Standard number: GB/T 38506-2020
Standard name: Determination of biochemical parameters in animal cell culture process
English name: Determination of biochemical parameters in animal cell culture process ||
tt||Standard format: PDF
Release time: 2020-03-06
Implementation time: 2020-03-06
Standard size: 986K
Standard introduction: This standard specifies the determination method of cell density, cell viability, pH value, dissolved oxygen, osmotic pressure, glucose content, bacterial endotoxin and lactic acid content in the process of animal cell scale culture.
This standard is applicable to the detection of biochemical parameters in the scale culture of Chinese hamster ovary cells (CHO), mouse myeloma cells (NSO, SP2/0), African green monkey kidney cells (Vero), baby hamster kidney cells (BHK-21), canine kidney epithelial cells (MDCK), and porcine testicular cells (ST). It can also be used as a reference for the detection of biochemical parameters in the scale culture of other eukaryotic cells.
This standard was drafted in accordance with the rules given in GB/T1.12009. ||
tt||This standard was proposed and managed by the National Technical Committee for Biochemical Testing Standardization (SAC/TC387). The drafting units of this standard are: Biological Research Institute of China Testing Technology Research Institute, Innovent Biologics (Suzhou) Co., Ltd., Huapai Biotechnology Group Co., Ltd., Shanghai Institute of Metrology and Testing Technology, Shanghai Zhangjiang Biotechnology Co., Ltd., Sichuan University, Chengdu Biotech Biotechnology Co., Ltd., Shanghai Maitai Junao Biotechnology Co., Ltd., Taizhou Maiboteike Pharmaceutical Co., Ltd., and Shanghai Biobio Pharmaceutical Co., Ltd.
Main drafters of this standard: Zhou Lihua, Zhou Kaisong, Song Hang, Qiu Wenying, Li Jianfeng, Liu Gang, Huang Lin, Ma Lixia, Li Zhenhua, Yang Kun, Li Yan, Jiang Zijing, Fang Pengfei, Cui Likai, Hao Weiwei, Ye Deping, Liang Zhijie, Zhang Dapeng, Zhang Liyan, Guo Huaizu, Xu Jin, Guo Qingcheng, Dai Jianxin
This standard specifies the determination methods of cell density, cell viability, pH value, dissolved oxygen, osmotic pressure, glucose content, bacterial endotoxin and lactic acid content in the process of animal cell scale culture. This standard is applicable to the detection of biochemical parameters in the process of scale culture of Chinese hamster ovary cells (CHO), mouse myeloma cells (NSO, SP2/0), African green monkey kidney cells (Vero), baby hamster kidney cells (BHK-21), canine kidney epithelial cells (MDCK) and porcine testicular cells (ST). The detection of biochemical parameters in the process of scale culture of other eukaryotic cells can also be used as a reference.

ICS07.080
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National Standard of the People's Republic of China
GB/T38506—2020
Determination of biochemical parameters in animal cell culture process2020-03-06Issued
State Administration for Market Regulation
National Standardization Administration
2020-03-06Implementation
This standard was drafted in accordance with the rules given in GB/T1.1—2009iiKAacJouaKAa
GB/T38506—2020
This standard was proposed and managed by the National Technical Committee for Standardization of Biochemical Testing (SAC/TC387). The drafting units of this standard are: Biological Research Institute of China Testing Technology Research Institute, Innovent Biologics (Suzhou) Co., Ltd., Huapai Biotechnology Group Co., Ltd., Shanghai Institute of Metrology and Testing Technology, Shanghai Zhangjiang Biotechnology Co., Ltd., Sichuan University, Chengdu Biotech Biotechnology Co., Ltd., Shanghai Maitaijunao Biotechnology Co., Ltd., Taizhou Maiboteike Pharmaceutical Co., Ltd., and Shanghai Biobio Pharmaceutical Co., Ltd.
The main drafters of this standard are: Zhou Lihua, Zhou Kaisong, Song Hang, Qiu Wenying, Li Jianfeng, Liu Gang, Huang Lin, Ma Lixia, Li Zhenhua, Yang Kun, Li Yan, Jiang Zijing, Fang Pengfei, Cui Likai, Hao Weiwei, Ye Deping, Liang Zhijie, Zhang Dapeng, Zhang Liyan, Guo Huaizu, Xu Jin, Guo Qingcheng, and Dai Jianxin. 1
1Scope
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GB/T38506-—2020
Methods for the determination of biochemical parameters in animal cell culture This standard specifies the methods for the determination of cell density, cell viability, pH value, dissolved oxygen, osmotic pressure, glucose content, bacterial endotoxins and lactic acid content in animal cell scale culture. This standard is applicable to the detection of biochemical parameters in the scale culture of Chinese hamster ovary cells (CHO), mouse myeloma cells (NSO, SP2/0), African green monkey kidney cells (Vero), baby hamster kidney cells (BHK-21), canine kidney epithelial cells (MDCK), and porcine testicular cells (ST). The detection of biochemical parameters in the scale culture of other eukaryotic cells can also be used for reference. Normative references
The following documents are essential for the application of this document. For all dated references, only the dated version applies to this document. For any undated referenced document, its latest version (including all amendments) shall apply to this document WS/T350 Reference Method for Determination of Serum Glucose Pharmacopoeia of the People's Republic of China (2015 Edition) Part II (National Pharmacopoeia Commission) Pharmacopoeia of the People's Republic of China (2015 Edition) Part III (National Pharmacopoeia Commission) 3 Terms and Definitions, Abbreviations
3.1 Terms and Definitions
The following terms and definitions apply to this document. 3.1.1
Cell density
celldensity
The total number of cells in a unit volume of a sample after the cells have been cultured. 3.1.2
cellviability
Cell viability
The percentage of viable cells to the total number of cells during cell culture. 3.2
2 Abbreviations
The following abbreviations apply to this document.
ATP: Adenosine triphosphate NADH: Nicotinamide adenine dinucleotide phosphate NAD(P): Nicotinamide adenine dinucleotide phosphate NAD(P)H: Nicotinamide adenine dinucleotide phosphate phosphate)
OPC: Open Platform Communications PBS: Phosphate Buffered Saline GB/T38506—2020
4 General requirements
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4.1 Unless otherwise specified, only reagents confirmed as analytically pure or pharmaceutical grade and purified water or water for injection specified in Part II of the Pharmacopoeia of the People's Republic of China (2015 edition) shall be used in the analysis. 4.2 The quality of the reagents used in the test should avoid changes in the performance of the reagents due to expiration. Therefore, the quality of the reagents should be verified according to the reagent instructions or product quality inspection report before the determination.
Note: Reagents such as fluorescent dyes and buffers should not be stored for a long time. Trypan blue may cause the dead cell measurement result to be too high or too low due to mismatch. 4.3 In order to reduce or avoid the risk of contamination caused by sampling, online detection is recommended for the determination of various parameters in the cell culture process. 4.4 During online detection, the relevant equipment should be confirmed according to the manufacturer's installation confirmation plan and operation confirmation plan, and the plan should be documented. Before the measurement, the manufacturer and/or user should confirm the performance of the equipment. 4.5 During online detection, the electrode is directly inserted into the cell fermentation liquid for detection without sampling. During offline detection, sampling should be carried out in accordance with the requirements of aseptic operation. After sampling, the sample should be processed by centrifugation, dilution, etc. according to the requirements of the detection parameters and detection methods, and then the sample should be tested immediately. Samples that cannot be tested immediately should be placed at 2℃~8℃ and tested within no more than 6h. 5 Determination of cell density
Determination of suspension cell density
Hemocytometer
5.1.1.1 Principle
Place the suspension of the sample to be tested on the hemocytometer plate, count directly under the microscope, and then calculate the number of cells. 5.1.1.2
Reagents and materials
PBS buffer: can be prepared by yourself or purchased commercially. 5.1.1.3 Instruments and apparatus
Hemocytometer: 16 grids × 25 grids or 25 grids × 16 grids 5.1.1.3.1
5.1.1.3.2 Inverted microscope: magnification × 400. 5.1.1.3.3 Micropipette: 20μL~200μ, 100μ~1000μ. 5.1.1.4 Determination steps
Take 900uL PBS buffer into a centrifuge tube, add 100μL of cell suspension, perform 10-fold serial dilutions in sequence, and count the cells at appropriate dilutions so that the number of cells in each large square is 100 to 300. Place a cover glass on the center of the counting plate, blow up and down to mix gently, and use a micropipette to slowly add the cell suspension along the edge of the cover glass, leaving no bubbles or overflow, and let it stand for 2-3 minutes. Count the cells in the large square under a microscope, repeat the counting three times, and take the average. Note: In the actual detection process, the required PBS buffer and cell suspension amount can be adjusted according to the expected cell density. 5.1.1.5 Result calculation
Calculate according to formula (1) for a 16×25 grid blood cell counting plate and according to formula (2) for a 25×16 grid blood cell counting plate. N=A×10*×d×400
Where:
N_A×10*×d×400
N is the number of cells per milliliter of suspension, in pieces; A is the number of cells observed on the counting plate, in pieces; the total dilution multiple of the cell solution.
The calculation result is expressed as an integer.
5.1.2 Automatic image analysis method
5.1.2.1 Principle
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GB/T38506—2020
. (2)
The collected source image data is smoothed, processed by histogram reading, and then eroded to fill holes, grayscale image, contrast enhancement, edge detection, binary transformation, mathematical morphology and correction, a series of image processing counting and analysis are performed to obtain the exact number of cells.
5.1.2.2 Reagents
Trypan blue solution (0.4%, W/V): Weigh 4.00g of trypan blue, add a small amount of water to grind, dissolve in water and make up to 100mL, filter with qualitative filter paper, and store at 4℃. When used, dilute to 0.4% with PBS solution. Commercial products can be purchased directly as needed. 5.1.2.3 Instruments and Equipment
Automatic cell counter: mainly composed of four parts: image processing system, image preprocessing system, image segmentation and image counting 5.1.2.4 Analysis steps
Start the automatic cell counter and set the cell type. According to the linear range and sample volume requirements of the instrument, take an appropriate amount of cell suspension and add it to the trypan blue dye (mix the cell suspension and trypan blue dye in a 1:1 ratio), blow up and down, and mix gently. Take an appropriate amount of sample mixture and add it to the counting pool. According to the instrument operating instructions, the high-definition image analysis system automatically analyzes the cell density, viability and morphology. Note: Other equivalent dyes can be used, such as thiazole blue. 5.1.3 Capacitance method
5.1.3.1 Principle
According to the cell suspension concentration at the time of measurement, establish a linear relationship between the cell suspension concentration and the capacitance value, and calculate the K value. Input the constant K value into the online cell detector detection system to obtain the cell density. 5.1.3.2 Equipment
Capacitance method online cell detector: measurement range, 0pF~400pF; accuracy, 0.01pF. 5.1.3.3 Determination steps
5.1.3.3.1 Capacitance 0” point calibration of online cell detector Turn on the power, turn on and stabilize for 30 minutes, place the electrode in blank cell culture medium, and set the appropriate temperature, stirring speed and pH. All parameters must be consistent with the parameters in the cell culture process. After the capacitance data is stable, perform capacitance “0” point calibration. Note: The specific calibration operation shall be adjusted accordingly according to the manufacturer’s requirements. 5.1.3.3.2 Cell density and capacitance value positive correlation constant \K” are determined. Add the cell suspension (with a concentration of M) for cell culture inoculation into the bioreactor and detect the cell capacitance value P. Continuously use 3
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4 batches of cell suspension concentration and cell capacitance value data statistics, make a linear relationship diagram based on the statistical results, draw a standard curve, and the value of constant K in formula (3) can be obtained based on the linear relationship results. 5.1.3.3.3 Determination and result calculation
The constant K value is input into the online cell detector detection system, and the live cell density can be obtained online according to formula (3): M-KP
Where:
M—cell suspension density, unit is per milliliter (cells/mL); P——cell capacitance value, unit is pico-farad (pF); K—cell density and capacitance value positive correlation constant. The calculation result is expressed to two decimal places. 5.1.4 Raman spectroscopy
5.1.4.1 Principle
When monochromatic light is irradiated onto the sample, it is only related to the structure of the scattering molecules themselves. Analyze the scattered spectrum with different frequencies from the incident light to obtain information on molecular vibration and rotation, and conduct molecular structure research and qualitative and quantitative detection. 5.1.4.2 Equipment
5.1.4.2.1 Raman spectrometer, mainly composed of control system, laser light source, probe, optical components and imaging system. 5.1.4.2.2 Bioreactor.
5.1.4.3 Determination steps and result statistics
5.1.4.3.1
Light intensity calibration
According to the instrument operating instructions, connect the Raman probe to the Raman spectrometer through optical fiber to perform light intensity calibration. 5.1.4.3.2 Sterilization
Install the calibrated Raman probe on the bioreactor and sterilize it together with the bioreactor. For disposable reactors, the probe can be directly inserted into the corresponding hole.
5.1.4.3.3 Determination
According to the instrument operating instructions, start the Raman spectrometer, add culture medium, inoculate, run the Raman spectrum acquisition program, set the exposure time to 10s, scan 75 times, and it takes about 15 minutes to measure a sample. Note: The exposure time and the number of scans can be adjusted according to the requirements of different brands of instruments. 5.1.4.3.4 Result Statistics
Import the mathematical model of cell density. The Raman spectrometer automatically saves the spectrum in the specified folder after each measurement. The process parameter results are calculated through the mathematical model. The corresponding results are displayed on the screen and can be transmitted to the database system or process control system through OPC. 5.2 Determination of adherent cell density
5.2.1 Determination of adherent cell density using microsphere carriers and other cell culture carriers Use an appropriate amount of digestion solution (0.25% trypsin, etc. can be used) to digest the cells to make the cells completely suspended, and then determine according to 5.1.1 or 5.1.2. 4
5.2.2 Determination of adherent cell density using sheet carriers as cell culture carriersiiiKAa~cJouakAa
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Completely immerse the sheet carrier in crystal violet staining solution and lyse it at 37℃ for 30min~60min to lyse the cells, and then determine according to 5.1.1 or 5.1.2.
Determination of cell viability
6.1 Principle
The cell membrane of living cells is a selective membrane that only allows substances to pass selectively. After cell death, the cell membrane is damaged and the permeability increases. According to the difference in cell membrane permeability between living cells and dead cells, after adding dye, dead cells are stained, while living cells are not stained, so the percentage of living cells to the total number of cells can be obtained. 6.2 Reagents and materials
Same as 5.1.1.2 and 5.1.2.2.
6.3 Equipment
Same as 5.1.1.3.
Determination steps and result calculation
Pipette an appropriate amount of cell suspension, add 0.4% trypan blue solution, so that the final concentration of trypan blue is 0.04%, and count according to 5.1.1 or 5.1.2 within 3 minutes. The cells stained blue-black are counted as the number of dead cells D. The cell viability is calculated according to formula (4): CD
Where:
X cell viability;
C total number of cells, in pieces;
number of dead cells, in pieces.
The calculation result is expressed to two decimal places. Determination of pH
7.1 Principle
........(4)
By measuring the activity of hydrogen ions in the cell culture medium, according to the Nernst equation, the ion activity is proportional to the electrode potential. For the cell suspension to be tested, the relationship curve between the electrode potential and the activity is established, and the acidity, neutrality and alkalinity of the cell culture medium to be tested are obtained. 7.2
2 Reagents
pH calibration solution: Different pH calibration solutions can be selected according to needs. Note: pH calibration solutions can use pH4.01 standard buffer (potassium hydrogen phthalate), pH7.00 standard buffer (mixed phosphate) and pH9.18 standard buffer (borax).
7.3 Equipment
7.3.1 Online pH electrode and offline desktop pH meter: accuracy 0.1. 7.3.2 Micropipette: 20uL~200μL, 100μL1000μL. 5
GB/T38506—2020
7.4 Determination steps
7.4.1 General
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pH online detection can be carried out simultaneously with offline detection, and the results of offline detection can be used to review and calibrate the online detection results. 7.4.2 Online detection
Before online detection, the pH electrode needs to be calibrated. After the calibration is completed, the electrode is installed in the reactor and then detected. 7.4.3 Offline detection
Before offline detection, the offline pH meter should be calibrated. Take 5mL10mL of cell culture medium into a 15mL centrifuge tube and test it according to the physical inspection method (General Rule 0631) in Part III of the Pharmacopoeia of the People's Republic of China (2015 Edition). Avoid shaking the culture medium during the test. The absolute difference between two independent test results obtained under repeatability conditions shall not exceed 5% of the arithmetic mean, otherwise, the test should be repeated; the samples taken should be tested in time to avoid long-term exposure to the air, which may cause measurement errors caused by changes in sample pH. 7.5 Result calculation
Read directly after the test. The calculation result is expressed to two decimal places. 8 Determination of dissolved oxygenwwW.bzxz.Net
8.1 Principle
Oxygen is reduced by the working electrode through the diaphragm, generating a diffusion current proportional to the oxygen concentration. By measuring this current, the concentration of dissolved oxygen in the cell culture suspension is obtained. The offset of dissolved oxygen detection is large after offline, and online detection 2 equipment is generally used
Online dissolved oxygen electrode.
Measurement steps
8.3.1 Instrument calibration
Use the dissolved oxygen electrode to measure the dissolved oxygen in real time during the culture process. The equipment installed with the dissolved oxygen electrode must be sterilized before testing. After sterilization, the initial culture parameters must be set according to the culture conditions. After the control is stable, the dissolved oxygen electrode must be activated for more than 2 hours before calibration. According to the requirements of different manufacturers and models, 100% one-point or 0, 100% two-point calibration can be performed. 8.3.2 Measurement
After sterilization and calibration are completed, set the conditions according to the instrument operating instructions, and monitor the dissolved oxygen content of the culture medium in real time during the cell culture process. The absolute difference of the two independent measurement results obtained by the sample under repeatability conditions The value does not exceed 5% of the arithmetic mean, otherwise, the measurement should be re-measured.
8.4 Result calculation
Read directly after online detection. Calculation results are expressed to two decimal places. 6
9 Determination of osmotic pressure
9.1 Principle
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GB/T38506—2020
According to the osmotic pressure molar concentration and freezing point temperature drop of the solution Based on the linear relationship between the values, the freezing point of the solution is measured to determine the osmotic pressure of the solution.
9.2 Equipment
Freezing point osmotic pressure meter
9.3 Measurement steps
Osmotic pressure is detected offline. Take 2mL of the suspension, centrifuge at 150g~300g for 5min~10min, and take the supernatant.
The osmolarity determination method is determined by the physical examination method (Tong 0632) in the Pharmacopoeia of the People's Republic of China (2015 Bottle). The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 5% of the arithmetic mean, otherwise, a new measurement 9.4 result calculation is required
According to the "Pharmacopoeia of the People's Republic of China" (2015) Physical inspection method (Tong 0632) Qian results, the payment is expressed as an integer 10 Determination of grapes
10.1 Principle
Under the catalysis of hexokinase, glucose and
ATP undergo a phosphorylation reaction , to generate glucose-6-phosphate, which is dehydrogenated under the catalysis of glucose-6-phosphate dehydrogenase, to generate 6-phosphogluconic acid, and at the same time reduce NAD(P) to NADP)H. After NAD(P) is converted into NAD(P )H is accompanied by an increase in the molar extinction coefficient at 340nm. The change in the molar extinction coefficient is proportional to the glucose content. The glucose content in the cell suspension can be determined by detecting the molar extinction coefficient at 339nm. 10.2 Reagent
Glucose 6-phosphate dehydrogenase reagent.
0.11mol/L saturated Ba(OH) solution.
1.5% Tris-HCI stock solution.
1.2% Tris-Base stock solution.
0.2% Tris-albumin stock solution
1g/L benzoin dilution.
22g/LZnSO, solution.
10g/L glucose standard stock solution: Weigh 1.00g of glucose standard dried at 105°C to constant weight, dilute it and set it to volume in a 100mL10.2.8
volumetric flask.
90.5% phenolphthalein indicator: Precisely weigh 0.5g of phenol, dissolve it in absolute ethanol, and adjust the volume to 100mL. You can also purchase commercially produced 10.2.9
UV-visible spectrophotometer
GB/T38506-2020
micropipette: 20μL~200μL, 100μL~1000μL. 10.3.2
3 balance: sensitivity 0.01g.
10.4 Measurement steps
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Take 2mL of cell culture fluid, centrifuge at 150g~300g for 10min, and take the supernatant. Detect according to the method of WS/T350 or use biosensors. The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 5% of the arithmetic mean. Otherwise, re-measurement is required.
10.5 Result calculation
The calculation results are expressed to two decimal places
Determination of bacterial endotoxin
11.1 Principle
When the bacteria die or autolyse Endotoxins will be released, and bacterial endotoxins can be detected qualitatively or semi-quantitatively by using shark reagent to produce agglutination reaction with bacterial endotoxins.
11.2 Reagent
Gel Shark Reagent.
11.3 Equipment
11.3.1 Thermostat.
2 micropipette: 20μL~200μL, 100μL~1000μl. 11.3.2
3 vortex mixer.
11.4 Measurement steps
Take 2mL of cell culture fluid, centrifuge at 150g~300g for 5min~10min, and take the supernatant. It was carried out in accordance with the bacterial endotoxin detection method in Part Three of the Pharmacopoeia of the People's Republic of China (2015 edition) (General Chapter 1143). 11.5 Calculation of results
The results are calculated in accordance with the Microbial Inspection Method (General Chapter 1143) in Part Three of the "Pharmacopoeia of the People's Republic of China" (2015 edition). The calculation results are expressed to one decimal place.
2 Determination of lactic acid content
12.1 Principle
Lactic acid generates pyruvate under the action of lactate dehydrogenase, and at the same time reduces nicotinamide adenine dinucleotide to generate NADH and H+ , detect the production substrate and calculate the lactic acid content in the fermentation broth 12.2 reagent
lactic acid detection kit or equipment, different detection equipment is equipped with different reagents. 8
12.3 Equipment
According to different detection mechanisms, the equipment used includes biochemical analyzers, spectrophotometers, etc. 12.4
Measurement steps
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GB/T38506-2020
Take an appropriate amount of cell culture fluid, centrifuge it at 150g~300g for 10 minutes, take the supernatant and press it in the kit or equipment operating instructions for testing. The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 5% of the arithmetic mean. Otherwise, re-measure 12.5
Result Calculation
Calculate the result according to the instructions of the kit, and the calculation result is expressed to two decimal places. Test report
The test report should include the following content:
Sample name;
Cell type;
Sample number:
Technical basis;|| tt||Main instruments and equipment;
Test parameters;
Test temperature and environment;
Test date, test personnel,9
UV-visible spectrophotometer
GB/T38506—2020
Micropipette: 20μL~200μL, 100μL~1000μL. 10.3.2
3 Balance: sensitivity 0.01g.
10.4 Determination steps
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Take 2mL of cell culture medium, centrifuge at 150g~300g for 10min, and take the supernatant. Detect according to the WS/T350 method or use a biosensor. The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 5% of the arithmetic mean. Otherwise, re-determination is required.
10.5 Calculation of results
Calculation results are expressed to two decimal places
Determination of bacterial endotoxins
11.1 Principle
Endotoxins are released when bacteria die or autolyze. The bacterial endotoxins can be detected qualitatively or semi-quantitatively by using shark reagent to produce agglutination reaction with bacterial endotoxins.
11.2 Reagents
Gel shark reagent.
11.3 Equipment
11.3.1 Constant temperature box.
2 Micropipette: 20μL~200μL, 100μL~1000μl. 11.3.2
3 Vortex mixer.
11.4 Determination steps
Take 2mL of cell culture medium, centrifuge at 150g~300g for 5min~10min, and take the supernatant. The test was carried out in accordance with the microbiological test method (General Rule 1143) of Part III of the Pharmacopoeia of the People's Republic of China (2015 Edition). 11.5 Calculation of results
The results were calculated in accordance with the microbiological test method (General Rule 1143) of Part III of the Pharmacopoeia of the People's Republic of China (2015 Edition). The results were expressed to one decimal place.
2 Determination of lactic acid content
12.1 Principle
Lactic acid generates pyruvic acid under the action of lactate dehydrogenase, and at the same time reduces nicotinamide adenine dinucleotide to generate NADH and H+. The production substrate was detected to calculate the lactic acid content in the fermentation broth. 12.2 Reagents
Lactic acid detection kit or equipment, different detection equipment is equipped with different reagents. 8
12.3 Equipment
Depending on the different detection mechanisms, the equipment used includes biochemical analyzer, spectrophotometer, etc. 12.4
Determination steps
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Take an appropriate amount of cell culture medium, centrifuge at 150g~300g for 10 minutes, take the supernatant and test according to the operating instructions of the kit or equipment. The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 5% of the arithmetic mean. Otherwise, the measurement should be repeated. 12.5
Result calculation
Calculate the results according to the instructions of the kit, and express the calculated results to two decimal places. Test report
The test report should include the following contents:
Specimen name;
Cell type;
Specimen number:
Technical basis;
Main instruments and equipment;
Test parameters;
Test temperature and environment;
Test date, test personnel,9
UV-visible spectrophotometer
GB/T38506—2020
Micropipette: 20μL~200μL, 100μL~1000μL. 10.3.2
3 Balance: sensitivity 0.01g.
10.4 Determination steps
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Take 2mL of cell culture medium, centrifuge at 150g~300g for 10min, and take the supernatant. Detect according to the WS/T350 method or use a biosensor. The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 5% of the arithmetic mean. Otherwise, re-determination is required.
10.5 Calculation of results
Calculation results are expressed to two decimal places
Determination of bacterial endotoxins
11.1 Principle
Endotoxins are released when bacteria die or autolyze. The bacterial endotoxins can be detected qualitatively or semi-quantitatively by using shark reagent to produce agglutination reaction with bacterial endotoxins.
11.2 Reagents
Gel shark reagent.
11.3 Equipment
11.3.1 Constant temperature box.
2 Micropipette: 20μL~200μL, 100μL~1000μl. 11.3.2
3 Vortex mixer.
11.4 Determination steps
Take 2mL of cell culture medium, centrifuge at 150g~300g for 5min~10min, and take the supernatant. The test was carried out in accordance with the microbiological test method (General Rule 1143) of Part III of the Pharmacopoeia of the People's Republic of China (2015 Edition). 11.5 Calculation of results
The results were calculated in accordance with the microbiological test method (General Rule 1143) of Part III of the Pharmacopoeia of the People's Republic of China (2015 Edition). The results were expressed to one decimal place.
2 Determination of lactic acid content
12.1 Principle
Lactic acid generates pyruvic acid under the action of lactate dehydrogenase, and at the same time reduces nicotinamide adenine dinucleotide to generate NADH and H+. The production substrate was detected to calculate the lactic acid content in the fermentation broth. 12.2 Reagents
Lactic acid detection kit or equipment, different detection equipment is equipped with different reagents. 8
12.3 Equipment
Depending on the different detection mechanisms, the equipment used includes biochemical analyzer, spectrophotometer, etc. 12.4
Determination steps
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Take an appropriate amount of cell culture medium, centrifuge at 150g~300g for 10 minutes, take the supernatant and test according to the operating instructions of the kit or equipment. The absolute difference between two independent measurement results obtained under repeatability conditions shall not exceed 5% of the arithmetic mean. Otherwise, the measurement should be repeated. 12.5
Result calculation
Calculate the results according to the instructions of the kit, and express the calculated results to two decimal places. Test report
The test report should include the following contents:
Specimen name;
Cell type;
Specimen number:
Technical basis;
Main instruments and equipment;
Test parameters;
Test temperature and environment;
Test date, test personnel,
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