Gene copy number, gene configuration and LC/HC mRNA ratio impact on antibody productivity and product quality in targeted integration CHO cell lines.

The augmentation of transgene copy numbers is a prevalent approach presumed to enhance transcriptional activity and product yield. CHO cell lines engineered via targeted integration (TI) offer an advantageous platform for investigating the interplay between gene copy number, mRNA abundance, product yield, and product quality. Our investigation revealed that incrementally elevating the gene copy numbers of both IgG heavy chain (HC) and light chain (LC) concurrently resulted in the attainment of plateaus in mRNA levels and product titers, notably occurring beyond four to five gene copies integrated at the same TI site. Furthermore, maintaining a fixed gene copy number while varying the position of genes within the vector influenced the LC/HC mRNA ratio, which subsequently exerted a substantial impact on product titer. Moreover, manipulation of the LC/HC gene ratio through the introduction of surplus LC gene copies led to heightened LC mRNA expression and a reduction in the levels of high molecular weight species. It is noteworthy that the effects of excess LC on product titer were dependent on the specific molecule under consideration. The strategic utilization of PCR tags enabled precise quantification of transcription from each expression slot within the vector, facilitating the identification of highly expressive and less expressive slots. Collectively, these findings significantly enhance our understanding of stable antibody production in TI CHO cell lines.

Strategies to improve CHO cell culture performance: Targeted deletion of amino acid catabolism and apoptosis genes paired with growth inhibitor supplementation.

Chinese hamster ovary (CHO) cells are the predominant host of choice for recombinant monoclonal antibody (mAb) expression. Recent advancements in gene editing technology have enabled engineering new CHO hosts with higher growth, viability, or productivity. One approach involved knock out (KO) of BCAT1 gene, which codes for the first enzyme in the branched chain amino acid (BCAA) catabolism pathway; BCAT1 KO reduced accumulation of growth inhibitory short chain fatty acid (SCFA) byproducts and improved culture growth and titer when used in conjunction with high-end pH-controlled delivery of glucose (HiPDOG) technology and SCFA supplementation during production. Accumulation of SCFAs in the culture media is critical for metabolic shift toward higher specific productivity and hence titer. Here we describe knocking out BCKDHa/b genes (2XKO), which act downstream of the BCAT1, in a BAX/BAK KO CHO host cell line background to reduce accumulation of growth-inhibitory molecules in culture. Evaluation of the new 4XKO CHO cell lines in fed-batch production cultures (without HiPDOG) revealed that partial KO of BCKDHa/b genes in an apoptosis-resistant (BAX/BAK KO) background can achieve higher viabilities and mAb titers. This was evident when SCFAs were added to boost productivity as such additives negatively impacted culture viability in the WT but not BAX/BAK KO cells during batch production. Altogether, our findings suggest that SCFA addbacks can significantly increase productivity and mAb titers in the context of apoptosis-attenuated CHO cells with partial KO of BCAA genes. Such engineered CHO hosts can offer productivity advantages for expressing biotherapeutics in an industrial setting.

SPEED-MODE cell line development (CLD): Reducing Chinese hamster ovary (CHO) CLD timelines via earlier suspension adaptation and maximizing time spent in the exponential growth phase.

Chinese hamster ovary (CHO) cells are the preferred system for expression of therapeutic proteins and the majority of all biotherapeutics are being expressed by these cell lines. CHO expression systems are readily scalable, resistant to human adventitious agents, and have desirable post-translational modifications, such as glycosylation. Regardless, drug development as a whole is a very costly, complicated, and time-consuming process. Therefore, any improvements that result in reducing timelines are valuable and can provide patients with life-saving drugs earlier. Here we report an effective method (termed SPEED-MODE, herein) to speed up the Cell line Development (CLD) process in a targeted integration (TI) CHO CLD system. Our findings show that (1) earlier single cell cloning (SCC) of transfection pools, (2) speeding up initial titer screening turnaround time, (3) starting suspension adaptation of cultures sooner, and (4) maximizing the time CHO cultures spend in the exponential growth phase can reduce CLD timelines from ~4 to ~3 months. Interestingly, SPEED-MODE timelines closely match the theoretical minimum timeline for CHO CLD assuming that CHO cell division is the rate limiting factor. Clones obtained from SPEED-MODE CLD yielded comparable titer and product quality to those obtained via a standard CLD process. Hence, SPEED-MODE CLD is advantageous for manufacturing biotherapeutics in an industrial setting as it can significantly reduce CLD timelines without compromising titer or product quality.