CN111198199A - CART cell cryopreservation liquid detection method - Google Patents
CART cell cryopreservation liquid detection method Download PDFInfo
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- CN111198199A CN111198199A CN201811362945.2A CN201811362945A CN111198199A CN 111198199 A CN111198199 A CN 111198199A CN 201811362945 A CN201811362945 A CN 201811362945A CN 111198199 A CN111198199 A CN 111198199A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N25/00—Investigating or analyzing materials by the use of thermal means
- G01N25/02—Investigating or analyzing materials by the use of thermal means by investigating changes of state or changes of phase; by investigating sintering
- G01N25/04—Investigating or analyzing materials by the use of thermal means by investigating changes of state or changes of phase; by investigating sintering of melting point; of freezing point; of softening point
- G01N25/06—Analysis by measuring change of freezing point
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for detecting a frozen stock solution of an expression CART cell, which comprises the following steps: and (5) detecting physical and chemical properties and safety. A series of strictly detected CART frozen stock solutions have the characteristics of uniformity, stability and safety.
Description
Technical Field
The invention relates to the field of medical biology, in particular to a method for detecting a frozen stock solution of an expression CART cell.
Background
Due to the complexity and changeability of biotechnological products, the technical sector is difficult to realize the overall quality control of the products only by the quality verification of the final products, so to ensure the safety, effectiveness and controllable quality of the products, the developing units must perform the quality control of the whole process from raw materials (including bacteria, seeds or cell banks), production processes, stock solutions, semi-finished products, finished products to storage conditions, and the like. How to establish a complete quality control system is a complex system engineering for biopharmaceuticals, and needs to be continuously explored in practice.
The quality control of the whole process is required for biological products, and the establishment and the perfection of quality standards are the most important links in the process. Compared with chemical drugs, the biological products have a plurality of verification projects and complex verification methods, and according to the current technical standard in China, more than 20 conventional verification projects are available for recombinant products. In recent years, with the supplementary revision of national biological product regulations and the implementation of GMP, the quality standards of domestic biotechnological drugs are more standardized and the implementation is more in place.
The CART cells are used as a living preparation and have more uncertainties, as an individualized cell preparation, the ratio of T cells in the production process of each CART cell is different, the activation degree of the T cells is different, the infection efficiency of the CAR-expressing virus is different, the proliferation rate of the CART cells cultured in vitro is different, the functional effect of the CART cells in the targeted tumor cells of each patient is different (such as different secretion amounts of INF-gamma and CD107a and different cell killing rates), so a whole set of release standards of the CART cells are needed in the industrial production stage of the CART cells, and the patients can be guaranteed to obtain qualified CART cells for reinfusion to eliminate the tumor cells in vivo. The final property of CART cell frozen stock solution as a CART preparation determines whether the quality control of the whole CART cell is successful or not by the perfection of the detection.
Disclosure of Invention
For the reasons, the invention provides a set of standard detection methods for detecting CART cell frozen stock solution, and the specific contents are shown in the following table.
TABLE 1 CART cell cryopreservation liquid safety assay
The invention has the following beneficial effects:
1) the method is simple to operate, and the prepared CART is good in safety and high in uniformity.
2) The instruments used in the detection method are conventional small instruments in a laboratory, and large instruments are not needed.
3) The detection method is easy to popularize, and lays a foundation for the industrial production of CART.
Detailed Description
Examples
The present invention is described in further detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations which become apparent in light of the teachings provided herein. The invention details several special detection implementation methods, and the conventional detection can refer to the method of Chinese pharmacopoeia.
Example 1 measurement of cell cryopreservation solution pH
The pH value of the virus liquid is detected according to the operating procedure of Mettler FE20pH in a meter of Mettler FE20pH which is qualified by measurement. Before the measurement, the instrument was calibrated with pH4.01 buffer, pH7.00 buffer and pH9.21 buffer previously set at 25 ℃, and after the calibration, the sample to be measured was measured and the measurement was repeated 3 times.
Example 2 detection of osmotic pressure of cell cryopreservation solution
Osmolality is typically determined indirectly by measuring the freezing point depression of a solution. In an ideal dilute solution, the freezing point depression is in accordance with the relationship of Δ Tf — K f × m, where Δ Tf is the freezing point depression, K f is the freezing point depression constant (186 when water is the solvent), and m is the molarity. And the osmotic pressure conforms to the relationship of P ═ K × m, wherein P is the osmotic pressure, K is the osmotic pressure constant, and m is the molarity of the solution. Since the concentrations in the two formulas are equal, the osmolality of the solution can be determined by freezing point depression. Osmometry mols-meters, which are usually designed using the principle of freezing point depression, usually consist of a refrigeration system, a heat-sensitive probe for measuring the current or potential difference and an oscillator (or metal probe). During measurement, the probe is dipped into the center of the test solution and is lowered into a cooling tank of the instrument. And starting a refrigerating system, when the temperature of the test solution is reduced to be below a freezing point, inducing the solution to be frozen by using an oscillator (or a metal probe) by using the instrument, and automatically recording the temperature of the freezing point depression. The measured value displayed by the instrument can be the temperature of the freezing point depression and also the osmolality.
Of course, the above description is not limited to the above examples, and the undescribed technical features of the present invention can be implemented by or using the prior art, and will not be described herein again; the above embodiments are merely for illustrating the technical solutions of the present invention and not for limiting the present invention, and the present invention has been described in detail with reference to the preferred embodiments, and those skilled in the art should understand that those skilled in the art can make changes, modifications, additions or substitutions within the spirit and scope of the present invention without departing from the spirit of the present invention, and shall also fall within the scope of the claims of the present invention.
Claims (8)
1. A release detection of CART cells comprises physicochemical property and safety detection.
2. Physicochemical property measurements according to claim 1 comprising appearance, PH and osmolarity measurements.
3. The safety assay of claim 1 comprising bacteria, fungi, mycoplasma, endotoxins.
4. The visual inspection standard of claim 2 which is a yellowish clear liquid visible to the naked eye.
5. The appearance test PH standard of claim 2 is between 6.5 and 7.5.
6. The osmolarity standard according to claim 2 is 1550 to 1850 mOsmol/kg.
7. Performing a standard bacterial, fungal, mycoplasma detection as negative according to claim 3; the endotoxin content was < 0.5 EU/ml.
8. The results according to claims 1-7 are all in compliance with the execution criteria to be released.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811362945.2A CN111198199A (en) | 2018-11-16 | 2018-11-16 | CART cell cryopreservation liquid detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811362945.2A CN111198199A (en) | 2018-11-16 | 2018-11-16 | CART cell cryopreservation liquid detection method |
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| Publication Number | Publication Date |
|---|---|
| CN111198199A true CN111198199A (en) | 2020-05-26 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201811362945.2A Pending CN111198199A (en) | 2018-11-16 | 2018-11-16 | CART cell cryopreservation liquid detection method |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103575866A (en) * | 2012-08-08 | 2014-02-12 | 四川奇力制药有限公司 | Quality control method of potassium chloride and sodium chloride injection |
| CN104721228A (en) * | 2015-04-03 | 2015-06-24 | 舟山医院 | Method for treating lung cancer by improving autogenous DC-CIK immunocytes |
| CN106795548A (en) * | 2014-09-03 | 2017-05-31 | 生物蛋白有限公司 | Found and Production conditions active biological protein in identical eukaryotic produces host |
| CN107148967A (en) * | 2016-11-09 | 2017-09-12 | 深圳宾德生物技术有限公司 | A kind of antigenspecific T lymphocyte frozen stock solution and its preparation method and application |
| CN108721331A (en) * | 2018-04-27 | 2018-11-02 | 上海恒润达生生物科技有限公司 | A kind of Chimeric antigen receptor T cell preparation and its Cord blood formulation preparation method |
-
2018
- 2018-11-16 CN CN201811362945.2A patent/CN111198199A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103575866A (en) * | 2012-08-08 | 2014-02-12 | 四川奇力制药有限公司 | Quality control method of potassium chloride and sodium chloride injection |
| CN106795548A (en) * | 2014-09-03 | 2017-05-31 | 生物蛋白有限公司 | Found and Production conditions active biological protein in identical eukaryotic produces host |
| CN104721228A (en) * | 2015-04-03 | 2015-06-24 | 舟山医院 | Method for treating lung cancer by improving autogenous DC-CIK immunocytes |
| CN107148967A (en) * | 2016-11-09 | 2017-09-12 | 深圳宾德生物技术有限公司 | A kind of antigenspecific T lymphocyte frozen stock solution and its preparation method and application |
| CN108721331A (en) * | 2018-04-27 | 2018-11-02 | 上海恒润达生生物科技有限公司 | A kind of Chimeric antigen receptor T cell preparation and its Cord blood formulation preparation method |
Non-Patent Citations (2)
| Title |
|---|
| 中国医药生物技术协会: "嵌合抗原受体修饰 T 细胞（CAR-T 细胞）制剂制备质量管理规范", 《中国医药生物技术》 * |
| 林 阳等: "细胞内液型胶体液PHS的研制及质量控制", 《药品检验》 * |
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Address after: 201210 9th floor, building 1, Lane 1238, Zhangjiang Road, Pudong New Area, Shanghai Applicant after: Shanghai Hengrun Dasheng Biotechnology Co.,Ltd. Address before: 201210 9th floor, building 1, Lane 1238, Zhangjiang Road, Pudong New Area, Shanghai Applicant before: SHANGHAI HRAIN BIOTECHNOLOGY Co.,Ltd. |
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