Factors affecting product association as a mechanism of host‐cell protein persistence in bioprocessing

中国仓鼠卵巢细胞 单克隆抗体 机制(生物学) 持久性(不连续性) 亲缘关系 生物 化学 细胞培养 抗体 色谱法 生物化学 计算生物学 遗传学 岩土工程 哲学 工程类 认识论
作者
Young Hoon Oh,Matthew L. Becker,Kerri M. Mendola,Leila H. Choe,Lie Min,Kelvin H. Lee,Yinges Yigzaw,Alexander Seay,Jerome Bill,Xuanwen Li,David J. Roush,Steven M. Cramer,Stefano Menegatti,Abraham M. Lenhoff
出处
期刊:Biotechnology and Bioengineering [Wiley]
卷期号:121 (4): 1283-1296 被引量:4
标识
DOI:10.1002/bit.28658
摘要

Abstract Product association of host‐cell proteins (HCPs) to monoclonal antibodies (mAbs) is widely regarded as a mechanism that can enable HCP persistence through multiple purification steps and even into the final drug substance. Discussion of this mechanism often implies that the existence or extent of persistence is directly related to the strength of binding but actual measurements of the binding affinity of such interactions remain sparse. Two separate avenues of investigation of HCP‐mAb binding are reported here. One is the measurement of the affinity of binding of individual, commonly persistent Chinese hamster ovary (CHO) HCPs to each of a set of mAbs, and the other uses quantitative proteomic measurements to assess binding of HCPs in a null CHO harvested cell culture fluid (HCCF) to mAbs produced in the same cell line. The individual HCP measurements show that the binding affinities of individual HCPs to different mAbs can vary appreciably but are rarely very high, with only weak pH dependence. The measurements on the null HCCF allow estimation of individual HCP‐mAb affinities; these are typically weaker than those seen in affinity measurements on isolated HCPs. Instead, the extent of binding appears correlated with the initial abundance of individual HCPs in the HCCF and the forms of the HCPs in the solution, i.e., whether HCPs are present as free molecules or as parts of large aggregates. Separate protein A chromatography experiments performed by feeding different fractions of a mAb‐containing HCCF obtained by size‐exclusion chromatography (SEC) showed clear differences in the number and identity of HCPs found in the protein A eluate. These results indicate a significant role for HCP‐mAb association in determining HCP persistence through protein A chromatography, presumably through binding of HCP‐mAb complexes to the resin. Overall, the results illustrate the importance of considering more fully the biophysical context of HCP‐product association in assessing the factors that may affect the phenomenon and determine its implications. Knowledge of the abundances and the forms of individual or aggregated HCPs in HCCF are particularly significant, emphasizing the integration of upstream and downstream bioprocessing and the importance of understanding the collective properties of HCPs in addition to just the biophysical properties of individual HCPs.
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