WO2019079970A1 - 一种治疗性抗体的放行检测方法 - Google Patents

一种治疗性抗体的放行检测方法

Info

Publication number
WO2019079970A1
WO2019079970A1 PCT/CN2017/107472 CN2017107472W WO2019079970A1 WO 2019079970 A1 WO2019079970 A1 WO 2019079970A1 CN 2017107472 W CN2017107472 W CN 2017107472W WO 2019079970 A1 WO2019079970 A1 WO 2019079970A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
cell
buffer
plate
washing
Prior art date
Application number
PCT/CN2017/107472
Other languages
English (en)
French (fr)
Inventor
贾晓青
陈敏
黄岗
周伟昌
陈智胜
Original Assignee
上海药明生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海药明生物技术有限公司 filed Critical 上海药明生物技术有限公司
Priority to PCT/CN2017/107472 priority Critical patent/WO2019079970A1/zh
Priority to TW107137281A priority patent/TWI821205B/zh
Publication of WO2019079970A1 publication Critical patent/WO2019079970A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates generally to the field of life sciences, and in particular to the establishment of quality control and release standard methods in antibody production processes.
  • PD-1 programmed death 1
  • PD-1 programmed death 1
  • Immunomodulation targeting PD-1 has important implications for anti-tumor, anti-infective, anti-autoimmune diseases and organ transplant survival. Its ligand PD-L1 can also be used as a target, and the corresponding antibodies can also play the same role. Therefore, therapeutic antibodies can be used in the clinic to inhibit PD-1 or PD-L1 immunomodulation to inhibit disease.
  • a feature of the combination of PD-1 and PD-L1 is that they can be either rapidly bound or rapidly dissociated during elution, so that the combination of the two occurs in a "homogenous" state without elution. Can more accurately simulate the in vivo situation (biomimic).
  • Electrochemiluminescence is a specific chemiluminescence reaction initiated by electrochemistry on the surface of an electrode.
  • the new generation of labeled immunoassay technology combines the high sensitivity of luminescence analysis with the high specificity of antigen-antibody reaction, and therefore has a wide range of applications.
  • the invention establishes the release method and the release standard in the antibody production process by using the electrochemiluminescence technology, and the method is different from the conventional FACS-based cell binding assay, the steps are more concise, the data is more stable and accurate, and the therapeutic antibody production process is favorable. Quality control in the middle.
  • a method for antibody release uses electrochemiluminescence to detect the cell binding activity of a test antibody; and four parameters are determined by the antibody concentration and the electrochemiluminescence intensity of the test article. Fitting, calculating biological activity, and obtaining release criteria. This method confirms the biological activity release assay that can be used for antibodies by methodology.
  • the antibody comprises an antibody drug, preferably a therapeutic antibody drug.
  • test antibody as described above includes an anti-PD-1 antibody or an anti-PD-L1 antibody, preferably including an anti-PD-1 therapeutic antibody or an anti-PD-L1 therapeutic antibody.
  • the step of releasing the anti-PD-1 antibody comprises:
  • PD-1 antibody is preferably a PD-1 therapeutic antibody
  • the reading buffer is preferably an MSD read buffer
  • the step of releasing the anti-PD-L1 antibody comprises:
  • PD-L1 antibody is preferably a PD-L1 therapeutic antibody
  • the reading buffer is preferably an MSD read buffer
  • an electrochemiluminescence detection of PD-1 antibody activity is also provided.
  • the method of the method, the steps of the method comprising:
  • PD-1 antibody is preferably a PD-1 therapeutic antibody
  • the reading buffer is preferably MSD read buffer
  • a method of detecting the activity of a PD-L1 antibody by an electrochemiluminescence technique comprising the steps of:
  • PD-L1 antibody is preferably a PD-L1 therapeutic antibody.
  • the reading buffer is preferably MSD read buffer
  • the step of releasing the PD-1 antibody or the step of the activity detecting method or the step of releasing the PD-L1 antibody or the step of detecting the activity may further comprise:
  • step a before step a), add a blocking solution to the cell plate and incubate for about 1 to 3 hours.
  • the cell plates were then washed with wash buffer.
  • x represents an independent variable
  • A represents the left platform
  • B represents the curvature parameter
  • C represents the half-inhibition amount
  • the unit is ng/mL
  • D represents the right platform.
  • test release standard is one or more of the following:
  • the biological activity of the control is between 75% and 125%.
  • the blocking solution is a buffer containing 1% to 5% by volume of bovine serum albumin or 10% to 30% fetal bovine serum, preferably 15% to 30%. % fetal bovine serum buffer or buffer containing 1% to 3% bovine serum albumin in a volume.
  • the wash buffer is a PBS buffer.
  • the cell plate is an MSD 96-well plate, more preferably a MSD high-binding 96-well plate.
  • the cell is a CHO cell that expresses PD-1 or PDL1.
  • the cell density in the cell plate is from 2.5E4/well to 20E4/well, preferably from 3.5E4/well to 7.5E4/well, more preferably from 3.5E4/well to 5E4/well.
  • the concentration of the PD-1 recombinant protein or PD-L1 recombinant protein is from 50 ng/mL to 200 ng/mL, preferably from 80 ng/mL to 160 ng/mL.
  • the detection secondary antibody has a concentration of from 500 ng/mL to 1000 ng/mL, preferably 1000 ng/mL.
  • the incubation time in step b) is from 60 minutes to 90 minutes, preferably 90 minutes.
  • the recombinant protein is a protein fused to a tag, wherein the tag is preferably a murine Fc.
  • the detection antibody is a sputum-conjugated anti-mouse IgG.
  • Figure 1 shows the specificity of the biological activity release method for anti-PD-1 therapeutic antibodies.
  • the abscissa is the concentration of the product and the reference, the ordinate is the ECL signal value, the blue circle is labeled as the reference product, the red box is labeled as the control, the orange diamond is labeled as the reference Rituximab, and the green triangle is labeled as the reference. Xolair.
  • Figure 2 shows a linear fit of the biological activity release method of anti-PD-1 therapeutic antibodies.
  • Figure 3 shows a histogram between cell plating concentration and ECL signal values.
  • Figure 4 shows a dose dependent histogram between PD-L1 concentration and ECL signal values.
  • Figure 5 is a graph showing the results of activity of stress samples under different conditions.
  • test article refers to a test sample to be identified that is to be determined.
  • the test antibody comprises a biologically active antibody sample to be detected which is produced in industrial production, and specifically includes a PD-1 antibody or a PD-L1 antibody.
  • antibody as used herein includes whole antibodies and any antigen-binding fragments (ie, “antigen-binding portions") or single chains thereof.
  • An “antibody” comprises at least two heavy (H) chains and two proteins linked to each other by a disulfide bond, a light chain (L) chain or an antigen binding portion thereof.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain CL.
  • VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed in more conserved regions called framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four FRs arranged in sequence from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.
  • antibody refers to an immunoglobulin or a fragment or derivative thereof, and includes any polypeptide comprising an antigen binding site, whether or not it is produced in vitro or in vivo.
  • antibody includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies.
  • antibody also includes antibody fragments that retain antigen binding function, such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments, such as those that specifically bind PD-1 or PD-L1. ability. Typically, such fragments will contain antigen-binding fragments.
  • therapeutic antibody As used herein, the terms “therapeutic antibody”, “therapeutic antibody” are used interchangeably and include, but are not limited to, antibodies that have been used in clinical or clinical studies, as well as potential antibodies of clinical utility that are being developed, The antibody can be administered to a subject to treat a disease; the term “therapeutic antibody” or “therapeutic antibody” also includes a detection antibody that can be used for in vitro diagnostics, in vitro studies, in vitro development, and the antibody can be used for Cytological levels or immunodetection.
  • programmed death 1 As used herein, the terms “programmed death 1”, “programmed cell death 1”, “protein PD-1”, “PD-1”, “PD1”, “PDCD1”, “hPD-1” and “ hPD-F” is used interchangeably and includes variants, isoforms, species homologs of human PD-1, and analogs having at least one common epitope of PD-1.
  • programmed death ligand 1 As used herein, the terms “programmed death ligand 1", “PD-ligand 1”, “PD-L1”, “PD L1”, “B7 homolog 1”, “B7-H1”, “ CD274" is used interchangeably and includes variants, isoforms, species homologs of human PD-L1, and analogs having at least one common epitope of PD-L1.
  • CV refers to a coefficient of variation that is an absolute value that reflects the degree of data dispersion.
  • the size of the data is not only affected by the degree of dispersion of the variable values, but also by the average level of the variable values.
  • the ratio of the standard deviation to the mean is called the coefficient of variation.
  • the coefficient of variation eliminates the effect of different units and/or mean numbers on the degree of variability in two or more data.
  • recombinant protein refers to the use of genetic recombination techniques to obtain a recombinant vector linked to a gene fragment that can be translated into a protein of interest, which is then transferred to a host cell that expresses the protein of interest to express a particular recombination. Protein molecule.
  • reading buffer refers to a buffer for readings, including but not limited to MSD read buffer.
  • release As used herein, the terms “release”, “real-time release”, “release detection”, “implementation release detection,” “RTR”, and “RTRT” are used interchangeably to refer to the evaluation and assurance of intermediates based on process data. / or the ability of the finished product to achieve acceptable quality; it typically combines the measured material properties and process control data to assess and assure the ability of the intermediate product or final product quality.
  • the reference, control, and test article were diluted by gradient or targeted dilutions into 10 or 11 concentration points as 10 or 11 points on the standard curve.
  • the quality control product is the same as the reference product, but is diluted separately from the reference product.
  • Continuously cultured cells or newly resuscitated cryopreserved cells are suitable for this method.
  • Cryopreserved cells PD-1/CHO-S cells were taken out from a liquid nitrogen tank and quickly thawed in a 37 ° C water bath. The cell suspension in the cryotube was added to a CHO medium containing about N x 9 mL (N means cell count), and gently mixed by pipetting. Discard the supernatant by centrifugation.
  • Blocking 150 ⁇ L of blocking solution (10% FBS/PBS or 3% BSA/PBS) was added to the MSD high binding 96 well. The MSD plate was placed on a shaker at 150-220 rpm and incubated for 2 ⁇ 1 hour at room temperature (this step can be omitted if no closure is required).
  • Example 2 Prepare the reference product, the quality control product, and the test solution according to the method described in Example 1.
  • the PD-L1-mFc stock solution was diluted to a final concentration of 800 ng/mL and vortexed to mix.
  • the secondary antibody (MSD SULFO-TAG Goat Anti-mouse Antibody) storage solution was diluted to a final concentration of 2.857 ⁇ g/mL, and vortexed and mixed. See Example 2 for preparation of the PD-1/CHO-S cell line.
  • PD-1/CHO-S cell plating 30 ⁇ L/well was added to the MSD high binding plate. A mixture of the reference product and the test antibody, PD-L1-mFc and the detection antibody was added to the MSD high-binding plate containing PD-1/CHO-S cells at 70 ⁇ L/well, and the total volume per well was 100 ⁇ L.
  • the relative activity of the control substance and the sample is calculated as follows:
  • the C value, ie the IC 50 value, is calculated according to the four parameters fitting of the reference product, the quality control product and the test sample, and the biological activity of the test product is calculated by the following formula.
  • the experiment is effective only if the quality control meets all of the following criteria.
  • double double holes all include a double hole CV ⁇ 30% of the linear segment
  • the biological activity of the control is between 75% and 125%;
  • the slope ratio of the curve is between 0.7 and 1.4; the high platform ratio is between 0.8 and 1.25.
  • the CV of all linear segments of the test sample is ⁇ 30%.
  • the irrelevant Isotype-matching antibody did not inhibit the binding of PD-L1 to the PD-1 expressing CHO cell line, and the control sample had no specific biological activity (Fig. 1). This method has good specificity for investigating the biological activity of anti-PD-1 products.
  • the average recoveries for the 50%, 71%, 100%, 141%, and 200% target concentration samples ranged from 80% to 120% (Table 1).
  • test results showed good linearity in the range of 50% to 200% target concentration, and the accuracy and precision of the results met the acceptable standards. Therefore, the test has an effective range of 50% to 200% target concentration range.
  • the parameters of PD-L1 concentration, test incubation time, cell density, detection antibody concentration and cell generation were within the study range, and the QC recovery rate was 80% ⁇ recovery ⁇ 120%.
  • the test is carried out by at least two experimental personnel.
  • the recovery rate of the quality control products is between 80% and 120%, which meets the requirements of release, which proves that the release method meets the long-term requirements. Inspection of quality control.
  • the experimental results demonstrate that the method is applicable to the biological activity methods of various stages of the laboratory against PD1 antibodies, including drug stocks, finished products. , biological samples for the stability of samples and intermediates.
  • Cell type CHO cells expressing PD-1 or PD-L1, respectively, and exploring cell density range: 6.25E3/well to 20E4/well. Results See Figure 3. Cells from 2.5E4/well to 20E4/well were suitable for the experiment.
  • Exploration range 60 to 90 minutes, incubation time is suitable for the experiment.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本发明公开了一种可用于抗体类药物放行的基于电化学发光技术的生物学活性方法，其特征在于，该方法利用电化学发光法能检测供试品抗体的生物学活性（细胞结合方法），通过对所述供试品抗体浓度和电化学发光强度进行四参数拟合，计算生物学活性。该方法通过方法学确认，能用于其中抗体类药物的放行检测。抗体类药物和供试品抗体包括抗PD-1抗体或抗PD-L1抗体。

Description

一种治疗性抗体的放行检测方法 技术领域
本发明总体上涉及生命科学领域，具体涉及抗体生产工艺中质量控制和放行标准方法的建立。
背景技术
PD-1(programmed death 1)程序性死亡受体1，是一种重要的免疫抑制分子。以PD-1为靶点的免疫调节对抗肿瘤、抗感染、抗自身免疫性疾病及器官移植存活等均有重要的意义。其配体PD-L1也可作为靶点，相应的抗体也可以起到相同的作用。因此临床中可采用治疗性抗体抑制PD-1或PD-L1免疫调节从而抑制疾病。
PD-1和PD-L1的结合的一个特点是它们既可以快速结合也可以在洗脱时快速解离，所以使两者的结合发生在没有洗脱的“均质化(homogenous)”状态方能更准确模拟体内情形(biomimic)。以前的方法，比如流式细胞检测(FACS)或表面等离子体共振(SPR)的细胞结合测定都不是均质化实验，不能满足这样的要求。并且由于这些方法的灵敏度和特异性不足，并不适用于治疗性抗体生产工艺中制定治疗性抗体放行标准，因此亟需进一步优化检测方法以期获得高灵敏度读数的检测方法。
电化学发光(ECL)是一种在电极表面由电化学引发的特异性化学发光反应。新一代的标记免疫测定技术，既兼有发光分析的高灵敏度和抗原抗体反应的高度特异性，因此，具有广泛的应用价值。本发明利用电化学发光技术建立了抗体生产工艺中放行方法及其放行标准，该方法不同于常规的基于FACS的细胞结合测定，其步骤更简洁、数据更稳定准确，有利于治疗性抗体生产工艺中的质量控制。
发明内容
本发明的一个方面，提供了一种抗体放行的方法，所述方法利用电化学发光法检测供试品抗体的细胞结合活性；通过对所述供试品抗体浓度和电化学发光强度进行四参数拟合，计算生物学活性，获得放行标准。该方法通过方法学确认能用于抗体的生物学活性放行检测。
在本发明的一个实施例中，所述抗体包括抗体类药物，优选包括治疗性抗体药物。
如前所述的供试品抗体包括抗PD-1抗体或抗PD-L1抗体，优选包括抗PD-1治疗性抗体或抗PD-L1治疗性抗体。
在本发明的一个实施例中，所述抗PD-1抗体的放行方法的步骤包括：
a)将细胞表面表达PD-1的细胞铺于细胞板中，之后不需要洗涤；
b)加入PD-L1重组蛋白、检测抗体、和所述PD-1抗体，孵育，之后不需要洗涤；其中所述PD-1抗体优选为PD-1治疗性抗体；
c)加入无表面活性剂的读数缓冲液；所述读数缓冲液优选为MSD read缓冲液；
d)使用设备读板，得到电化学发光强度。
在本发明的一个实施例中，所述抗PD-L1抗体的放行方法的步骤包括：
a)将细胞表面表达PD-L1的细胞铺于细胞板中，之后不需要洗涤；
b)加入PD-1重组蛋白、检测抗体、和所述抗PD-L1抗体，孵育，之后不需要洗涤；其中所述PD-L1抗体优选为PD-L1治疗性抗体；
c)加入无表面活性剂的读数缓冲液；所述读数缓冲液优选为MSD read缓冲液；
d)使用设备读板，得到电化学发光强度。
在本发明的另一方面，还提供了一种电化学发光检测PD-1抗体活 性的方法，所述方法的步骤包括：
a)将细胞表面表达PD-1的细胞铺于细胞板中，之后不用缓冲液洗涤；
b)加入PD-L1重组蛋白、检测抗体、和所述PD-1抗体，孵育，之后不用缓冲液洗涤；其中所述PD-1抗体优选为PD-1治疗性抗体；
c)加入无表面活性剂的读数缓冲液，之后不用缓冲液洗涤；所述读数缓冲液优先为MSD read缓冲液；
d)使用设备读板，得到电化学发光强度。
在本发明的另一方面，还提供了一种电化学发光技术检测PD-L1抗体活性的方法，所述方法的步骤包括：
a)将细胞表面表达PD-L1的细胞铺于细胞板中，之后不用缓冲液洗涤；
b)加入PD-1重组蛋白、检测抗体、和所述PD-L1治疗性抗体，孵育，之后不用缓冲液洗涤；其中所述PD-L1抗体优选为PD-L1治疗性抗体。
c)加入无表面活性剂的读数缓冲液，之后不用缓冲液洗涤；所述读数缓冲液优先为MSD read缓冲液；
d)使用设备读板，得到电化学发光强度。
在本发明的一个实施例中，前述PD-1抗体的放行方法或活性检测方法的步骤或PD-L1抗体的放行方法或活性检测的步骤或还可以包括：
a')在步骤a)之前，在细胞板中加入封闭液，孵育约1至3小时，之
后用洗涤缓冲液洗涤细胞板。
如前所述任一方法，其中，对所述供试品抗体浓度和电化学发光强度利用公式(1)进行四参数拟合，根据参考品、质控品及供试品四参数拟合计算得到C值，即IC50值，利用公式(2)计算得到供试品相对活性：
Figure PCTCN2017107472-appb-000001
Figure PCTCN2017107472-appb-000002
其中，x表示自变量，A表示左平台，B表示曲率参数，C表示半数抑制量，单位为ng/mL，D表示右平台。
在本发明的一个实施例中，供试品放行可接受标准为以下中的一种或以上：
1)A参数/D参数≥2；
2)平行性分析：曲线的斜率比值在0.7至1.4之间；高平台比值在0.8至1.25之间；
4)所有线形段复孔的CV≤30％；
5)质控品的生物学活性在75％至125％之间
6)四参数拟合S型曲线的R2≥0.95。
在本发明的一个实施例中，所述封闭液为含体积浓度为1％至5％的牛血清白蛋白的缓冲液或10％至30％胎牛血清的缓冲液，优选为15％至30％胎牛血清的缓冲液或含体积浓度为1％至3％牛血清白蛋白的缓冲液。
在本发明的一个实施例中，所述洗涤缓冲液为PBS缓冲液。
在本发明的一个实施例中，所述细胞板为MSD 96孔板，更优选为MSD高结合的96孔板。
在本发明的一个实施例中，所述细胞为表达PD-1或PDL1的CHO细胞。
在本发明的一个实施例中，细胞板中的细胞密度为2.5E4/孔至20E4/孔，优选为3.5E4/孔至7.5E4/孔，更优选为3.5E4/孔至5E4/孔。
在本发明的一个实施例中，所述PD-1重组蛋白或PD-L1重组蛋白的浓度为50ng/mL至200ng/mL，优选为80ng/mL至160ng/mL。
在本发明的一个实施例中，所述检测二抗的浓度为500ng/mL至1000ng/mL，优选为1000ng/mL。
在本发明的一个实施例中，所述步骤b)中的孵育时间为60分钟至90分钟，优选为90分钟。
在本发明的一个实施例中，所述重组蛋白为融合了标签的蛋白质，其中所述标签优选为鼠源Fc。
在本发明的一个实施例中，所述检测抗体为钌结合的抗小鼠IgG。
附图说明
图1示出抗PD-1治疗性抗体的生物学活性放行方法的专属性。其中横坐标为产品和对照品的浓度，纵坐标为ECL信号值，蓝色圆圈标记为参考品，红色方框标记为质控品，橘色菱形标记为对照品Rituximab，绿色三角形标记为对照品Xolair。
图2示出抗PD-1治疗性抗体的生物学活性放行方法的线性拟合图。
图3示出细胞铺板浓度与ECL信号值之间的柱状图。
图4示出PD-L1浓度与ECL信号值之间剂量依赖柱状图。
图5示出不同条件处理下的应激样品的活性结果图。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
在详细描述本发明之前，应当理解，本发明不限于特定的生物***或细胞类型。还应当理解，本文使用的术语仅用于描述特定实施例的目的，而不是限制性的。如本说明书和所附权利要求中所使用的，单数形式“一个”，“一”和“该”除非另有明确规定，包括复数指示物。因此，例如，提及“细胞”包括两个或多个细胞或细胞的整个培养物的组合；对于“多核苷酸”的提及实际上包括该多核苷酸的许多拷贝。除非在本说明书的提醒中另有定义，本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同的含义。
在本文中所用的术语“供试品”是指待鉴别、待测定的供测试样品。在本发明中，供试品抗体包括在工业生产中被生产出的待检测生物学活性的抗体样本，具体包括PD-1抗体或PD-L1抗体。
本文所用的术语“抗体”包括全抗体和任何抗原结合片段(即“抗原结合部分”)或其单链。“抗体”包含至少两个重(H)链和两条通过二硫键相互连接的轻链(L)链或其抗原结合部分的蛋白质。每个重链由重链可变区(本文缩写为VH)和重链恒定区组成。重链恒定区由三个结构域CH1，CH2和CH3组成。每个轻链由轻链可变区(本文缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区域可以进一步细分为高变区域，称为互补决定区(CDR)，散布于更保守的区域，称为框架区(FR)。每个VH和VL由以下顺序从氨基末端到羧基末端排列的三个CDR和四个FR组成：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。其中术语“抗体”是指免疫球蛋白或其片段或衍生物，并且包括包含抗原结合位点的任何多肽，不管其是否在体外或体内产生。该术语包括但不限于多克隆、单克隆、单特异性、多特异性、非特异性、人源化、单链、嵌合、合成、重组、杂交、突变和接枝抗体。术语“抗体”还包括保留抗原结合功能的抗体片段，例如Fab、F(ab')2、Fv、scFv、Fd、dAb和其他抗体片段，例如特异性结合PD-1或PD-L1的 能力。通常，这样的片段将包含抗原结合片段。
如本文中所使用，术语“治疗类抗体”、“治疗性抗体”可互换使用，包括但不限于已经用于临床或临床研究的抗体，还包括正在研发的具有临床使用价值的潜在抗体，所述抗体可被施用于受试者，治疗某种疾病；术语“治疗类抗体”或“治疗性抗体”还包括可用于体外诊断、体外研究、体外研发的检测性抗体，所述抗体可用于细胞学水平或免疫检测。
如本文中所使用，术语“程序性死亡1”、“程序性细胞死亡1”、“蛋白PD-1”、“PD-1”、“PD1”、“PDCD1”、“hPD-1”和“hPD-F”可互换使用，并且包括变体、同种型、人PD-1的物种同源物和具有PD-1的至少一个共同表位的类似物。
如本文中所使用，术语“程序性死亡配体1”、“PD-配体1”、“PD-L1”、“PD L1”、“B7同源物1”、“B7-H1”、“CD274”可互换使用，包括变体、同种型、人PD-L1的物种同源物和具有PD-L1的至少一个共同表位的类似物。
如本文中所使用，术语“CV”是指变异系数，是反映数据离散程度的绝对值。其数据大小不仅受变量值离散程度的影响，而且还受变量值平均水平大小的影响。标准差与平均数的比值称为变异系数。变异系数可以消除单位和(或)平均数不同对两个或多个资料变异程度比较的影响。变异系数的计算公式为：变异系数CV＝(标准偏差SD/平均值Mean)×100％。
如本文中所使用，术语“重组蛋白”是指应用基因重组技术，获得连接有可以翻译成目的蛋白的基因片段的重组载体，之后将其转入可以表达目的蛋白的宿主细胞从而表达特定的重组蛋白分子。
如本文中所使用，术语“读数缓冲液”是指用于读数的缓冲液，包括但不限于MSD read缓冲液。
如本文中所使用，术语“放行”、“实时放行”、“放行检测”、“实施放行检测、“RTR”、“RTRT”可互换使用，是指根据工艺数据，评估和保证中间品和/或成品达到可接受质量的能力；其通常将被测量物料属性和工艺控制等的数据进行有效结合，据此评估和保证中间产品或最终成品质量的能力。
实施例
通过以下实施例进一步说明本发明。提供实施例仅用于说明目的，并且不应被解释为以任何方式限制本发明的范围或内容。
实施例1：参考品、质控品和供试品制备(关于抗PD-1抗体)
根据蛋白标示量，用分析培养基将参考品、质控品和供试品通过梯度稀释或者靶向稀释成10个或11个浓度点，作为标准曲线上的10个或11个点。其中质控品与参考品是同一种，但是与参考品独立稀释。
实施例2：PD-1/CHO-S细胞株的准备
连续培养的细胞或新复苏的冻存细胞都适用于该方法。
1、连续培养的细胞：将细胞离心后弃上清。
2、冻存细胞：从液氮罐中拿出PD-1/CHO-S细胞，并快速放入37℃水浴锅中解冻。将冻存管中的细胞悬液加入到含约N×9mL(N表示细胞支数)CHO培养基中，轻柔地吹打混匀。离心弃去上清。
3、用冷的DPBS将细胞洗涤一次。离心后弃上清。根据所需要的细胞悬液体积，加入分析培养基重悬细胞。取细胞悬液进行细胞计数，确定细胞密度及细胞活率。用分析培养基调整细胞密度至16.7E5/mL，将调整浓度的细胞悬液混匀。
实施例3：测试方法
1、封闭：将150μL封闭液(10％FBS/PBS或3％BSA/PBS)加入到MSD高结合96孔中。MSD板置于振荡器上以150～220转/分钟的转速振荡，室温孵育2±1小时(如果不需要封闭，此步可以省略)。
2、按照实施例1中所述的方法制备参考品、质控品及供试品溶液。将PD-L1-mFc存储液稀释至终浓度为800ng/mL，涡旋混匀。将检测二抗(MSD SULFO-TAG Goat Anti-mouse Antibody)存储液稀释至终浓度为2.857μg/mL，涡旋混匀。PD-1/CHO-S细胞株的准备参见实施例2。
3、将稀释好的参考品及供试品，稀释好的800ng/mL PD-L1-mFc和稀释好的2.857μg/mL检测抗体以1:1:2(v:v:v)的比例(例如45μL:45μL:90μL)转移至U形底96孔板中(转移板)的第二列至第十二列中，混匀。在第一列中加入以1:1(v:v)混合(例如90μL:90μL)的分析培养基和检测抗体，作为背景。2至8℃冰箱避光保存。
4、用DPBS或1×PBS将MSD高结合96孔板洗3遍(如果不需要封闭MSD板，则此步省略)。
5、PD-1/CHO-S细胞铺板：以30μL/孔加入到MSD高结合板中。将参考品及供试品抗体、PD-L1-mFc和检测抗体的混合物以70μL/孔加入到含有PD-1/CHO-S细胞的MSD高结合板中，每孔的总体积为100μL。
6、将MSD板置于室温或22～28℃恒温培养箱中避光孵育60至90分钟。
7、向MSD板的每孔中加入100μL的2×不含表面活性剂的Read Buffer。
8、用MSD MESOTM SECTORS 600或同等设备读板。
9、数据分析：
虽然每次实验同时操作多块板，但是每块板独立计算。
用四参数对抗体浓度和电化学发光强度进行拟合
Figure PCTCN2017107472-appb-000003
其中，x＝自变量
A＝左平台
B＝曲率参数
C＝半数抑制量(ng/mL)
D＝右平台
平行性分析：根据参考品及供试品四参数拟合计算得到B值，即四参数拟合曲线的斜率，利用下列公式计算得到供试品或质控品的曲线斜率的比值(Slope Ratio)：
Figure PCTCN2017107472-appb-000004
质控品与样品的相对活性计算如下：根据参考品、质控品及供试品四参数拟合计算得到C值，即IC50值，利用下列公式计算得到供试品生物学活性。
Figure PCTCN2017107472-appb-000005
10、在一个具体实施例中，***适用性
只有当质控品满足以下所有标准，实验才有效，
a)A参数/D参数≥2；
b)平行性分析：曲线的斜率比在0.7至1.4之间；高平台比 在0.8至1.25之间；
c)双复孔都包括在线形段的复孔CV≤30％；
d)质控品的生物学活性在75％至125％之间；
e)四参数拟合S型曲线的R2≥0.95；
如果质控品的结果不满足以上的标准，实验被认为是无效的。
参考品可接受标准
A参数/D参数≥2
所有线性段复孔CV≤30％.
四参数拟合S型曲线的R2≥0.95
供试品可接受标准
A参数/D参数≥2
平行性：曲线的斜率比在0.7～1.4之间；高平台比在0.8至1.25之间
供试品的所有线性段复孔的CV≤30％.
四参数拟合S型曲线的R2≥0.95
实施例4：方法确认
1、专属性
不相关的Isotype匹配抗体不能抑制PD-L1与表达PD-1的CHO细胞株的结合，对照样品无特异性的生物学活性(图1)。该方法对考察抗PD-1产品的生物学活性具有良好的特异性。
2、准确度
50％、71％、100％、141％以及200％目标浓度样品的平均回收率介于80％至120％之间(表1)。
3、精密度
50％、71％、100％、141％以及200％目标浓度样品，分别独立8次；
中间精密度检测结果每个样品RSD均小于25％(表1)。
表1.准确度和精确度结果列表
Figure PCTCN2017107472-appb-000006
4、线性
使用回收率研究的数据，预期相对活性与实测相对活性线性拟合情况较好，Pearson相关系数≥0.95(图2)。
5、试验有效范围
试验结果在50％至200％目标浓度范围内回收率显示了良好的线性，而且结果的准确性与精密度都符合可接受标准。因此，该试验有效范围为50％至200％目标浓度范围。
6、耐用性
PD-L1浓度、试验孵育时间、细胞密度、检测抗体浓度和细胞代次等参数在研究范围内，QC回收率均满足80％≤回收率≤120％
7、应激样品
热处理、酸处理(大约pH 3)和碱处理(大约pH 11)的应激样品的活性相对于未处理的对照明显下降(图5)。
8、长期的质量控制
在至少6个月的时间内，由至少两名实验人员进行检测，在此过程中，质控品的回收率均在80％至120％之间，符合放行的要求，证明本放行方法满足长期质量控制的考察。
9、结论
实验结果，包括专属性、准确度、精密度、试验有效范围以及线性、耐用性和应激样品分析，证明该方法适用于抗PD1抗体的实验室各阶段的生物活性方法，包括药物原液，成品，稳定性样品及中间体的生物学活性检测。
实施例5：方法参数的优化
1、细胞密度
细胞类型：分别表达PD-1或PD-L1的CHO细胞，探索细胞密度范围：6.25E3/孔～20E4/孔。结果参见图3，2.5E4/孔～20E4/孔的细胞均适用于实验。
2、PD-L1/PD-1浓度
探索浓度范围：4至2000ng/mL。结果参见图4，100±50ng/mL的PD-L1均适用于实验。
3、其他参数
3.1、检测二抗浓度
探索浓度范围：100ng/mL至2000ng/mL。750±250ng/mL的检测二抗浓度均适用于实验。
3.2、试验孵育时间
探索范围：60至90分钟，孵育时间均适用于实验。
3.3、封闭液
探索条件：1％BSA至3％BSA、15％至30％的FBS/培养基、无封闭。三者均适用于该实验。
3.4、分析缓冲液
探索条件：1％BSA、10％至30％FBS/培养基、10％至30％FBS/PBS。三者均适用于该实验。
3.5、冻存细胞和连续培养细胞的比较
结果：均适合于实验。
通过引用并入
本文引用的每个专利文献和科学文献的全部公开内容通过引用并入本文用于所有目的。
等效
本发明可以在不脱离其基本特征的情况下以其他具体形式实施。因此，前述实施例被认为是说明性的，而不是对本文所述的本发明的限制。本发明的范围由所附权利要求书而不是由前述说明书表示，并且意在将落入权利要求书的等同物的含义和范围内的所有改变包括在其中。

Claims (20)

  1. 一种用于抗体生物学活性放行方法，其特征在于，所述方法利用电化学发光法检测供试品抗体的细胞结合活性，通过对所述供试品抗体浓度和电化学发光强度进行四参数拟合，计算生物学活性，该方法通过方法学确认能用于抗体的生物学活性放行检测。
  2. 如权利要求1所述的方法，其特征在于，所述抗体包括抗体类药物。
  3. 如权利要求1或2所述的方法，其特征在于，所述供试品抗体包括抗PD-1抗体或抗PD-L1抗体。
  4. 如权利要求3所述的方法，其特征在于，所述抗PD-1抗体的生物学活性放行方法的步骤包括：
    a)将细胞表面表达PD-1的细胞铺于细胞板中，之后不需要洗涤；
    b)加入PD-L1重组蛋白、检测抗体、和所述抗PD-1抗体，孵育，之后不需要洗涤；
    c)加入无表面活性剂的读数缓冲液；
    d)使用设备读板，得到电化学发光强度。
  5. 如权利要求3所述的方法，其特征在于，所述抗PD-L1抗体的生物学活性放行方法的步骤包括：
    a)将细胞表面表达PD-L1的细胞铺于细胞板中，之后不需要洗涤；
    b)加入PD-1重组蛋白、检测抗体、和所述抗PD-L1抗体，孵育，之后不需要洗涤；
    c)加入无表面活性剂的读数缓冲液；
    d)使用设备读板，得到电化学发光强度。
  6. 一种利用电化学发光技术检测抗PD-1抗体的细胞活性的方法，其特征在于，所述方法的步骤包括：
    a)将细胞表面表达PD-1的细胞铺于细胞板中，之后不需要洗涤；
    b)加入PD-L1重组蛋白、检测抗体、和所述抗PD-1抗体，孵育，之后不需要用洗涤；
    c)加入无表面活性剂的读数缓冲液；
    d)使用设备读板，得到电化学发光强度。
  7. 一种利用电化学发光技术检测抗PD-L1抗体的细胞活性方法，其特征在于，所述方法的步骤包括：
    a)将细胞表面表达PD-L1的细胞铺于细胞板中，之后不需要液洗涤；
    b)加入PD-1重组蛋白、检测抗体、和所述PD-L1单克隆抗体，孵育，之后不需要洗涤；
    c)加入无表面活性剂的读数缓冲液；
    d)使用设备读板，得到电化学发光强度。
  8. 如权利要求4至7任一项所述的方法，其特征在于，所述步骤还可以包括：
    a')在步骤a)之前，在细胞板中加入封闭液，孵育约1至3小时，之后用缓冲液洗涤细胞板。
  9. 如权利要求1至7任一项所述的方法，其特征在于，
    对所述供试品抗体浓度和电化学发光强度利用公式(1)进行四参数拟合，根据参考品、质控品及供试品四参数拟合计算得到C值，即IC50值，利用公式(2)计算得到供试品相对活性：
    Figure PCTCN2017107472-appb-100001
    Figure PCTCN2017107472-appb-100002
    其中，x表示自变量，A表示左平台，B表示曲率参数，C表示半数抑制量，所述半数抑制量的单位为ng/mL，D表示右平台。
  10. 如权利要求9所述的方法，其特征在于，***适应性标准为以下标准中的一个以上：
    a)A参数/D参数≥2；
    b)平行性分析：曲线的斜率比在0.7至1.4之间；高平台比在0.8至1.25之间；
    c)所有线性段的复孔CV≤30％；
    d)所述质控品的生物学活性在75％至125％之间；
    e)四参数拟合S型曲线的R2≥0.95。
  11. 如权利要求8所述的方法，其特征在于，所述封闭液为含体积浓度为1％至5％的牛血清白蛋白的缓冲液或10％至30％胎牛血清的缓冲液，优选为15％至30％胎牛血清的缓冲液或含体积浓度为1％至3％牛血清白蛋白的缓冲液。
  12. 如权利要求11所述的方法，其特征在于，所述缓冲液为PBS缓冲液或基础培养基。
  13. 如权利要求4至7任一项所述的方法，其特征在于，所述细胞板为96孔板，优选为MSD高结合的96孔板。
  14. 如权利要求4至7任一项所述的方法，其特征在于，所述细胞为表达PD-1或PD-L1的CHO细胞。
  15. 如权利要求4至7任一项所述的方法，其特征在于，细胞板中的细胞密度为2.5E4/孔至20E4/孔，优选为3.5E4/孔至7.5E4/孔，更优选为3.5E4/孔至5E4/孔.
  16. 如权利要求4至7任一项所述的方法，其特征在于，所述PD-1重组蛋白或PD-L1重组蛋白的浓度为50ng/mL至200ng/mL，优选为80ng/mL至160ng/mL。
  17. 如权利要求4至7任一项所述的方法，其特征在于，所述检测抗体的浓度为500ng/mL至1000ng/mL，优选为1000ng/mL。
  18. 如权利要求4至7任一项所述的方法，其特征在于，步骤b)中的孵育时间为60分钟至90分钟，优选为90分钟。
  19. 如权利要求4至7任一项所述的方法，其特征在于，所述重组蛋白为融合了标签的蛋白质，其中所述标签优选为鼠源Fc。
  20. 如权利要求4至7任一项所述的方法，其特征在于，所述检测抗体为钌结合的抗小鼠IgG。
PCT/CN2017/107472 2017-10-24 2017-10-24 一种治疗性抗体的放行检测方法 WO2019079970A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2017/107472 WO2019079970A1 (zh) 2017-10-24 2017-10-24 一种治疗性抗体的放行检测方法
TW107137281A TWI821205B (zh) 2017-10-24 2018-10-23 一種治療性抗體的放行檢測方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/107472 WO2019079970A1 (zh) 2017-10-24 2017-10-24 一种治疗性抗体的放行检测方法

Publications (1)

Publication Number Publication Date
WO2019079970A1 true WO2019079970A1 (zh) 2019-05-02

Family

ID=66247070

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/107472 WO2019079970A1 (zh) 2017-10-24 2017-10-24 一种治疗性抗体的放行检测方法

Country Status (2)

Country Link
TW (1) TWI821205B (zh)
WO (1) WO2019079970A1 (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0522677A1 (en) * 1991-07-10 1993-01-13 TDK Corporation Method of measuring concentration of immunoreactant using electrochemiluminescence
CN103777024A (zh) * 2007-05-08 2014-05-07 霍夫曼-拉罗奇有限公司 用于检测特异性免疫球蛋白g类抗体的方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9535074B2 (en) * 2014-09-08 2017-01-03 Merck Sharp & Dohme Corp. Immunoassay for soluble PD-L1

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0522677A1 (en) * 1991-07-10 1993-01-13 TDK Corporation Method of measuring concentration of immunoreactant using electrochemiluminescence
CN103777024A (zh) * 2007-05-08 2014-05-07 霍夫曼-拉罗奇有限公司 用于检测特异性免疫球蛋白g类抗体的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ABRAHAM, R. ET AL.: "Determination of Binding Constants of Diabodies Directed against Prostate-Specific Antigen Using Electrochemiluminescence-Based Immunoassays", JOURNAL OF MOLECULAR RECOGNITION, vol. 9, no. 5-6, 31 December 1996 (1996-12-31), pages 456 - 461, XP002381122, DOI: doi:10.1002/(SICI)1099-1352(199634/12)9:5/6<456::AID-JMR282>3.0.CO;2-8 *
DAI, YUE ET AL.: "Evaluation of the Methodology for Thyrotropin Receptor Antibody Measurement with Roche Electro Chemiluminescence System", LABELED IMMUNOASSAYS AND CLINICAL MEDICINE, vol. 22, 31 January 2015 (2015-01-31), pages 52 - 55 *

Also Published As

Publication number Publication date
TWI821205B (zh) 2023-11-11
TW201923348A (zh) 2019-06-16

Similar Documents

Publication Publication Date Title
JP2023071948A (ja) ヒトプログラム死リガンド１（ｐｄ－ｌ１）に結合する抗体
JP6336911B2 (ja) アドレノメジュリンアッセイおよび成熟アドレノメジュリンの測定方法
US6998241B2 (en) Antibody pair screening methods
JP2021517239A (ja) 神経変性を検出するためのアッセイ
JP2017535273A (ja) 免疫チェックポイントの調節因子を評価するためのシステム及び方法
TW201938584A (zh) 抗pla2-gib抗體及其用途
CN116396384B (zh) 针对小鼠脂联素的兔单克隆抗体及其应用和双抗夹心法酶联免疫检测试剂盒
JP6977105B2 (ja) Ｉｇｆ−１ｒ抗体および癌の診断のためのその使用
CN104067127A (zh) 用于***癌分析的组合物和方法
JP7010833B2 (ja) Ｉｌ－２１抗体及びその使用
WO2018227643A1 (zh) 一种用于脂肪性肝炎检测的靶标志物gp73及检测应用方法
CN109696461B (zh) 一种治疗性抗体的放行检测方法
EP2968460B1 (en) Protein formulations
US20210277149A1 (en) Anti-idiotype antibodies and methods of using the same
TW201927820A (zh) 抗ｐｄ-ｌ１抗體及使用其來偵測ｐｄ-ｌ１之方法
TWI821205B (zh) 一種治療性抗體的放行檢測方法
US11174310B2 (en) Disulfide-type HMGB1-specific antibody, method for measuring disulfide-type HMGB1 and kit for said measurement, and measurement method capable of quantitating all of HMGB1 molecules including reduced HMGB1, disulfide-type HMGB1 and thrombin-cleavable HMGB1 and kit for said measurement
WO2021030450A1 (en) Novel lox-1 antibody compositions, lox1 neutralization assay and methods of treatment using same
JP7086607B2 (ja) Ｉｇｆ－１ｒ抗体および癌の診断のためのその使用
US11913951B2 (en) High throughput method for measuring the protease activity of complement C3 convertase
WO2023068248A1 (ja) Ｉ型コラーゲン架橋ｎ－テロペプチドの免疫測定方法及び免疫測定キット、並びに抗体又はその抗体断片
TW201718656A (zh) 用於結合紫杉醇之以抗體為主的親和性試劑
CN116008555A (zh) 一种检测单克隆抗体对rankl结合力的方法及其应用
JP2024520552A (ja) 抗ｃｄ７９ｂ抗体に対する抗イディオタイプ抗体
KR20240067082A (ko) 포스포-타우 항체 및 사용 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17929962

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17929962

Country of ref document: EP

Kind code of ref document: A1