Pegylation of staphylococcus aureus protein a ligands for improved efficiency in monoclonal antibody chromatography purification
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Authors
Rdultowski, Mariusz
Issue Date
2024-12
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Chemical engineering
Alternative Title
Abstract
Staphylococcus aureus protein A (SPA) affinity chromatography is extensively used in the purification of monoclonal antibody (mAb) therapeutics due to the specific interaction between the SPA ligand and the mAb Fc region, providing effective removal of impurities generated during the cell culture production phase of the process. However, the resulting eluate must still undergo further treatment through a series of polishing chromatography steps in order to adequately reduce impurity levels to an acceptable range specified by regulatory agencies before preparation of the drug substance and drug product. Conjugation of SPA ligands with polyethylene glycol (PEG), a non-toxic, inert, highly tunable, and water-soluble polymer, through a process called PEGylation has been previously studied in the context its potential to reduce non-specific binding events and its impact on mAb binding. The PEGylated SPA media was observed to reduce host cell protein content while retaining mAb yield during host cell culture fluid (HCCF) purification, increasing the selectivity of the resin for mAbs. SPA ligand PEGylation strategies were investigated in more detail in this work to gain a better understanding of the relationship between the excluded volume generated by the conjugated PEG and the binding behavior and purification efficiency of the PEGylated SPA media. In the first aim, characterization of commercially available CaptivA PriMAB SPA media was conducted to assess the changes in physical properties and binding behaviors of resins after PEGylation through the N-terminal conjugation of linear PEG chains ranging in size between 21.5 and 41.4 kDa, along with a branched 20.5 kDa PEG chain. PEGylation resulted in the alteration of the pore size distribution of the media as a function of PEG size, and varying impacts on the antibody binding capacities of the resins. HCCF purification with respect to host cell protein and residual DNA removal was most efficient with the 21.5 kDa PEGylated resin, although the amount of eluted aggregate content trended inversely with the volume of conjugated PEG per ligand. The 39% increase in selectivity for mAbs observed from HCP content reduction for the 21.5 kDa PEGylated resin compared to the unmodified media reinforces the viability of PEGylation as a means to improve the selectivity of SPA media for mAbs.
The second aim addresses the use of an alternative PEG attachment site. A novel SPA media based on a Z-domain dimer with an A104C point mutation was conjugated through a thiol-maleimide reaction to assess the impact of the PEG binding site on media antibody binding characteristics and HCCF purification efficiency. Maleimide activated PEGs differing in size between 5.4 and 20.6 kDa were assessed. The 5.4 kDa PEGylated media generally exhibited minimal impact on the binding capacity and dissociation constant of the media. Increasing conjugated PEG size resulted in reduced extents of PEGylation, but reduced the antibody binding capacity of the media and the antibody binding strength to the ligand. The 5.4 kDa PEGylated media was most effective in reducing impurities during HCCF purification while retaining reasonable mAb yields, indicating that the extent of PEGylation reaction may play a significant role in the reduction of nonspecific binding events. When comparing the 5.4 kDa PEGylated media to the unmodified media, a 98% increase in selectivity for mAbs through HCP content reduction and a 53% increase in selectivity for mAbs through rDNA content reduction are both observed with the PEGylated variant, further supporting PEGylation as a strategy to effectively improve selectivities of SPA medias.
In the third aim, molecular dynamics simulations of PEGylated B domains of SPA were used to gain insight into the orientation and space occupied by PEG and the polymer’s impact on the binding region of SPA on a molecular level. Simulations were conducted using linear 5 kDa, linear 20 kDa, and branched 20 kDa PEG chains attached at the N-terminus of SPA, as well as a linear 5 kDa PEG chain attached to the thiol group of a mutated B domain variant of SPA from alanine to cysteine at the 46th residue (A46C) which mimics the mutation investigated in Aim 2. Results indicated that the increased linear PEG size exhibited larger volume occupancy around the antibody Fc binding region of SPA. Additionally, PEG attachment at the thiol site showed no overlap with the SPA-mAb Fc binding region, reflecting the minimal influence on binding capacity observed in Aim 2. Impacts on the change in Gibbs free energy of the antibody Fc-PEG complex were estimated from the N-terminus PEGylated SPA simulations, while the thiol-targeted 5 kDa PEG revealed no effect due to the lack of occupation of the SPA-antibody binding region.
Overall, this work advanced the understanding of the performance of media with PEGylated SPA ligands, and provides insight into the relationship between the extent of reaction, size of conjugated PEG, and conjugated PEG volume on media performance and impurity clearance for the further optimization of PEGylation in protein A affinity chromatography.
Description
December 2024
School of Engineering
School of Engineering
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY