Elucidating uptake and metabolic fate of dipeptides in CHO cell cultures using 13C labeling experiments and kinetic modeling.

The rapidly growing market of biologics including monoclonal antibodies has stimulated the need to improve biomanufacturing processes including mammalian host systems such as Chinese Hamster Ovary (CHO) cells. Cell culture media formulations continue to be enhanced to enable intensified cell culture processes and optimize cell culture performance. Amino acids, major components of cell culture media, are consumed in large amounts by CHO cells. Due to their low solubility and poor stability, certain amino acids including tyrosine, leucine, and phenylalanine can pose major challenges leading to suboptimal bioprocess performance. Dipeptides have the potential to replace amino acids in culture media. However, very little is known about the cleavage, uptake, and utilization kinetics of dipeptides in CHO cell cultures. In this study, replacing amino acids, including leucine and tyrosine by their respective dipeptides including but not limited to Ala-Leu and Gly-Tyr, supported similar cell growth, antibody production, and lactate profiles. Using 13C labeling techniques and spent media studies, dipeptides were shown to undergo both intracellular and extracellular cleavage in cultures. Extracellular cleavage increased with the culture duration, indicating cleavage by host cell proteins that are likely secreted and accumulate in cell culture over time. A kinetic model was built and for the first time, integrated with 13C labeling experiments to estimate dipeptide utilization rates, in CHO cell cultures. Dipeptides with alanine at the N-terminus had a higher utilization rate than dipeptides with alanine at the C-terminus and dipeptides with glycine instead of alanine at N-terminus. Simultaneous supplementation of more than one dipeptide in culture led to reduction in individual dipeptide utilization rates indicating that dipeptides compete for the same cleavage enzymes, transporters, or both. Dipeptide utilization rates in culture and cleavage rates in cell-free experiments appeared to follow Michaelis-Menten kinetics, reaching a maximum at higher dipeptide concentrations. Dipeptide utilization behavior was found to be similar in cell-free and cell culture environments, paving the way for future testing approaches for dipeptides in cell-free environments prior to use in large-scale bioreactors. Thus, this study provides a deeper understanding of the fate of dipeptides in CHO cell cultures through an integration of cell culture, 13C labeling, and kinetic modeling approaches providing insights in how to best use dipeptides in media formulations for robust and optimal mammalian cell culture performance.

Chemical inhibitors of hexokinase-2 enzyme reduce lactate accumulation, alter glycosylation processing, and produce altered glycoforms in CHO cell cultures.

Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by-product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase-2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6-phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and N-glycosylation. Five inhibitors of HK2 enzyme at different concentrations were evaluated, of which 2-deoxy- d-glucose (2DG) and 5-thio- d-glucose (5TG) successfully reduced lactate accumulation with only limited impacts on CHO cell growth. Individual 2DG and 5TG supplementation led to a 35%-45% decrease in peak lactate, while their combined supplementation resulted in a 60% decrease in peak lactate. Inhibitor supplementation led to at least 50% decrease in moles of lactate produced per mol of glucose consumed. Recombinant EPO-Fc titers peaked earlier relative to the end of culture duration in supplemented cultures leading to at least 11% and as high as 32% increase in final EPO-Fc titers. Asparagine, pyruvate, and serine consumption rates also increased in the exponential growth phase in 2DG and 5TG treated cultures, thus, rewiring central carbon metabolism due to low glycolytic fluxes. N-glycan analysis of EPO-Fc revealed an increase in high mannose glycans from 5% in control cultures to 25% and 37% in 2DG and 5TG-supplemented cultures, respectively. Inhibitor supplementation also led to a decrease in bi-, tri-, and tetra-antennary structures and up to 50% lower EPO-Fc sialylation. Interestingly, addition of 2DG led to the incorporation of 2-deoxy-hexose (2DH) on EPO-Fc N-glycans and addition of 5TG resulted in the first-ever observed N-glycan incorporation of 5-thio-hexose (5TH). Six percent to 23% of N-glycans included 5TH moieties, most likely 5-thio-mannose and/or 5-thio-galactose and/or possibly 5-thio-N-acetylglucosamine, and 14%-33% of N-glycans included 2DH moieties, most likely 2-deoxy-mannose and/or 2-deoxy-galactose, for cultures treated with different concentrations of 5TG and 2DG, respectively. Our study is the first to evaluate the impact of these glucose analogs on CHO cell growth, protein production, cell metabolism, N-glycosylation processing, and formation of alternative glycoforms.

Cell-culture process optimization via model-based predictions of metabolism and protein glycosylation.

In order to meet the rising demand for biologics and become competitive on the developing biosimilar market, there is a need for process intensification of biomanufacturing processes. Process development of biologics has historically relied on extensive experimentation to develop and optimize biopharmaceutical manufacturing. Experimentation to optimize media formulations, feeding schedules, bioreactor operations and bioreactor scale up is expensive, labor intensive and time consuming. Mathematical modeling frameworks have the potential to enable process intensification while reducing the experimental burden. This review focuses on mathematical modeling of cellular metabolism and N-linked glycosylation as applied to upstream manufacturing of biologics. We review developments in the field of modeling cellular metabolism of mammalian cells using kinetic and stoichiometric modeling frameworks along with their applications to simulate, optimize and improve mechanistic understanding of the process. Interest in modeling N-linked glycosylation has led to the creation of various types of parametric and non-parametric models. Most published studies on mammalian cell metabolism have performed experiments in shake flasks where the pH and dissolved oxygen cannot be controlled. Efforts to understand and model the effect of bioreactor-specific parameters such as pH, dissolved oxygen, temperature, and bioreactor heterogeneity are critically reviewed. Most modeling efforts have focused on the Chinese Hamster Ovary (CHO) cells, which are most commonly used to produce monoclonal antibodies (mAbs). However, these modeling approaches can be generalized and applied to any mammalian cell-based manufacturing platform. Current and potential future applications of these models for Vero cell-based vaccine manufacturing, CAR-T cell therapies, and viral vector manufacturing are also discussed. We offer specific recommendations for improving the applicability of these models to industrially relevant processes.

The potential liver donor with tuberculosis: A fresh look at international recommendations based on a survey of practice in Indian liver transplant centres.

Background The western recommendations for the use of organs from liver donors with tuberculosis (TB) come from an environment where the burden of disease is low and cadaveric organ donation rates are high-in complete contrast to the Indian scenario, where these recommendations may be too restrictive. Methods A questionnaire relating to current practice on the use of organs from liver donors with TB was sent to all liver transplant centres in India. Results Responses were obtained from 94% of centres. Two-thirds accepted organs from deceased donors with TB in the elective setting, especially for recipients with a high MELD (Model for end-stage liver disease) score. The proportion rose by 1.5 times in the setting of acute liver failure. Two-thirds advised anti-TB treatment (ATT) for corresponding recipients, and the remaining advised isonicotinic acid hydrazide (INH) prophylaxis. Untreated living donors with TB were not accepted. Half the respondents accepted living donors after completion of ATT, and did not treat recipients postoperatively. The remainder accepted them after 8 weeks of treatment and advised INH prophylaxis or ATT for recipients. Conclusions That this practice has not impacted recipient outcomes suggests that the guidelines for management of liver donors and recipients may need to be altered for populations endemic for TB.

Hepatic arterial communicating arcades-Case series and review of literature.

This case series describes the hepatic arterial communicating arcades and their importance in the endovascular management of hepatic artery pseudoaneurysm, pediatric post liver transplant lobar arterial occlusion, and lobar arterial stenosis due to gall bladder carcinoma. We describe new types of arterial communicating arcades which have not been described earlier.

Diaphragmatic herniation following donor hepatectomy for living donor liver transplantation: a serious complication not given due recognition.

A clear appreciation of benefits and risks associated with living donor hepatectomy is important to facilitate counselling for the donor, family, and recipient in preparation for living donor liver transplant (LDLT). We report a life-threatening complication occurring in one of our live liver donors at 12 weeks following hemi-liver donation. We experienced five donor complications among our first 50 LDLT: Clavien Grade 1, n=1; Clavien grade 2, n=3; and Clavien grade 3B, n=1. The one with Clavien grade 3B had a life-threatening diaphragmatic hernia occurring 12 weeks following hepatectomy. This was promptly recognized and emergency surgery was performed. The donor is well at 1-year follow-up. Here we provide a review of reported instances of diaphragmatic hernia following donor hepatectomy with an attempt to elucidate the pathophysiology behind such occurrence. Life-threatening donor risk needs to be balanced with recipient benefit and risk on a tripartite basis during the counselling process for LDLT. With increasing use of LDLT, we need to be aware of such life-threatening complication. Preventive measures in this regard and counselling for such complication should be incorporated into routine work-up for potential live liver donor.

Utilization of microbial oil obtained from crude glycerol for the production of polyol and its subsequent conversion to polyurethane foams.

We have demonstrated possible use of microbial oil in biopolymer industries. Microbial oil was produced from biodiesel based crude glycerol and subsequently converted into polyol. Fermentation of crude glycerol in a batch bioreactor using Rhodosporidium toruloides ATCC 10788 produced 18.69g/L of lipid at the end of 7days. The microbial oil was then chemically converted to polyol and characterized using FT-IR and 1H NMR. For comparison, canola oil and palm oil were also converted into their respective polyols. The hydroxyl numbers of polyols from canola, palm and microbial oil were found to be 266.86, 222.32 and 230.30 (mgKOH/g of sample) respectively. All the polyols were further converted into rigid and semi-rigid polyurethanes (maintaining the molar -NCO/-OH ratio of 1.1) to examine their suitability in polymer applications. Conversion of microbial lipid to polyurethane foam also provides a new route for the production of polymers using biodiesel based crude glycerol.

Esophageal exclusion and bypass for corrosive injury: The lessons learnt.

While it is always preferable to excise and replace the diseased esophagus in corrosive injuries, the surgeon may be compelled to exclude and bypass it by a substernal conduit in select situations wherein excision is technically hazardous. This case illustrates the lessons learnt from a potentially life-threatening complication of bipolar esophageal exclusion.