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LV10053B - Process for preparing a concentrate of blood coagulation factor viii-von willebrand factor complex from total plasma - Google Patents

Process for preparing a concentrate of blood coagulation factor viii-von willebrand factor complex from total plasma Download PDF

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Publication number
LV10053B
LV10053B LVP-92-496A LV920496A LV10053B LV 10053 B LV10053 B LV 10053B LV 920496 A LV920496 A LV 920496A LV 10053 B LV10053 B LV 10053B
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Prior art keywords
quot
treatment
concentration
buffer
chromatography
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LVP-92-496A
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English (en)
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LV10053A (lv
Inventor
Thierry Burnouf
Miryana Burnouf
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Centre Regional De Transfusion
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Publication of LV10053A publication Critical patent/LV10053A/lv
Publication of LV10053B publication Critical patent/LV10053B/lv

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hematology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Crystals, And After-Treatments Of Crystals (AREA)
  • Compounds Of Unknown Constitution (AREA)

Description

LV 10053 CĪI0C0E nOJiyqEHHH KOHIIEHTPATA KOMflJIEKCA "ΦΑΚΤΟΡ-ΥΙΙΙ + ΦΑΚΤΟΡ $OH-BHJIJIEEPAHflA”
OnncaHne
HacTOHmee μ3ο6ρθτθηηθ KacaeTca MeTOfla πρηγοτοβλθηηη H3 nejiBHOfi njia3Mbi KOHiļeHTpaTa KOMiuieKca "OaKTop-yiIl + φ3κτορ oČJiaflaiomero bhcokom cnem^tmecKofi βκτηβηοοτβιο .
JleneHHe γθμοΦηληη A ηηβθκι^ηθη φΒΚτορβ-νΐϋ ηβληθτοη odbinHoft npaKTHKoK h TpedyeT ncnoJib30BaHhh npenapaTOB bmcokoh nncTOTbi, ητοόΗ c οαηομ ctopohh, υμθηβιπητβ pMCtc 3arpH3HeHHH npenapaTa BHpycaMH h c ,r(pyrofi, nocKOJibKy ηηβθκκμη ηθο6χοαημο yacTO ποβτορητβ, ητοόι,ι m6e-aiaTB ηθκθλβτθλβηηχ HMMyHHbix nocjieflCTBufi, onacHbix ajih nauneHTa npw HaKonjieHHH 0CTaT0WHbix 33γρη3ηθηηη npenapaTa. - 2 - Β τθχηη^θοκμχ ueHTpax y?«e πρμμθηηιοτ pa3JWHHbie μθτολη očpačoTKH njia3Mbi κροΒΗ nejioBena c iļejibio o^hctkh ®aKTopa-yiII. 3τμ μθτολη bkjiio-uaioT ocaffiAeHne 3arpfl3HHiomHx προτθηηοβ c ncnojib30BaHneM pana χημη-yecKHx peareHTOB, rejib-npoHMKaiomyio χροΜβτοΓρβφΗΐο, χροΜβτοΓρβφκιο no HMMyHHOMy Cp01lļCTByj o6MeHHyiO ΧρΟΜβΤΟΓρβφΗΚ), a TaK«e BCeB03M0ffiHbie co-neTaHMH 3thx BecbMa pa3JinnHbix μθτολοβ. B njia3Me φ3κτορ-ΥΜ npncyTCTByeT jrniiib b Hedojibum KOJwqecTBax, no3TOMy rjiaBHon npodJieMofi, KOTopyio ηθο6χοαημο peuiHTb, ηβληθτοη npo-H3B0flHTejibH0CTb npoiļecca οηκοτκΗ. Κρομθ τογο, φθκτορ-ΥΙΙΙ npejļCTaBJiaeT codoft HecTadnjībHbiii προτθηη, npmieM TaKoft, κοτορκΗ μοηθτ 6ητβ 3κτιίβμ-Ρ0Β3Η flpyrHMH COflepHtamHMHCH Β ΚρΟΒΚ φΒΚΤΟρβΜΗ. OflHaKO, B aKTHBnpOBaH-hom cocroHHnn oh yTpanHBaeT cbok> JienedHyio iļeHHOCTb. TaKHM odpa30M, flJiH noflCTyna κ peuieHMio flaHHoft npodJieMbi HeotoflHMbi cnemiajībHO npncno-codJieHHbie MeTOflbi οηηοτκΗ, a TaKHe τοηΗΟβ OKOH^aTejibHoe fl03npoBaHne. 3aflBHTeJieM yie pa3pa6oTaH μθτοα o^hctkh c ncnojib30BaHneM hoho- ΟβΜΘΗΗΟΓΟ ΧρΟΜβΤΟΓ^φίφΟΒβΗΗΗ, H3JI0?KeHHblft B naTeHTHOii 3aflBKe ^paHUHH N2 88 07530. JiaHHbiii μθτολ odecnemīBaeT noJiyweHHe KOHiļeHTpaTa φ3κτορ8-yui BbicoKofi CTeneHH tjhctoth.
OjļHaKO corjiacHO 3T0My H3BecTH0My MeTOfly, KaK θτο npaKTHKyeTCH. H flPyrHMH M3r0T0BHT6JIHMH, BbinyCKaiOlllMMM aHaJIOrH^HblH npenapaT, HCnOJIb-3yeMbiw McxoflHbiM MaTepnaji npeflCTaBJiHJi co6oh KpHOOca»fleHHyio φρ3κΐ1Ηΐο njiaeMH. Ha CTaflun KpHoocajRfleHHH TepneTca 30-40% ®aKTopa-yfll, κοτορπη ocTaeTCH β οοβθτλθηηοη kmakocīh βθρχηθγο cjioh.
EbiJio 6u 3aMaHynBO pa3pa<5oTaTb μθτολ πρηγοτοβλθηηη μοκομογο npe-napaīa H3 iļejibHOH njia3Mbi, ηθ noflBeprHyTofi KpHoocasfleHHFo c iļejibio orpa-HHneHHH nOTepb Φ3ΚΤ0Ρ3-ΥΙΙΙ. ΚρΟΜβ ΤΓΟ, ΠΟΛΟδΗΗΗ ΜΘΤΟΛ HBHJICfl 6bl BecbMa Bbiroj^HbiM ynpomeHneM fljifl HeoflHHaKOBO ocHameHHbix ueHTpoB-jiadopaTopHK, BbinycKaiomHx npenapaT, rfle MHorfla 6biBaeT TpyflHO ocymecTBMTb Kpnooca?K-ΛΘΗΗΘ. Βοτ noweMy 33ηβμτθλβ pa3padoTaJi npocTofi μθτολ βηαθλθηηη omimeH-ηογο φ3κτορ3-Υ111 H3 ueJibHOH njia3Mbi, iiTO Π03Β0ληθτ nojiy^HTL· cTadmibHbin KOHueHTpaT bucokom mhctoth, npuneM flaHHbifi μθτοα xapaKTepn3yeTCH oneHb χοροΐϋΗΜ βμχο,ιιομ npoflyKTa.
TaKHM odpa30M, HacTOHUļee μ3ο6ρθτθηηθ KacaeTCH πρηγοτοβλθημη KOHiļeHTpaTa φ3κτορ3-ΥΙΙ1 H3 pejibHon njia3MU. θτοτ npopecc BKJiio^aeT oyn-CTKy, yflajieHne cocTaBJiflioinnx προτροΜόκΗΟΒΟΓΟ KOMnjieKca (φβκτορΗ Π, ΥΠ, IX, X), a TaKHie OnHCTKy aHHOHOOdMeHHbIM Χρ0Μ3Τ0Γρ3φΜρ0Β3ΗΙ4βΜ, ΠΟ-3bojihk)ihhm nepe3 Bbīdop rejiH y[ BbiMbiBaromero pacTBopa nojiynnTb κομπλθκο "φ3κτορ-Υΐυ + φ3κτορ φοΗ-BMJiJiedpaHfla" bhcokoh «hctotu. - 3 - LV 10053 3TOT ΜΘΤΟΛ ΜΟΙΚΗΟ ΤβΚΗΙΘ HCn0JIb30BaTb flJIH nOJiyiieHHH (πρΗ yCJI0BHH flonojiHHTejibHbix onepaiļHii ouhctkh) οπηιιιθηηηχ pacTBopoB Tarae ,npyrnx ΠΡΟΤΘΗΗΟΒ nJia3MbI, Τ3ΚΗΧ KaK φΗόρΜΗΟΓΘΗ, φΗόρΟΗΘΚΤΗΗ, aJIbdyMHH, HMMy-HOrJIOdyjIHHbI H aHTHTpOM(5HH-Ul.
HaHHbin Μθτο,η pa3padoTaH πρμμθηητθλβηο k njia3Me kpobh nejioBeica, HO OH paBHbIM 0dpa30M npHJIOMM Μ K nJia3Me 85ΗΒΟΤΗΟΓΟ npOHCXO®fleHHH.
CorjiacHO MeTOfly, OTBe^aromeMy HacTOHiiteMy η3ο6ρθτθηηιο, b Ka^ecTBe ηοχοληογο MaTepnajia mojkho Hcnojib30BaTb uejibHyio njia3My, ho npH btom ηθο6χο,ε;ημο ογοβορητβ, πτο 3Ta njia3Ma ηβληθτοη οβθμθη hjih 3aMopo®eH-hom c Lļejibio ee coxpaHeHHH, ho tojibko ηθ KpHooca?meHHOH. Ilejiecoodpa3-ho, ^odbi 3Ta nJia3Ma dbuia HaKonneHa b npHcyTCTBHH aHTHKoaryjiHHTa hjih ®e CTadHJiH3npyioinero pacTBopa. OdbinHo jļjiji θτοη iļejra πρημθηηιοτ hh-TpaTO-fleKCTpo30Hī)OC®aTHyio CMecb. IIejiecoodpa3HO TaK®e HcnoJib30BaTb jirodon j[pyroK οπθηηΦηπθοκηη CTadHJiH3aTop ®aKTopa-yiIl, Jindo ββοαημηη b 3Ty CMecb, Jivido 3aMeHfliomHH ee. B KaqecTBe pacTBopa, CTadmin3npyiomero ηοχο^ηηη MaTepnaji nJia3Mbi, u;ejiecoodpa3HO HcnojiB30BaTB pacTBop, coflepffiamHH CMecb 0,2-2 u mji-·*· re-napnHa, 1-5 mm 3THJieH,D;HaMHHTeTpayKcycHOM khcjioth m 1-10 mm flnxjiopHfla KaJIbUHfl, C B03M0IHHM JļOdaBJieHHeM rJIK)K03bI flO KOHLļeHTpaiiHH 5-60 Γ.Λ.-1. Μθτολ, OTBe^aioiiiHH HacTonmeMy H3odpeTeHHio b KaqecTBe HamnbHOH CTaflHH BKJnoyaeT npefl-oca®fleHwe, HBJiHiomeecfi KOMdHHauHen oca®fleHHH xjio-pnflOM dapHH h aflcopdHpoBaHHH Ha reJie rHflpoKCHfla ajnoMHHHH.
Uejiecoodpa3Ho odpadaTbrnaTb xjioph,bom dapHH njiasMy, BejiimHHa pH κοτοροΜ flOBefleHa λο 6,5, ποοτθπθηηο BBOflfl χπορΗβ dapHH ito Tex nop, nona He dy,EieT flocTHrHyTa OKOH^aTeJibHan ero KOHiļeHTpaiļHH 0,08 M, ripu HenpepbiBHOM nepeMeuiHBaHHH c nocjieflyiomeft odpadoTKOM ^eHτpHφyΓnpoBa-ημθμ πρκ 5-10°C c uejibto ycTpaHeHHH ocanmeHHbix προτθηηοβ h, HaKOHeu, oTdopoM HajļocaflotiHOH μηλκοοτη. OcaimeHHbie προτθηηη qejiecoodpa3Ho OTOdpaTb flJIH nOJiyiieHHH H3 ΗΗΧ npOTpOMdHHOBOrO KOMnJieKCa. 3aTeM ocymecTBJiHK)T κοητβκτηροββηηθ Haflocaflo^HOH ικηακοοτη c 3%-hhm rejieM rHflpoKCHfla ajnoMHHHH (πρκ pH = 6,5), a,nlcopdHpyiomero oc-TaBuiHeca 3arpH3HHiomHe προτθηηη. 3īa odpadoTKa conpoBOffiflaeTCH oxJia®-ΑθΗΗβΜ βο 5-8°C b KpnocTaTe, ^eHτpHφyΓHpoBaHHeM npn 5°C h OTdopoM HaflocafloyHOM makocth, KOTopyio Bbiflep®HBaiOT npn 5-8°C.
Vi3 ynOMHHyTOH HaflOCaflOnHOH «HflKOCTH HeOdXO^HMO y,HaJIHTb COJIH. 0TO MOffiHO OCymeCTBHTb JIHdO yJIbTpaφHJIL·TpHpOBaHHeM B npHCyTCTBHH dy-Φθρηογο pacTBopa, Hcnojib3yeMoro Ha nocjieAyioiiieM CTaflHH o^ihctkm xpo-μετογρ3Φηροβ3ηηθμ, c flodaBJieHHeM κ 3T0My pacTBopy 0,5-2 U MJI~^ re-napHHa, JiHdo χροΜ3τοτρ3φΗροΒ3ΗΗθΜ Ha ycTaHOBKe sephadex.G 25 TaK®e - 4 - C HCn0JIb30BaHHeM γΠΟΜΗΗγΤΟΓΟ βγφβρΗΟΓΟ paCTBOpa. flajiee, μθτολ, OTBenaKMUHM HacT0HiueMy Η3θόρβτβΗΜΐο, BKJiionaeT pa3-flejieHne c Mcnojib30BaHH9M aHnoHoodMOHHoro χρομβτογρβΦηροβθηηη. B na-τθητηοΑ 33ΗΒΚβ ipaHiļHM P 88 07530 3aHBMT0Jib y?Re onydJiHKOBaji βηηβλθη-Hbie hm npeHMyinecTBa pn^a cmoji c hh3ko$ HOHoodMeHHOH cnocodHOCTbio h nopaMH dojibiuoro pa3Mepa, ητο b flaJībHefiuieM odecnenHBaeT y,n;epiHBaHHe MOJieKyji, BecbMa KpynHbix no BeJinnHHe. TaKoe "npoflJieHHoe" yn;epiKaHHe MOJieKyjI n03B0JIHeT 0dpa30BbIBaTb CBH3H 3ΤΗΧ MOJieKyJI CO CMOJIOH, OTJIH-gaioīpecfl cjiadon γηβροΦο6ηοοτμ) m noflodpaTb TaKyio HOHHyio cnjiy dyφep-ηογο pacTBopa, KOTopan flonycKaeT H3dMpaTejibHoe flecopdHpoBaHne τθχ MJIH ΗΗΗΧ CBH3aHHbIX TaKHM 0dpa30M MOJieKyJI.
Cmojih flaHHoro THFia nocTynafOT b npofla»y nofl odipM ηβημθηοββηημ Fractogel. MoffiHO HCn0JIb30BaTb flEAE-Fractogel 650 ( R) a TaKie T-HJIH Ū-MAE-Fractogel φηρΜΗ Merck. HeKOTOpbie H3 3ΤΗΧ CMOJI odbBIHO flO-CTynHbi b Ton μοαμΦη^ημη, KOTopyio Hx nocTaBipK xapaKTepH3yeT KaK "mynajibueBHflHbie CMOJibi". CTpyKTypa hx MaTpup MOfli#MUMpoBaHHe c pejibio yBeJinneHHfl CBH3biBaiome$ ποβθρχηοοτη no οτηοιιιθημιο k nojioiKMTejibHbiM 3a-pnflaM, hto cnocodcTByeT yBejiHgeHmo padonefi θμκοοτη rejia. Ι^ΦθρημΚ pacTBop χροΜ3τοΓρ3φΜηβσκοη kojiohkh npeflCTaBJineT codofi dyφepHbI¾ pacTBop Ha οοηοβθ ijHTpaTa HaTpun h xjiopnAa ηοτρηη, πρπηβΜ 3HaneHHe pH pacTBopa 0TperyjiHp0BaH0 flo 7. Iļejiecoodpa3HO, HTOdbi θτοτ pacTBop coflepfflaji xjiopHfl KaJībUHH b KOjmnecTBe 0,5-6 mm, jih3hh b ko-jmnecTBe 2-4 r.h ιμιηηηη b KOJiH^ecTBe 8-11 r.ji"·*·. 0cymecTBJieHHe npopecca b flaJībHeniueM 3aKjnonaeTCH b 3a,D;aHH0M yBeJiHneHHH KOHueHTpa-u,m ynoMHHyīoro xjiopnjļa HaTpHH.
OcymecTBJieHne MeTOfla o^hctkh corjiacHo M3odpeTeHHio BKJiionaeT ββθ-Αθηηθ npefl-ognineHHoro npenapaTa, onncaHHoro Bbiuie, b χροΜ3τοΓρ3φΗ-necKyio KOJioHKy. Πρπ 3anaHHbix ycjioBHHx (0,11 M xjiopnfla HaTpHH) KOJioHKa b coctohhhm CBH3biBaTb οηθηβ KpynHbie MOJieKyjibi, HanpnMep, KOMnjieKC "φΒκτορ φοΗ-BnjuiedpaHfla + φ3κτορ-ΥΙΙΓ no3BOJiHH φHόpHHOΓeHy, ajibdyMHHy, HMMyHorjiodyjmHaM, aHTHTpoMdHHy-UI h φHdpoHeκτHHy BbiMbiBaTbCH ΒΜβοτβ c φωΐΒΤρΒΤΟΜ.
CorjiacHo npeflnogTHTejibHOMy BapnaHTy ocyinecTBJieHHH H3odpeTeHHH, Lļejlb KOTOpOTO COCTOHT B ,Π,ΟΟΤΗΙΚΘΗΗΗ OnTHMaJIbHOii 3φφβΚΤΜΒΗ00ΤΗ BblfleJie-hm φ3ΚΤορ3-ΥΙΙ], HOHHyio cnjiy dyφepHOΓO pacTBopa χροΜ3τθΓρ3φη^8θκοη kojiohkh 3a ΟβΗΗ npneM cpa3y yBejiHgHBaioT flo KOHLļeHTpaiļHH xjiopH,i];a HaT-Phh 0,27 M. CorjiacHo ,DipyroMy BapHaHTy ocymecTBJieHHH H3odpeTeHHfl yno-MHHyT'afl CTaflHH βμμμβθηηη npeflBapneTCH πρβ,ιιπροΜΗΒκοίί nyīeM yBejiHneHHH - 5 - LV 10053 M0HH0M CHJIbl paCTBOpa flO KOHlļeHTpaiļHH XJIOPMfla HaTpHfl 0,13 M C uejlbio yflajieHHH Φη6ροηθκτμη3. ΚοΜΠΛβκο "φ3κτορ-ΥΙΙ1 + φΒκτορ φθΗ-BHJiJiedpaHfla", ,D;ecopdHpyeMbiH h BbiMbiBaeMbiči b βτμχ ycjiOBHHX, o6jiaflaeT οπβηΗφΜηθοκοΜ βκτηβηοοτβιο, co-CTaBJiHromefi no μθηβιιιθη Mepe 5-10 u μγ-^. Odipn βηχοα npo,o;yKTa npn ocymeTCBJieHHn npoLļecca cocTaBJineT no MeHbiueii Mepe 350 u Ha 1 ji nc-χοβΗοη njia3Mbi h MOieT dbiTb flOBefleH no μθηβιιιθη Mepe λο 500 u Ha 1 ji npn BBefleHHH b HCxoflHyio njia3My onHcaHHofi Bbiuie CTadHJM3HpyiomeH CMecn. B 3aBHcnM0CTn ot odJiacTH nocjieflyiomero πρΗΜβΗβΗΗΗ pacTBop, co-flepffiauinn KOMnjieKC "φβκτορ-νΐΙΙ + φβκτορ φθΗ-BMjuiedpaHfla", mokho nofl-BeprHyTb flonojiHMTejibHon onepapnu οηΗοτκκ, b nacTHocTM, κοηι(θητρηρο-BaHHio n onncTKe μθτολομ ποβτορηογο xpoMaτoΓpaφHpoBaHHfl. AHajiornnHo npeflbiflymeMy noBTopņoe xpoMaTorpa®npoBaHne Tanse mojrho peajM30BaTb Ha CMOJiax MapoK HEAE- hjih T- U MAE) Fractogel η τ.π. Mohho TaKse ncnojib30BaTb h HHbie HOCHTejiH, cyJIbφaτ fleKCTpaHa, HMM0dHJW3HpoBaH-HblM renapHH, Cyj^OnponMJIOBhie CMOJIbI, CMOJIbI C ΧΜΜΜΗβΟΚΗΜ HJIH MMMyH-HblM CpOflCTBOM.
HonOJIHHTeJIbHOe ΧρθΜ3ΤΟΓρ3φΗρθΒ3ΗΗβ npOBOflHT C HCn0JIb30BaHHeM τογο ie ochobhoto (Sy®epHoro pacTBopia, ητο h paHee, CorJiacHO npefl-nonTMTejibHOMy BapnaHTy ocymecTBJieHHH η3θ6ρθτθηηη, iiejib κοτοροτο 3a-KJironaeTCH b bo3mojkho dojiee bmcokoh οτθπβηη KOHueHTpupoBaHHfl, flonoji-HHTeJIbHOe ΧΡ0Μ3Τ0Γρ3φΗρ0Β3ΗΗβ npOBOflfīT B ΤβΧ Κθ ΟΒΜΗΧ yCJIOBHflX, <iTO η nepBoe c θληηοτβθηηοΚ ογοβορκομ yBejmneHHfl mohhom chjih dyφepHOΓO pacTBopa cpa3y flo 0,27 M xjiopnfla Ηβτρηπ. CorJiacHO flpyroMy BapwaHTy ocymecTBJieHMH η3ο6ρθτθηηη nepBHtiHoe yBejmneHHe hohhom chjim 6y®epHO-ro pacTBopa flo 0,13 M xjiopnfla HaTpun npw3BaH0 yflajiHTB ocīaTonHbie 3a- rpH3HHI0mHe ΠρΟΤβΗΗΗ, a ΠΟΟΛβ^Κ)ΙΙ1ββ (ΒΤΟρΗΗΗΟβ) ΠΟΒΗΙΙΙΘΗΗβ HOHHOM CH- jibi pacTBopa flo KOHiļeHTpaiļHH xjiopHfla HaTpMH 0,27 Μ προΒΟβπτ ajth no-BbiuieHHB ocodo nHCToro KOHneHTpaTa KOMnjieKca ’^κτορ-ΥΙΙΙ + φ3κτορ Φοη-BHJiJiedpaHfla", kotophh TaKHM odpa30M npHTdpeTaeT cneL^HnecKyio aKTHB-hoctb, paBHyio no MeHBuiefi Mepe 10-20 u μγ"^.
HpyrHe προτβΜΗΗ, coflepffiamHecfl b nepBOM Φηλβτρ3τθ kojiohkh, Ta-KHe KaK HMMyHOrJIOdyJIHHbI, aJIbdyMHH^ aHTHTpOMdHH-Ul, φHdpHHOΓeH H φHdpo ΗβΚΤΗΗ, TaKffie MOflSHO OnMCTHTB H KOHL[eHTpHpOBaTb C Η0Π0ΛΒ30Β3ΗΗΘΜ odbin ΗΗΧ Χρ0Μ3Τ0Γρ3φΗηθ0ΚΗΧ ΜβΤΟβΌΒ. Μβτο,ιι, oTBenaiomHH HacTOHmeMy H3odpeTeHMio, npeflycMaTpHBaeT TaKffie odpadoTKy no ,π,θ33κτηβηροβ3ηηιο BMpycoB, BbinojiHHeMyio KaK odbmHo. B cjiy nae Mcnojib30BaHHH χημμηθοκογο peareHTa, HanpHMep, ecjiH npeflycMOTpeHa odpadoTKa c ynacTHeM pacTBopMTejiH-fleTepreHTa, to ee u,eJiecoodpa3Ho -. .6 - ΠρΟΒΟβΜΤΒ nepefl Ka»flbIM Χρ&ίέΐΟΓρβφΗρΟΒβΗΗΘΜ, HO TaK, UTOdbl B flajlL·-ηθμιιιθμ rapaHTHpoBaTb yflajieHHe ne3aKTHBnpyiomHx peareHTOB. ΠρβΛΜβΤΟΜ HaCTOHmerO Η3Ό6ρβΤβΗΜΗ HBJIflIOTCH TaKHte KOHIļeHTpaiļMH KOMriJieKca "φβκτορ-νΐΙΙ + φβκτορ φοΗ-BHJiJiedpaHfla", a TaKae KOHueHTpa-Uhh flpyrwx προτβΗΗΟΒ njia3Mbi, nojiyqaeMbix c HcnoJib30BaHM8M onHcaHHoro Bbime MeTOfla. ^·.;· ynoMHHyTHe KOHueHTpaiļHH ycTaHOBJieHbi β οοοτβθτοτβημ co CTaHflap-ΤβΜΗ φ3ρΜ8ΚΟΠΘΗ H MOryT dbīfcb VHCn0JIb30BaHbI flJIfl jiewedHbix pejieH.
Hn»ecJieflyiomHe npHMepH ,BJiJiiocTpHpyK)T nBa BapnaHTa ocyinecTBJieHHH H3odpeTeHHH, “He-BbixoflflmHe sa npeflejibi odJiacTH ero πρημθηθηηη. npHMep 1
BepyT 250 mji cBeatefi nJia3Mbi hjih 33μορομθηηοη njia3Mbi, npeflBapM-TejibHO pacTonjieHHOM flo '22-25°C. flJia3My HaKaruiHBaioT b npncyTCTBHH aHTMKoaryjiHHTa-CTadnjiH3aTopa (HanpHMep, ηΗτρ3το-Α8κοτρο3θφοοφ3ΤΗθΓθ) h flOBOflHT pH flo 6,5 yKcycHOH khcjiotoh. a) npe^-o^HCTKa K njiasMe nocpeflCTBOM nepHCTajībTimecKoro Hacoca nodaBJiniOT 20 mji 1 M paCTBOpa xjiopHfla jdapHjj^pg pH = 6,5 no oKoimaTejibHOH KOHLjeHTpa-Khh xjiopHfla 0,08 M. Xji0pnflj5apHfl.BBOflflT co cKopocTbio 4-8 mji. mhh”*, a 3aTeM CMecb ποηΛβρϊΗΒβΒτ^^οοτοΗΗΗΗ nepeMeuiHBaHHH β τθηθηηη 15 mhh.
Hajiee CMecb ηβΗτρΗφ^ρΗξ^ιοτ ripu gacTOTe BpauieHHH 2 700 mhh η πρκ TeMnepaType 8°C β τθ^θηηθ 20 mhh, nocjie <iero OTdHparoT HanocanogHyro «HflKOCTb. > 3aTeM Hafloca,iiowHyK) '«HflĶpcTb ancopdHpyioT Ha rejie rnnpoKCHna ajiro-MHHHfl MapKH Alhydrogel C 3%-HOK KOHIieHTpaUHeM AI (0H)g, COdJIIOflaH ' cooTHonieHHe 2,3 r rejin Ha 1 ji njia3MH. BejiHgHHy pH ηοεοηπτ no 6,5 η ocyinecTBJiflioT oxjiaHtneHHe b KpnocTaTe no 5°C. flajiee CMecb ^eHτpHφyΓH-pyioT npH 5°C h 2 700 mhh“* β τθ^θηηθ 20 mhh, nocjie gero OTdnpaioT Ha-nocanogHyio κηακοοτβ, KOTopyro. coxpaHHK)T npH 5°C.
Tanan odpadoTKa no3BOJifleT. ycTpaHHTb κομποηθητη προτρομ6ηηοβογο KOMnJieKca (φ8ΚτορΗ Π, ΥΠ, LX, X), KOTopbift mokho codpaTb b KagecTBe caMocTOHTejibHoro nponyKTa η ,ο^ηοτητβ η3Βθοτηημη μθτοαβμη . JIyguiHe pe3yjibTaTbi mokho' noJiygHTb KOMČHHHpoBaHHeM nsyx yKa3aHHbix ΜθτοηοΒ odpadoTKH, β to βρθμα nan odpadoTKa npenapaTOM Aihydrogei caMa no cede HenocTaTonHa ^jia nojiHoro ycTpaHeHHH npoTpoMdHHa n npyrnx κομποηθητοβ KOMnJieKca ppsbAv a OTflejibHo B3HTan odpadoTKa xjiopnnoM dapHH-ynycKaeT Ηθκοτοροβ KOJiHgecTBo φ3κτορ8-Χ, npoTpoMdHHa h, HaKOHeu, φ8ΚΤ0ρ3-ΥΠ. - 7 - LV 10053
CočpaHHyio τβκμμ odpa30M HaflocaflogHyīo MHflKocTb o^ninaroT ot cojiefi yjlΒΤρ3φΐυΐ5Τρ0Β3ΗΗβΜ B npMCyTCTBHH ΤΟΓΟ Ηίθ dyφepHOΓO paCTBOpa, KOTOpbIH MCnOJIb3yiOT Ha nOCJieflyiOmeH CTaflHH Χρ0Μ8Τ0Γρ3φπρ0Β3ΗΗΗ (CM.HHSe), C flO-daBJieHHeM renapHHa b KOJimiecTBe 1 u mjT* hjih χρομ3τογρ3Φμροβ3ηιίθμ
Ha KOJiOHKe sephadex g 25 c ncnoJib30BaHneM τογο κβ 6γφ6ρΗ0Γ0 pacTBopa. 3aTeM npuMeHHfOT 00EraHyK> o6padOTKy pacTBopHTejieM-fleTepreHTOM c uejibio fle3aKTHBHpoBaHHfl BHpycoB b TeyeHHe 6 n πρκ 24°C. Μβτο,η TaKOM odpadOTKH πρηβθ,γιθη b onncaHHH η3ο6ρθτθημη k EBponeficKofi naTeHTHoffi 3a-ηβκθ P 0 131 740. 6) OtiHCTKa Χρ0Μ3Τ0Γρ3φΗρ0Β3ΗΗβΜ
Hafloca/i;otiHyio χηακοοτβ, npouiefluiyio npefl-oyHCTKy n Rmnm, noflBep-raioT otiHCTKe χροΜ3τοΓρ3φκροΒ3ΗΗβΜ. Jļnfl θτογο πρημθηηκτ KOJiOHKy THna K 26/30 (Η3Γ0Τ0ΒΗΤΘΛΒ φΐφΜβ Pharmacia-Uppsala UJBeiļHfl) flKaMeTpOM 2,6 cm m pado^eft bucotoA 30 cm, H3 κοτορυχ 10 cm 3anojiHeHbi cmojioh HEAE-Fractogel TSK 650 ( r ) φκρΜΗ Merck. BBOflHMbiii b K0Ji0HKy npoMbiBO^Hbiū dyφepHbIH pacTBop ημθθτ cJieflyiomHH cocTaB: 10 mM UHTpaTa HaTpun, 1 mM xjiopHfla KajībUHH, 9 r.Ji.-^ rjiniflīHa, 3 r.Ji.”^ JiH3HHa; κ 3T0My dyφepHOMy pacTBopy flodaBJintoT XJiopnfl HaTpun ΛΟ ΚΟΗΘ^ίΗΟΗ KOHIļeHTpaiiHH 0,11 Μ H flOBOflHT pH flO 7.
OdpadaTbiBaeMyio npody bboaht b K0Ji0HKy co cKopocTbio 100 mji.u.
KojioHKy npoMbiBafOT dyφepHbIM pacTBopoM ajih yflajieHHH Heaflcopdupo-ββηηηχ προτβΗΗΟΒ, BKJirouaiomMX Φμ6ρμηογθη, ajibdyMHH, HMMyHorjiodyjiHHH, aHTHTpoMdHH-111 η Φη6ρηηογθη, a TaKHte areHTbi, fle3aKTHBHpyioiiine BnpycH. 3aTeM flecopdHpyK)T κομπλθκο Μφ3κτορ-Υ111 + φ3κτορ φθΗ-BnjiJiedpaHfla" nocpejņcTBOM ποβηιπθηηη hohhoh chjih pacTBopa (dyφepHOΓO pacTBopa) flo KOHIļeHTpaiļMM HCn0JIb3yeM0r0 flJIH 3Τ0Γ0 XJIOpHfla HaTPHH, paBHOH 0,27 M.
CoflepjKaiiiHŪ φ3Κτορ-ΥΙΙΙ pacTBop, nojiy<ieHHuii flaHHbiM μθτολομ, odJiafla-θτ οπβΐίΗφΗ^βοκοίί aKTHBHocTbio nopflflKa 5-10 ι u μγ'^, npiraeM θΦΦθκτηβ-HOCTb OMHCTKH ΠΟ ΟΤΗΟΙϋΘΗΗΚ) Κ ΠΛ33ΜΘ, BBOflHMOH B KOJIOHKy, COCTaBJIfleT 60-80%. HJIfl KOHUeHTpaiļHfi OdOHX φΒΚΤΟρΟΒ, T.e. φ3ΚΤ0ρ3-ΥΙΙ] Η φ3ΚΤ0ρ3 φΟΗ-BmiJiedpaHfla, BcerRa HadjiioflaeTCfl οτηοιπθηηθ, djiM3Koe κ ιυ/ιυ npimeM φaκτop φοΗ-BMJiJiedpaHfla BbipaiKaioT b eflWHHijax cc^aKTopa pHCTopeTHHa. 4HCTOTy ynoMHHyToro pacTBopa, coflepjKamero φ3κτορ-ΥΙΙΙ, eņe mokho cjierna yjiymiiMTb ββθαθηηθμ αοποληητθλβηογο χρομβτογρβΦη^θοκογο pa3fle-λθηηη, b pe3yjibTaTe κοτοροτο nojiy^aioT npoflyKT, noflJiejKaiiiHH nocjieflyio-meMy KOHiļeHTpHpoBaHHio. HanpHMep, depyT BTopyro KOJioHKy co cmojioh HEAE-Fractogei codjnoflan Te xe ycjioBHH 3anpaBKH, uto η Bbime, h ocymecTBJin-k)t npefl-npoMbiBKy 0,13 M pacTBopoM xjiopwfla HaTpun, ycTpaHflKMnyio ocTaTO^- - 8 -
Hbie 3arpH3Hfnoipie προτθζηη. 3aTeM noBbuiaioT ζοηηυιο czjiy pacTBopa ao KOHlļeHTpaiļHH ΧΛΟΡΗΑβ ΗΑΤρΖΗ 0,27 C IļeJIbK)· AeCOp6npOBaHHH Z BbIMbIBaHZfl ΟΖΖΉΘΗΗΟΓΟ AO BbICOKOŽ ΟΤΘΠΘΗΖ Z ΚΟΗΑΘΗΤρζρΟΒΑΗΗΟΓΟ ΚΟΜΠΛΘΚΟβ ”φ3ΚΤ0ρ- ym + OaKTop $0H-BHAAe<5paHAa''. Βτοργίο OnepaiļHK) KOHIJOHTpzpOBaHZH ΧρΟΜΑΤΟΓρΑφζρΟΒΑΗΖΘΜ MOffiHO 3a-ΜβΗΖΤΒ Υ·ΪΙΒΤρ3φΖ.ΠΒΤρζρθΒ3ΗΖΘΜ.
CorjiacHo ο(5ηζηομυ BapzaHTy ocymecTBJieHZH HacTonmero Ζ3ο6ρθτθηζη nepByiO CTaAHIO OZZTCKZ ΧΡ0Μ3Τ0Γρ3φζρ0ΒΑΗΖΘΜ ΠΡΟΒΟΑΗΤ Ha CMOJie flEAE-Fractogei. OAHaico BecbMa χοροιιιζβ pe3yjibTaTbi <5biJiz TaK®e nojiyweHbi c zcn0JIb30BaHZeM ΗΟΒΗΧ CMOJI φζρΜΗ Merck TaKZX KaK TMAE-Fractogel (3Aecb TMAE 03HazaeT Tpz-MeTZJiaMZH0-3TZJi) zjiz JļMAE-Fractogei UMAE-di- MAE), 3KBHBaJieHTHbIX ΠΟ CBOHCTBaM CMOJie flEAE-Fractogel. ΠρΖΜΘΗΗ-κ)τ z HOBbie cmojih T.H. "mynajibiieBZAHoro” Tzna, pa3padoTaHHbie φζρΜΟΖ Merck, KOTopbie B.MroJiJiep npeACTaBZJi Ha κοηΦθρθηαζζ no izakocthož Χρ0Μ3Τ0Γρ3φΜΜ, ΠρΟΧΟΑΗΒΙϋβΗ B ΟΤΟΚΓΟΑΒΜβ B ΖίΟΗΘ 1989 ΓΟΑ&· [ļpHMep 2 Βτοροζ BapnaHT ocymecTBJieHZH HacTonmero Ζ30<5ρθτθηζη Tarae πο-3Β0ΑΗΘΤ nOJiynZTb KOHLļeHTpaTbl φζόρΖΗΟΓΘΗΒ Μ φΖ(5ρ0ΗΘΚΤΖΗΑ ΠΡΖ ΟΑΗΟΒρβ-μθηηομ πολυζθηζζ KOHLļeHTpaTa, coAepjRaiuero φ3κτορ-Υ111 η φΑκτορ Φοη-BzjiAedpaHAa, πρζ ηθοκοαβκο ποηζηθηηομ βηχοαθ npoAyKTa, ηο πρζ ηθμηογο ΥΛΥΖιαθηηοζ οπΘΑζφζζθοκοίί βκτζβηοοτη cooTBeTCTByiomero κομπλθκοα. ΤθχηοαογηζθοκηΚ npOLtecc aHajiorzgeH npoiļeccy npzMepa 1, 3a zckjho-ζθηζθμ onepauzz βημμβαηζη KOMnjieicca "φΑκτορ-ΥΙΙΙ + φaκτop φοΗ-BzjiJie-dpaHAa" H3 nepBOZ kojiohkz co cmojioK flEAE-Fractogel. Πρζ 3T0M φζόρΖΗΟΓβΗ, aHTZTpOM(5ZH-IIJ, aJIb(5yMZH Z ZMMyHOrJIOdyJIZ-Hbl ΗΘ yAepffiHBaK)TCfl KOJIOHKOŽ Z BblMblBaiOTCH B φΖΛΒΤΡΒΤ. Κομπλθκο "φ3κτορ-ΥΙΙ1 + φΑΚΤορ φθΗ-BzjiJiečpaHAa" μοκηο ο^ζοτζτβ z KOHlļeHTpzpOBaTb Ha BTOpOŽ CTaAHZ MeTOAOM Χρ0Μ3Τ0Γρ3φΖΖΘ0Κ0Γ0 pa3Ae-jieHZH Ha KOJioHKe, coAepffiamež CMOJiy HEAE- Fractogei βγφβρΗΗζ pacTBOp, zoHHan czjia κοτοροΓΟ οοοτΒθτοτΒγβτ KOHueHTpaiļzz xjiopzAa HaTpzfl 0,17 M, ΗΘ AOnyCKai0llteŽ 0ΒΗ3ΗΒ3ΗΖΗ OCTaTOZHbDC 3arpH3HHI0mZX ΠρΟΤΘΖΗΟΒ. Υβθλζ-zeHze zohhoz czjih ao KOHneHTpaijzz xjiopzAa Ηβτρζπ 0,27 M očecnenzBaeT Aecop6zpoBaHze z βημηβαηζθ KOMnjieKca "φΑκτορ-ΥΙΙΙ + φΑκτορ φθΗ-Bzjuie-6paHAa". TaKZM očpa30M nojiynaioT npenapaT co οπθΐίζφζζβσκοζ ακτζβηοοτβκ) 10-20 ΐυ μγ-·*·. flajiee MOfflHO OtiZCTZTb z KOHiļeHTpzpoBaTb φζόρζΗΟΓθΗ z φΖδρΟΗΘΚΤΖΗ - 9 - LV 10053 C HCn0JIb30BaHHeM H3BeCTHbIX ΧρΟΜΒΤΟΓρβφΗΐίθΟΚΗΧ MeTOflOB flJIfl· nOJiyneHHH pacTBopoB, πρΗΓΟβΗΗΧ no CBoeMy KaqecTBy ajih jienedHbix uejien h onHcaH-hhx b naTeHTHOH 3anBKe ipaHLļHH N? 38 07530. ΠρΟΗΗΘ ΠΡΟΤΘΗΗΗ nJia3MH MOffiHO OHHCTHTb H KOHlļeHTpupOBaTb C HC-nojiB30BaHHeM ο6μηηηχ μθτολοβ pa3flejieHHH. ΠρΗΜΘρ 3 np0H3B0flHTeJIbH0CTb OUHCTKH MOJRHO ΘΙΚΘ ČOJiee yJiymilHTb CTa0HJIH3H- poBaHHeM ηοχοληοη iuia3MH, npeflBapuTejibHO noflBeprHyTOH oTTaHBaHHio flo 25°C, nocpeflCTBOM flodaBJieHHH cJieflyioiiteft ομθοη: renapnH 1 mji-^ 3THJieHflHaMMHTeTpayKcycHafl _n KHCJiOTa 2 mM hjih 0,74 r.Ji 1 xjiopnfl KajībiļMfl 6 mM hjih 0,67 r.ji. B 3Ty CMecb moikho τβκκβ ββθοτη flonoJiHHTeJibHO rjnoK03y (KOHueHTpa iļm 5-60 r.Ji "*). flocjie 3τογο pH noHHiKaiOT flo 6,5 flodaBJieHHeM yKcycHOH khcjioth. Hajiee BbinojiHHioT onepauHH oiihctkh to^ho Tan ae, KaK θτο H3JioateHO b npHMepax 1 hjih 2. B 3ΤΗΧ yCJIOBHHX OdlIļHH BbIXOfl npOflyKTa OnHCTKH COCTaBJIHeT ΠΟ ΜΘΗΒ uieH Mepe 500 u φβκτορβ-ΥΙΙΙ: C Ha 1 ji ηοχοαηοη njia3Mbi. 6to cooTBeTCTByeT npoHBJieHHio cyMMapHOH 3κτηβηοοτη φ3Κτορ3-Υ1Ε πρπ οπΘΐίΗφΗΗβοκοπ aKTHBHocTH nOCJieflHerO , paBHOH 10-30 u : C Ha 1 ΜΓ npoTeHHa. - 10 - LV 10053
$0PMyJIA H30EPETEHHH 1. Cnocod nojiyweHHH KOHijeHTpaTa KOMnjieKca ”φ3κτορ-ΥΙΙ1 + φ3Κτορ φθΗ-BMJIJiedpaHfla", odjsflaiomero BbICOKOH ΟΠθρΗφΗΜΘΟΚΟίί aKTHBHOCTbK), οτ-jiHuaiomHMCH τθμ, ητο c iļejiM) ynpomeHMH npopecca uejibHyio njia3My nofl-BepraioT πρθαοπμοτκθ nocpeflCTBOM AByxcTyneHnaTOH odpadoTKH cHanana XJiopHflOM dapnn, a 3aTeM γηαροκομαομ θπιομμηηη, a Tanse onncTice xpoMa-Τ0Γρ3φΗρ0Β3ΗΗΘΜ Ha aHHOHOOdMeHHOM ΓΘΠΘ, OdecnenUBaiOmeM CBH3bIBaHHe H y,E[epHtHBaHHe οπθηβ KpynHbix MOJieKyji. 2. Cnocod no n.l h OTjmnaioiitHHCH τθμ, ητο KeJibHafl njia3Ma npefl-CTaBJineT codofi cBe»yio hjw 3aMopoateHHyio njia3My.
Cnocod no π.π. 1 h 2 h OTJinnaiomHMCfl τθμ, ητο b njia3My bboaht CTadnjiH3npyioiiiyio ομθοβ, coflepffiau^io 0,2-2 u renapnHa, 1-5 mM 9th-jieHflnaMMHTeTpayKcycHoii khcjioth h 1-10 mM xjiopn,qa Kajībnufl. 4. Cnocod no π.3 n oTJinnaiomHMCH τθμ, ητο cTadMJiM3Hpytoinafl CMecb BKJītonaeT TaKHte rjnoK03y npw KOHqeHTpaiļHH ποοπθαηθμ 5-60 r.Ji. 5. Cnocod no π.π. 1-4, h OTJinnaiomHiicH τθμ, ητο npeflonHCTKa bkjiio-naeT cjieflyiomne onepaiļHH: a) ocaffifleHHe xjiopnflOM dapnn c nocjieflyioiuHM qeHTp^yrnpoBaHneM npn HH3K0M TeMnepaType h oTdopoM Ha,E[ocaflowHOH makocth; d) aflcopdnpoBaHHe Ha rejie rHflpoKCHAa ajnoMHHHH c nocjie,Ei;yK)iHHM LieHTp&Kī)yrHpoBaHHeM npw hh3koh TeMnepaType h OTdopoM HaflocaflonHofi JKHflKOCTH; b) odpadoTKy c pejibio yflajieHHfl cojiefi. 6. Cnocod no π.5 h OTjmnaioiiļHficfl τθμ, uto ocaifleHne xjiopnflOM dapnn ocymecTBJiHK)T ββθαθηηθμ pacTBOpa c KOHLļeHTpaiJ[Hefi xjiopH,qa dapnn 1 M H ΒΘΛΗΠΗΗΟΗ pH = 6,5 ΠρΗ ΗΘΠρβρΗΒΗΟΜ ΠθρθΜΘΠΙΗΒΘΗΗΗ, ΠΟΟΠΘ ΠΘΓΟ προβοαητ αθητρ^γηροβθημθ ηρη 5-10°C, a 3aTeM OTdnpaioT HaflocajDio^-HyiO ffiHflKOCTb. 7. Cnocod no n.n. 5 h 6 h OTJinwaiomHHCH τθμ, ητο aflcopdnpoBaHHe Ha rejie rnflpoKCHjņa θλιομηηηη ocymecTBJiHFOT npn KOHneHTpaiļHH γθλη 3% h βθληπμηθ pH = 6,5 c nocjiejtjnomnM pe3KHM oxjia?KAeHHeM ao 5®C, ρθητρη-®yrnpoBaHHeM npw 5°C h OTdopoM οοβθτπθηηοη khakocth βθρχηθγο cjioh. 8. Cnocod no nyHKTaM 5-7, oTJmnaroutHHCfl τθμ, ητο odpadoTKy c uejibio ycTpaHeHHH cojiefi προβοαητ yJIbτpaφHJIbτpHpoBaHHeM b npncyTCTBHH dyφepH0Γ0 pacTBopa, κοτορκη Ha nocjieAyiotueii CTaflHH npoLļecca bboaht b - 11 - xpoMaTorpa(ī)HtiecKyio kojiohkjt, πρκηθΜ κ 3T0My pacTBopy npeflBapHTejibHO AOČaBJiHiOT renapHH. 9. Cnocod no nyHKTaM 5-7, OTJinnaiomHHCH τθμ, ητο odpadoTKy c uteJiBio ycTpaHeHHH cojien npoBOflHT μθτοαομ χρομθτο^Φηροβθηηη Ha κο-λοηκθ sephadex G 25, coflepffiameH tot εθ 6y$epHbiii pacTBop, kotophh ncnojib3yiOT Ha nocjieflyiomeH CTaflHH ohhctkh μθτοαομ χρομθτογρθΦηη, πρπ-ueM κ 3T0My pacTBopy npeflBapHTejibHO flodaBJieH renapHH. 10. Cnocod no nyHKTaM 1-9, OTjmnaioiiiHficH τθμ, ητο Hafloca.iionHyK) KMflKocTb njia3Mbi, noflBeprHyTOH npefl-onncTKe, bboaht b χρομθτογρθΦη-necKyio KOJiOHKy, coflepEamyio θηηοηοο(5μθηηηη rejib, He BbinycKaioiiiHH οηθηβ KpynHbie MOJieKyjibi, ho flonycKaiomHH βμμηβθηηθ Φη6ρηηογθη3, ajibdyMHHa, HMMyH0rJI06yJIHH0B, aHTHTpOM6HHa-ll] Η φΗ6ρθΗΘΚΤΗΗ3 B φMJΪL·Tpaτ. 11. Cnocod no nyHKTaM 1-10, OTJiHnaioipficfl τθμ, ητο xpoMaTorpa-φηηθΟΚΗΗ rejib npeflCTaBJineT codoH rejib THna bhhhjioboto nojiHMepa, κ φyHκ^HOHaJIbHbIM rpynnaM κοτοροτο πρηκρθπλθηη rpynnHpoBKH HEAE hjih εθ T- hjih H-MAE. 12. Cnocod no n.n. 11 h OTJiHnaiomHHCH τθμ, ητο nojiHMepHbin rejib npeflCTaBJifleT codon. npoayKT MapKH Fractogei. 13. Cnocod no nyHKTaM 10-12, OTJiwiaiomHficH τθμ, ^to dyφepHbIM pacTBop ajih 3anojiHeHHH χροΜθτοτρθφΗηθοκοΗ kojiohkh npeflCTaBJineT codon 6yφepHbIH pacTBop Ha οοηοβθ uHTpaTa HaTpHH h xjiopHfla KajībijHH, coflep-EaiUHH rJIHIļHH H JIH3HH, ΠρΗΜΘΜ K 9T0My paCTBOpy flOdaBJIHIOT TaĶEe XJIOpHfl HaTpHH flO KOHBļeHTpanHH 0,11 Μ H flOBOflHT pH ΛΟ 7. 14. Cnocod no n.n. 13, OTJiHnaiomHiicfl τθμ, ητο dy®epHHH pacTBop co^epEKT 8-11 r.Ji"·*· rjiHUHHa h 2-4 r.Ji"^ jiH3HHa. 15. Cnocod no n.n. 10-14, OTJiwiaromHiicH τθμ, ijto HOHHyra CHJiy dy0epHoro pacTBopa yBejiH^HBai0T ^o KOHueHTpaiļHH xjiopnna HaTpHH 0,27 M c nejibio flecopdnpoBaHHH KOMnjieKca "φΗκτορ-ΥΙΙΙ + φ3κτορ φθΗ-BHjuiedpaH-fla" H3 χροΜβτοΓρβφΗϊίθοκοίί kojiohkh.
16. Cnocod no nyHKTaM 10-14, OTJiHnaiomHiicH τθμ, ητο HOHHyio cnjiy dyφepHOΓO pacTBopa noBbimaioT flo KOHiļeHTpauHH xjiopHfla HaTpHH 0,13 M c nejibio HCKJiioneHHH φHdpoHeκτHHa, a 3aTeM flo 0,27 M c nejibio flecopdn-poBaHHH KOMnjieKca "φβκτορ-ΥΙΠ + φ3κτορ φoH-BHJIJIedpaH(^a,, H3 xpoMaTO- Γρ3φπηθΟΚΟΗ KOJIOHKH. 17. Cnocod no nyHKT3M 1-16, OTJiHnafoutHficH τθμ, ητο KOMnjieKC "φβκτορ-ΥΙΠ + φθκτορ φοΗ-BHJiJiedpaHfla'’, flecopdupoBaHHHfi Η3 xpoMaTO-ΓΡ3φΗηβοκοΗ kojiohkh, noflBepraioT aonoJiHHTeJibHon onepanHH o^ihctkh h KOHueHTpHpoBaHHH xpoMaτoΓpaφHpoBaHHeM. - 12 - LV 10053 18. Cnoco6 no nyHKTaM 1-17, OTJiHnaroinHHCH τθμ, ητο flonoJiHHTejib-HyfO 0Čpa60TKy Χρ0Μ3Τ0Γρ3φΗΡ0Β3ΗΗΘΜ ΠρΟΒΟβΗΤ Ha Η0Η000ΜΘΗΗ0Η CMOJie, BbīdpaHHofi H3 rpynnbi cmoji, BKJHonaiomeH flEAE- (hjih ?κθ T- H-MAE) Fracto-gel HMM06HJIH3HP0BaHHblii aMHHOreKCHJI, HMM06HJIH3Hp0BaHHblil aMMHOreK-chji, HMMo6Hjm3HpoBaHHbiii renapHH, cyjib(|)aT .ņeKCTpaHa, cyj^onponHJi, a Tanie CMOJibi, odjiaflaiomne xHMHnecKHM hjih HMMyHHbiM cpohctbom. 19. Cnocod no n.n. ļ7 h 18, OTJiHnaroiHHKcfl τθμ, ητο αοποπηητθλβ-Hyio odpadoTKy χροΜΒτοηρ3φΗροΒ3ΗΗθΜ προΒΟ,ιι,ΗΤ c Hcnojib30BaHHeM ^φβρ-ηογο pacTBopa c κοηηθητρθηηθη xjiopHfla HaTpHH, oTperyjmpoBaHHoft ao 0,11 M, npnneM 3Ta odpačoTKa BKJiionaeT flBa cymecTBeHHbix ποβμιιιθηηη hohhoh CHJībi pacTBopa: CHanajia flo 0,13 M xjiopHfla ηθτρηη, a 3aTeM ao 0,27 M. 20. Cnocod no n.n. 17 h 18, OTjmnaiotuHHCH τθμ, ητο flonojiHHTejib-Hyio o6pa6oTKy xpoMaTorpa®HpoBaHHeM npoBOflHT c Hcnojib30BaHHeM 6yφep-ηογο pacTBopa c KOHiļeHTpaiļHeH xjiopHfla HaTpHH, 0Tperyjinp0BaHH0H flo 0,17 Μ, πρπΗθΜ 3Ta odpaČOTica 3aKJiionaeTCH b oflHOKpaTHOM ποβηιιιθηηη hohhoh CHJībi pacTBopa cpa3y flo 0,27 M xJiopHfla HaTpHH. 21. Cnocoč no n.n. 1-14, OTJiHnafomHHCH τθμ, ητο προτθηηη, coflep-latpecn b χροΜΒΤΟΓρΒφκηθοκοΜ Φη;ιβτρ3τθ, nojiyneHHOM corjiacHO n.10, noflBepraioT flonoJiHHTejibHofi onepaiļHH o^hctkh η κοηηθητρηροββηηη. 22. Cnocoč no n.n. 1-21, OTJiHtiaioinHHCH τθμ, ητο nepefl Kanmon χροΜ3τοτρ3φΗ^θοκοΗ onepaiļHeii προβοαητ o6bmHyio odpadoTKy no fle3aKTH-BHpoBaHHio BHpycoB c Hcnojib30BaHHeM pacTBopHTejiH-fleTepreHTa. - 13 - LV 10053 Ρβφθρβτ M3o6peTeHHe KacaeTCH μθτο,ι^ πρμγοτοβλθηηη KOHijaHTpaTa κοΜίυιβκ-ca '^aKTop-yffl + $aKTop-yiU φοΗ-BHJiJiedpaHfla", odjiaflaioiiiero bhcokoh cne Ι^ΗφΗ^ΘΟΚΟΗ aKTHBHOCTBIO, H3 LjeJIbHOf! (He KpHOOCaffifleHHOH) IUia3MH. Μθτο,π, BKJirouaeT npesfle Bcero npefl-o<iHCTKy, npeflCTaBJiflioii^K) co6oh flByxcTyneHwaTyK> odpa6oTKy BHauajie χλορη^ομ 6apnfl, a 3aTeM rnflpoKCH-flOM aJlIOMHHHH.
MeTOfl, κροΜβ τογο, BKjno^aeT nocjieflyiomyK) om4CTKy χροΜ3τθΓρ3φκρο BaHHeM Ha 3ηηοηοο6μθηηο{ι ομολθ THna JļEAE- Fractogei. Μθτοα BKJiro^aeT onepaLļHio fle3aKTHBHpoBaHHfl BnpycoB nocpeflCTBOM o6pa6oTKn c ncnojib30BaHHeM pacTBopHTejiH-fleTepreHTa. Μθτοα πο3βοληθτ name BbmejiHTb H3 toh ®e ruia3Mbi Φη6ρηηογθη, aJIbdyMHH, HMMyHOrJIOdyJIHHtI, aHTHTpOMdHH-Ul, φΗόρΟΗΘΚΤΗΗ H npOTpOMdHHO BblK ΚΟΜΠΛΘΚΟ.
Pa3JitmHbie KOHiļeHTpaiļHH, noJiyyaeMbie c ncnojib30BaHneM MeTOfla ot-Be^aiomero H3o6peTeHHH, npurofiHbi, b uacTHOCTM, jļjih jie*ie0Hbix uejiefl.

Claims (21)

  1. LV 10053 Izgudrojuma formula. . 1. Kompleksa "faktors ΥΙΙΙ+fon Villebranda faktors" koncentrāta, kuram piemīt augsta specifiskā aktivitāte, iegūšanas paņēmiens, atšķiras ar to, ka lai vienkāršotu procesu, veselu plazmu pakļauj iepriekšējai tīrīšanai ar div-pakāpju apstrādi vispirms ar bārija hloridu, tad alumīnija hidroksīdu, kā arī tīrīšanai ar hromatogrāfēšanu uz anjonapmainasgela, kurš nodrošina ļoti lielo molekulu noturēšanu un saistīšanu.
  2. 2. Paņēmiens saskaņā ar 1.p., atšķiras ar to, ka veselā plazma ir svaiga vai sasaldēta plazma.
  3. 3. Paņēmiens saskaņā ar 1.vai 2.p., atšķiras ar to, ka plazmā ievada stabilizējošu maisījumu, kurš satur 0,2 - 2 U ml ^ heparīna, 1-5mM etilēndiamīn-tetraetiķskābi un 1 - 10 mM kalcija hloridu.
  4. 4. Paņēmiens saskaņā ar 3.p., atšķiras ar to, ka stabilizējošais maisījums ietver arī glukozi ar koncentrāciju 5 - 60 g.l.
  5. 5. Paņēmiens saskaņā ar 1 - 4 p.p., atšķiras ar to, ka iepriekšējā tīrīšana ietver šādas operācijas: a) nogulsnēšana ar bārija hloridu ar sekojošu centrifugēšanu pie zemas temperatūras un virsnogulšņu šķidruma noņemšanu; b) adsorbēšana uz alumīnija hidroksīda gela ar sekojošu centrifugēšanu pie zemas temperatūras un virsnogulšņu šķidruma noņemšanu; c) apstrāde sāļu aizvadīšanai,
  6. 6. Paņēmiens saskaņā ar 5.p., atšķiras ar to, ka nogulsnēšanu ar bārija hloridu veic ievadot šķīdumu ar bāpija hlorīda koncentrāciju 1 M un pH=6,5 nepārtraukti maisot, pēc tam veic centrifugēšanu pie 5 - 10°C, tad novāc virsnogulšņu šķidrumu.
  7. 7. Paņēmiens saskaņā ar 5. vai 6.p., atšķiras ar to, ka adsorbēšanu uz alumīnija hidroksīda gela veic pie gela koncentrācijas 3 % un pH=6,5 ar sekojošu strauju atdzesēšanu līdz 5°C, centrifugēšanu pie 5°C un virsējā slāņa gaišākā šķidruma aizvākšanu.
  8. 8. Paņēmiens saskaņā ar 5-7 p.,atšķiras ar to, ka apstrādi, lai aizvāktu sāļus, veic ar ultrafiltrēšānu buferšķīduma klātbūtnē, kuru nākošajā procesa stadijā ievada hromatogrāfijas kolonnā pie kam šim šķīdumam iepriekš 2 pievieno heparinu.
  9. 9. Paņēmiens saskaņā ar 5 - 7 p., atšķiras ar to, ka apstrādi, lai aizvā^ktu sāļus veic ar hromatografēšanas metodi kolonnā Sephadex G 25, kura satur to pasu buferšķidumu, kuru izmanto nākošajā tīrīšanas stadijā ar hromato -grāfžsanas metodi, pie kam šim šķīdumam iepriekš pievienots heparins.
  10. 10. Paņēmiens saskaņā ar1-9p.,atšķiras ar to, ka plazmas, kura pakļauta iepriekšējai tīrīšanai, virsnogulšņu šķidrumu ievada hromatogrāfias kolonnā, kura saturanjonapmainās gelu, kurš neizlaiž ļoti lielas molekulas, bet »····· pieļauj fibrinogena, albumina, imunogldbullnu, antitromblna-III un fibronektina izskalošanu filprātā
  11. 11. Paņēmiens saskaņā ar 1 - 10 p., a t š ķ i r a s ar to, ka hromatografis-kais gels ir vinilpolimēra tipa gels, kura funkcionālajām grupām piestiprināti DEAE vai T-vai D-MAE grupējumi.
  12. 12. Paņēmiens saskaņā ar 11.p., atšķiras ar to, ka polimēru gels ir markas Fractogel produkts.
  13. 13. Paņēmiens saskaņā ar 10 - .12 p., a t š ķ i r a s ar to, ka buferšķidums hromatogrāfija kolonnas, piepildīšanai ir buferšķidums uz nātrija citrāta un kal. cija hlorīda bāzes, kurš satur glicinu un lizinu, pie kam šim šķīdumam pievieno ari nātrija hlorīdu līdz koncentrācijai 0,11 M un noved pH līdz 7.
  14. 14. Paņēmiens saskaņā ar 13.p., a t š ķ i r a s ar to, ka buferšķidums satur 8-11 gl . ^ glicinu un 2 - 4 gl.. ^ lizinu.
  15. 15. Paņēmiens saskaņā ar 10 - 14p,, atšķiras ar to/· ka buferšķlduma jonu spēku palielina līdz nātrija hlorīda koncentrācijai 0,27 H^lai desorbētu kompleksu "faktors-ΥΙΙΙ. + fon Villebranda faktors" no· hromatogrāfija kolonnas .
  16. 16. Paņēmiens saskaņā ar 10 - 14 p., atšķiras ar to, ka buferšķlduma jonu spēku paaugstina līdz nātrija hlorīda koncentrācijai 0,13 M, lai izslēgtu fibronektinu, bet pēc tam līdz 0,27 M, lai desorbētu kompleksu "faktors-ΥΙΙΙ + fon Villebranda faktors" no hromatogrāfija kolonnas
  17. 17. Paņēmiens saskaņā ar 1-I6p., atšķiras ar to, ka kompleksu " f ak-•tors-ΥΙΙΙ + fon Villebranda faktors" kurš ir desorbēts no hromatogrāfija kolonnas. pakļauj papildus tīrīšanai un koncentrēšanai ar hromatogrāfēšanu.
  18. 18. Paņēmiens saskaņā ar 1-17p.,atšķiras ar to, ka papildus apstrādi ar hromatografēšanu veic uz jonapmainasl sveķiem, kurus izvēlas no sveķu grupas, ka$ satur DEAE-(vai ari T-D-MAE)Fractogel imobilizētu aminoheksilu, imobilizētu aminoheksilu, imobilizētu heparinu, dekstrāna sulfātu, sulfopropi-lu, kā ari sveķus, kuriem ir ķīmiska vai imūna tieksme, 19. ^Paņēmiens saskaņā ar 17. vai 18.p., atšķiras ar to, ka papildus apstrādi ar hromatografēšanu veic izmantojot buferšķidumu ar nātrija hlorīda koncentrāciju, kura noregulēta līdz 0,11 M, pie kam šī apstrāde ietver divas LV 10053 3 būtiskas šķīduma jonu spēka paaugstināšanas: vispirms l£dz 0,13 M nātrijā hlorīda, pēc tam līdz 0,27 M.
  19. 20. Paņēmiens saskaņa ar 17. un 18.p., atšķiras ar to, ka papildus apstrādi ar hromatogrāfēšanu veic, izmantojot buferšķidumu ar nātrija hlorīda koncentrāciju, noregulētu lidz 0,17 M, pie kam š£ apstrāde izpaužas kā vienreizēja šķiduma jonu spēka paaugstināšana lidz 0,27 M nātrija hlori-..da,
  20. 21. Paņēmiens saskaņā ar 1 - 14 p., atšķiras ar to, ka proteinus, kurus satur hromatografias filtrātsļ kurš iegūts saskaņā ar 10.p., pakļauj papildus t£r£šanai un koncentrēšanai.
  21. 22. Paņēmiens saskaņā ar 1 - 21 p., a t š ķ i r a s ar to, ka pirms katras hromatogrāfiskās operācijas veic parasto apstrādi virušu dezaktivēšanai, izmantojot šķīdinātāju-detergentu.
LVP-92-496A 1989-09-05 1992-12-24 Process for preparing a concentrate of blood coagulation factor viii-von willebrand factor complex from total plasma LV10053B (en)

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DE3904354A1 (de) * 1989-02-14 1990-08-16 Behringwerke Ag Pasteurisiertes, gereinigtes von willebrand-faktor-konzentrat und verfahren zu seiner herstellung
ES2042138T3 (es) * 1989-05-24 1993-12-01 Miles Inc. Filtracion en gel del factor viii tratado termicamente.
AT403991B (de) * 1990-04-04 1998-07-27 Atomic Austria Gmbh Alpinschi
AT403992B (de) * 1991-02-22 1998-07-27 Head Sport Ag Ski
DE4204694C3 (de) * 1992-02-01 1995-10-12 Octapharma Ag Verfahren zur Gewinnung von hochreinem, virusinaktiviertem Faktor VIII mittels Anionenaustauscher-Chromatographie
IT1256622B (it) * 1992-12-04 1995-12-12 Sclavo Spa Processo per l'estrazione del complesso fattore viii-fattore von willebrand (fviii:c-fvw) da plasma umano totale.
DE4430205A1 (de) * 1994-08-26 1996-02-29 Behringwerke Ag Zusammensetzungen, die als Gegenmittel für Blut-Antikoagulanzien geeignet sind und deren Verwendung
DE4435485C1 (de) * 1994-10-04 1996-03-21 Immuno Ag Verfahren zur Gewinnung von hochreinem von Willebrand-Faktor
AT403764B (de) 1996-03-15 1998-05-25 Immuno Ag Stabiler faktor viii/vwf-komplex
AT403765B (de) 1996-04-12 1998-05-25 Immuno Ag Verfahren zur herstellung einer präparation enthaltend einen hochgereinigten komplex
AT406373B (de) * 1997-02-27 2000-04-25 Immuno Ag Verfahren zur reinigung von faktor viii/vwf-komplex mittels kationenaustauscherchromatographie
CA2308610A1 (en) * 1997-11-05 1999-05-14 Welfide Corporation Heparin cofactor ii preparation and process therefor
DK0937735T3 (da) * 1998-02-11 2003-06-02 Zlb Bioplasma Ag Fremgangsmåde til fjernelse af kausalt agens/kausale agentia for overførbare spongiforme encephalopatier fra proteinopløsninger
ES2541470T3 (es) * 1999-02-22 2015-07-20 University Of Connecticut Formulaciones de factor VIII libres de albúmina
DE19937218A1 (de) * 1999-08-06 2001-02-08 Aventis Behring Gmbh Verfahren zur Reindarstellung der den Blutgerinnungsfaktor VII aktivierenden Protease, ihres Proenzyms oder eines Gemisches beider Proteine mittels Affinitätschromatographie
ES2229931B1 (es) * 2003-10-03 2006-01-16 Grifols, S.A. Composicion liquida bilogicamente estable de fviii, de fvw o del complejo fviii/fvw humanos.
RU2253475C1 (ru) * 2004-02-02 2005-06-10 Общество с ограниченной ответственностью "Гемоцентр" Способ получения препарата фактора viii
FR2874216B1 (fr) * 2004-08-16 2006-11-03 Lab Francais Du Fractionnement Procede de preparation d'un concentre de facteur von willebrand (fvw) par voie chromatographique et concentre de fvw susceptible d'etre ainsi obtenu
EP2385825B1 (en) 2008-11-07 2018-10-10 University of Connecticut Factor viii formulations
WO2013083858A1 (en) 2012-04-24 2013-06-13 Novo Nordisk A/S Compounds suitable for treatment of haemophilia
CN105705516B (zh) 2013-11-08 2019-11-01 杰特有限公司 浓缩von Willebrand因子或其络合物的方法
US9663553B2 (en) 2014-01-29 2017-05-30 Hemarus Therapeutics Limited Integrated process for the production of therapeutics (human albumin, immunoglobulins, clotting factor VIII and clotting factor IX) from human plasma
CN104231073B (zh) * 2014-09-25 2017-01-25 广东双林生物制药有限公司 一种人凝血因子viii的制备方法
CN105315365A (zh) * 2015-11-17 2016-02-10 上海洲跃生物科技有限公司 一种人抗凝血酶iii的制备方法

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CA2024667A1 (en) 1991-03-06
HUT56858A (en) 1991-10-28
HU905797D0 (en) 1991-03-28
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US5679776A (en) 1997-10-21
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