Transcriptome analysis of a CHO cell line expressing a recombinant therapeutic protein treated with inducers of protein expression.
The search for specific productivity (qP) determinants in Chinese hamster ovary (CHO) cells has been the focus of the biopharmaceutical cell line engineering efforts aimed at creating "super-producer" cell lines. In this study, we evaluated the impact of small-molecule inducers and temperature shift on recombinant protein production, and used transcriptomic analysis to define gene-phenotype correlations for qP in our biological system. Next-generation RNA Sequencing (RNA-Seq) analysis revealed that each individual inducer (caffeine, hexamethylene bisacetamide (HMBA) and sodium butyrate (NaBu)) or a combination treatment had a distinct impact on the gene expression program of the RANK-Fc cell line. Temperature shift to 31 °C impacted inducer action with respect to transcriptional changes and phenotypic cell line parameters. We showed that inducer treatment was able to increase expression level of the Fc- fusion mRNA and the selectable marker mRNA from 16% up to 45% of total mRNA in the cell. We further demonstrated that qP exhibited a strong positive linear correlation to transcript levels of both the RANK-Fc fusion protein and the dihydrofolate reductase (DHFR) selectable marker. In fact, these were 2 out of 7 transcripts with significant positive correlation to qP at both temperatures. Many more transcripts were anti- correlated to qP, and gene set enrichment analysis (GSEA) revealed that those were involved in cell cycle progression, transcription, mRNA processing, translation and protein folding. Therefore, we postulate that the transcript level of the recombinant protein is a major qP determinant in our biological system, while downregulation of routine activity within the cell is necessary to divert cellular resources towards recombinant protein production.
Drug Substance Development, Amgen Inc., Seattle, WA 98119, United States.
Dina
D
Fomina-Yadlin
Drug Substance Development, Amgen Inc., Seattle, WA 98119, United States.
Mirna
M
Mujacic
Drug Substance Development, Amgen Inc., Seattle, WA 98119, United States.
Kathy
K
Maggiora
Drug Substance Development, Amgen Inc., Seattle, WA 98119, United States.
Garrett
G
Quesnell
Drug Substance Development, Amgen Inc., Seattle, WA 98119, United States.
Ramsey
R
Saleem
Drug Substance Development, Amgen Inc., Seattle, WA 98119, United States. Electronic address: jeff.mcgrew@justbiotherapeutics.com.
Jeffrey T
JT
McGrew
D000081:Acetamides / Q000494:pharmacology; D000818:Animals; D020148:Butyric Acid / Q000494:pharmacology; D016466:CHO Cells; D002110:Caffeine / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling; D040901:Proteomics; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D013696:Temperature
D000081:Acetamides; D012333:RNA, Messenger; C517736:Rank-Fc; D011993:Recombinant Fusion Proteins; D020148:Butyric Acid; D002110:Caffeine; C014026:hexamethylene bisacetamide
Chinese hamster ovary; RNA-Seq; Recombinant protein expression; Small-molecule inducers; Specific productivity; Temperature shift