Optimization and application of proteomic methods for characterization of host cell protein impurities from Chinese hamster ovary cells
Author(s) | Valente, Kristin | |
Date Accessioned | 2022-09-14T16:43:15Z | |
Date Available | 2022-09-14T16:43:15Z | |
Publication Date | 2014 | |
SWORD Update | 2022-08-11T19:08:30Z | |
Abstract | Biopharmaceuticals, including monoclonal antibodies (mAbs) and other therapeutic proteins, are typically secreted by Chinese hamster ovary (CHO) cells along with hundreds of endogenous host cell protein (HCP) impurities that must be removed from the therapeutic product for patient safety. Identification and characterization of these extracellular CHO HCPs by proteomic techniques, such as two-dimensional electrophoresis (2DE) and shotgun methods, can aid process design, resulting in improved biopharmaceutical manufacturing operations. ☐ Sample preparation protocols that maximize total protein recovery are fundamental to increasing the utility of proteomic methods and extracellular CHO HCP recovery is particularly challenging due to the relatively low protein concentration. In this work, design of experiment (DOE) methods are presented for optimizing sample preparation parameters, and favorable conditions are demonstrated to vary across different sample types. Additionally, we developed protocols that improve extracellular CHO HCP capture to facilitate analysis by 2DE and shotgun workflows, with optimized precipitation conditions demonstrated to differ between the two techniques. ☐ As biopharmaceutical manufacturing moves towards continuous bioprocessing, it is important to consider the impact of extended culture of CHO cells and therefore this work monitors the extracellular CHO HCP impurity profile over 500 days of culture. Many extracellular CHO HCPs evade clearance during downstream purification due to product association with mAbs or by exhibiting similar retention characteristics to mAbs on chromatographic media. This work characterizes the methods by which specific HCP impurities co-purify with mAbs by exploring mAb association in varied solution conditions and evaluating the retention characteristics of extracellular HCPs across polishing chromatography operations. ☐ In this work, difficult-to-remove HCPs are defined as impurities that exhibit (1) variable expression with cell age, (2) product association with mAbs, or (3) similar retention to mAbs on chromatographic media. In total, 116 difficult-to-remove HCPs were identified, with 10 of these HCPs challenging purification by all three methods. Short interfering RNA (siRNA) technology was applied to reduce expression of one of these HCPs. Because the difficult-to-remove HCPs presented here may persist across purification operations into the final drug product, further investigation is needed to ensure their removal during bioprocessing and improve the safety of biopharmaceuticals. | en_US |
Advisor | Lee, Kelvin H. | |
Advisor | Lenhoff, Abraham M. | |
Degree | Ph.D. | |
Department | University of Delaware, Department of Chemical and Biomolecular Engineering | |
DOI | https://doi.org/10.58088/zykt-kg21 | |
Unique Identifier | 1344391294 | |
URL | https://udspace.udel.edu/handle/19716/31358 | |
Language | en | |
Publisher | University of Delaware | en_US |
URI | https://login.udel.idm.oclc.org/login?url=https://www.proquest.com/dissertations-theses/optimization-application-proteomic-methods/docview/2708506162/se-2?accountid=10457 | |
Keywords | Biopharmaceuticals | |
Keywords | Biotechnology | |
Keywords | Chinese hamster ovary | |
Keywords | Difficult-to-remove impurities | |
Keywords | Host cell proteins | |
Keywords | Proteomics | |
Title | Optimization and application of proteomic methods for characterization of host cell protein impurities from Chinese hamster ovary cells | en_US |
Type | Thesis | en_US |