Recombinant Spidroins Fully Replicate Primary Mechanical Properties of Natural Spider Silk

Dragline spider silk is among the strongest and toughest bio-based materials, capable of outperforming most synthetic polymers and even some metal alloys.1,2,3,4 These properties have gained spider silk a growing list of potential applications that, coupled with the impracticalities of spider farming, have driven a decades-long effort to produce recombinant spider silk proteins (spidroins) in engineered heterologous hosts.2 However, these efforts have so far been unable to yield synthetic silk fibers with mechanical properties equivalent to natural spider silk, largely due to an inability to stably produce highly repetitive, high molecular weight (MW) spidroins in heterologous hosts.1,5 Here we address these issues by combining synthetic biology techniques with split intein (SI)- mediated ligation for the bioproduction of spidroins with unprecedented MW (556 kDa), containing 192 repeat motifs of the Nephila clavipes MaSp1 dragline spidroin. Fibers spun from these synthetic spidroins display ultimate tensile strength (), modulus (E), extensibility (), and toughness (UT) of 1.03 +/- 0.11 GPa, 13.7 +/- 3.0 GPa, 18 +/- 6%, and 114 +/- 51 MJ/m3, respectively-equivalent to the performance of natural N. clavipes dragline silk.6 This work demonstrates for the first time that microbially produced synthetic silk fibers can match the performance of natural silk fibers by all common metrics (, E, , UT), providing a more dependable source of high-strength fibers to replace natural spider silks for mechanically demanding applications. Furthermore, our biosynthetic platform can be potentially expanded for the assembly and production of other protein-based materials with high MW and repetitive sequences that have so far been impossible to synthesize by genetic means alone

Elucidating dipeptide utilization and metabolism by CHO cells for improved cell culture performance

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

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Understanding mammalian cell metabolism and developing cell culture strategies for enhanced manufacturing of biotherapeutics

Mammalian cells, like Chinese hamster ovary (CHO) and human embryonic kidney (HEK293), are used as workhorses for biotherapeutics production owing to their ability to grow in large scale suspension cultures and produce high product titers with desirable product quality attributes (PQAs). However, bioprocessing faces challenges due to inefficient cell metabolism resulting in accumulation of toxic by-products, hindering cell proliferation, and causing cell death. Sub-optimal formulations of cell culture media (CCM), an expensive raw material, further reduces process yields. In this thesis, we studied the synergistic relationship between cell metabolism and CCM by employing bioanalytical tools to develop cell culture strategies for enhanced biotherapeutic production. Firstly, we identified inhibitory metabolites (IMs) secreted by mammalian cells using LC-MS/MS based metabolomics pipeline. Eight IMs were found to accumulate in cell culture supernatants at levels detrimental to cell growth and protein synthesis. Pathway mapping of IMs revealed amino acids (AAs) as chief contributors toward IMs buildup. A design of experiment (DOE) guided statistical framework was developed to modify AA levels for bioprocess enhancements. Reduced AA levels in CCM lowered accumulation of IMs. Next, due to low solubility and stability of traditional AAs, dipeptides were tested as alternative nutrients to problematic AAs like tyrosine and cysteine. Supplementation of dipeptides in CHO cultures supported biomass synthesis and protein production. 13C-Labeling experiments and kinetic modeling were performed to elucidate the utilization kinetics of dipeptides. We determined that dipeptides are cleaved both intracellularly and extracellularly and the cleavage rate depends on the structure, composition, and concentration of supplementation. Furthermore, Ala-Cys-Cys-Ala (ACCA) dipeptide dimer boosted growth and improved efficiency of glucose metabolism of CHO cells. High solubility of ACCA in basal medium simplified fed-batch processes by eliminating cysteine requirements from feed medium. Lastly, induction of cytotoxicity in HEK293 cultures during transient recombinant adeno-associated virus (rAAV) production was characterized. Analysis of rAAV-producing cells revealed caspase-mediated apoptosis as a likely mechanism of cellular death. Inhibition of caspases using small molecule, Z-VAD.fmk, alleviated cell death and increased full to empty capsids ratio, a key PQA for rAAV vectors. To sum up, cellular metabolism was investigated for CCM development to achieve superior biomanufacturing

Understanding mammalian cell metabolism and developing cell culture strategies for enhanced manufacturing of biotherapeutics

Mammalian cells, like Chinese hamster ovary (CHO) and human embryonic kidney (HEK293), are used as workhorses for biotherapeutics production owing to their ability to grow in large scale suspension cultures and produce high product titers with desirable product quality attributes (PQAs). However, bioprocessing faces challenges due to inefficient cell metabolism resulting in accumulation of toxic by-products, hindering cell proliferation, and causing cell death. Sub-optimal formulations of cell culture media (CCM), an expensive raw material, further reduces process yields. In this thesis, we studied the synergistic relationship between cell metabolism and CCM by employing bioanalytical tools to develop cell culture strategies for enhanced biotherapeutic production. Firstly, we identified inhibitory metabolites (IMs) secreted by mammalian cells using LC-MS/MS based metabolomics pipeline. Eight IMs were found to accumulate in cell culture supernatants at levels detrimental to cell growth and protein synthesis. Pathway mapping of IMs revealed amino acids (AAs) as chief contributors toward IMs buildup. A design of experiment (DOE) guided statistical framework was developed to modify AA levels for bioprocess enhancements. Reduced AA levels in CCM lowered accumulation of IMs. Next, due to low solubility and stability of traditional AAs, dipeptides were tested as alternative nutrients to problematic AAs like tyrosine and cysteine. Supplementation of dipeptides in CHO cultures supported biomass synthesis and protein production. 13C-Labeling experiments and kinetic modeling were performed to elucidate the utilization kinetics of dipeptides. We determined that dipeptides are cleaved both intracellularly and extracellularly and the cleavage rate depends on the structure, composition, and concentration of supplementation. Furthermore, Ala-Cys-Cys-Ala (ACCA) dipeptide dimer boosted growth and improved efficiency of glucose metabolism of CHO cells. High solubility of ACCA in basal medium simplified fed-batch processes by eliminating cysteine requirements from feed medium. Lastly, induction of cytotoxicity in HEK293 cultures during transient recombinant adeno-associated virus (rAAV) production was characterized. Analysis of rAAV-producing cells revealed caspase-mediated apoptosis as a likely mechanism of cellular death. Inhibition of caspases using small molecule, Z-VAD.fmk, alleviated cell death and increased full to empty capsids ratio, a key PQA for rAAV vectors. To sum up, cellular metabolism was investigated for CCM development to achieve superior biomanufacturing

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

Durable SARS-CoV-2 B cell immunity after mild or severe disease

AbstractMultiple studies have shown loss of SARS-CoV-2 specific antibodies over time after infection, raising concern that humoral immunity against the virus is not durable. If immunity wanes quickly, millions of people may be at risk for reinfection after recovery from COVID-19. However, memory B cells (MBC) could provide durable humoral immunity even if serum neutralizing antibody titers decline. We performed multi-dimensional flow cytometric analysis of S protein receptor binding domain (S-RBD)-specific MBC in cohorts of ambulatory COVID-19 patients with mild disease, and hospitalized patients with moderate to severe disease, at a median of 54 (39-104) days after onset of symptoms. We detected S-RBD-specific class-switched MBC in 13 out of 14 participants, including 4 of the 5 participants with lowest plasma levels of anti-S-RBD IgG and neutralizing antibodies. Resting MBC (rMBC) made up the largest proportion of S-RBD-specific class-switched MBC in both cohorts. FCRL5, a marker of functional memory when expressed on rMBC, was dramatically upregulated on S-RBD-specific rMBC. These data indicate that most SARS-CoV-2-infected individuals develop S-RBD-specific, class-switched MBC that phenotypically resemble germinal center-derived B cells induced by effective vaccination against other pathogens, providing evidence for durable B cell-mediated immunity against SARS-CoV-2 after recovery from mild or severe COVID-19 disease.Graphical Abstract</jats:sec