CELL ENGINEERING AND CULTURE MEDIA MODIFICATION STUDIES TO IMPROVE THE CELL CULTURE PERFORMANCE

Mammalian cell cultures have become a universal path for producing therapeutic proteins, monoclonal antibodies in particular, in biopharmaceutical industries. There is a constant attempt to improve the productivity of the cell cultures through research in the industry and educational institutions. The productivity of the cells can be improved through various techniques. The use of additives is the most economical option to pursue for improving the productivity of the cells. The additives, hydrolysates, were used in the cell culture at a final concentration of 0.4% and we have seen a two to threefold increase in the productivity of the cells. While soy hydrolysate doubled the productivity along with the cell growth, cotton hydrolysates have increased it three times when compared to the control flasks not supplemented with any hydrolysates. Five different lots of cotton hydrolysates were tested simultaneously as media supplements for a lot variability study. The cotton lots 100NTCR and 100PCHO performed the best among the five lots tested in terms of both cell growth and IgG productivity. Both these lots were selected to perform proteomics analysis on the hydrolysate samples to understand the mode of action of these hydrolysates when added to the cell cultures. Essential amino acids’ biosynthetic pathways were to be engineered into mammalian cells. In particular, the research described in the thesis targeted branched chain amino acids- Leucine, Isoleucine and Valine. All the genes were cloned into the mammalian expression vector pBUD4.1 and later transfected into CHO-K1 and HEK 293T cell lines and their expression was tested using a western blot. Five out of the nine genes to be expressed, have worked. This opens up new avenues to explore the machinery in the mammalian cell and lead to the synthesis of a minimal cell

Amino acid analysis to support a genome-scale nutrient minimization forecast algorithm for controlling essential amion acid levels in CHO cell cultures

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Design of experiments guided control of waste inhibitory by-products in high density CHO cell cultures

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Tracking amino acid metabolism in CHO cell cultures using stable isotope labeling assisted metabolomics

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Elucidating amino acid metabolism in CHO cells

CHO cells require complex media for cell growth and protein production. The major components of industrial media are amino acids, however, relatively little is known about the metabolism of amino acids in CHO cell cultures. Here, we applied advanced 13C-flux analysis tools to elucidate the metabolic flow of the amino acids in a fed-batch CHO culture that overproduced IgG. Carbon flows were tracked throughout the growth phase and changes in metabolism were quantified when cells transitioned from growth phase to stationary phase. In addition, we quantified how changes in amino acids profiles in the medium translated to changes in cell growth, protein production and product quality attributes. To trace each amino acid individually, custom media formulations were used, where each medium formulation was depleted of a specific amino acid. A labeled 13C variant of the depleted amino acid was then added to the medium at the desired concentration. CHO cells were then grown in fed-batch culture. As the cells metabolized the labeled amino acids, this resulted in a redistribution of 13C-atoms which we quantified using GC-MS for both extracellular metabolites (including lactate, amino acids and the IgG product) and intracellular metabolites (including free intracellular metabolites, cell proteins, lipids and carbohydrates). We then estimated metabolic fluxes using state-of-the-art 13C-metabolic flux analysis. This allowed us to calculate the fraction of each amino acid that was used for cell growth, protein production, lactate formation and energy generation. We also investigated the effects of labeling in both the batch and fed-batch stationary phase. Finally, we investigated the effects of varying amino acid concentrations. Each 13C-labeled amino acid was added to the medium at a lower or higher concentration compared to the base medium. 13C-metabolic flux analysis was again performed and changes in fluxes were compared in order to determine the precise impacts of amino acid concentration changes on the flux profiles. Taking all of this data together, we are now building a predictive kinetic model that relates how the metabolism of CHO cells can be predicted from amino acid profiles. In future work, model predictions will be experimentally validated as a means of optimizing the amino acid composition of industrial culture media

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

ELUCIDATING MAMMALIAN CELL METABOLISM USING ‘OMICS TO ENHANCE BIOPROCESS CAPABILITIES

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry towards production of biotherapeutics of great value to the mankind. These cells possess a unique feature of providing a human- compatible post-translational modification pattern which makes them the most suitable mammalian hosts for the industry. Understanding the metabolism of various simple and complex molecules present in their culture environment is essential in order to envision ways to improve the productivity of these cells. It is possible to decipher this metabolism through various ‘omics techniques such as metabolomics, proteomics and transcriptomics. In order to understand how CHO cells, utilize simple molecules such as amino acids, 13C-tracer metabolomics was enforced which is a technique helping researchers look using a magnifying glass in and around the cells for molecules of interest and their derivatives for a long time now. In the current study, we replaced each amino acid with its 13C enriched form to elucidate the path it takes in CHO cell cultures and subsequently utilized this knowledge towards various applications such as deducing an optimized amino acid nutrient composition for these cells to not just conserve these biomolecules, but also to limit the release of growth and productivity inhibiting molecules in high cell density cultures. Other techniques to understand cellular metabolism such as global metabolomics using LC-MS were employed to identify culture performance inhibitors. Successfully limiting their production in the cell culture using media design strategies such as design of experiments enabled expand the production window of these cell lines. Complex medium additives such as plant hydrolysates are often used to enhance culture performance although their mechanism of action in enhancing cell performance is not well understood. In this study, cottonseed hydrolysates were added to CHO batch cell cultures, enhancing cell growth, IgG titers, and productivities. Extracellular metabolomics coupled with TMT proteomics revealed several metabolic phases in the entire culture duration and clearly highlighted differences in key central metabolism players potentially driving the performance changes in the presence of cottonseed hydrolysate. As a raw material for CHO cells, cottonseed hydrolysate added alternate substrates such as galactose and peptides in addition to the predominant energy sources like glucose and free amino acids that are in the basal medium. As a result, these cultures demonstrated a shift from production to consumption and vice-versa in the metabolism of various glycolysis and TCA (Tricarboxylic Acid) cycle precursors/by-products such as serine, glycine, lactate, pyruvate, glutamate and aspartate suggesting reincorporation to combat nutrient deprivation. Quantitative proteomics revealed 5521 proteins and numerous differentially abundant proteins related to growth, metabolism, oxidative stress, protein productivity, and apoptosis/cell death at day 5 and day 6 in hydrolysate cultures. Differential abundance of amino acid transporter proteins and catabolism enzymes such as BCAT1 (Branched Chain Amino Acid Transaminase 1) and FAH (Fumarylacetoacetate Hydrolase) can alter availability and utilization of several amino acids. Also, pathways involved in growth including the polyamine biosynthesis through higher ODC1 (Ornithine decarboxylase 1) abundance and hippo signaling were upregulated and downregulated, respectively. Central metabolism rewiring was indicated by GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) downregulation, which corresponded with re-uptake of secreted lactate in the cottonseed-supplemented cultures. Overall, cottonseed hydrolysate supplementation modifies culture performance by altering cellular activities critical to growth and protein productivity including metabolism, transport, mitosis, transcription, translation, protein processing, and apoptosis. And finally, it is imperative to design more efficient CHO cell processes for the biopharmaceutical industry, in possession of the abovementioned wide range of information on cellular metabolism of CHO cells through ‘omics techniques. Graduate Advisor: Dr. Michael Betenbaugh Dissertation Readers: Dr. Marc Donohue, Dr. Alan Ston

CELL ENGINEERING AND CULTURE MEDIA MODIFICATION STUDIES TO IMPROVE THE CELL CULTURE PERFORMANCE

Mammalian cell cultures have become a universal path for producing therapeutic proteins, monoclonal antibodies in particular, in biopharmaceutical industries. There is a constant attempt to improve the productivity of the cell cultures through research in the industry and educational institutions. The productivity of the cells can be improved through various techniques. The use of additives is the most economical option to pursue for improving the productivity of the cells. The additives, hydrolysates, were used in the cell culture at a final concentration of 0.4% and we have seen a two to threefold increase in the productivity of the cells. While soy hydrolysate doubled the productivity along with the cell growth, cotton hydrolysates have increased it three times when compared to the control flasks not supplemented with any hydrolysates. Five different lots of cotton hydrolysates were tested simultaneously as media supplements for a lot variability study. The cotton lots 100NTCR and 100PCHO performed the best among the five lots tested in terms of both cell growth and IgG productivity. Both these lots were selected to perform proteomics analysis on the hydrolysate samples to understand the mode of action of these hydrolysates when added to the cell cultures. Essential amino acids’ biosynthetic pathways were to be engineered into mammalian cells. In particular, the research described in the thesis targeted branched chain amino acids- Leucine, Isoleucine and Valine. All the genes were cloned into the mammalian expression vector pBUD4.1 and later transfected into CHO-K1 and HEK 293T cell lines and their expression was tested using a western blot. Five out of the nine genes to be expressed, have worked. This opens up new avenues to explore the machinery in the mammalian cell and lead to the synthesis of a minimal cell

ELUCIDATING MAMMALIAN CELL METABOLISM USING ‘OMICS TO ENHANCE BIOPROCESS CAPABILITIES

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry towards production of biotherapeutics of great value to the mankind. These cells possess a unique feature of providing a human- compatible post-translational modification pattern which makes them the most suitable mammalian hosts for the industry. Understanding the metabolism of various simple and complex molecules present in their culture environment is essential in order to envision ways to improve the productivity of these cells. It is possible to decipher this metabolism through various ‘omics techniques such as metabolomics, proteomics and transcriptomics. In order to understand how CHO cells, utilize simple molecules such as amino acids, 13C-tracer metabolomics was enforced which is a technique helping researchers look using a magnifying glass in and around the cells for molecules of interest and their derivatives for a long time now. In the current study, we replaced each amino acid with its 13C enriched form to elucidate the path it takes in CHO cell cultures and subsequently utilized this knowledge towards various applications such as deducing an optimized amino acid nutrient composition for these cells to not just conserve these biomolecules, but also to limit the release of growth and productivity inhibiting molecules in high cell density cultures. Other techniques to understand cellular metabolism such as global metabolomics using LC-MS were employed to identify culture performance inhibitors. Successfully limiting their production in the cell culture using media design strategies such as design of experiments enabled expand the production window of these cell lines. Complex medium additives such as plant hydrolysates are often used to enhance culture performance although their mechanism of action in enhancing cell performance is not well understood. In this study, cottonseed hydrolysates were added to CHO batch cell cultures, enhancing cell growth, IgG titers, and productivities. Extracellular metabolomics coupled with TMT proteomics revealed several metabolic phases in the entire culture duration and clearly highlighted differences in key central metabolism players potentially driving the performance changes in the presence of cottonseed hydrolysate. As a raw material for CHO cells, cottonseed hydrolysate added alternate substrates such as galactose and peptides in addition to the predominant energy sources like glucose and free amino acids that are in the basal medium. As a result, these cultures demonstrated a shift from production to consumption and vice-versa in the metabolism of various glycolysis and TCA (Tricarboxylic Acid) cycle precursors/by-products such as serine, glycine, lactate, pyruvate, glutamate and aspartate suggesting reincorporation to combat nutrient deprivation. Quantitative proteomics revealed 5521 proteins and numerous differentially abundant proteins related to growth, metabolism, oxidative stress, protein productivity, and apoptosis/cell death at day 5 and day 6 in hydrolysate cultures. Differential abundance of amino acid transporter proteins and catabolism enzymes such as BCAT1 (Branched Chain Amino Acid Transaminase 1) and FAH (Fumarylacetoacetate Hydrolase) can alter availability and utilization of several amino acids. Also, pathways involved in growth including the polyamine biosynthesis through higher ODC1 (Ornithine decarboxylase 1) abundance and hippo signaling were upregulated and downregulated, respectively. Central metabolism rewiring was indicated by GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) downregulation, which corresponded with re-uptake of secreted lactate in the cottonseed-supplemented cultures. Overall, cottonseed hydrolysate supplementation modifies culture performance by altering cellular activities critical to growth and protein productivity including metabolism, transport, mitosis, transcription, translation, protein processing, and apoptosis. And finally, it is imperative to design more efficient CHO cell processes for the biopharmaceutical industry, in possession of the abovementioned wide range of information on cellular metabolism of CHO cells through ‘omics techniques. Graduate Advisor: Dr. Michael Betenbaugh Dissertation Readers: Dr. Marc Donohue, Dr. Alan Ston

Towards a universal CHO reference platform with epigenome characterization for the biotechnology community

Chinese Hamster ovary (CHO) cells are widely used both by academic researchers and in the biotechnology industry. However, comparing studies across a wide spectrum of labs in the CHO community has been challenging due to the different variants of host cell line, culture media and proteins of interest used in the individual laboratories. Unfortunately, unlike other communities, there is no standard CHO platform that can be used as a baseline for experimentation and evaluation, leading to a limited understanding of how a result or innovation from one group may be applied to another group. This limits the pace at which innovations in cell line development are achieved by the community. As a result, there is a growing need to create and establish a common platform with the goal of comparability and compatibility across the CHO bioprocessing community. The Advanced Mammalian Biomanufacturing Innovation Center (AMBIC) is a US based academic-industrial-government collaborative initiative dedicated to developing improved upstream biomanufacturing methods. Together, AMBIC’s five academic and sixteen industrial members are working together to implement a newly developed CHO based reference platform that has performance characteristics similar to what is used in the industry. Our initial goals have been to identify reference production and host cell lines together with a common platform medium used in the production of model recombinant protein targets, including antibodies and other targets. These cell lines are also being used to develop standardized processes that can be comparable across AMBIC sites and within the CHO community. In concert, these reference platforms are being applied to evaluate and understand CHO cell line capabilities and processing parameters for improving the production platform. Our progress in establishing a reference cell host and the partner media and processing parameters will be described, and the role of such a reference standard in helping to define the scope of other AMBIC research endeavors will also be delineated. We believe such a reference CHO platform will facilitate a robust and dynamic CHO research and development environment and hasten progress in cell culture engineering in coming decades