Protein Adsorption and Separation in Multimodal Anion Exchange Chromatography Media

Roberts, Joey, Chemical Engineering - School of Engineering and Applied Science, University of Virginia
Carta, Giorgio, EN-Chem Engr Dept, University of Virginia

Multimodal chromatography resins comprising ligands that combine charged and hydrophobic moieties provide advantages over their monomodal counterparts. In order to implement these resins in practical processes, it is necessary to understand how ligand chemistry and the physical properties of the resin matrix impact protein adsorption, selectivity, and transport.

This work focuses on multimodal anion exchange (MMAEX) resins combining a quaternary ammonium ion group with hydrophobic moieties. The first portion of this work includes physical characterization of four MMAEX resins with different ligand chemistries. Single-component systems of the relatively large proteins bovine serum albumin (BSA) monomer, BSA dimer, and thyroglobulin (Tg) are studied. Adsorption kinetics are shown to be controlled by pore diffusion for all four resins and proteins, but with diffusivities that decrease as the protein size increases.

The second portion of this work focuses on frontal separation performance for two multimodal anion exchange resins, Nuvia aPrime and Capto Adhere ImpRes, compared with the monomodal resin Nuvia HP-Q using mixtures of monomeric and dimeric bovine serum albumin as a model. Although preferential binding of the dimer over monomer is observed for all three resins, frontal separation results show better separation performance on Nuvia HP-Q compared to the multimodal resins at low ionic strength. Additionally, elution of the loaded protein from the multimodal resins shows increased composition of aggregates and oligomers that were not present in the feed mixture.

Aggregate formation in multimodal resins is investigated in the third portion. For the multimodal resins, adsorbing BSA in pure monomeric form at low ionic strength and desorbing in 1 M NaCl results in the formation of dimer and higher order oligomers to an extent that depends on the time of incubation and the load conditions. Longer incubation times, load buffers leading to stronger binding, and higher temperatures resulted in more extensive formation of oligomers. The oligomers appear to be formed directly on the chromatographic surface. Oligomer formation on the multimodal resins affects the separation of monomer-dimer mixtures by frontal chromatography impairing the ability of the dimer in the feed to displace the adsorbed monomer since the latter is gradually converted to more strongly bound species during loading.

Last, an analysis of the relationship between the number of plates measured with a small molecule tracer and the breakthrough curve of a strongly bound protein in short laboratory chromatography columns (1-5 cm) considering flow non-uniformity is presented. The model presented provides a tool to model experimental breakthrough data and to assess the degree of flow uniformity required to obtain meaningful dynamic binding capacity measurements using minicolumns in a high-throughput lab setting.

PHD (Doctor of Philosophy)
Protein chromatography, Multimodal anion exchange, Competitive adsorption, Protein aggregation
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