Jeffrey T. McGrew

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

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.

Gene expression measurements normalized to cell number reveal large scale differences due to cell size changes, transcriptional amplification and transcriptional repression in CHO cells.

Conventional approaches to differential gene expression comparisons assume equal cellular RNA content among experimental conditions. We demonstrate that this assumption should not be universally applied because total RNA yield from a set number of cells varies among experimental treatments of the same Chinese Hamster Ovary (CHO) cell line and among different CHO cell lines expressing recombinant proteins. Conventional normalization strategies mask these differences in cellular RNA content and, consequently, skew biological interpretation of differential expression results. On the contrary, normalization to synthetic spike-in RNA standards added proportional to cell numbers reveals these differences and allows detection of global transcriptional amplification/repression. We apply this normalization method to assess differential gene expression in cell lines of different sizes, as well as cells treated with a cell cycle inhibitor (CCI), an mTOR inhibitor (mTORI), or subjected to high osmolarity conditions. CCI treatment of CHO cells results in a cellular volume increase and global transcriptional amplification, while mTORI treatment causes global transcriptional repression without affecting cellular volume. Similarly to CCI treatment, high osmolarity increases cell size, total RNA content and antibody expression. Furthermore, we show the importance of spike-in normalization for studies involving multiple CHO cell lines and advocate normalization to spike-in controls prior to correlating gene expression to specific productivity (qP). Overall, our data support the need for cell number specific spike-in controls for all gene expression studies where cellular RNA content differs among experimental conditions.