Kit and method for quantitatively detecting residual amount of pichia host cell protein

Abstract

The present application belongs to the technical field of biological detection, and aims at the problems of existing detection methods, such as quantitative limit and low detection sensitivity. The present application discloses a kit and method for quantitatively detecting Pichia pastoris host cell protein residual amount. The present application introduces empty plasmid without recombinant human serum albumin gene into Pichia pastoris, and combines Protein A chromatography column and host cell protein coupling affinity column for purification, so that the prepared polyclonal antibody can be effectively used for quantitative detection of Pichia pastoris host cell protein residual amount in biological products. The kit composed of the polyclonal antibody has good accuracy and repeatability for quantitative detection of host cell protein residual amount in Pichia pastoris source biological products, and the quantitative limit is 0.2 ng / mL. The kit provided by the present application can satisfy quantitative detection of host cell protein residual amount in all Pichia pastoris source biological products.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a kit and method for quantitatively detecting residual protein in Pichia pastoris host cells. Background Technology

[0002] Pichia pastoris ( pichia pastoris Pichia pastoris, as one of the most commonly used expression systems, has been widely used in recombinant drug proteins and recombinant industrial enzymes. Similar to other hosts (such as CHO and Vero cells), Pichia pastoris, as a host, produces unique host cell proteins (HCPs) while efficiently expressing recombinant proteins. HCPs are a major impurity component in biopharmaceuticals. Residual HCPs are present at various stages of the recombinant biopharmaceutical manufacturing process. Their composition is complex and heterogeneous, and they are extremely difficult to completely remove. HCPs have potential immunogenicity, triggering immune responses. Reports indicate that residual HCPs in some recombinant biopharmaceuticals can lead to an "adjuvant effect," causing the body to produce anti-drug antibodies (ADAs) against the target drug protein, thereby reducing drug efficacy. Therefore, strict quality control of HCPs in each stage of the biopharmaceutical manufacturing process is necessary.

[0003] The main methods for detecting HCP in biopharmaceuticals include silver staining, HPLC, and enzyme-linked immunosorbent assay (ELISA). Silver staining has a maximum resolution of 2-10 ng and suffers from drawbacks such as inability to quantify, making it unsuitable for modern biopharmaceutical industry testing standards. HPLC offers high resolution and precise quantification, but the results are nonspecific, and data analysis is subjective. Furthermore, the small sample loading volume of HPLC makes it difficult to detect low-content HCP components. ELISA is currently the most widely used method for detecting HCP residues. Its principle lies in the specific binding reaction between antigen and antibody. The double-antibody sandwich ELISA method is characterized by good repeatability, high sensitivity, and simple operation, and has become a recognized method for HCP residue detection. This method is already used in the Chinese Pharmacopoeia, Volume III, for detecting host protein residues. Therefore, several commercially available universal HCP ELISA kits targeting different host cells have emerged, such as the CHO HCP ELISA kit. E. coli HCP ELISA kits, Vero HCP ELISA kits, etc., will help researchers to quickly and accurately assess the HCP content in biopharmaceuticals. Deng Chunping et al. used an Escherichia coli HCP ELISA detection kit to detect the HCP residues in recombinant basic fibroblast growth factor (rbFGF) stock solution and intermediate samples. The limit of detection and limit of quantitation were both 3.33 ng / mL.

[0004] However, due to manufacturing processes, commercially available general-purpose HCP ELISA kits often lack specificity for the actual components of HCP. Therefore, preparing HCP-specific antibodies is crucial for the reliability of ELISA kits. Currently, inventors have developed HCP ELISA kits with specific antibodies for targeted quantitative detection of host protein residues in recombinant biological products. Ke Zhi et al. established a double-antibody sandwich ELISA method for detecting CHO cell host protein residues in monoclonal antibody products, with a detection limit of 3.125 ng / mL. Patent CN105884886A discloses an antibody specifically recognizing HCP in rice endosperm cells and establishes an ELISA method for detecting rice HCP residues in recombinant proteins, with a detection limit as low as 1.25 ng / mL. Patent CN105021824A discloses an ELISA kit and method for detecting CHO host cell proteins, with a detection sensitivity of 0.3 ng / mL. Patent CN109384832A discloses a method for preparing a specific antibody to detect Hansenula polymorpha protein residues in recombinant vaccine products, and establishes an ELISA kit for detecting HCP residues with a detection limit of 2 ng / mL. Patent CN106957362A discloses an ELISA method for quantitatively detecting HCP residues in Pichia pastoris-derived human serum albumin, with a limit of quantification of 0.22 ng / mL.

[0005] Chemiluminescence immunoassay (CLIA) is a detection method based on the principle of ELISA, combined with a chemiluminescence analysis system. Compared with traditional ELISA methods, it has advantages such as wide linearity, high sensitivity, high accuracy, and short processing time, and is now widely used in the development of diagnostic reagents for several human diseases. For high-dose protein drugs, such as human serum albumin with a clinical injection dose of 10 g / dose, which is much higher than the doses of other types of injectable protein drugs, the control of impurity content in albumin preparations is particularly important. Currently, there are few reports on the detection of Pichia pastoris host protein residues using CLIA methods, and the published detection methods have problems with limits of quantitation and low detection sensitivity. Therefore, it is necessary to design a CLIA method with high detection sensitivity for the detection of Pichia pastoris HCP. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a kit and method for quantitatively detecting residual protein in Pichia pastoris host cells.

[0007] The first aspect of this invention provides a kit for quantitatively detecting residual Pichia pastoris host cell proteins, comprising a horseradish peroxidase-labeled polyclonal antibody that specifically recognizes Pichia pastoris host cell proteins.

[0008] Horseradish peroxidase-labeled polyclonal antibodies were obtained as follows:

[0009] Animals were immunized using Pichia pastoris host cell proteins to obtain antibody-containing animal serum.

[0010] The animal serum containing antibodies is purified to obtain polyclonal antibodies;

[0011] The purification process includes sequentially purifying the antibody-containing animal serum using a Protein A chromatography column and then using a host cell protein-coupled affinity column.

[0012] Horseradish peroxidase (HRP) labeling was performed on the polyclonal antibody to obtain HRP-labeled polyclonal antibody.

[0013] Preferably, the host cell protein-coupled affinity column is newly prepared according to the present invention and is obtained by the following method:

[0014] Take the coupling medium and wash it in coupling solution A at 0~4℃ for at least 30 min to obtain the washed coupling medium.

[0015] The host cell protein was replaced to a concentration of 5-10 mg / ml by coupling with solution B to obtain the ligand solution;

[0016] The washed coupling medium was diluted with coupling solution A. The diluted coupling medium was mixed with an equal volume of ligand solution and gently stirred overnight at 4°C. Then, the mixture was blocked and washed to obtain a host cell protein coupling affinity column.

[0017] Preferably, the coupling solution A is 1 mM HCl; and the coupling solution B is 0.1 M NaHCO3.

[0018] Preferably, the solid-liquid ratio of the washed medium to the coupling solution A is 1:1.

[0019] The preferred animal to be immunized is the New Zealand White rabbit.

[0020] Further preferred, the Pichia pastoris host cell protein is obtained by the following method:

[0021] (1) Construction of Pichia pastoris empty vector expression strain

[0022] Pichia pastoris GS115 strain was transformed by electroporation of linearized pPIC9K plasmid, and Pichia pastoris empty vector expression strain was obtained by screening.

[0023] (2) Preparation of Pichia pastoris host cell protein

[0024] After activating the Pichia pastoris empty vector expression strain on a plate, the Pichia pastoris host cell protein was obtained by fermentation induction and purification.

[0025] Preferably, the kit described above further includes antibody diluent, sample diluent, substrate solution A, substrate solution B, and PBST solution.

[0026] Preferably, the antibody diluent is a PBS-BSA solution, and the pH of the antibody diluent is 7.4;

[0027] The sample diluent was PBST solution with a concentration of 0.15 M and a pH of 7.4.

[0028] The formulation of substrate solution A is as follows: citric acid monohydrate 3.2 g / L, sodium acetate 7.0 g / L, urea peroxide 0.5 g / L;

[0029] The formulation of substrate solution B is as follows: 1.0 g / L citric acid monohydrate, 1.0 g / L sodium chloride, 0.4 g / L tetramethylbenzidine, and 0.1 g / L EDTA-Na2.

[0030] A second aspect of this invention provides a method for quantitatively detecting residual protein in Pichia pastoris host cells, comprising the following steps:

[0031] Using the above-mentioned kit, the sample to be tested was incubated.

[0032] The incubated samples were subjected to enzyme-linked immunosorbent assay (ELISA) to obtain absorbance values.

[0033] Based on the obtained absorbance value and the absorbance-protein concentration standard curve, the amount of residual host cell protein in the sample to be tested is obtained.

[0034] Preferably, the absorbance-protein concentration standard curve is obtained by the following method:

[0035] Pichia pastoris host cell protein solutions at concentrations of 0, 0.2, 0.5, 1.0, 2.5, 5.0, 12.5, 25, and 30 ng / mL were used as Pichia pastoris host cell protein standard solutions.

[0036] Multiple Pichia pastoris host cell protein standard solutions were subjected to enzyme-labeled detection to obtain multiple absorbance values;

[0037] Based on the obtained absorbance values ​​and the corresponding Pichia pastoris host cell protein concentrations, a standard curve of absorbance value versus protein concentration was plotted.

[0038] Preferably, the limit of quantification for residual Pichia pastoris host cell proteins in the kit is 0.2 ng / mL.

[0039] Compared with the prior art, the advantages of this invention are as follows:

[0040] (1) In preparing HCP antigen, the present invention introduces an empty plasmid without recombinant human serum albumin (rHSA) gene into Pichia pastoris to construct an empty plasmid strain without rHSA expression, and ensures that its insertion copy number is consistent with that of the engineered strain expressing rHSA, so as to ensure that the rHSA producing strain and the HCP producing strain are a single variable, and the fermentation method and purification method used are consistent with the rHSA production process, so that the prepared HCP can cover the HCP in the product, thereby improving the detection accuracy of the kit;

[0041] (2) This invention uses New Zealand white rabbits to prepare antibodies and combines Protein A chromatography column and host cell protein-coupled affinity column to purify the prepared antibodies. This can eliminate non-HCP related antibodies (i.e., captured animal autoimmune related antibodies - which can be understood as the autoantibodies produced by animals in response to foreign antigens from birth to the entire growth stage) in the protein A purified antibodies, thereby further improving the purity of HCP antibodies and thus improving the detection sensitivity of the kit. The detection capability of the kit of this invention is more than 30 times higher than that of commercial kits, so that the purity of the prepared Pichia pastoris-derived recombinant human serum albumin preparation can reach 99.9999999%, which can ensure the safety of injectable rHSA drugs.

[0042] (3) The limit of quantitation of the kit provided by the present invention is 0.2 ng / mL, which is much higher than that of commercial ELISA kits; it meets the quality control requirements in the industrial production process and provides controllable evaluation for the quality control of injectable rHSA drugs. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1The results of SDS-PAGE electrophoresis of Pichia pastoris HCP are shown. From left to right, the three lanes are reduced electrophoresis, non-reduced electrophoresis, and standard molecular weight markers (from top to bottom: 245 kD, 180 kD, 135 kD, 100 kD, 75 kD, 63 kD, 48 kD, 35 kD, 25 kD, 20 kD, 17 kD, 11 kD).

[0045] Figure 2 The results of electrophoresis of purified Pichia pastoris HCP antibodies are shown. Lanes 2, 3, and 4 from left to right are antibodies purified by protein A chromatography; lanes 5, 6, and 7 are antibodies purified a second time by HCP-coupled affinity chromatography; lane 1 is a standard molecular weight marker (from top to bottom: 112 kD, 66 kD, 45 kD, 35 kD, 25 kD, 18 kD).

[0046] Figure 3 The results of Western blotting analysis of Pichia pastoris HCP are shown. The three lanes from left to right are for reducing electrophoresis, non-reducing electrophoresis, and standard molecular weight markers (from top to bottom: 245 kD, 180 kD, 135 kD, 100 kD, 75 kD, 63 kD, 48 kD, 35 kD, 25 kD, 20 kD, 17 kD, 11 kD).

[0047] Figure 4 The linear range standard curve of the CLIA kit provided by this invention.

[0048] Figure 5 These are the standard curves for different batches of CLIA reagent kits of this invention.

[0049] Figure 6 The figures show the rHSA detection standard curves; the left figure shows the standard curve of the CLIA kit provided by this invention, and the right figure shows the standard curve of a commercially available ELISA kit. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0051] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0052] The reagents and equipment involved in the embodiments of this invention are as follows:

[0053] DEAE and Protein A chromatography media: GE;

[0054] Membrane packaging: Novasep;

[0055] Chromatography system: Novasep Corporation;

[0056] ELISA plate: Costa, 42592;

[0057] HRP: Meilumbio, MB6017;

[0058] TMB developer: Solarbio, PR1200;

[0059] 20×DAB: Solarbio, DA1010;

[0060] Freund's complete adjuvant: Sigma, F5881;

[0061] Freund's incomplete adjuvant: Sigma, F5506;

[0062] Human serum albumin: Chengdu Rongsheng (20%, 50mL, 10g / bottle);

[0063] Pichia pastoris-derived recombinant human serum albumin: Shenzhen Progene Pharmaceutical Technology Co., Ltd. (20%, 50mL, 10g / bottle);

[0064] BCA kit: Thermofisher, 23227;

[0065] Commercially available Pichia pastoris HCP ELISA kit: CYGNUS, F140;

[0066] Microplate reader: BioTek, SYNERGY H1;

[0067] Thermostatic oscillator: Hangzhou Aosheng, MB100-2A;

[0068] Example 1:

[0069] This invention provides a kit for quantitatively detecting residual Pichia pastoris host cell proteins, comprising a horseradish peroxidase-labeled polyclonal antibody that specifically recognizes Pichia pastoris host cell proteins.

[0070] Horseradish peroxidase-labeled polyclonal antibodies were obtained as follows:

[0071] Animals were immunized using Pichia pastoris host cell proteins to obtain antibody-containing animal serum.

[0072] The animal serum containing antibodies is purified to obtain polyclonal antibodies;

[0073] The purification process includes sequentially purifying the antibody-containing animal serum using a Protein A chromatography column and then using a host cell protein-coupled affinity column.

[0074] Horseradish peroxidase (HRP) labeling was performed on the polyclonal antibody to obtain HRP-labeled polyclonal antibody.

[0075] In this embodiment of the invention, the host cell protein-coupled affinity column is prepared by the following method:

[0076] Take the coupling medium and wash it in coupling solution A at 0~4℃ for at least 30 min to obtain the washed coupling medium.

[0077] The host cell protein was replaced to a concentration of 5-10 mg / ml by coupling with solution B to obtain the ligand solution;

[0078] The washed coupling medium was diluted with coupling solution A. The diluted coupling medium was mixed with an equal volume of ligand solution and gently stirred overnight at 4°C. Then, the mixture was blocked and washed to obtain a host cell protein coupling affinity column.

[0079] In this embodiment of the invention, coupling solution A is 1 mM HCl; coupling solution B is 0.1M NaHCO3.

[0080] In this embodiment of the invention, the solid-liquid ratio of the washed coupling medium to the coupling solution A is 1:1.

[0081] In this embodiment of the invention, the animal to be immunized is a New Zealand white rabbit.

[0082] In this embodiment of the invention, the Pichia pastoris host cell protein is obtained by the following method:

[0083] (1) Construction of Pichia pastoris empty vector expression strain

[0084] Pichia pastoris GS115 strain was transformed by electroporation of linearized pPIC9K plasmid, and Pichia pastoris empty vector expression strain was obtained by screening.

[0085] (2) Preparation of Pichia pastoris host cell protein

[0086] After activating the Pichia pastoris empty vector expression strain on a plate, the Pichia pastoris host cell protein was obtained by fermentation induction and purification.

[0087] The kit provided in this embodiment of the invention also includes antibody diluent, sample diluent, substrate solution A, substrate solution B, and PBST solution.

[0088] Specifically, the antibody diluent was a PBS-BSA solution (1% BSA) with a pH of 7.4.

[0089] The sample diluent was PBST solution with a concentration of 0.15 M and a pH of 7.4.

[0090] Based on 1L of ultrapure water, the formulation of substrate solution A is: 3.2 g / L citric acid monohydrate, 7.0 g / L sodium acetate, and 0.5 g / L urea peroxide;

[0091] Based on 1L of ultrapure water, the formulation of substrate solution B is as follows: 1.0 g / L citric acid monohydrate, 1.0 g / L sodium chloride, 0.4 g / L tetramethylbenzidine, and 0.1 g / L EDTA-Na2.

[0092] The preparation method of horseradish peroxidase-labeled polyclonal antibody in the embodiments of the present invention will be described in detail below.

[0093] 1. Construction of Pichia pastoris empty vector expression strain

[0094] Pichia pastoris was transformed by electroporation using linearized pPIC9K plasmid. Pichia Pastoris The GS115 strain was plated on MD plates (YNB 13.4 g / L; Glucose 20 g / L; D-biotin 0.4 mg / L; agar powder 15 g / L) and cultured at 28℃ for 2-3 days. After the emergence of single clones, the strains were transferred to YPD plates containing 4 mg / mL G418 antibiotic (Peptone 20 g / L; Yeast Extract 10 g / L; Glucose 10 g / L) and cultured at 28℃ for 2-3 days. After screening with 4 mg / mL G418, the insertion copy number of the pPIC9K plasmid was quantitatively analyzed by real-time PCR. Strains with the same insertion gene copy number as the recombinant human serum albumin (rHSA) expression strain in patent publication number WO2017190671A1 were selected to obtain the Pichia pastoris empty vector expression strain.

[0095] 2. Preparation of Pichia pastoris host cell protein

[0096] After plate activation of the Pichia pastoris empty vector expression strain, it was inoculated into a seed tank and cultured stepwise at 28°C. Finally, it was transferred to a 500 L fermenter. When the cell density OD... 600When the concentration reaches 700-800, methanol is added to initiate fermentation, which ends after 72 hours. The fermentation broth is centrifuged using a continuous flow centrifuge (12000×g), and the supernatant is collected. The supernatant is then filtered through a plate and frame filter press with a pore size of 1 mm. The obtained filtrate is ultrafiltered through a 30 kD membrane into 30 mM NaAc (pH 5.8) buffer, and then purified using DEAE chromatography media. Elution is performed using elution buffer (30 mM HAc, 0.2 mM NaCl, pH 5.8), and the eluent is collected. Finally, the eluent is concentrated using a 10 kD membrane and ultrafiltered into 20 mM PB solution to obtain Pichia pastoris host cell protein, i.e., Pichia pastoris-specific HCP; it is stored at 4°C. The SDS-PAGE electrophoresis results of Pichia pastoris-specific HCP are shown below. Figure 1 As shown.

[0097] 3. Preparation of Pichia pastoris-derived specific HCP antibody

[0098] The specific HCP concentration derived from Pichia pastoris was determined by dialyzing the specific HCP from Pichia pastoris into 10 mM PBS (pH 7.4) solution, concentrating it to a protein concentration ≥4 mg / ml, and then calibrating it using the BCA method.

[0099] 3.1 Preparation of Antibody-Containing Serum

[0100] 3.1.1 Immunity

[0101] New Zealand white rabbits (purchased from Vital Rivers), rats (purchased from Vital Rivers), and sheep (commissioned by Shandong Aileke Biotechnology Co., Ltd.) were selected for immunization. Pichia pastoris-derived specific HCP was diluted to 4 mg / ml with PBS solution, then emulsified with an equal volume of adjuvant. For the initial immunization, Freund's complete adjuvant was used, with a dose of 1 mg / animal, administered subcutaneously at multiple points on the back (8-10 points per animal). Three booster immunizations were given with the same dose, using Freund's incomplete adjuvant. Blood was collected one month after immunization to obtain serum from rabbits, mice, and sheep immunized with Pichia pastoris-derived specific HCP. To obtain highly effective antibodies, the conditions during antibody immunization were optimized as shown in Table 1.

[0102] Table 1. Optimization settings for antibody immunization process:

[0103] ;

[0104] 3.1.2 Serum titer test after rabbit triple immunization

[0105] ELISA was performed using microplates coated with Pichia pastoris-derived specific HCP (coating concentration of 0.25 µg / ml). The plates were incubated with rabbit serum immunized with Pichia pastoris-derived specific HCP diluted in PBS at 37°C for 30 min. After washing, HRP-labeled goat anti-rabbit antibody was added, and the plates were incubated at 37°C for 30 min. After washing, TMB substrate solution was added for color development, and the plates were incubated at 37°C for 12 min. Finally, stop solution (2M H₂SO₄) was added, and the OD was measured using a microplate reader. 450 The test results are shown in Table 2 below.

[0106] Table 2 OD of rabbit serum 450 Test results:

[0107] ;

[0108] 3.1.3 Serum titer test after second immunization in rats

[0109] ELISA was performed using microplates coated with Pichia pastoris-derived specific HCP (coating concentration of 0.25 µg / ml). The plates were incubated with mouse serum immunized with Pichia pastoris-derived specific HCP diluted in PBS at 37°C for 30 min. After washing, HRP-labeled goat anti-mouse antibody was added, and the plates were incubated at 37°C for 30 min. After washing, TMB substrate solution was added for color development, and the plates were incubated at 37°C for 15 min. Finally, stop solution was added, and the OD was measured using a microplate reader. 450 The test results are shown in Table 3 below.

[0110] Table 3 OD of mouse serum 450 Test results:

[0111] ;

[0112] 3.1.4 Serum titer test after the fourth immunization of sheep

[0113] ELISA was performed using microplates coated with Pichia pastoris-derived specific HCP (coating concentration of 0.25 µg / ml). The plates were incubated with sheep serum immunized with Pichia pastoris-derived specific HCP diluted in PBS at 37°C for 30 min. After washing, HRP-labeled voles anti-sheep antibody was added and incubated at 37°C for 30 min. After washing, TMB substrate solution was added for color development and incubated at 37°C for 15 min. Finally, stop solution was added, and OD was measured using a microplate reader. 450 The test results are shown in Table 4 below.

[0114] Table 4 OD of sheep serum 450 Test results:

[0115] ;

[0116] Based on the results in Tables 2-4, it can be seen that the efficacy of Pichia pastoris-derived HCP-specific rabbit serum is higher than that of mouse serum and sheep serum. Therefore, New Zealand white rabbits were selected as the HCP immunization animals. The obtained Pichia pastoris-derived HCP-specific rabbit serum will be used as the antibody-containing serum in this embodiment of the invention.

[0117] 3.2 Preparation of Polyclonal Antibodies

[0118] The obtained serum samples containing antibodies were diluted with Buffer A solution (20 mM PB, pH 8.0), filtered, and then purified using a Protein A chromatography column and an HCP-coupled affinity column. The specific steps are as follows:

[0119] Protein A purification by column chromatography:

[0120] (1) Equilibrate the Protein A chromatography media purification system with Buffer A (20 mM PB, pH 8.0) by 3-5 column volumes.

[0121] (2) Load the filtered serum sample at a rate of 5 mL / min and collect the permeate. After loading, continue to rinse the tubing with Buffer A for 5 column volumes.

[0122] (3) Elute the Protein A chromatography media purification system with Buffer B (20 mM PB, 0.5 M NaCl, pH 8.0) for 3-5 column volumes.

[0123] (4) Elute with 0.1 M citric acid, pH 3.0 at 3 mL / min and collect the eluent.

[0124] HCP-coupled affinity column purification:

[0125] (1) Preparation of HCP-coupled affinity column

[0126] ① Packing material preparation:

[0127] Take a known volume of coupling medium (the volume of the coupling medium is determined based on the amount of HCP) and wash it in coupling solution A (0~4℃) for at least 30 min. Displace the HCP to a concentration of 5~10 mg / ml using coupling solution B to obtain the ligand solution. Dilute the washed coupling medium with coupling solution A, specifically, the solid-liquid ratio of the washed coupling medium to coupling solution A should be 1:1; then mix the diluted coupling medium and the ligand solution in equal volumes and gently stir overnight at 4℃ to obtain the packing material.

[0128] The coupling medium is NHS-activated Bestarose 4FF (Berglon, catalog number: AA118307); the coupling A solution is 1 mM HCl, which is used to maintain the activity of the coupling medium; the coupling B solution is 0.1M NaHCO3, which is used to provide the pH conditions for the coupling reaction to occur.

[0129] ② After the ligand coupling is completed, the packing material needs to be sealed and cleaned:

[0130] Blocking: Remove the coupling supernatant and add blocking solution (0.1M Tris-HCl, pH 8.5), and block at room temperature for 4 hours.

[0131] Cleaning: Wash 3-6 times alternately with 0.1M Tris-HCl (pH 8-9) and 0.2M acetic acid (pH 3-6), each time using 3 times the column volume of material.

[0132] (2) Purification: Equilibrate the HCP-coupled affinity column purification system with Buffer A (20 mM PB, pH 8.0) by 3-5 column volumes.

[0133] (3) The sample collected after purification by Protein A chromatography was loaded at a rate of 5 mL / min, and the permeate was collected. After loading, the tubing was flushed with Buffer A for 5 column volumes. Then, the sample was eluted with elution buffer (0.1 M citric acid, pH 3.0) at a rate of 3 mL / min, and the elution fraction was collected. The polyclonal antibody, namely the Pichia pastoris-derived HCP antibody, was obtained.

[0134] 3.3 Antibody Detection

[0135] The Pichia pastoris-derived HCP antibody was dialyzed into PBS solution (10 mM, pH 7.4) and concentrated to a protein concentration ≥4 mg / ml. The antibody concentration was determined using the BCA method. Antibody purity was detected by SDS-PAGE electrophoresis, and the results are as follows: Figure 2 As shown in the figure. Analysis revealed that the purity of the Pichia pastoris-derived specific HCP antibody was ≥95.0%.

[0136] The Pichia pastoris-derived HCP-specific antibodies were detected by Western blotting. The primary antibody was an HCP polyclonal antibody, and the secondary antibody was an HRP-labeled goat anti-rabbit antibody. DAB staining was used for color development. The results are as follows: Figure 3 As shown. The Western Blot colorimetric image ( Figure 3 ) and SDA-PAGE electrophoresis results ( Figure 1 By comparison, the analysis showed that the antibodies could cover almost all antigens (HCP).

[0137] 4. Preparation of horseradish peroxidase (HRP)-labeled polyclonal antibodies

[0138] (1) Dissolve 1 part of HRP in pure water, add freshly prepared sodium periodate (100 mM) solution, protect from light, react at room temperature for 20 min, and then dialyze overnight through acetate buffer (pH 4.4);

[0139] (2) Take 4 portions of Pichia pastoris-derived specific HCP antibody, dialyze it with carbonate buffer (pH 9.5), mix it with the dialyzed HRP solution in (1), adjust the pH value to 9-10 with carbonate buffer, and react at room temperature with shaking in the dark for 3 hours.

[0140] (3) Add an appropriate amount of freshly prepared sodium borohydride solution (100 mM) and shake at room temperature for 30 min.

[0141] (4) Add saturated ammonium sulfate solution pre-cooled at 2-8 °C dropwise, incubate overnight at 2-8 °C, centrifuge to collect the precipitate, and discard the supernatant. Wash the precipitate once with 50% saturated ammonium sulfate / PBS solution, centrifuge again to discard the supernatant, collect the precipitate, dissolve it with PBS solution, transfer it to a dialysis bag, and dialyze it with PBS solution.

[0142] (5) Take the HRP-labeled antibody after dialysis and measure the OD. 403 / OD 280 The HRP-labeled antibody after dialysis is calculated based on the amount of labeling, and is thus an HRP-labeled polyclonal antibody.

[0143] Example 2

[0144] This invention provides a method for quantitatively detecting residual protein in Pichia pastoris host cells, comprising the following steps:

[0145] The test sample was incubated using the kit from Example 1 above (i.e., the CLIA kit).

[0146] The incubated samples were subjected to enzyme-linked immunosorbent assay (ELISA) to obtain absorbance values.

[0147] Based on the obtained absorbance value and the absorbance-protein concentration standard curve, the amount of residual host cell protein in the sample to be tested is obtained.

[0148] In this embodiment of the invention, the absorbance-protein concentration standard curve is obtained by the following method:

[0149] Pichia pastoris host cell protein solutions at concentrations of 0, 0.2, 0.5, 1.0, 2.5, 5.0, 12.5, 25, and 30 ng / mL were used as Pichia pastoris host cell protein standard solutions.

[0150] Multiple Pichia pastoris host cell protein standard solutions were subjected to enzyme-labeled detection to obtain multiple absorbance values;

[0151] Based on the obtained absorbance values ​​and the corresponding Pichia pastoris host cell protein concentrations, a standard curve of absorbance value versus protein concentration was plotted.

[0152] The detection method provided in the embodiments of the present invention will be described in detail below.

[0153] 1. Detection Method

[0154] (1) Take a certain amount of HRP-labeled polyclonal antibody, add an appropriate amount of PBS-BSA solution (1% BSA), mix well, and obtain HRP-labeled antibody solution.

[0155] (2) Add 50 μL of HCP standard / sample solution and 50 μL of PBST solution to each microplate, cover with a sealing film, and place the microplate in a constant temperature shaker and incubate at 25 °C and 400 rpm for 2 h.

[0156] (3) Clean the microplate by adding 300 μL of PBST solution to each well and repeating 5 times.

[0157] (4) Add 100 μL of HRP-labeled antibody solution to each well, seal the well, and incubate at 25 °C and 400 rpm for 1 h.

[0158] (5) Wash the plate again with 300 μL of PBST solution, repeat 5 times.

[0159] (6) Add 50 μL of substrate solution A and 50 μL of substrate solution B to each well;

[0160] The formulation of substrate solution A, based on 1L of ultrapure water, is as follows: citric acid monohydrate 3.2 g / L, sodium acetate 7.0 g / L, and urea peroxide 0.5 g / L.

[0161] Based on 1L of ultrapure water, the formulation of substrate solution B is as follows: 1.0 g / L citric acid monohydrate, 1.0 g / L sodium chloride, 0.4 g / L tetramethylbenzidine, and 0.1 g / L EDTA-Na2.

[0162] (7) Add stop solution (i.e., 2M H2SO4) and measure OD. 450 and OD 630 value.

[0163] Specifically, Pichia pastoris HCP solutions of 0, 0.2, 0.5, 1.0, 2.5, 5.0, 12.5, 25, and 30 ng / mL were used as Pichia pastoris HCP standard solutions. Different concentrations of Pichia pastoris HCP solutions were obtained by diluting the Pichia pastoris-derived specific HCP prepared in the embodiments of the present invention to a specific concentration using PBS solution. The 0 ng / mL Pichia pastoris HCP solution was a PBS solution.

[0164] Multiple Pichia pastoris HCP standard solutions were subjected to enzyme-linked assays to obtain multiple absorbance values; each HCP standard solution was measured in parallel replicates.

[0165] Using the concentrations and corresponding absorbance values ​​of Pichia pastoris HCP standard solutions, a standard curve and linear equation were plotted based on absorbance versus protein concentration. R0 2 The concentration should be ≥0.99. The concentration of residual Pichia pastoris host cell protein in the sample solution is calculated using the absorbance-protein concentration standard curve and the absorbance of the measured sample solution. Sample solution purity = , where C X The concentration of residual Pichia pastoris host cell protein (ng / mL), C A The concentration of rHSA in the sample is expressed in mg / mL.

[0166] 2. Linearity range verification

[0167] The drawn absorbance-protein concentration standard curve and linear equation are as follows: Figure 4 As shown. (Through) Figure 4 The linear equation is Y = 10876X + 10692, R0 2 =0.996. Analysis showed that the HCP content of this kit was within the range of 0~30 ng / mL, exhibiting good linearity.

[0168] 3. Accuracy Verification

[0169] Take 200 μL of 2.5 ng / mL standard, add 50 μL of human serum albumin injection, dilute the standard to a concentration of 2 ng / mL, perform 6 parallel tests, calculate the recovery rate of the solution and the RSD of the test results. The results are shown in Table 5.

[0170] Table 5. Accuracy verification test data:

[0171] ;

[0172] The results in Table 5 show that the HCP CLIA kit provided by this invention detects values ​​for samples that are close to the true values.

[0173] 4. Repeatability verification

[0174] Take a sample of Pichia pastoris-derived recombinant human serum albumin preparation, perform 6 parallel tests, and calculate the RSD of the 6 test results. The results are shown in Table 6.

[0175] Table 6 Repeatability verification test data:

[0176] ;

[0177] The results in Table 6 show that the CLIA kit used in this invention has an RSD of 7.0% for parallel detection of the same sample, which meets the requirement of ≤15.0%, indicating that the CLIA kit provided by this invention has good reproducibility.

[0178] 5. Limit of Quantification Validation

[0179] The diluted reference solution was tested in parallel six times, and the standard deviation of the luminescence values ​​for the six tests was calculated using the formula LOQ = 10δ / S, where LOQ is the limit of quantitation, δ is the standard deviation of the six luminescence values, and S is the slope of the standard curve. The detection and calculation results are shown in Table 7.

[0180] Table 7. Detection and calculation data for the limit of quantitation validation:

[0181] ;

[0182] By combining the results in Table 7 with the standard curve, the linear regression equation for the limit of quantitation was obtained: Y = 12400X + 7870. According to the LOQ formula, the limit of quantitation of the CLIA kit provided by this invention is 0.2 ng / mL.

[0183] 6. Specificity verification

[0184] Blank solvent and human serum albumin injection were used as samples for testing. In addition, sample dilution was used as a negative control (NCT, 0 ng / mL HCP). The test results are shown in Table 8.

[0185] Table 8. Specificity verification test data:

[0186] ;

[0187] Table 8 shows that the detection values ​​of the blank solvent and human serum albumin injection are both lower than NCT, indicating that the kit has good specificity and does not specifically react with human albumin or impurities in the solvent.

[0188] 7. Durability verification

[0189] Take 400 μL of the 2.5 ng / mL standard and add 100 μL of diluent to dilute it to 2 ng / mL as the test sample. Take three different batches of the CLIA kit provided in Example 1 of this invention. The standard curves of the three different batches of CLIA kits are shown below. Figure 5 As shown in Table 9, the HCP content of the sample to be tested was detected, and the results are shown in Table 9.

[0190] Table 9. Durability test data:

[0191] ;

[0192] The results in Table 9 show that the recovery rates of the three different batches of kits for the same sample all met the requirements (70-125%), indicating that the kits and detection methods provided in this embodiment of the invention have good reliability.

[0193] 8. Detection of Pichia pastoris-derived specific rHSA host cell protein residues

[0194] The CLIA kit provided in Example 1 of this invention and the commercially available Pichia pastoris HCP ELISA kit were used to quantitatively detect HCP residues in Pichia pastoris-derived recombinant human serum albumin preparations. The standard curve is shown in [Figure 1]. Figure 6 The test results are shown in Tables 10 and 11.

[0195] Table 10 rHSA detection data of the CLIA kit of the present invention:

[0196] ;

[0197] Table 11. rHSA data detected by commercially available Pichia pastoris HCP ELISA kit:

[0198] ;

[0199] The results in Tables 10 and 11 show that the CLIA kit provided by this invention has a higher detection capability than commercially available universal ELISA kits, and its detection capability for HCP residues is more than 30 times higher than that of commercially available kits.

[0200] The above-described embodiments are merely preferred embodiments of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A kit for quantitatively detecting residual Pichia pastoris host cell proteins, characterized in that, This includes horseradish peroxidase-labeled polyclonal antibodies that specifically recognize Pichia pastoris host cell proteins; Horseradish peroxidase-labeled polyclonal antibodies were obtained as follows: Animals were immunized using Pichia pastoris host cell proteins to obtain antibody-containing animal serum. Pichia pastoris host cell proteins were obtained through the following methods: (1) Construction of Pichia pastoris empty vector expression strain Pichia pastoris GS115 strain was transformed by electroporation of linearized pPIC9K plasmid, and Pichia pastoris empty vector expression strain was obtained by screening. The copy number of the Pichia pastoris empty vector expression strain is the same as the copy number of the recombinant human serum albumin expression strain. (2) Preparation of Pichia pastoris host cell protein After activating the Pichia pastoris empty vector expression strain on a plate, the Pichia pastoris host cell protein was obtained by fermentation induction and purification. The animal serum containing antibodies is purified to obtain polyclonal antibodies; The purification process includes sequentially purifying the antibody-containing animal serum using a Protein A chromatography column and then using a host cell protein-coupled affinity column. Host cell protein-coupled affinity columns were prepared using the following method: Take the coupling medium and wash it in coupling solution A at 0~4℃ for at least 30 min to obtain the washed coupling medium. The host cell protein was replaced to a concentration of 5-10 mg / ml by coupling with solution B to obtain the ligand solution; The washed coupling medium was diluted with coupling solution A. The diluted coupling medium was mixed with an equal volume of ligand solution and gently stirred overnight at 4°C. Then, the mixture was blocked and washed to obtain a host cell protein coupling affinity column. The coupling solution A was 1 mM HCl; the coupling solution B was 0.1 M NaHCO3. Horseradish peroxidase labeling was performed on the polyclonal antibody to obtain horseradish peroxidase labeled polyclonal antibody. The limit of quantification for residual Pichia pastoris host cell proteins in the kit is 0.2 ng / mL.

2. The kit for quantitative detection of residual Pichia pastoris host cell protein according to claim 1, characterized in that, The solid-liquid ratio of the washed coupling medium to coupling solution A is 1:

1.

3. The kit for quantitative detection of residual Pichia pastoris host cell protein according to claim 1, characterized in that, The animals to be immunized are New Zealand white rabbits.

4. The kit for quantitative detection of residual Pichia pastoris host cell protein according to claim 1, characterized in that, It also includes antibody diluent, sample diluent, substrate solution A, substrate solution B, and PBST solution.

5. The kit for quantitative detection of residual Pichia pastoris host cell protein according to claim 4, characterized in that, The antibody diluent was a PBS-BSA solution with a pH of 7.

4. The sample diluent was PBST solution with a concentration of 0.15 M and a pH of 7.

4. The formulation of substrate solution A is as follows: citric acid monohydrate 3.2 g / L, sodium acetate 7.0 g / L, urea peroxide 0.5 g / L; The formulation of substrate solution B is as follows: 1.0 g / L citric acid monohydrate, 1.0 g / L sodium chloride, 0.4 g / L tetramethylbenzidine, and 0.1 g / L EDTA-Na2.

6. A method for quantitatively detecting residual protein in Pichia pastoris host cells, characterized in that, Includes the following steps: Using the kit according to any one of claims 1-5, the sample to be tested is incubated. The incubated samples were subjected to enzyme-linked immunosorbent assay (ELISA) to obtain absorbance values. Based on the obtained absorbance values ​​and the absorbance-protein concentration standard curve, the amount of residual host cell protein in the sample to be tested is obtained.

7. The method for quantitatively detecting residual Pichia pastoris host cell protein according to claim 6, characterized in that, The absorbance-protein concentration standard curve was obtained using the following method: Pichia pastoris host cell protein solutions at concentrations of 0, 0.2, 0.5, 1.0, 2.5, 5.0, 12.5, 25, and 30 ng / mL were used as Pichia pastoris host cell protein standard solutions. Multiple Pichia pastoris host cell protein standard solutions were subjected to enzyme-labeled detection to obtain multiple absorbance values; Based on the obtained absorbance values ​​and the corresponding Pichia pastoris host cell protein concentrations, a standard curve of absorbance value versus protein concentration was plotted.

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