Significant impact of mTORC1 and ATF4 pathways in CHO cell recombinant protein production induced by CDK4/6 inhibitor.

The CDK4/6 inhibitor has been shown to increase recombinant protein productivity in Chinese hamster ovary (CHO) cells. Therefore, we investigated the mechanism that couples cell-cycle inhibitor (CCI) treatment with protein productivity utilizing proteomics and phosphoproteomics. We identified mTORC1 as a critical early signaling event that preceded boosted productivity. Following CCI treatment, mTOR exhibited a transient increase in phosphorylation at a novel site that is also conserved in humans and mouse. Upstream of mTORC1, increased phosphorylation of AKT1S1 and decreased phosphorylation of RB1 may provide molecular links between CDK4/6 inhibition and mTORC1. Downstream, increased EIF4EBP1 phosphorylation was observed, which can mediate cap-dependent translation. In addition, the collective effect of increased phosphorylation of RPS6, increased phosphorylation of regulators of RNA polymerase I, and increased protein expression in the transfer RNA-aminoacylation pathway may contribute to enhancing the translational apparatus for increased productivity. In concert, an elevated stress response via GCN2/EIF2AK4-ATF4 axis persisted over the treatment course, which may link mTOR to downstream responses including the unfolded protein response and autophagy to enhance proper protein folding and secretion. Together, this comprehensive proteomics and phosphoproteomics characterization of CCI-treated CHO cells offers insights into understanding multiple aspects of signaling events resulting from CDK4/CDK6 inhibition.

A multiauxotrophic CHO cell line for the rapid isolation of producers of diverse or high levels of recombinant proteins.

Chinese Hamster Ovary (CHO) cells are widely used for the high-level production of recombinant proteins. We created a multiauxotrophic mutant of CHO-K1 cells, CHO8A, that is deficient in eight enzymatic steps in the purine/pyrimidine biosynthetic pathways. Prototrophy was restored by transfections with complementary DNA-based genes for the eight missing activities. CHO8A cells permit: (1) selection of transfectant clones that have incorporated genes for eight or more different polypeptides, suitable for engineering complex proteins, or pathways; and (2) the single-step selection of high producers of a particular protein. The latter is achieved by simultaneous use of eight vectors, each bearing one of the eight rescue genes and a cargo protein gene. Screening as few as 10 surviving colonies yielded high producers secreting mAbs at 84 picograms per cell per day or more. CHO8A was isolated by CRISPR-Cas9 knockout of 10 genes in the pathways to pyrimidines (Dhodh, Umps, Ctps1, Ctps2, and Tyms) and purines (Paics, Atic, Impdh1, Impdh2, and Gmps).

Differential effects on natural killer cell production by membrane-bound cytokine stimulations.

Robust manufacturing production of natural killer (NK) cells has been challenging in allogeneic NK cell-based therapy. Here, we compared the impact of cytokines on NK cell expansion by developing recombinant K562 feeder cell lines expressing membrane-bound cytokines, mIL15, mIL21, and 41BBL, individually or in combination. We found that 41BBL played a dominant role in promoting up to 500,000-fold of NK cell expansion after a 21-day culture process without inducing exhaustion. However, 41BBL stimulation reduced the overall cytotoxic activity of NK cells when combined with mIL15 and/or mIL21. Additionally, long-term stimulation with mIL15 and/or mIL21, but not 41BBL, increased CD56 expression and the CD56bright population, which is unexpectedly correlated with NK cell cytotoxicity. By conducting single-cell sequencing, we identified distinct subpopulations of NK cells induced by different cytokines, including an adaptive-like CD56bright CD16- CD49a+ subset induced by mIL15. Through gene expression analysis, we found that different cytokines modulated signaling pathways and target genes involved in cell cycle, senescence, self-renewal, migration, and maturation in different ways. Together, our study demonstrates that cytokine signaling pathways play distinct roles in NK cell expansion and differentiation, which sheds light on NK cell process designs to improve productivity and product quality.

A proteomics approach to decipher a sticky CHO situation.

Chinese hamster ovary (CHO) cells serve as protein therapeutics workhorses, so it is useful to understand what intrinsic properties make certain host cell lines and clones preferable for scale up and production of target proteins. In this study, two CHO host cell lines (H1, H2), and their respective clones were evaluated using comparative TMT-proteomics. The clones obtained from host H1 showed increased productivity (6.8 times higher) in comparison to clones from host H2. Based on fold-change analyses, we observed differential regulation in pathways including cell adhesion, aggregation, and cellular metabolism among others. In particular, the cellular adhesion pathway was downregulated in H1, in which podoplanin, an antiadhesion molecule, was upregulated the most in host H1 and associated clones. Phenotypically, these cells were less likely to aggregate and adhere to surfaces. In addition, enzymes involved in cellular metabolism such as isocitrate dehydrogenase (IDH) and mitochondrial-d-lactate dehydrogenase ( d-LDHm) were also found to be differentially regulated. IDH plays a key role in TCA cycle and isocitrate-alpha-ketoglutarate cycle while d-LDHm aids in the elimination of toxic metabolite methylglyoxal, involved in protein degradation. These findings will enhance our efforts towards understanding why certain CHO cell lines exhibit enhanced performance and perhaps provide future cell engineering targets.

Epigenetic comparison of CHO hosts and clones reveals divergent methylation and transcription patterns across lineages.

In this study, we examined DNA methylation and transcription profiles of recombinant clones derived from two different Chinese hamster ovary hosts. We found striking epigenetic differences between the clones, with global hypomethylation in the host 1 clones that produce bispecific antibody with higher productivity and complex assembly efficiency. Whereas the methylation patterns were found mostly inherited from the host, the host 1 clones exhibited continued demethylation reflected by the hypomethylation of newly emerged differential methylation regions (DMRs) even at the clone development stage. Several interconnected biological functions and pathways including cell adhesion, regulation of ion transport, and cholesterol biosynthesis were significantly altered between the clones at the RNA expression level and contained DMR in the promoter and/or gene-body of the transcripts, suggesting epigenetic regulation. Indeed, expression changes of epigenetic regulators were observed including writers (Dnmt1, Setdb1), readers (Mecp2), and erasers (Tet3, Kdm3a, Kdm1b/5c) involved in CpG methylation, histone methylation, and heterochromatin maintenance. In addition, we identified putative transcription factors that may be readers or effectors of the epigenetic regulation in these clones. By combining transcriptomics with DNA methylation data, we identified potential processes and factors that may contribute to the variability in cell physiology between different production hosts.

A TAF4 coactivator function for E proteins that involves enhanced TFIID binding.

The multisubunit TFIID plays a direct role in transcription initiation by binding to core promoter elements and directing preinitiation complex assembly. Although TFIID may also function as a coactivator through direct interactions with promoter-bound activators, mechanistic aspects of this poorly defined function remain unclear. Here, biochemical studies show a direct TFIID-E-protein interaction that (1) is mediated through interaction of a novel E-protein activation domain (activation domain 3 [AD3]) with the TAF homology (TAFH) domain of TAF4, (2) is critical for activation of a natural target gene by an E protein, and (3) mechanistically acts by enhancing TFIID binding to the core promoter. Complementary assays establish a gene-specific role for the TAFH domain in TFIID recruitment and activation of a large subset of genes in vivo. These results firmly establish TAF4 as a bona fide E-protein coactivator as well as a mechanism involving facilitated TFIID binding through direct interaction with an E-protein activation domain.

Characterization of Bispecific Antibody Production in Cell Cultures by Unique Mixed Mode Size Exclusion Chromatography.

Bispecific antibodies have received wide attention as promising immunotherapeutic agents because of their high specificity and the ability to target immune cells to tumors. However, analysis of bispecific antibodies is challenging because multiple forms of antibodies are potentially generated during production in cell culture. Most analyses of bispecific antibodies rely on liquid chromatography with mass spectrometry (LC-MS), which could miss detection or becomes less quantitative if those forms are not physically separated. Here, we report a novel and sensitive mixed mode size exclusion chromatography (MM SEC) coupled with multiangle light scattering (MALS) to analyze different forms of bispecific IgG molecules under native conditions. The method displayed great ability to separate various antibody forms with peak resolutions unmatched by other methods we tested, isolating desired bispecific molecules, parental homodimers, half molecules, and antibodies with mispaired light and heavy chains. Each peak was analyzed by online MALS and then identified and confirmed by intact and reduced LC-MS of isolated forms. MM SEC in this study performs by a novel mechanism through the interactions of resin with protein surface hydrophobic clusters distributed across CDRs of light chains. This novel MM SEC allows quantitative detection of even low abundance forms and provides a new tool for screening expression profiles of cell culture clones, monitoring purification, and evaluating drug substance purity.

A doubly auxotrophic CHO-K1 cell line for the production of recombinant monoclonal antibodies.

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production due to their hardiness, ease of transfection, and production of glycan structures similar to those in natural human monoclonal antibodies. To enhance the usefulness of CHO-K1 cells we developed a new selection system based on double auxotrophy. We used CRISPR-Cas9 to knockout the genes that encode the bifunctional enzymes catalyzing the last two steps in the de novo synthesis of pyrimidines and purines (uridine monophosphate synthase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase [ATIC], respectively). Survival of these doubly auxotrophic cells depends on the provision of sources of purines and pyrimidines or on the transfection and integration of open reading frames encoding these two enzymes. We successfully used one such double auxotroph (UA10) to select for stable transfectants carrying (a) the recombinant tumor necrosis factor-α receptor fusion protein etanercept and (b) the heavy and light chains of the anti-Her2 monoclonal antibody trastuzumab. Transfectant clones produced these recombinant proteins in a stable manner and in substantial amounts. The availability of this double auxotroph provides a rapid and efficient selection method for the serial or simultaneous transfer of genes for multiple polypeptides of choice into CHO cells using readily available purine- and pyrimidine-free commercial media.

Investigation of the Impact from IL-2, IL-7, and IL-15 on the Growth and Signaling of Activated CD4+ T Cells.

While CAR-T therapy is a growing and promising area of cancer research, it is limited by high cost and the difficulty of consistently culturing T-cells to therapeutically relevant concentrations ex-vivo. Cytokines IL-2, IL-7 and IL-15 have been found to stimulate the growth of T cells, however, the optimized combination of these three cytokines for T cell proliferation is unknown. In this study, we designed an integrated experimental and modeling approach to optimize cytokine supplementation for rapid expansion in clinical applications. We assessed the growth data for statistical improvements over no cytokine supplementation and used a systems biology approach to identify genes with the highest magnitude of expression change from control at several time points. Further, we developed a predictive mathematical model to project the growth rate for various cytokine combinations, and investigate genes and reactions regulated by cytokines in activated CD4+ T cells. The most favorable conditions from the T cell growth study and from the predictive model align to include the full range of IL-2 and IL-7 studied, and at lower levels of IL-15 (6 ng/mL or 36 ng/mL). The highest growth rates were observed where either IL-2 or IL-7 was at the highest concentration tested (15 ng/mL for IL-2 and 80 ng/mL for IL-7) while the other was at the lowest (1 ng/mL for IL-2 and 6 ng/mL for IL-7), or where both IL-2 and IL-7 concentrations are moderate-corresponding to condition keys 200, 020, and 110 respectively. This suggests a synergistic interaction of IL-2 and IL-7 with regards to promoting optimal proliferation and survival of the activated CD4+ T cells. Transcriptomic data analysis identified the genes and transcriptional regulators up/down-regulated by each of the cytokines IL-2, IL-7, and IL-15. It was found that the genes with persistent expressing changes were associated with major pathways involved in cell growth and proliferation. In addition to influencing T cell metabolism, the three cytokines were found to regulate specific genes involved in TCR, JAK/STAT, MAPK, AKT and PI3K-AKT signaling. The developed Fuzzy model that can predict the growth rate of activated CD4+ T cells for various combinations of cytokines, along with identified optimal cytokine cocktails and important genes found in transcriptomic data, can pave the way for optimizing activated CD4 T cells by regulating cytokines in the clinical setting.

Effect of Fc-Glycan Structure on the Conformational Stability of IgG Revealed by Hydrogen/Deuterium Exchange and Limited Proteolysis.

Human therapeutic immunoglobulin gamma (IgG) molecules contain an N-glycan on each of their Fc CH2 domains. These glycans include high-mannose, hybrid, and complex types. Recombinant IgG molecules containing high-mannose glycans have been shown to clear faster in human blood, and exhibit decreased thermal stability. The molecular mechanism behind these observations, however, is not well understood. In this work, we used hydrogen/deuterium exchange combined with mass spectrometry (HDX MS), as well as proteolytic degradation under a native-like condition, to assess the impact of different glycoforms on the molecular structure and stability of recombinant IgG1 and IgG2 molecules expressed from Chinese hamster ovary cells. Our HDX MS data indicate that the conformation of these IgG molecules was indeed influenced by the glycan structure. IgG molecules containing high-mannose and hybrid glycans showed more conformational flexibility in the CH2 domain. This conclusion was further supported by the analysis of glycopeptides released from these molecules by trypsin digestion under a native-like condition. The higher CH2 conformational flexibility of IgG molecules with high-mannose and hybrid glycans contributes to their decreased thermal stability. IgG molecules containing sialylated glycans in the CH2 domain exhibited similar enzymatic degradation behavior as high-mannose glycans, suggesting decreased CH2-domain stability compared to shorter complex glycans, likely resulting from steric effect that decreased the glycan-CH2 domain interaction.

Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures.

A deep-well plate enabled automated high-throughput cell line development platform.

Cell line development (CLD) plays a crucial role in the manufacturing process development of therapeutic biologics. Most biologics are produced in Chinese hamster ovary (CHO) cell. Because of the nature of random transgene integration in CHO genome and CHO's inherent plasticity, stable CHO transfectants usually have a vast diversity in productivity, growth, and product quality. Thus, we often must resort to screening a large number of cell pools and clones to increase the probability of identifying the ideal production cell line, which is a very laborious and resource-demanding process. Here we have developed a deep-well plate (DWP) enabled high throughput (DEHT) CLD platform using 24-well DWP (24DWP), liquid handler, and other automation components. This platform has capabilities covering the key steps of CLD including cell passaging, clone imaging and expansion, and fed-batch production. We are the first to demonstrate the suitability of 24DWP for CLD by confirming minimal well-to-well and plate-to-plate variability and the absence of well-to-well cross contamination. We also demonstrated that growth, production, and product quality of 24DWP cultures were comparable to those of conventional shake flask cultures. The DEHT platform enables scientists to screen five times more cultures than the conventional CLD platform, thus significantly decreases the resources needed to identify an ideal production cell line for biologics manufacturing.

Non-invasive UPR monitoring system and its applications in CHO production cultures.

Unfolded protein response (UPR) is the primary signaling network activated in response to the accumulation of unfolded and/or misfolded protein in the endoplasmic reticulum (ER). The expression of high levels of recombinant proteins in mammalian cell cultures has been linked to the increased UPR. However, the dynamics of different UPR-mediated events and their impact on cell performance and recombinant protein secretion during production remain poorly defined. Here, we have created a non-invasive UPR-responsive, fluorescence-based reporter system to detect and quantify specific UPR-mediated transcriptional activation of different intracellular signaling pathways. We have generated stable antibody-expressing CHO clones containing this UPR responsive system and established FACS-based methods for real-time, continuous monitoring of the endogenous UPR activation in live cultures. The results showed that the UPR activation is dynamically regulated during production culture. The clones differed in their UPR patterns; both the timing and the degree of UPR-induced transcriptional activation were linked to cell performance, such as growth, and viability. In addition, the cell culture environment, such as media composition and osmolarity, significantly impacted endogenous UPR activation. Taken together, these data demonstrate a utility of this UPR monitoring system in recombinant protein production processes and the observations increase our understanding of the critical role of UPR in regulating diverse phenotypes of the cells including growth, survival and recombinant protein secretion under different culture environments and processing conditions.

An In-Vitro Study of the Expansion and Transcriptomics of CD4+ and CD8+ Naïve and Memory T Cells Stimulated by IL-2, IL-7 and IL-15.

The growth of T cells ex vivo for the purpose of T cell therapies is a rate-limiting step in the overall process for cancer patients to achieve remission. Growing T cells is a fiscally-, time-, and resource-intensive process. Cytokines have been shown to accelerate the growth of T cells, specifically IL-2, IL-7, and IL-15. Here a design of experiments was conducted to optimize the growth rate of different naïve and memory T cell subsets using combinations of cytokines. Mathematical models were developed to study the impact of IL-2, IL-7, and IL-15 on the growth of T cells. The results show that CD4+ and CD8+ naïve T cells grew effectively using moderate IL-2 and IL-7 in combination, and IL-7, respectively. CD4+ and CD8+ memory cells favored moderate IL-2 and IL-15 in combination and moderate IL-7 and IL-15 in combination, respectively. A statistically significant interaction was observed between IL-2 and IL-7 in the growth data of CD4+ naïve T cells, while the interaction between IL-7 and IL-15 was found for CD8+ naïve T cells. The important genes and related signaling pathways and metabolic reactions were identified from the RNA sequencing data for each of the four subsets stimulated by each of the three cytokines. This systematic investigation lays the groundwork for studying other T cell subsets.

Identification of RNA content of CHO-derived extracellular vesicles from a production process.

Recent studies have unveiled the unique roles of extracellular vesicles (EVs) in various cellular processes including protein degradation, transport, and intercellular communication. However, the EVs of Chinese hamster ovary (CHO) cells, the workhorse of biologics manufacturing, have not been well-characterized despite their significant roles in protein production. Herein, we successfully isolated CHO EVs from CHO fed-batch cultures and identified their messenger RNA (mRNA) and micro RNA (miRNA) contents through next-generation sequencing. We found that mRNAs corresponding to oxidative phosphorylation were highly enriched in microvesicles (large EVs) but absent in exosomes (small EVs). We also found that both large EVs and small EVs had enriched mRNA species corresponding to key signaling pathways for cell proliferation, survival, and growth, including the TGFß and PI3K/Akt pathways. In addition, the enrichment of miR-196a-5p in both small EVs and large EVs suggests an anti-apoptotic and proliferative function for EVs through intercellular communication. The identification of these mRNAs and miRNAs associated with cell growth and survival sheds light on the potential role of extracellular vesicles in the context of biologics manufacturing and may help further optimize CHO biologics production.

Chromosomal instability drives convergent and divergent evolution toward advantageous inherited traits in mammalian CHO bioproduction lineages.

Genetic instability of Chinese hamster ovary (CHO) cells is implicated in production inconsistency through poorly defined mechanisms. Using a multi-omics approach, we analyzed the variations of CHO lineages derived from CHO-K1 cells. We identify an equilibrium between random genetic variation of the CHO genome and heritable traits driven by culture conditions, selection criteria, and genetic linkage. These inherited changes are associated with the selection pressures related to serum removal, suspension culture transition, protein expression, and secretion. We observed that a haploid reduction of a Chromosome 2 region after serum-free, suspension adaptation, was consistently inherited, suggesting common adaptation mechanisms. Genetic variations also included ∼200 insertions/deletions, ∼1000 single-nucleotide polymorphisms, and ∼300-2000 copy number variations, which were exacerbated after gene editing. In addition, heterochromatic chromosomes were preferentially lost as cells continuously evolved. Together, these observations demonstrate a highly plastic signature for adapted CHO cells and paves the way towards future host cell engineering.

Retrospective assessment of clonality of a legacy cell line by analytical subcloning of the master cell bank.

In recent years, assurance of clonality of the production cell line has been emphasized by health authorities during review of regulatory submissions. When insufficient assurance of clonality is provided, augmented control strategies may be required for a commercial production process. In this study, we conducted a retrospective assessment of clonality of a legacy cell line through analysis of subclones from the master cell bank (MCB). Twenty-four subclones were randomly selected based on a predetermined acceptance sampling plan. All these subclones share a conserved integration junction, thus providing a high level of assurance that the cell population in the MCB was derived from a single progenitor cell. However, Southern blot analysis indicates that at least four subpopulations possibly exist in the MCB. Additional characterization of these four subpopulations demonstrated that the resulting changes in product quality attributes of some subclones are not related to the genetic heterogeneity observed in Southern blot hybridization. Furthermore, process consistency, process comparability, and analytical comparability have been demonstrated in batches produced across varying manufacturing processes, scales, facilities, cell banks, and cell ages. Finally, process and product consistency together with a high level of assurance of clonal origin of the MCB helped clear the hurdle for regulatory approval without requirement of additional control strategies.

Improvement of the efficiency and quality in developing a new CHO host cell line.

Chinese hamster ovary (CHO) cells are a ubiquitous tool for industrial therapeutic recombinant protein production. However, consistently generating high-producing clones remains a major challenge during the cell line development process. The glutamine synthetase (GS) and dihydrofolate reductase (DHFR) selection systems are commonly used CHO expression platforms based on controlling the balance of expression between the transgenic and endogenous GS or DHFR genes. Since the expression of the endogenous selection gene in CHO hosts can interfere with selection, generating a corresponding null CHO cell line is required to improve selection stringency, productivity, and stability. However, the efficiency of generating bi-allelic genetic knockouts using conventional protocols is very low (<5%). This significantly affects clone screening efficiency and reduces the chance of identifying robust knockout host cell lines. In this study, we use the GS expression system as an example to improve the genome editing process with zinc finger nucleases (ZFNs), resulting in improved GS-knockout efficiency of up to 46.8%. Furthermore, we demonstrate a process capable of enriching knockout CHO hosts with robust bioprocess traits. This integrated host development process yields a larger number of GS-knockout hosts with desired growth and recombinant protein expression characteristics.

Modulating tyrosine sulfation of recombinant antibodies in CHO cell culture by host selection and sodium chlorate supplementation.

BACKGROUND: Tyrosine sulfation is a post-translational modification found on many surface receptors and plays an important role in cell-cell and cell-matrix interactions. However, tyrosine sulfation of therapeutic antibodies has only been reported very recently. Because of potential potency and immunogenicity concerns, tyrosine sulfation needs to be controlled during the manufacturing process. METHODS AND RESULTS: In this study, we explored methods to modulate antibody tyrosine sulfation during cell line development and upstream production process. We found that tyrosine sulfation levels were significantly different in various Chinese hamster ovary (CHO) cell lines due to differential expression of genes in the sulfation pathway including tyrosylprotein sulfotransferase 2 (TPST2) and the sulfation substrate transporter SLC35B2. We also screened chemical inhibitors to reduce tyrosine sulfation in CHO culture and found that sodium chlorate could significantly inhibit tyrosine sulfation while having minimal impact on cell growth and antibody production. We further confirmed this finding in a standard fed-batch production assay. Sodium chlorate at 16 mM markedly inhibited tyrosine sulfation by more than 50% and had no significant impact on antibody titer or quality. CONCLUSION: These data suggest that we can control tyrosine sulfation by selecting CHO cell lines based on the expression level of TPST2 and SLC35B2 or adding sodium chlorate in upstream production process.

Improving Antibody Production in Stably Transfected CHO Cells by CRISPR-Cas9-Mediated Inactivation of Genes Identified in a Large-Scale Screen with Chinese Hamster-Specific siRNAs.

The Chinese hamster ovary (CHO) cell line is commonly used for the production of biotherapeutics. As cell productivity directly affects the cost of production, methods are developed to manipulate the expression of specific genes that are known to be involved in protein synthesis, folding, and secretion to increase productivity. However, there are no large-scale CHO-specific functional screens to identify novel gene targets that impact the production of secreted recombinant proteins. Here, a large-scale, CHO cell-specific small interfering RNA screen is performed to identify genes that consistently enhance antibody production when silenced in a panel of seven CHO cell lines. Four genes, namely, Cyp1a2, Atp5s, Dgki, and P3h2, are identified, and then selected for CRISPR-Cas9 knockout validation in recombinant CHO cell lines. Single knockout of Cyp1a2, Atp5s, or Dgki, but not P3h2, results in a more than 90% increase in specific antibody productivity. Overall, the knockout of Cyp1a2 demonstrates the most significant improvement of antibody production, with a minimal impact on cell growth.