Journal watch: Unlocking efficiency in column chromatography with packed bed supporting inserts

Open Access:
Nature Communications

ABSTRACT

To purify increasing amounts of biotherapeutics more efficiently, the use of high flow rates or greater resin bed heights during downstream chromatography steps is a tantalizing option. A limitation of utilizing high flow rates is the differential pressure generated by packed chromatography resin beds. As a resin bed height increases, the resin is susceptible to compression. By increasing the permeability of a packed resin bed through control of the hydraulic radius, column pressure-flow dynamics can be improved. Chromatography column performance using a commercially available Protein A resin was assessed with and without OMEGA, a column insert designed to modulate the hydraulic radius of the column by providing vertical supports through the packed resin bed. OMEGA was shown to reduce the effective hydraulic radius of packed resin beds, increase the permeability of packed columns by 44%–73%, and yield a 42%–50% decrease in pressure differential across the resin bed at a comparable linear velocity. The structural support provided by OMEGA enables higher operational flow rates and increased resin bed height without impact to either dynamic binding capacity or purified product quality. With the OMEGA column insert, scale-up hurdles are mitigated, and faster downstream processing times are unlocked across column geometries.

INTRODUCTION

As cell culture titers of biotherapeutics are driven to higher levels, a greater burden is placed on downstream processing to achieve a faster and more cost-effective operation. Leveraging higher flow rates and larger scale chromatography columns are approaches that may be used to reduce downstream processing time and control costs by minimizing manufacturing suite time; however, chromatography resins impose pressure limitations, restricting flow rates to well under the maximum capacity of manufacturing suite liquid handlers. Due to the compressible nature of resin chromatography beads, increases in flow rate, resin bed height, or column diameters result in dramatic increases in pressure differential (ΔCP) within the column (Stickel and Fotopoulos, 2001). This column ΔCP must be avoided to prevent damage to chromatography resin beds and associated processing equipment. Despite the capacity for increased flux with liquid handlers, elevated flow rates have remained inaccessible.

The development of resin-based chromatography steps for operation at the manufacturing-scale typically begins with columns of inner diameters (ID) of 1–5 cm. At the commercial manufacturing-scale, a larger volume of resin is needed, which requires the use of larger columns. The increase in column size is typically accomplished by increasing column diameter rather than bed height; commercial offerings for column hardware can reach up to 200 cm in inner diameter, while the resin bed height is typically limited to 30 cm to avoid compression (Łącki, 2018). Despite maintaining bed height when transitioning from development-scale to manufacturing-scale columns, column ΔCP hurdles are commonly encountered necessitating a reduction in flow rate. Increasing inner diameter results in a dramatic change in column aspect ratio and an increase in the hydraulic radius. In the case of an open channel, an increase in hydraulic radius increases the volumetric flow rate of a fluid channel due to a reduction in drag; however, when applied to a packed resin bed, an increase in hydraulic radius often requires a reduction in volumetric flow rate to avoid excessive column ΔCP. This is partially attributable to wall effects: resin near the center of a cylindrical column experiencing compression due to the lack of frictional support provided by the column walls (Colby, O’Neill, and Middelberg, 1996; Soriano, Titchener-Hooker, and Shamlou, 1997; Parker, Mehta, and Caro, 1987; Guiochon et al., 1999). The compression at the center of a packed resin bed results in decreased permeability and a flow velocity gradient across the diameter of the column. As the column diameter and hydraulic radius increases, a greater proportion of the resin bed is susceptible to compression resulting in decreased permeability (Prentice et al., 2020). With decreased permeability, a column’s resolving power and theoretical plate count are reduced (“Consolidation of Particle Beds and Packing of Chromatographic Columns,” n.d.).

Performance enhancements of modern resin-based chromatography operations have included improvements to resin matrices and advanced ligand chemistries. These improvements have yielded superior stability and packing performance relative to traditional chromatography resins yet are still susceptible to performance degradation resulting from compression. One existing substitute for resin-based chromatography is monolithic columns, which eliminate column packing and can operate at higher flow rates. The major drawbacks of monolithic columns are the limited choice of column chemistries and dimensions as well as their lower binding capacities relative to chromatography resins (Rathore, Kumar, and Kateja, 2018). An emerging alternative to resin chromatography in downstream processing is membrane-based adsorption technology (Qu et al., 2023). Membrane adsorbers are capable of operation at much higher flow rates; however, due to the limited binding capacity of membrane adsorbers available on the market today, an increased number of processing cycles are required relative to resin chromatography to process the same amount of biotherapeutic (Winderl, Neumann, and Hubbuch, 2021; Liu et al., 2011). At this time, it is uncertain if wide-scale adoption of membrane absorption technologies are viable at commercial scale operations (Qu et al., 2023). Strategies to enhance the performance of existing resin media include rapid cycling of short bed-height columns in batch mode (Kaltenbrunner et al., 2016; Guo, Jin, and Kanani, 2020), thereby increasing the productivity (grams of product processed per mL of resin per hour) of resin batch operations. However, rapid cycling in short bed-height resin batch chromatography lowers the resin capacity utilization, which makes inefficient use of resin lifetime and process buffers. Methods designed to use multiple loading flow rates to increase resin utilization capacity while maintaining high process productivity have also been reported (Kanani, 2018; 2019) and modeled (Guo, Jin, and Kanani, 2020). Continuous multicolumn chromatography has also been explored to process more load material using less resin (Kaltenbrunner et al., 2016), has been demonstrated to be feasible at the pilot scale (Ötes et al., 2018), and similarly modeled for process productivity (Guo, Jin, and Kanani, 2020). While the use of multiple flow rates and multiple short bed-height columns has great potential, these technologies do not fully address concerns with resin compression, and hurdles remain for their implementation at manufacturing scale. A technology that provides a performance upgrade to existing standard resin batch mode operations, while also maintaining compatibility for use with future continuous modes of operation is needed.

Reducing the compressibility of chromatography resins can be achieved by providing additional wall support with column inserts. When reducing the amount of compression experienced throughout a resin bed by use of column inserts consisting of concentric rings, simulations of packed columns have shown that the permeability of a packed resin bed is increased (Gerontas et al., 2015; Riess et al., 2022). According to Darcy’s Law, increased permeability enables the use of higher flow rates or increased resin bed heights without a corresponding increase in ∆CP across the resin bed. A concentric ring column insert design does not directly control the column’s hydraulic radius and imposes practical difficulties for column packing. By managing a column’s hydraulic radius through tuned column inserts, we hypothesize that permeability will be controllable independent of column geometry. With regulated resin bed permeability, higher flow rates may be achievable while maintaining the resin’s performance characteristics. This would enable large-scale columns to have pressure-flow characteristics similar to small-scale columns while retaining the increased binding capacity associated with a scale-up in the volume of resin.

In this work, we evaluate a novel design for a column insert (OMEGA) that reduces the hydraulic radius of a resin bed by providing vertical wall supports throughout the full height of the bed. This degree of support addresses the unmet need for improved processing time, maintains resin bed integrity, and allows for simple column packing (Figure 1). As designed, OMEGA reduces the hydraulic radius in a manner that is decoupled from the column’s inner diameter. Effectively, the amount of support provided for each column geometry is scaled according to the inner diameter of the column. The degree of compression alleviated by OMEGA’s inclusion in a packed resin bed was measured across several column geometries. The resultant change in compression and its impact on permeability was assessed in this work. With and without the use of OMEGA within columns, the impact of permeability on operational flow rates and ∆CP was measured. Lastly, a model monoclonal antibody (mAb) was purified using a traditional chromatography column and a column supported with OMEGA inserts. Purification performance of the model mAb was analyzed for column operational characteristics as well as product quality.

For further reading please visit: 10.3389/fbioe.2025.1613174

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