CN105267964A - Protein preparation preservation method capable of reducing generation of protein aggregate - Google Patents
Protein preparation preservation method capable of reducing generation of protein aggregate Download PDFInfo
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- CN105267964A CN105267964A CN201410323054.1A CN201410323054A CN105267964A CN 105267964 A CN105267964 A CN 105267964A CN 201410323054 A CN201410323054 A CN 201410323054A CN 105267964 A CN105267964 A CN 105267964A
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Abstract
The invention discloses a protein preparation preservation method capable of reducing generation of protein aggregate. The method is characterized in that the refrigeration temperature of a protein preparation is -40 DEG C to -196 DEG C. The method can effectively reduce formation of protein dimer or multimer in freezing-thawing of the protein preparation.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of protein formulation store method.
Background technology
In in the past 10 years, get the Green Light listing therapeutic protein formulations new drug get more and more, and high concentration (more than 50mg/ml) be suitable for hypodermic protein formulation, the developing direction having become protein formulation because it is easy to use.Usually, the high dose protein drug dosage regimen General Requirements protein formulation concentration higher than 1mg/kg or 100mg/ dosage is used to reach tens more than mg/ml, but the protein formulation of high concentration easily protein aggregation occurs, and then affect the activity of medicine, pharmacokinetics and drug safety.
Research finds, when the protein solution of high concentration is stored in low temperature (lower than 0 DEG C), protein easily forms dimer or polymer in multigelation process.Further research finds, the formation of protein aggregation needs the collision between two molecules, and the formation relation of concentration and gathering depends on size and the binding mechanism of aggregation, protein aggregation can cause covalency (such as disulfide bond connection) or non-covalent (reversible or irreversible) to combine, and the irreversible aggrengation caused by Non-covalent binding occurs via the hydrophobic region exposed by temperature, machinery or chemical process that can change native protein conformation usually.
There will be a known multiple method at present makes protein formulation more stable.Such as, the heatshock protein described in European patent EP 0599344A by adding such as HSP25 improves the stability of protein.Report in Chinese patent CN101745103B and add satisfied fatty acid, L-arginine, reduced glutathion and oxidized form of glutathione to improve albumin stabilize.European patent EP 0025275A describes the salt of nitrogenous substrate as contained arginine, guanidine or imidazoles for stabilizing immunoglobulin.Other method also has: by adding polyethers (European patent EP 0018609A), by adding glycine, albumin and dextran sulfate (US Patent No. 4808705), by adding detergent Tween20 (German patent DE 2652636, British patent GB8514349), by adding dodecyl Polyethylene Glycol (Chinese patent CN101007840), by adding citrate buffer solution (international patent application WO9322335), by adding chelating agen (international patent application WO9115509) etc., thus improve the stability of protein solution.Although said method can make protein formulation have certain stability, but when not having a kind of method to be applicable to multigelation, still Protein requirement is stable, better can reduce the method that albumen aggressiveness generates when being especially dissolved in water again after protein freeze thawing again, less than 0 DEG C cryopreservation or lyophilizing, have not yet to see report.
Therefore, how effectively to reduce protein formulation is this area urgent problem in generation that is freezing and/or protein aggressiveness when thawing always.
Summary of the invention
Applicant of the present invention finds the generation of albumen aggressiveness and the cryogenic temperature substantial connection of preparation in protein formulation through large quantifier elimination, by regulating suitable cryogenic temperature, effectively can reduce the formation of albumen aggressiveness in protein formulation, thus completing the present invention.Particularly, the invention discloses:
1. a protein formulation store method, is characterized in that, described protein formulation cryogenic temperature is: subzero 40 degrees Celsius to subzero 196 degrees Celsius.
2. the method according to above-mentioned 1, is characterized in that, the cryogenic temperature of described protein formulation is: subzero 40 degrees Celsius to subzero 80 degrees Celsius.
3. the method according to above-mentioned 2, is characterized in that, the cryogenic temperature of described protein formulation is: subzero 80 degrees Celsius.
4., according to the arbitrary described method of above-mentioned 1-3, it is characterized in that, the thaw point of described protein formulation is: 4 degrees Celsius to 37 degrees Celsius.
5., according to the arbitrary described method of above-mentioned 1-3, it is characterized in that, the protein concentration of described preparation is greater than 5mg/ml.
6. method according to above-mentioned 5, is characterized in that, the protein concentration of described preparation is greater than 50mg/ml.
7. according to the arbitrary described method of above-mentioned 1-3, it is characterized in that, described protein is antibody or fusion rotein.
8. method according to above-mentioned 7, is characterized in that, described protein is fusion rotein.
9. method according to above-mentioned 8, is characterized in that, described protein is TNFR-FC fusion rotein.
General protein formulation approximately less than-20 DEG C can be completely freezing, therefore people generally can by protein formulation freezen protective under the environment of-20 DEG C.The course of defrosting of protein formulation is then the reverse procedure of protein frozen process, if the too high increase likely causing albumen aggressiveness of the temperature of thawing, but the temperature of thawing is lower simultaneously, so thawing time possibility that can longly also have albumen aggressiveness to increase, general 4-37 degree of selecting thaws.
Our research worker finds through large quantifier elimination, and protein formulation is along with the increase of cryogenic temperature, and albumen aggressiveness also can increase.By further research, we find, protein formulation cryogenic temperature is subzero 40 degrees Celsius to subzero 196 degrees Celsius, preferably subzero 40 degrees Celsius to subzero 80 degrees Celsius, most preferably subzero 80 degrees Celsius, can effectively reduce protein formulation freezing and/or thaw time protein aggressiveness generation.Specifically see specific embodiments of the invention.
Albumen aggressiveness of the present invention refers to: molecular weight is at least the protein aggregate of monomeric protein molecular weight 2 times, and its content can measure by SEC-HPLC method.
Detailed description of the invention
Following examples, experimental example further illustrate of the present invention, should not be construed as limitation of the present invention.
Detecting instrument and method
Measure by current edition " Chinese Pharmacopoeia " three annex III B item " high performance liquid chromatography ".
High pressure liquid chromatograph: DionexUltimate3000
Chromatographic column: hydrophilic silica gels size exclusion chromatograph post, exclusion limit 500kD, aperture
, granularity 5 μm, column internal diameter 7.8mm, column length 30cm; Select TskgelG3000SWXL
Mobile phase is 200mmol/L phosphate buffer, pH6.6-7.0; Applied sample amount 10 ~ 40 μ g, determined wavelength 280nm.Calculate by areas of peak normalization method
The albumen aggressiveness of embodiment 1 protein formulation under different cryogenic temperature generates experiment
Sample 1:TNFR-FC protein solution (concentration 39mg/ml) (source Suzhou Alphamab Co., Ltd.)
Get 5 1000ml centrifuge bottles, 550ml sample 1 is poured respectively at centrifuge bottle, respectively these centrifuge bottles installing albumen are placed in-20 DEG C ,-40 DEG C ,-70 DEG C ,-80 DEG C refrigerators and liquid nitrogen (-196 DEG C) is freezing, be placed on after liquid nitrogen freezing is good-20 degree refrigerators in 48 hours, then respectively above-mentioned freezing protein solution is thawed in 30 degree of water-baths, then detect the albumen aggressiveness generation situation of the protein solution that respectively thaws by SEC-HPLC method, testing result is in table 1:
Albumen aggressiveness under the different cryogenic temperature of table 1 generates experimental result
As can be seen from Table 1, subzero 40 degrees Celsius are carried out frozen to protein solution under subzero 196 degrees celsius, its albumen aggressiveness increase all generates few than subzero 20 degrees Celsius of frozen albumen aggressiveness of routine, and especially when carrying out freezing for-80 DEG C, albumen aggressiveness decreases nearly 36%.
The albumen aggressiveness of embodiment 2 variable concentrations protein formulation under different cryogenic temperature generates experiment
Sample 2:TNFR-FC protein solution (concentration 5mg/ml) (source Suzhou Alphamab Co., Ltd.)
Sample 3:TNFR-FC protein solution (concentration 50mg/ml) (source Suzhou Alphamab Co., Ltd.)
By 3 500ml centrifuge bottles, sample 2 protein solution of 200ml is poured respectively at centrifuge bottle, simultaneously, take 3 500ml centrifuge bottles again, then pour sample 3 protein solution of 200ml at centrifuge bottle respectively into, then respectively the centrifuge bottle that above-mentioned 6 install protein solution is placed in-20 degree ,-40 degree, in the refrigerator of-80 degree, freeze overnight.After freezing, be placed in room-temperature water bath and thaw.Detect by SEC-HPLC method the aggressiveness situation of solution of having thawed, simultaneously with SEC-HPLC method detect not through frozen raw sample 2 and sample 3 solution aggressiveness situation in contrast, testing result is in table 3.
The albumen aggressiveness of table 3 variable concentrations protein formulation under different cryogenic temperature generates information slip
As can be seen from the above table the protein solution-20 DEG C of 5mg/ml frozen after the thaw albumen aggressiveness of liquid add 28% than the albumen aggressiveness of original solution, and-80 DEG C frozen after the white aggressiveness of the liquid eggs that thaws only add 9% than original solution; In addition, the protein solution-80 DEG C of 50mg/ml freezing after thaw the white aggressiveness of liquid eggs be only-20 DEG C freezing after to thaw 65% of the white aggressiveness of liquid eggs.Visible compare conventional-20 DEG C frozen, freezen protective protein solution under-40 DEG C or-80 DEG C of conditions, the generation of albumen aggressiveness all has minimizing, and the higher effect of protein solution concentration is more obvious.
Claims (9)
1. a protein formulation store method, is characterized in that, described protein formulation cryogenic temperature is: subzero 40 degrees Celsius to subzero 196 degrees Celsius.
2. method according to claim 1, is characterized in that, the cryogenic temperature of described protein formulation is: subzero 40 degrees Celsius to subzero 80 degrees Celsius.
3. method according to claim 1, is characterized in that, the cryogenic temperature of described protein formulation is: subzero 80 degrees Celsius.
4., according to the arbitrary described method of claim 1-3, it is characterized in that, the thaw point of described protein formulation is: 4 degrees Celsius to 37 degrees Celsius.
5., according to the arbitrary described method of claim 1-3, it is characterized in that, the protein concentration of described preparation is greater than 5mg/ml.
6. method according to claim 5, it is characterized in that, the protein concentration of described preparation is greater than 50mg/ml.
7. according to the arbitrary described method of claim 1-3, it is characterized in that, described protein is antibody or fusion rotein.
8. method according to claim 7, it is characterized in that, described protein is fusion rotein.
9. method according to claim 8, it is characterized in that, described protein is TNFR-FC fusion rotein.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410323054.1A CN105267964A (en) | 2014-07-08 | 2014-07-08 | Protein preparation preservation method capable of reducing generation of protein aggregate |
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| CN201410323054.1A CN105267964A (en) | 2014-07-08 | 2014-07-08 | Protein preparation preservation method capable of reducing generation of protein aggregate |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111574583A (en) * | 2020-04-10 | 2020-08-25 | 上海海路生物技术有限公司 | Protein renaturation reagent and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131676A (en) * | 2013-01-04 | 2013-06-05 | 天津耀宇生物技术有限公司 | Silkworm recombinant baculovirus showing recombinant human tumor necrosis factor receptor-Fc fusion protein gene, and preparing method and application thereof |
| CN103446583A (en) * | 2013-03-21 | 2013-12-18 | 百奥泰生物科技(广州)有限公司 | Human antibody preparation for treating TNF (tumour necrosis factor)-alpha related diseases |
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2014
- 2014-07-08 CN CN201410323054.1A patent/CN105267964A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131676A (en) * | 2013-01-04 | 2013-06-05 | 天津耀宇生物技术有限公司 | Silkworm recombinant baculovirus showing recombinant human tumor necrosis factor receptor-Fc fusion protein gene, and preparing method and application thereof |
| CN103446583A (en) * | 2013-03-21 | 2013-12-18 | 百奥泰生物科技(广州)有限公司 | Human antibody preparation for treating TNF (tumour necrosis factor)-alpha related diseases |
Non-Patent Citations (1)
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| 江滨等主编: "《抗肿瘤药物临床应用指南》", 31 May 2005, 中国协和医科大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111574583A (en) * | 2020-04-10 | 2020-08-25 | 上海海路生物技术有限公司 | Protein renaturation reagent and its application |
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Address after: Shanghai city 201203 libing road Pudong New Area Zhangjiang hi tech Park No. 399 Applicant after: Three country Kin Pharmaceutical (Shanghai) Limited by Share Ltd Address before: Shanghai city 201203 libing road Pudong New Area Zhangjiang hi tech Park No. 399 Applicant before: Shanghai CP Guojian Pharmaceutical Co., Ltd. |
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Application publication date: 20160127 |