CN117427173A - Globulin protective agent - Google Patents
Globulin protective agent Download PDFInfo
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- CN117427173A CN117427173A CN202311392632.2A CN202311392632A CN117427173A CN 117427173 A CN117427173 A CN 117427173A CN 202311392632 A CN202311392632 A CN 202311392632A CN 117427173 A CN117427173 A CN 117427173A
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- globulin
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- alanine
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- isoleucine
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- 102000006395 Globulins Human genes 0.000 title claims abstract description 79
- 108010044091 Globulins Proteins 0.000 title claims abstract description 79
- 239000003223 protective agent Substances 0.000 title claims abstract description 44
- 108060003951 Immunoglobulin Proteins 0.000 claims abstract description 68
- 102000018358 immunoglobulin Human genes 0.000 claims abstract description 68
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims abstract description 63
- 235000004279 alanine Nutrition 0.000 claims abstract description 63
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 claims abstract description 45
- 229960000310 isoleucine Drugs 0.000 claims abstract description 45
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 claims abstract description 45
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims abstract description 21
- 229920000053 polysorbate 80 Polymers 0.000 claims abstract description 21
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 claims abstract description 18
- 229940068968 polysorbate 80 Drugs 0.000 claims abstract description 18
- 238000002360 preparation method Methods 0.000 claims description 43
- 235000014705 isoleucine Nutrition 0.000 claims description 41
- 108090000623 proteins and genes Proteins 0.000 claims description 37
- 210000002381 plasma Anatomy 0.000 claims description 11
- 210000002966 serum Anatomy 0.000 claims description 4
- 229960003767 alanine Drugs 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 claims description 3
- 230000004151 fermentation Effects 0.000 claims description 3
- 239000000654 additive Substances 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 claims description 2
- 238000005215 recombination Methods 0.000 claims description 2
- 238000009826 distribution Methods 0.000 abstract description 24
- 230000006641 stabilisation Effects 0.000 abstract description 11
- 238000011105 stabilization Methods 0.000 abstract description 11
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000011534 incubation Methods 0.000 abstract description 3
- 239000010836 blood and blood product Substances 0.000 abstract description 2
- 229940125691 blood product Drugs 0.000 abstract description 2
- 230000003204 osmotic effect Effects 0.000 abstract description 2
- 230000001105 regulatory effect Effects 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 46
- 102000004169 proteins and genes Human genes 0.000 description 35
- 235000018102 proteins Nutrition 0.000 description 34
- 230000000694 effects Effects 0.000 description 29
- 229920000642 polymer Polymers 0.000 description 25
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 22
- 239000000203 mixture Substances 0.000 description 21
- 238000009472 formulation Methods 0.000 description 16
- 238000000034 method Methods 0.000 description 15
- 230000001681 protective effect Effects 0.000 description 12
- 239000004471 Glycine Substances 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 229940024606 amino acid Drugs 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 10
- 206010043376 Tetanus Diseases 0.000 description 9
- 238000010438 heat treatment Methods 0.000 description 9
- 238000009928 pasteurization Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 229940072221 immunoglobulins Drugs 0.000 description 7
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 6
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 6
- 239000002244 precipitate Substances 0.000 description 6
- 238000013112 stability test Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000000539 dimer Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000002195 synergetic effect Effects 0.000 description 4
- 101000623895 Bos taurus Mucin-15 Proteins 0.000 description 3
- 206010037742 Rabies Diseases 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000012869 ethanol precipitation Methods 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 238000010253 intravenous injection Methods 0.000 description 3
- 230000010494 opalescence Effects 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 208000006454 hepatitis Diseases 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 208000005252 hepatitis A Diseases 0.000 description 2
- 208000002672 hepatitis B Diseases 0.000 description 2
- 238000011835 investigation Methods 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 229960005367 tetanus antitoxin Drugs 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000002391 anti-complement effect Effects 0.000 description 1
- 108010008730 anticomplement Proteins 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001879 gelation Methods 0.000 description 1
- 230000008570 general process Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229950008882 polysorbate Drugs 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000011085 pressure filtration Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000012430 stability testing Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229940036116 tetanus immunoglobulin Drugs 0.000 description 1
- 238000007669 thermal treatment Methods 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39516—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum from serum, plasma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention relates to the technical field of blood product production, in particular to a globulin protective agent which comprises 0.01-1.0M alanine, 0.0-0.2M isoleucine and 20-120 mu g/ml polysorbate 80. Adding the immunoglobulin protective agent into 3-250g/L immunoglobulin, and regulating pH value to 3.5-7.50 to obtain immunoglobulin product. The globulin protectant is a sugar-free prescription, so that the clinical application range is widened; the ideal molecular size distribution of the product before and after heat stabilization is ensured, and the molecular size distribution after heat stabilization is also high; can also be used for low pH incubation and special globulin potency protection. The protective agent is suitable for the prescription of the immunoglobulin, can ensure the stability of immunoglobulin products and the applicability of human bodies (including factors such as pH, osmotic pressure, toxicity and the like), and has ideal application and popularization values.
Description
Technical Field
The invention relates to the technical field of blood product production, in particular to a globulin protective agent.
Background
The immunoglobulin product is prepared from healthy human blood plasma by separating and purifying with low temperature ethanol protein separation method (pressure filtration separation method), removing anticomplement activity, and inactivating virus. As most of the production methods of the globulin are low-temperature ethanol centrifugation, the product yield is low, the stability is poor, and a protective agent needs to be added into the finished globulin product.
Chinese patent CN101972479B discloses a preparation process of intravenous injection human immunoglobulin, after the immunoglobulin is obtained, maltose or glycine is added into protein liquid, and the pH value is regulated by using 1mol/L HCl, so that the maltose content is 10+/-1% or the glycine content is 2+/-0.2%, the pH value is 3.8-4.4, and the protein content is 8+/-3%, thus obtaining the prepared protein liquid. The active ingredient of intravenous injection human immunoglobulin is protein, of which more than 95% are immunoglobulin. This technique uses maltose or glycine as a stabilizer, but uses sugar, alcohol and glycine as a protective agent has drawbacks in the effect of protecting globulin, for example, poor molecular size distribution after thermal stabilization. The use of sugar as a protective agent is not beneficial to the clinical use of sugar-contraindicated patients, can cause renal failure clinically, and has high polymer content after heat stabilization. The content of polymers is higher after the glycine protectant is thermally stabilized. Imported globulin products also use proline as a protective agent, but the use of proline presents a patent barrier, monopolized by foreign products. And proline is used as a protective agent to have a better protective effect on globulin at normal temperature or low temperature, but has a poorer protective effect at a high Wen Ji heat stability. Therefore, there is a need to develop a novel globulin protectant to obtain more desirable globulin-protecting effects while reducing the production costs.
Disclosure of Invention
The invention aims to provide a globulin protective agent to solve the technical problem that the existing globulin protective agent has an unsatisfactory protective effect on globulin under the conditions of normal temperature, low temperature and high temperature.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a globulin protectant comprises alanine, isoleucine and polysorbate 80 at working concentrations of 0.01-1.0M, 0-0.2M and 20-120 μg/ml, respectively.
The technical proposal also provides the application of the globulin protectant as the additive of immunoglobulin products
The principle adopting the technical scheme has the beneficial effects that:
the inventors found that the use of a combination of alanine and isoleucine can produce a desirable and comprehensive protective effect on immunoglobulin preparations by a large number of screening of various amino acids. In addition to the two amino acids and glycine and proline used in this scheme, methionine, acetylchromic acid and arginine do not have protective effect during use and cannot protect immunoglobulin products. Glycine, which itself has a certain protective effect after heat treatment of the immunoglobulin preparation, also shows a defect in its protective effect on the protein. However, the use of a combination of alanine and isoleucine still ensures that the molecular size distribution of the formulation is above 90% after heat treatment. Thus, the use of a combination of alanine and isoleucine not only protects the finished product, but also works well after thermal stabilization, especially for the molecular size distribution. In addition, the use of a combination of alanine and isoleucine can also be used for specific globulin potency protection, for example: hepatitis b, tetanus, hepatitis a, rabies or neocrown antibodies, etc. The protective agent of the scheme is suitable for an immunoglobulin prescription, and can ensure the stability of immunoglobulin products and the applicability of human bodies (such as pH, osmotic pressure, toxicity and the like).
Further, the globulin protectant is mixed with an immunoglobulin to form a globulin preparation; the concentration of immunoglobulin in the globulin preparation is 3-250g/L, and pH value is 3.5-7.50.
Further, the working concentration of the alanine and the isoleucine is 0.1-0.3M and 0.05-0.15M respectively; the concentration of immunoglobulin in the globulin preparation is 3-150g/L, and pH value is 4.5-5.5.
Further, the working concentration of the alanine and the isoleucine is 0.1-0.19M and 0.06-0.15M respectively; the concentration of immunoglobulin in the globulin preparation is 40-100g/L, and pH value is 5.0-5.5.
Further, the working concentrations of alanine and isoleucine were 0.15M and 0.1M, respectively; the concentration of immunoglobulin in the globulin preparation was 50g/L and the pH was 5.5.
Further, the working concentration of alanine is 0.1-0.3M; the concentration of immunoglobulin in the globulin preparation is 100-250g/L, and pH value is 6.0-6.5.
Further, the working concentration of alanine was 0.25M; the concentration of immunoglobulin in the globulin preparation was 100g/L and the pH was 6.5.
Further, polysorbate 80 was used at a working concentration of 60 μg/ml.
Further, the immunoglobulin is extracted from plasma or serum, or is prepared by a genetic recombination technique, or is prepared by a fermentation technique.
The technical principle of the technical scheme is as follows:
alanine is added into the finished product of the globulin as a protective agent or alanine and isoleucine are added into the finished product of the globulin as the protective agent, so that the polymer content of the globulin is maintained at a lower level after the finished product is heated, and the quality of the product is effectively ensured. When alanine is used as a protective agent, the protein concentration of the globulin preparation is required to be maintained at about 100g/L to effectively ensure the inhibition of the increasing trend of the polymer content. At lower protein concentrations, the protective effect of alanine was slightly poorer. When alanine and isoleucine are used, the protective effect on the preparation with the globulin concentration of 40-100g/L is good, and the alanine and the isoleucine show obvious synergistic effect. Alanine and isoleucine, when used in combination with other amino acids, respectively, did not show similar synergy.
The protective agent adopting the technical scheme can protect immunoglobulin from various sources. In particular immunoglobulins obtainable by extraction from plasma or serum, which are complex in composition and have up to hundreds of thousands of different antibody molecules, the IgG subtype distribution in the obtained preparation is consistent with the in vivo ratio. While most protectants of the prior art are only capable of acting on immunoglobulin preparations of relatively simple composition, e.g., recombinant antibody fragments (incomplete immunoglobulins), including only a small subset of immunoglobulins, with a single composition.
To sum up, the beneficial effects of this technical scheme lie in:
(1) The formula is a sugar-free formula: the preparation prescription developed in this time does not contain sugar alcohol, has wider clinical application range, lower side effect and safer product, and is particularly suitable for diabetics.
(2) The globulin product added with the protective agent has clear and transparent appearance and no opalescence.
(3) The protective agent of the scheme has the advantages of high molecular size distribution of finished products, high molecular size distribution after heat stabilization and good heat resistance.
(4) The globulin product added with the protective agent has qualified high-temperature stability and long-term stability inspection, and meets the three requirements of the Chinese pharmacopoeia of 2020 edition.
(5) The protective effect on specific antibodies is good, such as hepatitis B, tetanus, hepatitis A, rabies and new crown antibody, and the non-reduction of the super-immune titer is ensured.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available.
Example 1:
the protective agent of the technical scheme can be applied to the protection of immunoglobulin (such as antibody), wherein the antibody can be hepatitis B, tetanus, hepatitis A, rabies or neocrown antibody. The immunoglobulin can be derived from human or other animal blood plasma and serum, or from conventional gene recombination technology and fermentation technology, and can be obtained by conventional purification treatment.
After obtaining the immunoglobulin, a protecting agent is added and the pH is adjusted to obtain the finished immunoglobulin product (i.e., the immunoglobulin preparation). The components and the contents in the finished product are as follows: 3-250g/L immunoglobulin, 0.01-1.0M alanine, 0.0-0.2M isoleucine, 0-120 μg/ml polysorbate 80 (Tween 80), pH3.5-7.50.
When the concentration of immunoglobulin in the final immunoglobulin product is 100-250g/L, the protectant can be selected from 0.1-0.3M alanine, 20-120 μg/ml polysorbate 80 (Tween 80), and pH6.0-6.5. The formulation of the protective agent of alanine and polysorbate 80 is more suitable for a finished product with the concentration of immunoglobulin of 100-250g/L, preferably with the concentration of immunoglobulin of 100-200g/L, more preferably with the concentration of immunoglobulin of 100-150g/L, and the effect is optimal when the concentration of immunoglobulin is 100 g/L. In the above case, the optimum concentration of the protecting agent is: 0.25M alanine and polysorbate 80 60. Mu.g/ml, pH 6.5 is preferred.
When the concentration of immunoglobulin in the finished immunoglobulin product is 3-150g/L, the protecting agent may be selected from the group consisting of: 0.1-0.3M alanine (more preferably 0.1-0.19M), 0.05-0.15M isoleucine (more preferably 0.06-0.15M), 20-120 μg/ml polysorbate 80 (Tween 80), pH4.5-5.5. The protective agent formula of alanine, isoleucine and polysorbate 80 is more suitable for finished products with the concentration of immunoglobulin of 40-100g/L, preferably 40-50g/L, and the effect is optimal when the concentration of the immunoglobulin is 50 g/L. In the above case, the optimum concentration of the protecting agent is: 0.15M alanine, 0.1M isoleucine and 60. Mu.g/ml polysorbate 80, preferably at a pH of 5.0-5.5, more preferably at a pH of 5.5.
The immunoglobulin content is too high, the viscosity of the globulin product is high, and the opalescence is heavy; the globulin preparation is unstable at high temperature with too low a content. The concentration of the globulin adopted by the technical scheme is 3-250g/L, and different types of protective agents are added according to different conditions of the concentration of the globulin, so that the maintenance effect on the stability of the finished product is ensured.
The effect of the protective agent is described in detail in the following experiments with immunoglobulins purified from healthy human plasma. Wherein, purifying human immunoglobulin from healthy human blood plasma is a conventional method in the prior art, and the general process is as follows: separating the qualified plasma of healthy people step by low temperature ethanol precipitation method to obtain component I precipitate, component II+III precipitate, component III precipitate, and component II precipitate, and performing pasteurization, primary precipitation, secondary precipitation, TMAE chromatography, and ultrafiltration concentration to obtain immunoglobulin IgG stock solution. Wherein, the low-temperature ethanol precipitation method is to carry out precipitation by adjusting pH, conductivity, ethanol concentration, temperature and protein concentration; pasteurization is performed using glycine as a protectant and keeping the temperature at 60℃for 10 hours (the acquisition of the precipitate of fraction II and pasteurization can be seen in the prior patent "2021114428378" Process for pasteurization of globulins with a combination of protectants "steps 1-7). TMAE chromatography parameters are pH 5.0-6.00, protein concentration 10-50 g/L, conductivity not higher than 2.00ms/cm, and pore diameter of ultrafiltration membrane package 10-50 KD. And carrying out a protective agent effect experiment by using the prepared immunoglobulin IgG stock solution. The human immunoglobulin is obtained by obtaining a component II precipitate by a low-temperature ethanol precipitation method, and then carrying out pasteurization and chromatographic treatment. It should be noted that the subject of the present protocol is a protective agent that protects the final immunoglobulin product, rather than the intermediate product during pasteurization. The protein protectant added during pasteurization may be gradually removed from the globulin during subsequent processing of the pasteurization (e.g., chromatography). However, the protective agent added to the finished globulin product is not removed later and is applied to the human body together with the globulin, so that the safety and effectiveness of the protective agent are very important.
In addition to conventional immunoglobulins, the inventors have also tried to use the present solution of the globulin protectant in other specific types of immunoglobulins (e.g., tetanus antibodies) to obtain the desired appearance, molecular size distribution, and thermally stable appearance, molecular size distribution, potency. The following experimental data mainly illustrate the effects of common immunoglobulins.
The formulated amount of alanine and isoleucine (or other candidate protectant) was dissolved with water for injection, the formulated amount of immunoglobulin was added with the dissolved protectant solution, and the formulated amount of polysorbate 80 was added, and the pH of the system was adjusted to the specified level to obtain an globulin product (finished product). The composition and content of the substances in the globulin preparations are shown in Table 1. Finished product detection is carried out according to three intravenous injection human immunoglobulin (pH 4) in Chinese pharmacopoeia from aspects of appearance, molecular size distribution, appearance after thermal stabilization, molecular size distribution, potency protection and the like.
Appearance inspection: the method is carried out by referring to the three parts of the 2020 edition of Chinese pharmacopoeia, and the appearance standard is as follows: colorless or pale yellow clear liquid, which can be slightly opalescent, should not appear cloudy. The experimental results are shown in table 1.
The thermal stability test process is as follows: after the sample is placed in a water bath at 57+/-0.5 ℃ for 4 hours, the sample is observed by naked eyes by a visible foreign matter inspection device without gelation or floccule, and the molecular size distribution of the sample is detected. The experimental results are shown in tables 2 and 3.
The detection process of the molecular size distribution comprises the following steps: the method is carried out by referring to rule 3122 of three general principles of Chinese pharmacopoeia of 2020 edition, and a sample can be divided into three main peaks, namely a polymer, a dimer and a monomer by a liquid chromatographic column, wherein a certain amount of cracking bodies can exist in the sample. Wherein the molecular size distribution is the sum of dimer and monomer. If cleavage is present, the sum of the molecular size distribution and the multimer is not 100%.
The high-temperature stability investigation and detection process comprises the following steps: the samples were subjected to investigation at 40℃for 4 months. The experimental results are shown in table 5.
The procedure for specific globulin titer measurement (tetanus antibody specific activity) was: the tetanus antitoxin titer is measured by referring to a 2020 edition of Chinese pharmacopoeia 'four-part rule 3508 mouse test method, the protein content is detected by referring to 2020 edition of Chinese pharmacopoeia' three-part rule 0731 first method, and the ratio of the tetanus antitoxin titer to the protein content is the specific activity of the tetanus antibody. The plasma is collected from healthy plasma donors according to the national approved procedure, the plasma with high tetanus antibody titer is screened out, and then the tetanus immunoglobulin is obtained through separation and purification. The experimental results are shown in table 4.
Table 1: the external appearance before and after the thermal stability test of different protectant formulations (heat treatment condition: heat preservation in water bath at 57+ -0.5 ℃ C. For 4 hours; polysorbate 80. Mu.g/ml is added into each formulation)
As can be seen from the data in Table 1, the use of different protectants ensures that the product meets the requirements in terms of appearance before and after thermal stabilization. The protein concentration in the preparation is too high, which causes opalescence, so that the protein concentration of the product is preferably controlled below 250g/L to ensure the optimal appearance. In addition, if the concentration of the protein is too low, the thermal stability of the globulin is affected, and if the concentration of the globulin is controlled to 50g/L or more, the concentration of the protein does not greatly affect the thermal stability of the globulin, and the molecular size distribution before and after the thermal stability does not greatly change. Therefore, the product appearance and the molecular size distribution after heat stabilization are comprehensively considered, and the concentration of the globulin of 50-150g/L is adopted as the optimal range.
Table 2: the molecular size distribution of the products after heat stabilization treatment using different protectants (heat treatment conditions: heat preservation in a water bath at 57+ -0.5 ℃ C. For 4 hours; immunoglobulin concentration is set to 50 g/L; polysorbate 80 60. Mu.g/ml is added to each formulation; the selected pH conditions are the optimum pH for the formulation after testing)
From the data in Table 2, it can be seen that the combination of alanine and isoleucine ensures that the product has an ideal molecular size distribution after heat treatment, and that the stability of the product is ideal. If alanine is used in combination with other amino acids than isoleucine or isoleucine is used in combination with other amino acids than alanine, the effect of maintaining the molecular size distribution of the heat-treated product is poor. In addition, alanine and isoleucine are used alone, and the effect of maintaining the molecular size distribution of the heat-treated product is not satisfactory. This demonstrates that the combination of alanine and isoleucine gives the most desirable synergistic effect. More specifically, comparing the experimental data of No. 3 (alanine 0.25M), no. 8 (alanine 0.15 m+isoleucine 0.1M) and No. 10 (isoleucine 0.25M), it can be seen that the total amount (concentration) of the protectant used by the three sets of experiments is consistent. Alanine and isoleucine are used independently, and the polymer content after the heat stability test can be only maintained to be about 30%, and the total dosage of alanine and isoleucine which are the same are used in combination, so that the polymer content after the heat stability test can be reduced to below 5%. This demonstrates that the combination of alanine and isoleucine provides a synergistic effect in maintaining the stability of the globulin preparation, as would be unexpected to one skilled in the art. Alanine and other amino acids in combination (numbers 5-7, 9) were not effective to significantly reduce the polymer content after the heat stability test relative to the formulation of number 3 at the same total dose. Isoleucine in combination with other amino acids (numbers 11-12) was not effective to significantly reduce the polymer content after heat stability testing at the same total dose relative to the formulation of number 10. This suggests that the combined use of alanine and isoleucine is more beneficial than other amino acid combinations, beyond expectations.
Table 3: detection results of molecular size distribution after thermal stabilization of the composition and content of substances in the globulin products (thermal treatment conditions: 57.+ -. 0.5 ℃ water bath for 4 hours)
In addition to the test of table 3 above, a globulin preparation containing a protective agent was formulated and tested for thermal stability as follows:
the content of detected polymers was 60% after incubation in a water bath at 57.+ -. 0.5 ℃ for 4 hours at 100g/L maltose, 80. Mu.g/ml polysorbate, pH4.2, protein concentration 50 g/L. If the protein concentration is raised to 100g/L, 150g/L or 200g/L, and the temperature is kept for 4 hours in a water bath at 57+/-0.5 ℃, the phenomenon of complete coagulation of the globulin product can occur.
The content of detected polymers was 20% after incubation in a water bath at 57.+ -. 0.5 ℃ for 4 hours at glycine 0.25M, polysorbate 80 60. Mu.g/ml, pH4.8, protein concentration 50 g/L. If the protein concentration was raised to 200g/L, the detected multimer content was 75%.
From the above experimental data, it can be seen that:
glycine or maltose is used as a protective agent for the finished globulin product, and the effect of maintaining the polymer content after the finished product is heated is not ideal for the case of low protein concentration. Particularly, after the protein concentration is increased, the finished product is heated and even the polymer content is increased sharply, and even solidification occurs. The technical scheme adopts alanine or the combination of alanine and isoleucine, so that the problem that the polymer content of the finished product is too high after being heated under the condition of higher protein concentration can be solved; and has better protection effect than other types of protective agents under the condition of smaller concentration of globulin. Alanine alone as a protective agent can maintain a multimeric content of 30% or less and a combination of alanine and isoleucine as a protective agent can maintain a multimeric content of 40% or less even at protein concentrations as high as 150g/L and 200 g/L. Alanine or a combination of alanine and isoleucine has a more desirable effect than maltose and glycine, which is an amino acid, at a high protein concentration, and shows a slightly better effect at a high protein concentration of alanine. And the combined use of alanine and isoleucine can also improve the protective performance of the protective agent under the condition of lower protein concentration.
More specifically, alanine alone was used as a protein protectant, and in the case of a preparation having a large protein concentration (e.g., 100g/L, table 3 Nos. 8 and 9), the pH of the preparation was maintained at a level of 6.0 to 6.5, and the polymer content was not excessively high (about 7%) after heat treatment of the preparation. However, when the protein concentration of the preparation is too high (e.g., 200g/L, table 3 No. 11) or too low (e.g., 50g/L, table 3 No. 12), the polymer content of the preparation after heat treatment may be too high. This demonstrates that alanine alone, under specific conditions (protein concentration in the formulation is around 100 g/L), has a better quality maintenance effect of the globulin formulation, improving the overall stability of the product. When the protein concentration in the preparation changes, the polymer content of the preparation after heat treatment may increase rapidly. Such a phenomenon was not expected by the inventors before the experimental results were seen.
More specifically, if isoleucine and alanine are used in combination, this limitation of the concentration of the formulation is markedly eliminated. As shown in Table 3, nos. 2, 13 and 14, when isoleucine and alanine were used as the protein protectant at a protein concentration of 40g/L to 100g/L in the preparation, the polymer content could be maintained at a relatively low level (about 10% and below). In particular, the synergistic effect of isoleucine and alanine was maintained at very low levels (about 3%) at protein concentrations of 50g/L in the formulation. Thus, the combined use of isoleucine and alanine not only synergistically increases the protective effect of globulin, but also overcomes the negative effect of protein concentration on the polymer content after heat treatment. In this case, the degree of freedom in formulating the globulin preparation is higher, and various types of globulin preparations having a protein content of 50g/L to 100g/L can be formulated without considering the influence of the protein concentration in this range on the quality of the product.
In addition, the pH value of the preparation is very important for the action and effect of the combined protective agent of alanine and isoleucine, the most suitable pH value is 5.0, and other pH values are selected to cause the increase of the content of the polymer. If the pH is set to 6.5 (No. 7), the polymer content will be increased to 40% or more, and the polymer content will be increased by more than 10 times relative to the pH of No. 2, which is 5.0. The setting of pH4.2 (No. 1) also resulted in a rise in the polymer content to about 10%, which was approximately 3-fold higher than the pH of No. 2, 5.0. The above data illustrates that the protectant of the present embodiment is particularly sensitive to pH and that slightly adjusting pH results in significant amounts of effect that are more than the inventors expect. The protective agent of this embodiment preferably exerts its effect by maintaining the pH at a level of 4.5 to 5.5, in particular at a level of 5.0.
In addition, the total concentration of the combined protectant has a certain influence on the exertion of the effect of the protectant. The effect is best when the total concentration of alanine and isoleucine is 0.25M. At optimal pH conditions, if the total concentration of alanine and isoleucine exceeds 0.25M (Table 3, no. 5), this also results in an increase in the polymer content.
Table 4: variation of specific activity of tetanus antibody (globulin concentration 50g/L, polysorbate 80 concentration 60. Mu.g/ml) stored at 40℃under different prescription conditions
In Table 4, the differences in specific activities of tetanus antibodies (1.67 and 1.61) in the two formulations were due to differences in formulation on day 0. From the experimental data in table 4, both formulations were ideal for protecting the antibody titers in the globulin preparations, and the titers were not reduced during 24 days of placement. Although the different formulations did not have a significant effect on potency, increased multimers of the 0.25M alanine preparation as a protectant were found during the stability high temperature study, and the preparation was subjected to a molecular size distribution experiment after 4 months at high temperature. The experimental results show that under the condition that only alanine is used as a protective agent, the ratio of dimer to monomer is reduced to 92% after 4 months of high-temperature experiment, the ratio of polymer to 8%, and the stability is poor, and the ratio of dimer to monomer can be effectively maintained when two amino acids are used in combination, and the experimental results are shown in Table 5 in detail.
Table 5: high temperature stability test results (treatment conditions: 40 ℃ C. For 4 months; concentration of globulin 50g/L, concentration of polysorbate 80 60. Mu.g/ml)
The foregoing is merely exemplary of the present invention, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present invention, and these should also be regarded as the protection scope of the present invention, which does not affect the effect of the implementation of the present invention and the practical applicability of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (10)
1. A globulin protectant characterized by: comprises alanine, isoleucine and polysorbate 80 with working concentration of 0.01-1.0M, 0-0.2M and 20-120 μg/ml respectively.
2. A globulin protectant according to claim 1, wherein: mixing the globulin protectant with an immunoglobulin to form a globulin preparation; the concentration of immunoglobulin in the globulin preparation is 3-250g/L, and pH value is 3.5-7.50.
3. A globulin protectant according to claim 2, wherein: the working concentration of the alanine and the isoleucine is respectively 0.1-0.3M and 0.05-0.15M; the concentration of immunoglobulin in the globulin preparation is 3-150g/L, and pH value is 4.5-5.5.
4. A globulin protectant according to claim 3, wherein: the working concentration of the alanine and the isoleucine is 0.1-0.19M and 0.06-0.15M respectively; the concentration of immunoglobulin in the globulin preparation is 40-100g/L, and pH value is 5.0-5.5.
5. The globulin protectant of claim 4, wherein: the working concentrations of alanine and isoleucine were 0.15M and 0.1M, respectively; the concentration of immunoglobulin in the globulin preparation was 50g/L and the pH was 5.5.
6. A globulin protectant according to claim 2, wherein: the working concentration of the alanine is 0.1-0.3M; the concentration of immunoglobulin in the globulin preparation is 100-250g/L, and pH value is 6.0-6.5.
7. The globulin protectant of claim 6, wherein: the working concentration of alanine was 0.25M; the concentration of immunoglobulin in the globulin preparation was 100g/L and the pH was 6.5.
8. A globulin protectant according to claim 1, wherein: the working concentration of polysorbate 80 was 60 μg/ml.
9. A globulin protectant according to any one of claims 4-8, wherein: the immunoglobulin is extracted from blood plasma or serum, or is prepared by a gene recombination technology or is prepared by a fermentation technology.
10. Use of an immunoglobulin protecting agent according to claim 9 as an additive to immunoglobulin preparations.
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