Advantages Of Affinity Chromatography

Chapter 4

Optimisation of a Protein A Chromatography Process

Introduction

The optimisation of a chromatography, we know, is vital to the efficiency of a separation process. Protein A chromatography by virtue of its incredible ability to achieve high purity of biopharmaceutical products has made itself indispensable for downstream purification of biomolecules (Hammond et al.)

Even though many variations of different chromatographic separations have been employed as ‘capture’ steps over the years, Protein A affinity chromatography has come to be used as the method of choice for direct ‘capture’ and purification of monoclonal antibodies. The highly specific binding between the Fc-region of an antibody and Protein A, as elucidated to in Chapter

Due to the varying pressures that protein A chromatography columns are exposed to during processing the integrity of the material may sometimes be compromised. This increased pressure is readily seen with increased flow rates and hence put a greater stress on the system to attain optimised affinity is required. This can be readily seen with the beaded agarose support material (Cube Biotech)

Fig. 25 above conveys increased agarose concentration and also increased cross linking. This increased concentration and cross linking is responsible for increased durability and make it somewhat robust.

Support materials must be able to withstand the back pressures encountered during normal separations without compressing. While most commercial packing materials meet this requirement, the build-up of particulate contaminants may restrict column flow and lead to high backpressures. Under these pressures, soft porous gel supports such as agarose beads will compress and increase the pressure even further causing collapse of the support structure. More mechanically stable supports (e.g. silica and heavily cross-linked polymers) are able to withstand these high pressures, but the build-up of particulate contaminants should be avoided if at all