Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography

Abstract

Recent studies using simple model systems have demonstrated that Continuous Countercurrent Tangential Chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an “after binder” to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (~0.67 g/L) and one with high titer (~6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to that obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products.

Continuous desalting of refolded protein solution improves capturing in ion exchange chromatography: A seamless process

“…Not only FDA has addressed this topic [12,13], it also raised the interest of numerous researchers in both academia and industry [14,15]. Even it is not realized on a large production scale yet, various different strategies for continuous biomanufacturing have been developed in recent years [8,[16][17][18][19][20][21].…”

“…Not only FDA has addressed this topic [12,13], it also raised the interest of numerous researchers in both academia and industry [14,15]. Even it is not realized on a large production scale yet, various different strategies for continuous biomanufacturing have been developed in recent years [8,[16][17][18][19][20][21].In this work, we describe a novel alternative desalting process using microporous ion exchange resins, which are typically employed for water demineralization and are relatively inexpensive [22]. In the current work, this type of resin is referred to as "micropore resin" due to its small pore sizes of approximately 1-1.5 nm according to manufacturer information.…”

Perspectives on integrated continuous bioprocessing—opportunities and challenges

“…in the form of a slurry which flows sequentially through a series of static mixers and hollow fiber membrane modules [30 ]. The micro-porous hollow fiber membranes retain the chromatographic resin while letting proteins and buffer components pass into the permeate.…”

“…The buffers used in the binding, washing, elution, and regeneration steps flow countercurrent to the resin slurry in a multi-stage configuration within each step, similar to the use of countercurrent staging in classical mixer-settler systems for extraction (with the hollow fiber membrane replacing the settler for the 'phase' separation). In contrast to the periodic behavior of multi-column chromatography systems, CCTC provides truly continuous steady-state operation, with the product obtained from the elution step at constant concentration and quality due to the uniformity in residence time in the binding, washing, and elution stages [30 ].…”

Experiment and simulation of an aqueous two-phase extraction process for the purification of a monoclonal antibody

“…Since most downstream processing unit operations, especially Protein A chromatography, are conventionally carried out in a discontinuous manner, there is a need for continuously operating process steps [2,3]. Aqueous two-phase extraction (ATPE) has been reported for successful purification of mAbs and other biological compounds [4,5,6,7,8,9,10,11,12,13].…”