Optimizing Bed Height
When packing a fixed bed chromatography column, the question of the optimum bed height for the purification step often arises. Due to the requirement for rapid development of a downstream process and the desire for generic purification processes utilizing platform technologies, the residence time for a particular class or type of biologic such as a biotherapeutic antibody is typically fixed, and bed heights are often selected somewhere within the region of 10–20 cm.
However, as the resolution of an affinity column is often bed-height independent, the evaluation of a number of packed bed-height strategies should be assessed. An important factor to consider early in the purification development is the rigidity of the base matrix. Rigid affinity adsorbents such as porous glass generate low back pressures over a wide range of flow velocities (>1,000 cm/h). Compressible chromatography media such as agarose adsorbents typically have recommended operating flow rates of 150–300 cm/h and may become volumetric flow-rate limiting with increased bed heights.
Two assumptions of commercial affinity adsorbents are made: first, the target protein binds with high affinity such that the interactions are not disrupted by increasing linear flow rate prior to elution; and second, the Kd is sufficiently slow that loss of the target protein during the washing phase is insignificant.
The duration of the remaining chromatography steps after loading the desired volume of feedstock, washing, elution, and reequilibration are therefore solely dependent on the volumetric flow rate through the column and the pressure limitations of the adsorbent and the chromatography equipment. At first glance, the shortest possible bed height looks to be the most favorable approach for the bed height of the column.
The volumetric flow rate (L/h) through a short column at a fixed linear velocity will be greater, reducing purification times and overcoming potential pressure limitations of the adsorbent. In addition, as the volume of a packed bed increases fourfold with every doubling of the column diameter, the volume of adsorbent lost in reducing the bed height may be readily accommodated by increasing the column diameter.
Bed heights with compressible adsorbents have been shown to have significant impact on the cost of goods (as the increased bed height impacts the maximum flow, which, in turn, influences the cycle time). Consider two small-scale affinity columns of differing bed heights but with similar volumes (Figure 1). At a linear flow rate of 150 cm/h (a typical maximum recommended operating flow rate for a 6% cross-linked agarose affinity adsorbent), the volumetric flow rate for a column with a 25 cm bed height is 120 mL/h (Figure 1A). However, a column with a theoretical bed height of 1 cm can accommodate a volumetric flow rate of almost 3,000 mL/h at the same linear flow rate (Figure 1B).
Although increasing column bed height results in higher flow resistance and increased back pressure, affinity columns may be reliably and successfully scaled up by adjusting the flow velocity through the column to ensure a constant residence time (doubling of the bed height is accompanied by a doubling of the flow rate in a fixed diameter column), and, in some cases, target proteins with relatively fast dissociation rates (Kd, s-1) and dissociation during column washing may benefit from a longer bed height to permit opportunity for re-binding while impurities are washed from the adsorbent. As the volumetric flow rate is usually adjusted to maintain the desired residence or contact time with the adsorbent, the duration of the loading step therefore becomes independent of column dimensions.