Chromatography-Free Antibody Purification by Affinity-Phase Separation

Abstract The objective of this NIH Phase II SBIR proposal is to demonstrate the commercial feasibility of IsoTag™, a novel cost-effective method to purify monoclonal antibodies (mAbs) that eliminates the need for Protein A (PrA) chromatography. This objective will be achieved by using a recombinant fusion comprised of a stimulus responsive elastin-like polypeptide (ELP) that can undergo a reversible soluble to insoluble phase transition, and the Z-domain (ZD) derived from PrA that binds the constant region of mAbs with high affinity and specificity. This proposal is motivated by the fact that the first and critical step of mAb purification —PrA affinity chromatography— has not kept pace with improvements in the recombinant expression level of mAbs, creating a bottleneck in their production. IsoTag™, which combines affinity capture of mAbs and their isolation by triggered phase separation with microfiltration, addresses this bottleneck. Upon addition of the ELP-ZD fusion to clarified cell culture harvest, the ELP-ZD binds the mAb. Upon triggering the phase transition of the mAb/ELP-ZD with a small amount of kosmotropic salt, the mAb/ELP-ZD complex undergoes liquid-liquid phase separation into micron-sized droplets, allowing it to be separated from all soluble contaminants by continuous tangential flow filtration, Next, the pH is lowered to ~4.0, which causes the ZD to lose its affinity for the mAb. The pure, eluted mAb is in the soluble phase and the ELP-ZD can be regenerated from the insoluble phase. In the NIH funded Phase I of this project, we have fully optimized the ELP-ZD and the process parameters for IsoTag™ purification. We have shown that at the bench scale, IsoTag™ can be used to successfully isolate mAb from mammalian cell culture with yields and contaminant removal that outperform commercially available alternatives (GE Predictor Plate and Thermo Fisher Nab Spin Column). Having established technical feasibility and developed a research use only, bench scale product, we plan to validate IsoTag™andapos;s commercial feasibility for large-scale mAb purification in this Phase II proposal. Our Specific Aims to achieve this objective are: (1) We will optimize microbial production of the ELP-ZD fusion using fermentation and develop a scalable purification process. We will then perform a 30 L pilot production run and begin developing an ISO9001 quality control management protocol. (2) We will validate an assay for quantifying residual ELP-ZD. (3) We will perform side-by-side purification of the NISTmAb at 30 L scale to compare IsoTag™ to leading PrA resins, MAbSelect SuRe and Amsphere A3. Last, we will use BioSolve and Matlab software to develop a model for an interface that takes a customerandapos;s inputs and calculates a personalized cost savings by switching to IsoTag™. The outcome of this project will be compelling data that encourages customer conversion. Isolereandapos;s vision is that IsoTag™ will be a plug-and-play downstream process that allows the industry to achieve fully integrated, continuous manufacturing where improved productivity and lower cost can make mAbs globally accessible.PROJECT NARRATIVE Monoclonal antibodies (mAb) are an important and fast growing drug class, but the first phase in their purification process is a capture step via Protein A chromatography, which requires expensive resin and equipment making it difficult and time consuming to execute at commercial scale. Our IsoTag™ technology will provide an innovative, non-chromatographic alternative that combines affinity/phase separation with microfiltration. IsoTag™ will create a paradigm shift in the bioprocessing industry with its lower cost and continuous process that offers faster mass transfer, milder elution conditions, and enhanced productivity.