A novel Protein A alternative stationary phase based on the variable heavy chain fragment of immune camelid antibody was evaluated and compared to commonly used commercial Protein A resins. The parameters evaluated were elution pH, equilibrium isotherm, dynamic binding capacity, and host cell protein clearance using a set of Chinese hamster ovary-derived monoclonal antibodies and Fc-fusion proteins. Linear retention experiments were used to compare the specificity of these resins for both non-IgG model proteins as well as antibodies and Fc-fusion proteins. The experimental results showed that the new camelid antibody resin behaved very similarly to Protein A resins in terms of retention of non-IgG model proteins and IgG-based molecules. Dynamic binding capacity was found to be comparable for Fc-fusion proteins and slightly lower for antibodies. Host cell protein clearance profiles were also similar under preparative conditions using complex biological feeds. Finally, the binding mechanism was explored by using different mobile-phase modifiers in linear pH gradient retention experiments.
Recently, a novel technique was developed for the rapid identification of affinity ligands against a diverse set of targets using the variable heavy-chain (VHH) region of single-chain antibodies found in the Camelidae family.25 These molecules possess good specificity because of their enlarged hypervariable region and have very high physical and thermal stability because of their single-domain nature.25,26 Recombinantly expressed VHH fragments can be used as affinity ligands and have applications in laboratory-scale immunoaffinity and immunoperfusion chromatography.27 Their potential application at industrial scale became more promising after it was proven that these antibody fragments can be expressed efficiently in microorganisms such as the yeast Saccharomyces cerevisiae.28
This technology has been commercialized by the Bio Affinity Company (Naarden, The Netherlands) to generate ligands (called CaptureSelect ligands) that can be customized for any purification challenge. Unlike other proteinaceous ligands (such as Protein A), these ligands have the distinct advantage of being stable in strongly alkaline solutions.29–31 One such ligand was generated against the Fc-region of human IgGs. This was shown to bind to all human IgG subclasses and no cross-reactivity was found with bovine or mouse IgG.30 This ligand has the additional advantage of being specific for human IgGs only and unlike Protein A, it can bind IgG3s as well. In 2007, the ligand was immobilized on a highly cross-linked agarose-based backbone through a long, hydrophilic spacer arm and marketed through GE Healthcare (Uppsala, Sweden) as IgSelect affinity medium.32 Although this resin can potentially be an attractive and manufacturing-friendly alternative to Protein A chromatography, very limited data exists so far on the performance of this new resin.29
This article provides the first comprehensive evaluation of this new ligand for MAb purification. Using several industrial MAbs and Fc-fusion proteins, the performance of this resin was compared to two of the most commonly used commercial Protein A resins: MabSelect from GE Healthcare and ProSep-vA High Capacity from Millipore (Billerica, MA). These two Protein A resins are on two different backbones (agarose versus controlled pore glass) and were specifically chosen to represent a wide spectrum of Protein A resins. The parameters evaluated were binding affinity, dynamic binding capacity, selectivity, and binding thermodynamics. Finally, the binding mechanism of IgSelect resin also was explored by linear gradient retention experiments with different mobile-phase modifiers.