CN112285224A

CN112285224A - Quality standard detection method of anti-PD-1 monoclonal antibody medicine

Info

Publication number: CN112285224A
Application number: CN202011066726.7A
Authority: CN
Inventors: 朱吉安
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-10-02
Filing date: 2020-10-02
Publication date: 2021-01-29

Abstract

The invention provides a quality standard detection method of an anti-PD-1 monoclonal antibody medicine, and relates to the field of biological medicine detection in the technical field of microbial fermentation. The quality standard detection method comprises the steps of identification of a sample to be detected, purity analysis, protein content detection and related impurity content detection. The method has strong specificity, good linearity, high precision and accuracy, and has important significance for quality control and clinical application of the anti-PD-1 monoclonal antibody.

Description

Quality standard detection method of anti-PD-1 monoclonal antibody medicine

The technical field is as follows:

the invention relates to the field of biological drug detection, in particular to a quality standard detection method of an anti-PD-1 monoclonal antibody drug.

Background art:

programmed death receptor-1 (PD-1) and programmed death ligand-1 (PD-L1) are a pair of immune co-inhibitory molecules. The PD-1/PD-L1 signal channel is closely related to the immune escape of tumors and viruses, and the medicament taking PD-1/PD-L1 as a target reactivates the anti-tumor immunity of the body and obtains good and lasting curative effect in various tumors, thereby having good application prospect.

PD-1 is an immune co-inhibitory molecule belonging to the family of CD28, consisting of 268 amino acids, belonging to the type I transmembrane protein, comprising 1 IgV-like region, 1 IsC-like region, 1 transmembrane hydrophobic region and 1 intracellular region consisting of 30 amino acids. PD-1 is mainly expressed on the membrane surface of T cells, B cells, NK cells and various tumor cells. PD-1 has 2 ligands, PD-L1 and PD-L2, belonging to the B7 family of transmembrane molecules. PD-L1 is a transmembrane protein composed of 290 amino acid subunits, the extracellular segment is two immunoglobulin constant regions (IgC) and an IgV-like structure domain, and the extracellular segment is mainly expressed in hematopoietic cells such as mature CD4+ T cells, CD8+ T cells, monocytes, macrophages, B cells, dendritic cells and the like, and the PD-L1 molecule is highly expressed in various tumor cells. PD-L2 is a transmembrane protein composed of 274 amino acid residues, has high similarity with PD-L1, but has certain difference, such as the in vivo tissue distribution of PD-L2 has limitation, and is only expressed on the membrane surface of macrophages, dendritic cells and some B cell subsets.

The PD-1/PD-L1 signaling pathway plays an important role in T cell immune processes. T cells under-express PD-1 at rest, whereas activation causes up-regulation of PD-1 expression. PD-1 as an inhibitory co-receptor can inhibit the activity of T cells after interacting with PD-L1, so that the cells are blocked in the G0/G1 phase, thereby inhibiting the proliferation of the T cells, preventing the T cells from differentiating into plasma cells and inducing the apoptosis of the T cells. This avoids the immortalization of T cells and maintains them in homeostasis. The negative feedback regulation effect of the T cells involved in the PD-1/PD-L1 signal channel plays a crucial role in clearing antigens and maintaining the balance of the organism.

PD-1/PD-L1 is used as a negative co-stimulatory molecule, and high expression of the molecule can be found in various tumors, such as melanoma, non-small cell lung cancer, ovarian cancer, renal cell carcinoma and the like. When tumors occur, the PD-L1 on the surface of the tumor cells interacts with PD-1 receptors on the surface of T cells, so that the immune response of the T cells can be inhibited, and the immune escape of the tumors occurs. The anti-PD-1 monoclonal antibody blocks a PD-1/PD-L1 signal channel through the binding of PD-1, thereby restoring T cell function.

At present, the development of anti-PD-1 or anti-PD-L1 monoclonal antibody medicaments has become a hotspot in tumor treatment, and the quality detection of the anti-PD-1 monoclonal antibody medicaments is an essential work in the process of producing the anti-PD-1 monoclonal antibody medicaments. Therefore, the invention provides a quality standard detection method of an anti-PD-1 monoclonal antibody medicament, aiming at the identification of the anti-PD-1 monoclonal antibody medicament, an isoelectric point detection method and a peptide map analysis method based on UPLC (ultra high performance liquid chromatography) separation are established; aiming at the analysis of product purity, detection methods such as SEC-HPLC (size exclusion chromatography), non-reduction CE-SDS (capillary gel electrophoresis), reduction CE-SDS, CEX-HPLC (cation exchange high performance liquid chromatography) and the like are established; aiming at the quantification of the anti-PD-1 monoclonal antibody medicine, a protein content determination method is established; aiming at the detection of related impurity content, an ELISA method is established to analyze Protein A residue and host Protein (HCP) residue, and qPCR is established to detect CHO host cell DNA residue.

The invention content is as follows:

the invention aims to provide a quality standard detection method of an anti-PD-1 monoclonal antibody medicament, which has strong specificity, good linearity, high precision and high accuracy and has important significance for quality control and clinical application of the anti-PD-1 monoclonal antibody.

The invention provides a quality standard detection method of an anti-PD-1 monoclonal antibody medicament, which is characterized by comprising the following steps of:

(1) and (3) identification: comprises isoelectric point determination and peptide spectrogram analysis of a sample to be detected;

(2) purity analysis: the method comprises monomer purity detection, main peak purity detection, light chain and heavy chain purity detection and charge heteroplasmon content detection of a sample to be detected;

(3) and (3) protein content detection: detecting a sample to be detected by using an ultraviolet spectrophotometry;

(4) and (3) detecting the content of related impurities: comprises detecting the residual quantity of Protein A, HCP and CHO host cell DNA in a sample to be detected;

the sample to be detected is an anti-PD-1 monoclonal antibody drug.

Preferably, the isoelectric point is determined by focusing and separating heteroplasms with different charges according to their isoelectric points by the CIEF method.

The method comprises the following steps:

performing capillary isoelectric focusing analysis with PA/MDQ capillary electrophoresis apparatus and neutral coating capillary assembly, collecting 3M urea CIEF gel 80 μ l and 3-10 carrier ampholyte (P)5. mu.l of harmalyte3-10) 100mM anolyte (H)₃PO₄) Mixing 10 μ l, 200mM catholyte (NaOH)15 μ l, isoelectric point markers (including polypeptide markers pI5.85 and pI8.18)4 μ l and 10mg/mL sample to obtain sample mixture; loading the sample mixed solution into a capillary, performing focusing separation at room temperature, injecting sample for 99s under 173kPa, wherein the focusing condition is that the focusing voltage is 25kV, and maintaining for 10min, and the separation condition is that 100mM ammonia water is adopted as migration liquid, the voltage is 25kV, and the maintaining for 30 min; detecting with ultraviolet absorption detector at 280nm wavelength, and recording isoelectric focusing spectrum.

Preferably, the peptide spectrogram analysis is to hydrolyze a sample to be detected by using Lys-C enzyme by utilizing an intracellular protease enzyme digestion-reverse phase chromatography, and obtain the peptide spectrogram by gradient elution of a reverse phase chromatographic column, and the method comprises the following steps of:

taking a sample to be detected, diluting the sample to be detected to 1mg/mL by using a urea buffer solution, carrying out denaturation for 10min at 68 ℃, adding DTT, reducing for 45min at 36 ℃, wherein the volume ratio of DTT to the sample to be detected is 1: 50; adding IAA, and reacting for 30min in a dark place, wherein the volume ratio of the IAA to the sample to be detected is 1: 40; adding DTT to terminate the reaction, wherein the volume ratio of DTT to the sample to be detected is 1: 20; replacement of the prourosin buffer with NH₄HCO₃Adding Lys-C enzyme, and carrying out water bath at 37 ℃ for 10h, wherein the volume ratio of the Lys-C enzyme to a sample to be detected is 1: 20; terminating the reaction by 5% TFA to obtain a digestion product; and detecting and analyzing the obtained enzyme digestion product by using a reversed-phase high performance liquid chromatography, wherein the chromatographic conditions are as follows: the mobile phase A is 0.1 percent aqueous solution of formic acid; the mobile phase B is 0.1 percent formic acid acetonitrile solution; gradient elution (5-120min, 2% B → 85% B), flow rate of 0.5mL/min, detection wavelength 214nm, to obtain peptide spectrogram.

Specifically, the peptide spectrogram analysis steps are as follows:

taking a sample to be detected with the concentration of 10mg/mL, diluting the sample to be detected to 1mg/mL by using 5mol/L urea buffer solution, performing denaturation for 10min at 68 ℃, adding 1mol/L DTT, and reducing for 45min at 36 ℃, wherein the volume ratio of the DTT to the sample to be detected is 1: 50; adding 1mol/L IAA for alkylation, and reacting for 30min in a dark place, wherein the volume ratio of the IAA to the sample to be detected is 1: 40; adding 1mol/L DTT to terminate the reaction, wherein the volume ratio of the DTT to the sample to be detected is 1: 20; the prourosin buffer was replaced to 20m using a 10KD centrifugal concentrator tubemol/L NH₄HCO₃Adding 1mg/mL Lys-C enzyme, carrying out water bath at 37 ℃, carrying out enzyme digestion and hydrolysis for 10 hours, wherein the volume ratio of the Lys-C enzyme to a sample to be detected is 1: 20; terminating the reaction by 5% TFA to obtain a digestion product; and detecting and analyzing the obtained enzyme digestion product by using a reversed-phase high performance liquid chromatography, wherein the chromatographic conditions are as follows: the mobile phase A is 0.1 percent aqueous solution of formic acid; the mobile phase B is 0.1 percent formic acid acetonitrile solution; gradient elution (5-120min, 2% B → 85% B), flow rate of 0.5mL/min, detection wavelength 214nm, to obtain peptide spectrogram.

Preferably, the monomer purity detection is implemented by separating a sample to be detected by using a size exclusion chromatographic column by using a SEC-HPLC method, and comprises the following steps:

diluting a sample to be detected to 10mg/mL by using a mobile phase, wherein the mobile phase is a mixed solution of 50mmol/L potassium phosphate and 300mmol/L sodium chloride, the pH value is 6.8, the loading amount is 10 mu L, the flow rate is 0.8mL/min, detection is carried out at 280nm, and the monomer content is calculated by adopting a peak area normalization method.

Preferably, the main peak purity detection is performed by using a non-reducing CE-SDS method, and comprises the following steps:

diluting a sample to be detected to 5.0mg/mL by using ultrapure water, taking 30 mu l of the solution, adding 5 mu l of mercaptoethanol and 70 mu l of SDS sample buffer solution, uniformly mixing, carrying out water bath at 70 ℃ for 10min, and carrying out sample loading detection; the sample amount is 50 mul, the separation voltage is 15kV, the capillary temperature is 30 ℃, the detection wavelength is 220nm, and the peak area percentage of the main peak, namely the purity of the main peak, is calculated by an area normalization method.

Preferably, the purity of the light chain and the heavy chain is detected by a reduction CE-SDS method, which comprises the following steps:

diluting a sample to be detected to 5.0mg/mL by using ultrapure water, taking 30 mu L of the solution, adding 5 mu L of 0.1mol/L N-ethylmaleimide and 95 mu L of SDS sample buffer solution, uniformly mixing, carrying out water bath at 70 ℃ for 10min, and carrying out loading detection; the sample amount was 50. mu.l, the separation voltage was 15kV, the capillary temperature was 30 ℃ and the detection wavelength was 220nm, and the relative area percentages of the heavy and light chains, i.e., the light and heavy chain purities, were calculated by area normalization.

Preferably, the charge heteroplasmon content detection is by CEX-HPLC method, comprising the following steps:

diluting a sample to be detected to 1mg/mL by using a mobile phase A, wherein the sample loading amount is 25 mul, the mobile phase A is 0.05M phosphate, the pH value is 4.0, the mobile phase B is a mixed solution of 0.05M phosphate and 0.5M sodium chloride, the pH value is 10.0, the flow rate is 0.8mL/min, gradient elution is carried out, the content of a main peak, an acidic peak and a basic peak of the sample to be detected is calculated by adopting a peak area normalization method, wherein the content of the main peak, the acidic peak and the basic peak of the sample to be detected is 50% within 30 minutes, and the content of a charge heteroplasmon is the detection wavelength of.

Preferably, the detection of the residual amount of Protein A comprises the following steps:

adding 100 mul/well of Protein A standard substance with gradient concentration of 0.1-2.0ppm and sample to be tested into a 96-well plate of chicken anti-Protein A polyclonal antibody coated in advance, and washing the plate; adding a chicken anti-Protein A polyclonal antibody marked by biotin, incubating for 2h at 40 ℃, and washing the plate; adding streptavidin coupled with horseradish peroxidase, and washing the plate; adding TMB solution, standing at room temperature for developing for 30min, stopping reaction with stop solution, reading light absorption value 450/650nm on a microplate reader, drawing a standard curve by using a four-parameter fitting regression model, and calculating the concentration of Protein A in the sample to be measured.

Preferably, the HCP residual amount detection comprises the steps of:

respectively adding an HCP standard substance with the gradient concentration of 0.1-20.0ppm and a sample to be detected to a 96-well plate coated with an anti-HCP capture antibody in advance, and washing the plate; adding a goat anti-HCP polyclonal antibody coupled with horseradish peroxidase, incubating at 37 ℃ for 2.5h, and washing the plate; adding HRP-coupled streptavidin, and washing the plate; adding TMB substrate solution, standing at room temperature for color development for 30 min; and (3) terminating the reaction by using a stop solution, reading a light absorption value of 450/650nm on an enzyme-labeling instrument, drawing a standard curve by using a four-parameter fitting regression model, and calculating the concentration of HCP in the sample to be detected.

Preferably, the detection of the DNA residue of the CHO host cell is to detect the DNA residue of the CHO host cell in a sample to be detected by using fluorescent quantitative PCR, and comprises the following steps:

s1, preparation of a DNA standard: extracting CHO cell genome DNA, EcoRI and BamHI double enzyme digestion, purifying and recovering enzyme digestion fragments, detecting A260/A280, and calculatingThe concentration and purity of the purified product was 10 by dilution with Tris-EDTA⁰、10¹、10²、10³、10⁴And 10⁵A pg/mL standard;

s2.qPCR amplification and standard curve drawing: the reaction system comprises 0.5 mu M upstream and downstream primers 1.2 mu l each, 5U Taq DNA polymerase 0.5 mu l, dNTPs 2.0 mu l, DNA buffer 3 mu l, template DNA 0.5 mu l, ddH₂O16.6. mu.l; the reaction conditions are as follows: pre-denaturation at 95 ℃ for 40 s; 45 cycles of 95 ℃ for 8min, 54 ℃ for 1min and 72 ℃ for 1 min; drawing a standard curve by taking the Ct value as a vertical coordinate and taking the logarithm value of the template DNA concentration as a horizontal coordinate;

the upstream primer is as follows: 5'-CCAGGCATTGGTGGCAC-3', respectively;

the downstream primer is as follows: 5 '-AGACAGGGTTTCTCTGT-3';

s3, detecting the DNA residue of the CHO host cell of the sample to be detected: and (4) extracting the DNA of the sample to be detected, performing qPCR amplification by using the reaction system and the reaction conditions in the step S2, and calculating the DNA concentration of the CHO host cell in the sample to be detected according to the standard curve.

PD-1 of the present invention includes but is not limited to the following patent applications: 201610656318.4.

the invention also provides the application of the quality standard detection method in the quality control of anti-PD-1 monoclonal antibody medicines.

The invention has the beneficial effects that:

the invention aims to provide a quality standard detection method of an anti-PD-1 monoclonal antibody medicament, which has strong specificity, good linearity, high precision and accuracy, can comprehensively and accurately detect the quality of the anti-PD-1 monoclonal antibody medicament, and has important significance for quality control and clinical application of the anti-PD-1 monoclonal antibody.

Drawings

FIG. 1 is an isobologram of the reference 1.1 of example 1;

FIG. 2 is an iso-focal spectrum of sample 1 of example 1, 1;

FIG. 3 is an iso-focal spectrum of sample 2 of example 1.1;

FIG. 4 is an iso-focal spectrum of sample 3 of example 1.1;

FIG. 5 is a comparison of the peptide profile analysis of sample 1 of example 1 at 1.2 with that of a reference;

FIG. 6 is a comparison of peptide profile analysis of sample 2 of example 1.2 with a reference;

FIG. 7 is a comparison of peptide profile analysis of sample 3 of example 1.2 with that of a reference;

FIG. 8 is a SEC chromatogram of sample 1 from example 2;

FIG. 9 is a SEC chromatogram of sample 2 from example 2;

FIG. 10 is a SEC chromatogram of sample 3 from example 2;

FIG. 11 is a non-reducing CE-SDS chromatogram of 2.2 in example 2, and FIG. A, B, C is a non-reducing CE-SDS chromatogram of samples 1-3, respectively;

FIG. 12 is a reduced CE-SDS chromatogram of 2.3 from example 2, with FIGS. A, B, C being reduced CE-SDS chromatograms for samples 1-3, respectively;

FIG. 13 is a CEX-HPLC chromatogram of 2.4 in example 2, with FIG. A, B, C being the CEX-HPLC chromatograms of samples 1-3, respectively;

FIG. 14 is a qPCR standard curve of 4.3 in example 4.

Detailed Description

In order to make the technical means, the original characteristics, the achieved purposes and the effects of the invention easily understood, the invention is further explained with the following embodiments, but the following embodiments are only the preferred embodiments of the invention, and not all embodiments. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified, and the drugs, reagents and the like used in the following examples are commercially available without otherwise specified.

Example 1

Identification of anti-PD-1 monoclonal antibody drugs:

1.1 isoelectric Point determination

Capillary isoelectric focusing analysis was performed using PA/MDQ capillary electrophoresis apparatus and neutral coated capillary assembly, using 80. mu.l of 3M urea CIEF gel, 5. mu.l of 3-10 carrier ampholyte (Pharmalyte3-10), and 100mM anolyte (H)₃PO₄) Mixing 10 μ l, 200mM catholyte (NaOH)15 μ l, isoelectric point markers (including polypeptide markers pI5.85 and pI8.18)4 μ l and 10mg/mL sample or reference to obtain sample mixture; loading the sample mixed solution into a capillary, performing focusing separation at room temperature, injecting sample for 99s under 173kPa, wherein the focusing condition is that the focusing voltage is 25kV, and maintaining for 10min, and the separation condition is that 100mM ammonia water is adopted as migration liquid, the voltage is 25kV, and the maintaining for 30 min; detecting with ultraviolet absorption detector at 280nm wavelength, and recording isoelectric focusing spectrum.

Samples to be tested (3 batches of anti-PD-1 monoclonal antibody stock solution produced by the company, the batch numbers are 3055S150525Y08, 3055S150630Y08 and 3055S150717Y08 respectively, and the sample names are respectively sample 1-3) and reference products (WBP3055STD, batch number 3055SD150630K01M 01).

As shown in FIGS. 1-4, the isoelectric points of the 3 batches of stock solutions are respectively 7.05, 6.99 and 7.02, which are the main peaks of the stock solutions, and are substantially identical to those of the reference product (isoelectric point of 7.08).

1.2 peptide Spectroscopy analysis

Taking 10mg/mL of a sample to be detected or a reference product (the sample is the same as that in 1.1), diluting the sample to be detected to 1mg/mL by using 5mol/L urea buffer solution, performing denaturation for 10min at 68 ℃, adding 1mol/L DTT (dithiothreitol) to perform reduction for 45min at 36 ℃, wherein the volume ratio of the DTT to the sample to be detected is 1: 50; adding 1mol/L IAA (iodoacetamide) for alkylation, and reacting for 30min in a dark place, wherein the volume ratio of the IAA to a sample to be detected is 1: 40; adding 1mol/L DTT to terminate the reaction, wherein the volume ratio of the DTT to the sample to be detected is 1: 20; replacing the original urea buffer solution with 20mmol/L NH4HCO3 by using a 10KD centrifugal concentration tube, adding 1mg/mL Lys-C enzyme, carrying out enzyme digestion and hydrolysis for 10h in water bath at 37 ℃, wherein the volume ratio of the Lys-C enzyme to a sample to be detected is 1: 20; terminating the reaction by 5% TFA (trifluoroacetic acid) to obtain an enzyme digestion product; the obtained enzyme digestion product is detected and analyzed by reversed phase high performance liquid chromatography, and the chromatographic conditions are as follows:

the mobile phase A is 0.1 percent aqueous solution of formic acid; the mobile phase B is 0.1 percent formic acid acetonitrile solution; gradient elution (5-120min, 2% B → 85% B), flow rate of 0.5mL/min, detection wavelength 214nm, to obtain peptide spectrogram.

According to the protein digestion product map, the main peptide fragment chromatographic peaks (

peak

1, 2 and 3) of 3 CDR regions are selected as main investigation and reference standards of peptide map analysis.

The detection results are shown in FIGS. 5-7, and the peptide spectra of 3 batches of stock solutions are substantially consistent with the reference.

Example 2

Purity analysis of anti-PD-1 monoclonal antibody drugs:

2.1 monomer purity detection

The sample to be tested (same sample as in 1.1 of example 1) was separated using a size exclusion chromatography column. Diluting a sample to be detected to 10mg/mL by using a mobile phase, wherein the mobile phase is a mixed solution of 50mmol/L potassium phosphate and 300mmol/L sodium chloride, p H is 6.8, the loading amount is 10 mu L, the flow rate is 0.8mL/min, detection is carried out at 280nm, and the monomer content is calculated by adopting a peak area normalization method;

the results of the measurements are shown in FIGS. 8-10, and the monomer purities of samples 1-3 were 99.2%, 99.3% and 99.3%, respectively.

2.2 Main Peak purity detection

Diluting a sample to be detected (the same as the sample in 1.1 of example 1) to 5.0mg/mL by using non-reduction CE-SDS method and ultrapure water, taking 30 mu l of the solution, adding 5 mu l of mercaptoethanol and 70 mu l of SDS sample buffer solution, uniformly mixing, carrying out water bath at 70 ℃ for 10min, and carrying out loading detection; the sample amount is 50 mul, the separation voltage is 15kV, the capillary temperature is 30 ℃, the detection wavelength is 220nm, and the peak area percentage of the main peak, namely the purity of the main peak, is calculated by an area normalization method.

The detection results are shown in FIG. 11, the purity contents of the main peaks of non-reduced CE-SDS of samples 1-3 are 99.3%, 99.2% and 99.2%, respectively, and there is no significant difference among 3 batches of stock solutions.

2.3 light and heavy chain purity assays

The purity of the light chain and the heavy chain is detected by a reducing CE-SDS method, which comprises the following steps:

diluting a sample to be detected (the same as the sample in 1.1 of example 1) to 5.0mg/mL by using ultrapure water, taking 30 mu L of the solution, adding 5 mu L of 0.1mol/L N-ethylmaleimide and 95 mu L of SDS sample buffer solution, uniformly mixing, carrying out water bath at 70 ℃ for 10min, and carrying out loading detection; the sampling amount is 50 mul, the separation voltage is 15kV, the capillary temperature is 30 ℃, the detection wavelength is 220nm, and the relative area percentage of the heavy chain and the light chain, namely the purity of the light chain and the heavy chain, is calculated by an area normalization method;

the results are shown in FIG. 12, and the purity of the reduced CE-SDS (light chain + heavy chain%) of samples 1-3 is shown in Table 1, and there is no significant difference between 3 batches of stock solutions.

Table 1: purity test results of light chain and heavy chain

2.4 detection of Charge heteroplasmon content

Monoclonal antibodies exist as different charge isomers due to post-translational modifications of amino acids, such as oxidation, deamidation, glycosylation, incomplete C-terminal lysine deletion and N-terminal Q cyclization, and thus require monitoring of the charge isomers of the sample to control product quality.

A sample to be detected (the same as the sample in 1.1 of example 1) is diluted to 1mg/mL by a mobile phase A by using a CEX-HPLC method and a TSK sp-5pw chromatographic column, the loading amount is 25 μ l, the mobile phase A is 0.05M phosphate, the pH value is 4.0, the mobile phase B is a mixed solution of 0.05M phosphate and 0.5M sodium chloride, the pH value is 10.0, the flow rate is 0.8mL/min, gradient elution is carried out, the mobile phase B is linearly increased from 5% to 50% within 30 minutes, the detection wavelength is 280nm, and the main peak, acidic peak and alkaline peak contents, namely the charge heteroplasmon content, of the sample to be detected are calculated by a peak area normalization method.

The detection results are shown in fig. 13, the main peak contents of samples 1-3 are 56.1%, 55.7% and 56.3%, the specific charge heteroplasmon distribution is shown in table 2, and no obvious difference exists among the three batches of stock solutions.

Table 2: detection result of content of charge heteroplasmon

Batch number	Sample name	Main Peak (%)	Acid Peak (%)	Basic Peak (%)
					3055S150525Y08	Sample	1	56.1	21.0	22.8
3055S150630Y08	Sample	2	55.7	20.8							23.5
					3055S150717Y08	Sample	3	56.3	19.8	24.0

Example 3

Detecting the protein content of the anti-PD-1 monoclonal antibody medicament:

the detection method comprises the following steps: the direct sample to be detected or the reference product (the same as the sample in 1.1 of the embodiment 1) is detected by an enzyme-labeling instrument and an ultraviolet spectrophotometry, and the protein molecule contains benzene-ring-containing amino acids such as tyrosine, tryptophan, phenylalanine and the like, so that the maximum absorption peak is formed near the wavelength of 280 nm. According to Lambert-beer's law, the absorbance value (A) of a protein solution at a certain fixed ultraviolet wavelength has a certain relationship with the extinction coefficient (epsilon) of the protein, the protein concentration (C) and the optical length (L): a ═ epsilon LC. Therefore, the concentration of the protein solution can be calculated from the absorbance value at a fixed wavelength, the optical path length of the cuvette (10mm), and the extinction coefficient of the protein substance (theoretical extinction coefficient. epsilon. of the protein is 1.685).

3.1 precision

The protein concentration was measured in 5 replicates (randomly 5 reference samples) by 3 qualified analysts, and the mean value of the 15 data and their RSD were calculated as described above. The results are shown in Table 3.

Table 3: protein content and precision results

The results show that: the protein content of 5 samples is 30.32-30.81mg/mL, and the RSD is 0.5%, which shows that the precision of the method is good.

3.2 accuracy

The anti-PD-1 monoclonal antibody drugs having known protein contents of 10.3mg/mL, 20.5mg/mL, and 30.1mg/mL were used as samples to be tested (i.e., sample A, BC, anti-PD-1 monoclonal antibody injection, lot Nos.: 20150701, 20150802, 20150903) and tested 1 time each by 3 analysts, and the results of the experiments were statistically shown in Table 4.

Table 4: protein content and accuracy results

The result shows that the RSD of the 3-time detection result of each sample to be detected is less than 2 percent, which indicates that the method can meet the accuracy requirement.

Example 4

Detecting the content of related impurities of the anti-PD-1 monoclonal antibody medicament:

4.1 detection of residual amount of Protein A

The detection method comprises the following steps: sandwich enzyme-linked immunosorbent assay (ELISA), adding 100 mul/well Protein A standard substance with gradient concentration of 0.1, 0.4, 0.8, 1.2, 1.6, 2.0ppm and sample to be tested into a 96-well plate coated with chicken anti-Protein A polyclonal antibody with concentration of 1.2 mug/mL in advance, and washing the plate for 3 times by PBS solution; adding 100 mul/hole of chicken anti-Protein A polyclonal antibody with the concentration of 0.8 mug/mL biotin labeling, incubating for 2h at 40 ℃, and washing the plate for 3 times by PBS solution; adding 100 mul/hole of horseradish peroxidase-coupled streptavidin with the concentration of 1.0 mug/mL, and washing the plate for 3 times by using PBS solution; adding 100 mu l/hole TMB solution, standing and developing for 30min at room temperature, stopping the reaction by using 100 mu l/hole stop solution, reading the light absorption value of 450/650nm on a microplate reader, drawing a standard curve by using a four-parameter fitting regression model (weight factor Y) through SoftMax Pro computer software, and calculating the concentration of Protein A in the sample according to the OD value of the sample to be detected and the standard curve.

4.1.1 precision

The assay was performed in 5 replicates (randomly 5 reference samples) by 3 qualified analysts, who tested the Protein a concentration as described above and calculated the mean of the 15 data and their RSD. The results are shown in Table 5.

Table 5: protein A concentration and precision results

The results show that: the Protein A content of 5 samples is less than 1.6ppm, and the RSD is 3.03%, which shows that the method has good precision and the residual quantity of the Protein A meets the standard.

4.1.2 accuracy

anti-PD-1 monoclonal antibody drugs known to contain Protein A at concentrations of 0.32ppm, 0.61ppm, and 1.54ppm were used as test samples 4 to 6 and tested 1 time each by 3 analysts, and the results of the experiments were counted as shown in Table 6.

Table 6: protein A concentration and accuracy results

The result shows that the RSD of the 3-time detection result of each sample to be detected is less than 5 percent, which indicates that the method can meet the accuracy requirement.

4.2 detection of the amount of residual CHO Host Cell Protein (HCP)

The detection method comprises the following steps: sandwich enzyme-linked immunosorbent assay (ELISA), a HCP standard substance with the gradient concentration of 0.1, 1.0, 2.0, 5.0, 10.0 and 20.0ppm of 100 mul/hole and a sample to be detected are respectively added on a 96-well plate which is pre-coated with an anti-HCP capture antibody with the concentration of 1.5 mug/mL, and the plate is washed for 3 times by PBS solution; adding 100 mul/well of horseradish peroxidase-coupled goat anti-HCP polyclonal antibody with the concentration of 1.0 mug/mL, incubating for 2.5h at 37 ℃, and washing the plate for 3 times by using PBS solution; adding HRP coupled streptavidin with the concentration of 1.0 mu g/mL at a concentration of 100 mu l/hole, and washing the plate for 3 times by using PBS solution; adding 100 mul/hole TMB substrate solution, standing at room temperature for 30min for color development; the reaction was stopped with 100. mu.l/well of stop solution, the absorbance was read on a microplate reader at 450/650nm, a standard curve was drawn by SoftMax Pro computer software using a four parameter fitting regression model (weight factor Y), and the concentration of HCP in the sample was calculated from the standard curve.

4.2.1 precision

5 aliquots (randomly 5 aliquots of the reference sample) were tested in parallel by 3 qualified analysts, each of whom was tested for HCP concentration as described above and the mean of the 15 data and their RSD calculated. The results are shown in Table 7.

Table 7: HCP concentration and precision results

The results show that: the HCP content of 5 samples was 0.94ppm and the RSD was 1.26%, indicating that the precision of the method was good and the HCP residual level met the standard.

4.2.2 accuracy

anti-PD-1 monoclonal antibody drugs known to contain HCPs at concentrations of 1ppm, 2ppm, and 3ppm were used as test samples 7 to 9 and tested 1 time each by 3 analysts, and the results of the experiments were counted as shown in Table 8.

Table 8: HCP concentration and accuracy results

4.3 detection of DNA residue in CHO host cell

The detection method comprises the following steps: the method for detecting the DNA residue of the CHO host cell in the sample to be detected by utilizing the fluorescent quantitative PCR comprises the following steps:

s1, preparation of a DNA standard: extracting CHO cell genome DNA, EcoRI and BamHI double enzyme digestion by DNeasy Tissue Kit, purifying and recovering enzyme digestion fragments, detecting A260/A280, calculating the concentration and purity of the purified product, diluting the product with Tris-EDTA to 10 respectively⁰、10¹、10²、10³、10⁴And 10⁵A pg/mL standard;

s2.qPCR amplification and standard curve drawing: the reaction system comprises 0.5 mu M upstream and downstream primers 1.2 mu l each, 5U Taq DNA polymerase 0.5 mu l, dNTPs 2.0 mu l, DNA buffer 3 mu l, template DNA 0.5 mu l, ddH₂O16.6. mu.l; the reaction conditions are as follows:pre-denaturation at 95 ℃ for 40 s; 45 cycles of 95 ℃ for 8min, 54 ℃ for 1min and 72 ℃ for 1 min; drawing a standard curve by taking the Ct value as the ordinate and the logarithm value of the template DNA concentration as the abscissa, as shown in FIG. 14;

the upstream primer is as follows: 5'-CCAGGCATTGGTGGCAC-3', respectively;

the downstream primer is as follows: 5 '-AGACAGGGTTTCTCTGT-3';

4.3.1 specificity

The kit is used for extracting the genomic DNA of human umbilical vein vascular endothelial cells (HUVECs), human embryonic Kidney 293cells (HEK 293) and milk Hamster Kidney cells 21(Baby Hamster systemic Kidney kit, BHK21), the DNA of the extracted HUVECs, HEK293 and BHK21 cells is used as template DNA, and ddH is used for a blank control group₂O was detected by the qPCR method described above instead of the template DNA to evaluate the specificity of the method.

The detection result shows that the qPCR result taking the DNA of the HUVECs, the HEK293 cell and the BHK21 cell as the template is basically consistent with that of a blank control group, and no specific amplification curve exists, so that the method has good specificity and can be used for detecting the DNA residue of the CHO host cell.

4.3.2 precision

The DNA residue of CHO host cells was determined in 5 aliquots (randomly 5 aliquots of 5 reference samples, 5mL each) by 3 qualified analysts in parallel according to the above method, and the average of the 15 data and their RSD were calculated. The results are shown in Table 9.

Table 9: residual DNA and precision results of CHO host cells

The results show that: the residual amount of the DNA of the CHO host cell measured by 5 samples is 0.94ppm, and the RSD is 0.17 percent, which shows that the precision of the method is good, and the residual amount of the DNA of the CHO host cell meets the standard.

4.3.3 accuracy

The anti-PD-1 monoclonal antibody drugs known to contain CHO host cell DNA residues of 5pg/dose, 15pg/dose, and 20pg/dose were used as samples to be tested 10-12, which were tested 1 time each by 3 analysts, and the results of the experiments were counted as shown in Table 10.

Table 10: CHO host cell residual and accuracy results

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the realization of those skilled in the art; where combinations of features are mutually inconsistent or impractical, such combinations should not be considered as being absent and not within the scope of the claimed invention.

Claims

1. A quality standard detection method for an anti-PD-1 monoclonal antibody drug is characterized by comprising the following steps:

the sample to be detected is an anti-PD-1 monoclonal antibody drug.

2. The method of claim 1, wherein the isoelectric point measurement comprises the steps of:

mixing urea CIEF glue, 3-10 carrier ampholyte, 100mM anolyte, 200mM catholyte, isoelectric point markers and 10mg/mL samples to be detected to prepare a sample mixed solution; loading the sample mixed solution into a capillary, performing focusing separation at room temperature, injecting sample for 99s under 173kPa, wherein the focusing condition is that the focusing voltage is 25kV, and maintaining for 10min, and the separation condition is that 100mM ammonia water is adopted as migration liquid, the voltage is 25kV, and the maintaining for 30 min; detecting by using an ultraviolet absorption detector at the wavelength of 280nm, and recording an isoelectric focusing spectrum;

the 3-10 carrier ampholyte is Pharmalyte (3-10); the anolyte is H₃PO₄(ii) a The catholyte is NaOH; the isoelectric point markers comprise polypeptide markers pI5.85 and pI 8.18;

the peptide spectrogram analysis comprises the following steps:

taking a sample to be detected, diluting the sample to be detected to 1mg/mL by using a urea buffer solution, carrying out denaturation for 10min at 68 ℃, adding DTT, reducing for 45min at 36 ℃, wherein the volume ratio of DTT to the sample to be detected is 1: 50; adding IAA, and reacting for 30min in a dark place, wherein the volume ratio of the IAA to the sample to be detected is 1: 40; adding DTT to terminate the reaction, wherein the volume ratio of DTT to the sample to be detected is 1: 20; replacement of the prourosin buffer with NH₄HCO₃Adding Lys-C enzyme, and carrying out water bath at 37 ℃ for 10h, wherein the volume ratio of the Lys-C enzyme to a sample to be detected is 1: 20; terminating the reaction by 5% TFA to obtain a digestion product; and detecting and analyzing the obtained enzyme digestion product by using a reversed-phase high performance liquid chromatography, wherein the chromatographic conditions are as follows: the mobile phase A is 0.1 percent aqueous solution of formic acid; the mobile phase B is 0.1 percent formic acid acetonitrile solution; gradient elution (5-120min, 2% B → 85%)B) The flow rate is 0.5mL/min, and the detection wavelength is 214nm, so that a peptide spectrogram is obtained.

3. The method according to claim 2, wherein the 3-10 carrier ampholyte is Pharmalyte (3-10); the anolyte is H₃PO₄(ii) a The catholyte is NaOH; the isoelectric point markers comprise polypeptide markers pI5.85 and pI 8.18.

4. The method for detecting quality standards according to claim 1, wherein the monomer purity detection comprises the following steps:

diluting a sample to be detected to 10mg/mL by using a mobile phase, wherein the mobile phase is a mixed solution of 50mmol/L potassium phosphate and 300mmol/L sodium chloride, the pH value is 6.8, the loading amount is 10 mu L, the flow rate is 0.8mL/min, the detection is carried out at 280nm, and the monomer content is calculated by adopting a peak area normalization method;

the main peak purity detection is realized by using a non-reduction CE-SDS method, and comprises the following steps:

diluting a sample to be detected to 5.0mg/mL by using ultrapure water, taking 30 mu l of the solution, adding 5 mu l of mercaptoethanol and 70 mu l of SDS sample buffer solution, uniformly mixing, carrying out water bath at 70 ℃ for 10min, and carrying out sample loading detection; the sample introduction amount is 50 mul, the separation voltage is 15kV, the capillary temperature is 30 ℃, the detection wavelength is 220nm, and the peak area percentage of the main peak, namely the main peak purity, is calculated by an area normalization method;

the purity detection of the light chain and the heavy chain is realized by a reduction CE-SDS method, and the method comprises the following steps:

diluting a sample to be detected to 5.0mg/mL by using ultrapure water, taking 30 mu L of the solution, adding 5 mu L of 0.1mol/L N-ethylmaleimide and 95 mu L of SDS sample buffer solution, uniformly mixing, carrying out water bath at 70 ℃ for 10min, and carrying out loading detection; the sampling amount is 50 mul, the separation voltage is 15kV, the capillary temperature is 30 ℃, the detection wavelength is 220nm, and the relative area percentage of the heavy chain and the light chain, namely the purity of the light chain and the heavy chain, is calculated by an area normalization method;

the charge heteroplasmon content detection is realized by using a CEX-HPLC method, and comprises the following steps:

5. The method for detecting quality standards according to claim 1, wherein the detection of the residual quantity of Protein A comprises the following steps:

respectively adding a Protein A standard substance with gradient concentration of 0.1-2.0ppm and a sample to be detected into a 96-well plate which is coated with a chicken anti-Protein A polyclonal antibody in advance, and washing the plate; adding a chicken anti-Protein A polyclonal antibody marked by biotin, incubating for 2h at 40 ℃, and washing the plate; adding streptavidin coupled with horseradish peroxidase, and washing the plate; adding TMB solution, standing at room temperature for developing for 30min, stopping reaction with stop solution, reading light absorption value 450/650nm on a microplate reader, drawing a standard curve by using a four-parameter fitting regression model, and calculating the concentration of Protein A in the sample to be measured.

6. The method of claim 1, wherein the HCP residual assay comprises the steps of:

7. The method for detecting quality standards according to claim 1, wherein the detection of the residual amount of CHO host cell DNA comprises the following steps:

s1, preparation of a DNA standard: extracting CHO cell genome DNA, EcoRI and BamHI double enzyme digestion, purifying and recovering enzyme digestion fragments, detecting A260/A280, calculating the concentration and purity of the purified product, diluting the product to 10 by Tris-EDTA⁰、10¹、10²、10³、10⁴And 10⁵A pg/mL standard;

the upstream primer is as follows: 5'-CCAGGCATTGGTGGCAC-3', respectively;

the downstream primer is as follows: 5 '-AGACAGGGTTTCTCTGT-3';

8. Use of the quality standard detection method of any one of claims 1 to 7 for quality control of anti-PD-1 monoclonal antibody drugs.