# Systems Biology of Recombinant 2G12 and 353/11 mAb Production in CHO-K1 Cell Lines at Phosphoproteome Level

## TL;DR

This study explores how different monoclonal antibodies affect the phosphoproteome of CHO cells, revealing insights into cellular stress and production efficiency.

## Contribution

The paper introduces a phosphoproteomics analysis of CHO cells producing different mAbs, revealing novel phosphorylation dynamics linked to production challenges.

## Key findings

- Significant phosphorylation changes were observed in nuclear proteins of 2G12-producing cells.
- Tunicamycin treatment altered phosphorylation patterns, including effects on eukaryotic elongation factor 2 kinase.
- Phosphorylation dynamics suggest regulation of nuclear pathways by 2G12 mAb expression.

## Abstract

Background: Chinese hamster ovary (CHO) cells are extensively used in the pharmaceutical industry for producing complex proteins, primarily because of their ability to perform human-like post-translational modifications. However, the efficiency of high-quality protein production can vary significantly for monoclonal antibody-producing cell lines, within the CHO host cell lines or by extrinsic factors. Methods: To investigate the complex cellular mechanisms underlying this variability, a phosphoproteomics analysis was performed using label-free quantitative liquid chromatography after a phosphopeptide enrichment of recombinant CHO cells producing two different antibodies and a tunicamycin treatment experiment. Using MaxQuant and Perseus for data analysis, we identified 2109 proteins and quantified 4059 phosphosites. Results: Significant phosphorylation dynamics were observed in nuclear proteins of cells producing the difficult-to-produce 2G12 mAb. It suggests that the expression of 2G12 regulates nuclear pathways based on increases and decreases in phosphorylation abundance. Furthermore, a substantial number of changes in the phosphorylation pattern related to tunicamycin treatment have been detected. TM treatment affects, among other phosphoproteins, the eukaryotic elongation factor 2 kinase (Eef2k). Conclusions: The alterations in the phosphorylation landscape of key proteins involved in cellular processes highlight the mechanisms behind stress-induced cellular responses.