Cellular responses to individual amino-acid depletion in antibody-expressing and parental CHO cell lines

• Published in: Biotechnology and bioengineering 2014-05, Vol.111 (5), p.965-979
• Main Authors: Fomina-Yadlin, Dina, Gosink, John J., McCoy, Rebecca, Follstad, Brian, Morris, Arvia, Russell, Chris B., McGrew, Jeffrey T.
• Format: Article
• Language: English
• Subjects: Amino acids; Amino Acids - metabolism; amino-acid depletion; Animals; Antibodies - metabolism; Bioengineering; Biotechnology; Cell culture; Cell Culture Techniques - methods; Cell cycle; cell cycle arrest; Cell Physiological Phenomena - physiology; Cellular; cellular signaling; chinese hamster ovary; CHO Cells; cholesterol biosynthesis; Cricetinae; Cricetulus; Culture Media - chemistry; Culture Media - metabolism; Depletion; Gene expression; Leucine; lipid metabolism; Lipid Metabolism - physiology; Lipids; Metabolism; microarray; nonessential amino acids; pathway analysis; Pathways; production conditions; Recombinant; Recombinant Proteins - metabolism; Signal transduction
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.25155

ABSTRACT Depletion of two nonessential amino acids, asparagine (Asn) and glutamine (Gln), occurred during a fed‐batch production process with a CHO cell line expressing a recombinant antibody. This depletion coincided with growth suppression and the onset of the stationary phase. Experimental withdrawal of Asn led to cell cycle arrest of cell line A in G0/G1 phase. On a mechanistic level, withdrawal of either Asn or Gln stimulated the amino‐acid response (AAR) pathway, indicating that depletion of nonessential amino acids can induce AAR in this cell line. Compared to withdrawal of an essential amino acid, leucine (Leu), withdrawal of either Asn or Gln induced fewer changes in downstream effectors of mammalian target of rapamycin (mTOR) signaling involved in regulation of global protein synthesis. Global transcriptional analysis followed by pathway analysis revealed that the cultures experienced a down‐regulation of cell‐cycle progression, DNA replication and nucleotide biosynthesis in an E2F‐dependent manner, as well as a down‐regulation of lipid metabolism in a SREBP1/2‐dependent manner as a result of individual amino‐acid withdrawal. Timing and magnitude of observed phenotypic and transcriptional responses to amino‐acid withdrawal differed between essential (Leu) and nonessential (Asn and Gln) amino acids examined. Observed responses were similar in parental (CS9 and CHOK‐1) and two other antibody‐producing CHO cell lines, but the magnitude of the transcriptional response was both cell‐line and amino‐acid dependent. Overall, these results suggest that depletion of nonessential amino acids in cell culture plays a role in the onset of the stationary phase of production process and offer mechanistic insights into the observed growth attenuation phenotype. Biotechnol. Biotechnol. Bioeng. 2014;111: 965–979. © 2013 Wiley Periodicals, Inc. The authors' findings indicate that (1) Amino‐acid response (AAR) pathway is intact and functional in CHO cells, that (2) nonessential amino acids, specifically asparagine and glutamine, induce AAR and impact ATF4 signaling in parental and antibody‐expressing CHO cell lines examined, and that (3) stimulation of the AAR pathway by nonessential amino acids leads to growth suppression via induction of G1 cell cycle arrest.