Predicting favorable landing pads for targeted integrations in Chinese hamster ovary cell lines by learning stability characteristics from random transgene integrations.

Chinese Hamster Ovary (CHO) cell lines are considered to be the preferred platform for the production of biotherapeutics, but issues related to expression instability remain unresolved. In this study, we investigated potential causes for an unstable phenotype by comparing cell lines that express stably to such that undergo loss in titer across 10 passages. Factors related to transgene integrity and copy number as well as the genomic profile around the integration sites were analyzed. Horizon Discovery CHO-K1 (HD-BIOP3) derived production cell lines selected for phenotypes with low, medium or high copy number, each with stable and unstable transgene expression, were sequenced to capture changes at genomic and transcriptomic levels. The exact sites of the random integration events in each cell line were also identified, followed by profiling of the genomic, transcriptomic and epigenetic patterns around them. Based on the information deduced from these random integration events, genomic loci that potentially favor reliable and stable transgene expression were reported for use as targeted transgene integration sites. By comparing stable vs unstable phenotypes across these parameters, we could establish that expression stability may be controlled at three levels: 1) Good choice of integration site, 2) Ensuring integrity of transgene and observing concatemerization pattern after integration, and 3) Checking for potential stress related cellular processes. Genome wide favorable and unfavorable genomic loci for targeted transgene integration can be browsed at https://www.borthlabchoresources.boku.ac.at/.

The impact of anti-apoptotic gene Bcl-2∆ expression on CHO central metabolism.

Anti-apoptosis engineering is an established technique to prolong the viability of mammalian cell cultures used for industrial production of recombinant proteins. However, the effect of overexpressing anti-apoptotic proteins on central carbon metabolism has not been systematically studied. We transfected CHO-S cells to express Bcl-2∆, an engineered anti-apoptotic gene, and selected clones that differed in their Bcl-2∆ expression and caspase activity. (13)C metabolic flux analysis (MFA) was then applied to elucidate the metabolic alterations induced by Bcl-2∆. Expression of Bcl-2Δ reduced lactate accumulation by redirecting the fate of intracellular pyruvate toward mitochondrial oxidation during the lactate-producing phase, and it significantly increased lactate re-uptake during the lactate-consuming phase. This flux redistribution was associated with significant increases in biomass yield, peak viable cell density (VCD), and integrated VCD. Additionally, Bcl-2∆ expression was associated with significant increases in isocitrate dehydrogenase and NADH oxidase activities, both rate-controlling mitochondrial enzymes. This is the first comprehensive (13)C MFA study to demonstrate that expression of anti-apoptotic genes has a significant impact on intracellular metabolic fluxes, especially in controlling the fate of pyruvate carbon, which has important biotechnology applications for reducing lactate accumulation and enhancing productivity in mammalian cell cultures.

Characterization of the proteases involved in the N-terminal clipping of glucagon-like-peptide-1-antibody fusion proteins.

In an attempt to develop high producing mammalian cell lines expressing glucagon-like-peptide-1-antibody fusion proteins (GLP-1), we have noted that the N-terminal GLP-1 portion of the fusion protein was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. The majority of the N-terminal clipped product appeared to be due to the removal of the entire biologically active peptide (30 amino acids) from the intact molecule. A number of parameters that influenced the degradative process were investigated. Additionally, protease inhibitors specific for each class of protease were tested. Results suggested that one or more serine-threonine class of protease(s) were involved in this process and inhibitors that are specific for this class of protease, including benzamidine hydrochloride could significantly inhibit the proteolytic degradation of the fusion proteins. Identification of the specific proteases involved in this process by shotgun proteomics methodology will pave the way for engineering the CHOK1SV cell line which will serve as a superior host for the production of future fusion protein products.

Combining high-throughput screening of caspase activity with anti-apoptosis genes for development of robust CHO production cell lines.

A set of anti-apoptotic genes were over-expressed, either singly or in combination, in an effort to develop robust Chinese Hamster Ovary host cell lines suitable for manufacturing biotherapeutics. High-throughput screening of caspase 3/7 activity enabled a rapid selection of transfectants with reduced caspase activity relative to the host cell line. Transfectants with reduced caspase 3/7 activity were then tested for improved integrated viable cell count (IVCC), a function of peak viable cell density and longevity. The maximal level of improvement in IVCC could be achieved by over-expression of either single anti-apoptotic genes, e.g., Bcl-2Δ (a mutated variant of Bcl-2) or Bcl-XL, or a combination of two or three anti-apoptotic genes, e.g., E1B-19K, Aven, and XIAPΔ. These cell lines yielded higher transient antibody production and a greater number of stable clones with high antibody yields. In a 5 L fed-batch bioreactor system, BΔ31-1, a stable clone expressing Bcl-2Δ, had a product titer that was 180% as compared to an optimal clone (Con-1) from the control cell line. Although lactate accumulated to more than 5 g/L in the control culture, its concentration was reduced in the anti-apoptotic BΔ31-1 cultures to below 1 g/L, confirming our earlier findings that cells over-expressing anti-apoptotic genes consume the lactate that would otherwise accumulate as a by-product in the culture medium. To the best of our knowledge, this is the first study to use the high throughput caspase screening method to identify CHO host cell lines with superior anti-apoptotic characteristics.