A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells

Abstract

A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by upregulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial-and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production.

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

“…Proteomics data analysis was performed using Progenesis QI for Proteomics V 3.2 using the following parameters: minimum number of fragment ion matches per peptide = 3, minimum number of fragment ion matches per protein = 7, minimum number of unique peptides per protein = 2, maximum number of one missed cleavage for tryptic digestion, fixed modification = carbamidomethyl C, variable modifications = oxidation M and deamidation N and Q, false discovery rate (FDR) ≤ 1%. Data were searched against the Cricetulus griseus NCBI FASTA database (http://www.ncbi.nlm.nih.gov/assembly/GCF_000419365.1/, downloaded 12th June 2015) and its underlying Mus musculus mRNA FASTA database (Liu et al, ), to which the protein sequence of the Hi3 standard protein ( E. coli chaperone protein ClpB) was added. The cellular proteomes were normalized to their total proteome whereas data normalization for the conditioned media proteomes was performed in relation to the internal Hi3 standard peptides due to the volume‐based approach used.…”

“…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, ).…”

Quick generation of Raman spectroscopy based in-process glucose control to influence biopharmaceutical protein product quality during mammalian cell culture

“…Aside from end product glycation considerations, glucose concentration can have a cascading effect on the production culture environment, requiring careful design of a glucose operating space. Increasing glucose concentration has been shown to correlate with decreased integral viable cell density and increased specific productivity rates . Alternatively, high glucose concentration has been shown to cause oxidative stress through the generation of reactive oxygen species and increase the production rate of toxic metabolic byproduct lactate as the majority of glucose consumed by mammalian cells is converted to lactate .…”

Chemical composition and the potential for proteomic transformation in cancer, hypoxia, and hyperosmotic stress

“…The comparisons here use all proteins reported to have expression patterns in Cluster 1 (up) or Cluster 5 (down), or only the proteins with high expression differences (ratio ≤-0.2 or ≥0.2) at all time points. Source: SI Table S4 of Liu et al (2015). 4.21 osmol/kg vs 3.17 osmol/kg osmotic pressure (NaCl treatment).…”