CN104804078A - Method for filtering viruses in blood coagulation factor VIII of human serum - Google Patents
Method for filtering viruses in blood coagulation factor VIII of human serum Download PDFInfo
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- CN104804078A CN104804078A CN201510221518.2A CN201510221518A CN104804078A CN 104804078 A CN104804078 A CN 104804078A CN 201510221518 A CN201510221518 A CN 201510221518A CN 104804078 A CN104804078 A CN 104804078A
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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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Abstract
The invention discloses a method capable of enabling a blood coagulation factor VIII solution from human plasma to quickly flow through a nanofiltration material. Medically acceptable water-soluble divalent metal salt with the concentration being 50-500 mmol/L is added into the blood coagulation factor VIII aqueous solution of the human plasma with the concentration being 10-100 IU/ml and the pH value being 6.0-8.0, the solution passes through a filter with the pore diameter of 10-35 nanometers to remove viruses, industrial application of a nanofiltration method to virus removal is realized, added metal salt is removed through dialysis, and the natural structure of a blood coagulation factor VIII can be recovered. The titer attenuation of the blood coagulation factor VIII in a filtration process is lower than 10%, preferably, 6%, so that the method is remarkably superior to common S/D (solvent/detergent) and dry heat inactivation methods.
Description
Technical field
The present invention relates to Medical blood product and making method, specifically adopt the filtering material of nano aperture to cross the method for the virus filtered in human serum platelet cofactor Ⅰ.
Background technology
Human serum platelet cofactor Ⅰ (F VIII) refers to the blood products extracting from human plasma and obtain, and the blood coagulation disorders be mainly used in clinically caused by platelet cofactor Ⅰ shortage is corrected, the mainly treatment of hemophilia A.For guaranteeing the Clinical practice safety of product, process of producing product must carry out virus removal/inactivation step, main haematogenous virus comprises lipid-coated virus such as hepatitis B virus, hepatitis B virus (HBV), hepatitis C virus, virus of AIDS and human T-leukemia virus (HTLV-I); Nonenveloped virus, as hepatitis virus (HAV) and assays for parvovirus B 19 and TSE Protein virus cause anomaly creutzfeldt-Jacob disease (vCJD).
Manufacturing process must balancing safety demand (needs of virus removal and/or deactivation) and provide high yield, rational cost and effective highly purified the finished product, leading inactivation of virus/removal the technology of present use is pasteurization, dry heat treatment, S/D process and nanofiltration, on platelet cofactor Ⅰ goods, then general S/D and dry thermoinactivation, but S/D method and dry thermoinactivation can cause platelet cofactor Ⅰ to tire decay 12-15%, even higher.
Nanofiltration, also referred to as " virus filtration ", is have multi-layer microporous structure based on the film used, can removes virus by size exclusion.This technical qualification gentle and effectively protein isolate and virus, comprise coating and nonenveloped virus, can be used for fragile platelet cofactor Ⅰ and F VIII natural structure remained intact.Non-fat coating parvovirus effectively can be removed with the strainer of 10-35 nanometers, as: HAV/ pig parvoviral (PPV, the virus model of a tolerance)/TSE (molecular size is about 18-35nm).And platelet cofactor Ⅰ molecule is comparatively large, be considered to the strainer that effectively can not pass 10-35nm, and the strainer in larger space (such as >=35nm) effectively can not remove HAV, B19 and TSE.
When adopting the strainer of 10-50nm that platelet cofactor Ⅰ solution is carried out virus filtration, filtration velocity slowly, and easily causes filter stoppage, and whole filtration is interrupted, and effective filtration carrying capacity of strainer is very little, does not reach 5L/m
2.Filtration velocity is less than 1L/m
2h, therefore for platelet cofactor Ⅰ, the industrial filter method that do not adopt removes virus removal.
Summary of the invention
The object of the present invention is to provide a kind of human serum platelet cofactor Ⅰ solution that human plasma can be made to originate to flow through the method for nanofiltration materials fast, realize the industrial application adopting nanofiltration process except virus removal thus.
The present invention is 10-100IU/ml in concentration, pH adds the medically acceptable water-soluble divalent metal that concentration is 50-500mmol/L in the human serum platelet cofactor Ⅰ aqueous solution of 6.0-8.0, and solution removes virus removal by the strainer of 10-35 nano apertures.
In the present invention, water-soluble divalent metal can be calcium salt, zinc salt, tin salt, mantoquita, manganous salt etc., preferably water-soluble Ca salt or zinc salt.Water-soluble Ca salt comprises calcium chloride, calcium acetate, citrate of lime, caoxalate, calcium tartrate, calglucon or calcium lactate, preferably calcium chloride.Water-soluble zinc salt comprises zinc chloride, zinc sulfate, zinc acetate, zinc citrate, zinc oxalate, zinc tartrate, Zinc Gluconate or zinc lactate, preferably zinc chloride or zinc sulfate.
Calcium ion or zinc ion concentration are preferably 200-400mmol/L.
Also can add amino acid in solution, its concentration is 50-500mmol/L, preferably 200-500mmol/L.Amino acid is preferably one or more of L-arginine, L-Histidine or 1B.
In the human serum platelet cofactor Ⅰ aqueous solution, human serum platelet cofactor Ⅰ is tired and is preferably 20-70IU/ml.
The present invention passes through the effects such as the polar adsorption of divalent metal salt, and reduce the particle diameter of platelet cofactor Ⅰ, platelet cofactor Ⅰ can be made to carry out virus filtration by the strainer in 10-35nm aperture smoothly, strainer carrying capacity is higher than 15L/m
2, filtration velocity can more than 6L/m
2h, realizes the industrial application of nanofiltration process except virus removal, can remove the metal-salt added, can recover the natural structure of platelet cofactor Ⅰ by dialysis.The decay that filtration procedure platelet cofactor Ⅰ is tired is lower than 10%, and under preferable case, the decay that platelet cofactor Ⅰ is tired, lower than 6%, is obviously better than general S/D and dry thermoinactivation.
Embodiment
Embodiment 1: the filtration velocity of different calcium ionic concn
The preparation method of human serum platelet cofactor Ⅰ: obtain cryoprecipitate after blood plasma centrifugation, after aluminium hydroxide absorption or PEG process, after S/D inactivation of virus, chromatography, wash-out obtains the protein water soln containing platelet cofactor Ⅰ.
Concentration preparation listed by human serum platelet cofactor Ⅰ solution according to the form below, regulate pH to 6.95, first measure tiring of platelet cofactor Ⅰ solution, in room temperature, under operating pressure difference 2.5bar, filtered by the positioned inline of 200nm, 50nm and 20nm successively, carry out virus filtration to the platelet cofactor Ⅰ solution after preparation, after measuring filtration velocity and filtration, platelet cofactor Ⅰ is tired and is seen the following form:
See in table, calcium ion concn is in 200-400mmol/L best results, filtration velocity is 4-6 times that do not add ionic calcium soln, by measure filter after platelet cofactor Ⅰ tire and can see that platelet cofactor Ⅰ can completely by the strainer of 20nm, filtration procedure decay of tiring is less than 8%.
Embodiment 2: the filtration velocity of different platelet cofactor Ⅰ concentration
Human serum platelet cofactor Ⅰ preparation method: carry out according to embodiment 1.
L-histidine monohydrochloride 250mmol/L is added in human serum platelet cofactor Ⅰ solution, add calcium chloride 250mmol/L, regulate pH to 6.89, tire by gradient dilution platelet cofactor Ⅰ solution and be about 10-100IU/ml, in room temperature, under operating pressure difference 2.5bar, successively by the strainer of 200nm, 50nm and 20nm, carry out virus filtration to the platelet cofactor Ⅰ solution after preparing, after measuring filtration velocity and filtration, platelet cofactor Ⅰ is tired and is seen the following form:
See in table, concentration filtration efficiency when 20-70IU/ml of platelet cofactor Ⅰ is higher, platelet cofactor Ⅰ decay of tiring is less than 6%, although concentration is very fast lower than solution filtration velocity during 20IU/ml, but platelet cofactor Ⅰ is tired, decay is greater than 10%, and concentration is tired to decay higher than platelet cofactor Ⅰ during 70IU/ml and is also greater than 10%.
Embodiment 3: the filtration velocity of different aminoacids concentration
Human serum platelet cofactor Ⅰ preparation method: carry out according to embodiment 1.
Human serum platelet cofactor Ⅰ solution is prepared by ultrafiltration, add calcium chloride 250mmol/L, regulate pH to 6.95, final platelet cofactor Ⅰ solution is tired and is about 50-60IU/ml, add L-arginine hydrochloride by gradient, gradient scope is 50-400mmol/L, in room temperature, carry out the positioned inline adopting 200nm, 50nm and 15nm under operating pressure difference 2.5bar, carry out virus filtration to the platelet cofactor Ⅰ solution after preparation, after measuring filtration velocity and filtration, platelet cofactor Ⅰ is tired and is seen the following form:
See in table, add amino acid and to a certain degree can improve filtration velocity, amino acid concentration filtration velocity when more than 200mmol/L is better.
Embodiment 4: the filtration velocity of Different Zinc ionic concn
Human serum platelet cofactor Ⅰ preparation method: carry out according to embodiment 1.
Human serum platelet cofactor Ⅰ solution is prepared by ultrafiltration, add L-arginine hydrochloride 200mmol/L, regulate pH to 6.95, final platelet cofactor Ⅰ solution is tired and is about 30-35IU/ml, zinc chloride is added by gradient, gradient scope is 200-400mmol/L, at room temperature, and operating pressure difference 2.5bar, adopt the positioned inline of 100nm and 20nm, carry out virus filtration to the platelet cofactor Ⅰ solution after preparation, reduce calcium chloride concentration by dialysis after filtering, its representative result is listed in the table below:
See in table, zinc ion concentration is in 300-400mmol/L best results, and filtration velocity is about 3 times that do not add zine ion solution.
Claims (9)
1. the virus filtration method in human serum platelet cofactor Ⅰ, it is characterized in that, be 10-100IU/ml in concentration, pH adds the medically acceptable water-soluble divalent metal that concentration is 50-500mmol/L in the human serum platelet cofactor Ⅰ aqueous solution of 6.0-8.0, and solution removes virus removal by the strainer of 10-35 nano apertures.
2. virus filtration method according to claim 1, it is characterized in that, described water-soluble divalent metal is water-soluble Ca salt or zinc salt.
3. virus filtration method according to claim 2, it is characterized in that, described water-soluble Ca salt is calcium chloride, calcium acetate, citrate of lime, caoxalate, calcium tartrate, calglucon or calcium lactate.
4. virus filtration method according to claim 2, it is characterized in that, described water-soluble zinc salt is zinc chloride, zinc acetate, zinc citrate, zinc oxalate, zinc tartrate, Zinc Gluconate or zinc lactate.
5. virus filtration method according to claim 2, it is characterized in that, described water-soluble Ca salt or zinc salt concentration are 200-400mmol/L.
6. virus filtration method according to claim 1, it is characterized in that, in the described human serum platelet cofactor Ⅰ aqueous solution, tiring of human serum platelet cofactor Ⅰ is 20-70IU/ml.
7. virus filtration method according to claim 1, it is characterized in that, add amino acid in described solution, concentration is 50-500mmol/L.
8. virus filtration method according to claim 1, is characterized in that, described amino acid be L-arginine or its hydrochloride, L-Histidine or its hydrochloride, 1B or its hydrochloride one or more.
9. virus filtration method according to claim 7 or 8, is characterized in that, described aminoacids content is 200-500mmol/L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110997015A (en) * | 2017-08-23 | 2020-04-10 | 德国杰特贝林生物制品有限公司 | Methods for viral filtration of von Willebrand factor |
| CN115779683A (en) * | 2022-12-16 | 2023-03-14 | 康日百奥生物科技(苏州)有限公司 | Virus removal filtering method |
Citations (2)
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| WO2005040214A1 (en) * | 2003-10-23 | 2005-05-06 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Virally-safe factor viii with a low content of higher multimers |
| CN101480490B (en) * | 2008-01-08 | 2013-05-29 | 基立福有限公司 | Process for obtaining a concentrate of von Willebrand factor or a complex of factor VIII/von Willebrand factor and use of the same |
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- 2015-05-05 CN CN201510221518.2A patent/CN104804078B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005040214A1 (en) * | 2003-10-23 | 2005-05-06 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Virally-safe factor viii with a low content of higher multimers |
| CN101480490B (en) * | 2008-01-08 | 2013-05-29 | 基立福有限公司 | Process for obtaining a concentrate of von Willebrand factor or a complex of factor VIII/von Willebrand factor and use of the same |
Non-Patent Citations (2)
| Title |
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| KLAUS BONAZZA: "Ca2+ concentration-dependent conformational change of FVIII B-domain observed by atomic force microscopy", 《ANAL BIOANAL CHEM》 * |
| 戚茂飞等: "CaCl2对葡萄糖纳滤截留率的影响", 《南京工业大学学报（自然科学版）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110997015A (en) * | 2017-08-23 | 2020-04-10 | 德国杰特贝林生物制品有限公司 | Methods for viral filtration of von Willebrand factor |
| CN115779683A (en) * | 2022-12-16 | 2023-03-14 | 康日百奥生物科技(苏州)有限公司 | Virus removal filtering method |
| CN115779683B (en) * | 2022-12-16 | 2024-03-22 | 康日百奥生物科技(苏州)有限公司 | Virus-removing filtering method |
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