Standard number: GB/T 38172-2019
Standard name: Protein A affinity chromatography medium
English name: Protein A affinity chromatographic medium
Standard format: PDF
Release time: 2019-10-18
Implementation time: 2019-10-18
Standard size: 786K
Standard introduction: This standard specifies the technical requirements, detection methods, inspection rules, labels, markings, packaging and transportation of protein A affinity chromatography medium.
This standard applies to the production and detection of protein A affinity chromatography medium.
2 Normative reference documents
The following documents are indispensable for the application of this document. For all dated reference documents, only the dated version applies to this document. For all undated reference documents, the latest version (including all amendments) applies to this document. GB/T191 Pictorial marking for packaging, storage and transportation
GB/T5475 Sampling method for ion exchange resin
GB/T6682 Specification and test method for water used in analytical laboratories This standard was drafted in accordance with the rules given in GB/T1.1-2009
This standard was proposed and managed by the China National Institute of Standardization.
Drafting units of this standard: Institute of Process Engineering, Chinese Academy of Sciences, China National Institute of Standardization, Zhongke Senhui Microsphere Technology (Suzhou) Co., Ltd.
Main drafters of this standard: Huang Yongdong, Zhao Lan, Zhu Kai, Wu Xuexing, Ma Guanghui, Su Zhiguo, Gong Fangling, Ma Aijin, Yang Weixing, Wang Shaoyun
This standard specifies the technical requirements, test methods, inspection rules, labels, markings, packaging, transportation and storage of protein A affinity chromatography media. This standard applies to the production and testing of protein A affinity chromatography media.

ICS07.080
National Standard of the People's Republic of China
GB/T38172—2019
Protein A affinity chromatographic medium
Protein A affinity chromatographic medium2019-10-18Issued
State Administration for Market Regulation
Standardization Administration of the People's Republic of China
Implementation on 2019-10-18
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard was proposed and managed by the China National Institute of Standardization. GB/T38172—2019
Drafting organizations of this standard: Institute of Process Engineering, Chinese Academy of Sciences, China National Institute of Standardization, and Zhongke Senhui Microsphere Technology (Suzhou) Co., Ltd.
The main drafters of this standard are Huang Yongdong, Zhao Lan, Zhu Kai, Wu Xuexing, Ma Guanghui, Su Zhiguo, Gong Fangling, Ma Aijin, Yang Weixing, Wang Shaoyun and Qin Jia. wwW.bzxz.Net
1 Scope
Protein A affinity chromatography medium
GB/T38172—2019
This standard specifies the technical requirements, test methods, inspection rules, labeling, marking, packaging, transportation and storage of protein A affinity chromatography medium.
This standard applies to the production and testing of protein A affinity chromatography medium. 2
Normative references
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For all undated references, the latest version (including all amendments) applies to this document. Packaging, storage and transportation pictorial marking
GB/T 191
GB/T5475
GB/T6682
Terms and definitions
Ion exchange resin sampling method
Specifications and test methods for water in analytical laboratories The following terms and definitions apply to this document. 3.1
Protein A affinity chromatographic medium
protein A affinity chromatographic medium A type of chromatographic medium prepared by coupling recombinant protein A to agarose separation medium. Technical requirements
Appearance requirements
Protein A affinity chromatographic medium should be spherical and full, with a smooth surface, and should be transparent under an optical microscope. 4.2
Performance requirements
Should meet the requirements in Table 1.
Table 1 Main performance requirements of protein A affinity chromatography medium Item
Range Particle size ratio (W diameter)/%
Average particle size (a)/μm
Maximum flow rate/(cm/h)
Ligand density (p)/(mg/mL)
Dynamic binding capacity (Qio%)/(mg/ml.)
90.0±13.5||tt ||≥300
GB/T38172—2019
Total colony count/（CFU/mL)
5-Hydroxymethylfurfural shedding amount/（μg/mL）Table 1 (continued)
Ratio of the volume of sample particles with a particle size range of 45.0um~165.0μm to the volume of all sample particles8
The highest flow rate can be reached at a pressure of 0.10MPa. b
The adsorption amount of human immunoglobulin (h-IgG) per milliliter of medium 5 Detection method
Sample treatment
According to GB/T5475, 5mL of sample was directly extracted from the product and placed in a 50mL G3 sand core funnel and dried for 5min. Wash with grade 3 water that meets GB/T6682 for 5 times, 2min each time, and finally use a vacuum pump to dry for 5min at a pressure of 0.1MPa. Place the washed medium in a beaker and add grade 3 water to ensure that there is 2 cm of grade 3 water on the medium. After mixing, a mixed system of protein A affinity chromatography medium and water is obtained.
5.1.2 Sample observation
Use a plastic pipette to suck up the mixed system and place it on a glass slide, and adjust the microscope magnification. Taking more than 80% of the area in the field of view as the protein A affinity chromatography medium as the standard, use a plastic pipette to increase or decrease the protein A affinity chromatography medium on the slide. Finally, press it with a cover glass. Adjust the focal length of the optical microscope to make the image in the field of view clear. Take a photo of the protein A affinity chromatography medium and save it. Particle size
Sample processing
The method is the same as 5.1.1.
5.2.2 Sample detection
Set the laser particle size analyzer parameters as follows: the measurement particle type is universal, the dispersant type is water, the analysis mode is single peak mode, and add the sample for measurement.
5.2.3 Result calculation
5.2.3.1 Range particle size ratio
Calculate according to formula (1):
W particle size
...................(I)
W Where:
W Rager
Range particle size ratio, %;
W. —The ratio of the volume of particles with a particle size of k (um) to the volume of all sample particles, %. 5.2.3.2 The average particle size
is calculated according to formula (2):
Wherein:
d——the average particle size of a certain number of medium particles counted, in micrometers (μm); d,——the particle size of a single particle, in micrometers (μm); N—the number of medium particles counted
GB/T38172—2019
. (2)
The absolute difference between three independent measurement results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 5.3 Maximum flow rate
5.3.1 Sample treatment
The method is the same as 5.1.1
5.3.2 Sample column loading
Select a 1.60cm×20.00cm chromatography column, pour the mixed slurry of medium and water into the chromatography column, block the column outlet, let it stand, control the medium bed height to 10.00cm±0.20cm, and fill the upper end of the column with water. Open the column inlet, and continuously pass 10 column volumes of tertiary water into the column at a flow rate of 0.5mL/min. After the bed is stable, the test can be carried out. 5.3.3 Sample measurement
Connect the chromatography column to the medium and low pressure chromatography system. When measuring, set a certain flow rate (mL/min) from zero, maintain the flow rate for 5 minutes, and record the column pressure P (MPa) at this time. Continue to increase the flow rate and measure the column pressure at the corresponding flow rate. Until the pressure reaches 0.10MPa, the corresponding flow rate is recorded as V0.1. 5.3.4 Result calculation
Calculate according to formula (3):
Where:
Fmx—maximum flow rate, in centimeters per hour (cm/h); Uo.1
volume flow rate at 0.1oMPa pressure, in milliliters per minute (mL/min); cross-sectional area of ​​chromatographic column, in square centimeters (cm): conversion factor for converting minutes to hours.
The absolute difference of three independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 5.4 Ligand density
5.4.1 Solution preparation
20g/L boric acid solution
Accurately weigh 20.00g boric acid in a 250mL beaker, add grade 3 water to dissolve and dilute to 1000mL....(3)
GB/T38172—2019
5.4.1.2 Methyl red-bromocresol green indicator Accurately weigh 3mg methyl red and 1mg bromocresol green in a 50mL beaker, add ethanol to dissolve and dilute to 100mL. 5.4.2 Sample treatment
Treat the medium sample according to method 5.1.1.
Then carry out nitration treatment, accurately weigh 1.00g sample, transfer it to a dry 100mL Kjeldahl flask, add 50mg selenium powder and 5mL concentrated sulfuric acid. Heat it on an electric stove. First, heat slowly over low heat for 2 hours until the liquid turns transparent and light green, then switch to high heat and continue heating for 30 minutes. When the liquid becomes clear and transparent, stop heating and let it cool naturally to room temperature. Accurately weigh 10.0 mg of protein A and dissolve it in 1 mL of water. At the same time, prepare 10.00 g/mL, 6.00 mg/mL, 3.00 mg/mL, and 1.25 mg/mL protein A standard solutions, and take 1 mL of each for nitration treatment. 5.4.3 Sample determination
Accurately weigh 1 mL of the nitrated sample and transfer it to a dry 100 mL Kjeldahl nitrogen determination bottle. Add 10 mL of 40% sodium hydroxide solution and react in a steam bath. Collect the distillate with 25 mL of 20 g/L boric acid solution (add 5 drops of methyl red-bromocresol green indicator, light purple), and immerse the outlet of the condenser below the liquid level of the boric acid solution. When the volume of the boric acid solution reaches 45 mL, the outlet of the condenser leaves the liquid surface. Rinse the outlet of the condenser with deionized water until the final volume is 50 mL and stop collecting. Titrate the above boric acid solution with 0.05 mol/L hydrochloric acid standard solution until gray or blue-purple is the end point, and record the volume of the hydrochloric acid standard solution consumed. Establish a standard curve of protein A mass-consumed hydrochloric acid volume. At the same time, take 1.00 g of blank medium to perform the above operation as a blank control. 5.4.4 Calculation of results
Calculate according to formula (4):
Wherein:
--protein A affinity chromatography medium ligand density, in milligrams per milliliter (mg/mL); protein A mass measured on protein A affinity chromatography medium, in milligrams (mg); mi
protein A mass measured on blank medium, in milligrams (mg); protein A affinity chromatography medium mass, in grams (g): 1.4 Conversion factor for converting protein A affinity chromatography medium mass to volume. The absolute difference between three independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 5.5
Dynamic loading capacity
5.5.1 Solution preparation
5.5.1.1 Buffer A, pH 7.0
Accurately weigh 4.17 g disodium hydrogen phosphate dodecahydrate, 1.27 g sodium dihydrogen phosphate dihydrate and 8.78 g NaCl in a 500 mL beaker, add tertiary water to dissolve, adjust the pH to 7.0, and finally make up to 1000 mL, and filter with a 0.45 μm filter membrane. 5.5.1.2 Buffer B, pH 3.0
Accurately weigh 7.51 g glycine, add tertiary water to dissolve, adjust the pH to 3.0, and finally make up to 1000 mL, and filter with a 0.45 μm filter membrane.
5.5.1.3 h-IgG sample (purity>98%) GB/T38172—2019
Dilute the h-IgG protein with buffer A and measure it at 280nm using a spectrophotometer. The final concentration is diluted to 3.00mg/mL. The protein concentration is calculated using the absorbance coefficient [1.33mL/(mg·cm). 5.5.2 Sample treatment
The method is the same as 5.1.1.
5.5.3 Sample column loading
Select a Φ1.60cm×20.00cm chromatography column, pour the mixed slurry of the medium and water into the chromatography column, block the column outlet, and let it stand to control the medium bed height to 5.00cm±0.20cm, and fill the top of the column with water. Open the column inlet and continuously pass 10 column volumes of tertiary water into the column at a flow rate of 2.0 mL/min. The bed can be tested after it is stable. 5.5.4 Sample determination
Connect the chromatography column to the chromatography system and detect the ultraviolet (UV) signal at 280 nm to monitor the load determination process. The dynamic binding capacity is expressed as a 10% penetration value. Measure and record the 100% UV signal of the sample h-IgG, including the UV absorption of the IgG subclass that is not bound to the medium. Use buffer A to balance the chromatography column to the UV baseline at a flow rate of 2.0 mL/min, load the sample at a flow rate of 2.0 mL/min, and end the loading when the UV signal concentration of h-IgG in the flow-through curve is 10% of the sample concentration, and record the loading volume. After the loading is completed, use buffer A to balance the UV signal to the baseline at a flow rate of 2.0 mL/min; use buffer B to elute at a flow rate of 2.0 mL/min. Calculate the dynamic loading. Determination of breakthrough volume under non-retention conditions: Connect the column to the chromatography system and detect the UV signal at 280 nm. Load 10 μL of 1% acetone and record the volume when the UV signal appears. 5.5.5 Calculation of results
Calculate according to formula (5):
C. (Vr-V.)
Wherein:
Dynamic binding capacity, in milligrams per milliliter (mg/mL): C.
Starting protein concentration, in milligrams per milliliter (mg/mL); (5)
——The volume of the effluent when the protein concentration C at the column outlet reaches 10% of the protein concentration C at the inlet, in milliliters (mL); V.——The breakthrough volume under non-retention conditions, in milliliters (mL) Vgel medium volume, in milliliters (mL). The absolute difference of three independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 5.6 Total colony count
5.6.1 Sample pretreatment
The method is the same as 5.1.1.
5.6.2 Sample determination
Reference to the microbial count method for microbial limit test of non-sterile products in the Pharmacopoeia of the People's Republic of China (2015 edition) (1105). Take 1mL of the drained medium, add 1mL of grade 3 water, and mix the sample with a vortex mixer. Sterilize the conical flask containing 30mL of tryptic soy agar GB/T38172-2019
lipid medium at 121℃ for 20min and place it in a 40.0℃ thermostat. When the culture medium cools to 40.0℃, use a micropipette to draw 1mL of the mixed sample and add it to the conical flask, mix well, pour the mixture into the culture dish, and cover it. Let the mixture solidify at room temperature. Place the cultured blood in a constant temperature incubator at 35℃ for 5 days. 5.6.3 Calculation of results
Check the culture dish after the incubation period. The absolute difference between the three independent determination results obtained under repeatability conditions for counting colony forming units (CFU) should not exceed 5 CFU/mL suspension. 5.7 5-Hydroxymethylfurfural shedding amount
5.7.1 Solution preparation
5.7.1.110% (volume fraction) methanol aqueous solution Accurately measure 100mL of chromatographic pure anhydrous methanol, mix it evenly with 900mL of secondary water, and then filter it with a 0.22μm pore size filter membrane and remove bubbles by ultrasound.
5.7.1.2 5-Hydroxymethylfurfural standard stock solution Accurately weigh an appropriate amount of 5-Hydroxymethylfurfural standard substance into a 100mL volumetric flask. Dissolve it with 10mL of 10% methanol aqueous solution, make up to volume, and prepare a 0.20mg/mL standard stock solution.
5.7.1.31mmol/L hydrochloric acid solution pH3.0
Measure 0.083mL concentrated hydrochloric acid (12mol/L) and dilute to 1000mL, adjust the pH to 3.0. 5.7.1.4100mmol/L sodium hydroxide solution. pH13.0 Weigh 4.00g of sodium hydroxide solid, dissolve it in 100mL of tertiary water, cool to room temperature, and dilute to 1000mL with tertiary water, and adjust the pH to 13.0.
5.7.1.5 Standard working solution
Pipette 5μL, 15μL, 25μL, 40μL, 50μL, 100μL, 1mL, 2mL of 5-hydroxymethylfurfural standard stock solution into 100mL volumetric flasks respectively, dilute to scale with 10% methanol aqueous solution, and prepare 0.01g/mL, 0.03μg/mL, 0.05μg/mL, 0.08pg/mL0.10μg/mL, 0.20μg/mL, 1.00μg/mL, 2.00uμg/mL standard working solutions. Prepare and use immediately. 5.7.2 Sample treatment
The cleaning method is the same as 5.1.1. Take several 1.00g portions of the dried protein A affinity chromatography medium and place them in glass test tubes with lids. Take 10mL of pH3.0 and pH13.0 solutions respectively and place them in each test tube. The sample tubes are incubated at 40.0℃. After 7 days, transfer the supernatant to a clean test tube.
The supernatant is rotary evaporated (60℃, 50r/min), fixed to 2mL, and an equal volume of 12mol/L hydrochloric acid is added, and hydrolyzed at 100℃ for 1h. Fixed to 5mL. Filter with a 0.45μm filter membrane to obtain the sample solution. 5.7.3 Sample determination
High performance liquid chromatography conditions: The chromatographic column is selected as C1.5μm, 250mm×4.6mm (inner diameter). The mobile phase is 10% methanol aqueous solution. Flow rate 1.0mL/min. Detection wavelength 285nm. Column temperature 30℃. Injection volume 10μL The standard working solution and sample solution are tested under the above high performance liquid chromatography conditions. Record the retention time and peak area of ​​the standard working solution, and draw a standard working curve with peak area versus corresponding concentration. Use the retention time of the standard working solution to qualitatively characterize the sample, and record the peak area at the retention time, and use the standard working curve to quantitatively characterize the sample. 5.7.4 Calculation of results
Calculate according to formula (6):
Wherein:
The amount of 5-hydroxymethylfurfural released per milliliter of medium, in micrograms per milliliter (ug/mL); GB/T38172—2019
The concentration of 5-hydroxymethylfurfural in the sample solution obtained from the working curve, in micrograms per milliliter (uμg/mL); c
VThe final constant volume of the sample after hydrolysis with hydrochloric acid, in milliliters (mL); m
The mass of the protein A affinity chromatography medium weighed, in grams (g); 1.4 The conversion factor for converting the mass of the protein A affinity chromatography medium into volume. The absolute difference of three independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. Inspection rules
6.1 Batch
Products with uniform quality produced in the same process cycle are considered as one batch.6.2 Sampling
Perform according to the provisions of GB/T5475.
6.3 Factory inspection
Each batch of products shall be inspected before leaving the factory, and only qualified products can leave the factory. The factory inspection items are the particle size, maximum flow rate, ligand density, dynamic loading capacity and total colony count specified in Chapter 4. 6.4
Type inspection
6.4.1 During normal production, a type inspection shall be conducted every six months. A type inspection shall be conducted in any of the following circumstances: a) During trial production and appraisal of new products; after normal production, if there are major changes in raw materials, processes, and equipment that may affect product performance; b) When the product is resumed after being discontinued for more than six months; d) When the factory inspection results are significantly different from those of the last type inspection; e) When the supervisory agency makes a request. The type inspection items are all the items specified in Chapter 4. Judgment rules
6.5.1 Factory inspection judgment and re-inspection
Qualified products.
The factory inspection items meet the requirements of the items in 6.3, and the batch is judged to be qualified. If 1 to 2 items of the factory inspection items do not meet the requirements, they should be re-inspected. If there is still one item that does not meet the requirements after the re-inspection, the batch is judged to be unqualified. If more than 2 items of the factory inspection items do not meet the requirements, the batch is judged to be unqualified. 6.5.1.3
GB/T38172—2019
Type inspection judgment and re-inspection
Type inspection items meet the requirements of the items in 6.4 and are judged to be qualified. 6.5.2.1
Type inspection items 1 to 3 (including 3 items) do not meet the requirements, and they should be re-inspected. If there is still one item that does not meet the requirements after the re-inspection, the batch is judged to be unqualified.
6.5.2.2
The batch is unqualified.
6.5.2.3If more than 3 items of the type inspection items do not meet the requirements, the batch is judged to be unqualified. 7 Labels, signs, packaging, transportation and storage labels
should at least include the following:
product name;
model;
volume;
production batch number;
production organization;
production date;
validity period;
notes.
packaging, storage and transportation labels shall comply with the provisions of GB/T191. 7.3
Plastic bottles should be used for packaging, and the packaging materials should ensure that the product is not contaminated or leaked during transportation and storage. Transportation
Should be transported at room temperature, avoid overcooling or overheating, and take measures to prevent the product from losing water. 7.5
Should be stored at 4℃～8℃, with a validity period of five years. If the validity period exceeds the validity period, it can be re-tested according to the provisions of this standard. If the re-test results meet the requirements of the standard, it can still be used.
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