Protein Production In Mammalian Cells Research Articles

Comprehensive Comparison of Baculoviral and Plasmid Gene Delivery in Mammalian Cells.

(1) Recombinant protein production in mammalian cells is either based on transient transfection processes, often inefficient and underlying high batch-to-batch variability, or on laborious generation of stable cell lines. Alternatively, BacMam, a transduction process using the baculovirus, can be employed. (2) Six transfecting agents were compared to baculovirus transduction in terms of transient and stable protein expression characteristics of the model protein ACE2-eGFP using HEK293-6E, CHO-K1, and Vero cell lines. Furthermore, process optimization such as expression enhancement using sodium butyrate and TSA or baculovirus purification was assessed. (3) Baculovirus transduction efficiency was superior to all transfection agents for all cell lines. Transduced protein expression was moderate, but an 18-fold expression increase was achieved using the enhancer sodium butyrate. Ultracentrifugation of baculovirus from a 3.5 L bioreactor significantly improved the transduction efficiency and protein expression. Stable cell lines were obtained with each baculovirus transduction, yet stable cell line generation after transfection was highly unreliable. (4) This study demonstrated the superiority of the BacMam platform to standard transfections. The baculovirus efficiently transduced an array of cell lines both transiently and stably and achieved the highest efficiency for all tested cell lines. The feasibility of the scale-up of baculovirus production was demonstrated and the possibility of baculovirus purification was successfully explored.

Development of an in vitro screening system for synthetic signal peptide in mammalian cell-based protein production.

Optimizing appropriate signal peptides in mammalian cell-based protein production is crucial given that most recombinant proteins produced in mammalian cells are thought to be secreted proteins. Until now, most studies on signal peptide in mammalian cells have replaced native signal peptides with well-known heterologous signal peptides and bioinformatics-based signal peptides. In the present study, we successfully established an in vitro screening system for synthetic signal peptide in CHO cells by combining a degenerate codon-based oligonucleotides library, a site-specific integration system, and a FACS-based antibody detection assay. Three new signal peptides were screened using this new screening system, confirming to have structural properties as signal peptides by the SignalP web server, a neural network-based algorithm that quantifies the signal peptide-ness of amino acid sequences. The novel signal peptides selected in this study increased Fc-fusion protein production in CHO cells by increasing specific protein productivity, whereas they did not negatively affect cell growth. Particularly, the SP-#149 clone showed the highest qp, 0.73 ± 0.01pg/cell/day from day 1 to day 4, representing a 1.47-fold increase over the native signal peptide in a serum-free suspension culture mode. In addition, replacing native signal peptide with the novel signal peptides did not significantly affect sialylated N-glycan formation, N-terminal cleavage pattern, and biological function of Fc-fusion protein produced in CHO cells. The overall results indicate the utility of a novel in vitro screening system for synthetic signal peptide for mammalian cell-based protein production. KEY POINTS: • An in vitro screening system for synthetic signal peptide in mammalian cells was established • This system combined a degenerate codon-based library, site-specific integration, and a FACS-based detection assay • The novel signal peptides selected in this study could increase Fc-fusion protein production in mammalian cells.

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells.

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10‐R98S). Our data support that RPL10‐R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock‐in of RPL10‐R98S was associated with a 1.7‐ to 2.5‐fold increased production of four transiently expressed recombinant proteins and 1.7‐fold for one out of two stably expressed proteins. In CHO‐S cells, eGFP reached a 2‐fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10‐R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10‐R98S phenotypes can maximize its exploitability for the mammalian protein production industry.

Abstract 1837: A human artificial chromosome (HAC)-based system as a powerful tool for stable co-expression of multiple proteins and therapeutic IgGs in mammalian cells

Abstract Background: Currently, there is increasing demand for recombinant therapeutic proteins produced in mammalian cell lines, especially therapeutic antibodies. MUC16-directed IgG and bispecific diabodies are one such example that represents a promising strategy for treatment of ovarian cancer. HEK293 and CHO cells are mostly used for large-scale stable expression of therapeutic proteins, however low protein yield, transgene silencing, cell line instability, and the time-consuming process of cell line development represent serious challenges. Here, we are presenting an alternative system for stable, high yield expression of multiple genes including complex therapeutic antibodies (HC and LC genes) and other proteins within single HAC. HAC vector propagate independently in cells without integration in the host chromosomes and has unlimited cloning capacity. Furthermore, this particular HAC can be targeted by transcriptional transactivators like tetR-VP16, eliminating transgene silencing, thereby providing stable long-term high yield protein production in mammalian cells. Methods: The targeting vector containing single or multiple genes is loaded at the unique LoxP site of tetO-HAC propagated in HPRT-deficient CHO cells by Cre-LoxP mediated recombination. As a result of recombination, functional HPRT gene will be reconstituted and HPRT-positive colonies are selected after 3 weeks growth in HAT medium. In the next step, HAC with therapeutic transgene(s) is transferred to the different high productive cell lines via MMCT technique following by high yield protein(s) expression in spinner flask. The alphoid tetO-HAC carries Bsr gene, so Blasticidin is used to select for the recipient cells that have taken up the HAC after Microcell-mediated chromosome transfer (MMCT). Results: Targeting vector with His-tagged MUC16 BiTEs gene (or iBAC caring HC and LC of anti-MUC16 IgG) was inserted into the HAC propagated in CHO cells. HPRT gene reconstitution was confirmed by PCR using a set of specific primers. Expression of His-tagged MUC16 BiTEs or anti-MUC16 IgG in CHO cells was confirmed by western blot and SDS-PAGE analysis. The yield of Ni- purified MUC16 BiTEs was up to 6 mg/l in original CHO cells. Our data from cellular models confirmed high efficacy of Ni- purified MUC16 BiTEs against OVCAR3 tumor cells, - up to 80% of the tumor cells were killed by T-cells in the presence of MUC16 BiTEs protein. After that, HAC/MUC16 BiTEs was successfully transferred to high productive ExpiHEK293F cells by MMCT. Therapeutic HAC transferring was confirmed by expression DsRed transgene linked with MUC16BiTEs through IRES and FISH analysis. FISH reveals the HAC propagated independently in ExpiHEK293F cells. Conclusions: Our data shows that the HAC is the new promising system for stable, high yield production of multiple therapeutic proteins. Citation Format: Artem Kononenko, Ian Laster, Oladapo Yeku, David Spriggs. A human artificial chromosome (HAC)-based system as a powerful tool for stable co-expression of multiple proteins and therapeutic IgGs in mammalian cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1837.

Direct transgene expression and mAb production by using new model UCOEs on iPS and CHO cells

Background & Aim It has recently been shown that efficient and stable expression of transgenes from the A2UCOE is at least in part due to its resistance to DNA methylation- mediated silencing. Analysis of a deletion series from the CBX3 end of a fully functional 2.5kb A2UCOE where expression of an eGFP reporter gene is directly driven off the HNRPA2B1 promoter, revealed a 1.2kb and 1.7kb truncation that retained full UCOE activity following transduction of P19 and F9 cells in both undifferentiated and differentiated states. These 1.2-1.7 and a further deletion 0.5kb A2UCOEs, were also able to retain stable expression in murine embryonic stem cells and now in human iPS cells during differentiation into embryoid bodies and three different tissue specific cell types. In addition, from a practical perspective our finding that the 0.5-1.2-1.7A2UCOEs retain the same stability of expression as the larger 2.5kb A2UCOE model suggests that it can effectively replace the latter within therapeutic LV constructs allowing a greater capacity and also more safety for the gene of interest by cutting off the enhancer elements which carries a potential mutation risk. A further project now on human mAb production with recombinant CHO cells by using our new UCOE models has been started and the early results show that they are more productive than the control A2UCOEs and able to produce human recombinant mAbs in mg to g level even under two months of cultivation. The results already encouraging to provide a new better UCOE model for recombinant protein production in mammalian cells. Methods, Results & Conclusion Firstly, the new UCOE models were cloned into lentiviral vectors and then the vectors were produced by 293T cells. Then, human iPS cells were transfected by the vectors without differentiation. The expression of the UCOE models were observed over 42 days by measuring eGfP+ cell levels in flow cytometry every week. The experiments then were followed by differentiation of transduced iPS cells into three different tissue spesific cell types (pre and neuronal, hepatocyte and cardiomyocyte cell types). Secondly, the aimed mAb DNA sequences were produced by PCR, and clonned into our UCOE vectors and then they were transduced into the CHO group sub-cell clones which the recombinant protein production will be done and the mAbs will then be isolated from the cell culture and analysed by correction methods. After the correction analyses, the production of aimed IgG1 ve IgE/k group mAbs were produced in bioreactor in gram level.

Study of the mechanism for increased protein expression via transcription potency reduction of the selection marker.

Stable transfection of mammalian cells using various expression cassettes for exogenous gene expression has been well established. The impact of critical factors in these cassettes, such as promoter and enhancer elements, on recombinant protein production in mammalian cells has been studied extensively to optimize the expression efficiency. However, few studies on the correlation between the strength of selection marker and the expression of gene of interest (GOI) have been reported. Here we investigated the correlation between the strength of a widely used selection marker, glutamine synthetase (GS) gene, and gene of interest in which the expression of GOI is driven by mouse cytomegalovirus (mCMV) major immediate early (MIE) promoter whereas the expression of GS is controlled by SV40E (Simian vacuolating virus 40E) promoter. We used a green fluorescent protein and the adalimumab antibody (heavy and light chain) as two distinct examples for the gene of interest. We then decreased the expression of GS gene by engineering a specific region of its SV40E promoter in these expression cassettes. By comparing the expression of GS and GOI at transcription and translation level before and after the SV40E promoter was weakened, we found that lower GS expression due to weaker SV40E transcription correlated well with the higher expression of recombinant proteins, mainly by increasing the copy number of GS and GOI integration into host cell genome.

A High-Throughput System for Transient and Stable Protein Production in Mammalian Cells.

Recombinant protein expression and purification is an essential component of biomedical research and drug discovery. Advances in automation and laboratory robotics have enabled the development of highly parallel and rapid processes for cell culture and protein expression, purification, and analysis. Human embryonic kidney (HEK) cells and Chinese hamster ovary (CHO) cells have emerged as the standard host cell workhorses for producing recombinant secreted mammalian proteins by using both transient and stable production strategies. In this chapter we describe a fully automated custom platform, Protein Expression and Purification Platform (PEPP), used for transient protein production from HEK cells and stable protein production from CHO cells. Central to PEPP operation is a suite of custom robotic and instrumentation platforms designed and built at GNF, custom cell culture ware, and custom scheduling software referred to as Runtime. The PEPP platform enables cost-effective, facile, consistent production of proteins at quantities and quality useful for early stage drug discovery tasks such as screening, bioassays, protein engineering, and analytics.

Pichia pastoris: SEL RAGI UNTUK PRODUKSI PROTEIN REKOMBINAN

Pichia pastoris is a group of methylotropic yeast known as a host of expression and protein production which is widely used for biopharmaceutical-based drug production. This yeast can grow fast with a high cell density. Its genetic stability, high cell density, and stress resistance make the development process and scale-up of P. pastoris can increase to a scale of 200,000 liters of culture. In contrast to the expensive and complex development of recombinant protein production in mammalian cells, the development of production in P. pastoris is relatively simple and cheaper. The advantage of P. pastoris as an expression system is that it is able to use methanol as a carbon source by inducing the expression of alcohol oxidase oxidase (AOX) enzyme. Promoter used by this enzyme is also used as a strong promoter for the expression of proteins that we want. Unlike in bacterial and mammalian systems, recombinant protein production in Pichia cells is not contaminated with endotoxins or viruses so it is safer and simplifies the downstream processes in bioproduction. The level of endogenous protein in the low supernatant allows Pichia to cultivate with a high volumetric productivity, therefore the process of protein production becomes very economical. This review provides an overview of several things that must be considered in utilizing P. pastoris as an expression system including the selection of vectors, strains, vector integration mechanisms into the genome, glycosylation processes, and applications in industry.

Nanoscale characterization coupled to multi-parametric optimization of Hi5 cell transient gene expression.

Polyethylenimine (PEI)-based transient gene expression (TGE) is nowadays a well-established methodology for rapid protein production in mammalian cells, but it has been used to a much lower extent in insect cell lines. A fast and robust TGE methodology for suspension Hi5 (Trichoplusia ni) cells is presented. Significant differences in size and morphology of DNA:PEI polyplexes were observed in the different incubation solutions tested. Moreover, minimal complexing time (< 1min) between DNA and PEI in 150mM NaCl solution provided the highest transfection efficiency. Nanoscopic characterization by means of cryo-EM revealed that DNA:PEI polyplexes up to 300-400nm were the most efficient for transfection. TGE optimization was performed using eGFP as model protein by means of the combination of advanced statistical designs. A global optimal condition of 1.5 × 106 cell/mL, 2.1μg/mL of DNA, and 9.3μg/mL PEI was achieved through weighted-based optimization of transfection, production, and viability responses. Under these conditions, a 60% transfection and 0.8μg/106 transfected cell·day specific productivity were achieved. The TGE protocol developed for Hi5 cells provides a promising baculovirus-free and worthwhile approach to produce a wide variety of recombinant proteins in a short period of time.

Genetic disruption of multiple α1,2-mannosidases generates mammalian cells producing recombinant proteins with high-mannose–type N-glycans

Recombinant therapeutic proteins are becoming very important pharmaceutical agents for treating intractable diseases. Most biopharmaceutical proteins are produced in mammalian cells because this ensures correct folding and glycosylation for protein stability and function. However, protein production in mammalian cells has several drawbacks, including heterogeneity of glycans attached to the produced protein. In this study, we established cell lines with high-mannose-type N-linked, low-complexity glycans. We first knocked out two genes encoding Golgi mannosidases (MAN1A1 and MAN1A2) in HEK293 cells. Single knockout (KO) cells did not exhibit changes in N-glycan structures, whereas double KO cells displayed increased high-mannose-type and decreased complex-type glycans. In our effort to eliminate the remaining complex-type glycans, we found that knocking out a gene encoding the endoplasmic reticulum mannosidase I (MAN1B1) in the double KO cells reduced most of the complex-type glycans. In triple KO (MAN1A1, MAN1A2, and MAN1B1) cells, Man9GlcNAc2 and Man8GlcNAc2 were the major N-glycan structures. Therefore, we expressed two lysosomal enzymes, α-galactosidase-A and lysosomal acid lipase, in the triple KO cells and found that the glycans on these enzymes were sensitive to endoglycosidase H treatment. The N-glycan structures on recombinant proteins expressed in triple KO cells were simplified and changed from complex types to high-mannose types at the protein level. Our results indicate that the triple KO HEK293 cells are suitable for producing recombinant proteins, including lysosomal enzymes with high-mannose-type N-glycans.

Codon Optimization in the Production of Recombinant Biotherapeutics: Potential Risks and Considerations.

Biotherapeutics are increasingly becoming the mainstay in the treatment of a variety of human conditions, particularly in oncology and hematology. The production of therapeutic antibodies, cytokines, and fusion proteins have markedly accelerated these fields over the past decade and are probably the major contributor to improved patient outcomes. Today, most protein therapeutics are expressed as recombinant proteins in mammalian cell lines. An expression technology commonly used to increase protein levels involves codon optimization. This approach is possible because degeneracy of the genetic code enables most amino acids to be encoded by more than one synonymous codon and because codon usage can have a pronounced influence on levels of protein expression. Indeed, codon optimization has been reported to increase protein expression by> 1000-fold. The primary tactic of codon optimization is to increase the rate of translation elongation by overcoming limitations associated with species-specific differences in codon usage and transfer RNA (tRNA) abundance. However, in mammalian cells, assumptions underlying codon optimization appear to be poorly supported or unfounded. Moreover, because not all synonymous codon mutations are neutral, codon optimization can lead to alterations in protein conformation and function. This review discusses codon optimization for therapeutic protein production in mammalian cells.

Modulation of Luciferase Production in Melanoma Cells in vitro

Reporter proteins find increasing application in biomedical studies in vitro and in vivo. However, to correctly interpret the results based on their use, it is important to understand whether reporter protein production is modulated in model cells and in what conditions such modulation may occur. Reporter activity was studied in Mel IL melanoma cells transiently transfected with a pCpG vector-based plasmid construct expressing firefly luciferase. Luciferase expression quickly dropped during the first two culture passages, which were followed by a quasi-stable period, when luciferase expression relatively slightly decreased with time. Phases of maximal and minimal luciferase production, which corresponded to the exponential and stationary growth phases, respectively, were observed during batch culture. When the medium was changed, luciferase production was stimulated in the stationary, but not exponential, cell growth phase. Severe hypoxia (0.1% O2) decreased the luciferase amount, suggesting substantial modulation of cell metabolism in total and luciferase production in particular. The targeted drug vemurafenib suppressed the luciferase production in Mel IL cells, whereas DMSO, which is often used as a drug solvent in experiments with cells, stimulated the luciferase production. Based on the results, it was hypothesized that modulation of reporter protein production in mammalian cells reflects the adaptation of intracellular metabolism to external conditions and may be a source of incorrect interpretations of experiments using reporter proteins.

LARP4 mRNA codon-tRNA match contributes to LARP4 activity for ribosomal protein mRNA poly(A) tail length protection.

Messenger RNA function is controlled by the 3' poly(A) tail (PAT) and poly(A)-binding protein (PABP). La-related protein-4 (LARP4) binds poly(A) and PABP. LARP4 mRNA contains a translation-dependent, coding region determinant (CRD) of instability that limits its expression. Although the CRD comprises <10% of LARP4 codons, the mRNA levels vary >20 fold with synonymous CRD substitutions that accommodate tRNA dynamics. Separately, overexpression of the most limiting tRNA increases LARP4 levels and reveals its functional activity, net lengthening of the PATs of heterologous mRNAs with concomitant stabilization, including ribosomal protein (RP) mRNAs. Genetic deletion of cellular LARP4 decreases PAT length and RPmRNA stability. This LARP4 activity requires its PABP-interaction domain and the RNA-binding module which we show is sensitive to poly(A) 3'-termini, consistent with protection from deadenylation. The results indicate that LARP4 is a posttranscriptional regulator of ribosomal protein production in mammalian cells and suggest that this activity can be controlled by tRNA levels.

Correlation Between Expression of Recombinant Proteins and Abundance of H3K4Me3 on the Enhancer of Human Cytomegalovirus Major Immediate-Early Promoter.

Role of epigenetic regulation in the control of gene expression is well established. The impact of several epigenetic mechanisms, such as DNA methylation and histone acetylation, on recombinant protein production in mammalian cells has been investigated recently. Here we investigate the correlation between the selected epigenetic markers and five trastuzumab biosimilar-producing Chinese hamster ovary (CHO) cell lines in which the expression of trastuzumab is driven by human cytomegalovirus (HCMV) major immediate-early (MIE) promoter. We chose the producing clones in which transcription was the determinative step for the production of recombinant trastuzumab. We found that the abundance of trimethylation of histone 3 at lysine 4 (H3K4Me3) on the enhancer of HCMV MIE promoter correlated well with the relative titers of recombinant trastuzumab among the clones. Such close correlation was not observed between the recombinant protein and other epigenetic markers examined in our study. Our results demonstrate that the HCMV MIE enhancer-bound H3K4Me3 epigenetic marker may be used as the epigenetic indicator to predict the relative production of recombinant proteins between the producing CHO cell lines.

Genome-scale RNA interference screen identifies antizyme 1 (OAZ1) as a target for improvement of recombinant protein production in mammalian cells.

For the purpose of improving recombinant protein production from mammalian cells, an unbiased, high-throughput whole-genome RNA interference screen was conducted using human embryonic kidney 293 (HEK 293) cells expressing firefly luciferase. A 21,585 human genes were individually silenced with three different siRNAs for each gene. The screen identified 56 genes that led to the greatest improvement in luciferase expression. These genes were found to be included in several pathways involved in spliceosome formation and mRNA processing, transcription, metabolic processes, transport, and protein folding. The 10 genes that most enhanced protein expression when downregulated, were further confirmed by measuring the effect of their silencing on the expression of three additional recombinant proteins. Among the confirmed genes, OAZ1-the gene encoding the ornithine decarboxylase antizyme1-was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing OAZ1 caused an increase of the ornithine decarboxylase enzyme and the cellular levels of putrescine and spermidine; an indication that increased cellular polyamines enhances luciferase expression without affecting its transcription. The study shows that OAZ1 is a novel target for improving expression of recombinant proteins. The genome-scale screening performed in this work can establish the foundation for targeted design of an efficient mammalian cell platform for various biotechnological applications. Biotechnol. Bioeng. 2016;113: 2403-2415. © 2016 Wiley Periodicals, Inc.

Overexpression of YY1 increases the protein production in mammalian cells

The production of therapeutic antibodies using mammalian cells remains a high-priority in the biopharmaceutical manufacturing industry. Bioengineers have targeted different cellular processes, including transcription, translation, secretion and post-translational modifications, to overcome the metabolic bottlenecks limiting production capacity and create high-producing mammalian cell lines. The polycomb group (PcG) proteins belong to a family of chromatin regulators with important roles in multicellular development. By overexpressing and screening genes from the PcG family, we have identified an epigenetic key player for biopharmaceutical manufacturing enhancement: the transcription factor Yin Yang 1 (YY1). The overexpression of YY1 led to an increase in the production of several product genes (SEAP, VEGF165, IgG including Rituximab), provided that human YY1 (hYY1) was expressed in human cells (HeLa, HT-1080, HEK-293T, FreeStyle™ 293-F) and Chinese hamster ovary cell-derived YY1 (cYY1) was expressed in CHO cells (CHO-K1, CHO-easyC, FreeStyle™ CHO-S, CHO-B13-24, CHO-IgG1). Ectopic expression of cYY1 in the stable CHO-derived IgG producer cell lines CHO-B13-24 and CHO-IgG1 increased the antibody titer up to 6-fold, suggesting that epigenetic engineering of mammalian production cell lines could become a new strategy to improve the manufacturing of complex protein pharmaceuticals.

Novel cell engineering of the Unfolded Protein Response to achieve efficient therapeutic protein production cell line

Bioproduction of therapeutic proteins such as monoclonal antibodies (mAb) continues to be a fast growing sector of advanced manufacturing. An ever increasing repertoire of therapeutic proteins coupled with the emergence of biosimilars has led to increasing global demand for higher yielding, faster, and more cost-effective manufacturing process. Central to any good production platform is the capacity of the production cell line. A suitable production cell line exhibits physiological traits such as high specific productivity (qp), short doubling time, high peak cell density and efficient metabolism. The emergence of a suitable production cell line with all the aforementioned traits is an extremely rare event, as it requires that all facets of cellular transcription, translation, secretion and metabolic efficiency are both individually optimized and collectively synchronized into a system capable of high level protein expression. Cellular translation and secretion capacity is thought to be one of the major cellular bottlenecks limiting protein production in mammalian cells. The over-expression of complex recombinant proteins such as mAbs driven by strong viral promoters exerts considerable burden on the Endoplasmic Reticulum (ER) and Golgi apparatus. Increased ER stress triggers the Unfolded Protein Response (UPR), which may lead to cell apoptosis and thus elimination of cells with high expression of the Gene of Interest (GOI). In this thesis, we adopted a host cell engineering approach to expand cellular translation and secretion capacity prior to expressing the GOI, in order to increase the probability of isolating high producers. Through screening of a panel of mAb producing clonal cell lines using qPCR, the XBP1 mRNA splice ratio was identified as a suitable target to augment host cell translation and secretion machinery. A high ratio of spliced over unspliced XBP1 mRNA is required to overcome the negative regulatory effect of unspliced XBP1 protein. Spliced XBP1 mRNA translates into a potent transcription factor that up-regulates a wide range of folding, secretory and quality control proteins, which forms the cytoprotective branch of the UPR. A stable CHO host cell line engineered with a high splice ratio of XBP1 mRNA exhibited increased expression of various chaperone and secretary vesicle proteins without increasing UPR associated apoptotic markers. This engineered host cell line was compared against the parental cell line in transient mAb production studies. The XBP1 cell line displayed a 7.5 fold increase in qp over the control cell line and more than 4 fold increase in final volumetric productivity. Moreover, a 3-fold increase in the percentage of high producers was observed in the cell distributions post-transfection when using the XBP1 host cell line. mAb producing cells derived from the XBP1 host cell line also demonstrated competency in conventional stirred-tank bioreactors, showing promise of a commercially viable production platform. In conclusion, XBP1 mRNA splice ratio engineered host cells have expanded translation and secretion capacity to alleviate the ER stress experienced from mAb production. The augmented capacity enabled multi-fold improvements in qp and volumetric productivity and increased the frequency of high producers in the transformed pool. The engineered host cell line showed great promise to become a commercially viable production platform cell line to significantly reduce the time and resources associated with cell line development.

A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr.

Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.

Epigenetic regulatory elements: Recent advances in understanding their mode of action and use for recombinant protein production in mammalian cells.

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.

Development of protein-free medium for therapeutic protein production in mammalian cells: recent advances and perspectives

Mammalian cell culture media used for the manufacture of therapeutic protein have advanced systematically from serum-containing into animal-free, protein-free and chemically defined formulations over the past decades. Initially driven by patient safety concerns associated with the use of animal-derived medium components, and later by inconsistent cell culture performance due to variability in plant-derived raw material lots, many biologics manufacturers redirect their focus on the development of proprietary media formulations and implementation of well-controlled chemically defined raw materials in all cell culture media and feeds for production processes. This article will provide an overview of current trends and objectives of industrial medium development efforts for therapeutic protein production.