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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 nonuniformity is presented. For practical conditions, while axial dispersion has only a small impact on the

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).

BACKGROUND Multimodal ion exchangers, containing ligands combining charged and hydrophobic moieties, exhibit enhanced separation compared to monomodal ion exchangers under low loading conditions. Commercial applications often require high protein loads and such conditions have not been investigated extensively, especially for multimodal anion exchangers. This work compares protein adsorption and separation by frontal chromatography for a commercial monomodal (Nuvia HP‐Q) and two multimodal anion exchangers (Nuvia aPrime and Capto Adhere) using bovine serum albumin (BSA) monomer and dimer as a model. RESULTS Greater dimer–monomer selectivity is observed for Nuvia aPrime and Capto Adhere than for Nuvia HP‐Q at low protein loads. However, binary adsorption isotherms at high protein loads show higher binding capacity and greater selectivity for Nuvia HP‐Q, despite having the same ligand density. Higher salt concentrations reduce the binding strength in all cases, but this reduction is less pronounced for the multimodal resins. As a result, selectivity increases for the latter at higher salt concentrations. Confocal laser scanning microscopy shows that, during co‐adsorption, displacement of the monomer by the dimer occurs for each resin. However, displacement becomes very slow for the multimodal resins during sequential adsorption. The multimodal resins give poorer separation by frontal chromatography, accompanied by low recovery and substantial on‐column formation of protein aggregates. CONCLUSION The selectivity of multimodal anion exchangers for separation of the model proteins studied varies with salt concentration and protein load. At high protein loads, multimodal resins can result in on‐column aggregate formation negatively impacting the kinetics of competitive binding and separation. © 2022 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).