Three doses of COVID-19 mRNA vaccine induce class-switched antibody responses in inflammatory arthritis patients on immunomodulatory therapies.

Patients with inflammatory arthritis (IA) are at increased risk of severe COVID-19 due to medication-induced immunosuppression that impairs host defenses. The aim of this study was to assess antibody and B cell responses to COVID-19 mRNA vaccination in IA patients receiving immunomodulatory therapies. Adults with IA were enrolled through the Johns Hopkins Arthritis Center and compared with healthy controls (HC). Paired plasma and peripheral blood mononuclear cell (PBMC) samples were collected prior to and 30 days or 6 months following the first two doses of mRNA vaccines (D2; HC=77 and IA=31 patients), or 30 days following a third dose of mRNA vaccines (D3; HC=11 and IA=96 patients). Neutralizing antibody titers, total binding antibody titers, and B cell responses to vaccine and Omicron variants were analyzed. Anti-Spike (S) IgG and S-specific B cells developed appropriately in most IA patients following D3, with reduced responses to Omicron variants, and negligible effects of medication type or drug withholding. Neutralizing antibody responses were lower compared to healthy controls after both D2 and D3, with a small number of individuals demonstrating persistently undetectable neutralizing antibody levels. Most IA patients respond as well to mRNA COVID-19 vaccines as immunocompetent individuals by the third dose, with no evidence of improved responses following medication withholding. These data suggest that IA-associated immune impairment may not hinder immunity to COVID-19 mRNA vaccines in most individuals.

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.

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.

Cottonseed hydrolysate supplementation alters metabolic and proteomics responses in Chinese hamster ovary cell cultures.

Hydrolysates are used as media supplements although their role is not well characterized. In this study, cottonseed hydrolysates, which contained peptides and galactose as supplemental substrates, were added to Chinese hamster ovary (CHO) batch cultures, enhancing cell growth, immunoglobulin (IgG) titers, and productivities. Extracellular metabolomics coupled with tandem mass tag (TMT) proteomics revealed metabolic and proteomic changes in cottonseed-supplemented cultures. Shifts in production and consumption dynamics of glucose, glutamine, lactate, pyruvate, serine, glycine, glutamate, and aspartate suggest changes in tricarboxylic acid (TCA) and glycolysis metabolism following hydrolysate inputs. Quantitative proteomics revealed 5521 proteins and numerous changes in relative abundance of proteins related to growth, metabolism, oxidative stress, protein productivity, and apoptosis/cell death at day 5 and day 6. Differential abundance of amino acid transporter proteins and catabolism enzymes such as branched-chain-amino-acid aminotransferase (BCAT)1 and fumarylacetoacetase (FAH) can alter availability and utilization of several amino acids. Also, pathways involved in growth including the polyamine biosynthesis through higher ornithine decarboxylase (ODC1) abundance and hippo signaling were upregulated and downregulated, respectively. Central metabolism rewiring was indicated by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) downregulation, which corresponded with re-uptake of secreted lactate in the cottonseed-supplemented cultures. Overall, cottonseed hydrolysate supplementation modified culture performance by altering cellular activities critical to growth and protein productivity including metabolism, transport, mitosis, transcription, translation, protein processing, and apoptosis. HIGHLIGHTS: Cottonseed hydrolysate, as a medium additive, enhances Chinese hamster ovary (CHO) cell culture performance. Metabolite profiling and tandem mass tag (TMT) proteomics characterize its impact on CHO cells. Rewired nutrient utilization is observed via glycolysis, amino acid, and polyamine metabolism. Hippo signaling pathway impacts cell growth in the presence of cottonseed hydrolysate.

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.

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.

Chemical speciation of trace metals in mammalian cell culture media: looking under the hood to boost cellular performance and product quality.

Upstream process development seeks to optimize media formulations to promote robust cell culture conditions and regulate product quality attributes such as glycosylation, aggregation, and charge variants. Transition metal ions Mn, Fe, Cu, and Zn present in cell culture media have a significant impact on cell growth, metabolism and product quality. These metals and other media components can have different chemical associations or speciation in media that are poorly characterized but may significantly impact their properties and effect on cellular performance. Computer-based equilibrium models are a good starting point for exploring metal speciation, bioavailability and conditions where precipitation may occur. However, some equilibrium constants, especially for newly introduced medium components, have not been experimentally determined. Owing to concurrent physical and biological processes, speciation may also be controlled by reaction kinetics rather than by equilibrium. These factors highlight the importance of analytically interrogating medium speciation to gain insights into the complex interconnections between media components and bioprocess performance.

Identification of novel inhibitory metabolites and impact verification on growth and protein synthesis in mammalian cells.

Mammalian cells consume large amount of nutrients during growth and production. However, endogenous metabolic inefficiencies often prevent cells to fully utilize nutrients to support growth and protein production. Instead, significant fraction of fed nutrients is diverted into extracellular accumulation of waste by-products and metabolites, further inhibiting proliferation and protein synthesis. In this study, an LC-MS/MS based metabolomics pipeline was used to screen Chinese hamster ovary (CHO) extracellular metabolites. Six out of eight identified inhibitory metabolites, caused by the inefficient cell metabolism, were not previously studied in CHO cells: aconitic acid, 2-hydroxyisocaproic acid, methylsuccinic acid, cytidine monophosphate, trigonelline, and n-acetyl putrescine. When supplemented back into a fed-batch culture, significant reduction in cellular growth was observed in the presence of each metabolite and all the identified metabolites were shown to impact the glycosylation of a model secreted antibody, with seven of these also reducing CHO cellular productivity (titer) and all eight inhibiting the formation of mono-galactosylated biantennary (G1F) and biantennary galactosylated (G2F) N-glycans. These inhibitory metabolites further impact the metabolism of cells, leading to a significant reduction in CHO cellular growth and specific productivity in fed-batch culture (maximum reductions of 27.2% and 40.6% respectively). In-depth pathway analysis revealed that these metabolites are produced when cells utilize major energy sources such as glucose and select amino acids (tryptophan, arginine, isoleucine, and leucine) for growth, maintenance, and protein production. Furthermore, these novel inhibitory metabolites were observed to accumulate in multiple CHO cell lines (CHO-K1 and CHO-GS) as well as HEK293 cell line. This study provides a robust and holistic methodology to incorporate global metabolomic analysis into cell culture studies for elucidation and structural verification of novel metabolites that participate in key metabolic pathways to growth, production, and post-translational modification in biopharmaceutical production.

Elucidating the impact of cottonseed hydrolysates on CHO cell culture performance through transcriptomic analysis.

In order to evaluate the impact of plant-based hydrolysates on CHO cells, a transcriptomic study was undertaken using cottonseed hydrolysate and Illumina's NextSeq transcriptomics profiling for 2 days of a batch cell culture. While cottonseed hydrolysate extended cell growth and increased antibody titer, significant effects were seen on transcriptomic signatures of supplemented cultures when compared to untreated cultures, evaluated using fold change, gene ontology (GO), and KEGG pathway analysis. Transcription and other factors commonly associated with cell growth such as those of the Atf family and homeobox proteins were upregulated while genes in the Hippo signaling pathway were downregulated. Genes involved in anabolic pathways such as gluconeogenesis and those involving protein folding and translation elongation were upregulated. GO analysis of biological processes for cottonseed-supplemented cultures indicated enrichments in DNA replication, protein processing, and unfolded protein response while molecular functions associated with growth such as GTPases, ATP binding, and aminoacyl t-RNA ligase activity were also enriched. Cellular components associated with structural integrity such as actin cytoskeleton, microtubules, mitochondrion, and Lewy body were enriched. Enriched KEGG pathways include growth-associated pathways such as cell cycle, pI3K-AKT-mTOR, and cancer-related pathways as well as those enhancing glycan metabolism, purine metabolism, amino acid biosynthesis, and protein processing in the endoplasmic reticulum (ER). These transcriptomic profiles provide insights into the roles that hydrolysates such as cottonseed can play in altering CHO cell growth and other physiological characteristics as well as suggesting ways in which CHO cell culture may be modified for enhancing performance in biotechnology applications. KEY POINTS: • Hydrolysate-supplemented cultures increased mammalian cell growth and productivity. • Fold-change analysis revealed upregulation in transcription and translation. • Enriched GOs and KEGG pathways including cell cycle and metabolism were observed.

Expanded Chinese hamster organ and cell line proteomics profiling reveals tissue-specific functionalities.

Chinese hamster ovary (CHO) cells are the predominant production vehicle for biotherapeutics. Quantitative proteomics data were obtained from two CHO cell lines (CHO-S and CHO DG44) and compared with seven Chinese hamster (Cricetulus griseus) tissues (brain, heart, kidney, liver, lung, ovary and spleen) by tandem mass tag (TMT) labeling followed by mass spectrometry, providing a comprehensive hamster tissue and cell line proteomics atlas. Of the 8470 unique proteins identified, high similarity was observed between CHO-S and CHO DG44 and included increases in proteins involved in DNA replication, cell cycle, RNA processing, and chromosome processing. Alternatively, gene ontology and pathway analysis in tissues indicated increased protein intensities related to important tissue functionalities. Proteins enriched in the brain included those involved in acidic amino acid metabolism, Golgi apparatus, and ion and phospholipid transport. The lung showed enrichment in proteins involved in BCAA catabolism, ROS metabolism, vesicle trafficking, and lipid synthesis while the ovary exhibited enrichments in extracellular matrix and adhesion proteins. The heart proteome included vasoconstriction, complement activation, and lipoprotein metabolism enrichments. These detailed comparisons of CHO cell lines and hamster tissues will enhance understanding of the relationship between proteins and tissue function and pinpoint potential pathways of biotechnological relevance for future cell engineering.

Recombinant Antibody Production in CHO and NS0 Cells: Differences and Similarities.

The commercial production of monoclonal antibodies (mAbs) has revolutionized the treatment of many diseases, including cancer, multiple sclerosis, and rheumatoid arthritis. These biotherapeutics have the potential to generate a global annual revenue of more than US$150 billion. Two cell hosts are predominantly utilized to produce these mAbs: Chinese hamster ovary (CHO) cells and murine myeloma cells (NS0). By 2017, nearly one-quarter of all approved mAbs in the market were produced using the NS0 host cell line, and around two-thirds were produced in CHO cells. Several different expression platforms are available: CHO-GS (glutamine synthetase), CHO-DHFR (dihydrofolate reductase), NS0, and GS-NS0, which have been characterized with respect to cell line and process development. Even though the major components of the cell culture media are common for both CHO and NS0 cells, specific growth media have been modified based on individual cellular requirements, such as cholesterol for NS0 cells. Additionally, understanding genomic and metabolic differences between the two cell hosts from an 'omics perspective has created a reference for media composition and antibody quality improvements.