Post‐translational events of a model reporter protein proceed with higher fidelity and accuracy upon mild hypothermic culturing of Chinese hamster ovary cells

Chinese hamster ovary cells (CHO) are routinely used in industry to produce recombinant therapeutic proteins and a number of studies have reported increased recombinant mRNA levels at temperatures <37°C. Surprisingly, the effect of reduced temperature on mRNA translation in CHO cells has not been investigated despite this process being highly responsive to environmental stresses. The relationship between low temperature culturing of CHO cells and mRNA translation was therefore investigated using labeling studies and dual luciferase reporter gene technology. Global protein synthetic capacity was not greatly affected at 32°C but was diminished at lower temperatures. The expression of both cap‐dependent and cap‐independent (IRES driven) mRNA translated luciferase reporter gene activity was highest at 32°C on a per cell basis and this was partially accounted for by increased mRNA levels. Importantly, post‐translational events appear to proceed with higher fidelity and accuracy at 32 than 37°C resulting in increased yield of active protein as opposed to an increase in total polypeptide synthesis. Therefore at 32°C recombinant cap‐dependent mRNA translation appears sufficient to maintain recombinant protein yields on a per cell basis and this is associated with improved post‐translational processing. Biotechnol. Bioeng. 2010;105: 215–220. © 2009 Wiley Periodicals, Inc.     

The cold-shock response in mammalian cells: investigating the HeLa cell cold-shock proteome

In recent years there have been a number of reports that suggest the sub-physiological (<37 °C) temperature in vitro culturing of mammalian cells can result in enhanced heterologous protein production. Despite these reports, the mechanisms by which mammalian cells respond to such conditions are largely unknown. We therefore set out to use a model in vitro culture HeLa cell system to begin investigating the cold-shock response in mammalian cell systems. Sub-physiological temperature cultivation resulted in reduced growth and proliferation and a lower total cell protein content. Proteomic analysis confirmed that HeLa cells actively respond to sub-physiological temperature by up-regulating a number of proteins and immunoblot analysis confirmed that specific proteins are indeed up-regulated in a time and temperature dependent manner. Additional work is likely to improve our understanding of the cold-shock response in mammalian cells and identify candidate target proteins for cell engineering to further enhance heterologous protein production at sub-physiological temperatures.     

Determination of Chinese hamster ovary cell line stability and recombinant antibody expression during long-term culture

Chinese hamster ovary (CHO) cell lines are frequently used as hosts for the production of recombinant therapeutics, such as monoclonal antibodies, due to their ability to perform correct post-translational modifications. A potential issue when utilizing CHO cells for therapeutic protein production is the selection of cell lines that do not retain stable protein expression during long-term culture (LTC). Instability of expression impairs process yields, effective usage of time and money, and regulatory approval for the desired therapeutic. In this study, we investigated a model unstable GS-CHO cell line over a continuous period of approximately 100 generations to determine markers of mechanisms that underlie instability. In this cell line, stability of expression was retained for 40-50 generations after which time a 40% loss in antibody production was detected. The instability observed within the cell line was not due to a loss in recombinant gene copy number or decreased expression of mRNA encoding for recombinant antibody H or L chain, but was associated with lower cumulative cell time values and an apparent increased sensitivity to cellular stress (exemplified by increased mRNA expression of the stress-inducible gene GADD153). Changes were also noted in cellular metabolism during LTC (alterations to extracellular alanine accumulation, and enhanced rates of glucose and lactate utilization, during the exponential and decline phase of batch culture, respectively). Our data indicates the breadth of changes that may occur to recombinant CHO cells during LTC ranging from instability of recombinant target production at a post-mRNA level to metabolic events. Definition of the mechanisms, regulatory events, and linkages underpinning cellular phenotype changes require further detailed analysis at a molecular level.     

On the effect of transient expression of mutated elF2α and elF4E eukaryotic translation initiation factors on reporter gene expression in mammalian cells upon cold-shock

There are a growing number of reports on the beneficial effects of subphysiological temperature in vitro culturing (27–35°C) of mammalian cells on recombinant protein yield. However, this effect is not conserved across cell lines and target products, and our understanding of the molecular mechanism(s) responsible for increased recombinant protein yield upon reduced temperature culturing of mammalian cells is poor. What is known is that mammalian cells respond to cold-shock by attenuating global cap-dependent translation. Here, we have investigated the hypothesis that the cap-dependent attenuation of mRNA translation upon cold-stress of in vitro-cultured mammalian cells can be prevented, or at least alleviated, by overexpressing mutant translation initiation factors in Chinese hamster ovary and HeLa cells. We have shown that the transient coexpression of either an eIF2αSer⁵¹→Ala⁵¹ mutant or an eIF4ESer²⁰⁹→Glu²⁰⁹ mutant with firefly luciferase affects luciferase expression levels in a cell line and temperature dependent manner. Further, regardless of the coexpression of initiation factors, transient reporter gene expression was enhanced at subphysiological temperatures (<37°C), suggesting that reduced temperature cultivation can be used to improve the yield of recombinant protein during transient expression. The implications of these results upon cell engineering strategies involving manipulation of the translational apparatus for the enhancement of recombinant protein synthesis upon cold-shock are discussed. Joint first authors who contributed equally to this work     

Effects of glutamine and asparagine on recombinant antibody production using CHO‐GS cell lines

A unique and nontraditional approach using glutamine and asparagine supplements for CHO‐glutamine synthetase (GS) cell lines was studied. In our experiments, we found that a decrease in pH and an increase in cell death occurred in production phase of a GS cell line, leading to reduced antibody expression and lower antibody yields. The experimental results and the statistical analysis (ANOVA) indicated that additions of glutamine and asparagine in the basal and feed media were effective to buffer the cell culture pH, reduce lactate generation, maintain a higher cell viability profile, and improve antibody productivity. In bench‐top bioreactors, glutamine and asparagine supplementation helped to prevent cell death, improve antibody yield, and reduce base usage. Glutamine is normally excluded from culture media for GS cell lines to prevent the bypass of selection pressure. In this study, however, the addition of glutamine did not affect cell population homogeneity, protein quality, or decrease antibody yield of two GS cell lines. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1457–1468, 2014     

Effective temperature shift strategy development and scale confirmation for simultaneous optimization of protein productivity and quality in Chinese hamster ovary cells

Temperature shifts to lower culture temperatures are frequently employed in the manufacturing of protein therapeutics in mammalian cells to improve productivity, viability, or quality attributes. The direction and extent to which a temperature shift affects productivity and quality may vary depending on the expression host and characteristics of the expressed protein. We demonstrated here that two Chinese hamster ovary (CHO) clones expressing different human monoclonal antibodies responded differently to a temperature shift despite sharing a common parental CHO cell line. Within a single CHO line, we observed a nonlinear response to temperature shift. A moderate shift to 35°C significantly decreased final titer relative to the unshifted control while a larger shift to 32°C significantly increased final titer by 25%. Therefore, we proposed a systematic empirical approach to assess the utility of a temperature shift for faster implementation during process development. By testing multiple shift parameters, we identified optimum shift conditions in shake flasks and successfully translated findings to benchtop bioreactors and 1,000-L bioreactor scale. Significant differences in final antibody titer and charge variants were observed with temperature shift increments as small as Δ1.5°C. Acidic charge variants decreased monotonically with decreasing shift temperature in both cell lines; however, final antibody titer required simultaneous optimization of shift day and temperature. Overall, we were able to show that a systematic approach to identify temperature shift parameters at small scales is useful to optimize protein production and quality for efficient and confident translation to large-scale production.     

Effect of culture pH on recombinant antibody production by a new human cell line, F2N78, grown in suspension at 33.0 °C and 37.0 °C

The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0 °C and 37.0 °C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0–7.4. Lowering the culture temperature from 37.0 °C to 33.0 °C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0 °C. The highest maximum antibody concentration was obtained at 37.0 °C and pH 6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.     

Impacts on product quality attributes of monoclonal antibodies produced in CHO cell bioreactor cultures during intentional mycoplasma contamination events

A mycoplasma contamination event in a biomanufacturing facility can result in costly cleanups and potential drug shortages. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and penetrate the standard 0.2-µm filters used in the clarification of harvested cell culture fluid. Previously, we reported a study regarding the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G 1 (IgG1) antibody. Our previous work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Careful evaluation of certain identified process parameters over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before detection from a traditional method. In this report, we studied the changes in the IgG1 product quality produced by CHO cells considered to be induced by the M. arginini contamination events. We observed changes in critical quality attributes correlated with the duration of contamination, including increased acidic charge variants and high mannose species, which were further modeled using principal component analysis to explore the relationships among M. arginini contamination, CHO cell growth and metabolites, and IgG1 product quality attributes. Finally, partial least square models using NIR spectral data were used to establish predictions of high levels (≥10⁴ colony-forming unit [CFU/ml]) of M. arginini contamination, but prediction of levels below 10⁴ CFU/ml were not reliable. Contamination of CHO cells with M. arginini resulted in significant reduction of antibody product quality, highlighting the importance of rapid microbiological testing and mycoplasma testing during particularly long upstream bioprocesses to ensure product safety and quality.     

Energy-Based Analysis and Prediction of the Orientation between Light- and Heavy-Chain Antibody Variable Domains

Diversity in antibody structure is crucial to the ability of the adaptive immune system to recognize the tremendously diverse set of potential antigens. The diversity in structure is most apparent in the six hypervariable loops of the complementarity-determining regions. However, given that these loops occur at the interface of the heavy- and light-chain variable domains and form the antigen-binding site, the relative orientation of the heavy- and light-chain variable domains can create another source of structural diversity leading to changes in antigen binding. Here, we first reexamine the diversity of V_L:V_H orientations in existing antibody crystal structures using 153 nonredundant sequences, demonstrating that the variation in V_L:V_H orientation is greater than that expected from effects of crystal packing, antigen binding, or the presence of antibody constant regions and increases, on average, as sequence similarity decreases for residues in the interface between the domains. We developed a tool for predicting the relative orientations of the heavy- and light-chain variable domains using side-chain rotamer sampling in the interface and molecular-mechanics-based energy calculations. When using variable domain backbones from the crystal structures, the predicted orientation is very close (< 1 Å RMSD) to the crystallographically observed orientation in most cases, confirming that the V_L:V_H orientation is determined by the antibody sequence and suggesting an approach to predicting the relative orientation of the variable domains when building homology models of antibodies. When applied to antibody homology models generated from templates with 55-75% sequence identity, we predict the V_L:V_H orientation of 20 antibodies with an average/median RMSD of 2.1/1.6 Å to the crystal structures.     

Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors

The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.     

Early prediction of instability of Chinese hamster ovary cell lines expressing recombinant antibodies and antibody-fusion proteins

One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian cell line that maintains stability of production. Problems with process yield, lack of effective use of costly resources, and a possible delay in obtaining regulatory approval of the product may ensue otherwise. Therefore the stability of expression in a number of Chinese hamster ovary (CHO) derived production cell lines that were isolated using the glutamine synthetase (GS) selection system was investigated by defining a culture as unstable if the titer (which is a measure of productivity) of a cell line expressing an antibody or antibody-fusion protein declined by 20-30% or more as it underwent 55 population doublings. Using this criterion, a significant proportion of the GS-selected CHO production cell lines were observed to be unstable. Reduced antibody titers correlated with the gradual appearance of a secondary, less productive population of cells as detected with flow cytometric analysis of intracellular antibody content. Where tested, it was observed that the secondary population arose spontaneously from the parental population following multiple passages, which suggested inherent clonal instability. Moreover, the frequency of unstable clones decreased significantly if the host cell line from which the candidate production cell lines were derived was apoptotic-resistant. This data suggested that unstable cell lines were more prone to apoptosis, which was confirmed by the fact that unstable cell lines had higher levels of Annexin V and caspase 3 activities. This knowledge has been used to develop screening protocols that identify unstable CHO production cell lines at an early stage of the cell line development process, potentially reducing the cost of biotherapeutic development.     

Effect of temperature,pH, dissolved oxygen,and hydrolysate on the formation of triple light chain antibodies in cell culture

THIOMABs are recombinant antibodies with reactive cysteine residues used for forming THIOMAB–drug conjugates (TDCs). We recently reported a new impurity associated with THIOMABs: one of the engineered cysteines forms a disulfide bond with an extra light chain (LC) to generate a triple light chain antibody (3LC). In our previous investigations, increased LC expression increased 3LC levels, whereas increased glutathione (GSH) production decreased 3LC levels. In this work, on three stably transfected CHO cell lines, we investigated the effects of temperature, pH, dissolved oxygen (DO), and hydrolysate on 3LC formation during THIOMAB fed‐batch cell culture production. Although pH between 6.8 and 7.0 had no significant impact on 3LC formation, temperature at 35°C instead of 33 or 31°C generated the lowest 3LC values for two cell lines. The decreased 3LC level correlated with increased GSH production. We implemented a 35°C temperature process for large‐scale (2,000 L) production of a THIOMAB. This process reduced 3LC levels by ～50% compared with a 33°C temperature process. By contrast, DO and hydrolysate had modest effect on 3LC levels for the model cell line studied. Overall, we did not find significant changes in LC expression under the conditions tested, whereas changes in GSH production were more evident. By investigating the impact of bioreactor process and medium conditions on 3LC levels, we identified strategies that reduced 3LC levels. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Polymer‐mediated flocculation of transient CHO cultures as a simple,high throughput method to facilitate antibody discovery

Most biopharmaceutical drugs, especially monoclonal antibodies (mAbs), bispecific antibodies (BsAbs) and Fc‐fusion proteins, are expressed using Chinese Hamster Ovary (CHO) cell lines. CHO cells typically yield high product titers and high product quality. Unfortunately, CHO cell lines also generate high molecular weight (HMW) aggregates of the desired product during cell culture along with CHO host cell protein (HCP) and CHO DNA. These immunogenic species, co‐purified during Protein A purification, must be removed in a multi‐step purification process. Our colleagues have reported the use of a novel polymer‐mediated flocculation step to simultaneously reduce HMW, HCP and DNA from stable CHO cell cultures prior to Protein A purification. The objective of this study was to evaluate this novel “smart polymer” (SmP) in a high throughput antibody discovery workflow using transiently transfected CHO cultures. SmP treatment of 19 different molecules from four distinct molecular categories (human mAbs, murine mAbs, BsAbs and Fabs) with 0.1% SmP and 25 mM stimulus resulted in minimal loss of monomeric protein. Treatment with SmP also demonstrated a variable, concentration‐dependent removal of HMW aggregates after Protein A purification. SmP treatment also effectively reduced HCP levels at each step of mAb purification with final HCP levels being several fold lower than the untreated control. Interestingly, SmP treatment was able to significantly reduce high concentrations of artificially spiked levels of endotoxin in the cultures. In summary, adding a simple flocculation step to our existing transient CHO process reduced the downstream purification burden to remove impurities and improved final product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1393–1400, 2017     

Exploring cellular behavior under transient gene expression and its impact on mAb productivity and Fc‐glycosylation

Transient gene expression (TGE) is a methodology employed in bioprocessing for the fast provision of recombinant protein material. Mild hypothermia is often introduced to overcome the low yield typically achieved with TGE and improve specific protein productivity. It is therefore of interest to examine the impact of mild hypothermic temperatures on both the yield and quality of transiently expressed proteins and the relationship to changes in cellular processes and metabolism. In this study, we focus on the ability of a Chinese hamster ovary cell line to galactosylate a recombinant monoclonal antibody (mAb) product. Through experimentation and flux balance analysis, our results show that TGE in mild hypothermic conditions led to a 76% increase in q_P compared to TGE at 36.5°C in our system. This increase is accompanied by increased consumption of nutrients and amino acids, together with increased production of intracellular nucleotide sugar species, and higher rates of mAb galactosylation, despite a reduced rate of cell growth. The reduction in biomass accumulation allowed cells to redistribute their energy and resources toward mAb synthesis and Fc‐glycosylation. Interestingly, the higher capacity of cells to galactosylate the recombinant product in TGE at 32°C appears not to have been assisted by the upregulation of galactosyltransferases (GalTs), but by the increased expression of N‐acetylglucosaminyltransferase II (GnTII) in this cell line, which facilitated the production of bi‐antennary glycan structures for further processing.