Proteins In Chinese Hamster Ovary Cells Research Articles

Overexpression of YTHDF3 increases the specific productivity of the recombinant protein in CHO cells by promoting the translation process.

Due to their high-quality characteristics, Chinese hamster ovary (CHO) cells have become the most widely used and reliable host cells for the production of recombinant therapeutic proteins in the biomedical field. Previous studies have shown that the m6A reader YTHDF3, which contains the YTH domain, can affect a variety of biological processes by regulating the translation and stability of target mRNAs. This study investigates the effect of YTHDF3 on transgenic CHO cells. The results indicate that stable overexpression of YTHDF3 significantly enhances recombinant protein expression without affecting host cell growth. Transcriptome sequencing indicated that several genes, including translation initiation factor, translation extension factor, and ribosome assembly factor, were upregulated in CHO cells overexpressing YTHDF3. In addition, cycloheximide experiments confirmed that YTHDF3 enhanced transgene expression by promoting translation in CHO cells. In conclusion, the findings in this study provide a novel approach for mammalian cell engineering to increase protein productivity by regulating m6A.

Synergistic promotion of transient transgene expression in CHO cells by PDI/XBP-1s co-transfection and mild hypothermia.

Transient gene expression system is an important tool for rapid production of recombinant proteins in Chinese hamster ovary (CHO) cells. However, their low productivity is the main hurdle to overcome. An effective approach through which to obtain high protein yield involves targeting transcriptional, post-transcriptional events (PTEs), and culture conditions. Here, we investigated the effects of protein disulfide isomerase (PDI) and spliced X-box binding protein 1 (XBP-1s) co-overexpression combined with mild hypothermia on the transient yields of recombinant proteins in CHO cells. The results showed that the gene of interest (GOI) and the PDI/XBP-1s helper vector at a co-transfection ratio of 10:1 could obviously increase transient expression level of recombinant protein in CHO cells. However, PDI/XBP-1s overexpression had no significance effect on the mRNA levels of the recombinant protein, suggesting that it targeted PTEs. Moreover, the increased production was due to the enhancing of cell specific productivity, not related to cell growth, viability, and cell cycle. In addition, combined PDI/XBP-1s co-overexpression and mild hypothermia could further improve Adalimumab expression, compared to the control/37°C and PDI/XBP-1s/37°C, the Adalimumab volume yield of PDI/XBP-1s/33°C increased by 203% and 142%, respectively. Mild hypothermia resulted in 3.52- and 2.33-fold increase in the relative mRNA levels of PDI and XBP-1s, respectively. In conclusion, the combination of PDI/XBP-1s overexpression and culture temperature optimization can achieve higher transient expression of recombinant protein, which provides a synergetic strategy to improve transient production of recombinant protein in CHO cells.

A highly efficient transposon vector system for recombinant protein expression in CHO cells

Chinese hamster ovary (CHO) cells are the most widely used mammalian host cells for the production of recombinant therapeutic proteins. Expression vectors are a key factor in determining the expression level of gene of interest (GOI) in CHO cells. Here, two transposon vector systems were constructed by adding two different pairs of inverted terminal repeats (ITR-1 and ITR-2), and four proteins of interest (POI), including enhanced green fluorescence protein (eGFP), secreted alkaline phosphatase (SEAP), human serum albumin (HSA) and Adalimumab (Adm) were expressed in CHO cells. The stably transfected cell pools and single cell clones were screened, and the expression levels of POIs were detected by qPCR, Western blot and ELISA. The results showed that both transposon vectors could improve the expression levels of POI at both mRNA and protein levels, while the ITR-2 sequence was more effective. Moreover, the positive cell clone rate of transposon system was higher than that of the control. In conclusion, the ITR-2 transposon vector system can be effectively used for the expression of recombinant proteins in CHO cells.

Promoter from Chinese hamster elongation factor-1a gene and Epstein-Barr virus terminal repeats concatemer fragment maintain stable high-level expression of recombinant proteins.

The Chinese hamster ovary (CHO) cell line is the main host for the high-titer production of therapeutic and diagnostic proteins in the biopharmaceutical industry. In most cases, plasmids for efficient protein expression in CHO cells are based on the cytomegalovirus (CMV) promoter. The autologous Chinese hamster eukaryotic translation elongation factor 1α (EEF1A1) promoter is a viable alternative to the CMV promoter in industrial applications. The EEF1A1 promoter and its surrounding DNA regions proved to be effective at maintaining high-level and stable expression of recombinant proteins in CHO cells. EEF1A1-based plasmids' large size can lead to low transfection efficiency and hamper target gene amplification. We hypothesized that an efficient EEF1A1-based expression vector with a long terminal repeat fragment from the Epstein-Barr virus (EBVTR) could be truncated without affecting promoter strength or the long-term stability of target gene expression. We made a series of deletions in the downstream flanking region of the EEF1A1 gene, and then in its upstream flanking region. The resulting plasmids, which coded for the enhanced green fluorescent protein (eGFP), were tested for the level of eGFP expression in the populations of stably transfected CHO DG44 cells and the stability of eGFP expression in the long-term culture in the absence of selection agents. It was shown that in the presence of the EBVTR fragment, the entire downstream flanking region of the EEF1A1 gene could be excluded from the plasmid vector. Shortening of the upstream flanking region of the EEF1A1 gene to a length of 2.5 kbp also had no significant effect on the level of eGFP expression or long-term stability. The EBVTR fragment significantly increased expression stability for both the CMV and EEF1A1 promoter-based plasmids, and the expression level drop during the two-month culture was more significant for both CMV promoter-based plasmids. Target protein expression stability for the truncated plasmid, based on the EEF1A1 gene and EBVTR fragment, is sufficient for common biopharmaceutical applications, making these plasmid vectors a viable alternative to conventional CMV promoter-based vectors.

Cost-Effective Protein Production in CHO Cells Following Polyethylenimine-Mediated Gene Delivery Showcased by the Production and Crystallization of Antibody Fabs.

Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes.

Effects and mechanism of small molecule additives on recombinant protein in CHO cells.

Chinese hamster ovary (CHO) cells can produce proteins with complex structures and post-translational modifications which are similar to human-derived cells, and they have been the ideal host cells for the production of recombinant therapy proteins (RTPs). Nearly 70% of approved RTPs are produced by CHO cells. In recent years, a series of measures have been developed to increase the expression of RTPs to achieve the lower production cost during the process of large-scale industrial production of recombinant protein in CHO cells. Among of them, the addition of small molecule additives in the culture medium can improve the expression and production efficiency of recombinant proteins, and has become an effective and simple method. In this paper, the characteristics of CHO cells, the effect and mechanism of small molecule additives are reviewed. KEY POINTS: • Small molecular additives on the expression of RTPs in CHO cells are reviewed • Small molecular additives improve the yield of RTPs • Small molecular additives provide methods for the optimization of serum-free medium.

Expanding the CRISPR toolbox for Chinese hamster ovary cells with comprehensive tools for Mad7 genome editing.

The production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.

Effects and mechanisms of animal-free hydrolysates on recombination protein yields in CHO cells.

Chinese hamster ovary (CHO) cells are the commonly used cell lines for producing recombinant therapeutic proteins (RTPs) because they possess post-translational modifications similar to human cells. Culture media are necessary for cell growth, and their quality affects the yields and quality of RTPs. Due to safety concerns for the complex purification of RTPs, the development of serum-free media (SFM) is necessary for CHO cells. To meet the need for CHO cells with higher cell density and RTP productivity with consistent product quality in large-scale suspension cultures, the optimization of SFM through adding some enzymatic animal-free hydrolysates (AFHs) is preferred. The AFHs can improve cell culture performance and product yield of RTPs without affecting their quality. Here, the effect and mechanism of various AFHs in improving CHO cell culture performance and protein expression are reviewed. KEY POINTS: • AFHs that improve the recombinant protein yield of CHO cells are reviewed. • AFHs improve recombinant protein yield via influencing cell performance. • The AFHs do not affect the quality of recombinant protein in CHO cells. • AFHs can provide nutrients, block cell cycle, and reduce oxidative stress.

The Effect of microRNA on the Production of Recombinant Protein in CHO Cells and its Mechanism.

Recombinant protein production by mammalian cells is the initial step in the manufacture of many therapeutic proteins. Chinese hamster ovary (CHO) cells are the most common host system to produce recombinant therapeutic proteins (RTPs). However, it is still challenging to maintain high productivity ensuring the good quality of RTPs produced by CHO cells. MicroRNAs(miRNAs) are short regulatory non-coding RNAs that can regulate cellular behavior and complex phenotypes. It has been found that miRNAs can enhance the expression level of recombinant proteins in CHO cells by promoting proliferation, resisting apoptosis, and regulating metabolism. miRNAs also can affect the quality of RTPs. In this review, we will discuss the effect and mechanism of miRNA on the expression level and quality of recombinant proteins in CHO cells.

Combination of sodium butyrate and decitabine promotes transgene expression in CHO cells via apoptosis inhibition

Chinese hamster ovary (CHO) cells are currently the most widely used host cells for production of recombinant therapeutic proteins (RTPs). Small-molecule additives related to cell cycle apoptosis and autophagy regulation have been used to promote RTP production. By combining two small-molecule additives, positive synergistic effects on transgene expression were observed in CHO cells. In the present study, six small-molecule additives were used, including hydrocinnamic acid (HCA), sodium butyrate (NaB), lithium acetate (LiAc), sodium succinate dibasic hexahydrate (SDH), decitabine (DAC), and sodium propionate (SP). Experiments to test the effects of their pairwise combinations on two different recombinant CHO cell lines (rCHO) were designed using Design-Expert 12.0. Different effects of various pairs of small molecules on apoptosis- and autophagy-related protein expression were observed in the rCHOs. The results showed that compared to the control culture, NaB alone increased the volumetric yield and specific productivity (Qp) by 166% and 143%, respectively. The volumetric yield and Qp of NaB combined with DAC (Cg1)-treated cells increased by 178% and 212%, respectively. Cg1 selectively blocked the cells in the G0/G1 cell cycle stage. The relative expression levels of B-cell lymphoma 2 (Bcl-2), Beclin 1, and microtubule-associated protein light chain 3 (LC3B) in Cg1-treated CHO cells were significantly increased, while relative levels of cleaved caspase-3 expression were significantly decreased. In conclusion, Cg1 had the most obvious effect on RTP production and Qp in CHO cells, suggesting the Cg1 combination of small molecules may be used to improve the expression of recombinant protein in CHO cells.

Targeted integration into pseudo attP sites of CHO cells using CRISPR/Cas9

Chinese hamster ovary (CHO) cells are regarded as a prominent host for manufacturing therapeutic proteins. Although conventional strategies for generating recombinant proteins in CHO cells depend on the random integration of a gene of interest (GOI), these established techniques occasionally result in genetically heterogeneous cell lines, which causes diminished expression of the recombinant proteins in the long run. Production instability can be reduced by SSI and creates stable cell lines with a consistent expression of the GOI. In this experiment, we demonstrate the targeted incorporation of a reporter cassette in two PhiC31 pseudo attP sites of CHO cells exploiting the homology-directed repair (HDR) generated by the CRISPR/Cas9 platform. Genes encoding GFP and puromycin resistance marker were precisely inserted into these loci via CRISPR/Cas9. Stable cell lines were suitably produced following antibiotic selection. Junction PCR and fluorescence assay determined targeted integration and expression homogeneity of the reporter cassette, respectively. Taken together, our results indicate the possibility of these two PhiC31 pseudo attP sites as the target sites for site-specific integration of a transgene mediated by CRISPR/Cas9. Furthermore, higher knock-in efficiency and expression homogeneity was observed in the pseudo attP site associated with chromosome 6 compared to the pseudo attP site from chromosome 3.

Multiplex secretome engineering enhances recombinant protein production and purity

Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a “clean” Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predict that the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyze the HCP content of 6-protein, 11-protein, and 14-protein knockout clones. These cell lines exhibit a substantial reduction in total HCP content (40%-70%). We also observe higher productivity and improved growth characteristics in specific clones. The reduced HCP content facilitates purification of a monoclonal antibody. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

An Easy and Fast Method for Production of Chinese Hamster Ovary Cell Line Expressing and Secreting Human Recombinant Activin A.

Objective Growth factors are key elements of embryonic stem cell (ESC) research. Cell line development in eukaryotes is a time-consuming procedure which usually takes 12-18 months. Here, we report an easy and fast method with which production of Chinese hamster ovary (CHO) cells that express and secrete recombinant Activin A, as a major growth factor in endo/mesoderm differentiation of embryonic stem cells is achieved within 3-4 weeks. Materials and Methods In this experimental study, we cloned human Activin A into the pDONR/Zeo gateway entry vector using the BP reaction. Activin A was subcloned next into the pLIX_403 and pLenti6.3/TO/V5-DEST destination vectors by the LR reaction. The result was the production of constructs with which 293T cells were finally transfected for virus production. CHO cells were transduced using viral particles to produce a cell line that secretes the His6- Activin A fusion protein. Results We developed a quick protocol which saves up to 3-4 weeks of time for producing recombinant proteins in CHO cells. The recombinant cell line produced 90 mg/L of functional Activin A measured in human ESC line Royan H5 (RH5), during in vitro differentiation into meso-endoderm and definitive endoderm. ConclusionOur results showed no significant differences in functionality between commercial Activin A and the one produced using our novel protocol. This approach can be easily used for producing recombinant proteins in CHO.

A study on enhanced O-glycosylation strategy for improved production of recombinant human chorionic gonadotropin in Chinese hamster ovary cells

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that exists as a heterodimer comprised of an α subunit and β subunit linked with disulfide bridges. The β subunit contains four O-glycosylation sites. Previous studies have found that the translation of mRNA to polypeptides of the β subunit was a severely limiting step for the expression of recombinant hCG protein in Chinese hamster ovary (CHO) cells. The effects of O-glycosylation on recombinant hCG protein expression were assessed by adding O-glycan precursors and overexpressing and knocking down key regulatory genes of O-glycan precursor synthesis and O-glycan sugar chain synthesis or hydrolases. The results indicated that O-glycosylation was indeed limiting in the expression of recombinant hCG protein, and N-acetylgalactosamine (GalNAc) was the major limiting precursor. Glutamine-fructose-6-phosphate transaminase 2 (Gfat2) and Uridine diphosphate-glucose pyrophosphorylase 2 (Ugp2), key regulatory genes of O-glycan precursor synthesis, were overexpressed. Ugp2 overexpression significantly increased the recombinant hCG protein level by 1.92 times compared to that of the control. The LC–MS/MS analysis and Phaseolus vulgaris leucoagglutinin (PHA-L) lectin blot analysis showed that Ugp2 overexpression significantly increased the total galactosylation levels of intracellular proteins and the O-glycosylation of recombinant hCG protein. The stability of the hCG protein to trypsin digestion was also enhanced. Ugp2 is the major limiting enzyme of the O-glycan precursor synthesis in recombinant hCG protein production. Furthermore, the effects and mechanisms of the key genes of O-glycan sugar chain synthesis and hydrolases such as polypeptide N-acetylgalactosaminyltransferase1 (Galnt1), Core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase (C1galt1), O-linked N-acetylglucosamine transferase (Ogt) and Hexosaminidase (Hex), were evaluated. The results indicated that Galnt1 overexpression increased the recombinant hCG protein level by 1.57 times and improved the total galactosylation of intracellular proteins, O-glycosylation and the stability of recombinant hCG protein. Galnt1 is the major limiting enzyme of O-glycan sugar chain synthesis. Overexpression of Ugp2 and Galnt1 simultaneously improved the recombinant hCG protein level by 2.44 times, and both had synergistic effects. Based on the results of overexpression of Galnt1, the major limiting gene of O-Glycan chain synthesis, the precursors GalNAc and Gal were added and increased the recombinant hCG protein level by 3.68 times. This study revealed the major limiting factors of O-glycosylation of recombinant hCG protein in CHO cells and proposed an effective expression regulation strategy.

Clonal variation in productivity and proteolytic clipping of an Fc-fusion protein in CHO cells: Proteomic analysis suggests a role for defective protein folding and the UPR

Product degradation, such as clipping, is a common quality issue in the production of Fc-fusion proteins from Chinese hamster ovary (CHO) cells. Degradation of proteins is mainly due to the action of either intracellular or extracellular host cell proteases. This study was carried out to understand more fundamentally the intracellular events that may play a role in determining why cell lines from the same cell line development project can vary with regards to the extent of Fc-fusion protein clipping. The cell lines that displayed the highest levels of clipping also produced less product than the cell lines with a lower level of clipping. In this study we applied differential quantitative label-free LC–MS/MS proteomic analysis to group clonally-derived cell lines (CDCLs) based on the level of clipping of the Fc-fusion protein. The analysis was carried out over two times points in culture and clones were designated as either having ‘high’ or ‘low’ clipping phenotypes. We have identified 200 differentially expressed proteins using quantitative label-free LC–MS/MS analysis between the two experimental groups. Functional assessment of the resultant proteomic data using Gene Ontology analysis showed a significant enrichment of biological processes and molecular functions related to protein folding, response to unfolded protein and protein translation. The levels of several proteases were also increased. This study identified protein targets that could be modified using cell line engineering approaches to improve the quality of recombinant Fc-fusion protein production in the biopharmaceutical industry.

Generation and characterization of a bivalent protein boost for future clinical trials: HIV-1 subtypes CR01_AE and B gp120 antigens with a potent adjuvant.

The RV144 Phase III clinical trial with ALVAC-HIV prime and AIDSVAX B/E subtypes CRF01_AE (A244) and B (MN) gp120 boost vaccine regime in Thailand provided a foundation for the future development of improved vaccine strategies that may afford protection against the human immunodeficiency virus type 1 (HIV-1). Results from this trial showed that immune responses directed against specific regions V1V2 of the viral envelope (Env) glycoprotein gp120 of HIV-1, were inversely correlated to the risk of HIV-1 infection. Due to the low production of gp120 proteins in CHO cells (2–20 mg/L), cleavage sites in V1V2 loops (A244) and V3 loop (MN) causing heterogeneous antigen products, it was an urgent need to generate CHO cells harboring A244 gp120 with high production yields and an additional, homogenous and uncleaved subtype B gp120 protein to replace MN used in RV144 for the future clinical trials. Here we describe the generation of Chinese Hamster Ovary (CHO) cell lines stably expressing vaccine HIV-1 Env antigens for these purposes: one expressing an HIV-1 subtype CRF01_AE A244 Env gp120 protein (A244.AE) and one expressing an HIV-1 subtype B 6240 Env gp120 protein (6240.B) suitable for possible future manufacturing of Phase I clinical trial materials with cell culture expression levels of over 100 mg/L. The antigenic profiles of the molecules were elucidated by comprehensive approaches including analysis with a panel of well-characterized monoclonal antibodies recognizing critical epitopes using Biacore and ELISA, and glycosylation analysis by mass spectrometry, which confirmed previously identified glycosylation sites and revealed unknown sites of O-linked and N-linked glycosylations at non-consensus motifs. Overall, the vaccines given with MF59 adjuvant induced higher and more rapid antibody (Ab) responses as well as higher Ab avidity than groups given with aluminum hydroxide. Also, bivalent proteins (A244.AE and 6240.B) formulated with MF59 elicited distinct V2-specific Abs to the epitope previously shown to correlate with decreased risk of HIV-1 infection in the RV144 trial. All together, these results provide critical information allowing the consideration of these candidate gp120 proteins for future clinical evaluations in combination with a potent adjuvant.

Glycoengineering of Mammalian Expression Systems on a Cellular Level.

Mammalian expression systems such as Chinese hamster ovary (CHO), mouse myeloma (NS0), and human embryonic kidney (HEK) cells serve a critical role in the biotechnology industry as the production host of choice for recombinant protein therapeutics. Most of the recombinant biologics are glycoproteins that contain complex oligosaccharide or glycan attachments representing a principal component of product quality. Both N-glycans and O-glycans are present in these mammalian cells, but the engineering of N-linked glycosylation is of critical interest in industry and many efforts have been directed to improve this pathway. This is because altering the N-glycan composition can change the product quality of recombinant biotherapeutics in mammalian hosts. In addition, sialylation and fucosylation represent components of the glycosylation pathway that affect circulatory half-life and antibody-dependent cellular cytotoxicity, respectively. In this chapter, we first offer an overview of the glycosylation, sialylation, and fucosylation networks in mammalian cells, specifically CHO cells, which are extensively used in antibody production. Next, genetic engineering technologies used in CHO cells to modulate glycosylation pathways are described. We provide examples of their use in CHO cell engineering approaches to highlight these technologies further. Specifically, we describe efforts to overexpress glycosyltransferases and sialyltransfereases, and efforts to decrease sialidase cleavage and fucosylation. Finally, this chapter covers new strategies and future directions of CHO cell glycoengineering, such as the application of glycoproteomics, glycomics, and the integration of 'omics' approaches to identify, quantify, and characterize the glycosylated proteins in CHO cells. Graphical Abstract.

MiR-143 targets MAPK7 in CHO cells and induces a hyperproductive phenotype to enhance production of difficult-to-express proteins.

In recent years, the number of complex but clinically effective biologicals such as multi-specific antibody formats and fusion proteins has increased dramatically. However, compared to classical monoclonal antibodies (mAbs), these rather artificially designed therapeutic proteins have never undergone millions of years of evolution and thus often turn out to be difficult-to-express using mammalian expression systems such as Chinese hamster ovary (CHO) cells. To provide access to these sophisticated but effective drugs, host cell engineering of CHO production cell lines represents a promising approach to overcome low production yields. MicroRNAs (miRNAs) have recently gained much attention as next-generation cell engineering tools. However, only very little is known about the capability of miRNAs to specifically increase production of difficult-to-express proteins. In a previous study we identified miR-143 amongst others to improve protein production in CHO cells. Thus, the aim of the present study was to examine if miR-143 might be suitable to improve production of low yield protein candidates. Both transient and stable overexpression of miR-143 significantly improved protein production without negatively affecting cell growth and viability of different recombinant CHO cells. In addition, mitogen-activated protein kinase 7 (MAPK7) was identified as a putative target gene of miR-143-3p in CHO cells. Finally, siRNA-mediated knock-down of MAPK7 could be demonstrated to phenocopy pro-productive effects of miR-143. In summary, our data suggest that miR-143 might represent a novel genetic element to enhance production of difficult-to-express proteins in CHO cells which may be partly mediated by down-regulation of MAPK7. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1046-1058, 2017.

A high density CHO-S transient transfection system: Comparison of ExpiCHO and Expi293

Chinese Hamster Ovary (CHO) cells are the principal mammalian host used for stable cell line generation and biotherapeutic protein production. Until recently, production of milligrams to grams of protein in CHO transient systems was challenging. As such, Human Embryonic Kidney (HEK293) cells are the most common mammalian cell type used for transient transfection. The post-translational modifications (PTMs) of a protein are dictated in part by the cell line used for expression, and changes in PTMs have been shown to affect both the activity and biophysical properties of proteins. Therefore, it is potentially advantageous to keep the host cell type consistent throughout drug discovery and development. To this end, we compared the ExpiCHO system, a high density CHO-S transient transfection system, to the Expi293 and FreeStyle MAX CHO transient systems. Fourteen proteins were expressed in both the Expi293 and ExpiCHO systems. For a majority of proteins tested, the protein titers observed with the ExpiCHO system were higher than those seen with both the FreeStyle MAX CHO and Expi293 systems. Antibodies expressed using the ExpiCHO system had glycosylation patterns more similar to antibodies produced in stable CHO cell lines than Expi293-derived antibodies. However, culture duration and temperature were found to affect protein titer, monodispersity, enzyme activity, and PTMs and should be carefully selected when using the ExpiCHO system. The ExpiCHO transient transfection systems allows for facile production of milligrams to grams of protein in CHO cells and de-risks the transition from transient to stable material during drug development.

Multi-omic profiling of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production.

Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO-K1 cells under growth-limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO-producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)+, adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT-PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)+ and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post-translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time-course analysis of high- and low-producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity. Biotechnol. Bioeng. 2015;112: 2373–2387. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.