Elucidation of the CHO Super-Ome (CHO-SO) by Proteoinformatics

Abstract

Chinese hamster ovary (CHO) cells are the preferred host cell line for manufacturing a variety of complex biotherapeutic drugs including monoclonal antibodies. We performed a proteomics and bioinformatics analysis on the spent medium from adherent CHO cells. Supernatant from CHO-K1 culture was collected and subjected to in-solution digestion followed by LC/LC–MS/MS analysis, which allowed the identification of 3281 different host cell proteins (HCPs). To functionally categorize them, we applied multiple bioinformatics tools to the proteins identified in our study including SignalP, TargetP, SecretomeP, TMHMM, WoLF PSORT, and Phobius. This analysis provided information on the presence of signal peptides, transmembrane domains, and cellular localization and showed that both secreted and intracellular proteins were constituents of the supernatant. Identified proteins were shown to be localized to the secretory pathway including ones playing roles in cell growth, proliferation, and folding as well as those involved in protein degradation and removal. After combining proteins predicted to be secreted or having a signal peptide, we identified 1015 proteins, which we termed as CHO supernatant-ome (CHO-SO), or superome. As a part of this effort, we created a publically accessible web-based tool called GO–CHO to functionally categorize proteins found in CHO-SO and identify enriched molecular functions, biological processes, and cellular components. We also used a tool to evaluate the immunogenicity potential of high-abundance HCPs. Among enriched functions were catalytic activity and structural constituents of the cytoskeleton. Various transport related biological processes, such as vesicle mediated transport, were found to be highly enriched. Extracellular space and vesicular exosome associated proteins were found to be the most enriched cellular components. The superome also contained proteins secreted from both classical and nonclassical secretory pathways. The work and database described in our study will enable the CHO community to rapidly identify high-abundance HCPs in their cultures and therefore help assess process and purification methods used in the production of biologic drugs.

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

“…In addition to the recombinant protein of interest, host cells spend a substantial amount of resources on producing other proteins, such as host cell proteins133132. Indeed, our cell lines demonstrated that ~90% of the ribosomal occupancy is spent on proteins other than the desired IgG (Fig.…”

“…GO terms for CHO were obtained using GOCHO13 and terms under the parent term ‘Biological Process’ were extracted and converted into a GMT file according to GSEA manual. A ranked list of genes was made using the expression values obtained from Ribo-Seq quantification.…”

“…We can now begin to understand the molecular basis of protein expression thanks to recent efforts to sequence the genomes of cell lines utilized for recombinant expression123. Specifically, RNA sequencing (RNA-Seq)24567891011 and proteomics1213 have provided detailed insights into transcription and post-translational processes; however, the translation of endogenous and recombinant mRNA has remained poorly characterized. This is of particular concern since RNA-Seq analyses neglect the fact that translation control of the mRNA is an essential and highly regulated step in determining levels of an expressed protein14.…”

Proteomics in biomanufacturing control: Protein dynamics of CHO‐K1 cells and conditioned media during apoptosis and necrosis

“…LC‐MS based “omics”‐approaches are increasingly used to gain advanced understanding of biomanufacturing processes such as to study host cell proteins (HCPs), productivity during process scale‐up or cellular response to altered media composition (Farrell et al, ; Gao et al, ; Liu et al, ). The recent elucidation of the CHO cellular and secreted proteome further facilitates research in this area (Baycin‐Hizal et al, ; Kumar et al, ). Advanced LC‐MS technology has therefore also been used to study proteomics changes in relation to CHO cell viability.…”

Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

“…CHO cells are well adapted to growing in suspension with different media compositions2. For large-scale commercial production of mAbs, fed-batch culture has been widely used at large scales, up to 20,000 L working volume, because of its operational simplicity and high-titers.…”