SnapShot: N-Glycosylation Processing Pathways across Kingdoms.

Post-translational modification of proteins with carbohydrates shapes their localization and function. This SnapShot presents the core pathways from different organisms that install these complex and highly variable structures.

Elucidating uptake and metabolic fate of dipeptides in CHO cell cultures using 13C labeling experiments and kinetic modeling.

The rapidly growing market of biologics including monoclonal antibodies has stimulated the need to improve biomanufacturing processes including mammalian host systems such as Chinese Hamster Ovary (CHO) cells. Cell culture media formulations continue to be enhanced to enable intensified cell culture processes and optimize cell culture performance. Amino acids, major components of cell culture media, are consumed in large amounts by CHO cells. Due to their low solubility and poor stability, certain amino acids including tyrosine, leucine, and phenylalanine can pose major challenges leading to suboptimal bioprocess performance. Dipeptides have the potential to replace amino acids in culture media. However, very little is known about the cleavage, uptake, and utilization kinetics of dipeptides in CHO cell cultures. In this study, replacing amino acids, including leucine and tyrosine by their respective dipeptides including but not limited to Ala-Leu and Gly-Tyr, supported similar cell growth, antibody production, and lactate profiles. Using 13C labeling techniques and spent media studies, dipeptides were shown to undergo both intracellular and extracellular cleavage in cultures. Extracellular cleavage increased with the culture duration, indicating cleavage by host cell proteins that are likely secreted and accumulate in cell culture over time. A kinetic model was built and for the first time, integrated with 13C labeling experiments to estimate dipeptide utilization rates, in CHO cell cultures. Dipeptides with alanine at the N-terminus had a higher utilization rate than dipeptides with alanine at the C-terminus and dipeptides with glycine instead of alanine at N-terminus. Simultaneous supplementation of more than one dipeptide in culture led to reduction in individual dipeptide utilization rates indicating that dipeptides compete for the same cleavage enzymes, transporters, or both. Dipeptide utilization rates in culture and cleavage rates in cell-free experiments appeared to follow Michaelis-Menten kinetics, reaching a maximum at higher dipeptide concentrations. Dipeptide utilization behavior was found to be similar in cell-free and cell culture environments, paving the way for future testing approaches for dipeptides in cell-free environments prior to use in large-scale bioreactors. Thus, this study provides a deeper understanding of the fate of dipeptides in CHO cell cultures through an integration of cell culture, 13C labeling, and kinetic modeling approaches providing insights in how to best use dipeptides in media formulations for robust and optimal mammalian cell culture performance.

Reconstruction of reverse transsulfuration pathway enables cysteine biosynthesis and enhances resilience to oxidative stress in Chinese Hamster Ovary cells.

Cysteine is a critically important amino acid necessary for mammalian cell culture, playing key roles in nutrient supply, disulfide bond formation, and as a precursor to antioxidant molecules controlling cellular redox. Unfortunately, its low stability and solubility in solution make it especially problematic as an essential medium component that must be added to Chinese hamster ovary and other mammalian cell cultures. Therefore, CHO cells have been engineered to include the capacity of endogenously synthesizing cysteine by overexpressing multiple enzymes, including cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH) and glycine N-methyltransferase (GNMT) to reconstruct the reverse transsulfuration pathway and overcome a key metabolic bottleneck. Some limited cysteine biosynthesis was obtained by overexpressing CBS and CTH for converting homocysteine to cysteine but robust metabolic synthesis from methionine was only possibly after incorporating GNMT which likely represents a key bottleneck step in the cysteine biosynthesis pathway. CHO cells with the reconstructed pathway exhibit the strong capability to proliferate in cysteine-limited and cysteine-free batch and fed-batch cultures at levels comparable to wildtype cells with ample cysteine supplementation, providing a selectable marker for CHO cell engineering. GNMT overexpression led to the accumulation of sarcosine byproduct, but its accumulation did not affect cell growth. Furthermore, pathway reconstruction enhanced CHO cells' reduced and glutathione levels in cysteine-limited conditions compared to unmodified cells, and greatly enhanced survivability and maintenance of redox homeostasis under oxidative stress induced by addition of menadione in cysteine-deficient conditions. Such engineered CHO cell lines can potentially reduce or even eliminate the need to include cysteine in culture medium, which not only reduces the cost of mammalian media but also promises to transform media design by solving the challenges posed by low stability and solubility of cysteine and cystine in future mammalian biomanufacturing processes.

Overexpression of peroxisome proliferator-activated receptor γ co-activator-1⍺ (PGC-1⍺) in Chinese hamster ovary cells increases oxidative metabolism and IgG productivity.

Chinese hamster ovary (CHO) cells are used extensively to produce protein therapeutics, such as monoclonal antibodies (mAbs), in the biopharmaceutical industry. MAbs are large proteins that are energetically demanding to synthesize and secrete; therefore, high-producing CHO cell lines that are engineered for maximum metabolic efficiency are needed to meet increasing demands for mAb production. Previous studies have identified that high-producing cell lines possess a distinct metabolic phenotype when compared to low-producing cell lines. In particular, it was found that high mAb production is correlated to lactate consumption and elevated TCA cycle flux. We hypothesized that enhancing flux through the mitochondrial TCA cycle and oxidative phosphorylation would lead to increased mAb productivities and final titers. To test this hypothesis, we overexpressed peroxisome proliferator-activated receptor γ co-activator-1⍺ (PGC-1⍺), a gene that promotes mitochondrial metabolism, in an IgG-producing parental CHO cell line. Stable cell pools overexpressing PGC-1⍺ exhibited increased oxygen consumption, indicating increased mitochondrial metabolism, as well as increased mAb specific productivity compared to the parental line. We also performed 13C metabolic flux analysis (MFA) to quantify how PGC-1⍺ overexpression alters intracellular metabolic fluxes, revealing not only increased TCA cycle flux, but global upregulation of cellular metabolic activity. This study demonstrates the potential of rationally engineering the metabolism of industrial cell lines to improve overall mAb productivity and to increase the abundance of high-producing clones in stable cell pools.

Metabolic engineering of Synechococcus elongatus 7942 for enhanced sucrose biosynthesis.

The capability of cyanobacteria to produce sucrose from CO2 and light has a remarkable societal and biotechnological impact since sucrose can serve as a carbon and energy source for a variety of heterotrophic organisms and can be converted into value-added products. However, most metabolic engineering efforts have focused on understanding local pathway alterations that drive sucrose biosynthesis and secretion in cyanobacteria rather than analyzing the global flux re-routing that occurs following induction of sucrose production by salt stress. Here, we investigated global metabolic flux alterations in a sucrose-secreting (cscB-overexpressing) strain relative to its wild-type Synechococcus elongatus 7942 parental strain. We used targeted metabolomics, 13C metabolic flux analysis (MFA), and genome-scale modeling (GSM) as complementary approaches to elucidate differences in cellular resource allocation by quantifying metabolic profiles of three cyanobacterial cultures - wild-type S. elongatus 7942 without salt stress (WT), wild-type with salt stress (WT/NaCl), and the cscB-overexpressing strain with salt stress (cscB/NaCl) - all under photoautotrophic conditions. We quantified the substantial rewiring of metabolic fluxes in WT/NaCl and cscB/NaCl cultures relative to WT and identified a metabolic bottleneck limiting carbon fixation and sucrose biosynthesis. This bottleneck was subsequently mitigated through heterologous overexpression of glyceraldehyde-3-phosphate dehydrogenase in an engineered sucrose-secreting strain. Our study also demonstrates that combining 13C-MFA and GSM is a useful strategy to both extend the coverage of MFA beyond central metabolism and to improve the accuracy of flux predictions provided by GSM.

CHOGlycoNET: Comprehensive glycosylation reaction network for CHO cells.

Chinese hamster ovary (CHO) cells are extensively used for the production of glycoprotein therapeutics proteins, for which N-linked glycans are a critical quality attribute due to their influence on activity and immunogenicity. Manipulation of protein glycosylation is commonly achieved through cell or process engineering, which are often guided by mathematical models. However, each study considers a unique glycosylation reaction network that is tailored around the cell line and product at hand. Herein, we use 200 glycan datasets for both recombinantly produced and native proteins from different CHO cell lines to reconstruct a comprehensive reaction network, CHOGlycoNET, based on the individual minimal reaction networks describing each dataset. CHOGlycoNET is used to investigate the distribution of mannosidase and glycosyltransferase enzymes in the Golgi apparatus and identify key network reactions using machine learning and dimensionality reduction techniques. CHOGlycoNET can be used for accelerating glycomodel development and predicting the effect of glycoengineering strategies. Finally, CHOGlycoNET is wrapped in a SBML file to be used as a standalone model or in combination with CHO cell genome scale models.

Addressing amino acid-derived inhibitory metabolites and enhancing CHO cell culture performance through DOE-guided media modifications.

Previously, we identified six inhibitory metabolites (IMs) accumulating in Chinese hamster ovary (CHO) cultures using AMBIC 1.0 community reference medium that negatively impacted culture performance. The goal of the current study was to modify the medium to control IM accumulation through design of experiments (DOE). Initial over-supplementation of precursor amino acids (AAs) by 100% to 200% in the culture medium revealed positive correlations between initial AA concentrations and IM levels. A screening design identified 5 AA targets, Lys, Ile, Trp, Leu, Arg, as key contributors to IMs. Response surface design analysis was used to reduce initial AA levels between 13% and 33%, and these were then evaluated in batch and fed-batch cultures. Lowering AAs in basal and feed medium and reducing feed rate from 10% to 5% reduced inhibitory metabolites HICA and NAP by up to 50%, MSA by 30%, and CMP by 15%. These reductions were accompanied by a 13% to 40% improvement in peak viable cell densities and 7% to 50% enhancement in IgG production in batch and fed-batch processes, respectively. This study demonstrates the value of tuning specific AA levels in reference basal and feed media using statistical design methodologies to lower problematic IMs.

Design space determination to optimize DNA complexation and full capsid formation in transient rAAV manufacturing.

Recombinant adeno-associated virus (rAAV) vectors are a promising platform for in vivo gene therapies. However, cost-effective, well-characterized processes necessary to manufacture rAAV therapeutics are challenging to develop without an understanding of how process parameters (PPs) affect rAAV product quality attributes (PQAs). In this work, a central composite orthogonal experimental design was employed to examine the influence of four PPs for transient transfection complex formation (polyethylenimine:DNA [PEI:DNA] ratio, total DNA/cell, cocktail volume, and incubation time) on three rAAV PQAs related to capsid content (vector genome titer, vector genome:capsid particle ratio, and two-dimensional vector genome titer ratio). A regression model was established for each PQA using partial least squares, and a design space (DS) was defined in which Monte Carlo simulations predicted < 1% probability of failure (POF) to meet predetermined PQA specifications. Of the three PQAs, viral genome titer was most strongly correlated with changes in complexation PPs. The DS and acceptable PP ranges were largest when incubation time and cocktail volume were kept at mid-high setpoints, and PEI:DNA ratio and total DNA/cell were at low-mid setpoints. Verification experiments confirmed model predictive capability, and this work establishes a framework for studying other rAAV PPs and their relationship to PQAs.

Chemical inhibitors of hexokinase-2 enzyme reduce lactate accumulation, alter glycosylation processing, and produce altered glycoforms in CHO cell cultures.

Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by-product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase-2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6-phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and N-glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2-deoxy- d-glucose (2DG) and 5-thio- d-glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%-45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO-Fc titers peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO-Fc titers. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes. N-glycan analysis of EPO-Fc revealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG-supplemented cultures, respectively. Inhibitor supplementation also led to a decrease in bi-, tri-, and tetra-antennary structures and up to 50% lower EPO-Fc sialylation. Interestingly, addition of 2DG led to the incorporation of 2-deoxy-hexose (2DH) on EPO-Fc N-glycans and addition of 5TG resulted in the first-ever observed N-glycan incorporation of 5-thio-hexose (5TH). Six percent to 23% of N-glycans included 5TH moieties, most likely 5-thio-mannose and/or 5-thio-galactose and/or possibly 5-thio-N-acetylglucosamine, and 14%-33% of N-glycans included 2DH moieties, most likely 2-deoxy-mannose and/or 2-deoxy-galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism, N-glycosylation processing, and formation of alternative glycoforms.

Generation of reference cell lines, media, and a process platform for CHO cell biomanufacturing.

Due to the favorable attributes of Chinese hamster ovary (CHO) cells for therapeutic proteins and antibodies biomanufacturing, companies generate proprietary cells with desirable phenotypes. One key attribute is the ability to stably express multi-gram per liter titers in chemically defined media. Cell, media, and feed diversity has limited community efforts to translate knowledge. Moreover, academic, and nonprofit researchers generally cannot study "industrially relevant" CHO cells due to limited public availability, and the time and knowledge required to generate such cells. To address these issues, a university-industrial consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) has acquired two CHO "reference cell lines" from different lineages that express monoclonal antibodies. These reference cell lines have relevant production titers, key performance outcomes confirmed by multiple laboratories, and a detailed technology transfer protocol. In commercial media, titers over 2 g/L are reached. Fed-batch cultivation data from shake flask and scaled-down bioreactors is presented. Using productivity as the primary attribute, two academic sites aligned with tight reproducibility at each site. Further, a chemically defined media formulation was developed and evaluated in parallel to the commercial media. The goal of this work is to provide a universal, industrially relevant CHO culture platform to accelerate biomanufacturing innovation.

Comprehensive N- and O-Glycoproteomic Analysis of Multiple Chinese Hamster Ovary Host Cell Lines.

Glycoproteomic analysis of three Chinese hamster ovary (CHO) suspension host cell lines (CHO-K1, CHO-S, and CHO-Pro5) commonly utilized in biopharmaceutical settings for recombinant protein production is reported. Intracellular and secreted glycoproteins were examined. We utilized an immobilization and chemoenzymatic strategy in our analysis. Glycoproteins or glycopeptides were first immobilized through reductive amination, and the sialyl moieties were amidated for protection. The desired N- or O-glycans and glycopeptides were released from the immobilization resin by enzymatic or chemical digestion. Glycopeptides were studied by Orbitrap Liquid chromatography-mass spectrometry (LC/MS), and the released glycans were analyzed by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Differences were detected in the relative abundances of N- and O-glycopeptide types, their resident and released glycans, and their glycoprotein complexity. Ontogeny analysis revealed key differences in features, such as general metabolic and biosynthetic pathways, including glycosylation systems, as well as distributions in cellular compartments. Host cell lines and subfraction differences were observed in both N- and O-glycan and glycoprotein pools. Differences were observed in sialyl and fucosyl glycan distributions. Key differences were also observed among glycoproteins that are problematic contaminants in recombinant antibody production. The differences revealed in this study should inform the choice of cell lines best suited for a particular bioproduction application.

Association of Frailty, Age, and Biological Sex With Severe Acute Respiratory Syndrome Coronavirus 2 Messenger RNA Vaccine-Induced Immunity in Older Adults.

BACKGROUND: Male sex and old age are risk factors for severe coronavirus disease 2019, but the intersection of sex and aging on antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has not been characterized. METHODS: Plasma samples were collected from older adults (aged 75-98 years) before and after 3 doses of SARS-CoV-2 mRNA vaccination, and from younger adults (aged 18-74 years) post-dose 2, for comparison. Antibody binding to SARS-CoV-2 antigens (spike protein [S], S receptor-binding domain, and nucleocapsid), functional activity against S, and live-virus neutralization were measured against the vaccine virus and the Alpha, Delta, and Omicron variants of concern (VOCs). RESULTS: Vaccination induced greater antibody titers in older females than in older males, with both age and frailty associated with reduced antibody responses in males but not females. Responses declined significantly in the 6 months after the second dose. The third dose restored functional antibody responses and eliminated disparities caused by sex, age, and frailty in older adults. Responses to the VOCs, particularly the Omicron variant, were significantly reduced relative to the vaccine virus, with older males having lower titers to the VOCs than older females. Older adults had lower responses to the vaccine and VOC viruses than younger adults, with greater disparities in males than in females. CONCLUSIONS: Older and frail males may be more vulnerable to breakthrough infections owing to low antibody responses before receipt of a third vaccine dose. Promoting third dose coverage in older adults, especially males, is crucial to protecting this vulnerable population.

Significant impact of mTORC1 and ATF4 pathways in CHO cell recombinant protein production induced by CDK4/6 inhibitor.

The CDK4/6 inhibitor has been shown to increase recombinant protein productivity in Chinese hamster ovary (CHO) cells. Therefore, we investigated the mechanism that couples cell-cycle inhibitor (CCI) treatment with protein productivity utilizing proteomics and phosphoproteomics. We identified mTORC1 as a critical early signaling event that preceded boosted productivity. Following CCI treatment, mTOR exhibited a transient increase in phosphorylation at a novel site that is also conserved in humans and mouse. Upstream of mTORC1, increased phosphorylation of AKT1S1 and decreased phosphorylation of RB1 may provide molecular links between CDK4/6 inhibition and mTORC1. Downstream, increased EIF4EBP1 phosphorylation was observed, which can mediate cap-dependent translation. In addition, the collective effect of increased phosphorylation of RPS6, increased phosphorylation of regulators of RNA polymerase I, and increased protein expression in the transfer RNA-aminoacylation pathway may contribute to enhancing the translational apparatus for increased productivity. In concert, an elevated stress response via GCN2/EIF2AK4-ATF4 axis persisted over the treatment course, which may link mTOR to downstream responses including the unfolded protein response and autophagy to enhance proper protein folding and secretion. Together, this comprehensive proteomics and phosphoproteomics characterization of CCI-treated CHO cells offers insights into understanding multiple aspects of signaling events resulting from CDK4/CDK6 inhibition.

Engineering redox sensors into CHO cells enables near-real-time quantification of intracellular redox in bioprocesses.

The production of biologics that treat complex diseases, such as cancer, autoimmune, and infectious disease, requires careful monitoring and control of cell cultures. While bioprocess optimizations have dramatically improved production yields, a lack of analytical tools has made it challenging to identify accompanying intracellular improvements. Intracellular redox can diminish the growth and productivity of biologics-producing cells and adversely impact product quality profiles yet characterizing redox is challenging due to its complex and highly transient nature. In this study, we integrated a fluorescent thiol-based redox biosensor to monitor intracellular redox in one bisAb- and two monoclonal antibody-producing clonal cell lines in a 14-day fed-batch bioreactor. We characterized biosensor functionality using three fluorescence measurement techniques and determined sensor oxidation correlates with the intracellular ratio of reduced (GSH) and oxidized glutathione (GSSG), an important cellular antioxidant. Our fed-batch bioreactor studies showed that sensor expression minimally affected bioprocess outcomes, including growth, productivity, product quality attributes, or intracellular redox attributes, including mitochondrial reactive oxygen species and total cellular GSH levels in all cell lines tested. Biosensor measurements taken throughout the culture revealed that the intracellular environment in these cell lines became more reduced throughout the culture, with the exception of a high pH condition which became more oxidized. Our results demonstrate the potential of using biosensors to monitor intracellular changes in near-real-time with minimal process effects, thus potentially improving future bioprocess optimizations.

A proteomics approach to decipher a sticky CHO situation.

Chinese hamster ovary (CHO) cells serve as protein therapeutics workhorses, so it is useful to understand what intrinsic properties make certain host cell lines and clones preferable for scale up and production of target proteins. In this study, two CHO host cell lines (H1, H2), and their respective clones were evaluated using comparative TMT-proteomics. The clones obtained from host H1 showed increased productivity (6.8 times higher) in comparison to clones from host H2. Based on fold-change analyses, we observed differential regulation in pathways including cell adhesion, aggregation, and cellular metabolism among others. In particular, the cellular adhesion pathway was downregulated in H1, in which podoplanin, an antiadhesion molecule, was upregulated the most in host H1 and associated clones. Phenotypically, these cells were less likely to aggregate and adhere to surfaces. In addition, enzymes involved in cellular metabolism such as isocitrate dehydrogenase (IDH) and mitochondrial-d-lactate dehydrogenase ( d-LDHm) were also found to be differentially regulated. IDH plays a key role in TCA cycle and isocitrate-alpha-ketoglutarate cycle while d-LDHm aids in the elimination of toxic metabolite methylglyoxal, involved in protein degradation. These findings will enhance our efforts towards understanding why certain CHO cell lines exhibit enhanced performance and perhaps provide future cell engineering targets.

Epigenetic comparison of CHO hosts and clones reveals divergent methylation and transcription patterns across lineages.

In this study, we examined DNA methylation and transcription profiles of recombinant clones derived from two different Chinese hamster ovary hosts. We found striking epigenetic differences between the clones, with global hypomethylation in the host 1 clones that produce bispecific antibody with higher productivity and complex assembly efficiency. Whereas the methylation patterns were found mostly inherited from the host, the host 1 clones exhibited continued demethylation reflected by the hypomethylation of newly emerged differential methylation regions (DMRs) even at the clone development stage. Several interconnected biological functions and pathways including cell adhesion, regulation of ion transport, and cholesterol biosynthesis were significantly altered between the clones at the RNA expression level and contained DMR in the promoter and/or gene-body of the transcripts, suggesting epigenetic regulation. Indeed, expression changes of epigenetic regulators were observed including writers (Dnmt1, Setdb1), readers (Mecp2), and erasers (Tet3, Kdm3a, Kdm1b/5c) involved in CpG methylation, histone methylation, and heterochromatin maintenance. In addition, we identified putative transcription factors that may be readers or effectors of the epigenetic regulation in these clones. By combining transcriptomics with DNA methylation data, we identified potential processes and factors that may contribute to the variability in cell physiology between different production hosts.

Attenuation of glutamine synthetase selection marker improves product titer and reduces glutamine overflow in Chinese hamster ovary cells.

The glutamine synthetase (GS) expression system is commonly used to ensure stable transgene integration and amplification in Chinese hamster ovary (CHO) host lines. Transfected cell populations are typically grown in the presence of the GS inhibitor, methionine sulfoximine (MSX), to further select for increased transgene copy number. However, high levels of GS activity produce excess glutamine. We hypothesized that attenuating the GS promoter while keeping the strong IgG promoter on the GS-IgG expression vector would result in a more efficient cellular metabolic phenotype. Herein, we characterized CHO cell lines expressing GS from either an attenuated promoter or an SV40 promoter and selected with/without MSX. CHO cells with the attenuated GS promoter had higher IgG specific productivity and lower glutamine production compared to cells with SV40-driven GS expression. Selection with MSX increased both specific productivity and glutamine production, regardless of GS promoter strength. 13 C metabolic flux analysis (MFA) was performed to further assess metabolic differences between these cell lines. Interestingly, central carbon metabolism was unaltered by the attenuated GS promoter while the fate of glutamate and glutamine varied depending on promoter strength and selection conditions. This study highlights the ability to optimize the GS expression system to improve IgG production and reduce wasteful glutamine overflow, without significantly altering central metabolism. Additionally, a detailed supplementary analysis of two "lactate runaway" reactors provides insight into the poorly understood phenomenon of excess lactate production by some CHO cell cultures.

A genome-scale nutrient minimization forecast algorithm for controlling essential amino acid levels in CHO cell cultures.

Mammalian cell culture processes rely heavily on empirical knowledge in which process control remains a challenge due to the limited characterization/understanding of cell metabolism and inability to predict the cell behaviors. This study facilitates control of Chinese hamster ovary (CHO) processes through a forecast-based feeding approach that predicts multiple essential amino acids levels in the culture from easily acquired viable cell density data. Multiple cell growth behavior forecast extrapolation approaches are considered with logistic curve fitting found to be the most effective. Next, the nutrient-minimized CHO genome-scale model is combined with the growth forecast model to generate essential amino acid forecast profiles of multiple CHO batch cultures. Comparison of the forecast with the measurements suggests that this algorithm can accurately predict the concentration of most essential amino acids from cell density measurement with error mitigated by incorporating off-line amino acids concentration measurements. Finally, the forecast algorithm is applied to CHO fed-batch cultures to support amino acid feeding control to control the concentration of essential amino acids below 1-2 mM for lysine, leucine, and valine as a model over a 9-day fed batch culture while maintaining comparable growth behavior to an empirical-based culture. In turn, glycine production was elevated, alanine reduced and lactate production slightly lower in control cultures due to metabolic shifts in branched-chain amino acid degradation. With the advantage of requiring minimal measurement inputs while providing valuable and in-advance information of the system based on growth measurements, this genome model-based amino acid forecast algorithm represent a powerful and cost-effective tool to facilitate enhanced control over CHO and other mammalian cell-based bioprocesses.

The interplay of protein engineering and glycoengineering to fine-tune antibody glycosylation and its impact on effector functions.

The N-glycan pattern of an IgG antibody, attached at a conserved site within the fragment crystallizable (Fc) region, is a critical antibody quality attribute whose structural variability can also impact antibody function. For tailoring the Fc glycoprofile, glycoengineering in cell lines as well as Fc amino acid mutations have been applied. Multiple glycoengineered Chinese hamster ovary cell lines were generated, including defucosylated (FUT8KO), α-2,6-sialylated (ST6KI), and defucosylated α-2,6-sialylated (FUT8KOST6KI), expressing either a wild-type anti-CD20 IgG (WT) or phenylalanine to alanine (F241A) mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry characterization of antibody N-glycans revealed that the F241A mutation significantly increased galactosylation and sialylation content and glycan branching. Furthermore, overexpression of recombinant human α-2,6-sialyltransferase resulted in a predominance of α-2,6-sialylation rather than α-2,3-sialylation for both WT and heavily sialylated F241A antibody N-glycans. Interestingly, knocking out α-1,6-fucosyltransferase (FUT8KO), which removed core fucose, lowered the content of N-glycans with terminal Gal and increased levels of terminal GlcNAc and Man5 groups on WT antibody. Further complement-dependent cytotoxicity (CDC) analysis revealed that, regardless of the production cells, WT antibody samples have higher cytotoxic CDC activity with more exposed Gal residues compared to their individual F241A mutants. However, the FUT8KO WT antibody, with a large fraction of bi-GlcNAc structures (G0), displayed the lowest CDC activity of all WT antibody samples. Furthermore, for the F241A mutants, a higher CDC activity was observed for α-2,6- compared to α-2,3-sialylation. Antibody-dependent cellular cytotoxicity (ADCC) analysis revealed that the defucosylated WT and F241A mutants showed enhanced in vitro ADCC performance compared to their fucosylated counterparts, with the defucosylated WT antibodies displaying the highest overall ADCC activity, regardless of sialic acid substitution. Moreover, the FcγRIIIA receptor binding by antibodies did not always correspond directly with ADCC result. This study demonstrates that glycoengineering and protein engineering can both promote and inhibit antibody effector functions and represent practical approaches for varying glycan composition and functionalities during antibody development.

Metabolic analysis of the asparagine and glutamine dynamics in an industrial Chinese hamster ovary fed-batch process.

Chinese hamster ovary (CHO) cell lines are grown in cultures with varying asparagine and glutamine concentrations, but further study is needed to characterize the interplay between these amino acids. By following 13 C-glucose, 13 C-glutamine, and 13 C-asparagine tracers using metabolic flux analysis (MFA), CHO cell metabolism was characterized in an industrially relevant fed-batch process under glutamine supplemented and low glutamine conditions during early and late exponential growth. For both conditions MFA revealed glucose as the primary carbon source to the tricarboxylic acid (TCA) cycle followed by glutamine and asparagine as secondary sources. Early exponential phase CHO cells prefer glutamine over asparagine to support the TCA cycle under the glutamine supplemented condition, while asparagine was critical for TCA activity for the low glutamine condition. Overall TCA fluxes were similar for both conditions due to the trade-offs associated with reliance on glutamine and/or asparagine. However, glutamine supplementation increased fluxes to alanine, lactate and enrichment of glutathione, N-acetyl-glucosamine and pyrimidine-containing-molecules. The late exponential phase exhibited reduced central carbon metabolism dominated by glucose, while lactate reincorporation and aspartate uptake were preferred over glutamine and asparagine. These 13 C studies demonstrate that metabolic flux is process time dependent and can be modulated by varying feed composition.