Optimization of a ligand immobilization and azide group endcapping concept via “Click-Chemistry” for the preparation of adsorbents for antibody purification

Influence of different spacer arms on Mimetic Ligand™ A2P and B14 membranes for human IgG purification

“…2a strictly for comparison reasons. In case of the B14-2LP ligand-spacer combination, an earlier study for polymethacrylate beads had provided evidence that the amino groups incorporated in the spacer chain and the surface endcapping had lead to reduced IgG binding capacities due to secondary binding of feed impurities [26]. Comparable adsorbents with B14-TRZ ligand spacer combination lacked this drawback and even showed an improved IgG capture performance compared to commercial Protein A media, when operated at low bead height and fast flow rates [28].…”

“…1). A recent study describes the IgG capture performance of B14-ligand bound via triazole linkage (TRZ) [26,27] to a new polymethacrylate based adsorbent (FractoAIMs-3). It was shown that B14 is suitable for IgG 1 capture at low bed heights and high flow-rates from cell culture feed [28], which can be considered being ideal properties for a corresponding membrane adsorber.…”

Molecular modeling to rationalize ligand–support interactions in affinity chromatography

“…In particular, the use of a specific spacer, a 1,4-substituted [1,2,3]-triazole (TRZ), allowed improving the binding capacity of the affinity material when pluronic acid was present. Successive studies performed by Horak et al using the same spacer showed that the termination of the surface, in particular endcapping of epoxide and azide groups, can have a significant impact on the performances of the affinity material, avoiding the unwanted adsorption of non-target molecules [36]. These results clearly show that the optimization of the performances of an affinity material is a complex process, which involves the contextual optimization of ligand, spacer, and support surface.…”

Click chemistry: A route to designing and preparing pseudo-biospecific immunoadsorbent for IgG adsorption

“…It is clear that the click reaction of the azidated support, Sep-N 3 , with the alkyne-functionalized ligand, His-alkyne, can greatly promote the immobilization of ligand onto the support matrix, so significantly increasing the density of ligand covalently linked to the sepharose. The reason is that an alkyne and an azide moiety, a matched pair of reactive groups, can couple with each other to yield 1,2,3-triazole ring, which is in essence highly selective, fast and straight forward, and stable to wide ranges of reaction conditions [30,31], so that no side reaction can occur. As a highly selective click reaction between both "clickable" reactants, the Cu(I)-catalyzed azide-alkyne cycloaddition between Sep-N 3 and His-alkyne, can inhibit undesired reactions from occurring to a considerable extent, thus increasing the amount of ligands immobilized on the support in an expected way.…”

“…This click reaction is inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH [29,30]. These features make click chemistry particularly attractive in bioconjugation, such as the development of sepharose-based affinity chromatography agents [31,32]. Based on this idea, sepharose beads bearing alkyne and azide groups were prepared from a commercial "specialty" sepharose-amine in order to conveniently immobilize ligands, and the as-prepared "clickable" sepharose beads can be successfully used as precursors to functional sepharose matrices for affinity chromatography [30].…”