CS260968B1

CS260968B1 - A method for producing human leukocyte interferon

Info

Publication number: CS260968B1
Application number: CS843139A
Authority: CS
Inventors: Leonid P Korobicyn; Anatolij M Pivovarov; Svetlana V Goncarova; Irina N Kulikova; Jurij V Tjagotin
Original assignee: Leonid P Korobicyn; Anatolij M Pivovarov; Svetlana V Goncarova; Irina N Kulikova; Jurij V Tjagotin
Priority date: 1984-04-27
Filing date: 1984-04-27
Publication date: 1989-01-12
Also published as: CS313984A1

Abstract

Řešení se týká oblasti průmyslové biologie, Způsob výroby lidského leukocytárního interferonu spočívá v odstředění krve dároe, oddělení leukooytů, hemolýze zbývajících erytrocytň s chloridem amonným resuspendování usazených leukooytů v živném prostředí podle Eagla a oddělení cílového produktu. Přínosem jg, že odstředění použité krve se uskutečňuje při 1500 až 5000 g po dobu 35 až 45 minut, odebrání 30 až 75 % objemu vrohní části leukooyty obsahující erytrooytové hmoty, hemolýza se provede roztokem 0,80 až 0,84 chloridu amonného při poměru 1 t /3 až 50/.The solution relates to the field of industrial biology. The method of producing human leukocyte interferon consists in centrifuging donor blood, separating leukocytes, hemolysis of the remaining erythrocytes with ammonium chloride, resuspending settled leukocytes in a nutrient medium according to Eagle and separating the target product. The advantage is that the centrifugation of the used blood is carried out at 1500 to 5000 g for 35 to 45 minutes, removal of 30 to 75% of the volume of the upper part of the leukocyte containing erythrocyte mass, hemolysis is carried out with a solution of 0.80 to 0.84 ammonium chloride at a ratio of 1 t /3 to 50/.

Description

3ΠΗΟΑΗΗΕ H3OBPETEHHH K ABTOPCKOMY CBHflETEJlbCTBY 3aHBjieHo: 20,08.81 3a «BKa #> 3337658 / 28-13 MKH3 A 61 K 45/02

Abtopw: Jl.n.Kopo6HHbiH, Α.Μ.Πηβοβθροβ, CBroHHaposa, HHKynHKQBaη ΙΟ, Β.Τηγοτηη

3aaBHTenb: Bcčcoioshmh HayHHo-HccnenoBaTenbCKHn HHCTHTyT oco6o nHCTtix 6ho-npenapaTOB

HaaBaHne Η3θ6ρετβΗΗΗ: CIIOCOB ΠΟΠΠΕΗΗΗ HEJIOBEHECKOrO ΠΕίΙΚΟΙίΗΤΑΡΗΟΓΌ HHTEP-ΦΕΡΟΗΑ Η3θ6ρεΤβΗΗβ OTHOCHTCH Κ oCíiaCTH npOMbnnneHHOft 0ΗθηθΓΗΗ and ΗΜβΗΗΟ κ Cnoc-6am nojiyMeHHH ΗΗτερφβροΗβ M3 jieftKOííHTQB kpobh qefloBeKa. H3BecTeH cnoco6 nonyneHHH jieftKOnRrapHoro Ηκτερφεροκβ nyTeM BbiflenoHHH ň HT OB OB OB OB OB, HHflyKllHH HHTep4) epOHOO6pa3OBaHHB, 0HOCHHTe3a ΗΗΤβρφθρθΗ3 ,, BblflejieHHHΗΗΤ.βρφερθΗ3 (1). ΟΛΗβκο Bbixofl κηετοκ cocTaBJíaeT 10-1512 rpm nonynanHH ηεδκσηΗτοΒ what «epxa-mHXCH B UeJIbHOÍi KPOBH» lÍ3BecTeH There cnocoS nonyneHHH HenoBenecKOro neŇKonHTapHoro ΗΗτβρφεροκβnyTeM uenTpi ^ yr: npoBaHH «nottopcKOň KpOBH, oŤefleneHHH ηεϋκοηΜΤΟΒ, neMonnsa ocTaTouHbix 3pnTpou; HTOB xjiophctmm ημμοηηθμ, pecycneHRHpOBaHHH ocajjKa ηεκκοηΗ -tob b nHTaTenbHoň cpene Hrna, BMněHHH neneepro npoflyiera (2),

BbIXOfl ΜΗΤβρφβρΟΗβ Ηβ3Η3ΗΗΤεηβΗ H COCTaBJIHST 4 * 10ť ME / n C eflHHHUbloĎteMa nepepaSaTbiBaeMoň KpOBH.

Uenbio θ3θ6ρετβΗΗΗ HBnneTCH noBbnueHHe Bbrxona ueneBoro npoflyKTa.yKaaaHHaH u, enb nocTHraeTCH τβΜ, ητο πρκ ocymecTBneHHH cnoco6a nonyHeHHH

uenoBeuecKoro πβήκομΗτερΗΟΓΟ ΗΗτβρφβροΗβ rivet ^ eHτpHφyΓHpoBaHHH noHopcKoňKpOBH, OTfleJieHHB JieftKOUHTOB, reMOÍIH3a OCTaTOHHbIX SpHTpOnHTOB XnOpMCTbIM aM -μοηηθμ, pecycneHflHpoBaHHH ocaflKa πεήκοηκτοΒ b nHTaTenbHoň cPen Hma, Bbine-neHHH neneBoro nponyKTa oTJiHHHTenbHoň ocoGeHHocTbio nenaerca το, HTO-neHTpH φyΓHpoBa ^^ Ile nenbHoň kpobh ocymecTBnjnoT πρκ 1500- 5000g b TeneHHe 35-45 MHHyT 260968 OTDHpaioT 30-75% οδτ, εΜρ Bepxaen uacra neHKOHHTcopepJKaiiien sphtpouhthoh Maccbi, reMOjiH3 ocymecTBJiHWT 0,80-0,842! pacTBopoM xnopHpa tip cooTHonieHHH1: (3-50).

Cnoco6 nOHCHHeTCH CJieflywmHMH npHMepaMH. ΠρΗΜβρ 1

KpoBb flOHopoB co6HpamT b njiacTHKOBbie πβκετΜ, coflepjKanme pHTpaTHbien CTa6n-jiH3aTop. Cpa3y nocne c6opa KpoBb yeHTp ^ yrHpyioT np «4 ° C in 20 MHHyTπρπ 2000g, in 6μ οτρεηΗΤΒ nna3My ot sphtpohhtob. nocne ypaneHHH njia3MM co6h-np 10-15 patoT Buf fy coaf, nepeHocHT ero προ6κρκΗ b, vk pacTBo οορερκβπριε 1-pa 3THJieHflnaMHHTeTpaaueTaTa (3flTA) h * pccýcneHpHpytoT ČMecb BbipepKHBaroT '10 MHHyT NPH + 4 ° C h ueHTpH <|> yrHpyiOT πρπ 160 g b τεηεΗΗε 10 mhh. OcapoK-HHTOb peeycneHflHpyioT b, 0 mji nHTaTenbHoň cpepbi Hrna MEM, copcpwameH 10% ue-noBeuecKoň α, ίΒοροτκκ. K nonyueHHofí cycneH3HH κηετοκ po6aBnnmT 1/10 oSteMa3ΠΤΑ h cycneH3Hio CMeiiiHBaioT c 8 ofMaMH 0.83% NH4 Cl. Περε3 HecKonbKO MHHyTcMecb ueHTpH $ yrHpyioT πρκ 1 60g b τεηεΗκε 10 MHHyT. nonyneHHbiH ocaflos πεήκο-Hhtob pecycneHflHpyioT b cBe »e nHTaTenbHoň cpepe Hrna MEM no KOHeuHoň koh-

1 nHTpa kpobh ηπη 15% οτ this KonnuecTBa πεήκορΗτοΒ, κοτοροε copepacHTca βflOHOpCKOH κροΒΗ.

JleňKopHTbi, pecycneHnHpoBaHHbie β πητβτθπβηοη cpepe Hrna, o6pa6aTbiBaroT200 ΜΕ / μπ neňKOHHTapHoro nenoBenecKoro naτερφεροΗβ πρκ 37 ° C b τεηεΗκε 2 na-cob. 3aTeM κ cycneH3HH ε κ S S S H H H H H B py -25 -25 -25 -25 -25 -25 na -25 -25 -25 -25 H H H H H H H H H H H H -25 -25 -25 -25 -25 -25 -25 -25 -25 -25. Πο HCTeneHHH 3Toro BpeMeHH HeapcopOupo-BaHHbi Ha ε η HT H H H H H H H H g g g g g g g g g g g g g g. IIonyueHHbiB oapoK κηετοκ pecycneHflHpyioT b nHTaTenbHoň cpepe HrnaMEM β κοΗεηΗΟΗ KOHneHTpapHH 5 - 10χ10β κπ / μπ. nHTaTenbHan cpepa copepxHT 5% ηεηοΒεηεοκοΗ nna3Mbi, CycneH3Hic κηετοκ HHKyČHpawT πρκ 37 ° C πρπ περεΜειΠΗΒβΗΗΗb tchghho 16-20 qacoB. Πο 33ΒεριπεΗΗΗ yKaaaHHoro cpOKa KynbTypanbHyio wHpKocTb οτρεηΗΐοτ οτ κηε-τοκ-npopypeHTOB qeHTpH & yΓΗροΒΗΗκε πρκ 1500g β τεηεΗΗε 30 MHHyT. OcapoKκηετοκ OTGpacbiBawr and KynbTypanbHyw) KHP, KOCTb BbipepacHBamT β τεηεΗκε 3-4 cy-flow NPH + 4EC h pH pacTBopa, but poBepeHHoro 2.5 1 HC1 c h penbw ηηηκτηβηρηηBHpyca-HHflyKTopa and 33τεΜ β Hen προΒορητ onpeperteHne THTpa ΗΗτερφεροΗΗ. A6conioTHHH Bbixofl ΗΗτερφεροΗβ c 1 nHTpa κροΒΗ pocraraeT 4 * 10 * ME. ΠρκΜερ 2

KpoBb coSHpawx b nnacTHKoshie naKGThi THna, Cpa3y nocne c6opa KpoBb ^ GHτpHφyΓHpy ^ oτ πρκ 2000 * 100g β τεηεΗΗε40-5 MHHyT πρκ + 4σΟ · c penbw οτρεηεΗΗΗ nnasMbi οτ sphtpophtob. Ποοηε ypaneHHHnna3Mbi co6Hparar bgpxhhg 2/3 o6T> GMa 3pHTpopHTapAn Maccbi h pHyPHpyioT β3 oStGMax 0.83 NK4C1. Cmocb peHTp ^ yrHpyroT npH 500g β τεηεΗκε 40 MHHyT. Πο-, nynGHHbiH ocaflOK κηετοκ pecycneupHpyjoT β 10 μMM 0.83% NH4C1 h peHTpH ^γy-, rHpyioT πρκ 150g β τεπεκκε 20 MHHyT. nonyneHHbift ocaflOK πείίκορΗτοΒ pecycnenpH- pyioT β ΠΗτβτεηϊιΗΟΗ CPEP Hma MEM copep »Ameitech 4% nenoBenecKoň cbibopotkh bkohchhoh KOHHOHTpapHH 10y κπ / μπ 'Βωχορ ηεήκορΗΤΟΒ cocTaBnaeT 5,0χ10β κπετοκc 1 nHTpa κροΒΗ ηπη 80% οτ hx copepJKaHHH penbrioň kpobh b, h HHpyKpHio 6hochh -T03 HHTODdlGOOHa TBK JKO IOK J ΠΡΗΜΒΡΒ 1.

KpoBb οτ poHOpoB co6HparoT b nnacTHKOBbie πβκετΜ THna "ΓεΜεκοη", copepaca-ηιηθ pHTpaTHbien cTa6HHH3aTop. Cpa3y nocne c6opa kpobh ρεΗτρΗφνΓΗργιοτ npH 2000-100g β · τεπεΗΗε 40-5 MHHyT npH + 4'C c penbw οτρεηεΗΗΗ nna3Mbi οτ 3ριιτρορΗΤθΒ. 260968 nocne yflaneHHH rinaaMbi co6npa »T BepxHHe 2/3 oSteMa spHTpoflHTapMoon Maccbi h pe-cycneHflHpyioT b 25 o & beMax 0.83% NH ^ CI.hu; eHTpn4) 80% NH4 Cl hUeHTpH $ ympyroT πρκ 150g b leuHe 20 MHHyr. nonyneHHbiň ocanox neňKOflHTOB pe-cyčneHflupyioT b πητθτθπβηοη cpefle Hrna MEM, coflepxameň 4% HenoBenecKoň cmbo-POTKH B KOHeHHOňAKOHfleHTpaflHH 10 ΚΠ / ΜΠ. BblXOfl neňKOflHTOB COCTaBnHeT 5x10 ππετοκ c 1 nHTpa kpobh ηπη 80% οτ hx κορτική coflepaiaHHH b flenbHOň kpobh. Hh-flyKflHIO H 6HOCHHTe3 ΗΗΤ ρ ρ ρ 3 3 Κ Κ Κ Κ Κ Κ Κ e 3 3 3 3 3 3 3 3 3 A6 A6 1 1 1 1 HT HT HT HT HT HT HT HT * * ÍIpHMep 4.

KpoBb οτ flOHopoB coČHpároT b nnacTHKOBbie naxeTbi Tuna 'TeMaKOH "coflepacaflHeUHTpaŤHbiň eTa6KrtH3aTop, Cpaay nocne c6opa xposb u eHTpn (i) yrHpyioT πρκ 2000 -tlOOg b τεηβΗΗβ 40í5 MHHyT πρκ + 4 * C c nenbio OTfleneHHH nna3Mbi οτ apHTpoflHToB.Ilocne yflaneHHH nna3Mbi co6npaioT BepxHHe 2.3 oCteMa spHTpoflHTapHoň Maccbi h cycneHflHpyroT PE-b-50 oS BEMAX NE ^ Cl. CMecb BbiflepscHBaror 10 MHHyT NPH + 4EC fleHT- h (150 g NPH JjyrHpyioT b TeneHne 20 MHHyT. nonyneHHbiň ocapoK κπετοκ pecycneHflHpy ROT b ΠΗΤβτεπΒΗοή CPEP; nanana MnanaEMEMlep% %ň% neno %enec %ň%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% %HTHTHTHTHTHTHTHTHTHTHTHTHTHTHTHTHTHTHTHTHTHT 4HT 4 4 4 τ τ K K K K K K K K we K n K K K K K K K K K K c c c c c c c c c c Π Π

KpoBb οτ flOHopoB coSHparoT b nnacTHKOBbie πβκετΜ Tnna "ΓβΜβκοΗ1 'coflěpacaflHeAHTpaTHbiň cTa6nnH3aTop. Cpaay nocne oTÓopa κροββ ΗεήτρΗφντΗρνιοτ πρκ 1500 gigabytes τεηεΗΗβ '90 MHH. Πρκ 4 C C nenbro OTfleneHHH nna3Mbi οτ spHTpoflHTOB. NocneyflaneHHH nna3Mbi coĎHpawT BepxHHe 75% o6beMa apHTpoflHTapHoň Macau h peeycneH- flnpyioT ee b 3 o & beMax 0.84% NH 4 Cl, BbiflepwHBakiT CMecb npH + 4tfC 20 MHHyT hfleHTpHpyioT npH 250g β τεηεΗΗε 20 MHHyT. τεηβΗΗθ20 MHHyT. OcaflOK neňKOflHTOB pecycneHflHpyroT b riHTaTenbHoň cpefle flo KOHeflfloňKOHfleHTpaflHH 10? κπ / μπ. Bbixofl neňKOflHTOB cocTaBnneT 3χ10 κπετοκ * c 1 nHTpakpobh ηπη 50% οτ o6mero coflepacaHHH β αθηβηοη kpobh. HHflyKflHio η 6nocHHTe3ΗΗτερφεροκε προβοαητ τθκ we KaK β npHMepe first 6 A6conioTHbiíí Bbixofl Η ΗτερφεροΗ3 c 1 niiTpa kpobh cocTaBnneT 6x10 ΜΕ.

KpoBb οτ flOHopoB coĎHpaioT β nnacTHKOBbie naxeTbi THna nreMaKOH "coflepaca-me RHŤpaTHbiň cTa6nnH3aTop. CPAE nocne c6opa KpoBb ΑβΗτρκφνΓΗρνιοτ πρκ5000g β τεηεΗΗε MHHyT NPH 10 4 * 0 c flenbio OTfleneHHH tina3Mbi οτ apHTpoflHTap- Maccbi Hon. Nocne yflaneHHH nna3Mbi co6Hparor BepxHHe 30% oSteMa spHTpoflHTapHoň

Maccbi h pecycneHflHpyroT β 3 o & beMa. 0.80% NH4 Cl, ΑβΗτρκφνΓΗρνιοτ ripn 250g bTeneHHe 20 MHHyr h ocafloK neňKOflHTOB pecycneHRHpyioT ποΒτορΗΟ b 10 oCteMax0,83% NH4 Cl. JleftKÓflHTbi ocaacflawT ΑβΗτρκφνΓΗροΒβΗΗεΜ πρκ 150 g b Tenemie 20 MH-H HYT pecycneHflHpyroT nHTaTenbHOň cpefle.'Bbixofl neňKOflHTOB b c 1 nHTpa kpobhcoc.TaBnneT 5 xlO9 κηβτοκ ηπη 80% οτ hx eight sets coflepacaHHH b flenbHOň kpobh.HHflyKflHio η 6ηοοηητθ3 ΗΗΤβρφβροΗβ προβοαητ so ace KaK β npHMepe first A6conroTHbiň BbiKpfl ΗΗΤβρφβροΗβ C 1 nHTpa kpobh cocTasnaeT 3xlOe ME npeflnoMeHHbiň cnoc © πο3Βθππβτ hobmchtL · Bbixofl fleneBoro npoflyKTa flo 10- 12 * · l0tf ME / n KpOBH ΠΟ CpaBHeHHIO C HSBeCTHblM CnOCOSOM 4'10 * ME / Λ KpOBH.

ΦΟΡΜΥΕΑ H30BPETÉHHH «

Cnoco6 nonyneHHH HenosenecKoro neňKoflHTapHoro ΗΗτερφεροΗβ nyTGM ιχβΗτρΗ- ^ yrHpoeaHHH flOHopcKoň kpobh, OTfleneHHH neňKOflHTOB, reMonH3a ocTaroMHbix 260,968 -4- apHTpoqHTOB xnopucTbiM θμμοηηθμ, pecycneHflHpoBaHHH ocaflKa neňKOflHŤOB b πητη-TenbHoň cpefle Hma, BbifleneHHH neneBoro npoflyKTa, OTnHnatomHňcH τβΜ, ητο, cuejibio noBbnueHHB Bbixofla peneBoro npoflyKTa , iíeHTpmjjyrupoBaHHG penbHoíí kpobhocymecTBjíHioT πρκ 1500-5000 g b TeneHHe MHHyT 35 to 45, 30 to 75% oT6nparoT o6i> eMaBepxHeft nacTH ňeňKOflHTCoflepxcameň apHTpouHTHoň Maccbi, reMonH3 ocymecTBnntoT0,80-0,84% pacTBopoM xnopHfla ημμοηηη πρκ cooTHomeHHH 1: (3-50). HCTOHHHKH ΗΗφορΜΗΐχΗΗ, npHHHTbie BO BHHMaHHe πρκ 3KcnepTH3e: 1. AHanor-ConoBbeB B.fl., ΕβκτβΜΗροΒ Γ.Α. "ΗϋτερφβροΗ b τεορκπ h npaKTHKeMeflHyHHb", M., 1970. 2. ΠροτοτΗΠ In: Human Interferon Kauppinen H.-U., Myllyly '' G, - 6, 1978p. 1-13.

PE Φ EPAT

CnOCOE ΠΟΗΥΗΕΗΗΗ HEJIOBEHECKOrO ΠΕΗΚΟΗΗΤΑΡΗΟΓΟ ΗΗΤΕΡΦΕΡΟΗΑ

CnocoS nonyneHHH nenoBenecKoro neňKopHTapHoro ΗΗτερφθροΗΗ coctoht b fleHT p ^ yrHpoBaHHH ROHopcKoň kpobh r / jejieHHH JieňKouHTOB, reM0JiH3e ocTaTonHbixSpHTpOflHTOB XflOpHCTbIM aMMOHHeM, pecycneHflHpOBaHHH ocaflKa neňKOflHŤOB B-ΠΗΤ3 TenbHoň cpefle Hma, BbifleneHHH peneBoro npoflyKTa, Hobmm ΗΒηπετοΗ το, ητοΗεΗτρκφνΓΗροΒ3Ηκβ HenbHoň kpobh ocyinecTEnnioT πρκ 1500-5000 g b TeneHHe 35-45 MHHyT, oTSHpaiOT 30-75% oSteMa BepxHeft nacTH neňKOflHTCoflep®ameft spHTpoflHT · I hoh Maccbi, reMonH3. ocymecTBnntoT 0.80-0.84% pacTBopoM xnopnfla ημμοηηη πρκjcooTHomeHHH 1: (3-50). IIpH3HaHo H3o6peTeHHeM no pe3ynbraTaM 3KcnepTH3bi, ocymecTBneHHoft FocypapcT-BeHHbíM CPοΜΗΤβτοΜ CCCP nó .nenaM H3o6peTeHHn η οτκρΜτκή, 260968

Claims

SUMMARY OF THE INVENTION A process for the production of human leukocyte interferon by centrifuging donor blood by separating leucocyte by hermolysis of the remaining erythrocytes with ammonium chloride, resuspending the sedimented ileucoyls in an eagle broth by separating the target product, The blood is centrifuged at 1500-5000 g for 35-45 minutes to collect 30-75% of the leucoacetate-containing upper part of the erythrocyte mass, followed by a 0.80 to 0.84% ammonium chloride solution at a ratio of 1: 3. to 50 /. 260968