Ray Field

A high‐yielding CHO transient system: Coexpression of genes encoding EBNA‐1 and GS enhances transient protein expression

An efficient rapid protein expression system is crucial to support early drug development. Transient gene expression is an effective route, and to facilitate the use of the same host cells as for subsequent stable cell line development, we have created a high‐yielding Chinese hamster ovary (CHO) transient expression system. Suspension‐adapted CHO‐K1 host cells were engineered to express the gene encoding Epstein‐Barr virus (EBV) nuclear antigen‐1 (EBNA‐1) with and without the coexpression of the gene for glutamine synthetase (GS). Analysis of the transfectants indicated that coexpression of EBNA‐1 and GS enhanced transient expression of a recombinant antibody from a plasmid carrying an OriP DNA element compared to EBNA‐1‐only transfectants. This was confirmed with the retransfection of an EBNA‐1‐only cell line with a GS gene. The retransfected cell lines showed an increase in transient expression when compared with that of the EBNA‐1‐only parent. The transient expression process for the best CHO transient cell line was further developed to enhance protein expression and improve scalability by optimizing the transfection conditions and the cell culture process. This resulted in a scalable CHO transient expression system that is capable of expressing 2 g/L of recombinant proteins such as antibodies. This system can now rapidly provide gram amounts of recombinant antibody to supply preclinical development studies that has comparable product quality to antibody produced from a stably transfected CHO cell line.

Model-directed engineering of "difficult-to-express" monoclonal antibody production by Chinese hamster ovary cells.

Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some “difficult‐to‐express” (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high‐yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1λ MAbs varying >4‐fold in volumetric titer, we showed that MAb‐specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co‐expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co‐expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co‐expression increased qP and reduced cell growth. This study demonstrates that expression‐engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of different engineering interventions. Biotechnol. Bioeng. 2014;111: 372–385.

Improved Fermentation Processes for NS0 Cell Lines Expressing Human Antibodies and Glutamine Synthetase

To meet the increasing requirement for therapeutic antibodies to conduct clinical trials, an enhanced culture medium and fed‐batch process was developed for GS‐NS0 cell lines. This process was shown to produce high concentrations of monoclonal antibodies for several cell lines expressing different antibodies. Cells were adapted to growth in a glutamine‐ and serum‐free medium containing bovine serum albumin (BSA), cholesterol, and transferrin. A number of amino acids were found to be depleted during cell culture. The concentrations of these amino acids were increased, and further cell culture analyses were performed. This process of cell growth and analysis was repeated over multiple cycles until no depletion was detected. This resulted in an amino acid supplement that was shown to be generic and enhanced antibody productivity up to 5‐fold for the three cell lines tested. Transferrin was replaced using tropolone, a lipophilic iron chelator and ferric ammonium citrate. Cell growth was equivalent to that in transferrin‐containing medium over the wide ranges tested. A concentrated feed solution, based on the amino acid supplement and the components of the serum‐and protein‐free supplements, was formulated. Addition of this feed in response to metabolic requirements resulted in a harvest titer a further 2‐fold higher than the enhanced culture medium. Harvest antibody titers of up to 600 mg/L were achieved for three cell lines expressing different antibodies, representing an increase of 10‐fold over the starting concentrations.

Cell line specific control of polyethylenimine‐mediated transient transfection optimized with “Design of experiments” methodology

We describe a design of experiments (DoE) response surface modeling strategy to optimize the concentration of basal variables underpinning polyethylenimine (PEI) mediated transfection of different CHO‐K1 derived parental cell populations in a chemically defined medium, specifically the relative concentration of linear 25 kD PEI, host CHO cells and plasmid DNA. Using recombinant secreted alkaline phosphatase (SEAP) reporter activity as the modeled response, a discrete simple maximum was predicted for each CHO host cell population. Differences between the modeled optima derived from host cell specific differences in PEI cytotoxicity, such that the PEI:cell interaction effectively limited PEI‐DNA polyplex load at a relatively constant PEI:DNA ratio. However, across the three CHO host cell populations, SEAP reporter production was not proportional to plasmid DNA input at the host cell specific predicted basal variable optima. A 10‐fold variation in SEAP reporter output per mass of plasmid DNA delivered was observed. To determine the cellular basis of this difference in transient productivity, host CHO cells were transfected with fluorescently labeled polyplexes followed by flow cytometric analysis. Each CHO host cell population exhibited a distinct functional phenotype, varying in the extent of PEI‐DNA polyplex binding to the cell surface and degree of polyplex internalization. SEAP production was directly proportional to the level of polyplex internalization and heparan sulfate proteoglycan level. Taken together, these data show that choice of host CHO cell line is a critical parameter, which should rationally precede cell line specific transient production platform design using DoE methodology.

Predicting the expression of recombinant monoclonal antibodies in Chinese hamster ovary cells based on sequence features of the CDR3 domain.

Despite the development of high‐titer bioprocesses capable of producing >10 g L−1 of recombinant monoclonal antibody (MAb), some so called “difficult‐to‐express” (DTE) MAbs only reach much lower process titers. For widely utilized “platform” processes the only discrete variable is the protein coding sequence of the recombinant product. However, there has been little systematic study to identify the sequence parameters that affect expression. This information is vital, as it would allow us to rationally design genetic sequence and engineering strategies for optimal bioprocessing. We have therefore developed a new computational tool that enables prediction of MAb titer in Chinese hamster ovary (CHO) cells based on the recombinant coding sequence of the expressed MAb. Model construction utilized a panel of MAbs, which following a 10‐day fed‐batch transient production process varied in titer 5.6‐fold, allowing analysis of the sequence features that impact expression over a range of high and low MAb productivity. The model identified 18 light chain (LC)‐specific sequence features within complementarity determining region 3 (CDR3) capable of predicting MAb titer with a root mean square error of 0.585 relative expression units. Furthermore, we identify that CDR3 variation influences the rate of LC‐HC dimerization during MAb synthesis, which could be exploited to improve the production of DTE MAb variants via increasing the transfected LC:HC gene ratio. Taken together these data suggest that engineering intervention strategies to improve the expression of DTE recombinant products can be rationally implemented based on an identification of the sequence motifs that render a recombinant product DTE.

High-Yielding CHO Cell Pools for Rapid Production of Recombinant Antibodies

Cambridge Antibody Technology (CAT) has developed a system using GS-CHO transfectant pools to rapidly produce gram amounts of multiple IgGs for early characterisation studies and expedite drug development. The system involves screening a small number of independent pools by assessment of IgG harvest titre from terminal cultures or by flow cytometric analyses of intracellular IgG, which allows a more rapid ranking of pool performance. The highest-yielding pools are then expanded for production and can express up to 1.4 g/L at 5 L bioreactor scale in 7.5 weeks from transfection. Other GS-CHO transfectant pools have been scaled up to 50 L in disposable wavebags, and pools have been shown to be suitable for scale-up beyond 100 L, allowing the rapid production of tens of grams of IgG. The pools and manufacturing clonal cell lines at CAT use the same host cell type, expression system and production process therefore minimising the potential for differences in product characteristics at different stages of drug development. Productive pools have also been cloned out to identify high-yielding cell lines that show similar productivities to more conventionally isolated clonal cell lines, thereby potentially efficiently integrating rapid supply of antibody for early testing with manufacturing cell line development.

Functionalized micro-capillary film for the rapid at-line analysis of IgG aggregates in a cell culture bioreactor

A micro-capillary film has been developed that offers the potential for an at-line analytical tool for rapid aggregate analysis during biopharmaceutical antibody production. A non-porous walled micro-capillary film (NMCF) with cation exchange functionality was demonstrated to act as a chromatography medium that could be operated with high linear fluid velocities and was highly resistant to blockage by entrained particulates, including cells. The NMCF containing 19 parallel microcapillaries was prepared using a melt extrusion process from poly(ethylene-vinyl alcohol) copolymer (EVOH). The NMCF-EVOH was modified to have cation-exchange functionality (NMCF-EVOH-SP) and shown to differentially bind monomer and aggregated species of IgG antibody directly from a bioreactor. The use of NMCF-EVOH-SP to quantify aggregate concentrations in monoclonal antibody preparations in less than 20 minutes was demonstrated.

Rapid Estimation of Human Monoclonal Antibody (IgG4) Concentration in Cell Culture Supernatants

Typically, Enzyme Linked Immunosorbant Assay (ELISA) is used to measure antibody concentration in cell culture supernatants. ELISA assays are time consuming with low sample throughput. Consequently, product accumulation information is not rapidly available and analysis is often retrospective following fermenter harvesting. Furthermore, ELISA assays are often imprecise, with the coefficient of variation in excess of 10%. More rapid and precise methods were therefore investigated

Recombinant Human Antibody Therapeutics: Supply Strategies for Early and Clinical Development from CHO Cells

Human antibody therapeutics are effective tools in the treatment of serious diseases. Isolation of human antibodies using display technologies combined with rapid reformatting methods allow the ability to screen for those antibody variants that exhibit high level of expression in the preferred IgG format. Alteration of the IgG gene can also markedly improve expression, e.g. removal of unwanted cryptic splice sites, and when coupled with process optimisation transient IgG expression titres of ∼100 mg/L from suspension CHO cells are achievable at >10 L scale in a wavebag bioreactor. Such strategies allow early evaluation of multiple IgG constructs in vitro and in vivo by facilitating production of gram amounts of IgG from CHO cells prior to stable cell construction.

Impact of Change in Fermentation Process pH and Dissolved Carbon Dioxide Concentration on Secreted Antibody Structure and Cell Physiology of a GS-NS0 Cell Line Expressing a Human Antibody

Increased production of recombinant proteins from mammalian cells is a key focus of process development. Process pH is one factor well known to dramatically affect growth and productivity of some mammalian cells (1-3). Most mammalian cell culture systems use a bicarbonate based pH buffer system, in which pH setpoint is maintained by addition of carbon dioxide or bicarbonate. When pH set-point is lowered, more carbon dioxide is required to maintain pH, which may elevate the dissolved carbon dioxide (pCO2) to growth inhibitory levels (4). To counteract this, the bicarbonate concentration in the medium can also be reduced. Changes to pH and pCO2 have also been shown to affect protein glycosylation (5), which may affect protein function (6). This study describes the impact of changing process pH and pCO2 on a GS-NS0 cell line expressing a human monoclonal antibody.