KR20080054120A - Enzyme-linked immunosorbent assay method for residual host cell derived proteins in recombinant antibody drug production - Google Patents

Abstract

An enzyme-linked immunosorbent assay(ELISA) method is provided to detect residual host cell derived proteins inducible abnormal immune responses in patients in production of recombinant antibody drug from a Chinese Hamster Ovary(CHO) cell line by using polyclonal antibody against all proteins derived from host cell, so that stability and safety of use in products are improved. A polyclonal antibody for detecting host cell derived proteins secreted together with a target protein in the production of recombinant protein, anti-GPIIbIIIa antibody, is prepared with proteins separated among proteins of a null cell which does not express the recombinant protein under the same purification conditions as the recombinant protein as immunogen. A method for producing the polyclonal antibody comprises the steps of: (1) removing cells from the cultured medium of null CHO cell through depth filtration to obtain the supernatant; (2) purifying the supernatant under the same conditions as the recombinant protein; and (3) inoculating the purified fraction as a immunogen into an animal and obtaining an antibody from the immunized animal. An ELISA kit for detecting residual host cell derived proteins in production of recombinant antibody drug contains the polyclonal antibody.

Description

Enzyme-Linked Immunosorbent Assay Method for Residual Host Cell derived Proteins in Recombinant Antibody Drug Production}

1silver Figure shows a comparison of weak cation exchange chromatograms.

(a) target protein: anti-GPIIbIIIa antibody producing cells,

(b) Null cell derived host cell protein

2Is Figure 2 shows the results of SDS-PAGE stained with silver and indirect Western blot analysis for rabbit anti-HCP IgG production.

(a) silver stained SDS-PAGE,

(b) pre-immune test,

(c) Second and Third Immunized Serum Tests

Lane 1: marker, lane 2: non-reducing HCP, lane 3: reduced HCP, lane 4: non-reduced anti-GPIIbIIIa antibody, lane 5: anti-GPIIbIIIa antibody

3Indirect ELISA Analysis of Rabbit Anti-HCP IgG Production. This figure compares the results of immunoserum observation and anti-labeled protein antibody production on pre-immune serum.

4HRP-bound rabbit IgG stained SDS-PAGE and enzyme substrate reaction analysis results.

(a) Silver stained SDS-PAGE analysis

Lane 1: marker, lane 2, 3: HRP bound rabbit IgG, lane 4: rabbit IgG, lane 5, 6: activated HRP.

(b) enzyme-substrate reactions

Lane 1: marker, lane 2 and 3: HRP bound rabbit IgG

5Shows the HPLC analysis of HRP bound rabbit anti-HCP IgG in GFC column.

6Silver stained SDS-PAGE and Western blot analysis of silver HRP bound rabbit anti-HCP IgG.

(a) Silver stained SDS-PAGE analysis

(b) Western blot analysis using rabbit A, B, A + B IgG

Lane 1: marker, lane 2: non-reduced HCP, lane 3: reduced HCP, lane 4: non-reduced anti-GPIIbIIIa antibody, lane 5: reduced anti-GPIIbIIIa antibody

7silver Silver stained during anti-GPIIbIIIa antibody purification Figure shows the results of HCP identification by SDS-PAGE and Western blot analysis.

(a) Silver stained SDS-PAGE analysis

(b) Western blot analysis using HRP-rabbit anti-HCP IgG

(c) Western blot analysis, primary rabbit IgG and secondary peroxidase labeled goat anti-rabbit IgG,

(d) Western blot analysis, using HRP-rabbit IgG

Lane 1 and lane 9: marker, lanes 2-8: anti-GPIIbIIIa antibody clarification step sample, lane 10: non-reduced HCP

8silver The figure shows Sandwich ELISA analysis for HCP quantification.

(a) Anti-GPIIbIIIa Antibody Purification Process

(b) Anti-GPIIbIIIa Antibody 3 Batch Product

9Shows the result of ELISA analysis of HRP bound IgG stability.

(a) Stabilizer Addition

(b) no stabilizer added

Nicholson, IC et al., J. Immunol. 163 , 6898-6906, 1999

2. Lucas, C., et al., J. Immunol. Methods 113 , 113-122, 1998

Himanen, JP et el., Vaccine 11 , 970-973, 1993

4. Eaton, LC J. Pharm. Biomed. Anal. 7 , 633-638, 1989

5. Briggs, J. et al., Biotechniques 9 , 598-606, 1990

6. Kung, VT et al., Anal. Biochem . 187 , 220-227, 1990

7. Eaton, LC, J. Chromatogr . A 705 , 105-114, 1995

 Robey, F. A., In Methods in Protein Sequence Analysis, K. A. Walsh Eds .: Human Press, Clifton, NJ, pp67-78, 1986.

9. Jones, AJ and JV O'Connor, Dev. Biol. Stand . 59 , 175-180, 1985

10. Anicetti, VR et al., Trends. Biotechnol . 7 , 342-349, 1989

11. Garg, VK et al., Bioprocess Technol . 12 , 29-54, 1991

12. Anicetti, VR, Analytical Biotechnology: Capillary Electrophoresis and Chromatography, In ACS Symposium Series, Cs. Horvath and JG Nikelly Eds: American Chemical Society, Washington DC. No. 434 , 127-140, 1990

13. Anicetti, VR et al., J. Immunol. Methods 91 , 213-224, 1986

14. Bass, R., ICH Topic Q 2 B Validation of Analytical Procedures; Methodology, Step 4 Consensus Guidline 6 , ICH-Technical Coordination, 1996

The present invention relates to the production of an ELISA kit that can detect a production cell line-derived protein according to the manufacturing process in the production of recombinant biopharmaceuticals in a Chinese hamster ovary cell line, and more specifically, does not have a gene for producing a recombinant biopharmaceutical (target protein) After production of all cell-derived proteins from the production cell line remaining after the initial purification of the manufacturing process in a null cell mock culture medium, polyclonal antibodies can be prepared, and protein-derived cell-derived proteins can be detected using immunological reactions. ELISA methods and kits thereof.

Since the late 1990s, recombinant antibodies have emerged as an important material for drug development. There are currently 11 new therapeutic drugs for sale, licensed by the Food and Drug Administration (FDA), and 6 new antibodies for human administration for diagnostic purposes (Table 1). The market for antibody drugs is estimated to reach $ 500 million in 2010, but the market for antibody drugs has already exceeded $ 2 billion in 2003, and the market size is expected to increase exponentially (1). Recombinant biopharmaceuticals of this kind have to be secured as biological agents, and in recent years, the criteria are becoming increasingly difficult. The most important factor related to safety is the verification of pollutants during production. Pollutant identification tests by microorganisms, viruses, endotoxins, etc. have been conducted for a long time. In recent years, it has become an important item to verify how mixed a protein or DNA derived from a production cell line is in a product. These two items are important factors that can cause immune dysfunction in patients. Commercially available diagnostic reagents for the detection of residual contaminants in biopharmaceuticals are on the market, but these methods can only be calibrated for most contaminants and may change depending on the cultivation or purification process of the product. Not suitable for process specific contaminants. Therefore, it is appropriate to develop a method for calibrating contaminants derived from production cell lines for each unique product (7).

Chinese hamster ovary (CHO) cells, which are used in this study, and CHO cells of other products and other manufacturers, may be essentially the same cell lines, but the expression vectors for production are differently produced, and the media of use are different. There may be. Therefore, each manufacturer should establish its own cell line specific host-derived protein detection method through enzyme linked immunosorbent assay (ELISA) or threshold method. In this study, the detection method was established by ELISA. Regulations on protein derived from production cell lines are not specified in exact figures, but in “case by case” (8) and require sufficient theoretical evidence and data (9-11). Consideration should be given to differences in dose, use as analytical or diagnostic reagents and for therapeutic use, and differences in expression strains (prokaryotes vs. eukaryotes or nontransformation vs. transformation) (12). In general, methods for controlling protein-derived cell line-derived proteins in recombinant drugs include silver-stained SDS-PAGE, immunoblot, strict process validation clearance studies, and quantitative process specific HCPs. Host Cell-derived Protein Assays are being developed (9), which require high specificity assays for the calibration of production-derived cell lines. (7) Polyclonal antibodies are produced by process-specific immunogens that contain proteins derived from production cell lines that are most representative in their own purification processes (13). Sea sandwich method (13) is used a lot but not limited.

Accordingly, the present inventors obtain a representative production cell line-derived protein, that is, a host cell line-derived whole protein, which has undergone the same purification process of the target protein through culture and purification of a nonfunctional cell line in a Chinese hamster ovary cell line, which is used as an immunogen. The present invention was completed by developing an ELISA method for producing and producing polyclonal antibodies to verify and quantify the removal of protein derived from the cell line.

Table 1: FDA-Approved Antibody Drug List

An object of the present invention is to provide an antibody against a cell line-derived protein and an ELISA method using the same in the production of recombinant biopharmaceuticals in Chinese hamster ovary cell lines.

The present invention provides a polyclonal antibody for detecting a cell line-derived protein during recombinant protein production using a Chinese hamster ovary cell.

The present invention also provides a method for producing a polyclonal antibody for detecting cell line-derived protein in the production of recombinant protein using a Chinese hamster ovary cell line.

The present invention also provides an ELISA kit comprising the polyclonal antibody.

Hereinafter, the present invention will be described in detail.

The present invention provides a polyclonal antibody for detecting a cell line-derived protein and a method for producing the same in recombinant protein production using a Chinese hamster ovary cell. Specific manufacturing methods include the following steps:

1) removing the cells through depth filtration of a culture medium of a nonfunctional Chinese hamster ovary cell line that does not express recombinant protein and obtaining a supernatant;

2) purifying the supernatant of step 1 under the same conditions as the recombinant protein; And

3) inoculating an animal capable of obtaining an antibody using the purified fraction of step 2 as an immunogen.

The inventors cultured Chinese hamster ovary cells, which are null cells that do not produce recombinant antithrombogenic antibodies (anti-GPIIbIIIa antibodies), which are proteins of interest, and proteins that are excreted extracellularly. Therefore, the protein derived from the production cell line was also taken only in the culture medium to obtain only the extracellular discharge, and then it was not separated from the target protein by using weak anion exchange chromatography and weak cation exchange chromatography, which are the first steps of purification and purification of the target protein. The remaining cell line derived protein of the nonfunctional cell line was purified (FIG. 1). In other words, the production cell line-derived protein isolated under the same purification conditions as the protein of interest was used as an immunogen for the production of subsequent antibodies.

In order to produce polyclonal antibodies using the cell line-derived protein as an immunogen, the adjuvant is mixed with the cell line-derived protein of a null cell, which is an immunogen, and subcutaneously injected into two rabbits (A and B). Clonal antibodies were generated. This was first purified by column chromatography for all rabbit IgG in the serum after immunization, and only the specific anti-HCP IgG that reacts with the protein derived from the cell line in the rabbit IgG was purified second. After the chromophore labeling on the purified antibody, the degree of antibody production was confirmed by Western blot (FIG. 2) and indirect sandwich ELISA. As a result, 16.9% IgG in the serum of Rabbit A was obtained and 15.8% IgG in the serum of Rabbit B after one step of purification. Again, after 2 steps, IgG formed by protein produced from 9% of rabbit A IgG and 10% of rabbit B IgG, which was purified in step 1, was produced, was obtained (FIG. 3).

The present invention also provides an ELISA kit comprising the polyclonal antibody.

In order to develop a direct sandwich ELISA method using a specific rabbit anti-HCP IgG, which has been purified in two stages, the chromophore Horse radish peroxidase (HRP) is labeled with a specific anti-HCP IgG antibody that reacts with a protein derived from a cell line. , The binding of the antibody and the chromophore was confirmed by the size of the silver stained SDS-PAGE (Fig. 4), it was confirmed that the chromophore by the substrate treatment reacted with the chromophore. In addition, the content of the sample before and after the reaction was confirmed by HPLC analysis using a gel filtration chromatography column (FIG. 5).

As a result of detecting the detection degree of the cell line-derived protein by Western blot using the antibody, when the antibodies of Rabbit A and B were used together, it was confirmed that the antibody bound to the protein of the cell line-derived protein of 90% or more (FIG. 6). .

Meanwhile, after preparing samples for each purification step from the production of the target protein, Western blot and sandwich ELISA were performed using an antibody labeled with a chromophore to measure the content of protein (ng) produced per cell of the target protein. It was. As a result, in the column purification step of the protein of interest, it was confirmed that most protein-derived cell lines were qualitatively removed (FIG. 8).

In order to verify the sandwich ELISA method developed in the present invention, the test items were determined and the test method was planned according to the ICH Regulation (14). As a result, the ELISA method using the anti-HCP IgG of the present invention was specific, accurate, limit of detection, and quantitative. In the limit, better results were found (Table 2).

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the present invention, but not to limit the present invention.

Example 1 Nonfunctional Cell Line Culture

Null cells used in the present invention are donated from Chinese hamster ovary (CHO) cell lines (DG44, Professor Chasin, University of North Carolina State, USA) such as cell lines producing recombinant antithrombogenic antibody (anti-GPIIbIIIa antibody), the protein of interest. ), A cell lacking a gene encoding an antithrombotic antibody as a target protein. The culture was fed incubated for 14 days in 100 L culture solution by applying the same protein production conditions, and the culture was adjusted to pH 7.3 ± 0.1 using 20 mM sodium phosphate (pH 7.3).

Example 2 Purification of Proteins Derived from Non-functional Cells

Since the anti-GPIIb / IIIa antibody, a protein of interest, is secreted extracellularly, the cells derived from the producing cell line were also removed and subjected to supernatant two times through depth filtration to obtain only the extracellularly released (Millistak +, Millipore, USA). ). A supernatant was purified using an AKTA pilot (Amersham biosciences, Sweden) as follows to prepare a host-derived protein.

As a rescue, the culture was passed through weak anion exchange chromatography (DEAE-Sepharose Fast Flow, Amersham biosciences, Sweden) to collect only the protein flowing out without binding and purified in one step. The first stage preparative column was filled with 2.5 L solid phase in BPG 140/500 (Amersham biosciences, Sweden) and 20 mM sodium phosphate (pH 7.3) was used as the equilibrium solvent. Two-stage purification was performed using weak cation exchange chromatography (CM-Sepharose Fast Flow, Amersham biosciences, Sweden), and the two-stage purification column was filled with 10.7 L of solid phase in BPG 200/300 (Amersham biosciences, Sweden). 20 mM sodium acetate (pH 5.3) was used. 30 mM sodium acetate (pH 4.5) was used as the sample dilution solvent and 20 mM sodium acetate-1 M sodium chloride (pH 5.3) was used as the desorption solvent. The instrument used an AKTA pilot (Amersham biosciences, Sweden) and monitored using UNICORN software (Amersham biosciences, Sweden).

As a result of comparing the purified protein using the weak cation exchange chromatography of the protein of the target cell and the cell line-derived cell line as described above, the protein from the cell line-derived protein was partially separated from the purified region (NaCl concentration: 0.154 to 0.377 M). It was confirmed that it came out (Fig. 1). For this reason, when the protein cell line-derived protein is purified from a nonfunctional cell line, the cell line-derived protein is purified under the same conditions as the protein of interest, and then used as an immunogen to make an antibody thereafter. Purification of the weak cation exchange chromatography yielded a total of 11.31 g of the cell line derived protein from 16 L of 0.7 mg / ml of the sample.

Example 3 Polyclonal Antibody Production

The rabbit (rabbit), New Zealand White species to 2 (rabbit A, B, (weeks) purchased Orient Bio) a polyclonal antibody producing cell line-derived proteins as immunogens in experimental animals for the production was used. Before entering the experiment, Western blot was performed using pre-immune sera of the experimental animals to confirm whether the experimental animals were produced. As a result, experimental animals As a result of Western blotting with rabbit pre-immune serum, no response was observed in pre-immune serum to antibody-derived protein (anti-GPIIbIIIa antibody) or protein of interest (FIG. 2B). In the experiment, two 300 g males had a stabilization period of two weeks, and then 50 mg / dose of adjuvant (Sigma, USA) was added to the production cell line-derived protein, which is an immunogen, subcutaneously at 100 mg / dose every week. To generate polyclonal antibodies through tertiary immunity.

Example 4 Polyclonal Antibody Purification

Serum collected after the third immunization was purified using a HiTrap rProtein A Fast Flow (Amersham biosciences, Sweden) column as a first step purification of all rabbit IgG in the post-immune serum. 20 mM sodium phosphate (pH 7.0) was used as the equilibrium solvent, and 0.1 M glycine-hydrogen chloride (pH 2.7) was used as the desorption solvent. PH correction of the desorbed fraction was adjusted to neutral pH by using 1 M Tris-hydrogen chloride (pH 9.0). Two-stage purification was used to purify only specific anti-HCP IgG that reacts with the production cell line derived protein (HCP, Host Cell-derived Peptide) in rabbit IgG.

Example 5 Western Blot

Western blot was used as a qualitative analysis method to confirm the production and purification of the antibody. The total protein of each sample to be used for analysis was analyzed using a Modified Lowry protein assay (Pierce, USA). Assay samples were separated using SDS-PAGE of protein derived from the cell line (Process specific HCP) used as an immunogen, and then transferred to the PVDF membrane (Invitrogen, USA). The primary antibody was treated with a 1,000-fold dilution of rabbit IgG specific for the rabbit pre-immune serum, post-immune serum and cell line-derived protein, for 1 hour, and the secondary antibody was Peroxidase labeled goat anti-rabbit IgG (H +). L) (KPL, USA) was diluted 2,500-fold and treated for 1 hour. After the chromophore labeling on the purified antibody, it was treated by diluting 1,000-fold with the antibody. TMB membrane peroxidase substrate (KPL, USA) was used as a substrate of the chromosome. At this time, when the anti-GPIIb / IIIa antibody of the target protein was tested at the same time as a negative control group it was confirmed that it does not respond to the immune serum.

As a result, the third immunity process resulted in 10,000 : Serum was obtained after immunization having a titer of 1, and the production of the antibody was confirmed by reaction with the protein cell-derived protein through Western blot (FIG. 2) and ELISA (FIG. 3). Analytical samples were pre-immune serum, post-immune serum and cell line-derived proteins as rabbits as primary antibodies in an ELISA-only container (NUNC) on which a process-specific HCP used as an immunogen was immobilized. The rabbit IgG specific for each was diluted 1,000-fold and treated for 1 hour, and the secondary antibody was diluted 2,500-fold with Peroxidase labeled goat anti-rabbit IgG (H + L) (KPL, USA) and treated with ABTS after 1 hour. Color reaction was induced using a microwell peroxidase substrate (KPL, USA). After color development, the reaction was stopped using ABTS peroxidase stop solution (KPL, USA) and the absorbance was measured at ₄₀₅ nm. As a control experiment, the target protein, anti-GPIIb / IIIa antibody, was immobilized in an ELISA vessel, and the rest of the procedure proceeded in the same manner. As a result, 16.9% IgG in the serum of Rabbit A was obtained and 15.8% IgG in the serum of Rabbit B after one step of purification. Again, after 2 steps, IgG formed by protein produced from 9% of rabbit A IgG and 10% of rabbit B IgG, which were purified in step 1, was produced.

Example 6 Coloring Enzyme Labeling

In order to develop a direct sandwich ELISA method using a specific rabbit anti-HCP IgG after two-step purification, the antibody was labeled with Horse radish peroxidase (HRP) using a Peroxidase labeling kit (Roche, Switzerland). The binding of the chromophore with the silver was confirmed in size through the SDS-PAGE stained (Fig. 4), and the chromogenic enzyme (TMB membrane peroxidase substrate, KPL, USA) was confirmed to be developed through the treatment. In addition, the changes of the cell sample-derived protein specific rabbit anti-HCP IgG samples before and after the enzyme coupling reaction were analyzed by HPLC analysis using a gel filtration chromatography column, TSK 3000 column (TOSHO, Japan). It was confirmed (FIG. 5).

As a result of detecting the detection degree of the cell line-derived protein by Western blot using the antibody, when the antibodies of Rabbit A and B were used together, it was confirmed that the antibody bound to the protein of the cell line-derived protein of 90% or more (FIG. 6). .

Example 7 Sandwich ELISA

After purification, the labeled antibody (HRP conjugated rabbit anti-HCP IgG) was dissolved in 50 mM carbonate buffer (pH 9.6) and placed in 100 ml of 96 well plate (NUNC, USA) and coated at 4 ° C. overnight. Blocking was performed by dissolving 2% fetal calf serum in phosphate buffered saline (PBS) and adding 0.05% Tween 20 to it for 2 hours at room temperature. As the substrate of the chromophore, ABTS microwell peroxidase substrate (KPL, USA) was used. After color development, the reaction was stopped using ABTS peroxidase stop solution (KPL, USA) and absorbance was measured at OD ₄₀₅ nm.

Example 8 Measurement of Protein Content Derived from Production Cell Line in Antibody Drugs

Samples were prepared for each purification step from the culturing step of the protein production process, and the concentration was equalized to 1 mg / ml based on the target protein (see Korean Patent 10-2005-0026812). The content of the production cell line derived protein (ng) per mg of the target protein (anti-GPIIb / IIIa antibody) was measured.

In the third column purification step of the protein of interest (anti-GPIIb / IIIa antibody) in the production and purification of the target protein using the antibody labeled with the chromophore enzyme, the cell line derived protein was identified and quantified. It was confirmed that most of the protein derived from the cell line was qualitatively removed (FIG. 8A). In addition, the result of direct ELISA under the optimized conditions, after the third column purification step, the content of the production cell line-derived protein was 300 ppm or less, and the content of the production cell line-derived protein in the final product stock was 5.8 ppm. In order to confirm the reproducibility of the purification step for each batch of antibody drug production, the experiments were conducted on three batches of antibody drug product stocks, and all of them were found to be 10 ppm or less (FIG. 8B).

Example 9 Sandwich ELISA Method Verification

In order to verify the sandwich ELISA method developed in the present invention, the test items were determined according to the ICH regulations (14) and the test method was planned. Test items include specificity, linearity, accuracy, repeatability, interior precision, detection limit and quantitation limit. Respective% relative standard deviation, correlation coefficient (R ² ), and% average recovery were identified. In addition, the verification was performed by using a direct ELISA method using an antibody labeled with a chromophore on rabbit IgG purified using HiTrap rProtein A FF (Amersham Biosciences, Sweden) column, which is a primary purification column for post-immune serum purification.

Comparing the experimental results, it was confirmed that the ELISA method using specific anti-HCP IgG obtained through a total of two steps of purification process showed better results in specificity, accuracy, detection limit, and quantification limit (Table 2).

Table 2: Validation of the ELISA System of the Invention

¹ HRP-rabbit IgG ELISA system

ELISA system using ² HRP-rabbit anti-HCP IgG.

Example 10 Stability Test of Labeled Antibody

In order to confirm that the antibodies (HRP-linked rabbit anti-HCP IgG) produced through various purification procedures and chromophore labeling can show reliable values for a certain period of time, 1, 2, At 3 weeks intervals, the absorbance at OD ₄₀₅ nm was measured using a sandwich ELISA method. In addition, the maintenance of stability according to the use of stabilizers (stabilizer, Roche, Switzerland) was confirmed through a comparative experiment.

As a result, after the chromophore labeling, the sensitivity decreased as time passed, and the difference in absorbance from the lowest concentration to the highest concentration gradually decreased. This tendency was confirmed to decrease significantly when a stabilizer was added. Experimental results show that the stabilizer can be stored and used at 4 ° C. for at least one month (FIG. 9).

Commercially available analytes use whole host cell lines that do not reflect expression vectors as antigens, and do not go through a series of purification of target proteins to use them. Assays for cell line derived proteins may be less accurate. This shows limitations in the analysis of unique production cell line-derived proteins that may vary depending on the production method or the characteristics of the recombinant cell. Accordingly, the antibody of the present invention and the ELISA method using the same produce recombinant biopharmaceuticals by using the whole protein derived from the host cell line which has undergone the same purification process of the target protein as the antigen by culturing a nonfunctional cell line which does not have the gene of the protein of interest. At the same time, it can be used to improve the safety of the product by detecting more accurately the impurity protein from the production cell line which can cause the immune response to the patient.

Claims (6)

Polyclonal antibody for detecting cell line-derived protein secreted with the protein of interest when producing recombinant protein using Chinese hamster ovary cell (Chinese hamster ovary cell). [Claim 2] The polyclonal antibody according to claim 1, wherein the polyclonal antibody is prepared by using an immunogen as a protein which is isolated under the same purification conditions as that of the recombinant protein among proteins of a null cell which does not express the recombinant protein. The polyclonal antibody according to claim 1 or 2, wherein the recombinant protein is an anti-GPIIbIIIa antibody. 1) removing the cells through depth filtration of a culture medium of a nonfunctional Chinese hamster ovary cell line that does not express recombinant protein and obtaining a supernatant; 2) purifying the supernatant of step 1 under the same conditions as the recombinant protein; And 3) A method of producing the polyclonal antibody of claim 1, comprising inoculating a animal from which the purified fraction of step 2 can be obtained as an immunogen. An ELISA kit for detecting a cell line-derived protein during recombinant protein production using a Chinese hamster ovary cell comprising the polyclonal antibody of claim 1 or 2. 6. The ELISA kit of claim 5, wherein the recombinant protein is an anti-GPIIbIIIa antibody.

Images