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.

Differential binding of heavy chain variable domain 3 antigen binding fragments to protein A chromatography resins.

This work examines the binding of 15 different VH3 IgGs and their corresponding F(ab')2 fragments to two different protein A chromatography resins: MabSelect(®), which utilizes a recombinant protein A ligand, and MabSelect SuRe(®) (SuRe), which utilizes a tetrameric Z domain ligand. The results show that VH3 F(ab')2 fragments can exhibit a variety of binding behaviours for the two resins. Contrary to previously published data, a subset of these molecules show strong interaction with the Z domain of SuRe(®). Furthermore, the results show that sequence variability of residue 57 in the VH3 heavy chain CDR2 domain correlates with binding behaviour on MabSelect(®) and SuRe(®). Site-directed mutagenesis of this residue confers gain or loss of VH3 F(ab')2 binding to these resins in 3 mAbs, demonstrating that it plays a key role in both recombinant protein A and Z domain interaction. A fourth mAb with a longer CDR2 loop was not affected by mutation of residue 57, indicating that CDR2 domain length may alter the binding interface and lead to the involvement of other residues in protein A binding.