Rapid Whole Monoclonal Antibody Analysis by Mass Spectrometry: An Ultra Scale-Down Study of the Effect of Harvesting by Centrifugation on the Post-Translational Modification Profile
Rapid Whole Monoclonal Antibody Analysis by Mass Spectrometry: An Ultra Scale-Down Study of the Effect of Harvesting by Centrifugation on the Post-Translational Modification Profile
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DOI：
10.1002/bit.22790
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发表时间：
2010-09-01
影响因子：
3.8
通讯作者：
Hoare, M.
中科院分区：
文献类型：
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作者：
Reid, C. Q.;Tait, A.;Hoare, M.
随着越来越多的复杂生物制药（基于蛋白质的）产品的产生和生产的趋势，在稳健性和再现性方面表征产品和生产工艺的需求和要求都在增加。这对于来自哺乳动物细胞培养物的产品特别重要，这些产品具有大分子结构，并且通常具有复杂的翻译后修饰（PTM），这些修饰可能影响最终产品的有效性、稳定性和最终安全性。因此，了解生物工艺的操作条件如何影响这些PTM的分布和组成，以确保最终产品的质量和活性一致，这一点至关重要。在此，我们表征了典型的生物过程，并确定了（a）从哺乳动物细胞培养物中收获的时间，以及（B）通过使用超缩小模拟物，初步回收阶段的性质如何影响重组IgG（4）单克隆抗体上观察到的PTM的分布和组成。特别是，我们描述了使用快速全抗体分析质谱分析同时发生的变化，重链C-末端赖氨酸残基的切割和糖基化模式，以及HL二聚体的存在。发现收获时间对观察到的糖基化模式的范围有很大影响，但对C-末端赖氨酸裂解没有影响。培养时间对抗体分子上发现的不同聚糖部分的比率具有深远影响。短聚糖的比例增加（例如，（G 0 F）（2）20-35%），随着培养时间的延长，长聚糖的比例也相应降低（e.例如，在一个实施例中，(G2F)2 74%，G1 F/G2 F从15.2%降至7.8%）。超规模缩小模拟物显示，这些培养物的后续处理不会改变所研究的翻译后修饰，但确实增加了工艺流中存在的半抗体的比例。超规模缩小方法和质谱法全抗体分析的结合证明，可以在开发过程的早期快速确定加工对单克隆抗体详细分子结构的影响。在这项研究中，我们已经证明了这种分析适用于关键工艺设计决策（例如：例如，在一个实施例中，收获时间），但是该方法也可以用作关于平台方法用于制备新药候选物的适用性的选择标准。该方法还为生物工艺工程师提供了在发现阶段预测生物工艺将如何影响最终产品质量的手段。Biotechnol. Bioeng. 2010; 107：85-95. (C)2010 Wiley Periodicals，Inc.
With the trend towards the generation and production of increasing numbers of complex biopharmaceutical (protein based) products, there is an increased need and requirement to characterize both the product and production process in terms of robustness and reproducibility. This is of particular importance for products from mammalian cell culture which have large molecular structures and more often than not complex post-translational modifications (PTMs) that can impact the efficacy, stability and ultimately the safety of the final product. It is therefore vital to understand how the operating conditions of a bioprocess affect the distribution and make up of these PTMs to ensure a consistent quality and activity in the final product. Here we have characterized a typical bioprocess and determined (a) how the time of harvest from a mammalian cell culture and, (b) through the use of an ultra scale-down mimic how the nature of the primary recovery stages, affect the distribution and make up of the PTMs observed on a recombinant IgG(4) monoclonal antibody. In particular we describe the use of rapid whole antibody analysis by mass spectrometry to analyze simultaneously the changes that occur to the cleavage of heavy chain C-terminal lysine residues and the glycosylation pattern, as well as the presence of HL dimers. The time of harvest was found to have a large impact upon the range of glycosylation patterns observed, but not upon C-terminal lysine cleavage. The culture age had a profound impact on the ratio of different glycan moieties found on antibody molecules. The proportion of short glycans increased (e.g., (G0F)(2) 20-35%), with an associated decrease in the proportion of long glycans with culture age (e. g., (G2F) 2 74%, and G1F/G2F from 15.2% to 7.8%). Ultra scale-down mimics showed that subsequent processing of these cultures did not change the post-translational modifications investigated, but did increase the proportion of half antibodies present in the process stream. The combination of ultra scale-down methodology and whole antibody analysis by mass spectrometry has demonstrated that the effects of processing on the detailed molecular structure of a monoclonal antibody can be rapidly determined early in the development process. In this study we have demonstrated this analysis to be applicable to critical process design decisions (e. g., time of harvest) in terms of achieving a desired molecular structure, but this approach could also be applied as a selection criterion as to the suitability of a platform process for the preparation of a new drug candidate. Also the methodology provides means for bioprocess engineers to predict at the discovery phase how a bioprocess will impact upon the quality of the final product. Biotechnol. Bioeng. 2010; 107: 85-95. (C) 2010 Wiley Periodicals, Inc.