Proteomic Profiling of IgG1 Producing CHO Cells Using LC/LC-SPS-MS3: The Effects of Bioprocessing Conditions on Productivity and Product Quality

Lisa Strasser; Amy Farrell; Jenny T C Ho; Kai Scheffler; Ken Cook; Patrick Pankert; Peter Mowlds; Rosa Viner; Barry L Karger; Jonathan Bones

doi:10.3389/fbioe.2021.569045

Proteomic Profiling of IgG1 Producing CHO Cells Using LC/LC-SPS-MS³: The Effects of Bioprocessing Conditions on Productivity and Product Quality

Front Bioeng Biotechnol. 2021 Apr 9:9:569045. doi: 10.3389/fbioe.2021.569045. eCollection 2021.

Authors

Affiliations

¹ Characterization and Comparability Laboratory, National Institute for Bioprocessing Research and Training, Dublin, Ireland.
² Thermo Fisher Scientific, Hemel Hempstead, United Kingdom.
³ Thermo Fisher Scientific, Germering, Germany.
⁴ Thermo Fisher Scientific, Dreieich, Germany.
⁵ Thermo Fisher Scientific, San Jose, CA, United States.
⁶ Barnett Institute of Chemical and Biological Analysis, Northeastern University, Boston, MA, United States.
⁷ School of Chemical and Bioprocess Engineering, University College Dublin, Dublin, Ireland.

Abstract

The biopharmaceutical market is dominated by monoclonal antibodies, the majority of which are produced in Chinese hamster ovary (CHO) cell lines. Intense cell engineering, in combination with optimization of various process parameters results in increasing product titers. To enable further improvements in manufacturing processes, detailed information about how certain parameters affect cellular mechanisms in the production cells, and thereby also the expressed drug substance, is required. Therefore, in this study the effects of commonly applied changes in bioprocessing parameters on an anti-IL8 IgG1 producing CHO DP-12 cell line were investigated on the level of host cell proteome expression combined with product quality assessment of the expressed IgG1 monoclonal antibody. Applying shifts in temperature, pH and dissolved oxygen concentration, respectively, resulted in altered productivity and product quality. Furthermore, analysis of the cells using two-dimensional liquid chromatography-mass spectrometry employing tandem mass tag based isotopic quantitation and synchronous precursor selection-MS³ detection revealed substantial changes in the protein expression profiles of CHO cells. Pathway analysis indicated that applied bioprocessing conditions resulted in differential activation of oxidative phosphorylation. Additionally, activation of ERK5 and TNFR1 signaling suggested an affected cell cycle. Moreover, in-depth product characterization by means of charge variant analysis, peptide mapping, as well as structural and functional analysis, revealed posttranslational and structural changes in the expressed drug substance. Taken together, the present study allows the conclusion that, in anti-IL8 IgG1 producing CHO DP-12 cells, an improved energy metabolism achieved by lowering the cell culture pH is favorable when aiming towards high antibody production rates while maintaining product quality.

Keywords: CHO cells; SPS-MS3; TMT; bioprocess monitoring; monoclonal antibody; protein characterization; proteomic profiling.