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

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.

Simultaneous Monitoring of Monoclonal Antibody Variants by Strong Cation-Exchange Chromatography Hyphenated to Mass Spectrometry to Assess Quality Attributes of Rituximab-Based Biotherapeutics

“…We hypothesize that this heterogeneity arises from the use of different expression host cells and/or different culture conditions (temperature, pH, CO 2 concentration, time, bioreactor size, etc.) in the bioprocesses that may tune the glycosylation profile and the C-terminal lysine variants of these biotherapeutics [53][54][55]. However, despite the molecular heterogeneity of the proteoforms observed in the two biotherapeutics, no clinical differences between the two drugs in terms of toxicity were observed when prescribed for the treatment of diffuse large B-cell lymphoma [28,29].…”

Cottonseed hydrolysate supplementation alters metabolic and proteomics responses in Chinese hamster ovary cell cultures

“…[ 26–28 ] A wide range of proteomics techniques such as tandem mass tag (TMT), isobaric tags for relative and absolute quantitation (iTRAQ), and Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH‐MS) have been used to explore the impact of bioprocessing changes on CHO cells. [ 29–35 ]…”

Proteomic Landscape of Adeno-Associated Virus (AAV)-Producing HEK293 Cells

“…Riboflavin metabolism is known to be involved in mitochondrial beta-oxidation, and is therefore essential for energy production within the cells [ 23 ]. Apart from the necessity of energy production to maintain cell viability, it has also been shown that energy metabolism plays an important role during recombinant protein expression [ 5 ]. In the past, cellular production systems have been greatly engineered in order to modify cell metabolism, aiming towards higher specific productivity rather than biomass production [ 24 ].…”

“…Another interesting observation is the activation of oxidative phosphorylation. As mentioned before, we have shown previously that increased recombinant protein productivity in CHO cells can be achieved upon a higher level of oxidative phosphorylation [ 5 ]. Cellular energy metabolism can easily be targeted by changes in the bioprocessing conditions.…”

“…Proteomic profiling provides important insights into underlying cellular mechanisms involved in recombinant protein expression. In recent years, proteomics has been extensively used to study, for example, Chinese hamster ovary (CHO) cells, which are routinely used to produce monoclonal antibodies (mAbs) [ 4 , 5 ]. Using the outputs of these proteomic investigations, CHO cells have been actively engineered, and bioprocessing conditions optimized, in order to modify the cell cycle and energy metabolism of the cells, aiming towards increased levels of specific productivity [ 6 , 7 , 8 , 9 ].…”