Despite extensive research conducted to increase protein production from Chinese hamster ovary (CHO) cells, cellular bottlenecks often remain, hindering high yields. In this study, a transcriptomic ...analysis led to the identification of 32 genes that are consistently upregulated in high producer clones and thus might mediate high productivity. Candidate genes were associated with functions such as signaling, protein folding, cytoskeleton organization, and cell survival. We focused on two engineering targets, Erp27, which binds unfolded proteins and the Erp57 disulfide isomerase in the endoplasmic reticulum, and Foxa1, a pioneering transcription factor involved in organ development. Erp27 moderate overexpression increased production of an easy‐to‐express antibody, whereas Erp27 and Erp57 co‐overexpression increased cell density, viability, and the yield of difficult‐to‐express proteins. Foxa1 overexpression increased cell density, cell viability, and easy‐ and difficult‐to‐express protein yields, whereas it decreased reactive oxygen species late in fed‐batch cultures. Foxa1 overexpression upregulated two other candidate genes that increased the production of difficult‐ and/or easy‐to‐express proteins, namely Ca3, involved in protecting cells from oxidative stress, and Tagap, involved in signaling and cytoskeleton remodeling. Overall, several genes allowing to overcome CHO cell bottlenecks were identified, including Foxa1, which mediated multiple favorable metabolic changes that improve therapeutic protein yields.
In this work, the authors performed a transcriptomic analysis to identify genes upregulated in CHO cells producing therapeutic proteins at high levels. Among them, they identified candidate genes whose overexpression could improve the production of easy‐ and difficult‐to‐express therapeutic proteins. This included Erp27, an endoplasmic reticulum‐located protein that binds unfolded proteins and the Erp57 protein disulfide isomerase, and Foxa1, a pioneering transcription factor whose overexpression upregulated the expression of high production‐associated genes.
In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a ...guanosine triphosphate GTPase‐activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein‐expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by‐products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells.
Pourcel et al. observed that several cytoskeletal genes are upregulated in highly productive CHO cell clones. Actin alpha overexpression yielded altered cytoskeletal organization, which was accompanied by improved recombinant therapeutic production as well as decreased levels of toxic lactate metabolic by‐products. Cells spontaneously displaying low actin polymerization yielded higher protein production in suspension cultures, highlighting the importance of the cytoskeleton for the efficient production of easy‐ as well as difficult‐to‐express therapeutic proteins.
In the field of therapeutic antibody production, diversification of fed-batch strategies is flourishing in response to the market demand. All manufacturing approaches tend to follow the generally ...accepted dogma of increasing titer since it directly increases manufacturing output. While titer is influenced by the biomass (expressed as IVCD), the culture time and the cell-specific productivity (
q
P
), we changed independently each of these parameters to tune our process strategy towards adapted solutions to individual manufacturing needs. To do so, we worked separately on the increase of the IVCD as high seeding fed-batch capacity. Yet, as intensified fed-batch may not always be possible due to limited facility operational mode, we also separately increased the
q
P
with the addition of specific media additives. Both strategies improved titer by 100% in 14 days relative to the standard fed-batch process with moderate and acceptable changes in product quality attributes. Since intensified fed-batch could rival the cell-specific productivity of a conventional fed-batch, we developed novel hybrid strategies to either allow for acceptable seeding densities without compromising productivity, or alternatively, to push the productivity the furthest in order to reduce timelines.
The Chinese hamster ovary (CHO) cells used to produce biopharmaceutical proteins are known to contain type‐C endogenous retrovirus (ERV) sequences in their genome and to release retroviral‐like ...particles. Although evidence for their infectivity is missing, this has raised safety concerns. As the genomic origin of these particles remained unclear, we characterized type‐C ERV elements at the genome, transcriptome, and viral particle RNA levels. We identified 173 type‐C ERV sequences clustering into three functionally conserved groups. Transcripts from one type‐C ERV group were full‐length, with intact open reading frames, and cognate viral genome RNA was loaded into retroviral‐like particles, suggesting that this ERV group may produce functional viruses. CRISPR‐Cas9 genome editing was used to disrupt the gag gene of the expressed type‐C ERV group. Comparison of CRISPR‐derived mutations at the DNA and RNA level led to the identification of a single ERV as the main source of the release of RNA‐loaded viral particles. Clones bearing a Gag loss‐of‐function mutation in this ERV showed a reduction of RNA‐containing viral particle release down to detection limits, without compromising cell growth or therapeutic protein production. Overall, our study provides a strategy to mitigate potential viral particle contaminations resulting from ERVs during biopharmaceutical manufacturing.
Endogenous retroviruses in Chinese hamster ovary cells were characterized at the genome, transcriptome and viral particle level. Following CRISPR‐mediated mutagenesis, the authors identified a single endogenous retrovirus locus responsible for the budding of viral particles into the cell supernatant. Loss‐of‐function mutations in this particular locus reduced viral genome‐containing viral particles to detection limits, thus augmenting the safety profile of Chinese hamster ovary cells for biopharmaceutical production.
Bispecific antibodies (bsAb) represent the most promising class of therapeutics due to their multiple mode of action. Their development raised tremendous efforts with a special focus on the ...engineering of these complex molecules, especially when they adopt an asymmetrical format. Various strategies have been successful in improving heterodimerization through the introduction of point mutations to force the correct pairing of opposite chains using either steric, electrostatic steering effects or both. These great advances in protein engineering combined with the development of robust process purification methods to remove homodimers have contributed to the fast progress of bsAbs towards clinics. Nevertheless, there is still a lack of systematic and efficient production methods to fine tune the expression of correctly paired bsAbs. In this study, we have evaluated the effects of manipulating different molar ratios of three polypeptide chains constituting a model bsAb, co-transfected, expressed and assembled in a single Chinese Hamster Ovary cell line. Two parental clones showing different heterodimerization and productivity profiles were chosen. We demonstrated that addition of gene copies at specific ratios to these clones could bias towards the generation of sub-clones with either reduced proper assembly or tune expression for enhanced integral bsAb. Our results demonstrate that expression of bsAb can be influenced beyond the initial engineering stage.
Cover Image, Volume 114, Number 8, August 2017 Droz, Xuan; Harraghy, Niamh; Lançon, Etienne ...
Biotechnology and bioengineering,
August 2017, 2017-Aug, 2017-08-00, Letnik:
114, Številka:
8
Journal Article
Recenzirano
Cover Legend The cover image, by Xuan Droz et al., is based on the Article Automated microfluidic sorting of mammalian cells labeled with magnetic microparticles for those that efficiently express ...and secrete a protein of interest, DOI: 10.1002/bit.26270.
Successful generation of high producing cell lines requires the generation of cell clones expressing the recombinant protein at high levels and the characterization of the clones' ability to maintain ...stable expression levels. The use of cis‐acting epigenetic regulatory elements that improve this otherwise long and uncertain process has revolutionized recombinant protein production. Here we review and discuss new insights into the molecular mode of action of the matrix attachment regions (MARs) and ubiquitously‐acting chromatin opening elements (UCOEs), i.e. cis‐acting elements, and how these elements are being used to improve recombinant protein production. These elements can help maintain the chromatin environment of the transgene genomic integration locus in a transcriptionally favorable state, which increases the numbers of positive clones and the transgene expression levels. Moreover, the high producing clones tend to be more stable in long‐term cultures even in the absence of selection pressure. Therefore, by increasing the probability of isolating a high producing clone, as well as by increasing transcription efficiency and stability, these elements can significantly reduce the time and cost required for producing large quantities of recombinant proteins.
Expression of genes integrated in the genome of mammalian cells, so as for instance to produce therapeutic proteins, is typically limited by chromatin‐linked effects. These include epigenetic silencing and variegation, which decrease the amounts of protein produced and thereby increase their cost. Here, the authors review how the use of DNA sequences that act as epigenetic regulators can be used to prevent these unwanted effects, and thereby facilitate the production of therapeutic proteins.
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