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.

Depth filtration is widely used in downstream bioprocessing to remove particulate contaminants via depth straining and is therefore applied to harvest clarification and other processing steps. However, depth filtration also removes proteins via adsorption, which can contribute variously to impurity clearance and to reduction in product yield. The adsorption may occur on the different components of the depth filter, that is, filter aid, binder, and cellulose filter. We measured adsorption of several model proteins and therapeutic proteins onto filter aids, cellulose, and commercial depth filters at pH 5-8 and ionic strengths <50 mM and correlated the adsorption data to bulk measured properties such as surface area, morphology, surface charge density, and composition. We also explored the role of each depth filter component in the adsorption of proteins with different net charges, using confocal microscopy. Our findings show that a complete depth filter's maximum adsorptive capacity for proteins can be estimated by its protein monolayer coverage values, which are of order mg/m , depending on the protein size. Furthermore, the extent of adsorption of different proteins appears to depend on the nature of the resin binder and its extent of coating over the depth filter surface, particularly in masking the cation-exchanger-like capacity of the siliceous filter aids. In addition to guiding improved depth filter selection, the findings can be leveraged in inspiring a more intentional selection of components and design of depth filter construction for particular impurity removal targets.

A diverse array of experimental techniques was used to compare protein sorption and transport on agarose-based resins with and without dextran extenders. Van 't Hoff plots indicated similar trends for lysozyme on the resins, with enthalpically dominated sorption. Salt breakthrough curves demonstrated significant exclusion of salt from the dextran, consistent with Donnan equilibrium predictions. Polarization-sensitive confocal microscopy and fluorescence recovery after photobleaching (FRAP) exhibited little effect of dextran modification on the respective rotational and translation mobilities of adsorbed protein at equilibrium. Finally, confocal microscopy imaging of intraparticle elution behavior showed that slower elution can occur for dextran-modified resins.

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.

A combined pore blockage and cake filtration model was applied to the virus filtration of an Fc-fusion protein using the three commercially available filters, F-1, F-2, and F-3 in a range of buffer conditions including sodium-phosphate and tris-acetate buffers with and without 200 mM NaCl at pH 7.5. The fouling behaviors of the three filters for the feed solutions spiked with minute virus of mice were described well by this combined model for all the solution conditions. This suggests that fouling of the virus filters is dominated by the pore blockage mechanism during the initial stage of the filtration and transformed to the cake filtration mechanism during the later stage of the filtration. Both flux and transmembrane resistance can be described well by this model. The pore blockage rate and the rate of increase of protein layer resistance over blocked pores are found to be affected by membrane properties as well as the solution conditions resulting from the modulation of interactions between virus, protein, and membrane by the solution conditions. K E Y W O R D S cake filtration, Fc-fusion protein, pore blocking model, solution conditions, virus filtration SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section. How to cite this article: Namila F, Zhang D, Traylor S, et al. The effects of buffer condition on the fouling behavior of MVM virus filtration of an Fc-fusion protein. Biotechnology and