This article presents a case study illustrating the benefits of using a high throughput process development (HTPD) approach for choice of media and optimization of the polishing step of a monoclonal antibody in a two-step purification process. The most commonly used format for HTPD is the 96-well plate to facilitate rapid screening of chromatographic parameters. The use of the 96-well plate format enabled the efficient screening of three different media and optimization of chromatographic parameters to maximize yield at the desired purity level.
Biopharmaceutical manufacturers are under increasing pressure to develop and produce biopharmaceuticals cost-effectively and within tight timelines. The US Food and Drug Administration is also nudging the industry to implement its Quality by Design (QbD) initiative in the manufacturing processes. QbD stipulates a better understanding of the influence of raw materials and intermediates, and control of process parameters. This initiative also is applicable to the development of downstream processes in the manufacture of monoclonal antibodies (MAbs). In this area, a new approach of high throughput process development (HTPD) is emerging as a useful tool for compliance with QbD. HTPD uses a screening format to facilitate the identification of optimal experimental conditions by directed, rapid screening of experimental parameters. A large experimental space can be investigated and characterized in a very short timeframe, enabling a better understanding of the effect of process conditions.
Traditionally, MAbs are purified in a three-step process. First, the target is captured by chromatography with Protein A media, and two subsequent polishing steps are then performed according to a variety of protocols, often involving a combination of ion-exchange chromatography and hydrophobic interaction chromatography. For particular antibodies, it is possible to apply a more efficient two-step process that involves one capture step and a single polishing step. In the polishing step of a two-step process, a multimodal chromatography medium capable of several interactions (e.g., hydrophobic, ion-exchange interactions, hydrogen bonding) can be used, which allows the selective removal of impurities like antibody aggregates, host cell proteins (HCPs), and leaked ligands. The complexity of these media requires a more thorough process optimization study. The most commonly used format for HTPD is the 96-well plate to facilitate rapid screening of chromatographic parameters.
This case study illustrates the benefits of using the HTPD approach for choice of media and optimization of the polishing step in a two-step MAb purification process. The use of a 96-well plate format enabled efficient screening of three different media and optimization of chromatographic parameters to maximize yield at the desired purity level. The initial capture step was performed using a Protein A medium (MabSelect SuRe, GE Healthcare), which was then used diluted or undiluted for the specific experiments.1 Three chromatography media have been investigated in this study (Table 1).
Table 1. Chromatography media tested in this study
INITIAL SCREENING OF CHROMATOGRAPHIC CONDITIONS
Initial experiments focused on finding the ideal incubation times and MAb concentrations for the remaining experiments (Table 2). These experiments were performed using 96-well plates (PreDictor, GE Healthcare) filled with the three chromatography media being studied, which eliminated the need for the more time-consuming column chromatography.
Table 2. Initial screening conditions for the three chromatography media tested
The chromatography media in the 96-well plate were first equilibrated at specific pH and salt concentrations, and the sample was then added at four different incubation times. The flow-through fraction was collected in a collection plate using centrifugation at 300g and was analyzed using size-exclusion chromatography (SEC) to determine monomer and aggregate contents.
Initial screening on the two anion-exchange media showed that antibody binding only occurred at pH 8.5 and 0 mM NaCl. The binding kinetics for all three media were fast for the monomer. After 10 min, the binding was complete, while the binding of the aggregates was approximately three-times slower—at least 30 min was required to achieve complete binding. The monomer content increased slightly at longer incubation times, possibly because of the displacement of monomer by aggregates. It was concluded that an incubation time of 1 h and a protein concentration of 5.3 g/L were the most appropriate conditions.