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

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

doi:10.3389/fbioe.2021.569045

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.

Highlights

With an increasing number of approvals every year, the biopharmaceutical market is dominated by monoclonal antibodies which are most commonly produced in Chinese hamster ovary (CHO) cells (Kaplon and Reichert, 2018; Walsh, 2018)
When cells entered the stationary phase, conditions were changed, as outlined in Table 1, maintaining the central point of each experiment constant (37◦C, 85% dissolved oxygen (DO)) to generate a biological triplicate that was used for reproducibility assessment
The relative standard deviation of the measured IgG concentration on day 7 was found to be below 1% (n = 3), highlighting the excellent repeatability that can be obtained using wave-mixed bioreactors

Summary

Introduction

With an increasing number of approvals every year, the biopharmaceutical market is dominated by monoclonal antibodies (mAbs) which are most commonly produced in Chinese hamster ovary (CHO) cells (Kaplon and Reichert, 2018; Walsh, 2018). It was shown that an improved energy metabolism under hyperosmotic conditions favors specific productivity of CHO cells (Lee et al, 2003). While most of these studies focused on the effects of one factor at a time, more systemic approaches such as quality by design and design of experiments have been used to study the link between applied parameters whilst further pushing the limits of recombinant protein production (Looby et al, 2011; Nagashima et al, 2013; Weng et al, 2020)

Methods

Results

Conclusion