Predicting the Expression of Recombinant Monoclonal Antibodies in Chinese Hamster Ovary Cells Based on Sequence Features of the CDR3 Domain
Predicting the Expression of Recombinant Monoclonal Antibodies in Chinese Hamster Ovary Cells Based on Sequence Features of the CDR3 Domain
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DOI：
10.1002/btpr.1839
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发表时间：
2014-01-01
影响因子：
2.9
通讯作者：
Field, Ray
中科院分区：
文献类型：
--
作者：
Pybus, Leon P.;James, David C.;Field, Ray
尽管开发了能够生产 >10 g L-1 重组单克隆抗体 (MAb) 的高滴度生物工艺，但一些所谓的难以表达 (DTE) MAb 只能达到低得多的工艺滴度。对于广泛使用的平台过程，唯一的离散变量是重组产物的蛋白质编码序列。然而，很少有系统的研究来确定影响表达的序列参数。这些信息至关重要，因为它将使我们能够合理设计基因序列和工程策略以实现最佳生物加工。因此，我们开发了一种新的计算工具，能够根据表达的 MAb 的重组编码序列预测中国仓鼠卵巢 (CHO) 细胞中的 MAb 滴度。模型构建使用了一组 MAb，经过 10 天的分批补料瞬时生产过程后，滴度变化为 5.6 倍，从而可以分析影响一系列高和低 MAb 生产力表达的序列特征。该模型鉴定了互补决定区 3 (CDR3) 内的 18 个轻链 (LC) 特异性序列特征，能够以 0.585 相对表达单位的均方根误差预测 MAb 滴度。此外，我们发现 CDR3 变异会影响 MAb 合成过程中 LC-HC 二聚化的速率，这可用于通过增加转染的 LC:HC 基因比率来提高 DTE MAb 变体的产量。总而言之，这些数据表明，基于对产生重组产物 DTE 的序列基序的鉴定，可以合理地实施改善 DTE 重组产物表达的工程干预策略。 (c) 2013 年美国化学工程师生物技术研究所。编, 30:188-197, 2014
Despite the development of high-titer bioprocesses capable of producing >10 g L-1 of recombinant monoclonal antibody (MAb), some so called difficult-to-express (DTE) MAbs only reach much lower process titers. For widely utilized platform processes the only discrete variable is the protein coding sequence of the recombinant product. However, there has been little systematic study to identify the sequence parameters that affect expression. This information is vital, as it would allow us to rationally design genetic sequence and engineering strategies for optimal bioprocessing. We have therefore developed a new computational tool that enables prediction of MAb titer in Chinese hamster ovary (CHO) cells based on the recombinant coding sequence of the expressed MAb. Model construction utilized a panel of MAbs, which following a 10-day fed-batch transient production process varied in titer 5.6-fold, allowing analysis of the sequence features that impact expression over a range of high and low MAb productivity. The model identified 18 light chain (LC)-specific sequence features within complementarity determining region 3 (CDR3) capable of predicting MAb titer with a root mean square error of 0.585 relative expression units. Furthermore, we identify that CDR3 variation influences the rate of LC-HC dimerization during MAb synthesis, which could be exploited to improve the production of DTE MAb variants via increasing the transfected LC:HC gene ratio. Taken together these data suggest that engineering intervention strategies to improve the expression of DTE recombinant products can be rationally implemented based on an identification of the sequence motifs that render a recombinant product DTE. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:188-197, 2014