Improved protein A resin for antibody capture in a continuous countercurrent tangential chromatography system.

Continuous countercurrent tangential chromatography (CCTC) enables steady-state continuous bioprocessing with low-pressure operation and high productivity. CCTC has been applied to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest and postcapture polishing of mAb; however, these studies were performed with commercial chromatography resins designed for conventional column chromatography. In this study, a small particle size prototype agarose resin (20-25 µm) with lower cross-linking was co-developed with industrial partner Purolite and tested with CCTC. Due to increased binding capacity and faster kinetics, the resulting CCTC process showed more than a 2X increase in productivity, and a 2X reduction in buffer consumption over commercial protein A resins used in previous CCTC studies, as well as more than a 10X productivity increase versus conventional column operation. Single-pass tangential flow filtration was integrated with the CCTC system, enabling simple control of eluate concentration. A scale-up exercise was conducted to provide a quantitative comparison of CCTC and batch column chromatography. These results clearly demonstrate opportunities for using otherwise unpackable soft small particle size resins with CCTC as the core of a continuous bioprocessing platform.

Continuous countercurrent tangential chromatography for mixed mode post-capture operations in monoclonal antibody purification.

Continuous Countercurrent Tangential Chromatography (CCTC) has been shown to demonstrate significant advantages over column chromatography including higher productivity, lower operational pressure, disposable flow path, and lower resin use. Previous applications of CCTC have been limited to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest. In this present article, a CCTC system was designed and tested for a post-capture antibody purification step. Mixed mode cation exchange-hydrophobic interaction chromatography resins with two different particle sizes were used to reduce host cell protein (HCP), leached protein A, DNA, and aggregates from a mAb stream after a protein A operation. Product output from CCTC was obtained at a steady-state concentration in sharp contrast to the periodic output of product in multi-column systems. The results show up to 101g of mAb/L of resin/hr productivity, which is 10× higher than in a batch column. A 5% yield increase (95% with CCTC vs. 90% in batch column) resulted from optimizing elution pH within a narrow operational window (pH 4-4.5). Contaminant removal was found to be similar to conventional column performance. Data obtained with the smaller particle size resin showed faster binding kinetics leading to reduced CCTC system volume and increased productivity. Buffer and water usage were modeled to show potential for utilization of in-line mixing and buffer tank volume reduction. The experimental results were used to perform a scale up exercise that predicts a compact CCTC flow path for 500 and 2000L batches using commercially available membranes. These results demonstrate the potential of using CCTC for post-capture operations as an alternative to packed bed chromatography, and provide a framework for the design and development of an integrated continuous bioprocessing platform based on CCTC technology.