Protein adsorption and separation with monomodal and multimodal anion exchange chromatography resins. Part II. Mechanisms of protein aggregation on the chromatographic surface

Abstract

BACKGROUND Proteins on chromatographic surfaces can undergo conformational changes, leading to aggregate formation. Such effects have been reported previously for hydrophobic interaction and cation exchange chromatography. In Part I of this work, we examined protein adsorption behavior on monomodal (Nuvia HP‐Q) and multimodal anion exchangers (Nuvia aPrime and Capto Adhere), observing that aggregates are also formed on these multimodal resins. In this work we examine the underlying mechanisms and the conditions that affect aggregate formation. RESULTS Incubating the multimodal anion exchangers with pure bovine serum albumin monomer at low ionic strength results in the formation of dimer and higher‐order oligomers (up to hexamers) to an extent that depends on the incubation time. The percentage of total protein initially bound that is desorbed decreased from >85% with a 5 min incubation to <40% with 24 h incubation at pH 7.0, indicating that additional irreversibly bound species are formed on the resin surface. Load buffers leading to stronger binding (higher pH and/or lower salt) and higher temperatures (32 °C) result in more extensive aggregation. Minimal aggregation occurs, instead, for Nuvia HP‐Q. CONCLUSION BSA oligomers are formed directly on the surface of the multimodal resins studied and are eluted as discrete species at increasing salt concentrations. Aggregate formation is promoted by the ligand hydrophobicity and affects the separation of monomer–dimer mixtures by frontal chromatography, as the ability of the dimer in the feed to displace the adsorbed protein is impaired by gradual conversion of the monomer to stronger bound species. © 2022 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Time‐dependent sorption behavior of lentiviral vectors during anion‐exchange chromatography

“…The rate of product loss, defined by the Langmuir‐spreading model, is dependent on the proteins rigidity, binding strength, and the free surface available (Ueberbacher et al, 2008). Similar observations were also made during adsorption of bovine serum albumin (BSA) on multimodal AIEX resins (Roberts & Carta, 2022). Here, loss of recoverable material with time was attributed to an increase in irreversibly bound species that result from aggregation of BSA into higher order oligomers.…”

“…Unlike other viral vector systems where dual peak profiles can be due to empty capsids, both these peaks contain functionally active vector. Material detected in these peaks may have differences in structure or stability that impact contact time‐related losses, as is the case for proteins during HIC where adiabatic compressibility and binding strength impact recovery loss (Roberts & Carta, 2022; Ueberbacher et al, 2008). We wished to assess the impact of adsorption time on vector eluted at different NaCl concentrations and whether peak 1 and peak 2 differed in their rate of loss to the irreversibly bound state.…”

Bovine Serum Albumin and Myoglobin Separation by Ion-Exchange SMB

“…The breakthrough curve exhibiting a sharp increase in the outlet concentration followed by a significant tail in protein concentration can be attributed to several plausible reasons. In the literature, it is possible to find that aggregation can occur with this protein, especially when working in a fixed-bed column . Therefore, instead of only a monomer being adsorbed from the protein molecule in each site, it can also be dimers and higher-order aggregates.…”

“…In the literature, it is possible to find that aggregation can occur with this protein, especially when working in a fixed-bed column. 28 Therefore, instead of only a monomer being adsorbed from the protein molecule in each site, it can also be dimers and higher-order aggregates. This phenomenon is favored by the proximity of other molecules on the surface, i.e., higher surface loadings.…”

“…The mass balance in the adsorbent particle is described by the linear driving force (LDF) equation • Extract node (25) • Feed node (26) • Raffinate node (27) • Nodes between other columns (28) Node global mass balances Additionally, to the column mass balances, the mathematical model used in this work also includes the dead volumes of the system, represented by transfer lines in which the plug flow is assumed, to describe the experimental SMB operation more accurately.…”