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.

Kinetics of Trisulfide-to-Disulfide Conversion of Therapeutic IgG1 Monoclonal Antibodies Under Physiological Conditions: A Case Study of Casirivimab And Imdevimab.

The percentage of trisulfide variants is a product quality metric that is monitored during the manufacture of monoclonal antibody (mAb)-based therapeutics. Results from earlier preclinical studies revealed that trisulfide linkages in mAbs are rapidly converted to disulfides in circulation. In this study, casirivimab and imdevimab, which are both IgG1 subclass mAbs that target the non-overlapping epitopes in SARS-CoV2 Spike protein, are used as models to study the kinetics of trisulfide-to-disulfide conversion in vivo in human circulation. To determine the percentage of trisulfide variants in systemic circulation immediately after intravenous injection, both mAbs were immunoprecipitated from serum samples collected from COVID-19 patients that received this cocktail antibody treatment as part of a first-in-human study. The immunoprecipitated mAbs were then digested under non-reducing conditions and evaluated by liquid-chromatography-mass spectrometry (LC-MS). Significant reductions in the percentages of trisulfide variants were observed in serum samples as early as 1 hr after completion of the intravenous infusion. A flow-through dialysis model designed to mimic the redox potential of blood revealed a plausible chemical mechanism for the rapid trisulfide-to-disulfide conversion of IgG1 subclass mAbs under physiological conditions.

Feasibility of a Remote-based Nutrition Education and Culinary Skills Program for Young Adults With Autism Spectrum Disorder.

OBJECTIVE: To evaluate the feasibility of a remote-based nutrition education and culinary skills program for young adults with autism spectrum disorder (ASD). METHODS: Thirteen young adults with ASD (26.6 ± 4.4 years; 77% male) participated in a remote-based nutrition program twice a week over 12 weeks. Principles of Social Cognitive Theory were used to develop the curriculum, which focused on nutrition education and meal preparation. Feasibility was assessed through attendance records, retention rates, duration/frequency of participant engagement, session notes, and participant interviews. RESULTS: The program had a 13% refusal rate and a 7% attrition rate. Participants had a session attendance rate of 83%. Session notes revealed that the remote format offered several methods of participant communication and captured evidence of family support. Interview themes included increased nutrition-related knowledge, self-efficacy, and behavioral capability. CONCLUSIONS AND IMPLICATIONS: The remote-based nutrition program met feasibility benchmarks for adherence and retention and was well-accepted by participants. A remote-based format for nutrition interventions can reach a wider range of young adults with ASD; however, input from the target population should be considered in the development of the program.

Speed and need: twin development challenges in rapid response for a SARS-CoV-2 antibody cocktail.

Neutralizing antibodies are one available tool for the treatment of infectious diseases. Speed in developing monoclonal antibody treatments is an understood requirement for emerging infectious diseases, and need for COVID-19 treatments during the worldwide pandemic has provided additional urgency. Process development (at Regeneron) and technology transfer (within Regeneron and to Genentech) of casirivimab and imdevimab (REGEN-COV™ or Ronapreve™) manufacturing processes have addressed speed and need with selected purification and cell culture examples provided, respectively, for these two development challenges. This was achieved through three key pillars: (1) Regeneron's proprietary Velocisuite® technologies, (2) deep monoclonal antibody process and manufacturing knowledge at both companies, and (3) Regeneron's and Genentech's commitment to deliver therapeutics to patients in need. Combined with business processes and risk management, these pillars rapidly allowed casirivimab and imdevimab to move to clinical manufacturing and to production at Genentech in a first-time process transfer under compressed timelines between the companies.

Implementation of a school-based Fitbit program for youth with Autism Spectrum Disorder: A feasibility study.

BACKGROUND: School settings may be optimal for physical activity interventions for youth with Autism Spectrum Disorder (ASD). Additionally, consumer-based fitness trackers may encourage youth with ASD to increase their physical activity levels, however, no studies have examined whether a fitness tracker program would be feasible in youth with ASD. OBJECTIVE: To examine the feasibility of a 12-week school-based Fitbit© program for youth with ASD. METHODS: Six classroom teachers and their students (n = 45) were provided with Fitbit fitness trackers to wear over 12-weeks. Classroom teachers monitored student tracker use and completed open-ended surveys to describe both their experience and their students' experience with the fitness trackers. RESULTS: Out of the 45 eligible students, 42 (94%) opted to participate in the study. All six teachers and 32 (76%) of the 42 students wore the fitness tracker daily over 12 weeks. Teachers reported that students were most interested in tracking their daily steps, and the short batter life, and account set-up were the biggest challenges to students. All six teachers felt that this program could have long-term sustainability, especially if tracker use could be incorporated into school curriculum and classroom activities. CONCLUSIONS: A school-based Fitbit program appears to be both feasible, and well-accepted by students with ASD. Future work should evaluate the preliminary efficacy of this type of program.

Brief report: The impact of the COVID-19 pandemic on health behaviors in adolescents with Autism Spectrum Disorder.

BACKGROUND: There is concern that the COVID-19 pandemic may negatively affect health behaviors in youth, especially youth diagnosed with Autism Spectrum Disorder (ASD). OBJECTIVE: The purpose of this paper was to examine changes in physical activity, screen-time, and sleep in adolescents with ASD due to the COVID-19 pandemic. METHODS: Nine adolescents with ASD completed surveys measuring physical activity, screen-time, and sleep duration prior to and during the pandemic. RESULTS: A significant decrease in days of physical activity (4.17 vs 2.27; p = 0.0006), and a significant increase in hours of both weekday (3.69 vs 6.25; p = 0.007) and weekend screen-time (5.94 vs. 7.39; p = 0.004) was observed during the pandemic. No changes regarding sleep duration was observed. CONCLUSIONS: Although preliminary, results suggest that physical activity and screen-time may be negatively affected by the COVID-19 outbreak in youth with ASD. The development of interventions to promote health behaviors in ASD populations during long periods of less-structured time (quarantine) should be considered.

Transition of a Judo Program from In-Person to Remote Delivery During COVID-19 for Youth with Autism Spectrum Disorder.

OBJECTIVES: To examine the feasibility of a remote judo program for high school students with autism spectrum disorder (ASD) during the COVID-19 pandemic. METHODS: Nine high school students with ASD had been participating in an in-person judo program that transitioned to a livestream format once the stay-at-home orders were mandated. The students completed surveys regarding their experience with the remote sessions. Their classroom teacher completed a semi-structured interview regarding the advantages and disadvantages of the remote format. RESULTS: Eight out of the nine students attended 92% of the remote classes. All nine students reported feeling satisfied with the remote judo sessions. Reported benefits of the remote format included the structured routine during the pandemic and the opportunity for physical activity. Disadvantages included a lack of space and lack of one-on-one instruction. CONCLUSIONS: The remote judo program appeared to be both feasible and acceptable for youth with ASD.

Protective strategies and alcohol use among college students: ethnic and gender differences.

This study investigated differences in alcohol consumption and the use of protective strategies (i.e., eating and designated drivers) between European American, African American, and Hispanic American college students. Gender differences were also examined. The study sample was drawn from a large southeastern university (n = 567). Data analysis employed regression, factor analysis, and analysis of variance. Results indicate that European Americans students reported a higher incidence of "drunk" episodes per week than other racial/ethnic groups and greater use of specific pre-drinking behaviors such as protective strategies than their non-European American peers. No statistically significant differences were found between the drinking patterns between genders.

A general evidence-based sequence variant control limit for recombinant therapeutic protein development.

Sequence variants (SVs) resulting from unintended amino acid substitutions in recombinant therapeutic proteins have increasingly gained attention from both regulatory agencies and the biopharmaceutical industry given their potential impact on efficacy and safety. With well-optimized production systems, such sequence variants usually exist at very low levels in the final protein products due to the high fidelity of DNA replication and protein biosynthesis process in mammalian expression systems such as Chinese hamster ovary cell lines. However, their levels can be significantly elevated in cases where the selected production cell line has unexpected DNA mutations or the manufacturing process is not fully optimized, for example, if depletion of certain amino acids occurs in the cell culture media in bioreactors. Therefore, it is important to design and implement an effective monitoring and control strategy to prevent or minimize the possible risks of SVs during the early stage of product and process development. However, there is no well-established guidance from the regulatory agencies or consensus across the industry to assess and manage SV risks. A question frequently asked is: What levels of SVs can be considered acceptable during product and process development, but also have no negative effects on drug safety and efficacy in patients? To address this critical question, we have taken a holistic approach and conducted a comprehensive sequence variant analysis. To guide biologic development, a general SV control limit of 0.1% at individual amino acid sites was proposed and properly justified based on extensive literature review, SV benchmark survey of approved therapeutic proteins, and accumulated experience on SV control practice at Regeneron.

Osteoporosis: prevention and implications for social work practice and policy.

Osteoporosis is one of the major disorders of our time and is increasing at an alarming rate. It affects over 10 million women in the United States and is expected to affect 14 million by the year 2020. There are a number of risk factors for osteoporosis that are of a modifiable nature; however, many women do not take the necessary precautions to prevent the disease. The reasons that women do not actively engage in preventative and remedial strategies to deal with the potential for osteoporosis are complex. It is clear that women must have adequate knowledge to make informed decisions regarding osteoprotective behaviors. Social workers are in a unique position to provide interventions not only on a micro level (educating individual clients) but also on a macro level in terms of large-scale education campaigns focusing on nutrition and exercise both in children and in adults. This article examines the reasons for the failure of women to engage in osteoprotective behaviors by exploring barriers to prevention and their impact on social work policy, practice, and research.

Understanding and acting on the growing childhood and adolescent weight crisis: a role for social work.

The childhood and adolescent overweight and obesity rates are rising at an alarming rate. Numerous individual, family, community, and social factors contribute to overweight and obesity in children and are explored. If left unaddressed, the epidemic of childhood and adolescent overweight and obesity may lead to amplified problems for individual children--including acute and chronic physical and psychological complications--and for the larger social environment. National efforts by researchers in a myriad of disciplines are underway to address this the issue at the individual, family, and community levels. These efforts include many steps with which social workers should seek to align themselves in terms of their own research and collaborative research and several barriers that hold practice implications for social workers. The importance of social work intervention and collaboration within primary, secondary, and tertiary prevention levels is explored.

Behavioral health: Treatment and prevention of chronic disease and the implications for social work practice.

The public health problems in the new millennium are largely related to lifestyle. The illness industry has seen a large growth in the United States with health care expenditure accounting for 14% of the gross national product. The field of behavioral medicine seeks to include individual responsibility in the prevention of chronic disease. There are great possibilities for lifestyle change through behavioral interventions. This manuscript outlines various applications of behavioral techniques and interventions utilized for smoking and obesity. Prevention paradigms and implications for social workers are also outlined.

Engineering sialic acid synthetic ability into insect cells: identifying metabolic bottlenecks and devising strategies to overcome them.

Previous studies have indicated negligible levels of both sialylation and the precursor N-acetylneuraminic acid (Neu5Ac) in a number of insect cell lines grown in serum-free medium. The overexpression of the human sialic acid 9-phosphate synthase (SAS) in combination with N-acetylmannosamine (ManNAc) feeding has been shown to overcome this limitation. In this study we evaluated the potential bottlenecks in the sialic acid synthesis pathway in a Spodoptera frugiperda (Sf9) insect cell line and devised strategies to overcome them by overexpression of the enzymatic pathway enzymes combined with appropriate substrate feeding. Coexpression of SAS and UDP-GlcNAc 2-epimerase/ManNAc kinase, the bifunctional enzyme initiating sialic acid biosynthesis in mammals, resulted in Neu5Ac synthesis without use of any external media supplementation to demonstrate that Neu5Ac could be generated intracellularly in Sf9 cells using natural metabolic precursors. N-Acetylglucosamine (GlcNAc) feeding in combination with this coexpression resulted in much higher levels of Neu5Ac compared to levels obtained with ManNAc feeding with SAS expression alone. The lower Neu5Ac levels obtained with ManNAc feeding suggested limitations in the transport and phosphorylation of ManNAc. The bottleneck in phosphorylation was likely due to utilization of GlcNAc kinase for phosphorylation of ManNAc in insect cells and was overcome by expression of ManNAc kinase. The transport limitation was addressed by the addition of tetra-O-acetylated ManNAc, which is easily taken up by the cells. An alternative sialic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), could also be generated in insect cells, suggesting the potential for controlling not only the production of sialic acids but also the type of sialic acid generated. The levels of KDN could be increased with virtually no Neu5Ac generation when Sf9 cells were fed excess GlcNAc. The results of these studies may be used to enhance the sialylation of target glycoproteins in insect and other eukaryotic expression systems.

Expression of a functional Drosophila melanogaster N-acetylneuraminic acid (Neu5Ac) phosphate synthase gene: evidence for endogenous sialic acid biosynthetic ability in insects.

In this study, we report the first cloning and characterization of a N-acetylneuraminic acid phosphate synthase gene from Drosophila melanogaster, an insect in the protostome lineage. The gene is ubiquitously expressed at all stages of Drosophila development and in Schneider cells. Similar to the human homologue, the gene encodes an enzyme with dual substrate specificity that can use either N-acetylmannosamine 6-phosphate or mannose 6-phosphate to generate phosphorylated forms of both the sialic acids, N-acetylneuraminic acid and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, respectively, when expressed in either bacterial or baculoviral expression systems. The identification of a functional sialic acid synthase in Drosophila indicates that insects have the biosynthetic capability to produce sialic acids endogenously. Although sialylation is widely distributed in organisms of the deuterstome lineage, genetic evidence concerning the presence or absence of sialic acid metabolism in organisms of the protostome lineage has been lacking. Homology searches of the Drosophila genome identified putative orthologues of other genes required for sialylation of glycoconjugates.