重组嵌合抗CD20单克隆抗体一级结构确证及其糖链功能初步研究

发布时间：2018-01-21 20:40

本文关键词： 单克隆抗体结构确证糖链功能研究　出处：《中国药品生物制品检定所》2008年硕士论文　论文类型：学位论文

【摘要】： 单抗药物是近年来复合增长率最快的一类生物技术药物,全球年销售额从1997年的3.10亿美元上升到2004年的103亿美元,并预测2010年将达到303亿美元;其在生物制药产业中所占份额也从2000年的1/5上升到2004年的1/4,并将在2010年达到1/3,单抗药物具有巨大的经济价值和社会价值。为保证单抗药物的用药安全,必须对其进行严格的质量控制。单抗药品与重组细胞因子类药物不同,其分子量大、结构复杂、容易发生翻译后修饰,质量研究相对复杂,研究建立与之适应的质量控制方法尤为必要和重要。针对此问题,本论文选择一种具有代表性的重组嵌合抗CD20单抗作为研究对象,对其一级结构进行了确证,并就单抗所含糖链对其结构与功能的影响进行了初步研究。论文第一部分对单抗的一级结构进行了确证。首先用液质联用的方法测定了单抗轻、重链的精确分子量,相对误差均小于0.01%。通过分别测定脱糖前后单抗重链的分子量确定其糖链类型为岩藻糖化的双触角复杂型N-糖,糖链存在不均一性,非还原端未发生唾液酸化,糖含量介于2.0%与2.4%之间。单抗N-末端存在焦谷氨酸封闭,对其进行焦谷氨肽酶酶解后用Edman降解法测定了其N-末端氨基酸序列,测定结果与理论值一致。通过绘制单抗的质量肽图确证了单抗的氨基酸序列(氨基酸覆盖率97%)、鉴定了其糖基化位点(Asn301)、并确证了其中10对链内二硫键的配对方式。第二部分分析了糖链对单抗结构的影响,主要是对单抗铰链区空间结构及其二级结构的影响。实验发现去除糖链后单抗铰链区的木瓜蛋白酶酶切位点发生变化,说明糖链的切除对单抗铰链区的空间结构产生了影响。圆二色谱测定结果显示:糖链切除前后单抗的圆二色谱不一致,说明糖链的切除对单抗的二级结构产生了影响。第三部分分析了糖链对单抗生物学功能的影响,主要是对抗体CDC活性的影响。测定结果显示去除糖链后单抗的CDC活性基本消失,说明糖链对其十分重要。该活性的产生主要涉及2种蛋白间的相互作用:抗体与细胞表面CD20抗原的结合以及补体与抗原抗体复合物的结合,并进一步设计实验对糖链影响单抗CDC活性的机制进行了研究。应用竞争法测定了糖链切除前后抗体与CD20抗原的结合能力,结果显示糖链切除后抗原抗体结合能力下降约40%,说明糖链切除影响抗体与抗原的结合并进一步影响抗体的CDC活性。应用BIAcore直接测定抗体与补体的结合能力,结果显示抗体与补体未发生明显的相互作用,原因为游离抗体的补体结合位点未暴露,不能与补体结合。综上所述,本课题对单抗的一级结构进行了细致的确证,建立并完善了一系列单抗结构分析方法,可为重组单抗制品的质量控制提供参考;另一方面,通过分析单抗所含糖链对其结构与功能的影响揭示了糖链的重要性,为重组糖蛋白类药物质量标准的建立提供了依据。
[Abstract]:Monoclonal antibody drugs are a class of biotech drugs combined the fastest growth rate in recent years, the global annual sales rose from $310 million in 1997 to $10 billion 300 million in 2004, and forecast in 2010 will reach $30 billion 300 million; its share rose from 1/5 in 2000 to 1/4 in 2004 in the biopharmaceutical industry, and will reach in 2010 1/3, monoclonal antibody drugs has great economic value and social value.
In order to ensure the safety of monoclonal antibody drugs, must carry on the strict quality control of the drug and recombinant cytokines. Monoclonal antibody drugs are different, their high molecular weight, complex structure, easy occurrence of post-translational modifications, quality research is relatively complex, the research establishment and to adapt the quality control method is particularly necessary and important. In order to solve this problem. This paper selects a representative of recombinant chimeric anti CD20 monoclonal antibody as the research object, the basic structure was confirmed, and the effect of sugar chain mAb on its structure and functions were studied.
In the first part of the monoclonal antibody level structure were confirmed. The first method with liquid chromatography-mass spectrometry determination of monoclonal antibody light, accurate molecular weight of the heavy chain, the relative error is less than 0.01%. by determined the sugar chain type fucosylated biantennary complex type N- sugar sugar after the molecular weight of the heavy chain and monoclonal antibody respectively, the heterogeneity of the sugar chain, non reducing end did not occur between sialic acid, sugar content is between 2% and 2.4%. The mAb N- terminal pyroglutamic acid closed the pyroglutamate aminopeptidase after enzymatic hydrolysis by Edman degradation method of the N- terminal amino acid sequence was determined. The determination results are consistent with the theoretical value of the. The quality of rendering monoclonal antibody peptide mapping confirmed the amino acid sequence of monoclonal antibodies (amino acid coverage rate of 97%), the glycosylation sites were identified (Asn301), and confirmed 10 of them in the chain of two disulfide pairing.
The second part analyzes the influence on the structure of sugar chain monoclonal antibody, is the main influence on the spatial structure and the hinge region of mAb two level structure. The experimental results showed that the removal of the sugar chain after papain mAb hinge cutting site changes, suggest that excision of sugar chain against hinge region spatial structure influence. The determination results round two: sugar chain chromatography showed that monoclonal antibody before and after excision of round two was not consistent, that of the sugar chain resection of mAb two level structure of impact.
The third part analyzes the influence on the biological function of sugar chain monoclonal antibody, is the main influence on the activity of CDC antibody were measured. The results show that the removal of the sugar chain after mAb CDC activity disappeared, that it is very important to the sugar chain. The activity produced mainly involve 2 types of protein-protein interactions: a combination of antibody and cell surface CD20 antigen and complement and antigen antibody complexes, and further design experiments on the sugar chain CDC antibody activity mechanism was studied.
Binding capacity of sugar chain before and after excision of antibody and CD20 antigen was determined by competition law, results showed that the sugar chain after removal of antigen antibody binding capacity decreased by about 40%, indicating the removal of oligosaccharides combined influence of antibody and antigen antibody and further affect the activity of CDC. Combined with the ability of direct determination of antibody and complement of BIAcore applications, and the results showed that the antibody complement without obvious interactions, due to the free antibody complement binding sites is not exposed, and complement fixation.
To sum up, the primary structure of the monoclonal antibody in detail were established and improved analysis method of a series of monoclonal antibody structure, provide reference for quality control of recombinant monoclonal antibody products; on the other hand, through the analysis of monoclonal antibody of sugar chain on the structure and function of the influence and reveals the importance of the sugar chain. It provides a basis for recombinant glycoproteins drug quality standard.

【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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