Andrew J. Racher

On the Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody Production by CHO Cells

Monoclonal antibodies (Mab) are heterotetramers consisting of an equimolar ratio of heavy chain (HC) and light chain (LC) polypeptides. Accordingly, most recombinant Mab expression systems utilize an equimolar ratio of heavy chain (hc) to light chain (lc) genes encoded on either one or two plasmids. However, there is no evidence to suggest that this gene ratio is optimal for stable or transient production of recombinant Mab. In this study we have determined the optimal ratio of hc:lc genes for production of a recombinant IgG4 Mab, cB72.3, by Chinese hamster ovary (CHO) cells using both empirical and mathematical modeling approaches. Polyethyleneimine‐mediated transient expression of cB72.3 at varying ratios of hc:lc genes encoded on separate plasmids yielded an optimal Mab titer at a hc:lc gene ratio of 3:2; a conclusion confirmed by separate mathematical modeling of the Mab folding and assembly process using transient expression data. On the basis of this information, we hypothesized that utilization of hc genes at low hc:lc gene ratios is more efficient. To confirm this, cB72.3 Mab was transiently produced by CHO cells at constant hc and varying lc gene dose. Under these conditions, Mab yield was increased with a concomitant increase in lc gene dose. To determine if the above findings also apply to stably transfected CHO cells producing recombinant Mab, we compared the intra‐ and extracellular ratios of HC and LC polypeptides for three GS‐CHO cells lines transfected with a 1:1 ratio of hc:lc genes and selected for stable expression of the same recombinant Mab, cB72.3. Intra‐ and extracellular HC:LC polypeptide ratios ranged from 1:2 to 1:5, less than that observed on transient expression of the same Mab in parental CHO cells using the same vector. In conclusion, our data suggest that the optimal ratio of hc:lc genes used for transient and stable expression of Mab differ. In the case of the latter, we infer that optimal Mab production by stably transfected cells represents a compromise between HC abundance limiting productivity and the requirement for excess LC to render Mab folding and assembly more efficient.

Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate.

We previously compared changes in individual protein abundance between the proteomes of GS‐NS0 cell lines with varying rates of cell‐specific recombinant monoclonal antibody production (qMab). Here we extend analyses of our proteomic dataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS‐NS0 cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non‐ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS‐NS0 cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X‐box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS‐NS0 cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein production. We infer that individual cells within parental populations are more functionally equipped for high‐level recombinant protein production than others and that this bias could be used to select cells that are more likely to achieve high qMab.

Control of Culture Environment for Improved Polyethylenimine‐Mediated Transient Production of Recombinant Monoclonal Antibodies by CHO Cells

In this study we describe optimization of polyethylenimine (PEI)‐mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell‐specific production. The optimal conditions for transient transfection of suspension‐adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 μg 106 cells−1 mL−1 at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin‐like growth factor (LR3‐IGF) and a reduction in culture temperature to 32 °C were found to increase product titer 2‐ and 3‐fold, respectively. However, mild hypothermia and LR3‐IGF acted synergistically to increase product titer 11‐fold. Although increased product titer in the presence of LR3‐IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post‐transfection increased both the integral of viable cell concentration (IVC) and cell‐specific Mab production rate. For cultures maintained at 32 °C in the presence of LR3‐IGF, IVC and qMab were increased 4‐ and 2.5‐fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 °C in the presence of LR3‐IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3‐fold increase in accumulated Mab titer from ∼13 to ∼39 mg L−1. Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS‐CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended “activated hypothermic synthesis”.

Use of lactate dehydrogenase release to assess changes in culture viability

This study reports the use of lactate dehydrogenase release to monitor changes in culture viability in flask culture and fixed bed, porosphere bioreactor systems. Lactate dehydrogenase release shows good agreement with increase in non-viable cell numbers and decline in glucose utilisation in flask cultures. Studies with the immobilised system show that lactate dehydrogenase release can detect loss of viability which is not always indicated by a decrease in glucose utilisation. The data show that culture viability in a repeated-feed-and-harvest system is influenced markedly by both a) the medium change regime itself and b) the use of an immobilised bioreactor compared to a flask system for the same medium change regime.

Cell line-specific control of recombinant monoclonal antibody production by CHO cells.

In this study we compare the cellular control of recombinant human IgG4 monoclonal antibody (Mab) synthesis in different CHO cell lines. Based on comprehensive empirical analyses of mRNA and polypeptide synthetic intermediates we constructed cell line‐specific mathematical models of recombinant Mab manufacture in seven GS‐CHO cell lines varying in specific production rate (qMab) over 350‐fold. This comparative analysis revealed that control of qMab involved both genetic construct and cell line‐specific factors. With respect to the former, all cell lines exhibited excess production of light chain (LC) mRNA and polypeptide relative to heavy chain (HC) mediated by more rapid LC transcription and enhanced LC mRNA stability. Downstream of this, cell lines differed markedly in their relative rates of recombinant mRNA translation, Mab assembly and secretion although HC mRNA abundance and the rate of HC translation generally exerted most control over qMab—the latter being directly proportional to qMab. This study shows that (i) cell lines capable of high qMab exceed a threshold functional competency in all synthetic processes, (ii) the majority of cells in parental and transfected cell populations are functionally limited and (iii) cell engineering strategies to increase Mab production should be cell line specific. Biotechnol. Bioeng. 2010;106: 938–951.

Functional heterogeneity and heritability in CHO cell populations

In this study, we address the hypothesis that it is possible to exploit genetic/functional variation in parental Chinese hamster ovary (CHO) cell populations to isolate clonal derivatives that exhibit superior, heritable attributes for biomanufacturing—new parental cell lines which are inherently more “fit for purpose.” One‐hundred and ninety‐nine CHOK1SV clones were isolated from a donor CHOK1SV parental population by limiting dilution cloning and microplate image analysis, followed by primary analysis of variation in cell‐specific proliferation rate during extended deep‐well microplate suspension culture of individual clones to accelerate genetic drift in isolated cultures. A subset of 100 clones were comparatively evaluated for transient production of a recombinant monoclonal antibody (Mab) and green fluorescent protein following transfection of a plasmid vector encoding both genes. The heritability of both cell‐specific proliferation rate and Mab production was further assessed using a subset of 23 clones varying in functional capability that were subjected to cell culture regimes involving both cryopreservation and extended sub‐culture. These data showed that whilst differences in transient Mab production capability were not heritable per se, clones exhibiting heritable variation in specific proliferation rate, endocytotic transfectability and N‐glycan processing were identified. Finally, for clonal populations most “evolved” by extended sub‐culture in vitro we investigated the relationship between cellular protein biomass content, specific proliferation rate and cell surface N‐glycosylation. Rapid‐specific proliferation rate was inversely correlated to CHO cell size and protein content, and positively correlated to cell surface glycan content, although substantial clone‐specific variation in ability to accumulate cell biomass was evident. Taken together, our data reveal the dynamic nature of the CHO cell functional genome and the potential to evolve and isolate CHO cell variants with improved functional properties in vitro. Biotechnol. Bioeng. 2013; 110: 260–274.

Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: improving the efficiency of cell line generation.

Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide‐ranging transfectant population. This identification process is on the critical path for first‐in‐human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One‐hundred and seventy‐five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines.

Strategies for selecting Recombinant CHO cell lines for cGMP manufacturing: Realizing the potential in bioreactors

Manufacture of recombinant proteins from mammalian cell lines requires the use of bioreactor systems at scales of up to 20,000 L. The cost and complexity of such systems can prohibit their extensive use during the process to construct and select the manufacturing cell line. It is therefore common practice to develop a model of the production process in a small scale vessel, such as a shake‐flask, where lower costs, ease of handling, and higher throughput are possible. This model can then be used to select a small number of cell lines for further evaluation in bioreactor culture. Here, we extend our previous work investigating cell line construction strategies to assess how well the behavior of cell lines in such a shake‐flask assessment predicts behavior in the associated bioreactor production process. A panel of 29 GS‐CHO cell lines, all producing the same antibody, were selected to include a mixture of high and low producers from a pool of 175 transfectants. Assessment of this panel in 10 L bioreactor culture revealed wide variation in parameters including growth, productivity, and metabolite utilization. In general, those cell lines which were high producing in the bioreactor cultures had also been higher producing in an earlier shake‐flask assessment. However, some changes in rank position of the evaluated cell lines were seen between the two systems. A potential explanation of these observations is discussed and approaches to improve the predictability of assessments used for cell line selection are considered.

Studies on monoclonal antibody production by a hybridoma cell line (C1E3) immobilised in a fixed bed, porosphere culture system

The aim of this study was to investigate the potential of fixed beds of macroporous glass spheres as a production process for animal cell products. The growth, metabolism and monoclonal antibody expression of a mouse-mouse hybridoma cell line was investigated in order to both test the potential of and to optimise the system. After the initial growth phase, the culture went into a steady-state phase brought on by glutamine limitation. An event occurred after 120-160 h of steady-state operation which destabilised the culture, causing a decline in productivity, after which the culture recovered. This event was analysed in detail to determine its cause, and whether a major switch in metabolic function had occurred. The parameter which correlated most closely to antibody production rate was oxygen, but as this was kept constant in the void medium of the bed it has to be concluded that oxygen diffusion into the spheres was the regulatory factor. A comparison of the fixed bed and a flask culture identified interesting differences in glucose metabolism between the two systems. The data gave strong indications as to how the productivity of the fixed bed system can be further improved. This includes optimisation of the glutamine concentration and modifying the porous structure of the spheres to improve diffusion characteristics.

Rapid whole monoclonal antibody analysis by mass spectrometry: An Ultra scale-down study of the effect of harvesting by centrifugation on the post-translational modification profile

With the trend towards the generation and production of increasing numbers of complex biopharmaceutical (protein based) products, there is an increased need and requirement to characterize both the product and production process in terms of robustness and reproducibility. This is of particular importance for products from mammalian cell culture which have large molecular structures and more often than not complex post‐translational modifications (PTMs) that can impact the efficacy, stability and ultimately the safety of the final product. It is therefore vital to understand how the operating conditions of a bioprocess affect the distribution and make up of these PTMs to ensure a consistent quality and activity in the final product. Here we have characterized a typical bioprocess and determined (a) how the time of harvest from a mammalian cell culture and, (b) through the use of an ultra scale‐down mimic how the nature of the primary recovery stages, affect the distribution and make up of the PTMs observed on a recombinant IgG4 monoclonal antibody. In particular we describe the use of rapid whole antibody analysis by mass spectrometry to analyze simultaneously the changes that occur to the cleavage of heavy chain C‐terminal lysine residues and the glycosylation pattern, as well as the presence of HL dimers. The time of harvest was found to have a large impact upon the range of glycosylation patterns observed, but not upon C‐terminal lysine cleavage. The culture age had a profound impact on the ratio of different glycan moieties found on antibody molecules. The proportion of short glycans increased (e.g., (G0F)2 20–35%), with an associated decrease in the proportion of long glycans with culture age (e.g., (G2F)2 7–4%, and G1F/G2F from 15.2% to 7.8%). Ultra scale‐down mimics showed that subsequent processing of these cultures did not change the post‐translational modifications investigated, but did increase the proportion of half antibodies present in the process stream. The combination of ultra scale‐down methodology and whole antibody analysis by mass spectrometry has demonstrated that the effects of processing on the detailed molecular structure of a monoclonal antibody can be rapidly determined early in the development process. In this study we have demonstrated this analysis to be applicable to critical process design decisions (e.g., time of harvest) in terms of achieving a desired molecular structure, but this approach could also be applied as a selection criterion as to the suitability of a platform process for the preparation of a new drug candidate. Also the methodology provides means for bioprocess engineers to predict at the discovery phase how a bioprocess will impact upon the quality of the final product. Biotechnol. Bioeng. 2010;107: 85–95.