Monoclonal antibodies have long been purified using a platform approach. This platform mainly consists of three chromatography steps. These include a Protein A based affinity chromatography step followed by polishing steps. The purpose of the affinity chromatography is to capture the mAb, while the polishing steps remove any remaining impurities, such as residual DNA, host cell proteins, viruses and aggregates. There are options in the selected polishing steps, but often they involve cation exchange or hydrophobic interaction. Increasingly membrane chromatography is being used as a polishing step to remove any remaining contaminants.

Over the past decade, upstream bioprocessing has increased expression levels leading to higher antibody titers. As a result, downstream purification has had to develop higher binding capacity Protein A sorbents to keep pace with current expression levels. In addition, the increase in titer caused an increase in impurities, necessitating innovation in polishing steps to more efficiently remove these impurities.

Purification has traditionally been slower to innovate mainly due to the difficulty in changing existing downstream processes, the lack of a universal purification process, and the low capacity of mixed mode chromatography sorbents that has limited their use despite their high resolution.

To address these issues, Pall® Biotech recently presented a chromatography process which combines KANEKA KanCapA^♦ sorbent, Mustang® Q chromatography membrane and CMM HyperCel™ sorbent. This three-step process has been demonstrated to eliminate the need for diafiltration or dilution between chromatography steps.

The Steps:

1. KANEKA KanCapA Protein A Affinity Capture Step

KANEKA KanCapA Protein A affinity sorbent has been designed for high binding capacity with enhanced selectivity to reduce host cell proteins (HCP) and aggregates while eluting at a mild pH, which can reduce denaturation and improve process yields. KANEKA KanCapA sorbent comprises a recombinant Protein A ligand covalently attached to a rigid cellulose matrix. This ensures stability over a high number of cycles and provides high flow performance at low pressure, making the sorbent compatible with the process requirements for large scale mAb manufacture in both batch and continuous processing using multicolumn chromatography solutions.

2. Mustang Q Ion Exchange Membrane Chromatography Polishing Step

The use of scalable Mustang XT membrane chromatography capsules is a flexible solution for downstream processing that helps process developers improve their process economics. In many applications, the dynamic binding capacity of Mustang Q membrane is equivalent to or greater than the equivalent resin chemistries; and unlike sorbents, the dynamic binding capacity is independent of flow rate. In polishing applications, for the removal of low levels of contaminants, this means high flow rates (more than 10 MV/min) and low pressure drops that can be fully exploited with minimum membrane volume.

3. CMM HyperCel Cation Exchange Mixed-Mode Polishing Step

CMM HyperCel sorbent is industry scale compatible and offers capture and impurity removal at moderate salt conductivity. It provides separation performance typical of a mixed-mode resin, and more, provides capacity and selectivity in a very broad range of conditions of use. The specificity allows loading of this mixed mode sorbent from pH 5 to > pH 7 and means that only the elution conditions need to be optimized while maintaining the binding conditions for the previous anion exchange step. This leads to reduced process development time and resources. Specificities of CMM HyperCel sorbent make it the tool of choice for recombinant proteins and monoclonal antibodies which are difficult to purify.

To learn more about their three-step process, Pall Biotech is hosting a webinar demonstrating the benefits of their chromatography platform. Pall Biotech will demonstrate the performance of the CMM HyperCel sorbent within a streamlined batch process, which consistently provided purified mAb with high yield and purity ( > 95% yield with < 0.5% aggregates and < 15 ppm HCP).  The result of investigations will show the versatility of CMM HyperCel sorbent, and how it provides flexibility within the development of a process whilst delivering high yield and purity of mAbs and recombinant proteins.

To view the webinar on demand, please see “Streamline Your Protein Purification Platform.” 

Pall, Mustang and HyperCel are trademarks of Pall Corporation.  KANEKA KanCapA is a trademark of Kaneka Corporation. ®indicates a trademark registered in the USA. ™ indicates a common law trademark