CN112851813A - 一种抗pd-1抗体的纯化方法 - Google Patents
一种抗pd-1抗体的纯化方法 Download PDFInfo
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- CN112851813A CN112851813A CN202110241410.5A CN202110241410A CN112851813A CN 112851813 A CN112851813 A CN 112851813A CN 202110241410 A CN202110241410 A CN 202110241410A CN 112851813 A CN112851813 A CN 112851813A
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Abstract
本公开涉及一种抗PD‑1抗体的纯化方法。具体而言，该纯化方法包括使用亲和层析、病毒灭活、阴离子膜层析和阳离子交换层析等方法来去除污染物。该工艺可降低生产成本并提高抗体收率。
Description
技术领域
本公开属于生物工程领域，具体涉及一种抗PD-1抗体的纯化工艺，包括使用亲和层析、病毒灭活、阴离子膜层析和阳离子交换层析等方法来去除污染物。
技术背景
抗体类药物是目前最有前景的生物技术药物，具有靶向性好、特异性强、毒副作用小等优点，主要用于恶性肿瘤和自身免疫性等疾病治疗。全球单抗的市场规模持续增大，已成为各国生物制药的竞争焦点。
近年来，国内制药企业也纷纷加速布局抗体类药物。抗体类药物作为生物大分子，研发的技术难度大，研发周期长。但抗体类药物市场需求和激烈竞争迫使各企业必须加快抗体药物的研发速度和缩短研发周期。加快细胞株筛选和细胞培养工艺的优化，成为其中非常重要的环节。细胞株筛选和细胞培养工艺的优化，需要纯化出足量的高纯度样品用于产品质量的分析检测，从而评估细胞株和细胞培养工艺是否能满足要求。
现有技术中，对于抗体类药物纯化的工艺包含多个步骤，常用的有亲和层析、离子交换层析、疏水层析、超滤等。由于抗体类药物分子的理化性质比较相似，因此，许多公司会建立自己的抗体纯化工艺平台，将多种纯化步骤串联起来使用，以求工艺稳定，产率较高。如(Abhinav A.Shukla，Brian Hubbard，Tim Tressel，Tim Tressel，Sam Guhan，DuncanLow.Downstream processing of monoclonal antibodies-Application of platformapproaches[J].Journal of Chromatography B，2007，848:28-39.)公开了一种纯化工艺，发酵液经过澄清步骤去除细胞等杂质以获得上清液，然后利用亲和层析捕获上清液中的目标蛋白，通过精纯去除蛋白中的各类杂质，然后进行病毒灭活，将病毒滴度降低，最后的超滤将蛋白置换使其形成稳定的制剂缓冲液。
抗PD-1抗体是当前备受瞩目的一类抗体，全球有多家跨国药企正在开发，施贵宝制药公司的nivolumab和默沙东公司的pembrolizumab已于2014年上市，上市后销售额均突破十亿美元。WO2017054646A公开了一种抗PD-1抗体的序列，该抗PD-1抗体安全性良好，已报到的临床研究结果已经显示出其具有一定的抗肿瘤作用([J].Journal of ClinicalOncology 35(2017):e15572-e15572)。
由于抗体分子量大、结构复杂，不同抗体之间的差异性也较大，不同抗体与离子交换剂的结合力不仅与其携带的电荷的数目有关，还与抗体分子量的大小及电荷排列等也有一定关系。因此，针对不同的抗体，优化出合适的离子层析纯化工艺，尽可能降低生产成本并提高抗体收率，是目前需要迫切解决的技术问题。
发明内容
本公开提供了一种利用阳离子层析纯化包含抗体和污染物的组合物的方法，包括以下步骤：
1)上样：将所述组合物加载到阳离子交换材料上；
2)清洗：用平衡缓冲液清洗阳离子交换材料；
3)洗脱：用洗脱缓冲液从阳离子交换材料上洗脱下目的抗体；
4)稀释：收集洗脱液，稀释。
一些实施方案中，在所述阳离子交换层析前还包括以下步骤：a)亲和层析；b)病毒灭活；c)阴离子膜层析。
一些实施方案中，所述亲和层析所使用的填料选自UniMab 50、UniMab HC、UniMabPro、MabSelect SuRe、MabSelect SuRe LX、MabSelect PrismA、Amsphere、ProSep UltraPlus(PUP)。在一些具体的实施方案中，所述亲和层析所使用的填料为MabSelect PrismA。
一些实施方案中，本公开的抗体为单克隆抗体。一些具体实施方案中，本公开的抗体为鼠源抗体、嵌合抗体、人源化抗体、全人抗体、骆驼抗体。一些具体实施方案中，本公开的抗体为全长抗体。
一些具体实施方案中，本公开的抗体为抗PD-1抗体。在一些具体实施方案中，所述抗PD-1抗体的重链CDR1、CDR2、CDR3的氨基酸序列如SEQ ID NO：1-3所示，轻链CDR1、CDR2、CDR3的氨基酸序列如SEQ ID NO：4-6所示；重链氨基酸序列如SEQ ID NO：7所示，轻链氨基酸序列如SEQ ID NO：8所示。抗PD-1抗体可以为变体，变体的重链CDR 1-3分别与SEQ IDNO：1-3具有至少70％、80％、90％、95％、98％或99％的同一性，和/或轻链CDR1、CDR2、CDR3分别与SEQ ID NO：4-6具有至少70％、80％、90％、95％、98％或99％的同一性。在一些实施方案中，变体的重链可变区与SEQ ID NO：9中的重链可变区具有至少70％、80％、90％、95％、98％或99％的同一性，和/或轻链可变区与SEQ ID NO：10的轻链可变区具有至少70％、80％、90％、95％、98％或99％的同一性。在一些实施方案中，变体的重链与SEQ IDNO：7具有至少70％、80％、90％、95％、98％或99％的同一性，和/或轻链与SEQ ID NO：8具有至少70％、80％、90％、95％、98％或99％的同一性。在一些实施方案中，变体的重链CDR 1较之SEQ ID NO：1有4、3、2、1个氨基酸突变；和/或重链CDR 2较之SEQ ID NO：2有4、3、2、1个氨基酸突变；和/或重链CDR 3较之SEQ ID NO：3有4、3、2、1个氨基酸突变；和/或轻链CDR 1较之SEQ ID NO：4有4、3、2、1个氨基酸突变；和/或轻链CDR 2较之SEQ ID NO：5有4、3、2、1个氨基酸突变；和/或轻链CDR 3较之SEQ ID NO：6有4、3、2、1个氨基酸突变。
表1.抗PD-1抗体的CDR序列
重链可变区
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSS
SEQ ID NO：9
轻链可变区
DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIK
SEQ ID NO：10
重链
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
SEQ ID NO：7
轻链
DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO：8
本专利中的抗体编码规则均采用kabat规则。
本公开提供了一种亲和层析纯化抗体的方法，采用低pH的洗脱缓冲液冲洗亲和层析柱进行产品洗脱并收集洗脱组分后无需调节pH或者电导率，直接进行低pH病毒灭活，具有操作简单、速度快、洗脱条件温和的特点。
发明详述
一、术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
本公开中的“抗体”以最广义使用，明确覆盖单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、及抗体片段，只要它们展现出期望的结合特异性。术语“抗体”涵盖“免疫球蛋白”，包括但不限于人抗体(或重组人抗体)、鼠源抗体、人抗体、人源化抗体、嵌合抗体。
术语“免疫球蛋白”是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。本公开的抗体还涵盖多价抗体(例如，双、多特异性抗体)。以及，本公开的抗体可以和任意的多肽、药物通过共价、非共价键连接。
术语“抗体或其抗原结合”或“功能片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，以及与抗体结合的Fv片段ScFv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。本公开的术语“与PD-1结合”，指能与人PD-1相互作用。本公开的术语“抗原结合位点”指抗原上不连续的，由本公开抗体或抗原结合片段识别的三维空间位点。
术语“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab‘片段，F(ab’)2片段，以及与人PD-1结合的Fv片段sFv片段；包含本公开所述抗体的选自SEQ ID NO:1至SEQIDNO:6中的一个或多个CDR区。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。本公开的术语“与PD-1结合”，指能与人PD-1相互作用。本公开的术语“抗原结合位点”指抗原上不连续的，由本公开抗体或抗原结合片段识别的三维空间位点。
术语“人抗体”或“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗体，所涉及的技术和方法在本领域中是熟知的，诸如：
(1)从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；
(2)从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；
(3)从重组组合人抗体文库中分离的抗体；以及
(4)通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。
此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的针对抗原或其表位的单克隆抗体。制备时用抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个具体的实施方案中，所述的鼠源抗抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本公开的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-graftedantibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，如包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。
术语“抗PD-1抗体”、“针对PD-1的抗体”和“针对PD-1蛋白的抗体”指能够以足够的亲和力结合PD-1蛋白，使得所述抗体可在靶向PD-1蛋白中用作诊断剂和/或治疗剂。本文中使用的术语“结合PD-1蛋白”是指，在BIAcore测定(Pharmacia Biosensor AB，Uppsala，瑞典)中或在ELISA中抗体对PD-1蛋白的结合，其中将经纯化的PD-1蛋白或PD-1蛋白CHO转染子包被在微量滴定板上。
术语“污染物”是指与期望的抗体产物不同的物质。污染物包括但不限于：宿主细胞物质，如中国仓鼠卵巢蛋白质(CHOP)；浸出的蛋白A；核酸；期望抗体的变体、片段、聚集物或衍生物；其它多肽；内毒素；病毒污染物；细胞培养基成分。
术语“离子交换材料”是指作为将该不需要的物质或杂质(例如HCP或组织蛋白酶L酶原)与该抗体进行分离的基础来使用的离子材料。示例性的离子交换材料包括但不限于阴离子树脂和阳离子树脂。
术语“阴离子交换材料”是指具有共价结合的带正电基团，如季氨基基团的离子交换树脂。市售的阴离子交换树脂包括但不限于DEAE纤维素、TMAE、QAE SephadexTM、Fast Q琼脂糖凝胶TM、Poros 50HQ、Poros XQ、GE Qhp、GE Capto Q和GECapto Q ImpRes。
术语“阳离子交换材料”是指这样的离子交换材料，它具有共价结合的带负电的配体，并因此具有游离阳离子可供与它所接触到的溶液中的阳离子进行交换。有多种阳离子交换树脂为本领域所公知，包括但不限于其中共价结合基团是羧酸跟或磺酸跟的阳离子交换树脂。市售的阳离子交换树脂包括但不限于CMC-纤维素、SP-SephadexTM和Fast S-SepharoseTM、POROS HS、POROS XS、SP-SEPHAROSE FAST FLOW、Capto S ImpAct、NuviaHRS、Nuvia S、Fractogel EMD SO3-(M)和Eshmuno CPX。
术语“缓冲液”是指通过其酸-碱成对成分的作用来抵抗pH变化的溶液。用于生物试剂的缓冲溶液通常能够维持恒定浓度的氢离子，使得溶液的pH在生理范围内。传统的缓冲成分包括但不限于有机和无机的盐、酸和碱。Buffers.AGuide for the Preparationand Use of Buffers in Biological Systems，Gueffroy，D.，Ed.CalbiochemCorporation(1975)中记载了取决于例如期望的缓冲液pH而可采用的多种缓冲液。示例性的“缓冲液”包括但不限于醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸、草酸盐、乳酸盐、磷酸盐、柠檬酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲液。示例性的“柠檬酸(盐)缓冲液”是包括柠檬酸根离子的缓冲液。柠檬酸盐缓冲液的示例包括柠檬酸-柠檬酸钠、柠檬酸-柠檬酸钾、柠檬酸-柠檬酸钙、柠檬酸-柠檬酸镁等。示例性的柠檬酸盐缓冲液是柠檬酸-柠檬酸钠。
术语“平衡缓冲液”在本公开中是指用于在将包含感兴趣抗体和一种或多种污染物的组合物加载在离子交换材料上之前平衡离子交换材料的缓冲液。
术语“洗涤缓冲液”或“洗涤液”在本公开中是指用以从结合由抗体的给定材料(例如亲和层析材料)带走杂质的缓冲液。
术语“洗脱缓冲液”或“洗脱液”是指用以将抗体从经一种或多种洗涤物质洗涤过的给定材料(例如亲和层析材料、离子交换材料)解离下来的缓冲液。洗脱缓冲液起到解离抗体的作用。典型的洗脱物质是本领域公知的，它们可具有较高浓度的盐、游离亲和配体或类似物或者其他能促进靶物质(例如抗体)从给定材料的解离的物质。洗脱缓冲液的电导率和/或pH使得该靶物质从该给定材料洗脱下来。
术语“洗杂缓冲液”或“洗杂液”是指在加载组合物之后且在洗脱感兴趣蛋白质之前流过给定材料(例如离子交换材料)的缓冲液。洗杂缓冲液可用于自给定材料(例如离子交换材料)清除一种或多种污染物，基本上不洗脱期望抗体产物。
术语“再生缓冲液”可以用来再生层析柱，从而使它能被再次使用。再生缓冲液具有所需的能充分将所有污染物和感兴趣的多肽从层析柱上除去的导电率和/或pH。
术语“电导率”是指水溶液在两个电极之间传导电流的能力。在溶液中，电流通过离子运输来流动，随着水溶液中存在的离子的量越来越多，溶液会具有更高的电导率。电导率的度量的基本单位是西门子(或欧姆)、欧姆(mS/cm)，而且可以使用电导率计来测量，诸如各种型号的Orion电导率计。因为电解电导率是溶液中的离子携带电流的能力，所以溶液的电导率可以通过改变其中的离子浓度来改变。例如可以改变溶液中缓冲剂的浓度和/或盐(例如氧化纳、乙酸钠、或氧化钾)的浓度来实现期望的电导率。
术语“病毒灭活”包括使混合物中所含的病毒失去功能，或者将病毒从待纯化的混合物中除去。该病毒可发源于抗体生产、下游加工步骤或制造环境。使病毒失去功能或除去病毒的方法是本领域公知的，包括但不限于热灭活(巴氏杀菌)、pH灭活、溶剂/去污剂处理、化学灭活剂灭活(如β-丙内脂或者例如美国专利4,534,972等中所述的菲咯啉铜)、紫外光和伽马射线辐射。
术语“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
术语“约”或“基本上包含”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多10％的范围。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
具体实施方式
以下结合实施例进一步描述，但所述实施例并非限制的范围。
实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1
将含有抗PD-1单抗的细胞澄清液按照40g/L填料的量上样到MabSelect PrismA亲和层析柱，用pH 7.0的20mM磷酸+1M氯化钠缓冲液冲洗亲和层析柱5倍柱体积，采用pH 3.0的50mM柠檬酸缓冲液洗脱并收集亲和收集液，孵育90min，完成低pH病毒灭活。将低pH病毒灭活液回调pH至5.0，过滤后得到低pH病毒灭活中和液，然后按照150mg/mL填料的量上样到Capto Q层析柱，收集阴离子层析流穿液。将阴离子层析流穿液按照90mg/mL填料上样到Fractogel EMD SO3-(M)层析柱，用pH5.0的20mM柠檬酸缓冲液冲洗阳离子层析柱5倍柱体积，采用pH5.0的20mM柠檬酸+200mM氯化钠缓冲液冲洗阳离子层析柱进行产品洗脱并收集阳离子洗脱收集液。检测最终获得的蛋白总量和SEC纯度。
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Claims (7)
1.一种利用阳离子层析纯化包含抗体和污染物的组合物的方法，包括以下步骤：
1)上样：将所述组合物加载到阳离子交换材料上；
2)清洗：用平衡缓冲液清洗阳离子交换材料；
3)洗脱：用洗脱缓冲液从阳离子交换材料上洗脱下目的抗体；
4)稀释：收集洗脱液，稀释，
其特征在于，其中所述抗体为抗PD-1抗体或其抗原结合片段，并且，轻链可变区包含分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；重链可变区包含分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3。
2.如权利要求1所述的方法，其特征在于，在阳离子交换层析前还包括以下步骤：
a)亲和层析；
b)病毒灭活；
c)阴离子膜层析。
3.如权利要求2所述的方法，其特征在于，所述亲和层析所使用的填料选自UniMab 50、UniMab HC、UniMab Pro、MabSelect SuRe、MabSelect SuRe LX、MabSelect PrismA、Amsphere、ProSep Ultra Plus(PUP)，优选为MabSelect PrismA。
4.如权利要求1或2所述的方法，其特征在于，其中所述抗PD-1抗体或其抗原结合片段选自抗PD-1人源化抗体。
5.如权利要求1所述的方法，其特征在于，其中所述抗PD-1人源化抗体的轻链可变区序列为如SEQ ID NO:10所示的序列或其变体，所述的变体优选在轻链可变区有0-10的氨基酸变化，更优选为A43S的氨基酸变化；重链可变区序列为如SEQ ID NO:9所示的序列或其变体，所述变体优选在重链可变区有0-10的氨基酸变化，更优选为G44R的氨基酸变化。
6.如权利要求1所述的方法，其特征在于，其中所述的抗PD-1人源化抗体轻链序列为如SEQ ID NO:8所示的序列，重链序列为如SEQ ID NO:7所示的序列。
7.一种组合物，其包含由权利要求1-6任一项所述方法获得的抗体以及缓冲液。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110272491A (zh) * | 2018-03-13 | 2019-09-24 | 江苏恒瑞医药股份有限公司 | 一种抗pd-1抗体的纯化工艺 |
| CN115477702A (zh) * | 2022-10-27 | 2022-12-16 | 广州誉衡生物科技有限公司 | 一种抗pd-1抗体亲和层析纯化工艺 |
| WO2023030064A1 (zh) * | 2021-09-01 | 2023-03-09 | 四川科伦博泰生物医药股份有限公司 | 一种提高抗体药物偶联物产品质量的方法 |
| WO2023040940A1 (zh) * | 2021-09-15 | 2023-03-23 | 江苏恒瑞医药股份有限公司 | Pvrig/tigit结合蛋白联合免疫检查点抑制剂用于治疗癌症 |
-
2021
- 2021-03-04 CN CN202110241410.5A patent/CN112851813A/zh active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110272491A (zh) * | 2018-03-13 | 2019-09-24 | 江苏恒瑞医药股份有限公司 | 一种抗pd-1抗体的纯化工艺 |
| CN110272491B (zh) * | 2018-03-13 | 2023-01-24 | 江苏恒瑞医药股份有限公司 | 一种抗pd-1抗体的纯化工艺 |
| WO2023030064A1 (zh) * | 2021-09-01 | 2023-03-09 | 四川科伦博泰生物医药股份有限公司 | 一种提高抗体药物偶联物产品质量的方法 |
| WO2023040940A1 (zh) * | 2021-09-15 | 2023-03-23 | 江苏恒瑞医药股份有限公司 | Pvrig/tigit结合蛋白联合免疫检查点抑制剂用于治疗癌症 |
| CN115477702A (zh) * | 2022-10-27 | 2022-12-16 | 广州誉衡生物科技有限公司 | 一种抗pd-1抗体亲和层析纯化工艺 |
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