Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells

Monoclonal antibodies (mAbs) have emerged as the most promising category of recombinant proteins due to their high efficiency for the treatment of a wide range of human diseases. The complex nature of mAbs creates a great deal of challenges in both upstream and downstream manufacturing processes. Proportional expression and correct folding and assembly of the light chain and heavy chain are required for efficient production of the mAbs. In this regard, expression vector design has proven to have profound effects on the antibody expression level as well as its stability and quality. Here, we have explored the efficiency of different vector design strategies for the expression of a recombinant IgG1 antibody in Chinese hamster ovary (CHO) cells. The antibody expression level was analyzed in transient expression and stable cell pools followed by expression analysis on single-cell clones. While detectable amounts of antibody were observed in all three systems, dual-promoter single-vector system showed the highest expression level in transient and stable expression as well as the highest productivity among clonal cells. Our results here show the importance of vector design for successful production of whole mAbs in CHO cells.     

Enhancing expression level and stability of transgene mediated by episomal vector via buffering DNA methyltransferase in transfected CHO cells

Nonviral episomal vectors present attractive alternative vehicles for gene therapy applications. Previously, we have established a new type of nonviral episomal vector-mediated by the characteristic motifs of matrix attachment regions (MARs), which is driven by the cytomegalovirus (CMV) promoter. However, the CMV promoter is intrinsically susceptible to silencing, resulting in declined productivity during long-term culture. In this study, Chinese hamster ovary (CHO) cells and DNA methyltransferase-deficient (Dnmt3a-deficient) CHO cells were transfected with plasmid-mediated by MAR, or CHO cells were treated with the DNA methylation inhibitor 5-Aza-2′-deoxycytidine. Flow cytometry, plasmid rescue experiments, fluorescence in-situ hybridization (FISH), and bisulfite sequencing were performed to observe transgene expression, its state of existence, and the CpG methylation level of the CMV promoter. The results indicated that all DNA methylation inhibitor and methyltransferase deficient cells could increase transgene expression levels and stability in the presence or absence of selection pressure after a 60-generation culture. Plasmid rescue assay and FISH analysis showed that the vector still existed episomally after long-time culture. Moreover, a relatively lower CMV promoter methylation level was observed in Dnmt3a-deficient cell lines and CHO cells treated with 5-Aza-2′-deoxycytidine. In addition, Dnmt3a-deficient cells were superior to the DNA methylation inhibitor treatment regarding the transgene expression and long-term stability. Our study provides the first evidence that lower DNA methyltransferase can enhance expression level and stability of transgenes mediated by episomal vectors in transfected CHO cells.     

CRISPR/Cas9‐mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long‐term stability

CHO cells are the preferred host for the production of complex pharmaceutical proteins in the biopharmaceutical industry, and genome engineering of CHO cells would benefit product yield and stability. Here, we demonstrated the efficacy of a Dnmt3a‐deficient CHO cell line created by CRISPR/Cas9 genome editing technology through gene disruptions in Dnmt3a, which encode the proteins involved in DNA methyltransferases. The transgenes, which were driven by the 2 commonly used CMV and EF1α promoters, were evaluated for their expression level and stability. The methylation levels of CpG sites in the promoter regions and the global DNA were compared in the transfected cells. The Dnmt3a‐deficent CHO cell line based on Dnmt3a KO displayed an enhanced long‐term stability of transgene expression under the control of the CMV promoter in transfected cells in over 60 passages. Under the CMV promoter, the Dnmt3a‐deficent cell line with a high transgene expression displayed a low methylation rate in the promoter region and global DNA. Under the EF1α promoter, the Dnmt3a‐deficient and normal cell lines with low transgene expression exhibited high DNA methylation rates. These findings provide insight into cell line modification and design for improved recombinant protein production in CHO and other mammalian cells.     

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.     

Evaluating the efficiency of phiC31 integrase‐mediated monoclonal antibody expression in CHO cells

Traditional methods to generate CHO cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. In comparison, site‐specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. PhiC31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as Cre. In this study, using different ratios of phiC31 serine integrase, we evaluated the phiC31 mediated gene integration for expression of a humanized IgG1 antibody (mAb0014) in CHO‐S cells. Light chain (LC) and heavy chain (HC) genes were expressed in one operon under EF1α promoter and linked by internal ribosome entry site (IRES) element. The clonal selection was carried out by limiting dilution. Targeted integration approach increased recombinant protein yield and stability in cell pools. The productivity of targeted cell pools was about 4 mg/L and about 40 µg/L in the control cell pool. The number of integrated transgenes was about 19 fold higher than the control cells pools. Our results confirmed that the phiC31 integrase leads to mAb expression in more than 90% of colonies. The productivity of the PhiC31 integrated cell pools was stable for three months in the absence of selection as compared with conventional transfection methods. Hence, utilizing PhiC31 integrase can increase protein titer and decrease the required time for protein expression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1570–1576, 2016     

Calcium-phosphate mediated DNA transfer into HEK-293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Transfection and protein expression in mammalian cells

This is the first report of transient transfection of suspended cells with purified plasmid DNA in bioreactors or spinner flasks. DNA/calcium phosphate complexes were pumped or injected directly into stirred cultures of the immortalized human embryo kidney cell line 293 (HEK-293) which had been adapted to growth in suspension. We identified culture conditions suitable for this approach and modified the protocol for the generation of precipitate complexes, based on our earlier work. In order to stabilize the DNA-vehicle-complex in the culture medium, we identified pH ranges and ion-concentrations which prevent dissolution or aggregation of the precipitate particles. Such conditions maintained suspended fine particles in spinners or bioreactors for up to 6 hr. During that period, cells and precipitate complexes interacted sufficiently to allow DNA transfer and subsequent expression of recombinant protein. In a simple 5 day batch process, with a starting density of 0.3 × 10⁶ cells mL^-1, about 0.5 mg L^-1 of a recombinant tissue plasminogen activator variant was observed.     

Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells

Low‐level and unstable transgene expression are common issues using the CHO cell expression system. Matrix attachment regions (MARs) enhance transgene expression levels, but additional research is needed to improve their function and to determine their mechanism of action. MAR‐6 from CHO chromosomes actively mediates high and consistent gene expression. In this study, we compared the effects of two new MARs and MAR‐6 on transgene expression in recombinant CHO cells and found one potent MAR element that can significantly increase transgene expression. Two MARs, including the human CSP‐B MAR element and DHFR intron MAR element from CHO cells, were cloned and inserted downstream of the poly(A) site in a eukaryotic vector. The constructs were transfected into CHO cells, and the expression levels and stability of eGFP were detected by flow cytometry. The three MAR sequences can be ranked in terms of overall eGFP expression, in decreasing order, as follows: human CSP‐B, DHFR intron MAR element and MAR‐6. Additionally, as expected, the three MAR‐containing vectors showed higher transfection efficiencies and transient transgene expression in comparison with those of the non‐MAR‐containing vector. Bioinformatics analysis indicated that the NFAT and VIBP elements within MAR sequences may contribute to the enhancement of eGFP expression. In conclusion, the human CSP‐B MAR element can improve transgene expression and its effects may be related to the NFAT and VIBP elements.     

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high‐producing clones among a large population of low‐ and non‐productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)‐based methotrexate (MTX) selection and glutamine synthetase (GS)‐based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS‐CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L‐MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS‐knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (～2%) of bi‐allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine‐dependent growth of all GS‐knockout cell lines. Full evaluation of the GS‐knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two‐ to three‐fold through the use of GS‐knockout cells as parent cells. The selection stringency was significantly increased, as indicated by the large reduction of non‐producing and low‐producing cells after 25 µM L‐MSX selection, and resulted in a six‐fold efficiency improvement in identifying similar numbers of high‐productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS‐knockout cells on recombinant protein quality is also discussed. Biotechnol. Bioeng. 2012; 109:1007–1015. © 2011 Wiley Periodicals, Inc.     

A balanced expression of two chains of heterodimer protein, the human interleukin-12, improves high-level expression of the protein in CHO cells

Interleukin-12 (IL-12) comprises of p40 and p35 subunits that are encoded by genes on separate chromosomes and form p70 heterodimer, a bioactive protein, and free p40, an antagonist of IL-12. Balance expression of two subunits within cells would be the key for high-level of production of bioactive IL-12. Thinking about different expression efficiencies of two genes (p40 gene with higher efficiency), we selected two expression vectors with different efficiencies and inserted genes of p40 and p35 into them separately and co-transfected them into Chinese hamster ovary (CHO) cells. The high-level expression of IL-12 was obtained when p40 cDNA was inserted into pcDNA3 (lower expression vector) and p35 cDNA was inserted into pEE14 (higher expression vector), but using pEE14 for p40 cDNA and pcDNA3 for p35 cDNA, which was opposite to the optimal design, or pEE14 or pcDNA3 for both p35 cDNA and p40 cDNA did not obtain high-level of production of p70 heterodimer, the bioactive IL-12. We also observed that using two chemical reagents in combination, as a pressure selection method or amplification for the two vectors, markedly enhanced the IL-12 production, when compared with any one selection chemical. Our results indicated that the balance expressions of two chains of hetrodimer protein, such as p40 and p35 of IL-12, would be a better choice to obtain high-level of production of the proteins.     

A new Chinese hamster ovary cell line expressing α2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins

Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins. Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the α2,6-sialyltransferase (α2,6-ST) cDNA. Glycoproteins produced by such CHO cells display both α2,6- and α2,3-linked terminal sialic acid residues, similar to human glycoproteins. Here, we have established a CHO cell line stably expressing α2,6-ST, providing a universal host for further transfections of human genes. Several relevant parameters of the universal host cell line were studied, demonstrating that the α2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line. A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e. interferon-γ and the tissue inhibitor of metalloproteinases-1. Interferon-γ purified from the universal host carried 40.4% α2,6- and 59.6% α2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-γ produced by normal CHO cells.     

A comparison of the CHO/HGPRT+ and the L5178Y/TK+/− mutation assays using suspension treatment and soft agar cloning: Results for 10 chemicals

The mouse lymphoma assay (MLA) and Chinese hamster ovary (CHO) cell assay are sensitive indicators of mutagenicity. The CHO assay has been modified technically to permit treatment in suspension and soft agar cloning comparable to the MLA. This methodology eliminates the risk of metabolic cooperation and the trauma of trypsinization. In addition, a larger population of cells can be treated and cloned for mutant selection. In order to compare the effectiveness of the test systems, 10 chemicals were evaluated for the induction of forward mutations in the CHO and MLA. Several of these chemicals have been reported as clastogenic; therefore, abbreviated colony sizing was performed to gauge the extent of genetic damage to the MLA cells. Both test systems detected benzo[a]pyrene, mitomycin C, acridine orange, and proflavin, and, with the exception of proflavin, more large colonies were present than small colonies. The suspect clastogen, phenytoin, was not mutagenic in the MLA and produced inconclusive results in the CHO. Ethidium bromide, a clastogen and a bacterial mutagen, was not mutagenic in either the MLA or CHO. Four compounds (p-aminophenol, benzoin, methoxychlor, and pyrene) were positive in the MLA, generally inducing a large number of small colonies, while demonstrating no mutagenic activity in the CHO assay. They have also been shown to be generally nongenotoxic in other test systems. Overall, the modified CHO assay did not appear to be better than the MLA for the detection of mutagenic agents. However, the MLA does appear to have lower specificity.Abbreviations AO acridine orange - BAP benzo[a]pyrene - BZN benzoin - CHO Chinese hamster ovary cell assay - DPH diphenylhydantoin - EB ethidium bromide - EMS ethylmethanesulfonate - 3MC 3-methylcholanthrene - MLA mouse lymphoma asay - MMC mitomycin C - MXC methoxychlor - PAP p-aminophenol - PRO proflavin - PYR pyrene     

Flow cytometry: an improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry.

In previous work, we clarified the relationship between the productivity and stability of gene-amplified cells and the location of the amplified gene. The location of the amplified gene enabled us to classify resistant cells into two types. One type of resistant cell group, in which the amplified genes were observed near the telomeric region, was named the "telomere type." The other type of cell group, in which the amplified genes were observed in other chromosomal regions, was named the "other type." The phenotypes of these two types of cells are very different. In this experiment, using a fluorescein isothiocyanate-labeled methotrexate (F-MTX) reagent with flow cytometry, we were easily able to distinguish between highly productive cells and the other types of cells. The level of fluorescence differed according to the difference in resistance to MTX. Based on this new finding, highly productive gene-amplified cells could be isolated from heterogeneous gene-amplified cell pools more easily than by the method of limiting-dilution assay. The limiting-dilution method requires several months to obtain highly productive gene-amplified cells, while our flow-cytometry-based method of selection requires only a few weeks.     

Enhanced targeted DNA methylation of the CMV and endogenous promoters with dCas9-DNMT3A3L entails distinct subsequent histone modification changes in CHO cells

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available.In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells.Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level.