CA2285976A1

CA2285976A1 - Method for inactivating pathogens, especially viruses, in a biological material

Info

Publication number: CA2285976A1
Application number: CA002285976A
Authority: CA
Inventors: Hans-Peter Schwarz; Peter Turecek; Gerold Zerlauth
Original assignee: Individual
Current assignee: Baxter AG
Priority date: 1997-04-08
Filing date: 1998-04-06
Publication date: 1998-10-15
Also published as: HUP0000825A1; HUP0000825A3; ATA59497A; BR9807938A; ES2214701T3; NO994821D0; EP0973543A1; NO994821L; EP0973543B1; JP2001519790A; SK138999A3; AU739845B2; AU6711898A; AT405608B; DK0973543T3; DE59810716D1; WO1998044941A1; PT973543E

Abstract

The invention relates to a method for inactivating pathogens, especially viruses, in a biological material, by means of incubation with a chemical agent. The invention is characterized in that the incubation takes place in the presence of an eluotropic salt corresponding to an NaCl concentration of at least 200 mmole/l, preferably at least 300 mmole/l. The invention also relates to a preparation containing a prothrombin complex and purified by chromatography.

Description

RCV 6Y- _ ln- 5-'~9 : 9:49.AN1 :__ ~'~3 151'7 98 05-i SM1ART & BIGGAR:# 2 FILE,~tN-tiV THIS AMENDED
TRANSLATION
A Method for Inactivating ~'a.thagens, in Particular Viruses, in a Biological Mater~_a1.
The present invention relates to a method for ~.nactivating pathogens in a bialvgical material by incubati.an with a chemical agent.
A biolagiGal material is derived from organisms or body liquids or microorganisms.
Since a biological material may be contaminated with pathogens, such as, e.g., infectious molecules or microorganisms and viruses, and pyrogens, respectively, Various methods for inactivating or depleting, respectively, pathogens and pyragens, respectively, have already been developed.
Such methods znclude physical and/or chemical treatments, such ass, e.g., diverse filtrat7.on methods (e. g, m,anv-, die- ar ultrafiltration), heat treatment, treatment with an acid or a base, treatment with a detergent and/or an organic solvent as well as treatment with W light or with laser light. Also various combinations oL such methods for inactivating and dep7.eting, respectively, pathoger~s have been suggested in the prior art.
From EP ~ 7.97 554, e.g., a. method c~f depyrvgex~izing and inactivating viruses in a bis~logical or pharmaceutical product is known, which comprises a treatment with a virus-inactivating and depyrogenizing RCV t3Y: 10- 5-99 :, .):4~aA'N :_~4:3 1 51.'~ 98 (r~-~ ~MAR'f Xz 13IGG:lR:t~ 3 agent, such as, e.g., an amphiphilic subairance az~djor a solvent. on a solid phase on which the product has beer_ adsorbed. After this treatment, the virus-inactivating and depyrogenizing agent is separated from the solid phase, the adsorbed product ie washed arid finally eluted from the sr~lid frhaee.
From ~P 0 131 740, the treatment of a protein--containing composition in a solution with organic solvents, such as di- or trialkyl phosphates, optionally in the presence of a detergent (aolvent/det.ergent treatment) is known, whereby protein-compositions free from lipid-containing viruses can be obtained.
From AT patent 402,151, a heat treatment is known o~herein to a preparation present in an aqueous solution a tensile is admixed at. a concentration of at least by weight, prior to heating.
1~ further method for reducing or >~uppressing, respectively, undesired activities its. biological yr pharmaceutical products ie known from EF 0 083 999, The latter is based on an extended Contact faith a solution or suspension of a non-denaturing amphiphile. The depyrogenized product: is treated with a~x~ ion Exchanger 4o remove the amphiphile.
1~ disadvantage of many of these methods known frt~m the prior art is.the frequent accurrerce of losses of activity of the J.abile proteins, e.g. blood proteins, ft~ V BY : 1 ~> - 5- ~'~S : '~ : 4 i3A~1 _ ~-1 ~; . 1 ~ 1" J~; 05-~ SV1AR'1' &
B I GGAR : #t 4 contained in tk~e cac~posit i ons to be treated. In particular when carrying out a chromatographic purification step, inactivation of proteins occurs to a relatively large extent. A degradation of protBirls may also lead to an activation. Thus it is, e.g., known that factor VII is vexy easily activated during a chromatographic purification due to a.utoCatalytiC
processes to factvx villa vrhich factor i.s undesired because it is very labile.
A further disadvantage consists in the large amount of time and apparatus required for many methods, which greatly seduces their practicability and thus often.
makes their use unsuitable on a large-technical scale.
The present invention is based on the object of providing a mathod of effectively inactivating pathogens in biological materials, which method is protein-preserving, in particular labile blood protsins, whic:rl can be transferred easily onto a large-teGhnical scale and can be carrieii out ecanomicall~~. rn particular, in the method fvr a.n;~ctivating pathogens, a degradation and a possible activation o1- proteins sus~:eptible thereto is to be largely avoided.
The afore-mentianed object is achieved in that a method. is provided for inactivating' pathogens, in particular, viruses, in a biological material by incubatian with a chemical. agent, wherein the incubation is carried out in the presence of an RCV t3Y : l f)- 5_ -'3'~; 9 : ~.~A'\'1 ~ _ ~'~~ 1 51'? 98 05~ S'\1ART & t3I
GEAR ; # 5 eluotrvpic salt Corresponding to an NaC1 concenCration of at least 200 mmvlf 1, preferably at least 300 rnmol/l.
Inactivation of pathogens in solution offers some advantages over the treatment of ar. adsoxbent. Thus, e.g., the practicability of such a method in a homogenous, single-phase system is higher, and validation of the inacti~ratiQn step is better possible.
The better accessibility of pathogens in a relatively homogenous phase also seems to increase the efficiency of the method step.
The biological material preferably comprises a human protein and in particular is plasma or a plasma fraction ar is derived frdrn a cell culture. Preferably, the biolog~.cal material comprises a bland factor, such as factor XII, XI, VIII, V, von Willebrand factor or fibrinogen, in particular a vitamin K-dependent prote~.n, such as factor II, factor VII, factor IX, factr~r X, protein C, protein S or protein 2, respectively.
The proteins may be present as single factors, preferably in purified form, or in a complex mixture.
Tn a very particularlx preferred ernk~odiment, the biological materia3. camprisea at least; one factor of the prQthrombin complex and, in particular, is a prothrombin complex-containing fractioxx or a factor VII-containing material., e_g. after aryoprecipitation of plasma, one departs from the corresponding RCS BY- _ 1«- o-J~: 9:ylAb1 ~ _~4~i ~ ~1'~ '~B fW-~ SM1AR'i & BIGG.AR:# F.;
supernatant (cryosupernatant?.
~'he preparation according to the invention preferably is one having FEIB activity (Factor Eight Inhibitor Bypassing l~ctivity), i.e. a preparation which a.s suitable for treating factor VIII inhibitor patients.
The cell-culture-derived material preferably is a material comprising recombinantly prepared blood factors, among them factors of intrinsic or extrinsic coagulation, of fibrinolysis, of thrombolysis, or the inhibitors thereof, xn particular vitamin K-dependent blood factors. As the cells, the cells commonly used for the expression of recombinant proteins are suitable, preferably mammalian cells, such as, e.g., Vero, CHO ox ~HK cells. The corresponding proteins may be subjected to the method of the invention for inactivation of possibly present pathogens either directly fxom the crude cell extract, it may, howevex, also be a pre-purified cell ~ra~tiori.
The chemical agent is, e.g., a detergent (amphiph~.le, tenside?. which preferably is contained in an amount of at least 1m, more preferred more than 5%, most preferred more than 10%; yet also other chemical agents may be employed aCCOrding to the invention, in particular those of which a virucidal, bactericidal or depyrogeni2ing effect is already known, or mixtures of the most varying chemical agents.

RCL' BY : 1.~ - p -'~'J : '~ : W) AM ~ ~4 a 1 51'? ;)H ()5-> SMART & E31 GG.AR
: # 7 The croiCe is, however, limited by the fact that the riat~.vity of the biological material sh.al.l. not be substantially adversely affected. For an eranomical mode o~ procedure, a chemical is chosen which retains more than 505 of the biological activity of the material, based on the activity prior to incubation, preferably at least 70~s, in particular more than 85e.
Retention of the biological activity means that the proteins contained in tk~e biological material are able to~fulfill the function or the various functions naturally ascribed to them. This blploglcal activity may be determined and stated depending on the type of protein, e.g. by means of a standardized chromogenic test or by antigen determinaL.ivn.
Qptionally, the chemical agent is separated after incubation.
13y ~~detergellt~, generally a synthet:io, organic, surface-active Substance 1S to be understood.
Preferably, a r~on-ionic detergent is used in Lhe method according to the inve.~tion_ Non-ionic tensideb, such as polyether, in particular. alkyl phenol palyglycol ether, are i.a. prvduc:l;s c~f er_hoxylation of fatty acids, fatty acid amides, fatty amines, fatty alcohols, amine oxides, fatty acid esters of polyalcohols and sugar esters.
Such a tenside does not act denaturing oxi the proteins and preferably is selected from the group of RC1 BY' : _ 1 « - 5-~~- : :3 : 50A~~1 : _+'1 ~3 1 51 '~ 98 l)5-j SMART & B I
GGAR : #~ B
polysvrbate and triton. As the polysorbate, e.g. Tween~
is used.
If detergents are used as chemical agents, according to a preferred embodiment they aze used without the addition of other agents, in particular without the addition of toxic organic substances or solvents, such aa, e.g., TNBP. In this manner, a risk of contamination is minimized.
According to the method of the invention, the biological material is incubated with a chemical agent.
TncubatiQn means the contacting of the biological material with. a solution, suspension or emulsion of a chemical agent for a perxoc~ of time sufficiently long for inactivation of pathogens ar pyrogenes, respectively, possibly present, at a specific temperature. Contacting may be simply effected by allowing the mixture to stand for a defined period of time.
2ncubation is effected according to the present invention in the presence of are eluotropic salt. 8y "eluotropic salt" hereinafter the salt in mixture with chemical agent or the salt in a complex composition is to be understood, with the property of dissolving adsorbed substances otxt of solid or lic;uid-impregnated, also gel-type adsorbents and/or to displace them.
Preferably, the eluotropic salt is a desorption agent as is used in chromatographic methods. The adsorbed 6;CV f3Y : 1 ()- 5-~)J ; :3: 51 A!tt ~. . +'~B - 1 5t'? ;)8 1)~-~ SD1ART & B 1 GGAIt : #~ 9 s»bstance is i.a. sufficiently soluble in the presence of the eluatx'opic salt, i.c,. preferably conditions are Chosen which do not precipitate the biological material.
The type and Concentration of the salt or of the composition, respectively, is generally selected depending on the adscr~asnt used. The eluting effect of a salt depends, e.g., on the polarity of the solvent, i.e. it increases e.g. in the sequence ethanol -acetone - methanol - water. The adsorbent may also be a solid phase, in particular a matrix suitable tar. iQn exchange chromatography. In the composition captaining the eluotLOpic salt, also further additives, e.g.
fuwther salts, may be ccntaine3. PrGfera~bl.y, the composition is an aqueous campositior having a pH
ranging between s.0 and 8.0, preferably around 7Ø
In a px.~eferred embodiment, sodium chloride is used as the eluotxopic salt, yet also other a7.kaline or alkaline earth salts, among them CaCl~, may be used. A9 the eluotropic salts, alsr~ so-called chnotropic agents, such as, e.g., urea, rhodanides ar guanidinium, mayr be employed. The concentration of ttie salt is at least a 20ct mmol/1, pr2terably ~ 3U0 mmal/1. The upper limit for the concentration employed will depend in parrir_.ular on the salability of the respective sa~.t and, for Na~l, is e.g. around 2 malfl. Chaotropic substances, such as, e.g., urea, may be employed _ g _ 1ZCWSY ~ _ lU- ~-~'j~~ ° '-):51:'161 ~ -_ . +'~a3 L ,~ l'> a8 (io-j SMrIR'I' & F3lGGAR ; #1(>
optionally even up to a concentration of $ mol/1.
Incubation of the biological material with the chemical ager_t is effected far a period of time sufficiently long to inactivate pathogens possibly present, preferably for a period of between 10 min and h, most preferred between 1 h ar>d 5 h_ The time required for the method according tv the invention may be,determined by means of model viruses, such as ATV, Sindbig, TBE or hepatitis virusQS in a pre-assay.
Also the choice of temperature has an influence on the period of time to be employed. In the method of the invention, incubation preferably is carriEd out at roam temperature, e.g. in a temperature range a.f_ bQt.ween 15 and 45°C, in particular between 20 anal. 3p°C.
In the method according tc the invention, the biol.ogi:al material preferabJ.y is adsorbed on a solid carrier, purified, arid incubation is effected immediately after elution df the purified material.
~lutien and incubation may be carried out consecutively, they may, hove°,rer, also be effected s i, mu 1 ~. aneou» ly .
According to a further preferred embodiment, incubaGi.on is effected after a chroa~atogaaphic purification of a biological tt!aterial, the eluatt having been still further processed, e.g.' by centrifugation, filtration, or other physical methods.
Preferably, the solid carrier is a material _ g _ ~''C'V epi : 1W 5-9J : :j: ilAM :__ _+'1'~3 1 vl'? 98 Ot~-~ SMART' Xc BIGGAR:#11 suitable for chromatography, iii particular a material suitable for 7.on exchange chromatography, hydrophobic chrarnatography, or affinit~~ chxvmatography. Materials, such as sepharose~', 8uperdexf , Sephadex'~',. Spherodex°, '~oyopearle, or inorganic materials, such as hydroxyl apaCa.te, are aged.
As the ion exchanger, anion exchanger materials, such as, e.g., DEAE Sephacel~, DEAF-sephadex°, DEAE-Sepharose° CL6B, DEAF-Sepharr~se° Fart Flow, QAE-Sephadex°, Q-Sepharose0 fast plow, ~-Sep.mrose~' Ftigh Performance, DEAE-Tris Acryl, nEAE Spherodex°, ~-Hyper.-D tabtainable through Sepra~or?, DEAE-Toyopearl~, QAE-Tvyopearlf, Fractcgel~ ENm-TMAE ar other Fractogel materials may bA used.
A~ examples of hydrophobic chromatographic materials, butyl-Sepharoseo, octyl-Sepharose~, phenyl-Sepharosef, Fz'actogel~TSK-Butyl, t-Butyl-H:Cc.'. Support or TSx Gel Butyl Toyopearl~' ought to be mentioned.
The biological material may be directly adsorbed an the carrier from a comglax mixture and purified, the inactivation step may, however, also be preceded or followed by further steps of. purifying the material, further chromatographic purificatzan steps being preferred within the scope of the present invention.
By the method according to the invention, pathogens are inactivated. By pathogens, also fragments of, e.g., viruses, in particular also the isolated geriome or the RCS' t3Y : _ 1W ~ W'~ ~ ~I ~ S'~AM v +'l~ 1 512 98 U5~ SMART & B I GGAR : # 12 fragments thereof, are understood.
ThE pathøgens may be lipid-enveloped pathogens, such as, e.g., hepatitis B virus, or non-lipid enve~,opad pathogens, such as, e.g., hepatitis A virus.
At present, virus inactivation methods azc called effective if after applying the method to a sample of a biological material which had been admixed with a high dose of a test virus, e.g. HI virus car Sindbis virus as a model virus fc~r hepatitis viruses, viruses can no longer be detected in the sample, and the virus titer thus has been reduced to below the detection limit.
Detection and quantitation of nucleic acids may, e.g.;
be effected by means of a pCR method as described in AT
patent 401,062, or by direct titration, Ae a measure far inantivation, the so-called reduction factor ie known which, after a single addition of test virus, is calculated from the decad~.c logarithm of the quotient of initial and final v;.xos titers. From European Guideline EC III/8115~89-EN of the Commission of the European Communities, furthermore, the so-called total reduction factor is known. It. is calculated from the sum of the reduction factors of individual, subsequent inactivation measures.
Freferably, a further, independent step for inactivating or depleting pathogens, respectively, is carried out. For this, all methods known from the prior - ii -RC'V F3Y : ~~- p-~~~ . : '1: 5-~,'1~'1 _: _ _ 4W 1 51 '? 5313 05> Sb9AR't' &
131 GGAR : #t l a3 art are usable to minimize the risk of infection.
In particular, a filtration and/or a heat treatment is effected as a further step Eor' inact~.vation or depletion, respectively. _ As the filtration, preferably a nanofiltration is performed. A preferred heat treatment is carried out ~on the salzd biological material, e.g. on a lyophilisate having a controlled water content, e.g. a water content of between 5 and 8%', and at a temperature of between 50 and 80°C, as is described in EP-o X59 311.
In a preferred embodiment, a 2-step treatment with a detergent ag the chemical agent ~.s provided. In doing so, a detergent is used in a first step in an amount oL
at least 1~, preferably at Least 5%, mast preferred at leant 10%. In a second step, a further detergent is used in an amount of at least z0%, preferably at least 12%, moat preferred at least 14s. The detergent used may be the same one for both steps; howe~rer, also different detergents may be used. Quite generally, the risk of a virus infection lfter administration of a corresponding preparation can be greati.y reduced ox eliminated, respectively, by the combination of steps for virus inactivation.
According to the present invention, also a chramatographically purified preparation is provided which comprises an autodyx~amically aCtivatable blood factor having a portion of activated blood factor of - ~.2 -RCS' !3Y : 1 ~- ~-r ~'O:-) : 9 = 5'?AM ~ ._. ~W3 l 512 J8 p5-: SMART & B 1 GGAR : # 14 less than 50%, based on the coxa.tent of activated and non-activated blood Factor, preferably less than 40%, more preferred less than 30%, still more preferred less than ~Q%, further preferred less than 10%, mast preferred less Chan 1%, and a detergent content.
In particular, the preparation is a prothrombin complex containing preparation having a factor vlza nativity of less than 50%, based or: the content of activated and non-activated factor VII, preferably less than 10%, most preferred less than 1%. The detergent content of the preparation according to the invention is pr~aent in a pharmaceutically acceptable amount, preferably between l% and the detection limit of the detergent.
By ~~autodynamically activatable bland factor~~, according to the present invention a blood factor is to be understood which is autc~caGalytically activatable by surface contact or by processes, such as, e.g., chromatographic processes. In particular, such a blood factor is a blood factor selected fro~rn the group of factor VII, factor XII, factor XT and pre-kallil~rein.
In a further preferred embodiment, the preparation is free from serine protease inhibitors, such as, e.g., thrombin inhibitors, ar cofactors, such as, e.g., heparin. In a special embodiment, the freedom from sucks.
substances exists already during a chromatographic process.

RCS- BY' lU- ?-'~9 :g:53A!vl ~ +9:3 1. 51'? 98 U5-j SMART & BtC,GAR:~tlS
Therefore, the pre6erit invention also relates to correspox~ding preparatioils obtainable by the method according to the invention.
Ire trie preparation according to the invention, also further additives may be contained, e.g. substances, such as arnirla acids, which act in a stabilizing manner.
The present invenr.ion shall be explained in more datail by way of the Following examples without, howevex, being restricted thereto.
EXA1~F~8 1:
Detei,geat treatment of activated prothrombin complex I~ETHA in the p~reeenae of TWEEN~~-80 15 m3 of DEAF-Sephadex~ A-5~, from Fhaz-nacia, were incubated far 15 min at room temperature with 1 ml of a solution of 30 g/1 EJaCl in water until swelling.
Thereafter, the gel was separated from tree swell~.ng supernatant by centrifugation. There followed five washings of the gel wz.irh Z ml of 1-;uffer each (9 gel Nay FiPO,~ . 2H? O, 7 g/1 BTaCl, pH 7 - 0; and twa further washp.ngs with a buffer (7 g/1 Na3 c~itr3te .2~iz D, 7 g/1 NaCl) also by resuspension and centri.fugati~on.
30 ml of fresh frozen human citrated. plasma were thawed a>"- 0 to +4~C, and the oryopreclpitate incurred was separated by centriftag&tion at +2°C. The '"cryosupernatant" resulting therefrom was irzc.ubated with the washed DEF.E-Sephadex~, FEiHA being generated and adsorbed on the gel together with the factors of RCV ~3Y~ ~«- ?-~''~ : ~3:~i:3:~11 ~ 't'1z;- 1 ~IZ 98 t) i~-~ SilI.ART &
BIGCiAk:#LE.
tha prothrombin complex and inert protein. Thez~eafter, coadsarbed inert protein was removed from the DEAE gel by washing with a buffer (9 g/1 Nay HPO~ . 2Hz O, 7 gJl NaCl).
The buffer-moist gel/prote~.n complex was then suspended for i h at asqv with z.5 ml of a solution a~
150 mg/ml TWEEN~-80 arid 30 mg/ml NaCl. By the treatment with the solution of high iania strength, protein was desorbed tpgether with the factors of the prr~thrombin complex and pathogens possibly present. Subsequently, the suspension was diluted by adding 6.5 ml of water and readsaxbed for 2 h at roam temperature, the protein fraction bei.z~g readsorbed again" whereas components of the inactivated pathagez~ remained in sr~luticn togethex with the detergent. 'The gel/pratein complex was then washed five times, each with I m? of a solution of g/i h'aCl in water go ae to be detsrgent~free.
Ear elSaLa.on, the gel was treated under stirring with 0.7 ml of a solution of-. 30 g/1 NaCl in water. The eluate was then dialysed against distilled water, frozen, and lyophili2ed. Azter reconstitution of 4he lyophilisate, the FEIH~activity was determ:~neci according to AZ'-B 350 726.
preparation of FEIBA prepared in the same manner, yet without treatment witri a detergent, was used as the cor~>r xo 1 .
The analysis of the preparation obtained exhibited 1O _ 5 _ ~g : :3 : 53A~1 _:_ . ~'13 - 1 51 '? :3f3 05-~ SMART & B I GGAR : #

a specific activity of 3.2 U FEIBA/mg protein at a protein content of 16.6 mg/ml after reconstitution of the lyophilisate and was~comparabse with the method variant without detergent treatment, a specific activity of 2.8 U/mg protein being obLai.ned at a protein concentration of 16.5 mg/ml.
E~CI~MPLE Z
Detergent tr~atmeat at the de~orption of FETBA with ext~rid~d ineubatioxi time The prothrombin complex fraction was adsorbed on DEAE-Sephadex~ az~alogoug to Example 1, washed free from inert protein, subsequently ~.t was desorbed with a TWBEN°/NaCl solution. However, the protein fraction was kept for 2 or 3 hours, respectively, in the desarbed state under otherwise equal conditiorxs. thereafter it was worked up to the final. product ae described in example 1.
The analysis of these formulations yielded a specific activ~.ty of 2.5 U of FEIBA/mg of protein at a protein content of 16.6 mg/ml with 2 h of incubation in the presence of TWEENR'-84, and a specific activity of 2.3 T1 of FEIBA/mg of protein at a protein content of 17.4 mg/ml with 3 h of incubatic~n'with detergent.
Thus it could be demonstrated that also the extended contact time with the detergent was not connected w~.th any substantial inactivation of the active suk~stance or reduction of yi.eJ.d.

RCV BY ~ _ ~W W g~3; ~'O : 58A~I .;_. _ _ ~4:3 I ' 1 '' B8 05~ SMAR'C & B I
GGAR ; # 18 EX~1M8LE 3:
Dstergaat traatmeat of F'8I8A with readaorption on a differrut gel rEIB~r was prepared as described in Example 1, After the treatment and desorption with detergent, the solutiozz obtained was transferred irntr~ a co~ztairaer in which 15 mg of DE1~E-Sephadex° A-50 , from Phr~t~macia, were pre-incubated to swelling xn a solution of 30 g/1 Nacl anal subsequently were provided by ;~i.ve washings each with .Z n21 df a buffer ( 9 g/1 Nay HPO~ . 2Hz O, 7 g/1 NaCl, pH 7_0?, and two further washings with a buffer (7 g/1 Na3citrate.2Ha0, 7 g/1 NaCl~, each by re-suspension and centrifugatiar~. After a 1 h adsorption of the diluted protein complex for separat:i.rzg the detergent, working up was effected according to the process described in Example 1. The thus obtained final praduat had a yield of 95~ as compared to a FEI$A
prepared aaaarding to the standard vax'iant, i.e.
withcut treatment with detergent, and was of comparable specific activity.
BXAMFhE 4:
DetergGat treatment o~ activated pr~thrombia complex FEIBA i,n the preaeace of TWEENn-$0 at irmreaeed temperaturo 15 mg of DEAE-Sephadex° A-5o, from Pharmacies, were incubated for 15 min at room temperature with 1 ml of a solution of 30 g/1 NaCI in water until swelling.

RCV f3Y ' 1.~'- ?-O~3 : 8 : ,~ ,~M ~ . - ~4:3 1 . 51'' G~ OWj SMART & B I GGAR
; # 1:1 'fhereaftex~, the gel was segarated from the swelling supernatant by centrifugation. There followed five washings of the gel with 1 ml of buffer each (9 g/1 Na" HP01 . 2H~ O, 7 g/1 NaCl, pH 7. 0) arid two further washings with a buffer (7 g/1 Na3eitrate.~H~O, 7 g/1 NaCI) also by xesusp$nsion and centrifugation.
30 ml of fresh frozen human citrated plasma were thawed at 0 bis +4°C, and the cryoprecipitate incurred was separated by centrifugati4r~ at +2dC. The "cryo-superziatant" resultir~g therefrom was incubated with the washed DEAF-Sephadex'~, FEIBI~r being generated atZd ad-sorbed on the geJ. together with the factors of the pro-thrombin complex and inert protein. Thereafter, coad-sorbed inert protein was removed fxom the DEP.E gel by washing with a buffer (9g/1 Naa HPO~ .2Hx 0, 7 g/1 NaCl ) .
The buffer-moist gel/protein complex was then suspended with 1.~ ml of a solution of 1 mg/ml TWDEN~-80 and 30 mg/m1 NaCl for 1 h at room temperal:ure, the protein fraction and non specifically adsorbed impurities being desorbed. Subsequently, the gei was separated by filtration. By further addition of '~E'WEEN~'-80, tre protein :solution then was brought to a detergent concentration of 150 mg/m1, and subsequently was incubated either far x h at 25°c or for 1 h at 40°C
with stirring sv as to inactivate øny pathogens possibly present. Thereafter, it was diluted by the addition of 6.5 ml of water, and a freshly washed RCV BY- _ _ I«- 5-9:3 : g:S.I.A,~1 ~. +'1B 1 512 ~~8 (15y SMART & C31GGAR:#20 prepared DEAF-Sephadex~ A-50 gel was readsorbed. Then it was washed detergent-free by five waghinge with 1 ml each of a solution of 7 g/1 NaC1 in water, anc~ F-i~rAaJ.1_y the prepa,rati.on wary further worked ~p as dpacribed in example 1 The analysis of both variants of tx'ea~:mer~C at. 25°C
and at 40°C showed a specific activity of the fEI~3A
preparation comparable to that of a standard variant urithout vix-~: s inactivation. The yields were 'i~~ of the standard variant.
g7C7~PLE 5:
Detergent treatment of prothrombiri CompleaC iri th.e praaez~ce of Tw~:EN~-84 (at present ar~nsidared icy Applicant to be the best mode of carrying vut the invontioss) 30 ml of fresh froaen human citrated plasma were thawed at 0 to +4~C, and the cryoprecipitate incurred was separated by centrifugation at +2~C. The ~~tryo-supernatant~~ resulting therefrom was admixed with 2 rU
of heparinrml. Subsequently, the prpteins of the pro-thrombin complex were adsorbed v~rith T1SAE-Sephadexc° A-50 from Pharmacia, at a concentration of 0.5 mg/ml. The gel/protein complex was separated from the solution arid washed each With a buffer I (4 gll Na3citrate.2H20, 7 g/1 NaCl, 9 g/1 Na~HP0i.2Hz0, 500 IU of heparin/1, pH
7.5) azxd subsequEntly washed with buffer 2 (4 g/1 Na~citrate.2H=p, 7 g/1 NaCl, 500 TU of hepariri/l, pH

Rl i' BY : 1_« - > v ~3~') : ~ : >9 ,1M ~ .. +'1:.3 1 i l '1 JF3 0.5-> SiIIART
& 1B 1 GGAR : # '__> 1 7.5) -The washed gel wa$ then suspended for pathogen inactivation with 1.5 mJ_ of a solut:i.on containing 150 mg of TWEEN~-80/ml and. 3l1 mg of NaCIJml, for 1 h at 2~°C. By this treatment, the protAin fraction was deaorbed together with any pathogQns or pathUgen fragments possibly present, and in the cn:.~rse c~f incuk~at?on with the detergent, such pathcagens were inactivated. Subsequently, it was diluted with 5 mJ. caf water as described in example l, and the grotein fraction including tt~e active substance was readaorbed t4 the ion exchange matrix for 1 h at room tempex'ature.
Theri it was v~rashed five ti;ttes with 1 ml. of a suffer (4 g/1 Na, citrate; 7 g/i NaCI , 5C~ TU of heparin~'1, pH
'7.5) sc~ ass to be detergent-free, and eluted with a solution of 1 g/1 Na3 citrate.2I~~C7, 30 g/1 NaCl, 1,00 TU of heparin, pH 7Ø To the el.uate, 1 zU of heparin/ml was ad,.~,ixed. The prat~hrombin complex-containing solut=an was rebuffered against a buffer containing 4 g/1 Na3citrate.2H~G, 8 g/1 NaCl, pH 7.0, and lyoph~.li~ed. In the reCOtlsL:itvted, lyophilized p:rc~thrombin complex the protein content al.d the content of prot:rsroxbin complex factoz°s was tested; the result;
can tae t~ake~: from Table 1 , A Celt mixture wit.~lout TWEEN~' treatment was prepared ds the control. The analysis results can also be Oaken from Table 1.
2~ _ RC'V f3Y - _ 1 «- 5-:)9 : ~) : 55A)9 : . _ +'1W ~>1'~ 9~3 US-> S!41AR'f & f3I
GGAR : #'?2 Comparison of the activities of the prvthrombin complex frxctar~ after carrying out the Method acoazding to the invention and without that method V
f~

M

ra W

Q :

'~

f3a N N

~J N ' ,-t ii ~

Lt~ N CV
~

x H

o -1-' a e.~
~

V
...

to "~ cV
~

Gv N ~V
~

H

H

r~

~-I
rv O
~

rn o o ~

~ N

i o u° ~ o RCV BY : 1()- 5-;~9 ; 9: 5 iAYI : 'f''Ia3 - 1 51'~ '3f; (1?-j 541.ART & E31 CIGAR : #2a3 Tt has been. shown that no aubatantial change of the composition of the prvthrombin complex was effected by the detergent treatment.
EX~rMPLE 6:
Deirergm>;tt treatment of factor V== with TWEEt~1'~-80 as compared to vxrul: 3,xzsctivation of factor VYr according to a conventional method From human citrated plasma, the px~ot,tlr~ontbin complex graction containing the coagulation factors prothrombin, alight portions of factor VTT, factor IX
and factor x were separated as described in example 5.
The major portian of coagulation factor Vrr remaining in the supernatant after adsorption on rFRF Sephadex~' A-50 was then recovered by adsorption on aluminum hydroxide. To this end, 10 ml of a 2% axuminum hydrogel suspension were admixed per 1 1 supernatant after separation of the prothrombin complex and stirred at 4~C for 30 min. Subsec,~uently, the aluminum hydrax~.de/protein complex was separated by centrifugai<a,an at 5, 000 rpm far 10 ;nin at approximateJ.y 4~C in a Sorvall RC3B ratar H~aOOA, the supernatant was discarded, and the precipitate was suspended with 3.5 %
of the volume of the prothrombin campl.ex supernatant used for adsorpticn, in a solution of ~ g/1 of Na~citrste.2HaO axed 7 gf1 of Nafl, pH 7.5, and stirred fQr 30 miri. By thi~9, inert protein was desarbed from the aluminum hydroxide. The factor VTI rema~.ning on the RC\ t3Y : _ 1«- 5-:~~_ : 9: 55AM ~ . +4~ l >1'' :1F3 Ou--i SM_AR'1' & B 1 GGAR
: #24 aluminum hydroxide was pelletiaed by renes~red centrifugation as described abaue. The supernatant was discarded, and the precipitate was further used far further processing. For desorption of the protein fra,ctio7n, the aluminum hydroxide/~actor VII complex was stirred for 3Q rnin with 1 % by volume of the prvthrombirr complex supernatant of a ~.3 mal/1 phosphate buffer, pH 8 .6 (53 .4 g/1 of Na2 HPO' .2H2 O were adjusted to pH 8.& with a solut~.on of 41.1 gjl NaH~ PO~ Ha O) used for adsorption and containing 1 % of TWEEN~~-so. S2:bsequeatly, far pathogen irlactirration, detergent was added to a final concentration of 15 % of TWEEN~~BD, and then it was stirred for 1 h at 40°C.
Thereafter, the soluC:ion was cooled to approximately 22°C and diluted with 9 parts of aqua dest.. The factor VII fraction was then readsorbed on 1 g/1 D>;AE-Sephadex'~ A-50 under stirring for. ~. h at approximately 22°C. Then the gel/protein complex was washed detergent-free on the sintered suction. filter by washing threA times, with 100 ml each per l~.ter. c~f_ em~aloyed, diluted Z'WEENm solution, with a buffer conta.i,ning 4 g/1 Naj citrate.2H; O an~~'. 7 g NaCl/1, pH
7.5, containing 500 I1;1 of heparin/1. The elution Gf the factor vII fraction was effected by stirring of the ion exchanger protein complex and 1ao ml/1 of diluted TWEEN~ solution of a 85 g/1 Na~l containing solutio>Z
for 30 min at 22°C. In the eluate, subsequently the RCV' 131' : _ 1~- ~-~9 : 9: Sg.AM ~ ._ +ha3 1 512 9f3 05--~ SA9ART & 131 GGAR
: #'?5 faotr~r VII content wa~3 measured by means of a chromogenic factor VIZ test, (Tmmunock~rom Faktor VII: C, Z1~IT1N0 AG, Vienna, measured against the international prothrornbin complex standard), the protean content was c~uantitated according to the method of Pradford [Anal.
Biochem. '72 :248-254 {1976) ) arrd factor VIIa according to the method from US 683,682 (measured against the intez'national factot~ VIIa standard). The results can be taken from Table 2.
For a comparison, factor VIT was separated from the other proteins of the prvthrombir complex by adsorption on aluminum hydroxide, as described above, and in the adsorbed state it was treated according to EP 0 x.97 554 with the virus-inactivating agents from EP 0 1f1 740 with T'WEENe-80 and tri-(N-butyl?-phosphate (TNBP). To r_his end, the alhydrogel protein complex was stirred in an. aqueous solution of 1% TWEEN~-8o and o.3% tri-(N-butyl?-phosphate for 18 h at 4°C w'iCxx a volume of 50 ml/1 prothrombin complex supernatant. Su~asequently it was centr,i.fuged as described above to separate the aluminum hydroxide protein complex, and by wasx7i,r,g with 3 x 100 ml of a soll~r.ion of 4~ g/1 Na3 citrate.2H~ O, 7 g/1 NaCl, pH 7.5, it was freed from an excess of TwEEN~-80 a~td tri-(N-butyl.)-phosphate by resuspending.
Between each wash, thexe followed a palletizing of the aluminum hydroxide/protein complex by centrifugation.
Elution was carried out under the same conditions as ~.n RCS' f3Y ~ 10- 5-99 ; 9 : SE~AW1 : ~4a3 1 51 '? 98 05~ SMAKT & B I GGAR : #2G
the parallel test mixture according to the method of the invention. Likewise, the ana3.yses of the final pxoduct were carried out analogously. The results can be taken from Table 2.
T ~ 2 Factor VTIa activities after carrying out the method of the invention and after carrying out the method aCCOrding to EP 0 197 554.

l2C.'V 6Y v _ J U- ~>-X79 : .3: ,F>.hM : _ ~'IW 1 51'? '.3f1 05-~ SM~R'f & B I
C~G~1R : N'?7 rl H

i ''r U m tt~ H ~' s~

r1 ao .-r W ,"y H '-" C M

r.

U '.. ~'-W ~ H

N v-i ~ r ri ~

~

1 ~, N ID
V -V ~ ~'1 t~

~v (a I'1 +i V
~.

~t1 '-' O

w~ .IJ

m -a .--_ ~
~~

Q, H

~:

n ~ ~
a V I~ r1 r'7 n1 '-' U

N
~

U ~
r1 s~

a O at-~

ri w-) ~ C1 h O ~ rd rti rtl d ~7 H

Sa N e'~1 i~

~

I ~
O

Qf Gf f-1 iN
~ 0 W

W I=4 -ri t1 Gti kCV FIY~ lU- y-99 : g:;pE>A:M :. +'l~ l 51'? ,)8 ()5-~ SMART' & C31GGAR:#''>8 I~ has lean shown that by applying this method, i~he faotar VIIa content was marked7~y .i.noreased as anmp~red to the method aaoor_di_z~g to the ~.nvenU-zc~2l, yer. despite the complex treatment of factor v:t I. nc~ ac~ti.vatznn could be round. Moreover, with the method accord-ng to the invention, the specific act~.vity of the obtained product was higher than in the comparative preparation.
BXA~pLE 7:
S~amiqua~atit~tiv~ doterrn,i~a~,tioia of hepatitis G virus In the pathogen inactivation formulations of examples 1 to 6, the samples were drawn from each of the starting materials, supernatant after cryoprecipitatier~ or adsorption supernatant after separatioil o~ the coagulation factors Ir, r~ and X, as well as the corresgoc~dingly purified and concentrated cvaguJ.~atian factor preparations. U.5 ml of these rsamples were diluted 1 + 1. with physiological phosphate-saline buffer, and viruses possibly present were pelJ.etiz~d by ultracentritugation. The RNA was extracted from the viral pellets by means of the RN~zal reagent method (Bivtecx, Houston, Texas), and dissolved in sterile a. dart..
RT-SCR for h°patitis ~ virus (HGV)nucleic acids was ~:arried out with the primer pair NS5a 1 and NS5a 2 (Linnen, J. et al., Science 271: 5~5-5Q8 (1996)). The sequence of the primer used (obtai:~able from Hoehringer Mannheim, Germany) for N85a 1 was:

RCS' B1': _ 1«- 5-aJ : a:57AN : ..~9~<3 1 51? 98 05--j SY1AR7' X~
E31GG.AR:#2:3 5'CTCTTTGTGGTAGTAGCCGAGAGAT ~~, and for NSSa 2:
5'CGAAT'GA.pTCAGAGGACGGGGTAT 3'. The primers were labelled with a fluorescent dye, and the fluorescent amplicons resulting therefrom accord~.ng to Gha routine methods of common pevR protocols were analysed on an ,A,~3I
377-Sequencer of Applied Biogystem~s. tn order to ba able to exclude the presence of RT-PCR inhibitors iz~
the samples, the samples were spiked with hepatitis C
virus-RNA mimics and analyzed in a hepatitis C-PCR
carried out according to EP 0 '~14 988. Exclusively extracts which did not show any inhibition in the HCV-PCR were used as evaluatable for HaV-PCR. The intensity of the fluorescence was taken as a measuxe for the content of hepatitis G virws_ zt hag been shown that starting materials used for fx~actianation had highly positive signals prior to pathogen inactivation according' tQ the inventive method, i.e. t>.ad a high concentration. of HGV nucleic acid amplificates, whereas in the eluates after readsortpion and separation of irhe vixus-inactivating agents, rio HGV-RNA could be detected any longer.
Tn parallel assays without carryzng out a detergent treatment, the eluates as well ae the starting matera.als used v~rexe HGV-PCR-positive.
_ 28 _

Claims

Claims:

1. method for inactivating pathogens, in particular viruses, in a biological material by incubation with a chemical agent, characterized in that the biological material is adsorbed on a solid carrier and incubation is carried out with a chemcial agent in the presence of an eluotropic salt corresponding to a NaCl concentration of at least 200 mM, preferably at least 300 mM, whereby incubation is effected simultaneously with the elution or immediately after the elution of the biological material.

2. A method according to claim 1, characterized in that a detergent is used as the chemical agent, which is preferably contained in an amount of at least 1%, more preferred more than 5%, moat preferred more than 10%.

3. A method according to claim 1 or 2, characterised in that sodium chloride is used as the eluotropic salt.

4. A method according to any one of claims 1 to 3, characterized in that the incubation is carried out for a period of time of between 10 min and l0 h, most preferred between 1 h and 5 h.

5. A method according to any one ai claims 3 to 4, characterised in that plasma or a plasma fraction or material from a cell culture is used as the biological material.

6. A method according to any one of claims 1 to 5, characterized in that a biological material is used which comprises a blood factor, in particular a vitamin K-dependent protein.

7. A method according to any one of claims 1 to 6, characterized in that a biological material is used which is a prothrombin complex-containing fraction.

8. A method according to any one of claims 1 to 7, characterized in that the biological material is adsorbed on a solid carrier, is purified, and incubation is carried out after elution of the purified material.

9. A method according to claim 8, characterized in that elution and incubation are effected simultaneously.

10. A method according to claim 8 or 9, characterized in that a chromatographic material is used as the solid carried, in particular a material suitable for ion exchange chromatography or affinity chromatography.

11. A method according to any one of claims 1 to 10, characterized in that further step for purifying the material are carried out, in particular a chromatographic purification.

12. A method according to any one of claims 1 to 11, characterized in that a further step for inactivating or depleting, respectively, pathogens is carried out, in particular a filtration or a heat treatment.

13. A method according to any one of claims 1 to 12, characterized in that a non-ionic detergent selected from the group of Tween and Triton ie used as the chemical agent.

14. A chromatographically purified preparation, comprising an autodynamically activatable blood factor with a portion of less than 50%, based on the content of activated and non-activated blood factor, preferably less than 40%, more preferred less Ghan 30%, still more preferred less than 20%, further preferred less than 10%, mast preferred less than 1%, and a detergent content.

15. A preparation according to claim 14, characterized in that the blood factor is selected from the group of factor VII, factor XII, factor XI and pre-kallikrein.

16. A preparation according to any one of claims 14 or 15, characterized in that it contains a prothrombin complex with a factor VIIa activity of less than 50%, based on the content of activated and non-activated factor VII, preferably less than 10%, most preferred less than 1%.

17. A preparation according to any one of claims 14 to 15, characterised in that the preparation is free from serine protease inhibitors and the cofactors thereof, respectively.

18. 1a preparation according to any one of claims 14 to 17, obtainable by a method according to any one of claims 1 to 13.