Development of integrated continuous bioprocessing using Continuous Countercurrent Tangential Chromatography (CCTC) platform for capture and polishing of monoclonal antibodies

Several recent studies have demonstrated the potential of using continuous countercurrent tangential chromatography (CCTC) for the purification of monoclonal antibody products. CCTC operates with a flowing resin slurry, exploiting traditional approaches of countercurrent staging to achieve \u3e 10-fold increases in productivity compared to traditional batch columns in a truly steady-state low pressure (\u3c15 psi) unit operation that can be directly integrated into a fully continuous biomanufacturing process. The featured data shows productivity and product quality outcomes from Protein A capture, as well as Anion exchange and Cation exchange polishing steps for purification of a commercial mAb. Another unique aspect of CCTC is the ability to use small particle size resins with improved binding kinetics and better system productivity but without any increase in pressure. The data also features our strategy for integrating the unit operations into a single process train incorporating an integrated in-line sampling strategy. In addition we discuss the potential of this platform to enable efficient processing of sensitive biologics because of significantly reduced residence time (\u3c10 min from binding to elution), as well as rapid in-line buffer adjustments of eluted product

Continuous countercurrent tangential chromatography for purification of high value therapeutic proteins

One of the main challenges in the development of integrated continuous processes for the production of high value therapeutic proteins is the replacement of traditional column chromatography, which tends to dominate current downstream processing. Continuous Countercurrent Tangential Chromatography (CCTC) has been designed to provide truly continuous product capture and purification using a column-free system that overcomes many limitations of traditional column chromatography. All operations in CCTC are conducted on a moving slurry that is continuously pumped through a cascade of static mixers (for mixing and residence time) and hollow fiber membrane modules (for separation of the fluid phase from the resin particles). For example, host cell proteins and other impurities are removed in the permeate collected through the hollow fiber membrane in the washing step, while the bound product is retained by the membrane. Contacting in the individual steps is performed in a countercurrent fashion, with the flowing slurry moving from stage (module) 1 to stage 2 while the permeate moves in a countercurrent direction. This significantly increases the overall throughput while improving product yield and purification. All of the chromatographic operations are performed simultaneously, with a fraction of the resin being used for binding while the rest of the resin is in the washing, elution, or regeneration steps. Recent experimental studies (Dutta et al., 2015) have demonstrated that CCTC can be successfully used for initial capture of a monoclonal antibody product from clarified CHO cell cell culture fluid produced in a fed batch bioreactor. Host cell protein removal and antibody yields and purities were similar to that obtained with conventional batch columns, but the CCTC provided a several-fold greater productivity (g purified mAb per liter resin per hr). In contrast to multicolumn systems, CCTC provides true steady-state operation, with the product concentration remaining constant over a multi-hour run. The current talk examines the effects of different resins on the overall performance of the CCTC system. Experiments were performed using different monoclonal antibodies with several Protein A resins, including small particle size prototype resins, having different static binding capacities and particle diameters. Data were obtained for the binding kinetics, the critical filtrate flux needed to avoid fouling, and the extent of particle attrition due to continued recirculation of the resin through the peristaltic pumps. The use of resins with smaller particle diameters significantly reduced internal mass transfer limitations, leading to faster binding kinetics and greater overall productivity. The critical flux was very high (above 500 LMH) for all resins tested. Limited experiments were also performed using ion exchange resins in both bind-and-elute and flow-through mode. The results clearly demonstrate the versatility of Continuous Countercurrent Tangential Chromatography for the continuous purification of high value protein therapeutics like monoclonal antibodies

Accurate determination of key surface properties that determine the efficient separation of bovine milk BSA and LF proteins

The aim of this work is to accurately measure fundamental surface properties, i.e., zeta potential, isoelectric point and protein size that determine the optimal separation conditions of Bovine serum albumin and lactoferrin, two high added value food proteins whose similarity in weight makes their separation a scientific and technical challenge. The systematic study of these proteins’ surface properties was performed under different conditions: (i) 3.0 < pH < 10.0, (ii) electrolyte type: KCl, NaCl and CaCl2 and concentration (0.01–0.1 M KCl) and (iii) protein concentration in the range of 0.04–4.0 g L-1 for BSA and 0.01–1.0 g L-1 for LF with the objective of establishing the optimal separation conditions. Finally, the comparison of the experimental and theoretically calculated values revealed significant deviations under specific conditions, highlighting the simplicity of the theoretical assumptions and leading to the conclusion that the use of experimental surface properties is still needed for the correct design of food protein separation processes.Financial support from the Projects CTQ2011-25262, CTM2011- 23912 and CTQ2012- 31639 (Ministerio de Economía y Competitividad-MINECO/SPAIN and Fondo Europeo de Desarrollo Regional-FEDER) is gratefully acknowledged

Optimal Pharmacologic Treatment Strategies in Obesity and Type 2 Diabetes

The prevalence of obesity has increased to pandemic levels worldwide and is related to increased risk of morbidity and mortality. Metabolic comorbidities are commonly associated with obesity and include metabolic syndrome, pre-diabetes, and type 2 diabetes. Even if the prevalence of obesity remains stable until 2030, the anticipated numbers of people with diabetes will more than double as a consequence of population aging and urbanization. Weight reduction is integral in the prevention of diabetes among obese adults with pre-diabetes. Lifestyle intervention and weight reduction are also key in the management of type 2 diabetes. Weight loss is challenging for most obese patients, but for those with diabetes, it can pose an even greater challenge due to the weight gain associated with many treatment regimens. This article will review optimal treatment strategies for patients with comorbid obesity and type 2 diabetes. The role of anti-obesity agents in diabetes will also be reviewed. This literature review will provide readers with current strategies for the pharmacologic treatment of obesity and diabetes with a focus on the weight outcomes related to diabetes treatments

Pharmacological Treatment of Diabetic Peripheral Neuropathy

Pain modulation is a key treatment goal for diabetic peripheral neuropathy patients. Guidelines have recommended antidepressant, anticonvulsant, analgesic, and topical medications—both approved and off-label—to reduce pain in this population