CHO-S Cell Residual Protein Detection Kit and Its Preparation Method

Abstract

The present invention relates to the field of biomedical analysis, and discloses a CHO-S cell residual protein detection kit and a preparation method thereof, including an enzyme-labeled plate coated with an anti-HCP protein antibody, biotin-labeled IgG, streptavidin labeled with horseradish peroxidase, and an auxiliary preparation. The usage method of the kit includes the following steps S1: coating the enzyme-labeled plate with a CHO HCP antibody; S2: adding a sample to be detected and a standard solution to the coated enzyme-labeled plate, incubating and washing; S3: adding biotin-labeled IgG to the enzyme-labeled plate washed in S2, incubating and washing; S4: adding streptavidin labeled with horseradish peroxidase to the enzyme-labeled plate washed in S3, incubating and washing; S5: adding a chromogenic solution to the enzyme-labeled plate washed in S4 for color development, incubating, adding a termination solution to terminate the reaction, and measuring the absorbance value at a wavelength of 450 nm. The kit makes up for the blank of a special HCP detection kit, and has high specificity and high detection precision for HCP detection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical analysis, and particularly to a kit for detecting residual proteins in CHO-S cells and a preparation method thereof. Background Art

[0002] Full name of CHO cells: Chinese Hamster Ovarian (CHO) cells. The proteins expressed by these cells are closest to natural protein molecules. The products are secreted extracellularly but rarely secrete their own endogenous proteins, which is very beneficial for the separation and purification of target proteins. It is an engineering cell widely used in monoclonal antibody and recombinant protein biologics at present. The CHO cell expression system has advantages such as a clear expression genetic background, a perfect and stable expression system, and a relatively high protein expression level. Although the amount of endogenous proteins secreted by CHO is small, there are still a small amount of proteins remaining in the semi-finished and finished products of biologics. Host cell proteins (HCPs) remaining in biologics belong to heterologous proteins, including both the structural proteins of host cells and the growth factors secreted by host cells (passage cells). HCPs not only have the potential to induce the body to produce anti-HCP antibodies, causing allergic reactions in the body, but also may have an "adjuvant effect" that causes the body to produce antibodies against protein drugs, affecting the therapeutic effect of drugs. Therefore, biomedical enterprises must quantitatively determine the HCPs in the semi-finished or finished products during the quality control process.

[0003] Currently, the main method for HCP determination is the ELISA method. As a classic method, the ELISA method is widely used because of its high sensitivity and specificity. The 2020 edition of the pharmacopoeia stipulates that the residual amount of host cell proteins in drugs should be determined by enzyme-linked immunosorbent assay and should not be higher than 0.01% of the total protein. Commercial ELISA kits eliminate the differences in HCPs caused by different CHO cell lines during preparation, so as to be applicable to a series of recombinant CHO cell lines. However, studies have shown that the following problems exist in the current ELISA kits during use: 1. There are large deviations in the results between different detection kits; 2. There are large differences in the results measured after replacing the same detection reagent and standard product; 3. There are large differences in the composition and proportion of standard products. Therefore, there is an urgent need to develop a special kit for detecting residual proteins in CHO cells. Summary of the Invention

[0004] The present invention aims to provide a kit for detecting residual proteins in CHO-S cells and a preparation method thereof, so as to solve the problems of large differences in detection results, low coverage rate of host proteins, and low detection accuracy when using ELISA kits to detect CHO-S HCPs in the prior art.

[0005] To achieve the above object, the present invention adopts the following technical solutions: A CHO-S cell residual protein detection kit, comprising an enzyme-linked immunosorbent assay (ELISA) plate coated with an anti-HCP protein antibody, biotin-labeled IgG, streptavidin labeled with horseradish peroxidase, and auxiliary preparations.

[0006] On the other hand, the present technical solution also provides a preparation method of the CHO-S cell residual protein detection kit, comprising the following steps:

[0007] Step I: Take the CHO-S host cell protein immunogen, dialyze it overnight with PBS, and quantitatively detect the total protein amount.

[0008] Step II: Immunize part of the HCP immunogen in rabbits to produce antibodies, and lyophilize the remaining HCP immunogen and use it as a standard product.

[0009] Step III: After quantitatively detecting the total protein amount, adjust the protein concentration of the HCP immunogen to 2 - 20 mg / mL, and store it for later use at low temperature.

[0010] Step IV: Use the antibodies obtained in Step II for booster immunization to obtain anti-HCP protein antibodies, and coat the HCP antibodies on a 96-well plate.

[0011] Step V: Biotin-label the antibody. After stirring and dialyzing IgG with PBS overnight, label it with biotin on ice; replace with fresh PBS and continue dialysis to obtain biotin-labeled IgG, and store it for later use at low temperature.

[0012] Step VI: Detection application.

[0013] The principle and advantages of this solution are as follows: This technical solution obtains a special kit for detecting CHO-S cell residual protein, and the kit has high detection sensitivity and is very suitable for popularization and application. When preparing the kit, first dialyze the obtained HCP immunogen overnight to remove contaminated small molecules to avoid affecting the quantitative detection of the total protein amount by the BCA method; then immunize part of the HCP immunogen in rabbits to produce antibodies, and lyophilize the other part and send it back to Genrixbio as a CHO-S HCP ELISA standard product. To ensure the concentration of the HCP protein antibody, booster immunization is carried out. The concentration of the HCP protein antibody has an important impact on the detection accuracy of the kit. When preparing biotin-labeled IgG, by optimizing the operation method and parameters, it is possible to remove excess biotin, which is crucial for reducing the ELISA background interference to the lowest level and will significantly affect the sensitivity of the ELISA detection method. Through the optimization of antibody generation, protein concentration adjustment, and antibody biotin-labeling steps, this technical solution not only obtains a special kit for detecting CHO-S cell residual protein but also ensures the detection sensitivity of the kit.

[0014] Preferably, as an improvement, biotin-labeled IgG is diluted and used at a ratio of 1:200 to 6400, and the diluent is a phosphate-Tween buffer containing 0.2% to 1% BSA at pH 7.4.

[0015] In this technical solution, after biotin-labeled IgG is diluted at the above ratio, it can meet the subsequent detection requirements, and the diluent is preferably a phosphate-Tween buffer.

[0016] Preferably, as an improvement, the auxiliary preparation includes: carbonate buffer at pH 9.6, TMB chromogenic solution, and termination solution, and the termination solution is sulfuric acid or hydrochloric acid.

[0017] Preferably, as an improvement, in step I, the condition for dialysis with PBS overnight is stirring at 4°C for 24 h.

[0018] In this technical solution, the condition for dialysis with pre-cooled PBS buffer at pH 7.4 overnight is stirring at 4°C for 24 h to ensure the activity of HCP protein, promote the desorption of small molecules such as impurities, and reduce the impact on subsequent detection and quantification steps.

[0019] Preferably, as an improvement, in step IV, the immunized animal is expanded to a rabbit, and the immunization is performed by multi-site intramuscular injection. In the first injection, every 1 mL of immunogen is mixed with 1 mL of Freund's complete adjuvant; in subsequent injections, every 1 mL of immunogen is mixed with 1 mL of Freund's incomplete adjuvant; the immunization program lasts for 98 days.

[0020] In this technical solution, compared with other animals, rabbits provide high-quality anti-CHO-S HCP antibodies, and because rabbits are small in size and have less blood volume, multiple rabbits are used to increase individual diversity and obtain population diversity of immune antibodies. The above immunization program is the optimal condition verified by experiments.

[0021] Preferably, as an improvement, in step V, the molar ratio of biotin to IgG is 5 to 20:1.

[0022] In this technical solution, the above molar ratio of biotin to IgG can avoid the influence of residual free biotin molecules on the activity and detection of biotin-labeled antibodies during the purification process. In actual use, it can also be optimized to 10:1, and a smaller molar ratio can avoid the influence of residual free biotin molecules during the purification process.

[0023] Preferably, as an improvement, the method for using the CHO-S cell residual protein detection kit includes the following steps:

[0024] S1: Coating the enzyme-linked immunosorbent assay (ELISA) plate with CHO-S HCP antibody;

[0025] S2: Add the sample to be detected and the standard solution to the coated ELISA plate, incubate, and wash.

[0026] S3: Add biotin-labeled IgG to the ELISA plate washed in S2, incubate, and wash.

[0027] S4: Add horseradish peroxidase-labeled streptavidin to the ELISA plate washed in S3, incubate, and wash.

[0028] S5: Add the chromogenic solution to the ELISA plate washed in S4 for color development, incubate, add the stop solution to terminate the reaction, and measure the absorbance value at a wavelength of 450 nm.

[0029] In this technical solution, the kit has simple steps and convenient operation when in use.

[0030] Preferably, as an improvement, in step S5, the chromogenic solution is TMB or OPD.

[0031] In this technical solution, either TMB or OPD as the chromogenic solution can meet the color development requirements for detection. Brief Description of the Drawings

[0032] Figure 1 It is the determination result of the antiserum titer on the 42nd day in the embodiment of the present invention.

[0033] Figure 2 It is the determination result of the antiserum titer on the 70th day in the embodiment of the present invention.

[0034] Figure 3 It is a schematic diagram of eluting IgG from the MAbSelect affinity column.

[0035] Figure 4 It is an analysis chart of the antibody coverage rate of two-dimensional protein blotting.

[0036] Figure 5 It is the original gel image of the total protein detection of acidic to neutral pH value CHO-S HCP stained with Sypro Ruby.

[0037] Figure 6 It is the gel image of the total protein detection, cutting, and analysis of acidic to neutral pH value CHO-S HCP stained with Sypro Ruby.

[0038] Figure 7 It is the original gel image of the western blot analysis of the antibody coverage rate of acidic to neutral pH value HCP against CHO-S HCP.

[0039] Figure 8Gel images for Western blot analysis, cutting, and analysis of anti-CHO-S HCP antibody coverage of acidic to neutral pH HCP.

[0040] Figure 9 Original gel image of total protein staining of neutral to alkaline CHO-S HCP detected using Sypro Ruby staining.

[0041] Figure 10 Gel images for total protein detection, cutting, and analysis of neutral to alkaline pH CHO-S HCP using Sypro Ruby staining.

[0042] Figure 11 Original gel image of Western blot analysis of anti-CHO-S HCP antibody coverage of neutral to alkaline pH HCP.

[0043] Figure 12 Gel images for Western blot analysis, cutting, and anti-CHO-S HCP antibody coverage of neutral to alkaline pH HCP.

[0044] Figure 13 CHO-S HCP sandwich ELISA condition optimization --- coating and reporter antibodies (3 concentration combinations).

[0045] Figure 14 Four-parameter curve of CHO-S HCP sandwich ELISA condition optimization --- coating and reporter antibody combination 1.

[0046] Figure 15 Four-parameter curve of CHO-S HCP sandwich ELISA condition optimization --- coating and reporter antibody combination 2.

[0047] Figure 16 Four-parameter curve of CHO-S HCP sandwich ELISA condition optimization --- coating and reporter antibody combination 3.

[0048] Figure 17 Optimized sandwich ELISA conditions --- typical standard curve.

[0049] Figure 18 Fitting curve of the linear range of the kit in the examples of the present invention. Detailed Description of the Invention

[0050] The following is a further detailed description through specific embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art; the experimental methods used are all conventional methods; the materials, reagents, etc. used can all be obtained from commercial sources.

[0051] CHO-S cell residual protein detection kit, comprising an enzyme-labeled plate coated with anti-HCP protein antibody, biotin-labeled IgG, streptavidin labeled with horseradish peroxidase, and auxiliary preparations. The auxiliary preparations include: carbonate buffer solution with pH 9.6, TMB chromogenic solution, termination solution, and the termination solution is sulfuric acid or hydrochloric acid. Preparation method of the CHO-S cell residual protein detection kit, comprising the following steps:

[0052] Step I: Use CHO-S mother cells (purchasing manufacturer: Invitrogen (Shanghai) Trading Co., Ltd.; product name: CHO-S cells; product number: A115570) to perform water bath resuscitation at 37°C and then use them to prepare HCP antigen without human IgG. Take the CHO-S host cell protein (HCP) immunogen, dialyze it overnight with 4L PBS (stir at 4°C for 24h), and quantitatively detect the total protein amount; the dialysis uses a 45mm flat-width dialysis tube of VWR Scientific (Spectra / Por, product number: 25225-260, cut-off molecular weight is 12,000 - 14,000 daltons). The purpose of dialysis is to remove contaminating small molecules to avoid affecting the accuracy of the total protein content determination. The total protein concentration after dialysis is 3.5mg / mL.

[0053] Step II: Immunize part of the HCP immunogen in rabbits to produce antibodies, and lyophilize the remaining HCP immunogen and send it back to Genrixbio as the CHO-S HCP ELISA standard; use a human IgG ELISA detection preparation to detect possible human IgG contamination in the immunogen. The result shows that no obvious human IgG contamination is detected in the immunogen (ArrayBridge human IgG ELISA, product number: AB000303), and the quantitative limit of the analysis system is 1.1ng / mL IgG.

[0054] After quantitatively detecting the total protein amount, adjust the HCP immunogen protein concentration to 4mg / mL and store it at -20°C for later use;

[0055] Step IV: Use the antibody obtained in Step II for booster immunization to obtain anti-HCP protein antibody, and coat the HCP antibody on a 96-well plate.

[0056] Use 6 rabbits to produce CHO-S HCP antibody. According to past experience, compared with other breeds of rabbits, better quality anti-CHO-S HCP antibody can be produced. In addition, there are slight differences in the antibody sets produced by different rabbits, so using multiple rabbits can also generate a better diverse antibody population. The booster immunization procedure is as follows:

[0057] Dosage: One injection per rabbit, 4 mg per injection (New Zealand white rabbits, male, Oryctolagus spp, weighing 2 - 3 kg at the start of immunization).

[0058] For the first injection, 1 mL of immunogen was mixed with 1 mL of Sigma - Aldrich complete Freund's adjuvant. For subsequent injections, 1 mL of immunogen was mixed with 1 mL of Sigma - Aldrich incomplete Freund's adjuvant (both from Sigma - Aldrich). Immunization was carried out by multi - site intramuscular injection (IM) (three injections dorsally and three additional injections ventrally). Each injection contained 2 mg of the injection solution, with a total of 4 mg of CHO - S HCP protein.

[0059] Immunization protocol: A multiple - immunization protocol over 98 days was used to obtain antibodies of optimal quality. The results are as Figure 1 、 2 shown. Figure 1 In the same large column, from left to right, they are 2000, 8000, 32000, 128000, 512000, 2048000 in turn; Figure 2 In the same large column, from left to right, they are 500, 2000, 8000, 32000, 128000, 512000 in turn; The antibody titer is defined as the highest dilution of the antibody serum when the OD reading by direct ELISA is greater than or equal to 0.5. The results showed that all six rabbits reached a titer (dilution) of 1:256,000, and 4 / 6 rabbits reached a titer (dilution) of 1:1,024,000.

[0060] Step V: Biotinylation of antibodies:

[0061] Purification: For large - scale IgG purification, 10 mL of antibody serum from each rabbit was mixed and filtered through a 0.45 μm filter. The anti - CHO - S HCP antibody was purified using a MAbSelect affinity column (GE Life Sciences, catalog number: 17 - 5474 - 01). The IgG binding capacity of the affinity column used for purification was 350 mg (resin volume 20 mL, column diameter 1.8 cm, column length 10 cm). The process was carried out under the following conditions: The sample was diluted 1:1 with PBS and loaded onto the MabSelect affinity column. The affinity column was washed with PBS until the absorbance at 280 nm was below 0.02. IgG was eluted with glycine (50 mM, pH 2.8), and the IgG fraction was collected when OD 280 reached 0.2, and elution was stopped when the peak OD280 was below 0.5.

[0062] The procedure is as follows: The flow rate was 2 mL / min

[0063] 0 - 10 minutes: Elution with 100% buffer A (PBS).

[0064] 10 - 50 minutes: Replace the 100% buffer A elution with buffer B (glycine, pH 2.8).

[0065] 50 - 60 minutes: Continue the 100% buffer B elution.

[0066] 60 - 70 minutes: Replace the 100% buffer B elution with 100% buffer A.

[0067] 70 - 90 minutes: Continue the 100% buffer A elution. The results are as Figure 3 shown. After IgG elution, immediately dialyze with 4 L of PBS at 4°C with stirring overnight. Immediate dialysis is to neutralize the acidic solution as soon as possible, thereby minimizing the acidic denaturation of IgG; there is no need to change the buffer.

[0068] Labeling: The above - purified IgG fraction is labeled with biotin. The biotin labeling uses the biotin labeling kit from Thermo Scientific (product numbers: PI - 21335, PI - 21435), and the biotin labeling reagent recommended by the manufacturer is used; the molar ratio of biotin:IgG is 20:1. After IgG is dialyzed with stirring overnight at 4°C with 4 liters of PBS in a dialysis tube (molecular cut - off is 10,000 daltons), it is labeled with biotin on ice for 2 hours. The next day, replace with 4 L of fresh PBS and continue dialysis for 24 hours. The above steps are repeated once. Remove the excess biotin as much as possible, which is crucial for minimizing the ELISA background interference and will significantly affect the sensitivity of the ELISA detection method. Recover the biotin - labeled IgG from the dialysis tube, quantitatively detect the protein concentration using a ultra - micro ultraviolet - visible light IgG Quantitation, and store it at - 20°C for later use.

[0069] Step VI: Detection application.

[0070] A method for using a kit for detecting residual proteins in CHO - S cells, comprising the following steps:

[0071] S1: Coat an enzyme - linked immunosorbent assay (ELISA) plate with CHO - S HCP antibody and block it with skim milk powder;

[0072] S2: Add the sample to be detected and the standard solution to the blocked ELISA plate, incubate, and wash;

[0073] S3: Add biotin - labeled IgG to the ELISA plate after washing in S2, incubate, and wash;

[0074] S4: Add streptavidin - horseradish peroxidase conjugate to the ELISA plate after washing in S3, incubate, and wash;

[0075] S5: Add chromogenic solution to the ELISA plate after washing in S4 for color development, incubate, add stop solution to terminate the reaction, and measure the absorbance value at a wavelength of 450 nm.

[0076] I. Coverage Verification

[0077] Experimental Example 1: Total Protein Detection Based on Two-Dimensional Gel and Western Blot Coverage Analysis

[0078] Combination Figure 4 As shown in the flowchart of coverage verification, the total protein of CHO-S HCP preparation was detected by isoelectric focusing electrophoresis of Bio-Rad; an 18-cm gradient gel strip (IPG) was used. The evaluation aim was to explore the optimal conditions to detect as many CHO-S HCP as possible. The solubility and repeatability of the gel should meet the requirements of 2-D Western Blot.

[0079] Experimental Example 2: Western Blot Analysis

[0080] Mix the antibody sera of 6 immunized rabbits, purify IgG with MabSelect and then perform Western Blot analysis. 1000 μg IgG was used for 2-D Western Blot analysis of acidic and basic HCP respectively. The reporting antibody was goat anti-rabbit IgG antibody (Rockland) conjugated with IRDye 800CW, and the terminal signal was detected using the Li-Cor Odyssey system. The PDQuest program (Bio-Rad, USA) was used to analyze the 2-D Western Blot pictures and 2-D total protein staining pictures with the same analysis parameter conditions. A 10%-20% SDS-Tris-Glycine gel was used, and then Sypro Ruby staining was performed. 1000 μg protein was loaded on an 18-cm IPG gel strip for isoelectric focusing electrophoresis separation, and Bio-Rad unstained standard was used. The primary antibody was anti-HCP IgG, and the secondary antibody was goat anti-rabbit IgG antibody (Rockland) conjugated with IRDye 800CW diluted 1:5000 times. The protein image was obtained using Bio-Rad ChemiDoc, and the protein immunoblot image was obtained using Li-Cor Odyssey. Coverage calculation method: The total number of protein spots of acidic and basic proteins recognized by anti-CHO-S HCP antibody was 187, and the total number of protein spots of acidic and basic proteins obtained by Sypro Ruby staining was 207, and the ratio was 90%. Combination Figures 5 - 12As shown in the figure, the Western blot analysis of the CHO-S HCP antibody coverage rate shows that the summary of the 2-D total protein staining and the 2-D Western Blot analysis of the anti-CHO-S HCP coverage rate is as follows: the total HCP protein analysis and the antibody-recognized protein spots show that the coverage rate of the anti-CHO-S HCP antibody in CHO-S HCP is 90%.

[0081] Experimental Example 3: Development and Research of a Specific Process for CHO-S HCP ELISA

[0082] A sensitive CHO-S HCP ELISA assay was developed based on the rabbit anti-CHO-S HCP antibody. The sandwich ELISA used the purified IgG from the pooled sera of 6 rabbits as the capture antibody, the biotinylated IgG in the sample as the reporting antibody for detection and the quantification of HCP from CHO-S biological products. The principle of the sandwich ELISA is that there are multiple epitopes on a large number of host cell proteins, resulting in more than one polyclonal antibody. Therefore, if the capture antibody occupies one of the epitopes, the other epitope will bind to the reporting antibody. The following is an example of the development process of the ELISA coating and reporting antibodies.

[0083] 1. Optimization of the Conditions for the CHO-S HCP Sandwich ELISA - Coating and Reporting Antibodies

[0084] The standard curves obtained using different concentrations of the secondary antibody are shown as Figures 13 - 17 shown, where Figure 14 is the curve at a secondary antibody concentration of 2.5 μg / ml, Figure 15 is the curve at a secondary antibody concentration of 5 μg / ml, Figure 16 is the curve at a secondary antibody concentration of 10 μg / ml, Figure 17 is a typical standard curve without a plateau obtained by adjusting the starting concentration. When the primary antibody is coated at 10 μg / ml, the optimal concentration of the secondary antibody is 10 μg / ml, and the best standard curve is obtained at this time.

[0085] I. Kit Performance Testing

[0086] Experimental Example 1: Specificity Testing

[0087] The test solution (108.3 mg / ml), buffer (GRl501-20190331-FB, FB), HCP standard solution (200 ng / ml), and bovine serum albumin solution (200 ng / ml) were diluted according to Table 1 of the validation protocol. The 10 mg / ml GRl501 stock solution was denoted as 10 mg / ml DS, the buffer diluted 21.66-fold from the stock solution was denoted as FB, the HCP standard solution was denoted as HCP, and the bovine serum albumin solution was denoted as BSA; one sample of each was prepared.

[0088] Table 1 Specificity Test Sample Solution Preparation

[0089]

[0090]

[0091] Note: Preparation of 200 ng / ml bovine serum albumin solution: Weigh 0.1 g of bovine serum albumin dry powder and dissolve it in 1 ml of ultrapure water to obtain a 100 mg / ml BSA stock solution, which is diluted 5×10 5 times before use to prepare the required solution.

[0092] Determination was carried out according to the "Standard Operating Procedure for Residual Host Cell Protein in CHO Cells (Exclusive Kit Method)", and duplicate wells were set. Calculate the recovery rate, and the recovery rate calculation formula is as follows:

[0093]

[0094] Acceptance criteria: The recovery rate of the standard HCP is between 80% and 120%, the recovery rate of bovine serum albumin (BSA) is <10%, and the FB HCP content is <1 ng / ml. The test results are shown in Table 2.

[0095] Table 2 Specificity Test Results

[0096]

[0097] Note: " / " indicates not added; "Range" indicates below the detection limit of the equipment

[0098] Experimental Example 2: Precision Test

[0099] 1. Repeatability

[0100] Prepare test samples at three levels: high concentration spike (HVS), medium concentration spike (MVS), and low concentration spike (LVS), with 3 replicates for each level. Carry out the test according to the "Standard Operating Procedure for Residual Host Cell Protein in CHO Cells (Exclusive Kit Method)", measure each sample once, and set duplicate wells. Statistically analyze the Average, SD, and RSD of the measurement results. Acceptance criteria: RSD ≤ 20%.

[0101] Table 3 Accuracy, Precision, Linearity and Range Test Sample Solution Preparation

[0102]

[0103] The test results are shown in Table 4.

[0104] Table 4 Repeatability Test Results

[0105]

[0106] 2. Personnel / Day Precision

[0107] Take the stock solution of GR1501 (GRl501 - 2019002DS, 108.3 mg / ml), denoted as DS, and dilute it to 10 mg / ml, 5 mg / ml, and 2.5 mg / ml with the diluent. Measure and calculate the HCP content in the results. The data for 3 days of the same batch of kits by the same person are the data for day precision, and the data for different personnel are used as the data for personnel precision. Acceptance criterion: RDS ≤ 30%. The results are shown in Tables 5 and 6.

[0108] Table 5 Results of Day Precision Experiment

[0109]

[0110] Table 6 Results of Personnel Precision Experiment

[0111]

[0112] Experimental Example 3: Accuracy Test

[0113] Refer to Table 3 to prepare test samples at three levels of HVS, MVS, and LVS on the same day, with 3 replicates for each level. Statistically analyze the recovery rate and the average recovery rate for each determination result at each concentration. Conduct according to the "Standard Operating Procedure for Residual CHO Cell Host Cell Proteins (Exclusive Kit Method)", perform each determination once, and set duplicate wells. Statistically analyze the recovery rate and the average recovery rate for each determination result at each concentration, and calculate according to the above recovery rate formula.

[0114] Acceptance criterion: The recovery rate and the average recovery rate are between 80% and 120%. The results are shown in Table 7.

[0115] Table 7 Results of Accuracy Experiment

[0116]

[0117]

[0118] Experimental Example 4: Limit of Quantification

[0119] Take the 1.111 ng / ml standard product and dilute it 3-fold downwards to 0.005 ng / ml. There are a total of 6 samples, as shown in the following table. Calculate the sample recovery rate. The previous point of the concentration point where the recovery rate is not within the acceptable range is the quantitation limit. Perform according to the "Standard Operating Procedure for Residual Host Cell Protein in CHO Cells (Exclusive Kit Method)", measure each sample once, and set duplicate wells. Calculate the sample recovery rate. The previous concentration point of the concentration point where the recovery rate is not within the acceptable range is the quantitation limit. Acceptance criteria: between 70% and 130%. As can be seen from the following table, the quantitation limit of this kit is 1.111 ng / ml.

[0120] Table 8

[0121]

[0122] Experimental Example Five: Detection Limit

[0123] Take the 1.111 ng / ml standard product and dilute it 3-fold downwards to 0.005 ng / ml. There are a total of 6 samples. Observe the mean result. The result shows that the previous point of the concentration point lower than the equipment detection limit is the detection limit. The results are shown in Table 9. The detection limit of the kit of the present invention is 0.123 ng / ml.

[0124] Table 9

[0125]

[0126] Experimental Example Six: Linear Range

[0127] According to the following table, add standard products with 5 concentration gradients to 10 mg / ml DS as test samples, specifically referring to Table 3, and calculate the recovery rates of the five concentration points. Use the added HCP content as the abscissa and the actually detected HCP content as the ordinate to fit a curve, and calculate the recovery rate according to the above recovery rate formula (range 80 - 120%). Acceptance criteria: linear R 2 ≥0.980, and the recovery rate should be between 80% and 120%. The obtained fitted curve is as Figure 18 shown, y = 0.9369x - 0.7984, R 2 = 0.9996.

[0128] Table 10

[0129]

[0130] Experimental Example Seven: Dilution Linearity

[0131] Take the GR1501 stock solution (GR1501-20190020S, 108.3 mg / ml), denoted as DS, dilute it to 10 mg / ml, 5 mg / ml, and 2.5 mg / ml with the diluent, measure it, and calculate the dilution change (i.e., compare with the previous dilution measurement result). Calculate the HCP content after multiplying the different dilution points by the dilution factor, and calculate the ratio of the results. Acceptance criteria: Starting from the minimum acceptable dilution factor (i.e., the results are consistent when diluted further down from this dilution factor), at least one dilution change of adjacent dilution factors should be between 70% and 130%.

[0132] Table 11 Dilution Linearity

[0133]

[0134] Note: The dilution change is the ratio of adjacent dilution concentrations. In Table 11, it refers to the result of 10 mg / ml in the table compared to the result of 5 mg / ml, and the result of 5 mg / ml compared to the result of 2.5 mg / ml.

[0135] Experimental Example VIII: Durability

[0136] Take the GR1501 stock solution (GR1501-2019002DS, 108.3 mg / ml), denoted as DS, dilute it to 10 mg / ml, 5 mg / ml, and 2.5 mg / ml with the diluent, measure it, calculate the HCP content of the result, calculate the RSD with the data of day-to-day precision. Acceptance criteria: RSD ≤ 30%. After experimental verification, the RSD is 14.2%, which can meet the durability requirements.

[0137] Table 12

[0138]

[0139] The present invention uses the Genrixbio CHO-S HCP preparation to establish a process-specific CHO-S HCP detection and evaluation system. The initial detection uses anti-human IgG ELISA, and the results show that there is no human IgG in the HCP preparation, and the starting materials for antibody production are good. Implement a 98-day antibody generation program to generate high-quality anti-CHO-S HCP antibodies.

[0140] The validation of the specificity test of the kit showed that the recoveries of HCP were 95% and 97%, the recoveries of BSA were 2% and 0%, and the recovery of FB was 0% (the result was lower than the detection limit of the equipment); this indicated that the kit was specific for HCP. In the precision test, the repeatability, operator precision, and inter-day precision of the actual sample were determined. Among them, the repeatability results showed that the RSDs of HVS, MVS, and LVS were 6.7%, 3.7%, and 2.8% respectively; the operator precision results showed that the RSD of DS was 5.3%; the inter-day precision results showed that the RSD of DS was 17.6%. The accuracy study of this kit showed that the recoveries of HVS, MVS, and LVS were 94%, 91%, and 103% respectively. The quantitative limit of the kit of the present invention was 1.111 ng / ml, and the detection limit was 0.123 ng / ml. The linear range study showed that the linear R 2 = 0.9996, and the recoveries in the linear range of 1 - 5 were 94%, 93%, 91%, 91%, and 103% respectively. The durability results showed that the RSD of DS was 14.2%. This result met the detection requirements for host cell protein residues produced by CHO-S cells.

[0141] The above are only examples of the present invention, and common general technical solutions and / or characteristics in the solutions are not described in detail here. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, and these should also be regarded as the protection scope of the present invention, which will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application shall be subject to the content of its claims, and the specific implementation manners described in the specification can be used to interpret the content of the claims.

Claims

1. CHO-S Cell Residual Protein Detection Kit, Characterized in that: It includes an enzyme-linked immunosorbent assay (ELISA) plate coated with anti-HCP protein antibody, biotin-labeled IgG, streptavidin labeled with horseradish peroxidase, and auxiliary preparations; Preparation method of the CHO-S cell residual protein detection kit, including the following steps: Step I: Take CHO-S host cell protein immunogen, dialyze it overnight with PBS, and quantitatively detect the total protein amount; Step II: Immunize part of the HCP immunogen in rabbits to produce antibodies, and lyophilize the remaining HCP immunogen and use it as a standard; Step III: After quantitatively detecting the total protein amount, adjust the protein concentration of the HCP immunogen to 4 mg / mL and store it for later use at low temperature; Step IV: Use the antibody obtained in Step II for booster immunization to obtain anti-HCP protein antibody, and coat the HCP antibody on a 96-well plate; Step V: Biotin-label the antibody. After stirring and dialyzing IgG with PBS overnight, label it with biotin on ice; replace with fresh PBS and continue dialysis to obtain biotin-labeled IgG, and store it for later use at low temperature; Step VI: Detection application; In Step IV, the animals for booster immunization are rabbits. The immunization is carried out by multi-site intramuscular injection. For the first injection, mix 1 mL of immunogen with 1 mL of Freund's complete adjuvant; for subsequent injections, mix 1 mL of immunogen with 1 mL of Freund's incomplete adjuvant; the immunization program lasts for 98 days, and each injection contains 4 mg of HCP immunogen protein.

2. The CHO-S cell residual protein detection kit according to claim 1, Characterized in that: The biotin-labeled IgG is used after being diluted at a ratio of 1:200 - 6400, and the diluent is a phosphate-Tween buffer containing 0.2% - 1% BSA with a pH of 7.

4.

3. The CHO-S cell residual protein detection kit according to claim 2, Characterized in that: The auxiliary preparations include: carbonate buffer with a pH of 9.6, TMB chromogenic solution, and termination solution. The termination solution is sulfuric acid or hydrochloric acid.

4. The CHO-S cell residual protein detection kit according to claim 3, Characterized in that: In Step I, the condition for dialysis with PBS overnight is to stir at 4°C for 24 h.

5. The CHO-S cell residual protein detection kit according to claim 4, Characterized in that: In Step V, the molar ratio of biotin to IgG is 5 - 20:

1.

6. A method for using the CHO-S cell residual protein detection kit according to any one of claims 1 - 3, Characterized in that, It includes the following steps: S1: Coat an ELISA plate with CHO-S HCP antibody; S2: Add the sample to be detected and the standard solution to the coated ELISA plate, incubate and wash; S3: Add biotin-labeled IgG to the ELISA plate after washing in S2, incubate and wash; S4: Add streptavidin labeled with horseradish peroxidase to the ELISA plate after washing in S3, incubate and wash; S5: Add chromogenic solution to the ELISA plate after washing in S4 for color development, incubate, add termination solution to terminate the reaction, and measure the absorbance value at a wavelength of 450 nm.

7. The method for using the CHO-S cell residual protein detection kit according to claim 6, characterized in that: in step S5, the chromogenic solution is TMB or OPD.

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