Polishing Complex Therapeutic Proteins - A new downstream purification platform using a salt-tolerant membrane adsorber. - BioPharm International


Polishing Complex Therapeutic Proteins
A new downstream purification platform using a salt-tolerant membrane adsorber.

BioPharm International
Volume 26, Issue 1, pp. 42-51

It has been noted for some time that an anion-exchange MA is an attractive alternative to anion-exchange columns when operated in flow-through mode to remove low levels of impurities, such as DNA, host-cell protein (HCP), and virus (5). Flow-through polishing columns are usually sized for speed to achieve desired flow rate and process time, using only a small fraction of the binding capacity available for impurity removal. Because MAs allow for a faster flow rate, a small MA device can replace a bigger column and still provide sufficient binding capacity for impurity clearance (6–8). Today, membrane chromatography has proven to be a robust alternative to Q column chromatography for polishing in flow-through mode, and multiple case studies have demonstrated the popularity of their implementation (9–13). Single-use MAs not only reduce process time, buffer usage, and floor space, but also eliminate the column packing and cleaning validation activities required for packed-bed columns. A detailed cost analysis showed that single-use Q membranes can be cost competitive compared with a reused Q Sepharose fast-flow column in a mAb process when its process capacity is sufficiently high (7). More recent analyses also show that using a disposable MA in flow-through mode provides comparable or a lower cost of goods (CoG) than using a packed-bed column (14, 15). Overall, for a flow-through polishing step, replacing a packed-bed column with a single-use MA can provide cost savings.

As with any other ion-exchange chromatography, conductivity and pH have significant effect on the performance of anion-exchange MA as a flow-through polishing unit operation. One report stated that to ensure sufficient impurity clearance, the ideal range for Sartobind Q flow-through step in a mAb process is 3–4 mS/cm at pH 7.0–7.2 (13). Similarly, another study using Q MA from Millipore found that to achieve > 1 log removal of host cell protein, the load had to be conditioned to pH 8.0 and a conductivity of < 4.0 mS/cm (6). The low salt tolerance of these Q MAs means that a dilution step prior to loading is often required to achieve desired impurity clearance, which increases process complexity. In recent years, efforts were made, both in academia and industry, to develop new types of anion-exchange MAs that have better salt tolerance, which will enable greater process flexibility and potentially lead to wider usage of MA flow-through polishing in the downstream processes. A systematic screening study by Riordan et al. identified three factors that contributed to salt tolerance of anion-exchange MA: ligand net charge, ligand density, and molecular structure of the ligand (16). Interestingly, the study also found that available hydrogens on the amine-binding group improved the salt tolerance of the ligand, indicating that primary amines might have better salt tolerance than quaternary amines.

Sartobind STIC (salt tolerant interaction chromatography, Sartorius Stedim Biotech, Goettingen, Germany) is a weak anion-exchange MA that is less sensitive to increasing salt concentration than standard Q membranes (17). It carries the polyallylamine ligand that provides high charge density and salt tolerance. The new double-porous membrane replaced the previous generation of membrane with hydrogel, which was shown to shrink and reduce binding site accessibility under high salt conditions (18). Sartobind STIC was shown to provide significantly higher binding capacity and higher LRV of model viruses compared with Sartobind Q in the presence of 150 mM NaCl (16.8 mS/cm) (17). This enhanced salt tolerance allows the MA polishing step to be conducted without load dilution, thus reducing process time and complexity.

This article describes the development work at Bayer to evaluate Sartobind STIC as a platform polishing unit operation for complex protein therapeutics. Specifically, the authors looked at product yield, and HCP and DNA removal by Sartobind STIC from very high salt intermediates, and how to improve the overall purification platform. Data suggest that using a salt tolerant MA enables the number of unit operations in a platform process to be reduced, potentially reducing cost of goods.

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