One‐step affinity capture and precipitation for improved purification of an industrial monoclonal antibody using Z‐ELP functionalized nanocages

Abstract

Protein A chromatography has been identified as a potential bottleneck in the monoclonal antibody production platform, leading to increased interest in non-chromatographic capture technologies. Affinity precipitation using environmentally responsive, Z-domain-elastin-like polypeptide (Z-ELP) fusion proteins has been shown to be a promising alternative. However, elevated temperature and salt concentrations necessary for precipitation resulted in decreased antibody monomer content and reduced purification capacity. To improve upon the existing technology, we reported an enhanced affinity precipitation of antibodies by conjugating Z-ELP to a 25 nm diameter, self-assembled E2 protein nanocage (Z-ELP-E2). The enlarged scale of aggregate formation and IgG-triggered crosslinking through multi-valent binding significantly outperformed traditional Z-ELP-based methods. In the current work, we sought to develop an affinity precipitation process capable of purifying industrial monoclonal antibodies (mAbs) at ambient temperature with minimal added salt. We discovered that the mAb-nanocage complex aggregated within 10 min at room temperature without the addition of salt due to the enhanced multi-valent cross-linking. After precipitating out of solution, the complex remained insoluble under all wash buffers tested, and only resolubilized after a low pH elution. Through optimization of key process steps, the affinity precipitation yield and impurity clearance met or exceeded protein A chromatography performance with 95% yield, 3.7 logs host cell protein reduction, and >5 logs of DNA reduction from mAb cell culture. Because of the operational flexibility afforded by this one-step affinity capture and precipitation process, the Z-ELP-E2 based approach has the potential to be a viable alternative to platform mAb purification.

High‐efficiency affinity precipitation of multiple industrial mAbs and Fc‐fusion proteins from cell culture harvests using Z‐ELP‐E2 nanocages

“…The goal of this study was to demonstrate the utility of the Z‐ELP‐E2 nanocage for the affinity precipitation of multiple mAbs and Fc‐fusion proteins with different molecular properties (Table ). Earlier results using a mAb designated “mAb C” from BMS demonstrated that a 3:1 Z‐domain:mAb molar ratio was optimal for precipitation at ambient temperature without any addition of salt (Swartz et al, ). To investigate whether this was true for other mAbs or Fc‐fusion proteins, Z‐ELP‐E2 nanocages were mixed with the purified target proteins at molar binding ratios ranging from 1:1 to 5:1 in PBS at 23°C (Figure a).…”

“…Z‐ELP[KV 8 F‐40]‐LPETG, a modified GGG‐E2 from Bacillus stearothermophilus , and sortase A from Staphylococcus aureus were expressed in E. coli and Z‐ELP was conjugated to E2 using the previously described procedures (Swartz et al, ). Briefly, Z‐ELP purified by inverse transition cycling (ITC; Meyer & Chilkoti, ), E2 partially purified by 70°C heating, and sortase A soluble lysate were mixed in a reaction buffer for 8 hr at 23°C and the ligation product was purified by ITC into phosphate‐buffered saline (PBS; 20 mM sodium phosphate, 150 mM sodium chloride, pH 7.2).…”

“…Previous work used an mAb designated “mAb C” in the current investigation (Swartz et al, ). All experiments using mAb C were repeated using the indicated procedures below.…”

“…mAb concentrations were determined using the theoretical extinction coefficient for each molecule. The supernatants were assumed to contain only mAb based on previous results (Swartz et al, ). For binding ratio experiments, 20 µM mAb samples were mixed with 20–100 µM Z‐ELP‐E2 (1:1 to 5:1 Z‐domain:mAb molar ratio) in PBS for 1 hr before centrifugation for mAbs A–D.…”

“…We performed high‐throughput process optimization using a model industrial mAb, and developed a simple, one‐step antibody affinity capture and precipitation platform for mAb purification from Chinese hamster ovary (CHO) cell culture fluids with equivalent yields and impurity clearance compared with Protein A chromatography (Swartz, Xu, Traylor, Li, & Chen, ). On mixing at a 3:1 Z‐domain:mAb molar ratio, >95% mAb was precipitated within minutes at ambient temperature without added salt due to extensive nanocage crosslinking.…”