Affinity chromatography is potentially the most selective method for protein purification and is often included as a primary capture step for the purification of a biotherapeutic antibody or enzyme. Purification development for the production of biopharmaceuticals is typically undertaken at small scale and then transferred to manufacturing scale after development and optimization of the purification step. Bed-height considerations and the impact of chromatography base matrix, flow rates, and manufacturing constraints that influence the packed bed-height strategies are essential for affinity purification of biopharmaceuticals.
Affinity chromatography is ideally suited to the purification of biopharmaceuticals due to the unique specificity characteristics of bound ligand and as such is likely to play an increasingly important role in the purification of biotherapeutic proteins.
Typically a significant percentage, 50–80%, of the total manufacturing cost of a therapeutic antibody may be incurred during downstream processing, and efficient affinity purification steps are essential to reduce cost of goods and ensure processing bottlenecks within the downstream manufacturing process do not impede market supply.
Several factors are usually considered when developing a purification step at small scale, including the dynamic capacity of the matrix (the volume of target protein that may be loaded onto the column before breakthrough occurs). Although the association rate (kobs) of a target protein to an immobilized ligand may be defined as kobs = Ka C + Kd (where C is the concentration of target protein and Ka and Kd are the association and dissociation rate constants, respectively), diffusion into the pores within the chromatography beads and mass transfer of the desired protein from the solute will also impact the dynamic binding capacity of a chromatography matrix at a particular flow rate.
Although models to assess film and pore diffusion into chromatography beads may be evaluated mathematically, the routine application of such methods may be technically challenging as mass transfer of the target protein from the solute may be dependent on a variety of factors. The percentage cross-linking, the size of the pores, and also the physical size of the target protein may impact the rate of diffusion into the chromatography beads, and other factors such as the flow rates, protein concentration, column length, temperature, buffer, conductivity, and pH may also have an impact on pore diffusion and the dynamic binding capacity of the adsorbent.
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.
Another factor that should be assessed early in the development phase is the proposed manufacturing scale required to supply sufficient quantities of product to the market. Due to the major investment required to purchase process-scale chromatography equipment, affinity purification steps are often scaled up using existing equipment available within a manufacturing facility. Therefore, increased bed heights may need to be considered under certain circumstances with these limitations in mind. Indeed another disadvantage of short bed heights for affinity chromatography at manufacturing scale is the major investment required for large diameter process-scale columns.
Chromatography columns with diameters of >100 cm are often custom manufactured and the use of short bed heights under utilizes the potential packed bed volume of the columns that could be used. However, when using rigid chromatography adsorbents, tall bed heights (Ž25 cm) should be considered to utilize the volume of the process-scale column at production scale (Figure 2). With compressible adsorbents, short bed heights (£10 cm) combined with increased diameter columns are favorable due to volumetric flow rate limitations imposed by the base matrix.