CA1282022C - Method for purifying an interferon - Google Patents

Abstract

ABSTRACT

A method of purifying a polypeptide having a physiological activity such as one having interferon activities from a culture mixture of a microorganism obtained by a recombinant DNA
technique and capable of producing the polypeptide is disclosed. The method comprised subjecting the cultured cells to extraction and purification in the presence of a salt of zinc or copper and poly-ethyleneimine thereby inhibiting decomposition and denaturation of the polypeptide. The extracted polypeptide can be further purified by column chromato-graphies using a column containing an anion exchange resin, column containing a cation exchange resin and column containing a gel filtration resin.

Description

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DESCRIPT~ON

MET80D FOR PURIFYIN~ AN INTERFERON
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Technical Field -This inve~tion relate to a method for purifying a physiologically aotiv~ polypeptide produced by a re~ombinant DNA technology without denatu~ation or decompo3ition by protea.~e~, more particularly, said polypeptide being produced by a microorganism transformed by a plasmid vector bearing a gene coding for a pQlypeptide having physiologicaL
activitie~. Thi~ Invention in particular provides an effective mathod for purifying a desired polypeptide from a culture mixture of a micxoorganism capable of producing a polypeptide having intexferon activitie~, -especially human immune (or gamma) interferon activities.

Interferon proteins have been classi~iecl into thre~ type_, alpha, beta and gamma (abbreviated to IFN a, IFN 3 and IFN-y re~pectively) based on antigeniG and structural differences. Gamma interfero~

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ha~ a number o~ characteri~tics that differe~iate it from alpha and beta interferon~. Am~ng these differ~nces are antigenic distinctivenes~ and greater activity with regard ~o Lmmunoregulation and anti-tumor e~fects. Human gamma inte~f~ron (r~ferre~ to herein as ~h-IFN-yn~ may b produced by T lymphocytes '~

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stimulated by mutagen~ or by antigens to which they are en~itized. I~ may also be obtained through cloning and expres ion tec~niques now well.kno~l to the art.
Recently, it haq become posqible by the progress in genetic engineering to produce many phy iologically active polypeptides from micro-organism~ or animal cells, although these substances have been produced by separation and purification from an organi~m. However, it cannot yet be said that a method has been established fQr extracti:ng and purifying the in~eQded ~ubstance with a purity suf~icient to be used ~or drugs and withou~ causing ~`
denaturation or decomposition.
~ Gamma interferon-containing cells, however obtained, are ~ollected and are disrupted by various mea~s such as osmotic shockl ultrasonic vibration, grinding or high shear disruption and the disrupted cell-gamma intexferon mixture is then processed to isolate the gamma interferon. The insoluble debriq i separated by centrifugation and the gamma interferon-containing supernatant is collected for purification.
Although disclo~ure has been made of certain technoLogy for such production methods, e.g. a method for 2S extracti~g and purifying the polypeptide produced by a recombinant microorgani m by using ~uanidine hydro-chloride a4~d urea (JapaneYe Patent Public DiRclosure No. 161321/1984 and U.S4 Patent No. 4,476,0~gl and -o~

a puiification method using a monoclonal. antibody (~a~anese Patent Public Disclosure No. 186995/1984), the intended substance is not always adequately purified without being subjected to denaturation and/
. or decomposition and without its activity being lost.
European Patent Application 0,087,686 dis-closes a three-step process for purifying human immune interferons from the cell-free supernatant or extract from the crude interferon source. In the first step (for naturally occurring interferon), an affinity column, such as Concanavalin-A Sepharose is used, followed by chromatography on a carboxymethyl silica column using an increasing salt gradient and finally, on a silica gel permeation column. If sufficient purity is not obtained, concen-tration and chromatography on either the TSK or CM column is used.
European Patent Application 0,063,4~2 discloses a purification process employing chromato-graphic methods using 1) Controlled Pore Glass beads;2) Concanavalin-A Sepharose ; 3) Heparin-Sepharose or Procion Red-agarose; and 4~ gel filtration.
European Patent Applications 0,107,498 and 0,077,670 disclose a purification scheme employing 1) polyethyleneimine precipitation; 2) pH precipitation of bacterial proteins; 3) concentration and dialysis;
4) chromatography on a) carboxymethyl cellulose;
~) a calcium phosphate gel; c) a carboxymethyl ~' *Trade-mark ~8~022 cellulo~e; and d) gel filtratio~ re~ins.
Tha~e purification process~s require A
multitude of steps, cause d~gradatio~ of the inter-feron by degradation or aggregation of the interferon molecule, or otherwise re~ult in a g~mma interferon product obtained in low yield or with low activity.
It goe~ without saying that a method for extracting and purifying the intended polypeptide from the culture mixture o~ the intenaed substance-producing microorganism without the activity o~ theintended sub~tanse b~ing lost and without being accompanied by denaturation is important for such uses as pharmaceuticals and that the establishment of such technology is useful from the viewpoint of ; 15 industry.
Such a purification method has been particu-larly desired for inter~eron, the employment of which in pharmaceuticals is now proceeding~ Interferons have anti-virus activity, but IFN y is expected to be useful as an anti-tumor agent and immune regulator because of its particularly strong cell growth inhibition. Furthermore, interferon activity has severaL sp~cificitiQs; ~or example, when inter~eron is used as a phanmaceuticaI, it is preerable to use interferon which originated from a human.
Furthermore, it i~ de3irable to e tablish processes for extractin~ and puri f yi~g interferon produced by genetic engineering.

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~321)Z2 Usually, in extracting and puriyi~g a polypeptide obtained fro~ recombinant microorganisms~
cultured microorganism~ are firRt kill~d~by using a bactericide (a nece~ary procass ~rom the viewpoint S of safety) and then the dead c~lls are di~rupted and subsequently subjected to extraotion. In these treatments, the inte~de~ polypeptide is sometimes de-naturated and its activity may be lost. Furthermore, these treatments are apt to activate protease included in the cells and sometim~ decompose the intended polypeptide.
A method in which a protein denatured and solubilized with a denaturing agent such as urea or guanidine hydrochloride is e~tracted and the denatur-ing agent is ~emoved in the course of purificationhas previously been disclosed for polypeptides purîfied from ceIls or recombinant microorganisms (as described before in Japanese Patent Public Disclosure No. 161321/1984, U.S. Patent No. 4,476,049, etc.)~ Rowaver, it is difficult to safely o~tain complete renaturation of the intended polypeptide even though the denaturing agents are removed.
Therefore, this method is not preferred if the intended polypeptide is used as a pharmaceutical becaus~, when tha partially den2tured polypeptide iR
mixed, it can become an antigen. On the other hand, i~ the purifica~ion method u~ing a monoclonal antibody which has also been raported las de~cribed b~fore in .

~8~X2 Japane~e Patent Public Disclosure ~o. 186995/1984, e c.), it can ba thou~ht that a denatur~d and unde~irable polypeptide or a pol~merized~polypsptide such as dimer and trimer may be bonded ~o the monoclon 1 antibody, depending upon the antlgenic determinant recognized with the monoclonal antibody used. Recently, a method for extracting h-IF~ r produced by recombinant Esch~richia coli in the presence of a protease inhibitor for the purpose of inhibiting decomposition of polypeptide with protease has be~n disclo3ed in the abovs mentioned U.S. Patent, but the guanidine hydrochloride used therein is also known as a protein denaturing agent ~see, for example, Japanese Patent Public Disclosure No. 161321/1984).
It is therefoxe expected that, although the decompo~
sition of polypeptide with protease can be inhibited, production of a denatured protein may well result.
It would be urther desirable to 1) pxovide a purification scheme to separate gamma interferon from the cell debri~ of the disrupted cells in which the gamma inter~eron was produced; 2) separate ga~ma interferon from cell con~aminant~ in high yields and with high purity and activity; ~) separate recombinant gamm~ interferon from cell contaminants;
and 4) separate gamma interferon from cell con-taminantq without substantially degrading the inter~eron. The pu~i~ication process de~cribed b~lou is such a proces~.

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The purification method of the present invention ef~icie~tly provide~ a polypeptide having the intended phy~iological activity in ubstantially S pure fonm, inhibiting decomposition of the polypeptide with protease and avoiding the denaturation of the polypeptid~. ~urthermore, although the present invention will achieve the purification of a poly-peptide having h~IFN-y activity, the method of the present invention i~ al~o applicahle to the extraction and purification of polypep~ide~ other than h-IFN-y, which have a si~e susceptible to protea~e decomposi-t.ion, such as Arg-Lys and Arg-Arg, and are produced by recombinant microorganism~
In the present invention, the above-described problems are ~olYed by adding one or more salts of zinc or copper and polyethyleneimine (abbreviated to PEI
below) in the extraction process. More specifically, the invention comprises suspending the culture cells of a recombinant microorganism in a buffer solution contai~ing one or more salts of zinc or copper, disrupting the cells, then adding PEI to the ~ -centrifuyed supernatant, and subsequently subjecting it to a suitable puri~ication proces3.
Various compound~ which can be used as the salts of zinc or copper include zinc chLoride, zinc sulfate, zinc ace~ate, zinc ace~ylac~ona~e, and copper sul~ate, but zinc chlorido, zinc acetat~ and copper .

~ Z ~Z ~'~2 sulfate ar~ preferred. There are diff~rences in the optimu~ concentration og salt dep~nding on the peptide-producing train, but it is generally in the range of 0.5 - 5 m~, more preerably 1 ~ 3 ~M in ~he ca e S of zinc salt~, and 0.01 - 3 m~, mora prefera~ly 0.25 - 1 mM in the ca~e of copper salts.
The~e salts are mixed with a buffer solution in the above-descrlbed conce~tration, the culture cell~ are suspe~ded in the resulting solution and then disrupted, and the ~up~rnata~t i~ obtained by centrifugation. PEI is added to the supernatant to achieve a final conrentration of 0.5 ~ he addition of PEI also unctions to precipitate a considerable amount of impure proteins. After PEI
addition, the supernatant i allowed to stand at a low temperature, for example about 4C. After centrifugation to remove the precipitates, ~he desired ~ubstance ls purified by a conventional method. ~he purification can be convenientLy carried out by combining several columns and dialyse~. In some cases, salting out may be involved in the proce~.
Specific embodLment-~ will be explained below in the ~xampl Prior to the present application, there wa~
a repo~t about the addition o~ a zinc salt to the cultur~ medium in IFN p~oduction with a Yi~W to increasing productivi~y ~Japaneso Patent Public Disclo~ur~ No. 14~597~1984). ~hat report wa~ however 2~
g int~nded to increa~e production in titer during the cultivation and dia not d~scribe th~ addition of the salt togei~hex with PEI in the extracting~step, as in the present invention. With regaxd to th~ use of S coppe~ compounds in the purification process, an example of the use of a copper-chl_late re~in column wa reported in Japanese Patent Public Disclosure No.
167597/1984. Howev~r, that invention related to a purification method for a preliminarily purified IFN
solution. Previou~ repor~s such a ~hese above invention~ are essentially dif~erent ~rom the pres~nt invention which is characterized by thc addition of the salts in the extraction stage for the purpose of purification without causing denaturation or decQmpo-sition of a protein throughout. Another object of the present invention is to provide a substantial}y pure polypeptide having h-IFN-y activity which can be obtained by the method of purification and extraction of the present invention.
The construction of W3110~pINST4 which is one of the ~train~ capable of producins a poly-peptide having h-IFN-y activity is disclosed in European Patent Application 0,134,673. The polypeptide produced by that ~train is called GIFl46 and is represented by the following amino acid seque~ce (I).

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Cy~ Tyr Cy~ Gln A~p Pro Tyr Val Lys Glu 20' A1a G1U A~n LeU LYR Lys ~yr Ph~ A~n~Ala Gly Hi~ Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly ~le Leu Ly~ As~ Trp Lyq 10Glu Glu Ser Asp Axg Lys Ile Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Le~ Phe - ~ 70 Lys Asn Phe Ly~ A p Asp Gln Sex ~$e Gln ~ sa Ly~ Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser A~n Lys Lys 20Lys Arg Asp A~p Phe Glu Lys Leu Thr Asn : Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg Ly~ Arg Ser Gln Met Leu Phe Arg 30Gly Arg Arg Ala Ser Gln (I) The GIF146-producing ~train is Escherichia c_ W3110 tra~formed by a plaqmid vector bearing a DNA fragm2n~ coding for ~he above-de . cribed GIF14 and repre sented by ~h~ DNA 3ecIuance .
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8;~ 2 TGC TAC TGC CAG ÇAC CCl~ TAC GTG AAG GAA
ACG ATG ACG GTC CTG GGT ATG CAC TTf:: CTT

GS~:T GAA AAC CTG AAG AAA TAC TTC AAC GC:T
CG~ CTT TTG GAC TTC TTT ATG AAG TTG CGA

GGT CAT TCT G*.C GTT GCT GAC AAC: GGT ACT
CCA GTA AGA CTG CAA CGA CTG TTG CCA TGA

C:TG TTC CTG GGT ATC CTG AAA AAC TGG AAA
GAC AAG GAC CCA TAG Gl~C TTT TTG ACC TTT

GAA GAA TCT GAC CGT A~ ATC ATG CAG TCT
CTT CTT AGA CTG GCA TTT TAG TAC GTC AGA

CAG ATt:: GTT TCT TTC TAC TTC AAG CTG TTC
GTC TAG CA~ AGA AAG AT(; AAG TTC GAC AAG

AAA A~C TTC ~G GAC GAC CP.G TCT ATC CAG
TTT TTG A~G TTC CTG CTG GTC AG~L TAG GTC

AAA TCT GTT GAA ACT ATC AA& GAA GAC ATG
TTT AGA CAA CTT q~GA TP G TTC t:TT CTG TAC:
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A~C GTT ~AG TTC TTC AAC TCT AAC A~G AAA

TTG CA~L TTC AAG ~G TTG AGA TTG TTC TTT

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AA& CGT GAC GAC TTC GAA AAG CT~ ~CT AAC
TT~ G Q CTG C~G AAG CTT TTC GAA TGA TTG

TAC TCT GTT ACT GAC CTT ~AT GTA CAG CGT
ATG AGA CAA TGA CTG GAA TTA CAT GTC GCA

AAA GCT ATC CAT GAA CTG ~TC CAG GTT ATG
TTT CG~ TAG GTA CTT GAC TAG GTC CAA TAC

GCT GAA CTG TCC CCG GCT GC~ AAA ACT GGT
CGA CTT GAC AGG GGC CGA CG~ TTT TGA CCA

AAG CGT AAA AGA TCT CAG ATG CTG TTC CGT
TTC GCA TTT TCT AGA GTC TAC GAC AAC GC~
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GGT CGT CGT GCT TCT CAG TAA
CC~ GCA GCA CGA AGA GTC ATT ~II) On the other hand, the strain (W311Q/pIN5T4N143~ capable of producing a polypeptide having h-IFN-y activity and represented by the following amino acid sequenc~ (IIII can be produced as explained below with re~erence to the Examp:Les. Thi~
polypeptide i~ r-ferr-d to a~ GIF143 her-und-r.

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, Gln Asp Pro Tyr Val Lys ~lu Ala Glu A~n Leu Lys Lys Tyr Phe Asn Ala Gly ~is Ser Asp Val Ala A~p Asn Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Ly Asp A~p Gln Ser Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser A~n Ly~ Lys Ly Arg Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu A~n Val Gln Arg Ly-~ Ala ILe Hi GLu Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys Arg ' y3 1S Arg Ser Gln Met Leu Phe Axg Gly Arg Arg Ala Ser Gln (III) In this amino acid sequence, Gln* repre~ents Gln or p-Gln.
Furthermore, the DNA rragment shown by the following DNA sequence coding for the polypeptide (GIF143~ may he used for production of the intended plasmid vector.

CAG GAC CCA TAC GTG AAG GAA GCT GAA AAC
GT~ CTG GGT ATG CAC TTC CTT CGA CTT TTG

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CTG AAG AAA TAC TTC AA(:: GCT G~T CAT TCT
GAC: TTC TTT ATG I~G TTG CG~ CCA GTA AGA

GAC GTT GCT GAC ~AC GGT ACT Cl'G TTC CTG
CTG CAA CGA CTG TTG CCA TGP. GAC AA~ GAC

GGT ATC CTG ~ AZ~C TGG AAA Gl~ GAA TCT
CCA TAG GAC TTT TTG At::C TTT CTT CTT AGA

GAC CGT AAA ATC: ATG 6AG TCT CAG A~TC GTT
CTG GCA TTT TAG TAC GTC AGA GTt:: TAG CAA

TCT TTC TAC TTC AAG CTG TTC AAA AAC TTC
AGA AAG ATG AAG TTC GAC AAG TTT TTG AAG

AAG GAC GP.C C~G TCT ATC CAG AAA TCT GTT
TTC CTG CTG Gq~C AGA TAG GTC TTT AGA CAA

G.~U ACT ATC AAG GAA GAC ATG AAC GTT AAG
CTT TGA T~G TTC CTT CTG TAC TTG CAA TTC

TTC TTC AAC TCT AAC AAG AAA AAG CGT GAC
AAt; AAG TTG AGA TTG TTC TTT TTC GCA CTG

GAC TTC GAA AAG CTT ACT AAC T~C TCT GTT
CTG ~G CTT TTC GAA TS~A TTG ATG AGA CAA

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. . -- , ~ . ' .' ,: ' ~82~122 ACT GAC CTT AAT GT~ ~AG CGT A~A GCT ATC
TG~ CTG GAA TTA CAT GTC GCA TTT CGA TAG

CAT GAA CTG ATC CAG GTT ATG GCT GAA CTG
GTA CTT GAC TAG GTC CAA TAC CGA CTT GAC

TCC CCG GCT GCT AAA ACT GGT AAG CGT AAA
AGG GGC CGA CG~ TTT TGA CCA TTC GC~ TTT

AGA TCT CAG ATG CTG TTC CGT GGT CGT CGT
TCT AGA GTC TAC GAC AAG GCA CCA GCA GCA
, GCT TCT CAG TAA
10. CGA AGA GTC ATT

rief Description of Drawinqs Figur~ a figure illustrating construc-tion scheme of plasmid vector pINST4N143 used for tran~formation of E~cherLchia coli to produce GIF143 15 which will be purified by the method of the pre~nt invention.
Figure 2 is a ~low diagram of a preferred embodLment of the gamm~ interferon purification proces~ of the present invention.
Figure 3 i5 a flow diagram of a more pre~erred embodLment of thi~ pu~ification proces~ Rhowing primarily chromatographic purification means.
Figure 4 i9 a flow diagram of a particularly ,....... .

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preferred embodiment of th~ present invention.
~ iqure S i~ a photograph which ~hows the protein bands of SDS-PAG~ obtalned by di~rupting the cells (W3110/pINST4N146) in a bu~fer solu ion contai~ing various concentration~ of zinc chloride and ~ubjecting the supernatant to SDS~PAGE.
Figure 6 i3 a photograph which shows the protein bands of SDS-PAGE obtainecl by using copper ~ulfate in p}ace of zinc chloride.
Figure 7 i5 a photogxaph which ~hows the protein band~ of SDS-PAGE obtained by di rupting the cells (W3110/pIN T4N143~ in a buffer solution containing variou~ conce~tra~ions of zinc chloride a~d subjecting the supernatant to SDS-PAGE.

The production is explained with reference to Fig. 1, in which the DNA fragment o~tained by AatII and BglII digestion of pGIF54, which is essentially the same plasmid as pGIF4 di~closed in Japanese Patent Public Disclosure No~ ~01995/1983, 20 i8 further treated with ~vaII to obtain an AvaII-BglI~
DNA fragment as shown in ~iy. 1. Then 7 the intended plasmid pIN5T4N143 is obtained by inserting in the presence of a DNA ligase a synthetic linker DNA
fragment represented by : 25 5' - AATTC~TGCAG - 3' 3' - GTACGTCCTG _ 5 ., .

~.Z8;~ 2 be~ween a~ AvalI site of the AvaII-~glII fragment above and an EcoRI si~e of a longer DNA ragment b~aring a tetracycline resistan~ gene (TCr) which is obtained by treating pIN5T4 (disclosed in Europea~
Patent Application 0,134,673) with BglII and EcoRI.
The resulting plasmid contain3 a gene coding for a polypeptide GIF143 corresponding to GIF146 from which a sequence of 3 amino acid xesidue~, i.e.
Cys-Tyx-Cys, at the N-end of GIF146 is elLminated.
Sub~equently, a host (~. coli W3llol i~ transformed with ~he plasmid according to a conventional mlethod to obtain a GIF-pxoducing transformed E~cherichia coli (W3110/pIN5T4N143).
In all figures, the purification proc~ss starts with the removal of nucleic acids fxom the supernatant resulting from centrifugation of homogenized gamma interferon-containing cells, prior steps being shown for clarity but not being part of the present invention.
: Although a purification will be described by using zinc chloride as the salt o~ zinc in the Exa~ples, the inve~tion i~ not limited to this salt.
Salts of zinc such as zinc sulfate and zinc acetate and salts of copper such as copper sulfate are also desirab}y used. Table I show~ the protease-inhibition ef~ects of variou~ metallic salt compou~d~, which were investigated by disrupting c~ll o~ the recombinant bacteria in bu~er solutio~s containing 1 ~M or 0.2 m~

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o~ the metallic compound a~d mea uring the stability of a polyp~ptide havi~g h-I~N~y activity contained in th~ sup~rnatank liquid a-~ a~ indicator of the effects.
A~ indicated in Table I, it wa~ found tha~ zinc sulfate, zinc acetate, zin~ ac2tylac~tonate and copper ~ulfate provide the desir~d eff~cts as well as zi~c chloride.

Table I: Protea~e dacompo~ition-inhibiti~g effect upon polypeptide by addition of Y.n, Cu and other metal salts Decomposition-inhibiting effect on polypeptide Metal Salt 1 mM 0.2 mM

None Zinc chloride ~ -Zinc ~ulfate +
~ Zinc ac~tate +
: Zir.c acetylacetonate +
Zinc salicylate ~ -Copper sulfate ~ ++ +
Ferrous sulfate ~ -Cobalt chloride - -Ammonium molybdate ~ -+ having decompo~ition-inhibiting effect - not having decomposition-inhibiting effect ., Fig. 5 (photograph) i3 aA SDS-PA~ patt~r~
showi~g the 3ta~ility of the polypeptide having : .
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3Z02~

h-IFN-y activity (the band of the protein shown by an arrow m rk2d a~ GIF146 in th~ figure~. The test ~ample were prepared by disrupting the ceils in a desired buffer con ai~ing a d~ferent concentration of zinc chloride. In Fig. 5 it can be seen that the polypeptide exhibiting h~IFN-y activity i~ stable i~
the preqence of 0.5 mM - 2 mM of zinc chLoride and susceptible to decomposition in the abs~nce or a lower concentration of zinc chloride. The band of 10 the protein shown by arrow ~" shows the polypeptide ~ ;
obtained by decompo~ition of GIF146 with protease during course of purification. The sampl~ shown by 2-1 was prepared by tha treatment described above : by uslny 2 mM of ZnC~2 and then further proceeding with purification. Furt~e~more, th~ sample qhown by 2-2 was prepared by proceeding with the purification after the treatment without using ZnCQ2. The results of a similar experimental run in which copper sulfate was used instead of zinc chloride:are shown in Fig. 6 (photograph). In this ca e, the desired re ults are observed in the range of 0.1 ~M - 4 mM. Although these ~alt~ show sufficient protease inhibition with higher concentration~, they are preexably used in a~ low a concentration a~ pos~ible because of the necessity to remove them Ln the purification process.
It i~ preferable that zinc chlorid~ i~ used in the range of 1 - 3 m~ and copper ~ulfate in th~ range o~ O.25 - 1 mM.
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Fig. 7 (photograph) was obtained with re~pect to a ~ample prel?ared by ~reating GIF143 ~roducing bacteria in the same manrler as fc7r E!ig . ~ . I n the figure, the band of t:he prot~in shown by an arrow indioated as GIF143 ic the polypep~ide having h-IFN-y activity an~ ~E~ shows the polypeptide obtained b~f partially decomposing GIF143 with protea e.
The primary classes of contaminantq in the disrupted cell/garoma inter~eron mixture are ~mall-size 10 particulate matter arld watçr-soluble fraction~ such a nucleic: acids, protease~, cell proteins, carbohydra~e~, :
lipids, cleaved intç~rferon fragments and interferon aggregate~ and other fra~nents resulting from disrup-tion of the cell in which the interferon was produced.
We have now discovered that gamma interferon can be obtained in high purity, with the retention of biological activity and with good yields, by procesC-ing the interferon-containing mixtures in a specific sequence a~ described ~low to minimize degradation of the interferon and to remove the contaminants fxom the inter~eron-containing mixture in a defined order.

Sub~tantially improved purity and activity are obtained by remov~ng the contaminants in the interferon-containing mixture in the following order:

1) nucleic acid3;
2) negatively charg~d proteases and contaninating cell proteins;

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3) po~itiv~ly charged protease~ and contaminating cell prot~in~; and 4) cleaved a~d aggregated interferon.

Thi~ ~equence of step~ is critical to obtaining S the desired results of this invent:ion. Provided that the listed contamina~ts are remo~ed in the ~pecified saquence, additional ~teps may be used to remove other contaminating materials ~uch as high molecular weight hydrophobic material~, if prese~t. The~e othex material~ may conveniently b~ removed either ater step 3 or after st0p 4 in a further step, step 5.
There are numerous methods, known to the art, to accomplish each of theqe separations. A~ stat~d above, those methods which can accomplish the sapara-lS tions under the m~ldest conditions, to minimizedegradation of the inte~feron, are the most desirable.
We have found that a pr~ferred method is to use an initia} polyethyleneimine precipitation followed by several chromatographic separations to remove the con~aminant~ i~ th~ order specified abo~e. The re~ins used in the chromatographic ~paratians and the order of theix use iq as ~ollows:

1) anion axchang~ resin;

2) cation exchange re~in; and 3) molecular ~ i2ve .

In addi~ion to th~ chromatographic separations, . ~

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~8~022 it i u3e~Ul to employ precipitation, filtration, concentration and dialysi~ procedure~.
In a pref~rred proc~dure th~ gamma interferon~
co~taining mixture is subjected to the following S procedure~:
1) nucleic acid removal using polyethylene-imine precipitation;
2~ negatively charged protease and contami-nating cell protei~ removal u~ing weakly basic anion ~xchange resin;
3) positively charged protease and contami-nating cell protein removal using weakly acidic cation exchange resin; and 4) cleaved and aggregated inter~eron and cell ~ragment removal using a molecular siev~.
: Filtration after each step, concentration after steps 3 and/or 4 and dialysis after step 5, are useful adjunctive procedures.
Thi~ ~oYel procedure has consi~tently produced gamma interferon having a purity of at least 95~ and a yield in exce~ of 5~.
An important featuxe of the present invention is the novel purification scheme, which is suitable for U9~ with gam~a interferon produced in any one of a nu~ber of ways such as from human cells grown in ~is~ue culture, fro~ leukocyt~ coll~cted from blood ~amples or through cloning tech~iques w~ll known in the art. Th~ purificatio~ schem~ is particulaxly .

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well suited for the purification of recombinant gamma interfex~ recovered from ~. coli cells. The cells are inactivat~d by one o~ the 3ta~dard m~thod3~ such as by th~ addition of a chemical ~iLl agent such a~
S chlorhexidine gluconate. Th~ inactivated cells are centrifuged, resuspended in a buf er and homogenized.
A convenient method for homogenizatlon of the gamma interferon-containing cells i5 high shear disruption using a Manton Gaulin ho~ogeni2er. The components of the di~rupted cells are separated by centrifugation into a precipitate and supernatant. The upernatant ~rom this process is a suitable ~ource for gamma interferon to be isolated and puxified by the method .
d~scribed herein.
lS The suspen~ion o~ the lysed cells comprises proteins, lipids, carb~hydrates and nucleic acids and insoluble cellular debris. Using conventional procedures, the water-insoluble components are separated from the water-soluble fraction of the~ cell which remains in the supernatant.
Xt i~ sometimes de~irable to provide cer ain preliminary processing steps prior to the extraction of the gamma interferon from the cells, such as procedures to minimize degradation of the interferon during processing. Any such preliminary proces~ing step~ may be used provided they do not inter~ere with the puri~ication sche~2 desc~ibed herein.
The multistep purification ~ch~me achieves .
' , ~ ' ` ' . . ' ' ~28 ~4 superior yield~ of pure interferon while maintaining biological activity. The sequence o~ separation steps i9 highly significant and i~ critical to~achieving the desirable results disclosed.
The order of removal of the contaminant~ from the interferon-containing mixture is a9 follow~:
a) removal of nucleic acids;
b) removal of negatively charged protease~ and contaminating cell protein;
c) removal of positively charged proteases and contaminating cell protein;
d) removal of low and high molecuiar weight impuxities, cleaved interferon and i~terferon aggre-gates.
For reasons presently unknown, removal of impurities in the order stated i5 critical to achieving high yields of purifLed gamma interferon with retention of biological activity. The individual steps u~ed for the r~moval o~ each class of impurities are 2Q conventional and known to the art. Due to the tendency of the gamma interferon to cleave or aggregate into inactive forms under harsh proces~ing condltions, purification step~ which can be conducted under the mildest proce33ing conditions are preferred.
The invention is further described utilizing specific pxoce~sing steps and conditions which have been found to mini~iz~ d~gradation of th~ in~arferon, but it should be xecognized that other con~entional .

'' :`" .
. ~. .

~a~oz2 2~

proce~sing step~ may be ~ubstituted for those disclosed provid~d that the equence of impurity removal remain~ -as described.
Unlesi otherwise ~tated in the following description~ pH values given may generally vary +0.5, preferably in the range +0.25 and most pre~erably +0.1.
Conductivity meaqurements may generally vary +5 mS, and are preferably held in the range ~3 mS. Operation are performed at a temperature in the range of from about 2 to about 15C.
The first step of the processing scheme involve¢ the removal of nucleic acld~. This removal i~ conveniently acc mpli~hed by addi~g polyethyle~e-imine to the supernatant ~rom the centriuged mixture of lysed gamma interferon-containing cells. Alter-natively, th~ polyethyleneimine solution may be added prior to homogenization of the cells, if desired.
The polyethyleneimine is added slowLy with stirring to a maximum concentration of about 0.8% and the mixture i9 allowed to settle for an appropriate periodi generally in the range of from about 30 to about 90 minutes. The mixture is then centrifuged and the supexnatant collected. Excellent results are obtaine~
when the polyethyleneimi~e is added as a 10% (v/v) solution in H20 in amount sufficisnt o rasult in the polyethyleneimine consi~ting of from a~out 0.7 to about 0.8~ (v/vl of the total ~olution. Th~ p~ of the solu~ion is 8 ~ 0.5, pr~farably + 0.1 and the temp~- -. '-' ' . ' . ' ' .

~8~ Z

rature iq held in the range of from about 2 to about 15C~ The protein concentr~tion in the 3upernatant i9 determined at this 3tag~ and at each ~urther proce.~sing stage by the sta~dard Coomas~ie blu~ -binding assay.
Another procedure for removal of the nucleic acid is by using chromatography on hydroxyapatite or immobilized PEI. Precipitation with protamine sulfate is another useful procedure.
A~ter removal of thQ nucleic acids, th~ gamma interferon-containing mixture is subject~d to a first protea~e removal step. ~he most convenient method for r~moving the protease~ i~ by chromatography of the supernatant from the nucleic acid removal step lS utilizi~g an anion ~xchang~ resin. Quaternary : aminoethyl, mixed amine or other intermediate base resins or a weak bas~ resin such as p-amino benzyl cellU108e i9 particularly useful.
Quaternary aminoethyl is a preferred anion exchange re in. The quat~rnary aminoethyl may be attached to a cross-linked dextran, cellu}ose, agarose or acrylic support. The pH of the supexnatant liquid is adjusted to 8.7 ~ 0.5, preferably ~-0.1, utilizing sodium hydroxide ox any othex convenient base. The conductivity of the solution is ad~usted to below 10 mS, preferably in the rang~ of rom abou~ 4 to about 8 mS, by the addition of d~ion$z~d ~2 The ~lution buf~r compri ~3 20 m~ 30dium ~8202 4-(2~hydroxyethyll~1-piperazine~propane sulfonate and 0.1% (v/v) 2-mercap~oetha~ol~ The pH o~ the buffer i.~ adju~ted to approxim~tely 8.7 wit~ 30dium hydroxide or other base. Qther buffers suitable for use in the same pH range may be subs~ituted fo~ the piperazine derivative and other antioxidant~ may be sub tituted for the mercaptoethano~.
The quaternary amino~thyl column is pre~
equilibrated with the buffer solution, the gamma interfexon-containi~g ~olution i9 added and tha adsorbed material elu~ed with the sam~ buffer.
Approximately the first two-thirds of the eluted protein solution, i.e., the first two-third~ of the volu~re, is pooled for transfer to the next purification step. The remaining one-third of the eluate may be rechromatographed on the same column equilibrated in the same manner. Approximately the first two-thirds of the protein flow-through is again pooledO The remàining solution may be further processed in the same manner. As previously, the - protein concentration is determined by a Coomassie blue binding a~.~ay.
An optionaI concen ~ation step may ~e employed at this point in the pu~ification. One convenient me~hod of conc2ntrating the ~olution is precipitation with a~monium ~ul~at~. The eluat~ from the quaternary aminoethyl column is pas~ed through a O.2 ~ filter and a~onium ~ulfa~e i~ add~d to a fi~aL concan~r,ation ~2~ 0 of ~rom about 40 to about 60~ saturation, with stirring, over a 5 - 10 minute periodO The suspension i~ allowed to ta~d for s~ver~l hour~ i~ an ice bath.
The precipitata i~ the~ oollected by centrifugation and ~ay be s ored at approximately -2QC until required for further processing.
~ hen required, the precipitate i3 dissolved in a solution comprising 20 mM Tris-~CQ and 0.1%
2-mercaptoethanol at a p~ of approximately 7.5 that ha3 been previously passed through a 10,000 molecular wei~ht cut-off filter. Th~ conduc~ivity of the solution i~ lowered to from about 3 to abo~t 5 mS by the addi~ion of a solution compri ing 1~ mM Tris-HCQ
and 0.1~ 2-mercaptoethanol at a p~ of approxLmately 7.5. The solution i~ pa~sed through a 0.2 ~ ~ilter and is ready for further processing. Other buffers suitable for use in the same pH ranqe may be sub-stituted for the Tris-HCQ and other antioxidants may be substituted:for the mercaptoethanol.
The po~itively charged proteases and other proteins in the solution are removed in the next processing step, which is conveniently accomplished utilizing a ca~ion exchange resin.
Excellent result-~ have been obtained using a carboxymethyl ca~ion ex~hange r~sin ~carboxymethyl attached to cros3-linked dextran, cellulose, agarose or acrylic support)~ The p~ of the ~olution from the previous process step i adjusted to abou ~2~2~

7~5 utilizing ~Q or other appropriate acid.
2-Mercaptoethanol or other Ruitable antioxi~ant is added to a concentration of about Ool~ tv/v~.
3eionized water containing 0.1% (v/v~ 2-mercapto-ethanol i~ also added to reduce the conductivity tobelow 20 mS, pr~ferably to the range of about 3 - 5 mS. The solution is filtered through a 0.2 micron filter in preparation for ~ub~e~uenk chromatography.
The cation exchange resin column is equili-brated with a uitable buffer such as a colution compri~ing 20 m~ Tris-~C~ and Ool~ 2-mercaptoethanol at a pH of approximately 7.5. After column equili-bra~ion by washin~ ~he column two or three tLmes wi~h the equilibra~i~g bu~fer and addition o~ the gamma interferon~containing solution, the solution iq eluted with approximately 13 to 15 column volumes of a gradient of sodium chloride dissolved in the equilibrating buffer. The sodium chloride content is increased from 0 to a maximum of approximately 0.5 in the buffer.
Appropriate fractions are collected and may be analyzed by gel electrop~oresis (SDS-PAGE), analytical HPLC and antivlral acti~ity. The purest fractions are pooled for further processing. The fractions contain-ing interferon of lower purity may be precipitated withapproxim~tely 40 to 60% ~aturation a~monium sul~ate, redi~301ve~, filtered and r~chro~a~ograph~d on - a carboxym~thyl colu~n a previou~ly de wribed.

8zc~z2 Fractio~s collected from the rechromatograph~d solutlon are analyzed and the purest fractions pooled with the fractions ob~ained from the first carboxy-methyl elutio~.
If the pre ence of high molecular weight hydrophobic impurity is det~cted by SDS-PAGE or other appropriate procedure, the eluate LS ~b j ected to optional chromatography to r2move such impuritiPs at this stage in th~ purifica~ion proce~s. A phanyl resi~ has been found to provid~ sàtis~actory re!sul 9-Octyl and butyl reslns may al50 b~ u~ed. The ~;olution from the previous proc~ssing step is iltered through a 0.2 ~ filter and sodiu~ chlorid~ added l0.5 0.75 ~) to raise the conductivity of the solution to approxi-mately 50 to 75 mS.
The bu~fer is a solution comprising 20 mMTris-HCQ, 0.1% (v/v) 2-mercap~oethanol and 500 to 850 mM, preerably 500 to 700 mM, sodium chloride or other salt to increase the conductivity to the appropriate 2 0 range ~
The column i9 pre-equilibrated with the buffer and the sample is loaded onto the column. From about 2 to about 4 column volumes of the buffer solution are added to the column. ~he adsorbed material is then eluted with at le~t one and preferably from about S to about 10 column volume3 of a solution comprisi~g 20 mM Tri~ , 100 m~ NaCQ and 0.1~ (v/v) 2-mercapto~thanol at a p~ o~ approxL~at~ly 7.5.

~' ~
,' ' :

3202'~

Appropriately sized ractions are collected and analyzed using SDS-PAGE, ~nalytic~l HPLC and antiviral activity. The pure3t fraction~ are pooled~
It is generally desirable to concentrate the S inter~eron-containing solution aft:er the phenyl-column chromatography. It is also generally desirable to concentrate the interferon-contaisling colution at this stage in tho e i~stances whexe the optional hydrophobic colu~n chromatography step ha~ not been u~ilized.
The protein conce~tration of the solution i~
determined by the CGomassie blue hindi~g assay. If the protein conce~tratlon is determined to be less than 0.2 mg/m~, the solution is preferably concentxated by ultra-filtration employing a 10,000 molecular weight cut-off membrane.
Further concentration may be accomplished by adding ammonium sulfate to the solution to a final ammonium ~ulfate concentration of from about 40 to about 60~ saturation with ~tirring over a 5 to }0 minute period. The suspension is allowed to sta~d in an ice bath after which the precipitate is collected ~y centrifugation. The precipitate is redissolved in a ~olution comprising 20 mM Tris-HCQ, 500 mM sodium chloride and 0.1~ 2-mercaptoethanol at a pH of about 7~5 that ha~ been previou~ly filtered throuqh a 10,000 molecular weigh~ cu~-o~f filter. The concentrated - olution i.~ pa3sed through a 0.2 ~ filter in pre-~202~2 3~

paration for the next purification step.
Low and high mQlecular weight impuri$ies and cleaved gamma int~rf~ron and intexferon ~ggregates are removed in a final chromatographic purifica ion step by passing the ga~ma int~r~eron-containing 501ution from the previous processi~g step through a gel filtration resin. The hydrophilic ~iltra~ion gel act~
a~ a molecular sieve to separate appropriate ized fractions from high and low molecular weight impurities contained in the olu~ion. A particularly u~eful filtration gel is a cros~-linked dextran based gel, identified by the trademark SE~HADEX ~-100, manu-factured by Pharmacia Fine Chemicals. The rs~in ha~ .
a ~ractionation molecular weight range of 4,000 to 150,000 or ylobular protein and peptide and 1, ooa to 100,000 for polysaccharides. Other resins having cut-off ranges of from about 1,000 to about 200,000 for proteins may also be used.
. The SEPHADEX G-l00 resin column is pre-equilibrated with a buffer solution comprising 20 mM
Tris HCQ, 500 m~ NaCQ and 0.1~ 2-mercaptoethanol at a pH of approximately 7.S. The adsorbed material is . eluted with the.buffex and appropriate fractions : collected. The protein concentration of each fraction is determined by a Cooma~si~ blue binding assay.
The ~raction~ are combLned on the ~a~i~ of purity as judged by 5DS-PAGE, analytical HPLC and antiviral --- activity.

: .

~L~3~

Alt~rnatively, the precipitate from the ammonium sulfate concentration step may be dissolved in a buffer ~olution o~ 20 mU sodi~L~ phosphate, S00 mM sodium chloride and û.1% ~v/v) 2-mercaptoethanol at S a pEI o~ a~out 7. S. The gamma interferon-containing solution is charged to a SEI?EIACRY~; S 200 g@l filtration column, preequilibrated with the same buffer (SE~HACRYL
S-200 is a trademark of Pharmacia Fine Chemicals for a resin of agarose cross-linked with acrylamide). The 10 final product i9 a clear to lightly hazy solution, colorless to light yellow in color~ The apparent molecular weight determined by SD5-PAGE is in the range of 17,000 to 19,500.
~he purified gamma interferon i~ dialysed against a buffer before use. A suitable buffer com-prises 20 mM sodium phosphate and 6 mM L-cysteine at a pH of about 6 . 8 . Another suitable buffer is 15 mM
sodium phosphate, 8 mM sodium citrate and 6 mM
L-cysteine HCQ at a pH of 5 . 0 . It is preferable to continue to dialys~ fo.r 8 hours or more and to con~inuously flush nitrogen through ~he system to minimize oxidation.
If neces~ary, the purified gamma interferon solution can be concentrated in the manner described above.
The pre-cent invention will now b~ explained with refe~enr~ to the r~er~nce exampla and the working Example~ 1-3.

.

:

~.X~3f~

~ of GIF143 ~E~
vector~
A GIFl43 expressio~ vector was p~oduced according to the following procedure~.
pGIFS4 (a plasmid equivalent to pGIF4 bearing a gene coding for GIF146) wa~ obtained from W~a02/pGIFS4 which was a dcm Escheri hia coli trans~or~ant (a strain lacking methylation of cytosine) according to a con-~entional method. pGIF54 (5 ~g) wa~ treated with 20 unit~ of AatIX a~d 20 unit~ o~ BglII to obtain a DNA
fr~qment of about 600 base pair~ bearing a part of GIF146 gene and a Qac W5 promoter. T~n, 0.5 ~g of the DNA ~ragment was cleaved by using S units o~ A~aII
to obta.in the fragment of about 400 base pair~ bearing lS a part of GIF146 gene. On the other hand, S ~g of pINST4 wa~ digested by using 20 unlts of EcoRI and 20 units of BglII to obtain the DNA fragment bearing a tetracycline-resistant gene, ~pp promoter, and a re-plication initiation site. Both the DNA fragment and 0.5 ~g of the chemically-synthe~ized linker sho~n in Fig. 1 ~synthesized by using a DNA synthesizer;
Applied Biosy~tem~ 380A) were subjected to mixed-ligation to obtain pIN5GN143.. W3110 was transformed by the obtained pINST4~143 by a conventional m~thod, for example, the method de~cribed in Japanese Paten~ Public Dicclosura ~o. 63395~1983, ~o obtain W3110/pIN5T4N143.
It wa~ confirmed that the obtained transformant wa a GIF143-producing ^qtrain by the following proo~dures.
W3110/pIN5T4N143 wa~ cultivat~d wi h shaki~g in 1.5 mQ of a medium contai~i~g poLypep one 3~, yeast extract 2%, glycProl 2%~ K~PO4 0.5~, ~gSO~-7~2O
O.010% and tetracycline 20 ~g/mQ in a 16.5 mm t~t tube at 30C (OD660 = 8), 0.5 ~Q of the culture mixture wa~ tran~f~rred to a~ Eppendor~ cup of 1.5 mQ
and centrifuged to collect the cells (1~,000 rp~, for 5 minutes). The cell~ ware ~u~p~nded in 0.5 mQ of PBS
solution (0.~ NaC~, 0.02~ RCQ~ ~.115% Na2~PO4, 0.02%
NaH2P04) contai~ing 1 mg/mQ lysozyme lm~-EiTA a.nd reacted at 0C ~or 30 minutes. The cells were th~n disrupted by repeating freeze-thawing treatntent 3 time~
and the supernatant.fraction was recovered by centri-fugation (10,000 rpm, for 10 minutes). The supernatant ~raction was investigated for anti-virus activity according to the method de~cribed in Japanese Patent Public Disclo~ure No. 201995/19~3. As a result, an anti-virus activity o~ 6 x 104 units/mQ was recognized. On th~ other hand, the cells obtained by collection in th~ same manner as be~ore wer~
dissolved in 200 ~Q of an SDS sampl~ q~lution (10 mM
pho~phate buffer solution co~taining 7 M urea~ 1~ SDS, 1~ 2-mercapto~thanol, pH 7.2) and then heated on a boiling watQr ba~h for 10 ~inuts~. Th~ r~sulting sample (20 ~Q~ wa~ i~olated by 13~ SDS-PAG~ and sub-jec~d ~o protein ~t~ni~g wi~h Coomas~ie blu~ R250~
.

~2~2Z

A~ a result, it wa~ co~fi~med that GIF143 protein (about 18 kd m~lecul~r weight3 in a yield corr ~po~ding to about 20~ of th~ total protein of ~35~E~L~ coli was produced.

Exam~le 1 Extraction and purification_of GIF146 -~
W3110/pIN5T4 which was a GIF146 producing strain was cultured wi~h aeratin~ a~d shaking i~ a medium contai~ing 3~ polypeptone, 2~ yea~t extract, 2~ glucose, 0.5~ KH~PO4~ 0.01~ MgSO4~7H2O and 20 ~g/mQ tetra-cycline ~or 24 hours~ The cells of the culture mixtur2 were completely killed with chlorohexLdine gluconate and cen~rifuged (8,000 rpm, for 10 minutes~ to obtain 800 g of tha W3110/pIN5T4 wet cell~. They were 15 suspended in 5.1 liters of a cooled 20 mM Tris-HCQ `:
buffer (abbreviate~ to "THB" hereinafter) having pH
of 7.4 and containing 1 mM ZnCQ2, disrupted by subjecting them to homogenizer MlS (manufacture~ by ~anton Gaulin CQ., Ltd.), cooled with ice and then centrifuged (7,000 rpm, for 20 min~tes) to obtain the supernatant. To the ~upernatant was added an aqueous 15% colution of polyethyleneimlne labbreviated to "PEI" h~reinafter) having a pH or 8.0 adjusted with HCQ so as to achieve a fin~l concentration of 0.75~.
The resulting solution wa~ 8 irred for 10 minuts~ and wa~ allowed to stand at 4C for 2 hour~. The pre-Gipitat~ produced wara removad by ~entrifugation 37 ~82~'~X

(?~ rpm, for 20 minute~) to obtain 4.7 liters of the supernatant. The supernatant wa3 subjected to a QAE.Sephadex A~25 (manufactured by Pharmacia Co., Ltd.) column equi}ibrated with a 20 m~ N-2-hydroxyethyl-piperazinyl-~'-3-propane sulfonate bufrer ~EPPS bufrer) of p~ 8.6 to obtain the non absorbed fraction. The fractio~ obtained was then subjected to a C~ Sepharose*
C~6B (manufactured by Pharmacia Co., Ltd.) column equilibrated with 20 mM THB of p~ 7.4 containins 0.1%
of ~-mercaptoethanol (~bbreviated to ~2-~En hexein-after) and the active fractio~ absorbed on the column was eluted with a linear concentratio~ gradient of 0 to 0.5 M ~aCQ to collect fractions which had a high interferon activity. The interferon activity was lS measured according tv the method described i~ Japanese Patent Public Disclosure No. 201995/1983. Ammonium sulfate W35 then added to the fraction so as to achieve a 50% saturation,and salting-out was then carried out, followed by centrifugation at 7,000 rpm 20 for 20 minutes to obtain th~ precipitate~ The pre- -cipitate was dissolved in a 20 mM sodium phosphate buffer (abbreviated to n20 mM PBS" hereinafter) of pH 7.4 containinq 0.3 NaC2 and 0.1~ 2-ME and subjected to a Sephacryl S-200 ~manufactured by Pharmacia Co., Ltd.) colum~ e~uilibratad with the same bu~fer solution to obtain 298 mg of the polypeptide corresponding to GIF146 a~ a final puri~ied product (~he specific acti~ity of interferon: 1~6 - 1.7 x 106 U/mg)~
* Trademark 38 ~2~ Z2 Analysis of the polypeptide by SDS-PAGE showed more than 99% purity and the position of the band by the si~me SDS-P~G~ was in agreement with that of GIP146 which was not decomposed. Furthermore, it was S confirmed that the polypeptide obtained had the same amino acid sequenc~ as the amino acid sequence (I) by an amino acid analysis. This shows the usefulness of the purification method of the present invention.
~hen S~phadex*G-lO0 (manufactured by Pharmacia Co., Ltd~j was employed instead of Sephacryl S-200 used in the previously described purification processes, sLmilar results were obtained. .

, Exam~le 2 -Puriication and extraction of GI~143 In the same manner as described in Example 1, W3110/pIN5T4Nl43 (refex to the Reference Example) was cultured, and the cells were killed with chloro-hexidine gluconate, and collected. The wet cells (300 g) were suspended in 2.1 liters or a 20 mM ~B
(pH 7.4~ containing 3 mM ZnCQ2 and disrupted by ho~ogenization, followed by centrifugation (7,000 rpm for 20 minutes) to obtain the supernatant. It was treated with PEI in the sa~e manner as in Example 1 to obtain 1,000 mQ of the supernatant. The liquid wa~ th~n absorbed on a G~ Sepharose CL6B column e~uili~rated with 20 m~ T~B (p~ 7.4) containing 0.1%
2-~E and eluted by a lin2ar concentration gradient of 7 * Trademark i~, .

39 ~2~3;2i~)ZX

a .1 - 0.8 M NaCQ. The active fraction waq diluted to 10-fold of the origin~l volume with 20 mM T~B
containing 0.1~ af 2-ME~ absorbed on a G~-Toyopearl*
column ~manufactured by Toyo Soda Co., Ltd.) which had been equilibrated with the same buffer solutlon, washed and then eluted with a line~r concentration gradient of 0.1 - 0.8 M NaCQ. The fractions having interferon activity were collected, and after ammonium sulfate was added to the combined fractions so as to achieve a 20% saturation, passed through a Butyl Toyopearl column (manufactured by Toyo Soda Co., I.td.).
The fractions passed through ~he column were collected and subjected to dialysis against distilled water to .
give 396 mg o~ protein as a ~inal product ~interferon specific activity: 4.8 x 106 U/mg protein).
As a result of the amino acid analysis and SDS-PAGE as described in Example 1, it was found that the obtained p~lypeptide (GIF143) had a purity o~ more than 99~ and had the same amino acid sequence as the above-described amino acid sequence ~III). Fur~hermore, none of the zinc compound added during the extractisn step could he detected even by atomic absorption spectrophotometry.

I. Cell Harvest, Protein Release and Polyethyleneimine Precipitation Inactivated E. coli (W3110/pINST4) cells 'l i' * Trademark ;;. . , ' ' . ' ` ' 32~)2 containing recombinant gamma interfero~ ara collected by ce~t~ifugationO C~ are resu pended in 20 ~M
Triq-Hc~ bufer (p~ 7.5) contai~ing 1 ~ ~nCQ2. The cell~ are disrupted in a hi~h pre~sure homogenizer.
The cell homogenate is cen~rifuged and the supernatant is collected. An aqueous 10~ (v/v) polyethyle~eimine (PEI~ solution adju~ted to p~ 8 with hydrochlor.ic acid i added to the supernatant to bring the final PEI
ooncentration to a maxlmum of 0.8%. The mixture is centrifuged and the supernatant is collecte~.

II.
The pH of the PEX supernatant is adjuqted to 8.7 wi~h 4 ~ NaOH. Deio~ized water i~ added to reduce the conductivity to below 10 mS. The batch is applied onto a QAE column at a loading of not greater ~han 50 grams of protein per liter of gel. The colum~ is equilibrated with a ~uffer of 20 mM sodium 4-(2-hydroxyethyl)-l piperazine-propane sulfonate and 0Ol~
2-mercaptoethanol at a p~ of 8.7 prior to loading.
~lution i~ perfo~med with the ~ame buf~er. The protein solution is collected and chromatographed in Stage III.

III. Carboxy~ethyl (CMI Column Chxomato~ra~hy The p~ of the protei~ eluate from Step 2 is adju~ted to 7.5 with 4 N HC~ and 2-mercaptoethanol is addad to a final concentration of 0.1~. The con-ductivity i~ adjusted to 20 mS or below by dilutin~

:' -~82~

with ultrafiltPred water containing 0.1% 2-mercapto-ethanol. Th2 solutio~ is pa3sed through a 0.2 ~ filter and charged onto a CM colu~n at a loading of not greater than 35 gram~ o prot~in per liter of gel~
The column is equilibxated with a buffer o~ 20 mM
Tris-HCQ and 0.1~ 2-mercaptoe~hanol at a p~ of 7.5 a~justed to a conductivity of 2a mS or below with sodium chloride prior to loading. The column i~ -washed with at least 2 column volume of the equilibrating buffer. The gamma interferon is eluted with a salt gradient in th~ range of 0 - 0.5 M UaCQ
dissolved in ths e~uilibrating buffer. .Fractio~s are combined as detexmined by sodium dodecyl sulfate-~polyacrylamide gel electrophoresis (SDS-PAGE).

IV. Phenyl Column Chromatography Chromatography on a phenyl column is performed after the presence~of higher molecular weight impurities is detected by SDS-PAGE. Sodium chloride is added to bring the conductivity to 50 - 75 mS before the solution iq ch~rged onto a phenyl col ~n at a loading o~ not greater than 15 grams of protein per liter of gel.
The column is e~uilibrated with a buffer of 20 mM
Tri -HCQ, 0.5 ~ NaCQ, 0.1~ 2-mercaptoethanol at a pH of 7.5 prior to loading. After the sample i~ loaded onto the column, it is fol}owed by at leaqt one bed volume of equilibrating b~lf~er. The colwmn i~ eluted with 20 m~ Tris-HC~, 0.15 M NaCQ J O . 1~ 2-mercaptoethanol .
' --~L~8~:~2 at a p~ of 7.5. ~ctive fractions are combined a~

detenmined by SDS~PA~E and antiviral assay.

V. ~
If the protein concentration of the combined carboxymethyl ~Step III) or phenyl. (Step IV) fractionsis less than 0.2 mg/mQ, the solution is concentrated by ultrafiltration employing a 10,000 ~.W. cut-off membra~e. Ammonium sulfate is added to a final con-centration of 40 to 60% saturation. The precipitate is collected by centrifugation and stored at about -20~C~ if required.

VI. SeE~adex G-100 Co}umn Chromato~raphY
The ~mmonium sulfate precipitate i5 dissolved in a buffer of 20 m~ Tris ~CQ, 0.5 M NaC~, 0.1~ 2 mercaptoethanol at a p~ o 7.5. The solution i5 centrifuged prior to passing throu~h a 0.2 ~ filter.
The filtered solution is charged onto a Sephadex G-100 column preoequilibrated with the same buffer. The load-ing is not greater than 3.5 grams of protein per liter 20 of geL . The column is eluted with the same buf fer and fractions are combined as determined by SDS-PAGE.

VII. Purified Gamma Interferon Dialysis Th2 combined Sephadex G-100 fractions are dialyzed against 15 mM sodium pho3phate, 8 mM sodium 25 citrato, 6 mM I.-cysteine ~;C~ at a pH of 5 . O . Dialysis is carried out with conti~uous spaxging of nitrogen .

through th~ buf~er with two changes of bu~fer at a min~mum of i~ive hour intar~als. If neces.~ary, the dialyzed solution is collcen~rated by ult~afiltration u~ing a 10, 000 mol~cular weight cut-off membrane to 5 a protein concentration grea'cer than 1 mg/mQ. The solution of purified gamma interferon i~ passed through a 0 . 2 ,u filter and stored at about -20C or below.
The purified gamma interferon may be stored for at least several months at temperatures of approxi~ately -20C to about -30C: by adding 50%
glycerol to the gamma interferon-containing solution~
The gamma interferon is prepar~d for use by filtering through a 0.2 ~ filter and dialyzing the solution against a solution comprising 20 mM sodium phosphate and 6 m~ L-cystein~ at a pH of about 6.8.
Alternatively, the dialysis solution is 15 mM sodium phosphata, 8 mM sodium citrate and 6 mM L-cysteine HC~, at a pH of about 5. After a dialysis period of at lea t 8 hour~ carried out under continuous nitrogen 20 sparging, the solution is preferably filtered through a 10,000 molecular weight cut-off filter.
Product obtained ha3 a purity of at least 95~
gamma interferon and a yieLd in excess of approximately $%- .

Claims (15)

1. A method of purifying human gamma interferon from a culture mixture of a microorganism obtained by a recombinant DNA technique and capable of producing the interferon, wherein a zinc salt or copper salt is added to the culture medium, the microorganism cells are disrupted and separated, and polyethyleneimine is added to precipitate proteins and nucleic acids;
wherein polyethyleneimine is added in such an amount that the concentration thereof is in the range from 0.5 to 1.5% (w/v);
which comprises the further steps of sequentially removing from the gamma interferon-containing solution:
negatively charged contaminating proteins;
positively charged contaminating proteins;
and high molecular weight materials.

2. A method according to claim 1, wherein said interferon is GIF146 or GIF143.

3. A method according to claim 2, wherein the microorganism cells recovered from the culture medium are suspended in a solution containing a zinc salt or copper salt and disrupted, and polyethyleneimine is added to the supernatant obtained by centrifugation of the suspension of the disrupted cells;
wherein said zinc salt is added in such an amount that the concentration thereof is in the range from 0.5 mM to 5 mM;
or said copper salt is added in such an amount that the concentration thereof is in the range from 0.05 mM to 3 mM;

and polyethyleneimine is added in such an amount that the concentration thereof is in the range from 0.5 to 1.1% (w/v).

4. A method as claimed in claim 3 which comprises the steps of sequentially removing A) negatively charged contaminating proteins by column chromatography with a weak base anion exchange resin;

B) positively charged contaminating proteins by column chromatography with a weak acid cation exchange resin;

C) low and high molecular weight materials by permeation chromatography with a gel filtration resin.

5. The method of claim 4 wherein the process includes the additional step of removing high molecular weight hydrophobic materials from the gamma interferon-containing solution either immediately after removal of the positively charged proteins or immediately after removal of the low and high molecular weight materials.

6. The method of claim 4 wherein the anion exchange resin is a quaternary aminoethyl resin; and/or the weak acid cation exchange resin is a carboxymethyl resin;
and/or the gel filtration resin is selected from SEPHADEX*
G-100 and SEPHACRYL*S-200; and/or the gamma interferon-containing solution is concentrated after step A.

7. The method of claim 6 wherein the gamma interferon-containing solution is concentrated by precipitation with ammonium sulfate.
* Trademark

8. The method of claim 6 wherein the gamma interferon-containing solution is concentrated by ultrafiltration, or by precipitation with ammonium sulfate, or by ultrafiltration followed by precipitation with ammonium sulfate.

9. The method of any one of claims 6 to 8 wherein the process includes a final step of dialysis of the gamma interferon-containing solution against cysteine-containing buffer in an oxygen-free environment.

10. A method as claimed in claim 3 comprising the further steps of:

1) centrifuging the mixture containing polyethyleneimine and zinc or copper salt to separate the resulting precipitate from the supernatant solution;

2) adsorbing the solution from step 1 on a column containing an anion exchange resin;

3) eluting the adsorbed material;

4) adsorbing the eluate from step 3 onto a column containing a cation exchange resin;

5) eluting the adsorbed material;

6) adsorbing the eluate from step 5 onto a column containing a gel filtration resin; and 7) eluting the adsorbed material.

11. The method of claim 10 wherein adsorbed material from step 2 is eluted with a buffer com-prising sodium 4-(2-hydroxyethyl)-1-piperazine-propane sulfonate and an antioxidant; and/or the adsorbed material from step 4 is eluted with a buffer comprising Tris-HC1 and an antioxidant; and/or the adsorbed material from step 6 is eluted with a buffer comprising Tris-HC1.

12. The method of claim 10 wherein the anion exchange resin is a quaternary aminoethyl resin; the cation exchange resin is a carboxymethyl resin; and the gel filtration resin is SEPHADEX*G-100.

13. The method of claim 11 wherein the anion exchange resin is a quaternary aminoethyl resin; the cation exchange resin is a carboxymethyl resin; and the gel filtration resin is SEPHADEX*G-100.

14. The method of claim 11 or claim 12 or claim 13 wherein the antioxidant is 2-mercapto-ethanol.

15. The method of claim 1, 4 or 10 wherein the microorganism is E. coli.
* Trademark