Optimization of a calcium-dependent Protein A-derived domain for mild antibody purification

Abstract

As reported here, we developed and optimized a purification matrix based on a Protein A-derived domain, ZCa, displaying calcium-dependent antibody binding. It provides an alternative to the acidic elution conditions of conventional Protein A affinity chromatography for purification of sensitive antibodies and other Fc-based molecules. We describe the multimerization of ZCa to generate a chromatography resin with higher binding capacity. The highest order multimeric variant, ZCaTetraCys, demonstrated a considerably high dynamic binding capacity (35 mg IgG/ml resin) while preserving the specificity for IgG. High recovery was obtained and host cell protein and DNA content in purified fractions proved to be comparable to commercial MabSelect SuRe and MabSelect PrismA. Various elution conditions for use of this domain in antibody purification were investigated. The purification data presented here revealed variations in the interaction of different subclasses of human IgG with ZCaTetraCys. This resulted in diverse elution properties for the different IgGs, where complete elution of all captured antibody for IgG2 and IgG4 was possible at neutral pH. This optimized protein ligand and the proposed purification method offer a unique strategy for effective and mild purification of antibodies and Fc-fusion proteins that cannot be purified under conventional acidic elution conditions due to aggregation formation or loss of function.

Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

“…Another essential contribution of this work is to promote a site-directed orientation of the ligand onto the BION. Different authors have evaluated the advantageous effects of a site-specific ligand immobilization on chromatographic beads − or nanoparticles. , On BION, accessible amino acids with affinity to iron oxide placed to a terminus of the protein can be used for this purpose . Venerando et al propose rules for the interaction of different proteins on iron oxides: proteins which have to change their tertiary structure to adapt to the surface during the binding process undergo conformational changes and, therefore, may lose their functionality .…”

Integrated continuous biomanufacturing on pilot scale for acid‐sensitive monoclonal antibodies

“…Efficient HCP removal was seen for both the capture step and the two polishing steps, with a ca 5‐log reduction in the final product as compared to the harvest (Figure 5b). After the capture step using the Z Ca resin, the impurity levels were reduced from >10 5 ppm to on average 300 ppm, meaning approximately a 3‐log reduction, which is expected from commercial Protein A resins (Cytiva, 2015b; Scheffel et al, 2019). A further decrease is demonstrated after CEX, with HCP levels of on average 13 ppm and finally 2 ppm after AEX, which is low in comparison to other mAb processes (Cytiva, 2015b).…”

“…N-Glycosylation analysis of purified mAb (AEX eluate) was performed with a GlycoWorks RapiFluor-MS N-Glycan kit and an ACQUITY UPLC H-Class system with a BEH Amide column (all Waters). The DNA content was measured in the harvest, capture, CEX and AEX eluate according to Scheffel et al (2019) after each sample had been stored at +4°C for a couple of days. The HCP levels were analyzed in the same samples as previously described (Scheffel et al, 2022) where the details of the analytical method for the detection of Tnbp can also be found.…”

Rational design of affinity ligands for bioseparation

“…Here, Hober's group successfully engineered the Z-domain to attain a calcium-dependent elution behavior in the presence of antibodies, therefore drastically reducing the harshness of the elution process [49]. Furthermore, the domain's multimerization was shown to possess significantly high dynamic binding capacities and to reduce host cell proteins and DNA content after purification of trastuzumab from CHO cell culture, when compared to the performance of commercial resins [50]. This multimerization feature of biological ligands in tandem is particularly interesting for affinity reagents because it is shown to improve performance taking advantage of avidity effects [51,52].…”