Determination of the origin of the N-terminal pyro-glutamate variation in monoclonal antibodies using model peptides
Determination of the origin of the N-terminal pyro-glutamate variation in monoclonal antibodies using model peptides
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DOI：
10.1002/bit.21260
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发表时间：
2007-06-15
影响因子：
3.8
通讯作者：
Cheng, Kuang-Chuan
中科院分区：
文献类型：
--
作者：
Dick, Lawrence W., Jr.;Kim, Catherine;Cheng, Kuang-Chuan
本工作的目的是确定重组蛋白和单克隆抗体中n端谷氨酰胺环化的原因。这种环化反应通常发生在抗体的轻链和/或重链的n端，并导致最终产物的异质性。利用两种模型肽和一种含有n端谷氨酰胺的抗体，研究了在不同实验条件和不同生物合成过程阶段下n端热-谷氨酸的形成。采用LC-MS分析分离和定量n端变异。实验证明，环化反应是自发的，高度依赖于温度和缓冲液的组成，而不依赖于ph。大多数生物制药过程中存在的条件加速了这种变体的形成。通常在抗体中观察到的n端谷氨酰胺到焦谷氨酸的几乎完全转化（约95%）似乎发生在生物反应器内，只有一小部分来自纯化、配方和分析准备。
The purpose of this work is to determine the, cause of the cyclization of the N-terminal glutamine in recombinant proteins and monoclonal antibodies. This cyclization reaction commonly occurs on the N-terminal of light and/or heavy chains of antibodies and leads to heterogeneity of the final product. Two model peptides and an antibody containing an N-terminal glutamine were used to investigate the formation of N-terminal pyro-glutamic acid under various experimental conditions and different stages of the biosynthetic process. LC-MS analysis was used to separate and quantify the N-terminal variants. Experiments prove that the cyclization reaction is spontaneous and highly dependent on temperature and buffer composition and less dependent on pH. The conditions presented in most biopharmaceutical processes accelerate the formation of this variant. The majority of the near complete conversion (> 95%) of N-terminal glutamine to pyro-glutamic acid commonly observed for antibodies appears to occur inside the bioreactor with only a small contribution from purification, formulation, and analytical preparation.