Protein A (ProA) chromatography is a highly selective capture and separation technique in use throughout the biopharmaceutical industry for the downstream purification of monoclonal antibodies (mAbs). We have previously demonstrated that chemical modification of ProA affinity ligands with polyethylene glycol (PEGylation) can increase the selectivity of ProA chromatography an order of magnitude or more by sterically suppressing non-specific mock contaminant binding interactions during processing. However, we have also observed that resin PEGylation results in the sacrifice of up to 37% of the static binding capacity of human IgG depending on the PEG molecular weight and reaction extent. Through detailed analysis of IgG mass transfer and binding kinetics, we observe a combination of fast and slow uptake kinetics within the unmodified and PEGylated Protein A resins. Here, we show that PEGylation is responsible for removing a portion of the slower binding capacity, which is hypothesized to represent 2:1 IgG:ProA interactions. This allows for the PEGylated resin, depending on modification extent, to retain its dynamic binding capacity in column breakthrough experiments. We further investigate the mechanism of the slower IgG uptake kinetics in the resins through time-series fluorescent imaging via confocal laser scanning microscopy (CLSM). Using a dual-label approach, we spatially resolve the adsorption front within the resin particles over time and use the results to develop a relationship between IgG effective diffusivity and the concentration of adsorbed protein. The relationship between uptake kinetics and resin pore size distribution is also investigated via inverse size exclusion chromatography performed before and after loading the resins with human IgG. We connect changes in pore size distribution to IgG and PEG excluded volume and examine the effects of porosity, tortuosity, and hindrance on resin performance. This work culminates in a mechanistic model to predict resin performance in a column based on fundamental mass transfer and kinetic parameters of the same resin obtained in batch measurements.

Mechanistic modeling of antibody transport, adsorption equilibrium, and uptake kinetics in protein A chromatography resins with PEGylated ligand

251st National Meeting (2016)

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