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. 2022 Feb 28;12(1):3280.
doi: 10.1038/s41598-022-06886-1.

Comparative systeomics to elucidate physiological differences between CHO and SP2/0 cell lines

Deniz Demirhan  1 , Amit Kumar  2 , Jie Zhu  3 , Pi Camilla Poulsen  4 , Natalia I Majewska  2   3 , Yinong Sebastian  5 , Raghothama Chaerkady  4 , Wen Yu  6 , Wei Zhu  5 , Li Zhuang  4 , Punit Shah  7 , Kristen Lekstrom  4 , Robert N Cole  8 , Hui Zhang  9 , Michael J Betenbaugh  2 , Michael A Bowen  10
Affiliations

Comparative systeomics to elucidate physiological differences between CHO and SP2/0 cell lines

Deniz Demirhan et al. Sci Rep. .

Abstract

Omics-based tools were coupled with bioinformatics for a systeomics analysis of two biopharma cell types: Chinese hamster ovary (M-CHO and CHO-K1) and SP2/0. Exponential and stationary phase samples revealed more than 10,000 transcripts and 6000 proteins across these two manufacturing cell lines. A statistical comparison of transcriptomics and proteomics data identified downregulated genes involved in protein folding, protein synthesis and protein metabolism, including PPIA-cyclophilin A, HSPD1, and EIF3K, in M-CHO compared to SP2/0 while cell cycle and actin cytoskeleton genes were reduced in SP2/0. KEGG pathway comparisons revealed glycerolipids, glycosphingolipids, ABC transporters, calcium signaling, cell adhesion, and secretion pathways depleted in M-CHO while retinol metabolism was upregulated. KEGG and IPA also indicated apoptosis, RNA degradation, and proteosomes enriched in CHO stationary phase. Alternatively, gene ontology analysis revealed an underrepresentation in ion and potassium channel activities, membrane proteins, and secretory granules including Stxbpt2, Syt1, Syt9, and Cma1 proteins in M-CHO. Additional enrichment strategies involving ultracentrifugation, biotinylation, and hydrazide chemistry identified over 4000 potential CHO membrane and secretory proteins, yet many secretory and membrane proteins were still depleted. This systeomics pipeline has revealed bottlenecks and potential opportunities for cell line engineering in CHO and SP2/0 to improve their production capabilities.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The experimental and bioinformatical workflow of SP2/0 and CHO cell line functional categorization.
Figure 2
Figure 2
(A) Number of genes identified in mRNA and/or protein levels (a) M-CHO exponential phase mRNA versus protein comparison (b) M-CHO stationary mRNA versus protein comparison (c) SP2/0 exponential mRNA versus protein comparison (d) SP2/0 stationary mRNA versus protein comparison (e) M-CHO exponential versus stationary mRNA comparison (f) M-CHO exponential and stationary protein comparison (g) SP2/0 exponential and stationary mRNA comparison (h) SP2/0 exponential and stationary protein comparison (B) (a–d) Comparison and correlation of mRNA and protein data in M-CHO or SP2/0 cell lines in exponential and/or stationary phases (C) (a–d) Comparison and correlation of mRNA and protein data between the M-CHO and SP2/0 cell lines in exponential or stationary phases (D) Downregulated proteins in M-CHO cells compared to SP2/0 cells.
Figure 3
Figure 3
(A) Heatmap of p-values from hypergeometric analysis of exponential and stationary phase M-CHO and SP2/0 cell lines and exponential phase CHO-K1 cell line (B) Heatmap of selected pathways from which are upregulated in SP2/0 exponential and SP2/0 stationary.
Figure 4
Figure 4
KEGG analysis of calcium signaling and pancreatic secretion pathways for (A) M-CHO (B) SP2/0 (C) CHO-K1 cell lines using proteomics and transcriptomics analysis. Orange stands for transcriptome and proteome data, magenta stands for only proteome data, green stands for only mRNA data and yellow stands for neither transcriptome nor proteome data (https://www.kegg.jp/kegg/kegg1.html).
Figure 5
Figure 5
Enrichment results of gene ontology analysis of (A) M-CHO molecular function (B) M-CHO biological process (C) SP2/0 molecular function (D) SP2/0 biological process (E) Secretory granule genes present in SP2/0 and CHO cells (F) Ingenuity Pathway Analysis of secretory granule genes present in SP2/0 cells.
Figure 6
Figure 6
CHO membranome analysis with cell surface biotinylation, glycoproteome and two step ultracentrifugation based enrichment methods.
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