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Reduced culture temperature is an increasingly popular practice to improve recombinant protein yields in CHO cells. Recent studies have attributed the enhancement of protein titers at sub-physiological temperatures to increased mRNA levels as well as extended stationary phase. We observed that reducing the culture temperature arrested cell growth,...
Context in source publication
Context 1
... master mix solutions were prepared by mixing 5 µL of TaqMan Gene Expression Master Mix (Applied Biosystems, Foster City, CA, USA) with 0.5 µL of 20X TaqMan custom gene expression assay per sample. The TaqMan primer and probe sequences are listed in Table 4. Triplicate qPCR reactions were loaded onto a 384-well plate containing 4.5 µL of sample and 5.5 µL of TaqMan master mix. ...
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Citations
... Given that transient recombinant protein production depends on a myriad of transfection and culture parameters, optimising these parameters directly influences the scalability and efficiency of recombinant protein production. Transfection parameters include seeding cell density (10), transfection reagent concentration (11), DNA concentration (12), complexation time, temperature, and volume (12), together with culture parameters such as medium replacements (7,13), serum deprivation (14), use of cell line maintenance antibiotic (15), incubation temperature (16,17), culture volume, harvesting time (18)(19)(20), and nutrient supplementation (21)(22)(23). Three main strategies are commonly used to optimise these parameters: the reductionist "one factor at a time" approach (24), stepwise optimisation from one condition to another (25), and the design of experiment approach (10), of which we took the single isolated factor approach. ...
Background
Optimizing recombinant antibody production is important for cost-effective therapeutics and diagnostics. With impact on commercialization, higher productivity beyond laboratory scales is highly sought, where efficient production can also accelerate antibody characterizations and investigations.
Methods
Investigating HEK293E cells for mammalian antibody production, various transfection and culture parameters were systematically analyzed for antibody light chain production before evaluating them for whole antibody production. Transfection parameters investigated include seeding cell density, the concentration of the transfection reagent and DNA, complexation time, temperature, and volume, as well as culture parameters such as medium replacement, serum deprivation, use of cell maintenance antibiotic, incubation temperature, medium volume, post-transfection harvest day, and common nutrient supplements.
Results
Using 2 mL adherent HEK293E cell culture transfections with 25 kDa linear polyethylenimine in the most optimized parameters, we demonstrated a ~2-fold production increase for light chain alone and for whole antibody production reaching 536 and 49 μg, respectively, in a cost-effective manner. With the addition of peptone, κ light chain increased by ~4-fold to 1032 μg, whereas whole antibody increased to a lesser extent by ~2.5-fold to 51 μg, with benefits potentially for antibodies limited by their light chains in production.
Conclusions
Our optimized findings show promise for a more efficient and convenient antibody production method through transfection and culture optimizations that can be incorporated to scale-up processes and with potential transferability to other mammalian-based recombinant protein production using HEK293E.
... Transient gene expression using mammalian cells has been established for decades (Baldi et al., 2012;Durocher et al., 2002;Nettleship et al., 2010;Pham et al., 2006). Several approaches have been described for the improvement of transient protein expression, in particular for difficult-to express proteins (Estes et al., 2015;Lin et al., 2015;Mason et al., 2014;Simone et al., 2003), namely decreasing both the expression temperature and coding DNA amount. ...
Serological assays are valuable tools to study SARS‐CoV‐2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS‐CoV‐2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS‐CoV‐2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID‐19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293‐E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme‐linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in‐depth characterization of SARS‐CoV‐2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS‐CoV‐2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high‐quality SARS‐CoV‐2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in‐depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID‐19 serologic tests.
... With regards to culture temperature, growing mammalian cells at sub-physiological temperatures has been shown to result in slowed or arrested growth and reduced metabolism (Trummer et al., 2006), cell-cycle arrest in the G1 phase as well as increased viability for prolonged periods of time (Fogolin et al., 2004), and reduced transcription / translation (Mason et al., 2014). It has also been reported to result in increased specific productivity rates (Yoon et al., 2003) and maintained or even improved product quality and titre (Fogolin et al., 2004;Trummer et al., 2006;Schneider et al., 2019). ...
... It has also been reported to result in increased specific productivity rates (Yoon et al., 2003) and maintained or even improved product quality and titre (Fogolin et al., 2004;Trummer et al., 2006;Schneider et al., 2019). Recent studies have suggested that improved protein titres at sub-physiological temperatures are due to an increase in mRNA half-life as well as extended stationary phase (Masterton et al., 2010;Mason et al., 2014). ...
... However, there are also reports where decreased culture temperature had little or no effect on protein expression (Yoon et al., 2003). Mason et al. (2014) have further reported that reduced culture temperature had a differential effect on protein and mRNA expression of closely related antibody mutants from stable cell lines, and they therefore propose that the effect of reduced culture temperature on titre is protein-dependent, with the accuracy of protein folding and assembly being improved at lower temperatures, and therefore enhancing the expression of proteins that have a propensity to misfold. ...
Recent improvements in volumetric antibody productivity (often in excess of 5 g/L) have been achieved by advances in cell lines and upstream processing, but often lead to harvest material becoming more difficult to recover. These intensified upstream operations require a renewed prioritisation of the integration of upstream and downstream process development to ensure product purification issues are taken into consideration, to avoid extensive and expensive clearance strategies downstream. Here, it was demonstrated that changes to upstream process parameters at the bioreactor stage of monoclonal antibody production affect product quantity and quality. Culture pH, temperature and seed density setpoints leading to high titre are commonly also linked to higher post-protein A HCP levels, reduced monomer percentages and increased percentages of undesirable glycan structures. To predict post-protein A product quality, several potential indicators that can be measured in harvest material (prior to using expensive purification resources) were explored, including culture viability and osmolality, revealing unexpectedly that culture viability could not be used for such a purpose, but that osmolality has the potential to be used as a product quality indicator. The impact of culture duration on product quality was also investigated and it was shown that as cultivation progressed and antibody titre increased, product quality declined, in one case due to post-protein A HCP levels increasing by 75% from day 14 to day 17 of culture. HCP identification by mass spectrometry was applied to this system to provide insights into cellular behaviour and HCP carryover during protein A purification. It showed increases in several classes of post-protein A HCPs (e.g. stress response proteins) as the culture progressed, particularly on days 15 and 17 of culture which were associated with significant increases in total HCP levels. This provides a new level of insight into HCPs that are retained during mAb purification which may be used to aide process development strategies.
... After approximately 4 days of cultivation, we decreased the temperature (confidential data) in the exponential phase in all described experiments to inhibit cell growth and shift cell physiology to mAb production. 7 At this time point, we also started with the fed batch phase. Two Novartis feeds (confidential data) were used during the processes and four additional feeds were developed and tested, as shown in Table 2. ...
The main goal in biosimilar development is to increase Chinese Hamster Ovary (CHO) viability and productivity while maintaining product quality. Despite media and feed optimization during process development, depletion of amino acids still occurs. The aim of the work was to optimize an existing industrial fed batch process by preventing shortage of amino acids and to gather knowledge about CHO metabolism. Several process outputs were evaluated such as cell metabolism, cell viability, monoclonal antibodies (mAbs) production, and product quality. First step was to develop and supplement an enriched feed containing depleted amino acids. Abundance of serine and glucose increased lactate production resulting in low viability and low productivity. In the next step, we developed an amino acid feed without serine to avoid the metabolic boost. Supplemented amino acids improved cell viability by 9%; however, mAb production did not increase significantly. In the final step, we limited glucose concentration (<5.55 mmol/L) in the cell culture to avoid the metabolic boost while supplementing an amino acid feed including serine. Data analysis showed that we were able to (a) replace depleted amino acids and avoid metabolic boost, (b) increase viability by 12%, (c) enhance mAb production by 0.5 g/L (total by approximately 10 g), and (d) extend the overall process time of an already developed bioprocess.
... Due to the complex structure of IgMs, folding and assembly can be a bottleneck in recombinant production. A reduction in cultivation temperature is sometimes beneficial in the production of 'difficult-to-express' proteins because the protein folding capacity and expression of chaperones from the endoplasmic reticulum is increased [36,37]. We hypothesized that the quantity of correctly-folded protein in the supernatant and intracellular aggregated IgM012_GL would be affected by cultivation temperature. ...
IgM antibodies are arousing considerable interest as biopharmaceuticals. Despite their immunotherapeutic potential, little is known about the impact of environmental conditions on product quantity and quality of these complex molecules. Process conditions influence the critical quality attributes (CQAs) of therapeutic proteins and thus are important parameters for biological safety and efficacy. Here, the results of a systematic study are presented that characterized the influence of temperature and pH on cell-specific productivity and IgM quality attributes. Biphasic temperature and pH shift experiments were performed as batch cultures in DASGIP® bioreactors under controlled conditions and defined by a specific design of experiment (DOE) approach. An internally-developed recombinant IgM producing CHO cell line was used. With respect to product quality, after an initial purification step efforts were focused on pentamer content, nucleic acid (NA) impurities and the glycosylation profile after an initial purification step. All quality attributes were evaluated by densitometric and chromatographic methods. The reduction of cultivation temperature severely reduced IgM titers, while pH variation had no impact. In contrast, IgM quality was not significantly influenced by bioprocessing parameters. Data revealed that an additional purification step is required to reduce the presence of NAs for in vivo applications. In conclusion, the results showed that for the chosen IgM model, IgM012_GL, variation in quality attributes is not caused by the environmental conditions of temperature and pH.
... It could be due to delayed induction of growth arrest phase. Similar results were reported where fed-batch cultivation with a temperature shift at the mid-exponential phase gave the best protein production, due to the optimal cell density [51,52]. Furthermore, the second temperature downshift to 28˚C at the mid-late stationary phase was found as the optimum point for misfolding level while having minimal effects on protein titer. ...
Cell culture process optimization is a critical solution to most of the challenges faced by the pharmaceutical manufacturing. One of the major problems encountered in large-scale production of therapeutic proteins is misfolded protein production. The accumulation of misfolded therapeutic proteins is an immunogenic signal and a risk factor for immunogenicity of the final product. The aim of this study was the statistical optimization of three-phasic temperature shift and timing for enhanced production of correctly folded Fc-fusion protein. The effect of culture temperatures were investigated using the biphasic culture system. Box–Behnken design was then used to compute temperature and time of shifting optimum. Response surface methodology revealed that maximum production with low level of misfolded protein was achieved at two-step temperature shift from 37°C to 30°C during the late logarithmic phase and 30°C to 28°C in the mid-stationary phase. The optimized condition gave the best results of 1860 mg L⁻¹ protein titer with 24.5% misfolding level. The validation experiments were carried out under optimal conditions with three replicates and the protein misfolding level was decreased by two times while productivity increased by ~ 1.3-fold. Large-scale production in 250 L bioreactor under the optimum conditions was also verified the effectiveness and the accuracy of the model. The results showed that by utilizing two-step temperature shift, productivity and the quality of target protein have been improved simultaneously. This model could be successfully applied to other products.
... For cell culture processes with CHO cells, mild hypothermia conditions in the range of 31 C to 34 C are typically used during the production phase. 44 In many cases, the reduction in temperature was associated with increased mRNA levels and growth cessation leading to enhancements in cell specific productivity and culture longevity, [45][46][47][48] but the effects are strongly cell line and product dependent. 49 The use of a temperature shift was also shown to be beneficial for maintaining glycoform quality in CHO cultures. ...
Inducible mammalian expression systems are becoming increasingly available and are not only useful for the production of cytotoxic/cytostatic products, but also confer the unique ability to uncouple the growth and production phases. In this work, we have specifically investigated how the cell culture state at the time of induction influences the cumate‐inducible expression of recombinant rituximab by a GS‐CHO cell line. To this end, cells grown in batch and fed‐batch cultures were induced at increasing cell densities (1 to 10 × 10 6 cells/mL). In batch, the cell specific productivity and the product yield were found to reduce with increasing cell density at induction. A dynamic feeding strategy using a concentrated nutrient solution applied prior and postinduction allowed to significantly increase the integral of viable cells and led to a 3‐fold increase in the volumetric productivity (1.2 g/L). The highest product yields were achieved for intermediate cell densities at induction, as cultures induced during the late exponential phase (10 × 10 6 cells/mL) were associated with a shortened production phase. The final glycosylation patterns remained however similar, irrespective of the cell density at induction. The kinetics of growth and production in a 2 L bioreactor were largely comparable to shake flasks for a similar cell density at induction. The degree of galactosylation was found to decrease over time, but the final glycan distribution at harvest was consistent to that of the shake flasks cultures. Taken together, our results provide useful insights for the rational development of fed‐batch cell culture processes involving inducible CHO cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2742, 2019
... The standard curve was generated using a purified mouse IgG antibody. The cell specific rate of production, Qp (pg/cell/day) (Brezinsky et al. 2003;Mason et al. 2014) was calculated using the following equation: ...
Introduction
Mammalian cells like Chinese hamster ovary (CHO) cells are routinely used for production of recombinant therapeutic proteins. Cells require a continuous supply of energy and nutrients to sustain high cell densities whilst expressing high titres of recombinant proteins. Cultured mammalian cells are primarily dependent on glucose and glutamine metabolism for energy production.
Objectives
The TCA cycle is the main source of energy production and its continuous flow is essential for cell survival. Modulated regulation of TCA cycle can affect ATP production and influence CHO cell productivity.
Methods
To determine the key metabolic reactions of the cycle associated with cell growth in CHO cells, we transiently silenced each gene of the TCA cycle using RNAi.
Results
Silencing of at least four TCA cycle genes was detrimental to CHO cell growth. With an exception of mitochondrial aconitase (or Aco2), all other genes were associated with ATP production reactions of the TCA cycle and their resulting substrates can be supplied by other anaplerotic and cataplerotic reactions. This study is the first of its kind to have established key role of aconitase gene in CHO cells. We further investigated the temporal effects of aconitase silencing on energy production, CHO cell metabolism, oxidative stress and recombinant protein production.
Conclusion
Transient silencing of mitochondrial aconitase inhibited cell growth, reduced ATP production, increased production of reactive oxygen species and reduced cell specific productivity of a recombinant CHO cell line by at least twofold.
... Since each antibody variant is expressed with a characteristic efficiency and minor changes in the framework or CDRs can have a major contribution, (Bentley et al., 1998) a rational design approach can not only be applied to improve antibody binding. It can also be utilized to overcome stability issues or expression challenges in antibody manufacturing (Seeliger, 2013;Mason et al., 2014;Seeliger et al., 2015;Popovic et al., 2017). Popovic et al., 2017 showed the improvement of expression by a single mutation identified by an in silico structure-based design approach. ...
Humanized monoclonal antibodies (mAbs) are among the most promising modern therapeutics, but defined engineering strategies are still not available. Antibody humanization often leads to a loss of affinity, as it is the case for our model antibody Ab2/3H6 (PDB entry 3BQU). Identifying appropriate back-to-mouse mutations is needed to restore binding affinity, but highly challenging. In order to get more insight, we have applied molecular dynamics simulations and correlated them to antibody binding and expression in wet lab experiments. In this study, we discuss six mAb variants and investigate a tyrosine conglomeration, an isopolar substitution and the improvement of antibody binding towards wildtype affinity. In the 3D structure of the mouse wildtype, residue R94h is surrounded by three tyrosines which form a so-called 'tyrosine cage'. We demonstrate that the tyrosine cage has a supporting function for the CDRh3 loop conformation. The isopolar substitution is not able to mimic the function appropriately. Finally, we show that additional light chain mutations can restore binding to wildtype-comparable level, and also improve the expression of the mAb significantly. We conclude that the variable light chain of Ab2/3H6 is of underestimated importance for the interaction with its antigen mAb 2F5.
... Reducing the culture temperature not only influences cell growth and protein expression but also influences glycosylation [18,24]. The effects of mild hypothermia on glycosylation appear to be highly dependent on cell line, protein types and temperature, thus explaining some of the contradictory observations reported in the literature. ...
... The temperature adjustments were applied as per the strategies explained in Table 1 Figs. 1 and 2, it can be seen that the main effect of mild hypothermia was a general reduction of the specific growth rates and an extension of the cell viability. The reduction in specific cell growth under reduced temperature conditions is in agreement with previous studies [18,33]. However, in the current study it was also observed that for cultures initially supplemented with Gln and the Reduced-Temperature regime, culture F, the viable cell density could not reach the same level as the control condition (culture B), even at the later stages of cultivation in contrast with previously reported results [29]. ...
... However, in the current study it was also observed that for cultures initially supplemented with Gln and the Reduced-Temperature regime, culture F, the viable cell density could not reach the same level as the control condition (culture B), even at the later stages of cultivation in contrast with previously reported results [29]. The observed prolonged period of high cell viability under the lower temperature conditions is in agreement with other studies [18,19]. ...
The impact of cell culture environment on the glycan distribution of a monoclonal antibody (mAb) has been investigated through a combination of experiments and modeling. A newly developed CHO DUXB cell line was cultivated at two levels of initial Glutamine (Gln) concentrations (0, 4 mM) and incubation temperatures of (33 and 37 °C) in batch operation mode. Hypothermia was applied either through the entire culture duration or only during the post-exponential phase. Beyond reducing cell growth and increasing productivity, hypothermia significantly altered the galactosylation index profiles as compared to control conditions. A novel semi-empirical dynamic model was proposed for elucidating the connections between the extracellular cell culture conditions to galactosylation index. The developed model is based on a simplified balance of nucleotides sugars and on the correlation between sugars’ levels to the galactosylation index (GI). The model predictions were found to be in a good agreement with the experimental data. The proposed empirical model is expected to be useful for controlling the glycoprofiles by manipulating culture conditions.
