Chromatography Optimization Strategy

Robust packing procedures can improve process performance and increase resin lifetime.
Mar 01, 2009
Volume 22, Issue 3


The optimization of a large-scale chromatography operation involved a strategy combining bench and pilot scale packing stability studies, and evaluation of hardware limitations at bench and large scales. 1 The effects of resin amount on column stability, hydrodynamic properties of compressible media, process resolution, and targeted recovery were evaluated at the bench scale. The process was scaled up for hardware and process performance evaluation. A robust packing procedure was developed at pilot scale to optimize system suitability, reduce the time required for packing, and improve process performance. The packing optimization provided an increased bed stability, which offered the basis to extend the number of chromatography cycles per packed column. The lifetime extension reduced resin costs per lot. The faster packing method and reduced frequency for packing reduced labor requirements for this step at large scale.

This case study relates to protein purification through ion exchange chromatography with a start of collection based on ultraviolet (UV) absorbance and end of collection based on reaching a volumetric endpoint. This approach mitigates the potential impact of impurities that elute on the back side of the main peak, but led to higher yield losses for runs, in which peak resolution was broader because of nonoptimized packing conditions.

Inconsistent performances of the chromatography step for the large-scale process led to high variability in process yields and purity. Therefore, basic process development techniques are required to assess and resolve the cause of variability and thereby improve and optimize the chromatography process.


Figure 1. Experimental bed height and predicted height at different aspect ratios
The quantity of resin required to achieve targeted compression on a packed bed is a factor that significantly affects the chromatography elution profile. Achieving targeted compression is critical to avoid headspace formation and overcompression in stainless-steel columns, in which this cannot be visually observed. Previously, the resin concentration on slurry was determined by the gravity settling (GS) method, which is a simple way to determine resin concentration as a volumetric ratio. The method calls for pouring a determined amount of homogenous slurry into graduated cylinders and allowing the resin to settle by gravity over an extended period, which is dependant on the storage solution. There are, however, certain drawbacks associated with this approach such as a long settling time before reading. The calibration resolution of the graduated cylinder and wall support effects provided misleading readings of bed volume. The resin concentration varied because of intricacies of the technique related to visual interpretation of the cylinder calibration resolution and an unstable resin settling using the previous storage solution.


An alternative to the GS method is the flow consolidation (FS) method, which takes into consideration the hydrodynamic properties of compressible media, showing a linear relationship between height and flow (Figure 1). The linearity between bed height and flow can be expressed in terms easily read on a chromatography column tube (Equation 1):

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