WO2020080978A2 - Procédé de production de substitut sanguin destiné à être utilisé en médecine vétérinaire - Google Patents

Procédé de production de substitut sanguin destiné à être utilisé en médecine vétérinaire Download PDF

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Publication number
WO2020080978A2
WO2020080978A2 PCT/RU2019/050191 RU2019050191W WO2020080978A2 WO 2020080978 A2 WO2020080978 A2 WO 2020080978A2 RU 2019050191 W RU2019050191 W RU 2019050191W WO 2020080978 A2 WO2020080978 A2 WO 2020080978A2
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WO
WIPO (PCT)
Prior art keywords
hemoglobin
kda
haemoglobin
polymerized
solution
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PCT/RU2019/050191
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English (en)
Russian (ru)
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WO2020080978A3 (fr
Inventor
Рахимджан Ахметджанович РОЗИЕВ
Владимир Александрович ХАРЛАМОВ
Ольга Борисовна Брускова
Ирина Григорьевна КОНДРАШЕВА
Елена Евгеньевна КАЛАШНИКОВА
Екатерина Валерьевна БОНДАРЕНКО
Кенес Тагаевич ЕРИМБЕТОВ
Анна Яковлевна ГОНЧАРОВА
Original Assignee
Рахимджан Ахметджанович РОЗИЕВ
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Priority claimed from RU2018136935A external-priority patent/RU2782076C2/ru
Application filed by Рахимджан Ахметджанович РОЗИЕВ filed Critical Рахимджан Ахметджанович РОЗИЕВ
Publication of WO2020080978A2 publication Critical patent/WO2020080978A2/fr
Publication of WO2020080978A3 publication Critical patent/WO2020080978A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/41—Porphyrin- or corrin-ring-containing peptides
    • A61K38/42—Haemoglobins; Myoglobins

Definitions

  • the invention relates to veterinary medicine, in particular to hematology, and relates to a method for producing a blood substitute based on polymerized hemoglobin with an oxygen transfer function.
  • the method includes obtaining deoxygenated hemoglobin, its polymerization and purification.
  • the invention can be used for the production of blood-replacing solutions comparable in efficiency of gas transport (for oxygen transfer) with red blood cells of animals.
  • For its preparation it was proposed to use the erythrocyte mass of human blood with a shelf life of not more than 36 days as a raw material, and this can adversely affect the reserves of clinical blood.
  • In the technology for the preparation of the preparation "Gelenpol” there is no stage of removal of low molecular weight hemoglobin (uncrosslinked tetramer), which may adversely affect the quality of treatment of patients.
  • a known method of obtaining a blood substitute / Patent RU2341286, 2007, A61K 38/42 / including the production of deoxygenated hemoglobin, its polymerization and purification.
  • the production of deoxygenated hemoglobin includes hemolysis by adding water to the erythrocyte mass, stroma separation, precipitation of non-heme proteins and their removal from the resulting hemoglobin solution.
  • Polymerization involves treating the resulting deoxygenated hemoglobin with modified glutaraldehyde and reducing with sodium borohydride, and purification involves diafiltration.
  • deoxygenated hemoglobin use leukocyte-free erythrocyte mass; non-heme protein precipitation is carried out by adding a concentrated sodium chloride solution to the hemoglobin solution. After removal of non-heme proteins, the hemoglobin solution is concentrated by ultrafiltration. Purification by diafiltration is carried out on cut-off membranes to obtain the finished product in the range of molecular weight not more than 450 kDa and not less than 100 kDa.
  • the disadvantage of this method is its complexity, a high degree of cost of resources and time to carry out the process.
  • This drawback is due to the fact that when receiving deoxygenated hemoglobin, the stage of hemoglobin concentration by ultrafiltration is used, which lengthens the process and leads to higher cost of the product.
  • modified glutaraldehyde is used, which also lengthens the process.
  • a known method of obtaining a blood substitute / Patent RU2361608, A61K 38/42, publ. 2008 / including the production of deoxygenated hemoglobin, its polymerization, purification and drying.
  • modification of glutaraldehyde is not required, therefore, the process is reduced by one step, and the use of a modifier of glutaraldehyde, glutamic acid, is excluded.
  • Diafiltration is carried out in glass reactors, instead of polymeric disposable containers, as described in the method according to the patent / RU 2203087 /, on cut-off membranes twice (two-stage diafiltration): by molecular weight 300 kDa, and then 100 kDa. In this case, the finished product is obtained with a molecular weight in the range of 192-320 kDa.
  • This method of obtaining a blood substitute is selected for the prototype.
  • the disadvantage of the prototype is the high degree of cost of resources and time for the process. This drawback is due to the fact that during two-stage diafiltration, the polymerized hemoglobin is cleaned twice on 300 kDa and 100 kDa cut-off membranes, which lengthens the process up to 40 hours, and also requires the use of significant volumes of water for injection used to prepare the wash buffer (up to 320 l).
  • the technical problem to be solved by the claimed invention is aimed at simplifying the technology for producing a blood substitute and obtaining a safe final product.
  • the technical result consists in reducing the filtration time of polymerized hemoglobin by 4 times, reducing the volume of the washing solution to 4 times, and reducing the pyrogenicity of the finished product.
  • a method for producing a blood substitute includes the production of deoxygenated hemoglobin, its polymerization, cleaning and drying.
  • the production of deoxygenated hemoglobin includes separation, hemolysis, stroma separation, precipitation of non-heme proteins and their removal from the resulting solution hemoglobin, its filtration.
  • Polymerization includes processing the obtained deoxygenated hemoglobin with unmodified glutaraldehyde, completion of the polymerization reaction is carried out by adding sodium borohydride; purification involves diafiltration.
  • a distinctive feature of the claimed method is that to obtain deoxygenated hemoglobin, a leukocyte-free erythrocyte mass is isolated by plasmapheresis, non-heme proteins are precipitated by adding dry sodium chloride to the hemoglobin solution to a final concentration of 0.6 wt%, the diafiltration step is carried out to purify the polymerized hemoglobin in one step on cut-off membranes with a molecular weight of 100 kDa, a finished product is obtained with the following molecular composition hemoglob on:
  • Example 1 A method of obtaining a blood substitute.
  • 6 L of water for injection (VDI) with a temperature of + 6-8 ° C is supplied from a collection tank located in the solution preparation room using a peristaltic pump. The hemolysis step takes place with stirring for 40 minutes.
  • the resulting hemolysate is pumped to sequential cartridge filters with a pore size of 50; twenty; 10; 5 and 1 ⁇ m, and further into the Flexel bio-container for the deposition of non-heme proteins with a volume of 10 l.
  • dry sodium chloride is supplied to the tank to a final concentration of 0.6 wt.%, In the amount of 0.028 kg.
  • the deposition process lasts 30 minutes.
  • the hemoglobin solution is pumped to the cartridge filters with a pore size of 5; 1 and 0.65 ⁇ m, arranged in series, and then to a sterilizing filter with a pore size of 0.22 ⁇ m and then to the reactor for the synthesis of polyhemoglobin.
  • a sterile nitrogen stream (rotameter control) is fed into the reactor to mix and deoxygenate the hemoglobin. Deoxygenation is carried out until the oxygen concentration in the equilibrium gas medium reaches 1.0-2, 0 vol.% (According to the reading of the oxygen concentration sensor in the reactor). Obtaining polymerized hemoglobin (Polymerization Stage).
  • the reaction mixture is subjected to purification by diafiltration on cut-off membranes for molecules with a molecular weight of 100 kDa and above.
  • the use of only one type of membrane instead of using two types of cut-off membranes with a molecular weight of 300 kDa, and then 100 kDa, is a distinctive feature of this method, compared with the method specified in patent RU2361608.
  • the solution is washed with 80 L of VDI to partially remove the modified hemoglobin (intramolecular crosslinked tetramer) and low molecular weight compounds, less than 64 kDa.
  • the resulting permeate is concentrated to 2 L and poured into a 5 L biocontainer.
  • the washing solution is sent to a wastewater treatment plant.
  • 0.231 L of a 40% sterile glucose solution is added to a bio-container with a polymerized hemoglobin solution by sterile connection with a 0.01 L connector of a 13.6% sterile ascorbic acid solution, 0.750 L of a 0.9% sterile NaCI solution, mixed. Then, the prepared solution is fed by a peristaltic pump to a sterilizing filtration unit with a pore size of 0.22 ⁇ m. The filtrate enters the storage tank to be fed into bottles by vials with further lyophilization of the preparation, for which a lyophilizer is used to ensure sterile powder is obtained.
  • Example 2 The decrease in the pyrogenicity of the finished product.
  • the pyrogenicity test of injection solutions and the substances from which they are made is based on measuring body temperature in rabbits before and after injection (OFS 42-0061-07).
  • the drug test was carried out on a group of three rabbits with an initial temperature of 38.5 - 39.5 ° C.
  • the body temperature is measured twice in each rabbit. Differences in temperature readings in the same animal should not exceed 0.2 ° C. Otherwise, the rabbit is excluded from the test. For the initial temperature take the value of the last measurement result.
  • test drug solution is administered intravenously to animals immediately after the second temperature measurement. Temperature measurements after administration of the test drug are carried out with an interval of not more than 30 minutes for three hours. A drug is deemed pyrogen-free if the sum of the individual maximum temperature increases is less than or equal to 1.2 ° C, and the individual temperature increase in none of the rabbits does not exceed 0.5 ° C.
  • test solution in a test dose was administered
  • the substance is pyrogenic.
  • the substance is pyrogen-free.
  • Example 3 Determination of the molecular composition of hemoglobin in the finished product (blood substitute).
  • the proposed method allows to obtain a blood substitute, the basis of which is polymerized hemoglobin from the blood of cattle with the following molecular distribution: - polymerized hemoglobin with a molecular weight of more than 64 kDa - from 75% to 95%;
  • the chromatograms (Fig. 1, 2, 3) show the molecular parameters of the preparation.
  • the profile of chromatograms in the retention time interval from 12 to 18 minutes corresponds to the fraction of polymerized hemoglobin with a molecular weight of more than 64 kDa.
  • the retention time interval from 18 to 22 minutes corresponds to a profile of modified hemoglobin with a molecular weight of 64 kDa.
  • Molecular parameters were determined by HPLC using a YMC-Pack Diol-200 column. The content of fractions in the preparation was evaluated by the normalization of peak areas.
  • FIG. 1-3 presents chromatograms of three experimental batches of the drug.
  • this method of obtaining a blood substitute contributes to a 4-fold reduction in the time of the diafiltration stage of polymerized hemoglobin to remove low molecular weight compounds with a molecular weight of less than 64 kDa and contributes to a 4-fold decrease in the volume of the wash solution.
  • a decrease in the volume of the washing solution favorably affects the pyrogenicity of the resulting preparation by reducing the likelihood of contamination of the product with the permissible amount of pyrogens in water (according to FS.2.2.0019.15), as well as the cost of the preparation.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention porte sur un procédé de production de substitut sanguin à partir d'hémoglobine polymérisée, une base de solutions de substitution de sang avec fonction de transfert d'oxygène pour s'utiliser en médecine vétérinaire, y compris la production de l'hémoglobine bovine, sa polymérisation et sa purification. Pour obtenir de l'hémoglobine désoxygénée on libère une masse érythrocytaire exempte de leucocytes au moyen de plasmaphérèse, la sédimentation de protéines non hemiques s'effectuant par l'ajout à la solution d'hémoglobine de chlorure de sodium à une concentration de 0,6 % en masse, l'étape de diafiltration pour la purification de l'hémoglobine polymérisée s'effectuant en un stade sur des membranes de dissociation pour une masse moléculaire de 100 kDa, on obtient un produit fini avec une teneur en hémoglobine polymérisée possédant une masse moléculaire de plus de 64 kDa dans une gamme de 75% à 95%. Le résultat technique consiste à réduire le temps de filtration d'hémoglobine polymérisée, réduire le volume de la solution de rinçage et réduire la pyrogénécité du produit fini.
PCT/RU2019/050191 2018-10-19 2019-10-21 Procédé de production de substitut sanguin destiné à être utilisé en médecine vétérinaire WO2020080978A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
RU2018136935A RU2782076C2 (ru) 2018-10-19 Способ получения кровезаменителя для применения в ветеринарии
RU2018136935 2018-10-19

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WO2020080978A2 true WO2020080978A2 (fr) 2020-04-23
WO2020080978A3 WO2020080978A3 (fr) 2020-06-18

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Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4826811A (en) * 1986-06-20 1989-05-02 Northfield Laboratories, Inc. Acellular red blood cell substitute
IL87708A (en) * 1988-09-08 1994-04-12 Technion Inst For Research And Hemoglobin-based blood substitute possessing a colloid oncotic pressure substantially similar to human blood and method for the preparation thereof
RU2341286C1 (ru) * 2007-07-25 2008-12-20 Общество с ограниченной ответственностью "Геленпол" Способ получения кровезаменителя и установка для осуществления способа
RU2361608C1 (ru) * 2008-03-18 2009-07-20 Общество С Ограниченной Ответственностью "Научно-Производственная Компания "Медбиофарм" Кровезаменитель с функцией переноса кислорода, фармацевтическая композиция (варианты)
US7989593B1 (en) * 2010-05-27 2011-08-02 Bing Lou Wong Method for the preparation of a high-temperature stable oxygen-carrier-containing pharmaceutical composition and the use thereof

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RU2018136935A3 (fr) 2022-04-25
RU2018136935A (ru) 2020-04-20

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