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Chinese hamster ovary (CHO) cells are used extensively to produce protein therapeutics, such as monoclonal antibodies (mAbs), in the biopharmaceutical industry. MAbs are large proteins that are energetically demanding to synthesize and secrete; therefore, high-producing CHO cell lines that are engineered for maximum metabolic efficiency are needed to meet increasing demands for mAb production. Previous studies have identified that high-producing cell lines possess a distinct metabolic phenotype when compared to low-producing cell lines. In particular, it was found that high mAb production is correlated to lactate consumption and elevated TCA cycle flux. We hypothesized that enhancing flux through the mitochondrial TCA cycle and oxidative phosphorylation would lead to increased mAb productivities and final titers. To test this hypothesis, we overexpressed peroxisome proliferator-activated receptor 𝛾 co-activator-1⍺ (PGC-1⍺), a gene that promotes mitochondrial metabolism, in an IgG-producing parental CHO cell line. Stable cell pools overexpressing PGC-1⍺ exhibited increased oxygen consumption, indicating increased mitochondrial metabolism, as well as increased mAb specific productivity compared to the parental line. We also performed 13C metabolic flux analysis (MFA) to quantify how PGC-1⍺ overexpression alters intracellular metabolic fluxes, revealing not only increased TCA cycle flux, but global upregulation of cellular metabolic activity. This study demonstrates the potential of rationally engineering the metabolism of industrial cell lines to improve overall mAb productivity and to increase the abundance of high-producing clones in stable cell pools.
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A Flexible Hybrid Site‐Specific Integration‐Based Expression System in CHO Cells for Higher‐Throughput Evaluation of Monoclonal Antibody Expression Cassettes
ABSTRACT The implementation of site‐specific integration (SSI) systems in Chinese hamster ovary (CHO) cells for the production of monoclonal antibodies (mAbs) can alleviate concerns associated with production instability and reduce cell line development timelines. SSI cell line performance is driven by the interaction between genomic integration location, clonal background, and the transgene expression cassette, requiring optimization of all three parameters to maximize productivity. Systematic comparison of these parameters has been hindered by SSI platforms involving low‐throughput enrichment strategies, such as cell sorting. This study presents a recombinase‐mediated cassette exchange (RMCE)‐capable SSI system that uses only chemical selection to enrich for transgene‐expressing RMCE pools in less than one month. The system was used to compare eight mAb expression cassettes containing two novel genetic regulatory elements, theAzin1CpG island and the Piggybac transposase 5’ terminal repeat, in various orientations to improve the expression of two therapeutic mAbs from two genomic loci. Similar patterns of productivity and mRNA expression were observed across sites and mAbs, and the best performing cassette universally increased mAb productivity by 7‐ to 11‐fold. This flexible system allows for higher‐throughput comparison of expression cassettes from a consistent clonal and transcriptional background to optimize RMCE‐derived cell lines for industrial production of mAbs.
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- Award ID(s):
- 2100502
- PAR ID:
- 10598659
- Publisher / Repository:
- Wiley-VCH
- Date Published:
- Journal Name:
- Biotechnology Journal
- Volume:
- 20
- Issue:
- 1
- ISSN:
- 1860-6768
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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AbstractThe pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal selection, media supplementation and genetic engineering of cells. However, improved cell productivity has inherent risk of impacting product quality attributes (PQA). PQAs may affect the products’ efficacy via stability, bioavailability, or in vivo bioactivity. Variations in manufacturing process may introduce heterogeneity in the products by altering the type and extent of N-glycosylation, which is a PQA of therapeutic proteins. We investigated the effect of different cell densities representing increasing process intensification in a perfusion cell culture on the production of an IgG1-κ monoclonal antibody from a CHO-K1 cell line. This antibody is glycosylated both on light chain and heavy chain. Our results showed that the contents of glycosylation of IgG1-κ mAb increased in G0F and fucosylated type glycans as a group, whereas sialylated type glycans decreased, for the mAb whole protein. Overall, significant differences were observed in amounts of G0F, G1F, G0, G2FS1, and G2FS2 type glycans across all process intensification levels. G2FS2 and G2 type N-glycans were predominantly quantifiable from light chain rather than heavy chain. It may be concluded that there is a potential impact to product quality attributes of therapeutic proteins during process intensification via perfusion cell culture that needs to be assessed. Since during perfusion cell culture the product is collected throughout the duration of the process, lot allocation needs careful attention to process parameters, as PQAs are affected by the critical process parameters (CPPs). Key points• Molecular integrity may suffer with increasing process intensity.• Galactosylated and sialylated N-glycans may decrease.• Perfusion culture appears to maintain protein charge structure.more » « less
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Abstract Chinese hamster ovary (CHO) cell lines are widely used to manufacture biopharmaceuticals. However, CHO cells are not an optimal expression host due to the intrinsic plasticity of the CHO genome. Genome plasticity can lead to chromosomal rearrangements, transgene exclusion, and phenotypic drift. A poorly understood genomic element of CHO cell line instability is extrachromosomal circular DNA (eccDNA) in gene expression and regulation. EccDNA can facilitate ultra-high gene expression and are found within many eukaryotes including humans, yeast, and plants. EccDNA confers genetic heterogeneity, providing selective advantages to individual cells in response to dynamic environments. In CHO cell cultures, maintaining genetic homogeneity is critical to ensuring consistent productivity and product quality. Understanding eccDNA structure, function, and microevolutionary dynamics under various culture conditions could reveal potential engineering targets for cell line optimization. In this study, eccDNA sequences were investigated at the beginning and end of two-week fed-batch cultures in an ambr ® 250 bioreactor under control and lactate-stressed conditions. This work characterized structure and function of eccDNA in a CHO-K1 clone. Gene annotation identified 1551 unique eccDNA genes including cancer driver genes and genes involved in protein production. Furthermore, RNA-seq data is integrated to identify transcriptionally active eccDNA genes.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1002/biot.202400520
