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. 2015 Nov 6;14(11):4687-703.
doi: 10.1021/acs.jproteome.5b00588. Epub 2015 Oct 13.

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

Amit Kumar  1   2 , Deniz Baycin-Hizal  3 , Daniel Wolozny  1 , Lasse Ebdrup Pedersen  4 , Nathan E Lewis  5   6 , Kelley Heffner  1 , Raghothama Chaerkady  7 , Robert N Cole  7 , Joseph Shiloach  2 , Hui Zhang  8 , Michael A Bowen  3 , Michael J Betenbaugh  1
Affiliations

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

Amit Kumar et al. J Proteome Res. .

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.

Keywords: CHO; host cell proteins; immunogenicity; ontology; proteomics; secretome; signal peptides.

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Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Overview of the process of obtaining functionally categorized CHO-SO through various filtering strategies and analysis techniques along with process of obtaining high abundance immunogenic CHO proteins.
Figure 2
Figure 2
Analysis of all CHO-SO genes (a) A histogram of number of proteins for each NSAF bin with an overlaid normal distribution curve. Two standard deviations (2.4689) from the mean (−13.8317) correspond to right-hand bound of 95% confidence interval with a Log2 NSAF value of −8.8939. (b) Ninety-two proteins with Log2 NSAF values greater than −8.8939 were used to build the pathway network in IPA. The figure shows network of genes corresponding to functions–cell survival and folding of proteins.
Figure 3
Figure 3
Results from bioinformatics analyses. (a) Number of positive hits as identified by different bioinformatics tools, which may also be a positive hit in another tool. (b) Proteins groups after combining positive results from SecretomeP, WoLF PSORT, and SPD as “Secreted Proteins”; positive results from SignalP, TargetP, and BLAST analysis on signal peptides database were combined as “Proteins with Signal peptides”; and positive results from TMHMM and Phobius were combined as “Membrane Proteins”.
Figure 4
Figure 4
An example output from the Web site used to find out GO of Chinese hamster and CHO cells. (a) The homepage of the Web site is accessible via http://ebdrup.biosustain.dtu.dk/gocho/. Clicking on “create a new data set” takes the user to a new page. (b) Users can input gene names (e.g., for which GO annotation identification is needed along with the species type on this page and submit the information). (c) The output shows gene symbol, gene name, and GO description as well as accession numbers for molecular function, biological processes, and cellular component categories.
Figure 5
Figure 5
Results from GO hypergeometric distribution analysis. The percentages shown are the percentage of gene in each GO function as compared to the total genes in the GO functions. (a) Top 15 molecular functions enriched in the 1015 filtered proteins. (b) Top 15 biological processes enriched in this same list. (c) Top 15 cellular components enriched. (d) Top 15 cellular components enriched in the CHO whole cell proteome.
Figure 6
Figure 6
Functional network categories overrepresented by the extracellular space and extracellular vesicular exosome proteins. Proteins related to “cell spreading”, “chemotaxis of cells”, and “exocytosis” functions are shown.
Figure 7
Figure 7
KEGG’s focal adhesion pathway displaying proteins in CHO-SO in green color, proteins in CHO supernatant but not in CHO-SO in orange color, proteins in CHO transcriptome-proteome data (but not in either CHO supernatant proteins or CHO-SO) in magenta color, and genes not found in any of the above data sets in yellow color.
See this image and copyright information in PMC

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