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

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.