Small-molecule control of antibody N-glycosylation in engineered mammalian cells.

N-linked glycosylation in monoclonal antibodies (mAbs) is crucial for structural and functional properties of mAb therapeutics, including stability, pharmacokinetics, safety and clinical efficacy. The biopharmaceutical industry currently lacks tools to precisely control N-glycosylation levels during mAb production. In this study, we engineered Chinese hamster ovary cells with synthetic genetic circuits to tune N-glycosylation of a stably expressed IgG. We knocked out two key glycosyltransferase genes, α-1,6-fucosyltransferase (FUT8) and ß-1,4-galactosyltransferase (ß4GALT1), genomically integrated circuits expressing synthetic glycosyltransferase genes under constitutive or inducible promoters and generated antibodies with concurrently desired fucosylation (0-97%) and galactosylation (0-87%) levels. Simultaneous and independent control of FUT8 and ß4GALT1 expression was achieved using orthogonal small molecule inducers. Effector function studies confirmed that glycosylation profile changes affected antibody binding to a cell surface receptor. Precise and rational modification of N-glycosylation will allow new recombinant protein therapeutics with tailored in vitro and in vivo effects for various biotechnological and biomedical applications.

Dissecting N-Glycosylation Dynamics in Chinese Hamster Ovary Cells Fed-batch Cultures using Time Course Omics Analyses.

N-linked glycosylation affects the potency, safety, immunogenicity, and pharmacokinetic clearance of several therapeutic proteins including monoclonal antibodies. A robust control strategy is needed to dial in appropriate glycosylation profile during the course of cell culture processes accurately. However, N-glycosylation dynamics remains insufficiently understood owing to the lack of integrative analyses of factors that influence the dynamics, including sugar nucleotide donors, glycosyltransferases, and glycosidases. Here, an integrative approach involving multi-dimensional omics analyses was employed to dissect the temporal dynamics of glycoforms produced during fed-batch cultures of CHO cells. Several pathways including glycolysis, tricarboxylic citric acid cycle, and nucleotide biosynthesis exhibited temporal dynamics over the cell culture period. The steps involving galactose and sialic acid addition were determined as temporal bottlenecks. Our results show that galactose, and not manganese, is able to mitigate the temporal bottleneck, despite both being known effectors of galactosylation. Furthermore, sialylation is limited by the galactosylated precursors and autoregulation of cytidine monophosphate-sialic acid biosynthesis.

Transient CHO expression platform for robust antibody production and its enhanced N-glycan sialylation on therapeutic glycoproteins.

Large-scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO-S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO-S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO-S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non-Fc N-linked glycans were expressed in transient ExpiCHO-S™, the glycan pattern was unexpectedly found to have few sialylated N-glycans, in contrast to glycans produced within a stable CHO expression system. To improve N-glycan sialylation in transient ExpiCHO-S™, we co-transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co-transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N-glycans during transient ExpiCHO-S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N-glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019.

Reactivity-driven cleanup of 2-Aminobenzamide derivatized oligosaccharides.

N-glycan profiling is commonly accomplished by the derivatization of the enzymatically released oligosaccharides with a fluorophore, thereby facilitating their analysis by hydrophilic-interaction liquid chromatography (HILIC). These fluorescent dyes are often present in large excess during derivatization reactions, and their removal is typically required to minimize chromatographic interference. Herein, we report a reactivity-driven 2-phase extraction protocol with the aldehyde reagent octanal, which demonstrated efficient 2-aminobenzamide cleanup as well as high derivatized N-glycan recovery. This cleanup method can be performed within minutes, and therefore provides an alternative sample preparation route for N-glycan profiling with improved time efficiency and operational simplicity.

Anesthesia report card - a customizable tool for performance improvement.

PURPOSE: Measuring and providing performance feedback to physicians has gained momentum not only as a way to comply with regulatory requirements, but also as a way to improve patient care. Measurement of structural, process, and outcome metrics in a reliable, evidence-based, specialty-specific manner maximizes the probability of improving physician performance. The manner in which feedback is provided influences whether the measurement tool will be successful in changing behavior. We created an innovative reporting tool template for anesthesiology practitioners designed to provide detailed, continuous feedback covering many aspects of clinical practice. METHODS: The literature regarding quality metric measurement and feedback strategies was examined to design a reporting tool that could provide high quality information and result in improved performance of clinical and academic tasks. A committee of department leaders and information technology professionals was tasked with determining the measurement criteria and infrastructure needed to generate these reports. Data was collected in a systematic, unbiased manner, and reports were populated with information from multiple databases and software systems. Feedback would be based on frequently updated information and allow for analysis of historical performance as well as comparison amongst peers. RESULTS: A template for an anesthesia report card was created. Categories included compliance, credentialing and qualifications, education, clinical and operating room responsibilities, and academic achievements. Physicians were able to choose to be evaluated in some of the categories and had to meet a minimum number of criteria within each category. This allowed for customization to each practitioner's practice. Criteria were derived from the measures of academic and clinical proficiency, as well as quality metrics. Criteria were objective measures and data gathering was often automated. Reports could be generated that were updated daily and provided historical information, and information about peers in the department and within each subspecialty group. CONCLUSIONS: We demonstrate the creation of an online anesthesia report card that incorporates metrics most likely to engender positive changes in practice and academic responsibilities. This tool provides timely and customized information for each anesthesia practitioner, designed to be easily modifiable to improve the quantity, quality, and substance of metrics being measured. Finally, our tool could serve as a template for a performance measuring tool that can be customizable to a wide variety of practice settings, and upon which both monetary and non-monetary incentives might be based in the future.