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.

E2 nanocages functionalized with Z-domain-elastin-like polypeptide affinity ligands (Z-ELP) using Sortase A (SrtA) ligation have been shown to be a promising scaffold for purifying monoclonal antibodies (mAbs) based on affinity precipitation. However, the reversible nature of SrtA reaction has been attributed to the low ligation efficiency (<25%) and has significantly limited the practical utility of the technology. Here, we reported an improved conjugation platform using the SpyTag/SpyCatcher pair to form a spontaneous isopeptide bond between SpyTag-E2 and Z-ELP-SpyCatcher fusion proteins of two different ELP chain-lengths. Using this system, E2 ligation efficiencies exceeding 90% were obtained with both 40- and 80-repeat Z-ELP-SpyCatcher fusions. This enabled the production of nanocages fully functionalized with Z-ELP for improved aggregation and mAb binding. Compared to the 50% decorated Z-ELP-E2 nanocages produced by SrtA ligation, the fully decorated Z-ELP-Spy-E2 nanocages exhibited a 10 °C lower transition temperature and a 2-fold higher mAb binding capacity. The improved transition property of the longer Z-ELP backbone allowed for >90% recovery of Z-ELP-Spy-E2 nanocages at room temperature using 0.1 M ammonium sulfate after mAb elution. The flexibility of customizing different affinity domains onto the SpyTag-E2 scaffold should expand our ability to purify other non-mAb target proteins based on affinity precipitation.

Affinity precipitation is an ideal alternative to chromatography for antibody purification because it combines the high selectivity of an affinity ligand with the operational benefits of precipitation. However, the widespread use of elastin-like polypeptide (ELP) capture scaffolds for antibody purification has been hindered by the high salt concentrations and temperatures necessary for efficient ELP aggregation. In this paper, we employed a tandem approach to enhance ELP aggregation by enlarging the dimension of the capturing scaffold and by creating IgG-triggered scaffold cross-linking. This was accomplished by covalently conjugating the Z-domain-ELP (Z-ELP) capturing scaffold to a 25 nm diameter E2 protein nanocage using Sortase A ligation. We demonstrated the isothermal recovery of IgG in the virtual absence of salt due to the significantly increased scaffold dimension and cross-linking from multivalent IgG-E2 interactions. Because IgG cross-linking is reversible at low pH, it may be feasible to achieve a high yielding IgG purification by isothermal phase separation using a simple pH trigger.

Affinity precipitation using Z-elastin-like polypeptide-functionalized E2 protein nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high-yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multivalent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A-D) and one Fc fusion protein (Fc A) with diverse molecular properties. A molar binding ratio of 3:1 Z:mAb was sufficient to precipitate >95% mAb in solution for all molecules evaluated at ambient temperature without added salt. The effect of solution pH on aggregation kinetics was studied using a simplified two-step model to investigate the protein interactions that occur during mAb-nanocage crosslinking and to determine the optimal solution pH for precipitation. After centrifugation, the pelleted mAb-nanocage complex remained insoluble and was capable of being washed at pH ≥ 5 and eluted with at pH < 4 with >90% mAb recovery for all molecules. The four mAbs and one Fc fusion were purified from cell culture using optimal process conditions, and >94% yield and >97% monomer content were obtained. mAb A-D purification resulted in a 99.9% reduction in host cell protein and >99.99% reduction in DNA from the cell culture fluids. Nanocage affinity precipitation was equivalent to or exceeded expected Protein A chromatography performance. This study highlights the benefits of nanoparticle crosslinking for enhanced affinity capture and presents a robust platform that can be applied to any target mAb or Fc-containing proteins with minimal optimization of process parameters.

Existing assays for the quantification of monoclonal antibody (mAb) cell culture titer often require expensive instruments or reagents and may be limited by the low-throughput or tedious protocols. Here, we developed a quick and cost-effective alternative assay based on mAb-induced cross-linking with Z-domain-ELP-E2 nanocages functionalized by SpyTag/SpyCatcher conjugation. After mixing mAb samples with a fixed nanoparticle concentration for 10 min, we found that the turbidity, measured by absorbance at 600 nm, exhibited a high-signal-to-background ratio and was proportional to the mAb concentration. A simple logarithmic regression was found to fit (R 2 = 0.99) the turbidity data for mAb concentrations between 100 and 1000 μg/mL. The optimized assay procedure was validated using two industrial mAb cell culture harvests, and a bridging study using Octet biolayer interferometry with Protein A sensors confirmed accurate and reproducible results. The assay procedure can be easily adapted to a high-throughput format for rapid mAb titer screening.