TWI821205B - A release detection method for therapeutic antibodies - Google Patents

A release detection method for therapeutic antibodies Download PDF

Info

Publication number
TWI821205B
TWI821205B TW107137281A TW107137281A TWI821205B TW I821205 B TWI821205 B TW I821205B TW 107137281 A TW107137281 A TW 107137281A TW 107137281 A TW107137281 A TW 107137281A TW I821205 B TWI821205 B TW I821205B
Authority
TW
Taiwan
Prior art keywords
antibody
cell
parameter
concentration
product
Prior art date
Application number
TW107137281A
Other languages
Chinese (zh)
Other versions
TW201923348A (en
Inventor
曉青 賈
陳敏
崗 黃
偉昌 周
陳智勝
Original Assignee
中國大陸商上海藥明生物技術有限公司
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 中國大陸商上海藥明生物技術有限公司 filed Critical 中國大陸商上海藥明生物技術有限公司
Publication of TW201923348A publication Critical patent/TW201923348A/en
Application granted granted Critical
Publication of TWI821205B publication Critical patent/TWI821205B/en

Links

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

Landscapes

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

Abstract

本發明揭示一種可用於抗體類藥物放行的基於電化學發光技術之生物學活性方法，其特徵在於，該方法利用電化學發光法能檢測供試品抗體之生物學活性(細胞結合方法)，藉由對該供試品抗體濃度及電化學發光強度進行四參數擬合，計算生物學活性。該方法藉由方法學確認，能用於其中抗體類藥物的放行檢測。抗體類藥物及供試品抗體包括抗PD-1抗體或抗PD-L1抗體。The present invention discloses a biological activity method based on electrochemiluminescence technology that can be used for the release of antibody drugs. It is characterized in that the method can use the electrochemiluminescence method to detect the biological activity of the test antibody (cell binding method). The biological activity was calculated by performing four-parameter fitting on the antibody concentration and electrochemical luminescence intensity of the test product. This method is methodologically confirmed and can be used for release testing of antibody drugs. Antibody drugs and test antibodies include anti-PD-1 antibodies or anti-PD-L1 antibodies.

Description

一種治療性抗體的放行檢測方法A release detection method for therapeutic antibodies

本發明大體上係關於生命科學領域，尤其係關於抗體生產製程中品質控制及放行標準方法的建立。The present invention relates generally to the field of life sciences, and in particular to the establishment of standard methods for quality control and release in antibody production processes.

PD-1 (programmed death 1)計劃性死亡受體1，為一種重要的免疫抑制分子。以PD-1為靶點之免疫調節對抗腫瘤、抗感染、抗自身免疫性疾病及器官移植存活等均有重要意義。其配位體PD-L1亦可作為靶點，相應抗體亦可起相同作用。因此臨床中可採用治療性抗體抑制PD-1或PD-L1免疫調節從而抑制疾病。PD-1 (programmed death 1) is an important immunosuppressive molecule. Immune regulation targeting PD-1 is of great significance in anti-tumor, anti-infection, anti-autoimmune diseases and organ transplant survival. Its ligand PD-L1 can also be used as a target, and the corresponding antibody can also play the same role. Therefore, therapeutic antibodies can be used in clinical practice to inhibit PD-1 or PD-L1 immune regulation to suppress the disease.

PD-1及PD-L1之結合的一個特點為其既可快速結合且亦可在溶離時快速解離，所以使兩者之結合發生在無溶離之「均質化(homogenous)」狀態方能更準確模擬體內情形(biomimic)。以前的方法，如流式細胞檢測(FACS)或表面電漿共振(SPR)之細胞結合測定均不為均質化實驗，不能滿足此類要求。且由於此等方法之靈敏度及特異性不足，並不適用於治療性抗體生產製程中制定治療性抗體放行標準，因此亟需進一步優化檢測方法以期獲得高靈敏度讀數之檢測方法。One of the characteristics of the combination of PD-1 and PD-L1 is that it can quickly combine and dissociate quickly during dissolution. Therefore, it is more accurate to make the combination of the two occur in a "homogenous" state without dissolution. Simulate in vivo conditions (biomimic). Previous methods, such as flow cytometry (FACS) or surface plasmon resonance (SPR) cell binding assays, are not homogenized experiments and cannot meet such requirements. Moreover, due to the insufficient sensitivity and specificity of these methods, they are not suitable for setting therapeutic antibody release standards in the therapeutic antibody production process. Therefore, there is an urgent need to further optimize the detection method in order to obtain a detection method with high sensitivity readings.

電化學發光(ECL)為一種在電極表面由電化學引發之特異性化學發光反應。新一代標記免疫測定技術，既兼有發光分析之高靈敏度及抗原抗體反應之高度特異性，因此，具有廣泛的應用價值。本發明利用電化學發光技術建立了抗體生產製程中放行方法及其放行標準，該方法不同於習知的基於FACS之細胞結合測定，其步驟更簡潔、資料更穩定準確，有利於治療性抗體生產製程中之品質控制。Electrochemiluminescence (ECL) is a specific chemiluminescence reaction induced by electrochemistry on the electrode surface. The new generation of labeled immunoassay technology combines the high sensitivity of luminescence analysis with the high specificity of antigen-antibody reactions. Therefore, it has wide application value. The present invention uses electrochemiluminescence technology to establish a release method and release standards in the antibody production process. This method is different from the conventional FACS-based cell binding assay. Its steps are simpler and the data is more stable and accurate, which is beneficial to the production of therapeutic antibodies. Quality control in the manufacturing process.

本發明之一個態樣提供一種抗體放行之方法，該方法利用電化學發光法檢測供試品抗體之細胞結合活性；藉由對該供試品抗體濃度及電化學發光強度進行四參數擬合，計算生物學活性，獲得放行標準。該方法藉由方法學確認能用於抗體之生物學活性放行檢測。One aspect of the present invention provides a method for releasing antibodies, which uses electrochemiluminescence to detect the cell-binding activity of a test antibody; by performing four-parameter fitting on the test antibody concentration and electrochemiluminescence intensity, Calculate biological activity and obtain release criteria. This method is methodologically confirmed to be suitable for biological activity release testing of antibodies.

在本發明之一個實施例中，該抗體包括抗體類藥物，較佳包括治療性抗體藥物。In one embodiment of the present invention, the antibody includes antibody drugs, preferably including therapeutic antibody drugs.

如前所述之供試品抗體包括抗PD-1抗體或抗PD-L1抗體，較佳包括抗PD-1治療性抗體或抗PD-L1治療性抗體。As mentioned above, the test product antibody includes an anti-PD-1 antibody or an anti-PD-L1 antibody, and preferably includes an anti-PD-1 therapeutic antibody or an anti-PD-L1 therapeutic antibody.

在本發明之一個實施例中，該抗PD-1抗體之放行方法的步驟包括： a) 將細胞表面表現PD-1之細胞鋪於細胞盤中，之後不需要洗滌； b) 加入PD-L1重組蛋白、檢測抗體、及該PD-1抗體，進行培育，之後不需要洗滌；其中該PD-1抗體較佳為PD-1治療性抗體； c) 加入無界面活性劑之讀數緩衝液；該讀數緩衝液較佳為MSD read緩衝液； d) 使用設備讀盤，得到電化學發光強度。In one embodiment of the present invention, the steps of the release method of the anti-PD-1 antibody include: a) Plate the cells expressing PD-1 on the cell surface in the cell plate, and do not need to wash them afterwards; b) Add PD-L1 recombinant protein, detection antibody, and the PD-1 antibody, and incubate without washing; the PD-1 antibody is preferably a PD-1 therapeutic antibody; c) Add surfactant-free reading buffer; the reading buffer is preferably MSD read buffer; d) Use the device to read the disk and obtain the electrochemical luminescence intensity.

在本發明之一個實施例中，該抗PD-L1抗體之放行方法的步驟包括： a) 將細胞表面表現PD-L1之細胞鋪於細胞盤中，之後不需要洗滌； b) 加入PD-1重組蛋白、檢測抗體、及該抗PD-L1抗體，進行培育，之後不需要洗滌；其中該PD-L1抗體較佳為PD-L1治療性抗體； c) 加入無界面活性劑之讀數緩衝液；該讀數緩衝液較佳為MSD read緩衝液； d) 使用設備讀盤，得到電化學發光強度。In one embodiment of the present invention, the steps of the release method of the anti-PD-L1 antibody include: a) Plate the cells expressing PD-L1 on the cell surface in the cell plate, and do not need to wash them afterwards; b) Add the PD-1 recombinant protein, detection antibody, and the anti-PD-L1 antibody, and incubate without washing; the PD-L1 antibody is preferably a PD-L1 therapeutic antibody; c) Add surfactant-free reading buffer; the reading buffer is preferably MSD read buffer; d) Use the device to read the disk and obtain the electrochemical luminescence intensity.

在本發明之另一態樣中，亦提供一種電化學發光檢測PD-1抗體活性之方法，該方法之步驟包括： a) 將細胞表面表現PD-1之細胞鋪於細胞盤中，之後不用緩衝液洗滌； b) 加入PD-L1重組蛋白、檢測抗體、及該PD-1抗體，進行培育，之後不用緩衝液洗滌；其中該PD-1抗體較佳為PD-1治療性抗體； c) 加入無界面活性劑之讀數緩衝液，之後不用緩衝液洗滌；該讀數緩衝液優先為MSD read緩衝液； d) 使用設備讀盤，得到電化學發光強度。In another aspect of the present invention, a method for electrochemiluminescence detection of PD-1 antibody activity is also provided. The steps of the method include: a) Plate the cells expressing PD-1 on the cell surface in the cell plate, and then wash them without buffer; b) Add the PD-L1 recombinant protein, detection antibody, and the PD-1 antibody, and incubate without washing with buffer; the PD-1 antibody is preferably a PD-1 therapeutic antibody; c) Add surfactant-free reading buffer, and then do not wash with buffer; the reading buffer is preferably MSD read buffer; d) Use the device to read the disk and obtain the electrochemical luminescence intensity.

在本發明之另一態樣中，亦提供一種電化學發光技術檢測PD-L1抗體活性之方法，該方法之步驟包括： a) 將細胞表面表現PD-L1之細胞鋪於細胞盤中，之後不用緩衝液洗滌； b) 加入PD-1重組蛋白、檢測抗體、及該PD-L1治療性抗體，進行培育，之後不用緩衝液洗滌；其中該PD-L1抗體較佳為PD-L1治療性抗體。 c) 加入無界面活性劑之讀數緩衝液，之後不用緩衝液洗滌；該讀數緩衝液優先為MSD read緩衝液； d) 使用設備讀盤，得到電化學發光強度。In another aspect of the present invention, a method for detecting PD-L1 antibody activity using electrochemiluminescence technology is also provided. The steps of the method include: a) Plate cells expressing PD-L1 on the cell surface in a cell plate, and then wash without buffer; b) Add the PD-1 recombinant protein, detection antibody, and the PD-L1 therapeutic antibody, and incubate without washing with buffer; the PD-L1 antibody is preferably a PD-L1 therapeutic antibody. c) Add surfactant-free reading buffer, and then do not wash with buffer; the reading buffer is preferably MSD read buffer; d) Use the device to read the disk and obtain the electrochemical luminescence intensity.

在本發明之一個實施例中，前述PD-1抗體之放行方法或活性檢測方法之步驟或PD-L1抗體之放行方法或活性檢測之步驟或亦可包括： a') 在步驟a)之前，在細胞盤中加入封閉液，培育約1至3小時，之後用洗滌緩衝液洗滌細胞盤。In one embodiment of the present invention, the steps of the release method or activity detection method of the PD-1 antibody or the steps of the release method or activity detection of the PD-L1 antibody may also include: a') Before step a), add blocking solution to the cell plate, incubate for about 1 to 3 hours, and then wash the cell plate with wash buffer.

如前所述之任一方法，其中，對該供試品抗體濃度及電化學發光強度利用公式(1)進行四參數擬合，根據參考品、質控品及供試品四參數擬合計算得到C值，亦即IC50 值，利用公式(2)計算得到供試品相對活性：(1) 其中，x表示自變量，A表示左平台，B表示曲率參數，C表示半數抑制量，單位為ng/mL，D表示右平台。Any method as described above, wherein the antibody concentration and electrochemical luminescence intensity of the test product are fitted using formula (1) to perform four-parameter fitting, and the calculation is based on the four-parameter fitting of the reference product, quality control product and test product. Obtain the C value, that is, the IC 50 value, and use formula (2) to calculate the relative activity of the test product: (1) Among them, x represents the independent variable, A represents the left platform, B represents the curvature parameter, C represents the half inhibition amount in ng/mL, and D represents the right platform.

在本發明之一個實施例中，供試品放行可接受標準為以下中之一者或以上： 1) A參數/D參數≥ 2； 2) 平行性分析：曲線之斜率比值在0.7至1.4之間；高平台比值在0.8至1.25之間； 4) 所有線形段複孔之CV ≤30%； 5) 質控品之生物學活性在75%至125%之間 6) 四參數擬合S型曲線之R2 ≥ 0.95。In one embodiment of the present invention, the acceptable standard for release of the test product is one or more of the following: 1) A parameter/D parameter ≥ 2; 2) Parallelism analysis: the slope ratio of the curve is between 0.7 and 1.4 between; the high plateau ratio is between 0.8 and 1.25; 4) The CV of multiple wells in all linear segments is ≤30%; 5) The biological activity of the quality control product is between 75% and 125% 6) Four-parameter fitting S-type R 2 of the curve ≥ 0.95.

在本發明之一個實施例中，該封閉液為含體積濃度為1%至5%牛血清白蛋白之緩衝液或10%至30%胎牛血清之緩衝液，較佳為15%至30%胎牛血清之緩衝液或含體積濃度為1%至3%牛血清白蛋白之緩衝液。In one embodiment of the present invention, the blocking solution is a buffer solution containing a volume concentration of 1% to 5% bovine serum albumin or 10% to 30% fetal bovine serum, preferably 15% to 30% Buffer solution of fetal calf serum or buffer solution containing bovine serum albumin with a volume concentration of 1% to 3%.

在本發明之一個實施例中，該洗滌緩衝液為PBS緩衝液。In one embodiment of the present invention, the washing buffer is PBS buffer.

在本發明之一個實施例中，該細胞盤為MSD 96孔盤，更佳為MSD高結合的96孔盤。In one embodiment of the present invention, the cell plate is an MSD 96-well plate, more preferably an MSD high-binding 96-well plate.

在本發明之一個實施例中，該細胞為表現PD-1或PDL1之CHO細胞。In one embodiment of the invention, the cells are CHO cells expressing PD-1 or PDL1.

在本發明之一個實施例中，細胞盤中之細胞密度為2.5E4/孔至20E4/孔，較佳為3.5E4/孔至7.5E4/孔，更佳為3.5E4/孔至5E4/孔。In one embodiment of the present invention, the cell density in the cell plate ranges from 2.5E4/well to 20E4/well, preferably from 3.5E4/well to 7.5E4/well, and more preferably from 3.5E4/well to 5E4/well.

在本發明之一個實施例中，該PD-1重組蛋白或PD-L1重組蛋白之濃度為50 ng/mL至200 ng/mL，較佳為80 ng/mL至160 ng/mL。In one embodiment of the present invention, the concentration of the PD-1 recombinant protein or PD-L1 recombinant protein is 50 ng/mL to 200 ng/mL, preferably 80 ng/mL to 160 ng/mL.

在本發明之一個實施例中，該檢測二抗之濃度為500 ng/mL至1000 ng/mL，較佳為1000 ng/mL。In one embodiment of the present invention, the concentration of the detection secondary antibody is 500 ng/mL to 1000 ng/mL, preferably 1000 ng/mL.

在本發明之一個實施例中，該步驟b)中之培育時間為60分鐘至90分鐘，較佳為90分鐘。In one embodiment of the present invention, the incubation time in step b) is 60 minutes to 90 minutes, preferably 90 minutes.

在本發明之一個實施例中，該重組蛋白為融合有標籤之蛋白質，其中該標籤較佳為鼠源Fc。In one embodiment of the present invention, the recombinant protein is a protein fused with a tag, wherein the tag is preferably murine Fc.

在本發明之一個實施例中，該檢測抗體為釕結合的抗小鼠IgG。In one embodiment of the invention, the detection antibody is a ruthenium-conjugated anti-mouse IgG.

相關申請案之交叉引用 本申請案主張2017年10月24日申請之PCT專利申請案序列號PCT/CN2017/107472之權益及優先權，其以全文引用的方式併入本文。Cross-references to related applications This application claims the rights and priority of the PCT patent application serial number PCT/CN2017/107472 filed on October 24, 2017, which is incorporated herein by reference in its entirety.

下面結合實施方式對本發明進行進一步詳細描述，給出之實施例僅為了闡明本發明，而非為了限制本發明之範圍。下述實施例中之實驗方法，如無特殊說明，均為習知方法。下述實施例中所用之材料、試劑等，如無特殊說明，均可自商業途徑得到。The present invention will be described in further detail below in conjunction with the embodiments. The examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples are all common methods unless otherwise specified. The materials, reagents, etc. used in the following examples can all be obtained from commercial sources unless otherwise specified.

在詳細描述本發明之前，應理解，本發明不限於特定生物系統或細胞類型。亦應理解，本文使用之術語僅用於描述特定實施例之目的，而非限制性的。如本說明書及所附申請專利範圍中所用，單數形式「一個」、「一」及「該」除非另有明確規定，包括複數個指示物。因此，例如，提及「細胞」包括兩個或更多個細胞或細胞之整個培養物的組合；提及之「多核苷酸」實際上包括該多核苷酸之許多複本。除非在本說明書之提醒中另有定義，本文使用之所有技術及科學術語具有與本發明之一般熟習此項技術者通常所理解相同的含義。Before the present invention is described in detail, it is to be understood that this invention is not limited to specific biological systems or cell types. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "cell" includes a combination of two or more cells or an entire culture of cells; reference to a "polynucleotide" actually includes many copies of that polynucleotide. Unless otherwise defined in the context of this specification, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

在本文中所用之術語「供試品」係指待鑑別、待測定之供測試樣品。在本發明中，供試品抗體包括在工業生產中生產的待檢測生物學活性之抗體樣本，具體包括PD-1抗體或PD-L1抗體。The term "test article" as used in this article refers to the test sample to be identified and measured. In the present invention, the test antibody includes an antibody sample whose biological activity is to be detected and is produced in industrial production, specifically including PD-1 antibody or PD-L1 antibody.

本文所用之術語「抗體」包括全抗體及任何抗原結合片段(亦即「抗原結合部分」)或其單鏈。「抗體」包含至少兩個重(H)鏈及兩條藉由二硫鍵相互連接之輕鏈(L)鏈或其抗原結合部分之蛋白質。各重鏈由重鏈可變區(本文縮寫為VH)及重鏈恆定區組成。重鏈恆定區由三個結構域CH1、CH2及CH3組成。各輕鏈由輕鏈可變區(本文縮寫為VL)及輕鏈恆定區組成。輕鏈恆定區由一個結構域CL組成。VH及VL區域可進一步細分為高變區域，稱為互補決定區(CDR)，散佈於更保守的區域，稱為框架區(FR)。各VH及VL由以下順序自胺基末端至羧基末端排列的三個CDR及四個FR組成：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重鏈及輕鏈之可變區含有與抗原相互作用之結合結構域。其中術語「抗體」係指免疫球蛋白或其片段或衍生物，且包括包含抗原結合位點之任何多肽，不管其是否在體外或體內產生。該術語包括但不限於多株、單株、單特異性、多特異性、非特異性、人源化、單鏈、嵌合、合成、重組、雜交、突變及接枝抗體。術語「抗體」亦包括保留抗原結合功能之抗體片段，例如Fab、F(ab')2、Fv、scFv、Fd、dAb及其他抗體片段，例如特異性結合PD-1或PD-L1之能力。通常，此類片段將包含抗原結合片段。The term "antibody" as used herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. "Antibody" is a protein containing at least two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain consists of a heavy chain variable region (herein abbreviated 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 (herein abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amine terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The term "antibody" refers to an immunoglobulin or a fragment or derivative thereof, and includes any polypeptide containing an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. The term "antibody" also includes antibody fragments that retain antigen-binding functionality, such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments, such as the ability to specifically bind to PD-1 or PD-L1. Typically, such fragments will comprise antigen-binding fragments.

如本文中所使用，術語「治療類抗體」、「治療性抗體」可互換使用，包括但不限於已用於臨床或臨床研究之抗體，亦包括正在研發的具有臨床使用價值之潛在抗體，該抗體可施用於受試者，治療某種疾病；術語「治療類抗體」或「治療性抗體」亦包括可用於體外診斷、體外研究、體外研發之檢測性抗體，該抗體可用於細胞學水準或免疫檢測。As used herein, the terms "therapeutic antibodies" and "therapeutic antibodies" are used interchangeably, including but not limited to antibodies that have been used in clinical or clinical research, and also include potential antibodies that are under development and have clinical use value. Antibodies can be administered to a subject to treat a certain disease; the term "therapeutic antibody" or "therapeutic antibody" also includes detection antibodies that can be used in in vitro diagnostics, in vitro research, and in vitro development. The antibodies can be used at the cytology level or Immunoassays.

如本文中所使用，術語「計劃性死亡1」、「計劃性細胞死亡1」、「蛋白PD-1」、「PD-1」、「PD1」、「PDCD1」、「hPD-1」及「hPD-F」可互換使用，且包括變體、同種型、人PD-1之物種同源物及具有PD-1之至少一個共同抗原決定基之類似物。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, isotypes, species homologues of human PD-1, and analogs that share at least one epitope of PD-1.

如本文中所使用，術語「計劃性死亡配位體1」、「PD-配位體1」、「PD-L1」、「PD L1」、「B7同源物1」、「B7-H1」、「CD274」可互換使用，包括變體、同種型、人PD-L1之物種同源物及具有PD-L1之至少一個共同抗原決定基之類似物。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, isotypes, species homologues of human PD-L1, and analogs that share at least one epitope in common with PD-L1.

如本文中所使用，術語「CV」係指變異係數，為反映資料離散程度之絕對值。其資料大小不僅受變量值離散程度影響，且亦受變量值平均水平大小影響。標準差與平均數之比值稱為變異係數。變異係數可消除單位及(或)平均數不同對兩個或更多個資料變異程度比較的影響。變異係數之計算公式為：變異係數CV =( 標準偏差SD / 平均值Mean )× 100%。As used in this article, the term "CV" refers to the coefficient of variation, which is an absolute value that reflects the dispersion of data. The data size is not only affected by the degree of dispersion of variable values, but also by the average level of variable values. The ratio of the standard deviation to the mean is called the coefficient of variation. The coefficient of variation can eliminate the influence of different units and/or averages on the comparison of the degree of variation of two or more data. The calculation formula for the coefficient of variation is: coefficient of variation CV = (standard deviation SD / mean mean) × 100%.

如本文中所使用，術語「重組蛋白」係指應用基因重組技術，獲得連接有可轉譯成目的蛋白之基因片段的重組載體，之後將其轉入可表現目的蛋白之宿主細胞從而表現特定重組蛋白分子。As used herein, the term "recombinant protein" refers to the application of genetic recombination technology to obtain a recombinant vector connected with a gene fragment that can be translated into a protein of interest, and then transfer it into a host cell that can express the protein of interest to express the specific recombinant protein. molecular.

如本文中所使用，術語「讀數緩衝液」係指用於讀數之緩衝液，包括但不限於MSD read緩衝液。As used herein, the term "read buffer" refers to a buffer used for reading, including but not limited to MSD read buffer.

如本文中所使用，術語「放行」、「實時放行」、「放行檢測」、「實施放行檢測、「RTR」、「RTRT」可互換使用，係指根據製程資料，評估及保證中間品及/或成品達到可接受品質的能力；其通常將所量測物料屬性及製程控制等的資料進行有效結合，據此評估及保證中間產品或最終成品品質之能力。As used herein, the terms "release," "real-time release," "release testing," "implementation release testing," "RTR," and "RTRT" are used interchangeably and refer to the evaluation and assurance of in-process products and/or based on process data. Or the ability of finished products to achieve acceptable quality; it usually effectively combines data such as measured material properties and process control to evaluate and ensure the quality of intermediate products or final products.

實施例 藉由以下實施例進一步說明本發明。提供實施例僅用於說明目的，且不應解釋為以任何方式限制本發明之範圍或內容。Example The present invention is further illustrated by the following examples. The examples are provided for illustrative purposes only and should not be construed as limiting the scope or content of the invention in any way.

實施例1：參考品、質控品及供試品製備(關於抗PD-1抗體) 根據蛋白標示量，用分析培養基將參考品、質控品及供試品藉由梯度稀釋或者靶向稀釋成10個或11個濃度點，作為標準曲線上之10個或11個點。其中質控品與參考品為同一種，但與參考品獨立稀釋。Example 1: Preparation of reference products, quality control products and test products (regarding anti-PD-1 antibodies) According to the labeled amount of protein, use the analytical medium to dilute the reference substance, quality control substance and test substance into 10 or 11 concentration points through gradient dilution or targeted dilution, which will be used as 10 or 11 points on the standard curve. The quality control product and the reference product are the same, but they are diluted independently from the reference product.

實施例2：PD-1/CHO-S細胞株之準備 連續培養的細胞或新復蘇的凍存細胞均適用於該方法。Example 2: Preparation of PD-1/CHO-S cell line This method is suitable for continuously cultured cells or newly thawed cryopreserved cells.

1、連續培養之細胞：將細胞離心後棄上清。1. Continuously cultured cells: Centrifuge the cells and discard the supernatant.

2、凍存細胞：自液氮罐中拿出PD-1/CHO-S細胞，並快速放入37℃水浴鍋中解凍。將凍存管中之細胞懸液加入至含約N×9 mL (N表示細胞支數)CHO培養基中，輕柔地吹打混勻。離心棄去上清。2. Cryopreserved cells: Take out the PD-1/CHO-S cells from the liquid nitrogen tank and quickly put them into a 37°C water bath to thaw. Add the cell suspension in the cryovial to the CHO medium containing approximately N×9 mL (N represents the number of cells), and mix by gentle pipetting. Centrifuge and discard the supernatant.

3、用冷DPBS將細胞洗滌一次。離心後棄上清。根據所需之細胞懸液體積，加入分析培養基重懸細胞。取細胞懸液進行細胞計數，確定細胞密度及細胞活率。用分析培養基調整細胞密度至16.7E5/mL，將調整濃度之細胞懸液混勻。3. Wash the cells once with cold DPBS. Discard the supernatant after centrifugation. According to the required cell suspension volume, add analysis medium to resuspend the cells. Take the cell suspension for cell counting to determine the cell density and cell viability. Use analytical medium to adjust the cell density to 16.7E5/mL, and mix the cell suspension with the adjusted concentration.

實施例3：測試方法 1、封閉：將150 µL封閉液(10%FBS/PBS或3%BSA/PBS)加入至MSD高結合96孔中。MSD盤置於震盪器上以150~220轉/分鐘之轉速震盪，室溫培育2±1小時(若不需要封閉，則此步可省略)。Example 3: Test method 1. Blocking: Add 150 µL of blocking solution (10% FBS/PBS or 3% BSA/PBS) to the MSD high binding 96-well. The MSD disk is placed on a shaker at a speed of 150~220 rpm and incubated at room temperature for 2±1 hours (if sealing is not required, this step can be omitted).

2、按照實施例1中所述之方法製備參考品、質控品及供試品溶液。將PD-L1-mFc存儲液稀釋至終濃度為800 ng/mL，渦旋混勻。將檢測二抗 (MSD SULFO-TAG Goat Anti-mouse Antibody)存儲液稀釋至終濃度為2.857 µg/mL，渦旋混勻。PD-1/CHO-S細胞株之準備參見實施例2。2. Prepare reference product, quality control product and test solution according to the method described in Example 1. Dilute the PD-L1-mFc stock solution to a final concentration of 800 ng/mL, and vortex to mix. Dilute the detection secondary antibody (MSD SULFO-TAG Goat Anti-mouse Antibody) storage solution to a final concentration of 2.857 µg/mL, and vortex to mix. For the preparation of PD-1/CHO-S cell line, please refer to Example 2.

3、將稀釋成之參考品及供試品、稀釋成之800 ng/mL PD-L1-mFc及稀釋成之2.857 µg/mL檢測抗體以1:1:2 (v:v:v)的比例(例如45 μL: 45 μL: 90 μL)轉移至U形底96孔盤中(轉移盤)之第二行至第十二行中，混勻。在第一行中加入以1:1 (v:v)混合(例如90 μL:90 μL)的分析培養基及檢測抗體，作為背景。2至8℃冰箱避光保存。3. Dilute the reference substance and test substance, the diluted 800 ng/mL PD-L1-mFc and the diluted 2.857 µg/mL detection antibody in a ratio of 1:1:2 (v:v:v) (For example, 45 μL: 45 μL: 90 μL) transfer to the second row to the twelfth row of a U-shaped bottom 96-well plate (transfer plate), and mix well. Add a 1:1 (v:v) mix (e.g. 90 μL:90 μL) of assay medium and detection antibody to the first row as background. Store in a refrigerator at 2 to 8°C away from light.

4、用DPBS或1×PBS將MSD高結合96孔盤洗滌3遍(若不需要封閉MSD盤，則此步省略)。4. Wash the MSD high binding 96-well plate 3 times with DPBS or 1×PBS (if the MSD plate does not need to be sealed, this step is omitted).

5、PD-1/CHO-S細胞鋪盤：以30 μL/孔加入至MSD高結合盤中。將參考品及供試品抗體、PD-L1-mFc及檢測抗體之混合物以70 μL/孔加入至含有PD-1/CHO-S細胞之MSD高結合盤中，每孔之總體積為100 μL。5. PD-1/CHO-S cell plating: Add 30 μL/well into the MSD high binding plate. Add the mixture of reference and test antibodies, PD-L1-mFc and detection antibodies to the MSD high-binding plate containing PD-1/CHO-S cells at 70 μL/well. The total volume of each well is 100 μL. .

6、將MSD盤置於室溫或22~28℃恆溫培養箱中避光培育60至90分鐘。6. Place the MSD disk in room temperature or a constant temperature incubator at 22~28°C and incubate in the dark for 60 to 90 minutes.

7、向MSD盤之每孔中加入100 μL的2×不含界面活性劑之Read Buffer。7. Add 100 μL of 2× Read Buffer without surfactant to each well of the MSD plate.

8、用MSD MESOTM SECTORS 600或同等設備讀盤。8. Use MSD MESOTM SECTORS 600 or equivalent equipment to read the disk.

9、資料分析： 雖然每次實驗同時操作多塊盤，但各盤獨立計算。9. Data analysis: Although each experiment operates multiple disks simultaneously, each disk is calculated independently.

用四參數對抗體濃度及電化學發光強度進行擬合 其中，x = 自變量 A = 左平台 B = 曲率參數 C = 半數抑制量(ng/mL) D = 右平台Four parameters were used to fit the antibody concentration and electrochemiluminescence intensity. Among them, x = independent variable A = left platform B = curvature parameter C = half inhibition amount (ng/mL) D = right platform

平行性分析：根據參考品及供試品四參數擬合計算得到B值，亦即四參數擬合曲線之斜率，利用下列公式計算得到供試品或質控品之曲線斜率比(Slope Ratio)： Parallelism analysis: Calculate the B value based on the four-parameter fitting of the reference product and the test product, which is the slope of the four-parameter fitting curve. Use the following formula to calculate the curve slope ratio (Slope Ratio) of the test product or quality control product. :

質控品與樣品之相對活性計算如下：根據參考品、質控品及供試品四參數擬合計算得到C值，亦即IC50 值，利用下列公式計算得到供試品生物學活性。 The relative activity of the quality control product and the sample is calculated as follows: The C value, that is, the IC 50 value, is calculated based on the four-parameter fitting of the reference product, quality control product, and test product. The biological activity of the test product is calculated using the following formula.

10、在一特定實施例中，系統適用性 僅當質控品滿足以下所有標準，實驗才有效， a) A參數/D參數≥2； b) 平行性分析：曲線之斜率比在0.7至1.4之間；高平台比在0.8至1.25之間； c) 雙複孔均包括在線形段之複孔CV≤30%； d) 質控品之生物學活性在75%至125%之間； e) 四參數擬合S型曲線之R2 ≥0.95； 若質控品之結果不滿足以上標準，則將實驗視為無效的。10. In a specific embodiment, the system applicability is only valid if the quality control product meets all the following standards, a) A parameter/D parameter ≥ 2; b) Parallelism analysis: the slope ratio of the curve is between 0.7 and 1.4 between; the high plateau ratio is between 0.8 and 1.25; c) Double wells include multiple wells in the linear section with CV ≤ 30%; d) The biological activity of the quality control product is between 75% and 125%; e ) The R 2 of the four-parameter fitted S-shaped curve is ≥0.95; if the results of the quality control product do not meet the above standards, the experiment will be considered invalid.

參考品可接受標準 A參數/D參數≥2 所有線性段複孔CV≤30% 四參數擬合S型曲線之R2 ≥0.95The reference product accepts the standard A parameters/D parameters ≥ 2, all linear segments, multiple hole CV ≤ 30%, R 2 of the four-parameter S-shaped curve fitting ≥ 0.95

供試品可接受標準 A參數/D參數≥2 平行性：曲線之斜率比在0.7~1.4之間；高平台比在0.8至1.25之間 供試品之所有線性段複孔之CV≤30% 四參數擬合S型曲線之R2 ≥0.95The acceptable standard A parameter/D parameter of the test product is ≥2. Parallelism: the slope ratio of the curve is between 0.7 and 1.4; the high plateau ratio is between 0.8 and 1.25. The CV of all linear segments of the multiple holes of the test product is ≤30%. Four-parameter fitting S-shaped curve R 2 ≥0.95

實施例4：方法確認Example 4: Method confirmation

1、專屬性 不相關的Isotype匹配抗體不能抑制PD-L1與表現PD-1之CHO細胞株之結合，對照樣品無特異性生物學活性(圖1)。該方法對考察抗PD-1產品之生物學活性具有良好特異性。1. Exclusiveness Irrelevant Isotype-matched antibodies were unable to inhibit the binding of PD-L1 to CHO cell lines expressing PD-1, and the control sample had no specific biological activity (Figure 1). This method has good specificity for examining the biological activity of anti-PD-1 products.

2、準確度 50%、71%、100%、141%以及200%目標濃度樣品之平均回收率介於80%至120%之間(表1)。2. Accuracy The average recoveries for 50%, 71%, 100%, 141% and 200% target concentration samples ranged from 80% to 120% (Table 1).

3、精密度 50%、71%、100%、141%以及200%目標濃度樣品，分別獨立8次；中間精密度檢測結果各樣品RSD均小於25%(表1)。 表1. 準確度及精確度結果清單 3. The target concentration samples with precision of 50%, 71%, 100%, 141% and 200% were independently tested 8 times respectively; the RSD of each sample in the intermediate precision test results was less than 25% (Table 1). Table 1. List of accuracy and precision results

4、線性 使用回收率研究之資料，預期相對活性與實測相對活性線性擬合情況較好，Pearson相關係數≥0.95(圖2)。4. Linear Using the data from the recovery study, the linear fit between the expected relative activity and the measured relative activity was good, with the Pearson correlation coefficient ≥0.95 (Figure 2).

5、試驗有效範圍 試驗結果在50%至200%目標濃度範圍內回收率顯示了良好線性，且結果之準確性與精密度均符合可接受標準。因此，該試驗有效範圍為50%至200%目標濃度範圍。5. Test effective range The test results showed good linearity in the recovery rate within the target concentration range of 50% to 200%, and the accuracy and precision of the results met acceptable standards. Therefore, the effective range of this test is 50% to 200% of the target concentration range.

6、耐用性 PD-L1濃度、試驗培育時間、細胞密度、檢測抗體濃度及細胞代次等參數在研究範圍內，QC回收率均滿足80%≤回收率≤120%。6. Durability Parameters such as PD-L1 concentration, test incubation time, cell density, detection antibody concentration, and cell passage were within the research range, and the QC recovery rate all met 80% ≤ recovery rate ≤ 120%.

7、應激樣品 熱處理、酸處理(大約pH 3)及鹼處理(大約pH 11)之應激樣品之活性相對於未處理之對照明顯下降(圖5)。7. Stress samples The activity of stressed samples treated with heat, acid (approximately pH 3) and alkali treatment (approximately pH 11) decreased significantly relative to the untreated control (Figure 5).

8、長期品質控制 在至少6個月之時間內，由至少兩名實驗人員進行檢測，在此過程中，質控品之回收率均在80%至120%之間，符合放行之要求，證明本放行方法滿足長期品質控制的考察。8. Long-term quality control Testing was carried out by at least two laboratory personnel for at least 6 months. During this process, the recovery rate of the quality control products was between 80% and 120%, which met the requirements for release and proved that this release method meets the long-term requirements. Quality control inspection.

9、結論 實驗結果，包括專屬性、準確度、精密度、試驗有效範圍以及線性、耐用性及應激樣品分析，證明該方法適用於抗PD1抗體之實驗室各階段的生物活性方法，包括藥物原液，成品，穩定性樣品及中間體之生物學活性檢測。9. Conclusion The experimental results, including specificity, accuracy, precision, experimental effective range, linearity, durability and stress sample analysis, prove that this method is suitable for bioactivity methods at all stages of the laboratory of anti-PD1 antibodies, including drug solutions and finished products. , Biological activity testing of stability samples and intermediates.

實施例5：方法參數之優化Example 5: Optimization of method parameters

1、細胞密度 細胞類型：分別表現PD-1或PD-L1之CHO細胞，探索細胞密度範圍：6.25E3/孔~20E4/孔。結果參見圖3，2.5E4/孔~20E4/孔之細胞均適用於實驗。1. Cell density Cell type: CHO cells expressing PD-1 or PD-L1 respectively. Explore the cell density range: 6.25E3/well ~ 20E4/well. The results are shown in Figure 3. Cells ranging from 2.5E4/well to 20E4/well are suitable for the experiment.

2、PD-L1/PD-1濃度 探索濃度範圍：4至2000 ng/mL。結果參見圖4，100 ± 50 ng/mL之PD-L1均適用於實驗。2. PD-L1/PD-1 concentration Explore the concentration range: 4 to 2000 ng/mL. The results are shown in Figure 4. PD-L1 at 100 ± 50 ng/mL is suitable for the experiment.

3、其他參數 3.1、檢測二抗濃度 探索濃度範圍：100 ng/mL 至2000 ng/mL。750 ± 250ng/mL之檢測二抗濃度均適用於實驗。 3.2、試驗培育時間 探索範圍：60至90分鐘，培育時間均適用於實驗。 3.3、封閉液 探索條件：1% BSA至3%BSA、15%至30%之FBS/培養基、無封閉。三者均適用於該實驗。 3.4、分析緩衝液 探索條件：1%BSA、10%至30%FBS/培養基、10%至30%FBS/PBS。三者均適用於該實驗。 3.5、凍存細胞及連續培養細胞之比較 結果：均適合於實驗。3. Other parameters 3.1. Detect secondary antibody concentration Explore the concentration range: 100 ng/mL to 2000 ng/mL. The detection secondary antibody concentration of 750 ± 250ng/mL is suitable for experiments. 3.2. Experimental cultivation time Exploration range: 60 to 90 minutes, all incubation times are applicable to experiments. 3.3. Sealing solution Exploration conditions: 1% BSA to 3% BSA, 15% to 30% FBS/medium, no blocking. All three are suitable for this experiment. 3.4. Analysis buffer Exploration conditions: 1% BSA, 10% to 30% FBS/medium, 10% to 30% FBS/PBS. All three are suitable for this experiment. 3.5. Comparison between cryopreserved cells and continuously cultured cells Results: All are suitable for experiments.

以引用之方式併入 本文引用之每一專利文獻及科學文獻之全部揭示內容出於所有目的以引用之方式併入本文。incorporated by reference The entire disclosure of each patent document and scientific document cited herein is incorporated by reference for all purposes.

等效物 本發明可在不脫離其基本特徵之情況下以其他具體形式實施。因此，將前述實施例視為說明性的，而非對本文所述之本發明的限制。本發明之範圍由所附申請專利範圍而非由前述說明書表示，且意在將落入申請專利範圍之等效物之含義及範圍內的所有改變包括在其中。equivalent The invention may be embodied in other specific forms without departing from its essential characteristics. Accordingly, the foregoing examples are to be considered illustrative and not limiting of the invention described herein. The scope of the present invention is indicated by the appended claims rather than the foregoing description, and all changes that fall within the meaning and range of equivalents of the claims are intended to be included therein.

圖1示出抗PD-1治療性抗體之生物學活性放行方法的專屬性。其中橫座標為產品及對照品之濃度，縱座標為ECL信號值，藍色圓圈標記為參考品，紅色方框標記為質控品，橘色菱形標記為對照品Rituximab，綠色三角形標記為對照品Xolair。 圖2示出抗PD-1治療性抗體之生物學活性放行方法之線性擬合圖。 圖3示出細胞鋪盤濃度與ECL信號值之間的柱狀圖。 圖4示出PD-L1濃度與ECL信號值之間劑量依賴柱狀圖。 圖5示出不同條件處理下之應激樣品之活性結果圖。Figure 1 illustrates 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 substance, the ordinate is the ECL signal value, the blue circle is marked as the reference substance, the red box is marked as the quality control substance, the orange diamond is marked as the control substance Rituximab, and the green triangle is marked as the control substance. Xolair. Figure 2 shows a linear fit plot of the biological activity release method for anti-PD-1 therapeutic antibodies. Figure 3 shows a histogram of cell plating concentration versus ECL signal value. Figure 4 shows a dose-dependent histogram between PD-L1 concentration and ECL signal value. Figure 5 shows the activity results of stress samples treated under different conditions.

Claims (23)

一種用於抗體生物學活性放行之方法，其特徵在於，該方法利用電化學發光法檢測供試品抗體之細胞結合活性，藉由對該供試品抗體濃度及電化學發光強度進行四參數擬合，計算生物學活性，該方法藉由方法學確認能用於抗體之生物學活性放行檢測，該方法包含：a)將細胞表面表現PD-1之細胞鋪於細胞盤中，之後不需要洗滌；b)加入PD-L1重組蛋白、檢測抗體及抗PD-1抗體，進行培育，之後不需要洗滌；c)加入無界面活性劑之讀數緩衝液；d)使用設備讀盤，得到電化學發光強度；e)對該供試品抗體濃度及電化學發光強度利用公式(1)進行四參數擬合，根據參考品、質控品及供試品四參數擬合計算得到C值，亦即IC50值，利用公式(2)計算得到供試品相對活性：
Figure 107137281-A0305-02-0018-1
Figure 107137281-A0305-02-0018-2
 其中，x表示自變量，A表示左平台，B表示曲率參數，C表示半數抑制量，該半數抑制量之單位為ng/mL，D表示右平台；或a)將細胞表面表現PD-L1之細胞鋪於細胞盤中，之後不需要洗滌；b)加入PD-1重組蛋白、檢測抗體及抗PD-L1抗體，進行培育，之後不需要洗滌； c)加入無界面活性劑之讀數緩衝液；d)使用設備讀盤，得到電化學發光強度；e)對該供試品抗體濃度及電化學發光強度利用公式(1)進行四參數擬合，根據參考品、質控品及供試品四參數擬合計算得到C值，亦即IC50值，利用公式(2)計算得到供試品相對活性：
Figure 107137281-A0305-02-0019-3
Figure 107137281-A0305-02-0019-4
 其中，x表示自變量，A表示左平台，B表示曲率參數，C表示半數抑制量，該半數抑制量之單位為ng/mL，D表示右平台。 A method for releasing the biological activity of antibodies, characterized in that the method uses electrochemiluminescence to detect the cell-binding activity of the test antibody, and performs a four-parameter simulation on the test antibody concentration and electrochemiluminescence intensity. Combined, the biological activity is calculated. This method is confirmed by methodology to be used for the biological activity release detection of antibodies. The method includes: a) plating cells expressing PD-1 on the cell surface in a cell plate, and no washing is required afterwards. ; b) Add PD-L1 recombinant protein, detection antibody and anti-PD-1 antibody, incubate, and no washing is required afterwards; c) Add surfactant-free reading buffer; d) Use equipment to read the plate to obtain electrochemiluminescence Intensity; e) Use formula (1) to perform four-parameter fitting on the antibody concentration and electrochemical luminescence intensity of the test product, and calculate the C value, that is, IC, based on the four-parameter fitting of the reference product, quality control product, and test product. 50 value, use formula (2) to calculate the relative activity of the test product:
Figure 107137281-A0305-02-0018-1
Figure 107137281-A0305-02-0018-2
 Among them, x represents the independent variable, A represents the left platform, B represents the curvature parameter, C represents the half inhibition amount, the unit of the half inhibition amount is ng/mL, and D represents the right platform; or a) The cell surface expresses PD-L1 The cells are spread in the cell plate and do not need to be washed afterwards; b) Add PD-1 recombinant protein, detection antibody and anti-PD-L1 antibody and incubate, and there is no need to wash afterwards; c) Add reading buffer without surfactant; d) Use the equipment to read the disk and obtain the electrochemical luminescence intensity; e) Use formula (1) to perform four-parameter fitting on the antibody concentration and electrochemical luminescence intensity of the test product. According to the four parameters of the reference product, quality control product and test product The C value, that is, the IC 50 value, is calculated by parameter fitting. The relative activity of the test product is calculated using formula (2):
Figure 107137281-A0305-02-0019-3
Figure 107137281-A0305-02-0019-4
 Among them, x represents the independent variable, A represents the left platform, B represents the curvature parameter, C represents the half inhibition amount, the unit of the half inhibition amount is ng/mL, and D represents the right platform. 一種利用電化學發光技術檢測抗PD-1抗體之細胞活性的方法，其特徵在於，該方法之步驟包括：a)將細胞表面表現PD-1之細胞鋪於細胞盤中，之後不需要洗滌；b)加入PD-L1重組蛋白、檢測抗體及抗PD-1抗體，進行培育，之後不需要用洗滌；c)加入無界面活性劑之讀數緩衝液；d)使用設備讀盤，得到電化學發光強度；e)對該供試品抗體濃度及電化學發光強度利用公式(1)進行四參數擬合，根據參考品、質控品及供試品四參數擬合計算得到C值，亦即IC50值，利用公式(2)計算得到供試品相對活性：
Figure 107137281-A0305-02-0019-5
Figure 107137281-A0305-02-0019-6
 其中，x表示自變量，A表示左平台，B表示曲率參數，C表示半數抑制量，該半數抑制量之單位為ng/mL，D表示右平台。 A method for detecting the cell activity of anti-PD-1 antibodies using electrochemiluminescence technology, which is characterized in that the steps of the method include: a) spreading cells expressing PD-1 on the cell surface in a cell dish, without washing afterwards; b) Add PD-L1 recombinant protein, detection antibody and anti-PD-1 antibody, and incubate without washing; c) Add surfactant-free reading buffer; d) Use equipment to read the plate to obtain electrochemiluminescence Intensity; e) Use formula (1) to perform four-parameter fitting on the antibody concentration and electrochemical luminescence intensity of the test product, and calculate the C value, that is, IC, based on the four-parameter fitting of the reference product, quality control product, and test product. 50 value, use formula (2) to calculate the relative activity of the test product:
Figure 107137281-A0305-02-0019-5
Figure 107137281-A0305-02-0019-6
 Among them, x represents the independent variable, A represents the left platform, B represents the curvature parameter, C represents the half inhibition amount, the unit of the half inhibition amount is ng/mL, and D represents the right platform. 一種利用電化學發光技術檢測抗PD-L1抗體之細胞活性的方法，其特徵在於，該方法之步驟包括：a)將細胞表面表現PD-L1之細胞鋪於細胞盤中，之後不需要液洗滌；b)加入PD-1重組蛋白、檢測抗體及PD-L1單株抗體，進行培育，之後不需要洗滌；c)加入無界面活性劑之讀數緩衝液；d)使用設備讀盤，得到電化學發光強度；e)對該供試品抗體濃度及電化學發光強度利用公式(1)進行四參數擬合，根據參考品、質控品及供試品四參數擬合計算得到C值，亦即IC50值，利用公式(2)計算得到供試品相對活性：
Figure 107137281-A0305-02-0020-7
Figure 107137281-A0305-02-0020-8
 其中，x表示自變量，A表示左平台，B表示曲率參數，C表示半數抑制量，該半數抑制量之單位為ng/mL，D表示右平台。 A method for detecting the cell activity of anti-PD-L1 antibodies using electrochemiluminescence technology, which is characterized in that the steps of the method include: a) spreading cells expressing PD-L1 on the cell surface in a cell plate, and then no liquid washing is required ; b) Add PD-1 recombinant protein, detection antibody and PD-L1 monoclonal antibody, incubate, and do not need to wash afterwards; c) Add surfactant-free reading buffer; d) Use equipment to read the plate, and obtain electrochemical results. Luminous intensity; e) Use formula (1) to perform four-parameter fitting on the antibody concentration and electrochemical luminescence intensity of the test product, and calculate the C value based on the four-parameter fitting of the reference product, quality control product and test product, that is, IC 50 value, use formula (2) to calculate the relative activity of the test product:
Figure 107137281-A0305-02-0020-7
Figure 107137281-A0305-02-0020-8
 Among them, x represents the independent variable, A represents the left platform, B represents the curvature parameter, C represents the half inhibition amount, the unit of the half inhibition amount is ng/mL, and D represents the right platform. 如請求項1至3中任一項之方法，其特徵在於，該步驟亦可包括：a')在步驟a)之前，在細胞盤中加入封閉液，培育約1至3小時，之後用緩衝液洗滌細胞盤。 The method according to any one of claims 1 to 3, characterized in that this step may also include: a') before step a), add blocking solution to the cell plate, incubate for about 1 to 3 hours, and then use buffer Wash the cell dish with liquid. 如請求項1至3中任一項之方法，其特徵在於，系統適應性標準為以下標準中之一者以上：a)A參數/D參數
Figure 107137281-A0305-02-0021-9
2；b)平行性分析：曲線之斜率比在0.7至1.4之間；高平台比在0.8至1.25之間；c)所有線性段之複孔CV
Figure 107137281-A0305-02-0021-10
30%；d)該質控品之生物學活性在75%至125%之間；e)四參數擬合S型曲線之R2
Figure 107137281-A0305-02-0021-11
0.95。 The method of any one of claims 1 to 3, characterized in that the system adaptability standard is one or more of the following standards: a) A parameter/D parameter
Figure 107137281-A0305-02-0021-9
2; b) Parallelism analysis: the slope ratio of the curve is between 0.7 and 1.4; the high plateau ratio is between 0.8 and 1.25; c) CV of multiple holes in all linear segments
Figure 107137281-A0305-02-0021-10
30%; d) The biological activity of the quality control product is between 75% and 125%; e) R 2 of the four-parameter fitted S-shaped curve
Figure 107137281-A0305-02-0021-11
0.95. 如請求項4之方法，其特徵在於，該封閉液為含體積濃度為1%至5%牛血清白蛋白之緩衝液或10%至30%胎牛血清之緩衝液。 The method of claim 4, wherein the blocking solution is a buffer containing a volume concentration of 1% to 5% bovine serum albumin or a buffer containing 10% to 30% fetal bovine serum. 如請求項6之方法，其特徵在於，該封閉液為含體積濃度為1%至3%牛血清白蛋白之緩衝液或15%至30%胎牛血清之緩衝液。 The method of claim 6, wherein the blocking solution is a buffer containing a volume concentration of 1% to 3% bovine serum albumin or a buffer containing 15% to 30% fetal bovine serum. 如請求項6之方法，其特徵在於，該緩衝液為PBS緩衝液或基礎培養基。 The method of claim 6, characterized in that the buffer is PBS buffer or basal culture medium. 如請求項1至3中任一項之方法，其特徵在於，該細胞盤為96孔盤。 The method according to any one of claims 1 to 3, characterized in that the cell plate is a 96-well plate. 如請求項9之方法，其特徵在於，該細胞盤為MSD高結合的96孔盤。 The method of claim 9, characterized in that the cell plate is a 96-well plate with high MSD binding. 如請求項1至3中任一項之方法，其特徵在於，該細胞為表現PD-1或PD-L1之CHO細胞。 The method according to any one of claims 1 to 3, characterized in that the cells are CHO cells expressing PD-1 or PD-L1. 如請求項1至3中任一項之方法，其特徵在於，細胞盤中之細胞密度為2.5E4/孔至20E4/孔。 The method according to any one of claims 1 to 3, characterized in that the cell density in the cell plate is 2.5E4/well to 20E4/well. 如請求項12之方法，其特徵在於，細胞盤中之細胞密度為3.5E4/孔至7.5E4/孔。 The method of claim 12, wherein the cell density in the cell plate ranges from 3.5E4/well to 7.5E4/well. 如請求項12之方法，其特徵在於，細胞盤中之細胞密度為3.5E4/孔至5E4/孔。 The method of claim 12, characterized in that the cell density in the cell plate is 3.5E4/well to 5E4/well. 如請求項1至3中任一項之方法，其特徵在於，該PD-1重組蛋白或PD-L1重組蛋白之濃度為50ng/mL至200ng/mL。 The method according to any one of claims 1 to 3, characterized in that the concentration of the PD-1 recombinant protein or PD-L1 recombinant protein is 50 ng/mL to 200 ng/mL. 如請求項15之方法，其特徵在於，該PD-1重組蛋白或PD-L1重組蛋白之濃度為80ng/mL至160ng/mL。 The method of claim 15, characterized in that the concentration of the PD-1 recombinant protein or PD-L1 recombinant protein is 80 ng/mL to 160 ng/mL. 如請求項1至3中任一項之方法，其特徵在於，該檢測抗體之濃度為500ng/mL至1000ng/mL。 The method according to any one of claims 1 to 3, characterized in that the concentration of the detection antibody is 500ng/mL to 1000ng/mL. 如請求項17之方法，其特徵在於，該檢測抗體之濃度為1000ng/mL。 The method of claim 17, characterized in that the concentration of the detection antibody is 1000ng/mL. 如請求項1至3中任一項之方法，其特徵在於，步驟b)中之培育時間為60分鐘至90分鐘。 The method of any one of claims 1 to 3, characterized in that the incubation time in step b) is 60 minutes to 90 minutes. 如請求項19之方法，其特徵在於，步驟b)中之培育時間為90分鐘。 The method of claim 19 is characterized in that the incubation time in step b) is 90 minutes. 如請求項1至3中任一項之方法，其特徵在於，該重組蛋白為融合有標籤之蛋白質。 The method according to any one of claims 1 to 3, characterized in that the recombinant protein is a protein fused with a tag. 如請求項21之方法，其特徵在於，該標籤為鼠源Fc。 The method of claim 21, characterized in that the label is mouse source Fc. 如請求項1至3中任一項之方法，其特徵在於，該檢測抗體為釕結合的抗小鼠IgG。 The method according to any one of claims 1 to 3, wherein the detection antibody is ruthenium-conjugated anti-mouse IgG.
TW107137281A 2017-10-24 2018-10-23 A release detection method for therapeutic antibodies TWI821205B (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
WOPCT/CN2017/107472 2017-10-24
PCT/CN2017/107472 WO2019079970A1 (en) 2017-10-24 2017-10-24 Release detection method for therapeutic antibody
??PCT/CN2017/107472 2017-10-24

Publications (2)

Publication Number Publication Date
TW201923348A TW201923348A (en) 2019-06-16
TWI821205B true TWI821205B (en) 2023-11-11

Family

ID=66247070

Family Applications (1)

Application Number Title Priority Date Filing Date
TW107137281A TWI821205B (en) 2017-10-24 2018-10-23 A release detection method for therapeutic antibodies

Country Status (2)

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

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160069900A1 (en) * 2014-09-08 2016-03-10 Gulesi Ayanoglu Immunoassay for soluble pd-l1

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69215983T2 (en) * 1991-07-10 1997-05-22 Tdk Corp Method for measuring the concentration of an immunoreactant using electrochemiluminescence
ATE519112T1 (en) * 2007-05-08 2011-08-15 Hoffmann La Roche METHOD FOR DETECTING SPECIFIC ANTIBODIES OF IMMUNOGLOBULIN CLASS G

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160069900A1 (en) * 2014-09-08 2016-03-10 Gulesi Ayanoglu Immunoassay for soluble pd-l1

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
期刊 Thompson et al., "Competitive Electrochemiluminescence Wash and No-Wash Immunoassays for Detection of Serum Antibodies to Smooth Brucella Strains", Clinical and Vaccine Immunology, 2009, 16(5), pp 765-771. *

Also Published As

Publication number Publication date
WO2019079970A1 (en) 2019-05-02
TW201923348A (en) 2019-06-16

Similar Documents

Publication Publication Date Title
JP6180931B2 (en) Novel antibodies for cancer diagnosis and / or prognosis
US10620211B2 (en) Histochemical assay for evaluating expression of programmed death ligand 1 (PD-L1)
CN106415269A (en) Direct immunohistochemistry assay
CN104067127A (en) Compositions and methods for prostate cancer analysis
CN110506209A (en) The Galectin-3 binding protein surveyed in urine is used to monitor the application method of lupus nephritis seriousness and progress
Congy-Jolivet et al. Production and characterization of chimeric anti-HLA monoclonal antibodies targeting public epitopes as tools for standardizations of the anti-HLA antibody detection
EP3885363A1 (en) Specific antibody for amh, and uses thereof
AU2018271338A1 (en) Protein formulations
JP7010833B2 (en) IL-21 antibody and its use
TWI821205B (en) A release detection method for therapeutic antibodies
CN109696461B (en) Release detection method of therapeutic antibody
WO2018227643A1 (en) Target marker gp73 for detecting steatohepatitis and detection application method
EP2707720B1 (en) Diagnosis and prognosis of triple negative breast and ovarian cancer
CN107367611A (en) The ELISA detection kit of epidermal growth factor acceptor third type mutant
US20210277149A1 (en) Anti-idiotype antibodies and methods of using the same
Fang et al. Bacterial coloration immunofluorescence strip for ultrasensitive rapid detection of bacterial antibodies and targeted antibody-secreting hybridomas
CN102863529A (en) VEGF (vascular endothelial growth factor) monoclonal antibody and fracture healing evaluation antibody chip with same
KR102350259B1 (en) IGF-IR Antibodies and Their Uses for Diagnosis of Cancer
CN116535511B (en) Immunohistochemical antibody for detecting PD-L1 and application thereof
CN117285624B (en) Anti-canine NT-proBNP bispecific antibody and kit
CN112430623B (en) Method for rapidly determining pharmaceutical biological activity of CGRP/CGRP receptor antibody
CN116008555A (en) Method for detecting binding force of monoclonal antibody to RANKL and application of method
CN113061640A (en) Method for stably and rapidly determining biological activity of anti-TIGIT monoclonal antibody drug
WO2018213680A1 (en) Diagnostic antibodies and methods of use
WO2007103538A2 (en) Methods of multiple antibody labeling and uses thereof