High throughput screening for resin regeneration

Abstract

As the biopharmaceutical industry is increasingly under cost pressure, the cost-efficiency and effectiveness of processes is of great importance. The downstream process is a major cost driver, with chromatography resin being an expensive raw material. This makes the frequent reuse of chromatography resin and the associated regeneration more important. The regeneration of the chromatography column is a balancing act, between avoiding a carry-over to the next batch by too weak cleaning conditions and a loss of ligands by too harsh conditions. Both conditions lead to a reduced number of cycles, increasing backpressure or decrease in performance of the chromatography column, resulting in massive costs due to additional consumption of chromatography resin.However, the regeneration procedure is often not considered in the process development scope. Instead, established conditions from literature are chosen. Regeneration requirements are influenced by various factors, including protein purity, chromatography step (capture or polishing), and resin properties. Consequently, multiple conditions must be tested for optimal results. While column-based execution is possible, it's resource-intensive and costly due to material and time demands.A high throughput screening was set up to screen for optimal regeneration conditions which is used in downstream development processes. Here, various chemicals as well as different chemical concentrations can be tested. Moreover, different combinations in number of steps can be tested as well based on an experimental design. Readout of the study is the remaining protein and ligand density determined by SDS-Page and HPLC. In the first step, the screening is carried out using TecanTM robot in 96-well plates, where 24 different conditions can be automatically tested simultaneously. The second step is a scale-up to RoboColumnsTM using a JanusTM robot, where the best conditions from the first attempt were verified. This experimental setup extends to parameters which are not demonstrated by a 96 well batch plate and furthermore it shows if the results are transferable. Employing this screening procedure an optimized regeneration condition on a capture column for aIVmodel protein expressed as inclusion body was selected. As a result of the preceding refolding procedure, the loaded refolded inclusion bodies encompass both accurately folded proteins and those with almost correct folding. This leads to the aggregation of these proteins on the column during the process.Such a screening enables better selection of regeneration conditions and therefore potentially saves money due to better resin usage.The screening setup is applicable for all CIEX resins and processes. The principle can be used for AIEX resins and processes as well as for all other chromatography types with adapted regeneration conditions. However, some analytical evaluation cannot be exactly transferred such as for HIC resin where no ligand density can be determined because no simple titration can be carried out.The developed procedure consisting of a screening in 96 well plates and optimization of selected conditions in RoboColumnsTM is ready to be used in standard process development of chromatography.