Enhancing protein productivities in CHO cells through adenosine uptake modulation - Novel insights into cellular growth and productivity regulation.

Maximizing production potential of recombinant proteins such as monoclonal antibodies (mAbs) in Chinese Hamster Ovary (CHO) cells is a key enabler of reducing cost of goods of biologics. In this study, we explored various strategies to utilize adenosine mediated effects in biologics manufacturing processes. Results show that supplementation of adenosine increases specific productivity by up to two-fold while also arresting cell growth. Introducing adenosine in intensified perfusion processes in a biphasic manner significantly enhanced overall productivity. Interestingly, adenosine effect was observed to be dependent on the cell growth state. Using specific receptor antagonists and inhibitors, we identified that ENTs (primarily Slc29a1) mediate the uptake of adenosine in CHO cell cultures. Transcriptomics data showed an inverse correlation between Slc29a1 expression levels and peak viable cell densities. Data suggests that in fed-batch cultures, adenosine can be produced extracellularly. Blocking Slc29a1 using ENT inhibitors such as DZD and DP alone or in combination with CD73 inhibitor, PSB12379, resulted in a twofold increase in peak viable cell densities as well as productivities in fed batch - a novel strategy that can be applied to biologics manufacturing processes. This is the first study that suggests that adenosine production/accumulation in CHO cell cultures can potentially regulate the transition of CHO cells from exponential to stationary phase. We also demonstrate strategies to leverage this regulatory mechanism to maximize the productivity potential of biologics manufacturing processes.

Dilution rates and their transition modes influence organic acid productivity and bacterial community structure on continuous meta-fermentation using complex microorganisms.

We investigated the effect of dilution rates (D) (0.05, 0.15, and 0.4 h-1) and its transition mode strategies (constant, up, and down modes) on organic acid productivity and bacterial community structure on continuous meta-fermentation using complex microorganisms. The number of bacterial species decreased with increasing D in the constant mode while up and down modes maintained high and low values, respectively, regardless of the changing D values. Caldibacillus hisashii was the predominant species in all modes at all D values, while other bacterial species, including Anaerosalibacter bizertensis and Clostridium cochlearium were predominant in only certain modes and D values. The highest total organic acid productivity of 3.16 g L-1 h-1 was obtained with 82.2% lactic acid selectivity at D = 0.4 h⁻1 in constant mode. Heterofermentation occurred in the up mode, while the down mode exhibited the maximum butyric acid productivity of 0.348 g L-1 h-1 with 43.8% selectivity at D = 0.05 h-1. The constant, up, and down modes showed the distinct main products of lactic, acetic and formic, and butyric acids, respectively. In this study, we proposed a new parameter of species-specific productivity (SSP) to estimate which species and how much a bacterium quantitatively contributes to the targeted organic acid productivity in continuous meta-fermentation. SSP was determined based on the abundance of functional genes encoding key enzymes from the results of 16S amplicon analysis. In conclusion, D values and their transition modes affect productivity by changing the bacterial community structure, and are a significant factor in establishing a highly productive process in continuous meta-fermentation.

Comparative study of commercial media to improve GMP manufacturing of recombinant human interferon ß-1a by CHO cells in perfusion bioreactor.

Chinese hamster ovary cells are the main cellular factories for production of a wide range of recombinant proteins in biopharmaceutical industry. Recombinant human Interferon beta-1a (rh-IFN ß-1a), as a cytokine is broadly used to treat multiple sclerosis. In this work, the cell line producing rh-IFN ß-1a was studied to improve cell density along with the specific expression. For this reason different cell culture experiments were done using different commercial serum-free media to find the appropriate media providing higher cell density. It was shown DMEMF12, DMEM:ProCHO5, and CHO-S-SFM II led to higher cell density and shorter doubling time. Next, using these media, fed-batch, and perfusion culture with temperature shift were implemented to investigate the best condition for industrial-scale manufacturing of rh-IFN ß-1a in terms of higher cell density and product expression yield. The results demonstrated that CHO-S-SFM II media and a thermally biphasic condition provide enhanced expression of rh-IFN ß-1a in perfusion bioreactor.

Stress-induced increase of monoclonal antibody production in CHO cells.

Monoclonal antibodies (mAbs) are of great interest to the biopharmaceutical industry due to their widely used application as human therapeutic and diagnostic agents. As such, mAb require to exhibit human-like glycolization patterns. Therefore, recombinant Chinese hamster ovary (CHO) cells are the favored production organisms; many relevant biopharmaceuticals are already produced by this cell type. To optimize the mAb yield in CHO DG44 cells a corelation between stress-induced cell size expansion and increased specific productivity was investigated. CO2 and macronutrient supply of the cells during a 12-day fed-batch cultivation process were tested as stress factors. Shake flasks (500 mL) and a small-scale bioreactor system (15 mL) were used for the cultivation experiments and compared in terms of their effect on cell diameter, integral viable cell concentration (IVCC), and cell-specific productivity. The achieved stress-induced increase in cell-specific productivity of up to 94.94.9%-134.4% correlates to a cell diameter shift of up to 7.34 µm. The highest final product titer of 4 g/L was reached by glucose oversupply during the batch phase of the process.

A comprehensive comparison of mixing and mass transfer in shake flasks and their relationship with MAb productivity of CHO cells.

The selection of highly recombinant protein (RP)-productive Chinese hamster ovary (CHO) cell lines is widely carried out in shake flasks. It is assumed that increases in the operating parameters in shake flasks lead to impairments in cell growth and RP production. These effects in cells metabolism are widely associated with high mass transfers and hydrodynamic stress. This study examined the impact of commonly used operational parameters on growth and specific productivity (qP) of two CHO cell lines differentially secreting a humanized anti-hIL8 monoclonal antibody (mAb) and cultured in 250 ml flasks. The evaluated parameters are filling volume (10, 15, and 20%), shaking frequency (60 and 120 revolutions per minute -rpm-), and orbital diameter (25.4 and 19 mm). The analysis of the oxygen transfer was done in terms of the measured volumetric mass transfer coefficient (kLa) and of the hydrodynamics in terms of power input per unit volume of liquid (P/V), the turbulent eddy length scale measured by the Kolmogorov's microscale of turbulence, the energy dissipation rate, the average shear stress, and the shear rate. Though almost all measured kinetic and stoichiometric parameters remained unchanged, mAb titer included, significant differences were found in maximum cell concentration, 10-45% higher in conditions with lower values of kLa and P/V. Changes in glucose metabolism contributing to qP were only shown in the higher producer cell line. Non-lethal responses to elevated oxygen transfer and shear stress might be present and must be considered when evaluating CHO cell cultures in shake flasks.

Compartment-specific 13C metabolic flux analysis reveals boosted NADPH availability coinciding with increased cell-specific productivity for IgG1 producing CHO cells after MTA treatment.

Increasing cell-specific productivities (CSPs) for the production of heterologous proteins in Chinese hamster ovary (CHO) cells is an omnipresent need in the biopharmaceutical industry. The novel additive 5'-deoxy-5'-(methylthio)adenosine (MTA), a chemical degradation product of S-(5'-adenosyl)-ʟ-methionine (SAM) and intermediate of polyamine biosynthesis, boosts the CSP of IgG1-producing CHO cells by 50%. Compartment-specific 13C flux analysis revealed a fundamental reprogramming of the central metabolism after MTA addition accompanied by cell-cycle arrest and increased cell volumes. Carbon fluxes into the pentose-phosphate pathway increased 22 fold in MTA-treated cells compared to that in non-MTA-treated reference cells. Most likely, cytosolic ATP inhibition of phosphofructokinase mediated the carbon detour. Mitochondrial shuttle activity of the α-ketoglurarate/malate antiporter (OGC) reversed, reducing cytosolic malate transport. In summary, NADPH supply in MTA-treated cells improved three fold compared to that in non-MTA-treated cells, which can be regarded as a major factor for explaining the boosted CSPs.

A Metabolomics Approach to Increasing Chinese Hamster Ovary (CHO) Cell Productivity.

Much progress has been made in improving the viable cell density of bioreactor cultures in monoclonal antibody production from Chinese hamster ovary (CHO) cells; however, specific productivity (qP) has not been increased to the same degree. In this work, we analyzed a library of 24 antibody-expressing CHO cell clones to identify metabolites that positively associate with qP and could be used for clone selection or medium supplementation. An initial library of 12 clones, each producing one of two antibodies, was analyzed using untargeted LC-MS experiments. Metabolic model-based annotation followed by correlation analysis detected 73 metabolites that significantly correlated with growth, qP, or both. Of these, metabolites in the alanine, aspartate, and glutamate metabolism pathway, and the TCA cycle showed the strongest association with qP. To evaluate whether these metabolites could be used as indicators to identify clones with potential for high productivity, we performed targeted LC-MS experiments on a second library of 12 clones expressing a third antibody. These experiments found that aspartate and cystine were positively correlated with qP, confirming the results from untargeted analysis. To investigate whether qP correlated metabolites reflected endogenous metabolic activity beneficial for productivity, several of these metabolites were tested as medium additives during cell culture. Medium supplementation with citrate improved qP by up to 490% and more than doubled the titer. Together, these studies demonstrate the potential for using metabolomics to discover novel metabolite additives that yield higher volumetric productivity in biologics production processes.

Production of butyrate and branched-chain amino acid catabolic byproducts by CHO cells in fed-batch culture enhances their specific productivity.

Chinese hamster ovary (CHO) cells in fed-batch cultures produce several metabolic byproducts derived from amino acid catabolism, some of which accumulate to growth inhibitory levels. Controlling the accumulation of these byproducts has been shown to significantly enhance cell proliferation. Interestingly, some of these byproducts have physiological roles that go beyond inhibition of cell proliferation. In this study, we show that, in CHO cell fed-batch cultures, branched-chain amino acid (BCAA) catabolism contributes to the formation of butyrate, a novel byproduct that is also a well-established specific productivity enhancer. We further show that other byproducts of BCAA catabolism, namely isovalerate and isobutyrate, which accumulate in CHO cell fed-batch cultures, also enhance specific productivity. Lastly, we show that the rate of production of these BCAA catabolic byproducts is negatively correlated with glucose uptake and lactate production rates. Thus, limiting glucose supply to suppress glucose uptake and lactate production, as in the case of fed-batch cultures employing high-end pH-controlled delivery of glucose (HiPDOG) technology, significantly enhances BCAA catabolic byproduct accumulation, resulting in higher specific productivities.

Optimization of Specific Productivity for Xylonic Acid Production by Gluconobacter oxydans Using Response Surface Methodology.

Large amounts of xylose cannot be efficiently metabolized and fermented due to strain limitations in lignocellulosic biorefinery. The conversion of xylose into high value chemicals can help to reduce the cost of commercialization. Therefore, xylonic acid with potential value in the construction industry offers a valuable alternative for xylose biorefinery. However, low productivity is the main challenge for xylonic acid fermentation. This study investigated the effect of three reaction parameters (agitation, aeration, and biomass concentration) on xylose acid production and optimized the key process parameters using response surface methodology The second order polynomial model was able to fit the experimental data by using multiple regression analysis. The maximum specific productivity was achieved with a value of 6.64 ± 0.20 g gx -1 h-1 at the optimal process parameters (agitation speed 728 rpm, aeration rate 7 L min-1, and biomass concentration 1.11 g L-1). These results may help to improve the production efficiency during xylose acid biotransformation from xylose.

A pooled CRISPR/AsCpf1 screen using paired gRNAs to induce genomic deletions in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are the most widely used host for the expression of therapeutic proteins. Recently, significant progress has been made due to advances in genome sequence and annotation quality to unravel the black box CHO. Nevertheless, in many cases the link between genotype and phenotype in the context of suspension cultivated production cell lines is still not fully understood. While frameshift approaches targeting coding genes are frequently used, the non-coding regions of the genome have received less attention with respect to such functional annotation. Importantly, for non-coding regions frameshift knock-out strategies are not feasible. In this study, we developed a CRISPR-mediated screening approach that performs full deletions of genomic regions to enable the functional study of both the translated and untranslated genome. An in silico pipeline for the computational high-throughput design of paired guide RNAs (pgRNAs) directing CRISPR/AsCpf1 was established and used to generate a library tackling process-related genes and long non-coding RNAs. Next generation sequencing analysis of the plasmid library revealed a sufficient, but highly variable pgRNA composition. Recombinase-mediated cassette exchange was applied for pgRNA library integration rather than viral transduction to ensure single copy representation of pgRNAs per cell. After transient AsCpf1 expression, cells were cultivated over two sequential batches to identify pgRNAs which massively affected growth and survival. By comparing pgRNA abundance, depleted candidates were identified and individually validated to verify their effect.

Mapping the molecular basis for growth related phenotypes in industrial producer CHO cell lines using differential proteomic analysis.

BACKGROUND: The ability to achieve high peak viable cell density earlier in CHO cell culture and maintain an extended cell viability throughout the production process is highly desirable to increase recombinant protein yields, reduce host cell impurities for downstream processing and reduce the cost of goods. In this study we implemented label-free LC-MS/MS proteomic profiling of IgG4 producing CHO cell lines throughout the duration of the cell culture to identify differentially expressed (DE) proteins and intracellular pathways associated with the high peak viable cell density (VCD) and extended culture VCD phenotypes. RESULTS: We identified key pathways in DNA replication, mitotic cell cycle and evasion of p53 mediated apoptosis in high peak VCD clonally derived cell lines (CDCLs). ER to Golgi vesicle mediated transport was found to be highly expressed in extended culture VCD CDCLs while networks involving endocytosis and oxidative stress response were significantly downregulated. CONCLUSION: This investigation highlights key pathways for targeted engineering to generate desirable CHO cell phenotypes for biotherapeutic production.

Differential expression of miRNAs and functional role of mir-200a in high and low productivity CHO cells expressing an Fc fusion protein.

OBJECTIVES: We used miRNA and proteomic profiling to understand intracellular pathways that contribute to high and low specific productivity (Qp) phenotypes in CHO clonally derived cell lines (CDCLs) from the same cell line generation project. RESULTS: Differentially expressed (DE) miRNAs were identified which are predicted to target several proteins associated with protein folding. MiR-200a was found to have a number of predicted targets associated with the unfolded protein response (UPR) which were shown to have decreased expression in high Qp CDCLs and have no detected change at the mRNA level. MiR-200a overexpression in a CHO CDCL was found to increase recombinant protein titer by 1.2 fold and Qp by 1.8 fold. CONCLUSION: These results may suggest a role for miR-200a in post-transcriptional regulation of the UPR, presenting miR-200a as a potential target for engineering industrially attractive CHO cell phenotypes.

The emerging role of cellular post-translational modifications in modulating growth and productivity of recombinant Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are one of the most commonly used host cell lines used for the production human therapeutic proteins. Much research over the past two decades has focussed on improving the growth, titre and cell specific productivity of CHO cells and in turn lowering the costs associated with production of recombinant proteins. CHO cell engineering has become of particular interest in recent years following the publication of the CHO cell genome and the availability of data relating to the proteome, transcriptome and metabolome of CHO cells. However, data relating to the cellular post-translational modification (PTMs) which can affect the functionality of CHO cellular proteins has only begun to be presented in recent years. PTMs are important to many cellular processes and can further alter proteins by increasing the complexity of proteins and their interactions. In this review, we describe the research presented from CHO cells to date related on three of the most important PTMs; glycosylation, phosphorylation and ubiquitination.

Understanding the effect of increased cell specific productivity on galactosylation of monoclonal antibodies produced using Chinese hamster ovary cells.

Achieving optimal productivity and desired product quality of the therapeutic monoclonal antibody (mAb) is one of the primary goals of process development. Across the various mAb programs at our company, we observed that increasing the specific productivity (qp) results in a decrease in the % galactosylation (%Gal) level on the protein. In order to gain further insight into this correlation, cells were cultured under different process conditions such as pH or media osmolality or in the presence of supplements such as sodium butyrate. A range of qp and N-glycan profiles were obtained with the greatest changes observed under high pH (lower qp, higher %Gal), higher osmolality (higher qp, lower %Gal) or sodium butyrate (moderately higher qp, moderately lower %Gal) conditions. Abundance of individual glycan species highlighted different bottlenecks in the N-glycosylation pathway depending on the treatment condition. Transcriptomics analysis was performed to identify changes in gene expression profiles that correlate with the inverse relationship between qp and %Gal. Results showed downregulation of Beta-1,4-galactosyltransferase 1 (B4GalT1), UDP-GlcNAc and Mn2+ transporter (slc35a3 and slc39a8 respectively) for the high osmolality conditions. Significant downregulation of slc39a8 (Mn2+ transporter) was observed for the sodium butyrate condition. No significant differences were observed for any of the genes in the N-glycosylation pathway under the high pH condition even though this condition showed highest %Gal. Together, data suggests that different treatments have distinct complex mechanisms by which the overall glycan levels of a mAb are influenced. Further studies based on these results will help build the knowledge necessary to design strategies to obtain the desired productivity and product quality of mAbs.

The Degree and Length of O-Glycosylation of Recombinant Proteins Produced in Pichia pastoris Depends on the Nature of the Protein and the Process Type.

The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N-linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O-glycosylation. O-glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O-mannosylation profiles in P. pastoris. Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O-glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, PGAP and PAOX1 . After purification and protein digestion, glycopeptides are analyzed by LC/ESI-MS. In the samples expressed with PGAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with PAOX1 . The results indicate that the O-glycosylation level is markedly higher when the protein is produced in a methanol-based expression system.

Methylthioadenosine (MTA) boosts cell-specific productivities of Chinese hamster ovary cultures: dosage effects on proliferation, cell cycle and gene expression.

A major goal for process and cell engineering in the biopharmaceutical industry is enhancing production through increasing volumetric and cell-specific productivities (CSP). Here, we present 5'-deoxy-5'-(methylthio)adenosine (MTA), the degradation product of S-(5'-adenosyl)-L-methionine (SAM), as a highly attractive native additive which can boost CSP by 79% when added to exponentially growing cells at a concentration of 250-300 µm. Notably, cell viability and cell size remain higher than in non-treated cultures. In addition, cell cycle arrests first in S-, then in G2-phase before levelling out compared to non-treated cultivations. Intensive differential gene analysis reveals that expression of genes for cytoskeleton mediated proteins and vesicle transport is amplified by treatment. Furthermore, the interaction of MTA with cell proliferation additionally stimulated recombinant protein formation. The results may serve as a promising starting point for further developments in process and cell engineering to boost productivity.

Increased MSX level improves biological productivity and production stability in multiple recombinant GS CHO cell lines.

Increasing cell culture productivity of recombinant proteins via process improvements is the primary focus for research groups within biologics manufacturing. Any recommendations to improve a manufacturing process obviously must be effective, but also be robust, scalable, and with product quality comparable to the original process. In this study, we report that three different GS-/- CHO cell lines developed in media containing a standard concentration of the selection agent methionine sulfoximine (MSX), but then exposed to increased MSX concentrations during seed train expansion, achieved titer increases of 10-19%. This result was observed in processes already considerably optimized. Expanding the cells with a higher MSX concentration improved cell line production stability with increased culture age. Production cultures in 500-L and 1000-L bioreactors replicated laboratory results using 5-L bioreactors, demonstrating process robustness and scalability. Furthermore, product quality attributes of the final drug substance using the higher MSX process were comparable with those from cells expanded in media with the standard selection MSX concentration. Subsequent mechanistic investigations confirmed that the cells were not altered at the genetic level in terms of integration profiles or gene copy number, nor transcriptional levels of glutamine synthetase, heavy chain, or light chain genes. This study provides an effective and applicable strategy to improve the productivity of therapeutic proteins for biologics manufacturing.

Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells.

The ever-growing biopharmaceutical industry relies on the production of recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. The traditional timelines of CHO cell line development can be significantly shortened by the use of targeted gene integration (TI). However, broad use of TI has been limited due to the low specific productivity (qP) of TI-generated clones. Here, we show a 10-fold increase in the qP of therapeutic glycoproteins in CHO cells through the development and optimization of a multicopy TI method. We used a recombinase-mediated cassette exchange (RMCE) platform to investigate the effect of gene copy number, 5' and 3' gene regulatory elements, and landing pad features on qP. We evaluated the limitations of multicopy expression from a single genomic site as well as multiple genomic sites and found that a transcriptional bottleneck can appear with an increase in gene dosage. We created a dual-RMCE system for simultaneous multicopy TI in two genomic sites and generated isogenic high-producing clones with qP of 12-14 pg/cell/day and product titer close to 1 g/L in fed-batch. Our study provides an extensive characterization of the multicopy TI method and elucidates the relationship between gene copy number and protein expression in mammalian cells. Moreover, it demonstrates that TI-generated CHO cells are capable of producing therapeutic proteins at levels that can support their industrial manufacture.

S-adenosylmethionine and methylthioadenosine boost cellular productivities of antibody forming Chinese hamster ovary cells.

The improvement of cell specific productivities for the formation of therapeutic proteins is an important step towards intensified production processes. Among others, the induction of the desired production phenotype via proper media additives is a feasible solution provided that said compounds adequately trigger metabolic and regulatory programs inside the cells. In this study, S-(5'-adenosyl)- l-methionine (SAM) and 5'-deoxy-5'-(methylthio)adenosine (MTA) were found to stimulate cell specific productivities up to approx. 50% while keeping viable cell densities transiently high and partially arresting the cell cycle in an anti-IL-8-producing CHO-DP12 cell line. Noteworthy, MTA turned out to be the chemical degradation product of the methyl group donor SAM and is consumed by the cells.

mRNA Transfection into CHO-Cells Reveals Production Bottlenecks.

Obtaining highly productive Chinese hamster ovary (CHO)-cell clones for the production of therapeutic proteins relies on multiple time-consuming selection steps. Several CHO-cell strains with high degrees of genomic and epigenetic variation are available. Each harbor potential advantages and disadvantages for any given product, particularly those considered difficult to express. A simple test system to quickly assess compatibility of cell line and product may therefore prove useful. Transient plasmid transfection falls short of the specific productivities of stable producer cells, making it unsuitable for the elucidation of high specific productivity bottlenecks. The aim of the study is to reach specific productivities approaching those of industrial production cell lines by transfection of in vitro transcribed mRNA. The system is characterized with respect to transfection efficacy (by quantitative PCR) and protein production (by flow cytometry and biolayer interferometry). Fluorescence of intracellular eGFP saturates at higher amounts of mRNA per cell, while the amount of secreted and intracellular EPO-Fc remain linearly correlated to the amount of mRNA taken up. Nevertheless, MS shows a severe reduction in N-glycosylation quality. This method allows for rapid elucidation of bottlenecks that would otherwise remain undetected until later during cell line development, giving insight into suitable strategies for preemptive targeted metabolic engineering and host cell line optimization.