The overall goal of this SBIR Phase I study is utilize our integrated modeling and experimental approach to computationally identify and experimentally develop a novel selection system in Chinese Hamster Ovary (CHO) cell line. The successful completion of this proof-of-concept study will demonstrate the utility of a systems biology based approach in rational design of novel selection systems in mammalian cell line protein production. Selection of high-producing mammalian cell lines represents a bottleneck in process development for the production of biopharmaceuticals. Production of therapeutic proteins in mammalian cell lines has been dominated by the use of selection markers that have metabolic origin. However, the current selection methods are hampered by a number of disadvantages, including extensive development timelines and cost. Our systems biology approach utilizes the knowledge of a whole cell metabolism and is capable to provide rational designs for identifying new selection systems. We aim to utilize our integrated computational and experimental approach to identify novel selection systems in CHO cell line and experimentally implement the most promising and advantageous candidate to validate our approach. We will perform this proof-of-concept study in three stages. In Aim I, we will identify essential metabolic reactions that are candidate targets for designing novel and superior selection systems using a computational model of CHO metabolism. We will rank-order and prioritize the candidate targets based on a number of criteria, including the predicted stringent specificity of the new selection system and improved cell physiology using our in-house computational tools. In Aim II, we will experimentally implement the top candidate selection system in the CHO-S cell line by: (a) creating an auxotrophic clone, (b) transiently transfecting cells with a selection vector that includes an antibody-expressing gene, and (c) selecting protein producing cell lines based on their auxotrophy. In Aim III, we will evaluate the development and implementation of a model-based selection system in CHO cells by comparing experimentally generated cell culture data with those calculated by the reconstructed model. Successful completion of this validation study will provide a demonstration of the scientific and technical feasibility of our integrated platform for design of new and superior metabolic selection systems in mammalian based protein production. PUBLIC HEALTH REVELANCE: Protein-based therapeutic products have contributed immensely to health care and constitute a large and growing percentage of the total pharmaceutical market. The majority of these FDA approved products are currently manufactured using mammalian cell culture systems. Mammalian cell line development for therapeutic protein production is an expensive and lengthy process. The need to engineer better performing selection systems to select stable protein producer cell lines calls for the use of a rational approach that combines modeling technologies with established experimental techniques to fundamentally change the way selection systems are developed. Reducing the cost and timelines of high producing cell lines using a superior selection system would ensure that the manufacturing of the next generation of medicines can be created in amounts large enough to meet patients' needs and at a price low enough that patients can afford them.
Public Health Relevance Statement: Project Narrative Protein-based therapeutic products have contributed immensely to health care and constitute a large and growing percentage of the total pharmaceutical market. The majority of these FDA approved products are currently manufactured using mammalian cell culture systems. Mammalian cell line development for therapeutic protein production is an expensive and lengthy process. The need to engineer better performing selection systems to select stable protein producer cell lines calls for the use of a rational approach that combines modeling technologies with established experimental techniques to fundamentally change the way selection systems are developed. Reducing the cost and timelines of high producing cell lines using a superior selection system would ensure that the manufacturing of the next generation of medicines can be created in amounts large enough to meet patients' needs and at a price low enough that patients can afford them.
Project Terms: 4-Amino-10-methylfolic Acid; 4-Amino-4-deoxy-10-methylpteroyl-L-glutamic Acid; 5,6,7,8-Tetrahydrofolate[{..}]NADP+ oxidoreductase; Alleles; Allelomorphs; Ammonia; Antibodies; Antibody Formation; Antibody Production; Antibody Response; Biologic Products; Biologic Sciences; Biological Agent; Biological Products; Biological Sciences; Biosynthetic Proteins; CHO Cells; Care, Health; Cell Culture System; Cell Culture Techniques; Cell Function; Cell Line; Cell Lines, Strains; Cell Process; Cell physiology; CellLine; Cells; Cellular Function; Cellular Physiology; Cellular Process; Characteristics; Chinese Hamster; Chinese Hamster Ovary Cell; Client; Clinical Trials, Phase I; Cloning; Computer Simulation; Computerized Models; DHFR; Data; Development; Dihydrofolate Dehydrogenase; Dihydrofolate Reductase; Disadvantaged; Drugs; EC 1.5.1.3; Early-Stage Clinical Trials; Engineering; Engineerings; Ensure; Enzyme Antagonist; Enzyme Inhibitor; Enzyme Inhibitor Agent; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Experimental Models; Experimental Models, Other; FDA approved; Folic Acid Reductase; Gene Amplification; Gene Targeting; Generalized Growth; Genes; Genital System, Female, Ovary; Genome; Glutamate Ammonia Ligase (ADP); Glutamate-Ammonia Ligase; Glutamine Synthetase; Goals; Growth; Hand functions; Healthcare; Heterogeneity, Population; Housing; Human; Human, General; Hybridomas; Intermediary Metabolism; Knowledge; L-Glutamate[{..}]ammonia ligase (ADP-forming); L-Glutamic acid, N-(4-(((2,4-diamino-6-pteridinyl)methyl)methylamino)benzoyl)-; Letters; Licensing; Life Sciences; METBL; Mammalian Cell; Mammals, Mice; Man (Taxonomy); Man, Modern; Marketing; Mathematical Model Simulation; Mathematical Models and Simulations; Measures; Medication; Medicine; Metabolic; Metabolic Processes; Metabolism; Methods; Methods and Techniques; Methods, Other; Methotrexate; Methotrexate Methylaminopterin; Methotrexatum; Metotrexato; Mice; Modeling; Models, Computer; Models, Experimental; Murine; Mus; Nucleic Acids; Ovary; PROV; Patients; Pattern; Pharmaceutic Preparations; Pharmaceutical Agent; Pharmaceutical Preparations; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Phase; Phase 1 Clinical Trials; Phase I Clinical Trials; Phase I Study; Phenotype; Physiology; Population; Population Heterogeneity; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Price; Process; Production; Productivity; Programs (PT); Programs [Publication Type]; Protein Modification; Protein Modification, Post-Translational; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteins; Provider; Public Health; Reaction; Recombinant Proteins; Recombinants; Research; SBIR; SBIRS (R43/44); Science of Medicine; Screening procedure; Series; Services; Simulate; Simulation, Computer based; Site; Small Business Innovation Research; Small Business Innovation Research Grant; Specificity; Staging; Subcellular Process; System; System, LOINC Axis 4; Systems Biology; Targetings, Gene; Techniques; Technology; Testing; Tetrahydrofolate Dehydrogenase; Therapeutic; Time; TimeLine; Tissue Growth; Transfection; Using hands; Zinc Finger Domain; Zinc Finger Motifs; Zinc Fingers; antibody biosynthesis; auxotrophy; base; biopharmaceutical; bioprocess; biotherapeutic agent; candidate selection; cell engineering; cellular engineering; computational modeling; computational models; computational simulation; computational tools; computer based models; computerized modeling; computerized simulation; computerized tools; cost; cultured cell line; deletion analysis; design; designing; diverse populations; drug/agent; feeding; flasks; gene product; glutamate synthetase; glutamine synthase; heterogeneous population; immunoglobulin biosynthesis; implantation; improved; in silico; meetings; model development; models and simulation; natural gene amplification; new therapeutics; next generation; next generation therapeutics; novel; novel therapeutics; nuclease; ontogeny; phase 1 study; phase 1 trial; phase I trial; pricing; product development; programs; protein folding; protocol, phase I; public health medicine (field); screening; screenings; simulation; success; therapeutic protein; uptake; validation studies; vector; virtual simulation
