Binder 1969 (Acta Pharmacol.Toxicol. (suppl. 2), 27:1-87).BlOAsp human insulin 64 104 (92-18) 15 B28Asp human insulin 53 104 (95-114) * Time to 50% disappearance of injection site (subcutaneously) in pigs. Method of Binder 1969 (Acta Pharmacol.Toxicol. (Suppl. 2), 27: 1-87).

** Museblodsglucoseassay ifølge europæisk farmacopi.** European blood glucose assay according to European Pharmacopoeia.

20 Det fremgår af ovenstående tabel 2, at tiden til 50% forsvinden af insulinanalogerne fra injektionsstedet er væsentligt reduceret i sammenligning med human insulin.It can be seen from Table 2 above that the time to 50% disappearance of the insulin analogues from the injection site is significantly reduced compared to human insulin.

Den biologiske styrke af insulinanalogerne er sammenlignelig med human insulin eller kun let reduceret.The biological potency of the insulin analogues is comparable to human insulin or only slightly reduced.