Optimized Selection Marker and CHO Host Cell Combinations for Generating High Monoclonal Antibody Producing Cell Lines.

There are several selection markers which are suitable for generating stably transfected Chinese hamster ovary (CHO) cell lines. Due to their different modes of action, each selection marker has its own optimal selection stringency in different host cells for obtaining high productivity. Using an internal ribosome entry site (IRES)-mediated tricistronic vector and a set of IRES variants with different strengths, the expression of five antibiotics resistance genes (ARGs) in CHO-K1 cells and dihydrofolate reductase (DHFR) in CHO DG44 cells is optimized to enhance the stringency of selection for high producing cells. There is an obvious optimal expression level for every selection marker, below or above which, the productivity is significantly lower. The enhanced productivity in ARG generated CHO K1 cells is due to selective integration of active site while the enhanced productivity in the amplified CHO DG44 cells results from increased gene copies. The high producing CHO K1 pools and clones generated using ARG exhibit better production stability than the amplified high producing CHO DG44 pools and clones. Loss of expression for the CHO K1 cell lines is due to loss of gene copies while for CHO DG44 is due to transcriptional silencing. mAb glycan profile also differed significantly between CHO K1 and CHO DG44 cell lines. These results would be helpful when developing optimized vectors for generating high mAb producing CHO cell lines.

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.

Impact of Signal Peptides on Furin-2A Mediated Monoclonal Antibody Secretion in CHO Cells.

Studies had shown the benefits of using furin-2A peptides for high monoclonal antibody (mAb) expression in mammalian cells. How signal peptides affect furin-2A mediated mAb secretion has yet to be investigated. The impact of signal peptides on mAb secretion in furin-2A based tricistronic vectors in CHO cells is evaluated. In each tricistronic vector, heavy chain (HC) is arranged as the first cistron and followed by a furin recognition sequence, a 2A peptide, light chain (LC), an internal ribosome entry site (IRES), and dihydrofolate reductase (DHFR). Signal peptides for HC and LC are either removed or changed in different vectors. The vectors with signal peptides on both HC and LC genes gIve the highest mAb secretion levels. Changing to signal peptides with different strengths on either HC or LC do not change the mAb secretion level. IgG is still secreted when the signal peptide on the LC gene is removed but at a lower level compared to the vectors containing signal peptides on both HC and LC genes. Removing the HC signal peptide results in almost no IgG secretion regardless of whether the downstream LC carries any signal peptide. Removing the furin cleavage site does not affect mAb secretion levels while removing the 2A sequence results in low mAb secretion. The results present here will be beneficial for designing furin-2A based vectors for expressing mAb in mammalian cells.

Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells.

BACKGROUND: Methylated CpG dinucleotides in promoters are associated with the loss of gene expression in recombinant Chinese hamster ovary (CHO) cells during large-scale commercial manufacturing. We evaluated a promoter devoid of CpG dinucleotides, CpGfree, in parallel with a similar CpG containing promoter, CpGrich, for their ability to maintain the expression of recombinant enhanced green fluorescent protein (EGFP) after 8 weeks of culturing. RESULTS: While the promoters gave similar transient expression levels, CpGfree clones had significantly higher average stable expression possibly due to increased resistance to early silencing during integration into the chromosome. A greater proportion of cells in clones generated using the CpGfree promoter were still expressing detectable levels of EGFP after 8 weeks but the relative expression levels measured at week 8 to those measured at week 0 did not improve compared to clones generated using the CpGrich promoter. Chromatin immunoprecipitation assays indicated that the repression of the CpGfree promoter was likely linked to histone deacetylation and methylation. Use of histone deacetylase inhibitors also managed to recover some of the lost expression. CONCLUSION: Using a promoter without CpG dinucleotides could mitigate the early gene silencing but did not improve longer-term expression stability as silencing due to histone modifications could still take place. The results presented here would aid in promoter selection and design for improved protein production in CHO and other mammalian cells.

Impact of hydrolysates on monoclonal antibody productivity, purification and quality in Chinese hamster ovary cells.

Plant and yeast derived hydrolysates are economical and efficient alternative medium supplements to improve mammalian cell culture performance. We supplemented two commercial Chinese hamster ovary (CHO) culture media with hydrolysates from four different sources, yeast, soybean, Ex-Cell CD (a chemically defined hydrolysate replacement) and wheat to improve the productivity of two cell lines expressing different monoclonal antibodies (mAbs). Yeast, soybean and Ex-Cell CD improved the final mAb titer by increasing the specific productivity (qP) and/or extension of the culture period. Wheat hydrolysates increased peak viable cell density but did not improve productivity. IgG recovery from protein A purification was not compromised for all cultures by adding yeast, soybean and Ex-Cell CD hydrolysates except for one sample from soybean supplemented culture. Adding these three hydrolysates neither increased the amount of host cell protein, DNA or aggregate impurity amounts nor affect their clearance after purification. Profiling of the glycan types revealed that yeast and soybean hydrolysates could affect the distribution of galactosylated glycans. Ex-Cell CD performed the best at maintaining glycan profile compared to the non-supplemented cultures. Overall, yeast performed the best at improving CHO culture growth and productivity without being detrimental to downstream protein A processes but could affect mAb product glycan distribution while Ex-Cell CD yielded lower titers but has less effect on glycosylation. The hydrolysate to use would thus depend on the requirements of each process and our results would provide a good reference for improving culture performance with hydrolysates or related studies.

IgG Aggregation Mechanism for CHO Cell Lines Expressing Excess Heavy Chains.

Aggregates in protein therapeutics like IgG monoclonal antibodies (mAb) are detrimental to product safety and efficacy. It has been reported that aggregates form in Chinese hamster ovary (CHO) cell lines expressing greater amount of heavy chain (HC) than light chain (LC). In this study, we observed that aggregates could form within the cells with excess HC and were partially secreted into the supernatant. The aggregates in the supernatant consisted of mainly HC and were partially dissociated under either reducing or denaturing conditions. Mutation of a predicted free cysteine on HC to prevent disulfide bonding did not reduce aggregation. Re-transfecting CHO cells with excess HC with more BiP, an important IgG molecular chaperone, partially reduced unwanted aggregates and fragments possibly by helping retain more incomplete products within the cell for either proper assembly or degradation. A second transfection of LC into CHO cells with excess HC to increase the LC expression to a level greater than the HC expression successfully removed all aggregates and fragments. mAb product aggregation in CHO cells with excess HC occur due to a combination of limited chaperones and LC:HC ratio. These results provide added insights to aggregate formation and would be useful for development of mAb cell lines with reduced aggregates.

Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.

Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing.

Impact of using different promoters and matrix attachment regions on recombinant protein expression level and stability in stably transfected CHO cells.

High expression level and long-term expression stability are required for therapeutic protein production in mammalian cells. Three commonly used promoters from the simian virus 40 (SV40), the CHO elongation factor 1α gene (EF1α), and the human cytomegalovirus major immediate early gene (CMV) and two matrix attachment regions from the chicken lysozyme gene (cMAR) and the human interferon ß (iMAR) were evaluated for enhancing recombinant gene expression level and stability in stably transfected CHO cells. In the absence of MAR elements, the SV40 promoter gave lower expression level but higher stability than the EF1α promoter and the CMV promoter. The inclusion of MAR elements did not increase the integrated gene copies for all promoters but did enhance expression level for only the SV40 promoter. The enhanced gene expression was due to an increase in mRNA levels. Neither MAR elements enhance gene expression stability during long-term culture. The combinations of SV40 promoter and MAR elements are the best for obtaining both high expression level and stability. The information presented here would be valuable to those developing vectors for generation of CHO cell lines with stable and high productivity.

Toward stable gene expression in CHO cells.

Maintaining high gene expression level during long-term culture is critical when producing therapeutic recombinant proteins using mammalian cells. Transcriptional silencing of promoters, most likely due to epigenetic events such as DNA methylation and histone modifications, is one of the major mechanisms causing production instability. Previous studies demonstrated that the core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene is effective to prevent DNA methylation. We generated one set of modified human cytomegalovirus (hCMV) promoters by insertion of one or two copies of IE in either forward or reverse orientations into different locations of the hCMV promoter. The modified hCMV with one copy of IE inserted between the hCMV enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability in CHO cells without comprising expression level when compared with the wild type hCMV. We also found that insertion of IE into a chimeric murine CMV (mCMV) enhancer and human elongation factor-1α core (hEF) promoter in reverse orientation did not enhance expression stability, indicating that the effect of IE on expression stability is possibly promoter specific.

Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells.

The human cytomegalovirus promoter (hCMV) is susceptible to gene silencing in CHO cells, most likely due to epigenetic events, such as DNA methylation and histone modifications. The core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene has been shown to prevent DNA methylation. A set of modified hCMV promoters was developed by inserting one or two copies of IE in either forward or reverse orientations either upstream of the hCMV enhancer, between the enhancer and core promoter (CP), or downstream of the CP. The modified hCMV with one copy of IE inserted between the enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability without compromising expression level when compared with the wild-type (WT) hCMV. A third of 18 EGFP expressing clones generated using MR1 retained 70% of their starting expression level after 8 weeks of culture in the absence of selection pressure, while none of 18 WT hCMV generated clones had expression above 50%. MR1 also improved antibody expression stability of methotrexate (MTX) amplified CHO cell lines. Stably transfected pools generated using MR1 maintained 62% of their original monoclonal antibody titer after 8 weeks of culture in the absence of MTX, compared to only 37% for WT hCMV pools. Low levels of CpG methylation within both WT hCMV and MR1 were observed in all the analyzed cell lines and the methylation levels did not correlate to the expression stability, suggesting IE enhances expression stability by other mechanisms other than preventing methylation.

Identifying and engineering promoters for high level and sustainable therapeutic recombinant protein production in cultured mammalian cells.

Promoters are essential on plasmid vectors to initiate transcription of the transgenes when generating therapeutic recombinant proteins expressing mammalian cell lines. High and sustained levels of gene expression are desired during therapeutic protein production while gene expression is useful for cell engineering. As many finely controlled promoters exhibit cell and product specificity, new promoters need to be identified, optimized and carefully evaluated before use. Suitable promoters can be identified using techniques ranging from simple molecular biology methods to modern high-throughput omics screenings. Promoter engineering is often required after identification to either obtain high and sustained expression or to provide a wider range of gene expression. This review discusses some of the available methods to identify and engineer promoters for therapeutic recombinant protein expression in mammalian cells.

An internal ribosome entry site (IRES) mutant library for tuning expression level of multiple genes in mammalian cells.

A set of mutated Encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) elements with varying strengths is generated by mutating the translation initiation codons of 10(th), 11(th), and 12(th) AUG to non-AUG triplets. They are able to control the relative expression of multiple genes over a wide range in mammalian cells in both transient and stable transfections. The relative strength of each IRES mutant remains similar in different mammalian cell lines and is not gene specific. The expressed proteins have correct molecular weights. Optimization of light chain over heavy chain expression by these IRES mutants enhances monoclonal antibody expression level and quality in stable transfections. Uses of this set of IRES mutants can be extended to other applications such as synthetic biology, investigating interactions between proteins and its complexes, cell engineering, multi-subunit protein production, gene therapy, and reprogramming of somatic cells into stem cells.

Comparison of internal ribosome entry site (IRES) and Furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells.

Four versions of tricistronic vectors expressing IgG1 light chain (LC), IgG1 heavy chain (HC), and dihydrofolate reductase (DHFR) in one transcript were designed to compare internal ribosome entry site (IRES) and furin-2A (F2A) for their influence on monoclonal antibody (mAb) expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells.

Control of IgG LC:HC ratio in stably transfected CHO cells and study of the impact on expression, aggregation, glycosylation and conformational stability.

Immunoglobulin G (IgG), the most common class of commercial monoclonal antibodies (mAbs), exists as multimers of two identical light chains (LC) and two identical heavy chains (HC) assembled together by disulfide bridges. Due to the kinetics of mAb assembly, it is suggested that expression of LC and HC in equal amounts is not optimal for IgG production. We designed a set of vectors using internal ribosome entry site (IRES) elements to control LC and HC expression. The intracellular LC:HC ratio of stable IgG expressing Chinese hamster ovary (CHO) cell pools can be controlled effectively at four different ratios of 3.43, 1.24, 1.12, and 0.32. The stable pools were used to study the impact of LC:HC ratio on mAb expression and quality. Gene amplification was most effective for pools with excess LC and generated the highest mAb titers among the transfected pools. When LC:HC ratio was greater than one, more than 97% of the secreted products were IgG monomers. The products also have similar N-glycosylation profiles and conformational stabilities at those ratios. For pools presented a lower LC:HC ratio of 0.32, monomers only constituted half of the product with the other half being aggregates and mAb fragments. High mannose-type N-glycans increased while fucosylated and galactosylated glycans decreased significantly at the lowest LC:HC ratio. Product stability was also adversely affected. The results obtained provide insights to the impact of different LC:HC ratios on stable mAb production and useful information for vector design during generation of mAb producing cell lines.

Post-transcriptional regulatory elements for enhancing transient gene expression levels in mammalian cells.

Low yield from transient gene expression in mammalian cells limits its application to areas where large amount of proteins are needed. One effective approach to enhance transient gene expression levels is to use post-transcriptional regulatory elements (PTREs). We have evaluated the effect of five PTREs on the transient gene expression of three proteins in two cell lines. Most of the elements increased expression but exhibited cell-specific and gene-specific effects. The tripartite leader sequence of human adenovirus mRNA linked with a major late promoter enhancer gave the most universal and highest enhancement of gene expression levels. It increased the expression of all three proteins in HEK293 cells and two proteins in CHO K1 cells by 3.6- to 7.6-fold. Combinations of multiple PTREs increased protein expression as much as 10.5-fold.

IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines.

A Tricistronic vector utilizing internal ribosome entry site (IRES) elements to express the light chain (LC), heavy chain (HC), and a neomycin phosphotransferase (NPT) selection marker from one transcript is designed for generation of mAb expressing CHO cell lines. As compared to the commonly used vectors, benefits of this design include: (1) minimized non-expressing clones, (2) enhanced stable mAb productivity without gene amplification, (3) control of LC and HC expression at defined ratios, and (4) consistent product quality. After optimization of the LC and HC arrangement and increasing selection stringency by weakening the NPT selection marker, this Tricistronic vector is able to generate stably transfected pools with specific productivity (qmAb) greater than 5pg/cell/day (pcd) and titers over 150mg/L. 5% of clones from these pools have qmAb greater than 20pcd and titers ranging from 300 to more than 500mg/L under non-optimized shake flask batch cultures using commercially available protein-free medium. The mAb produced by these clones have low aggregation and consistent glycosylation profiles. The entire process of transfection to high-expressing clones requires only 6 months. The IRES-mediated Tricistronic vector provides an attractive alternative to commonly used vectors for fast generation of mAb CHO cell lines with high productivity.

Evaluating post-transcriptional regulatory elements for enhancing transient gene expression levels in CHO K1 and HEK293 cells.

Five post-transcriptional regulatory elements, (i) the 50 untranslated region (UTR) of human heat shock protein 70 mRNA (Hsp70), (ii) the 163-bp long splice variant derived from the 50 UTR of vascular endothelial growth factor (SP163), and (iii) the tripartite leader sequence of human adenovirus mRNA linked with a major late promoter enhancer (TM), (iv) the first intron of human cytomegalovirus immediate early gene (Intron A), and (v) the post-transcriptional regulatory element derived from woodchuck hepatitis virus (WPRE), are evaluated for enhancing transient gene expression levels in two industrial cell lines, HEK293 and CHO K1 using firefly luciferase (Fluc), interferon gamma (IFN), and Trastuzamab monoclonal antibody. Except for the Hsp70 which has no effects, all other elements enhance expression but exhibit cell-specific and gene-specific effects. TM provides the most universal and highest enhancement of gene expression levels. It enhances the expression of all three proteins in HEK293 cells and two proteins, Flucand IFN in CHO K1 cells by 3.6- to 7.6-fold. The remaining elements enhance expression of one or more proteins in at least one cell line by 1.7- to 3.2-fold. Combining WPRE with either Intron A, SP163, or TMhas cumulative effects on gene expression. The combinations can increase Fluc expression by up to 10.5-fold in HEK293 cells. These results provide valuable information to improve vectors for high level transient gene expressions in HEK293 and CHO K1 cells.

Mutated polyadenylation signals for controlling expression levels of multiple genes in mammalian cells.

A set of mutated SV40 early polyadenylation signals (SV40pA) with varying strengths is generated by mutating the AATAAA sequence in the wild-type SV40pA. They are shown to control the expression level of a gene over a 10-fold range using luciferase reporter genes in transient transfection assays. The relative strength of these SV40pA variants remains similar under three commonly used mammalian promoters and in five mammalian cell lines. Application of SV40pA variants for controlling expression level of multiple genes is demonstrated in a study of monoclonal antibody (mAb) synthesis in mammalian cells. By using SV40pA variants of different strengths, the expression of light chain (LC) and heavy chain (HC) genes encoded in a single vector is independently altered which results in different ratios of LC to HC expression spanning a range from 0.24 to 16.42. The changes in gene expression are determined by measuring mRNA levels and intracellular LC and HC polypeptides. It is found that a substantial decrease of HC expression, which increases the LC/HC mRNA ratio, only slightly reduces mAb production. However, reducing the LC expression by a similar magnitude, which decreases the LC/HC mRNA ratio results in a sharp decline of mAb production to trace amounts. This set of SV40pA variants offers a new tool for accurate control of the relative expression levels of multiple genes. It will have wide-ranging applications in fields related to the study of biosynthesis of multi-subunit proteins, proteomic research on protein interactions, and multi-gene metabolic engineering.