Preparation and characterization of prototypes for multi-modal separation aimed for capture of positively charged biomolecules at high-salt conditions

Abstract

Several prototypes of aromatic (Ar) and non-aromatic (NoAr) cation-exchange ligands suitable for capture of proteins from high conductivity (ca. 30 mS/cm) mobile phases were coupled to Sepharose 6 Fast Flow. These new prototypes of multi-modal cation-exchangers were found by screening a diverse library of multi-modal ligands and selecting cation-exchangers resulting in elution of test proteins at high ionic-strength. Candidates were then tested with respect to breakthrough capacity of bovine serum albumin (BSA), human IgG and lysozyme in buffers adjusted to a high conductivity. By applying a salt-step or a pH-step the recoveries were also tested. We have found that aromatic multi-modal cation-exchanger ligands based on carboxylic acids seem to be optimal for the capture of proteins at high-salt conditions. Experimental evidence on the importance of the relative position of the aromatic group in order to improve the breakthrough capacity at high-salt conditions has been found. It was also found that an amide group on the alpha-carbon was essential for capture of proteins at high-salt conditions. Compared to a strong cation-exchanger such as SP Sepharose Fast Flow the best new multi-modal weak cation-exchangers have breakthrough capacities of BSA, human IgG and lysozyme that are 10-30 times higher at high-salt conditions. The new multi-modal cation-exchangers can also be used at normal cation-exchange conditions and with either a salt-step or a pH-step (to pH-values where the proteins are negatively charged) to accomplish elution of proteins. In addition, the functional performance of the new cation-exchangers was found to be intact after treatment in 1.0 M sodium hydroxide solution for 10 days. For BSA it was also possible to design cation-exchangers based on non-aromatic carboxyl acid ligands with high capacities at high-salt conditions. A common feature of these ligands is that they contain hydrogen acceptor groups close to the carboxylic group. Furthermore, it was also possible to obtain high breakthrough capacities for lysozyme and BSA of a strong cation-exchanger (SP Sepharose Fast Flow) if phenyl groups were attached to the beads. Varying the ligand ratio (SP/Phenyl) could be used for optimizing the function of mixed-ligand ion-exchange media.

Expanded bed adsorption/desorption of proteins with Streamline Direct CST I adsorbent

“…Streamline Direct CST I is a new type of cation exchanger with multimodal functional groups, which not only takes advantage of electrostatic interaction, but also takes advantage of hydrogen bond interaction and hydrophobic interaction to tightly bind proteins. In other words, the new type of multi-modal ligand on adsorbent is able to interact with proteins through various intermolecular forces to get a high binding capacity in high ionic strength and salt concentration feedstocks (Johansson et al, 2003a(Johansson et al, , 2003b.…”

Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR

“…Recent advances in the design of multimodal (MM) chromatographic systems have produced previously undescribed classes of chromatographic materials that can provide alternative and improved selectivities as compared to traditional single mode chromatographic materials (3)(4)(5)(6)(7)(8)(9). Johansson et al have developed a library of MM ligands that can be employed for the capture of charged proteins under high salt conditions (4,5).…”

“…Johansson et al have developed a library of MM ligands that can be employed for the capture of charged proteins under high salt conditions (4,5). Liu et al have developed a silica-based MM resin capable of weak anion-exchange and reversed-phase interactions for the simultaneous separation of acidic, basic, and neutral pharmaceutical compounds (9).…”

Design of Polymeric Porous Adsorbent Media and the Dynamic Behavior of Transport and Adsorption of Bioactive Molecules in Such Media

“…Cross-linked agarose with desired porosity and stability has been found to be a suitable base matrix and polysaccharides such as dextran have been found to be useful extenders [3]. Depending on the biomolecules to be captured, the affinity ligands in the IEC systems are either positively charged groups, e.g., amine groups, [3,6] or negatively charged groups, e.g., carboxyl, sulphonic acid, [3,7]. The binding between the extender and the base matrix and between the affinity ligands and the extender involves covalent bonds.…”