SBIR-STTR Award

The objective of this Phase I SBIR proposal is to develop a novel and cost-effective method for affinity purification of antibodies that eliminates the need for chromatography. This objective is motivated by the fact that monoclonal antibodies (mAbs) are the largest and fastest growing class of drugs, with approximately four new antibody drugs approved per year and a projected 2020 worldwide sales of nearly $125 billion. The significance of this SBIR application stems from the limitations of current mAb purification processes employed by industry, the first step of which is affinity-based chromatography using Protein A resin. Chromatography is notoriously difficult to scale up from bench to industry and requires complex, expensive equipment and chemical reagents. To address these time and cost limitations, we have developed a non- chromatographic technology that uses affinity-based phase separation as an alternative to chromatography. The core of this technology includes a mAb binding Z-domain (ZD) derived from staphylococcal Protein A. The ZD is recombinantly fused to a stimulus responsive elastin-like polypeptide (ELP); this fusion undergoes a soluble to insoluble phase transition that can be precisely tuned and is triggered by a small amount of heat (or isothermally with salt). Because host-cell proteins and other contaminants are not thermally sensitive, the phase transition of the covalent complex (mAb and ZD-ELP fusion) can be leveraged, producing pure mAbs after cycling through a few heated and cooled centrifugation steps. Unlike chromatography, our method is a batch process?and is hence easily scalable at lower cost? and it requires just three simple and inexpensive pieces of equipment: a centrifuge, a rotisserie, and a refrigerator. The central hypothesis of this proposal is that our non-chromatographic affinity-based aggregation method can be used to isolate antibodies secreted from mammalian expression systems with a level of purity comparable, if not superior to, Protein A chromatography. In preliminary studies, we have already demonstrated that ELP-ZD can be used to successfully isolate IgG from Expi293 cells. To achieve the objectives of this proposal, we will carry out the following specific aims: We will optimize the transition temperature by testing fusions with varied hydrophobicity and MW and select an ELP-ZD that transitions at room temperature. We will also optimize the number and configuration of ZDs, as assessed by the binding affinity and yield. We will quantify the removal of three major contaminants: host-cell proteins, DNA, and endotoxin. Finally, we will test the optimal ELP-ZD's ability to purify IgG therapeutics of different subclass as well as an Fc fusion, as these are the most common types of antibody-based therapeutics. This work is imperative for demonstrating and accelerating ELP-ZD's path to commercialization as an alternative to Protein A chromatography. The impact of this proposal will be the development of a highly scalable, non-chromatographic alternative to Protein A chromatography, which will greatly simplify and reduce the cost of purification of the fastest growing class of biologic drugs. !

Project Terms:
Address; Affinity; Affinity Chromatography; Amino Acids; Anions; Antibodies; Antibody Affinity; antibody engineering; Antibody Therapy; base; Behavior; Binding; Binding Proteins; Biological Assay; Biopolymers; Cations; Cell Culture Techniques; Cells; Centrifugation; Chemicals; Chimeric Proteins; Chromatography; Clinical; commercialization; Complex; cost; cost effective; Culture Media; Dependence; design; Development; DNA; Economics; Elastin; Endotoxins; Equipment; Escherichia coli; Excision; Exhibits; Foundations; Gold; High Pressure Liquid Chromatography; Human; Hydrophobicity; Immunoglobulin Constant Region; Immunoglobulin G; improved; Industrialization; Industry; innovation; Location; Mammalian Cell; Marketing; Measures; Mediating; Methods; Monoclonal Antibodies; novel; novel diagnostics; novel therapeutics; Outcome; Peptides; Pharmaceutical Preparations; Phase; Phase Transition; Plant Resins; Polymers; polypeptide; Process; Production; protein complex; Protein Engineering; Proteins; Reagent; Recombinants; Recovery; Research; research and development; RNA; Sales; scale up; Small Business Innovation Research Grant; Sodium Chloride; Specificity; Staphylococcal Protein A; stem; Stimulus; System; Technology; Temperature; Testing; Therapeutic; Therapeutic Human Experimentation; Therapeutic Monoclonal Antibodies; Time; tool; Transition Temperature; Variant; Vertebral column; Work;

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™'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 customer'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. Isolere'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.

Public Health Relevance Statement:
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.

Project Terms:
Address; Adoption; Affinity; Affinity Chromatography; Antibodies; Bacteria; Behavior; Binding; Biological Assay; bioprocess; Capital; Cell Culture Techniques; Cell Density; Cells; Centrifugation; Chimeric Proteins; Chromatography; Complex; Computer software; Consumption; cost; cost effective; Cost Savings; Cyclic GMP; Data; Development; DNA; Elastin; Endotoxins; Ensure; Equipment; Escherichia coli; Excision; Exhibits; Fermentation; Filtration; Formulation; Funding; Harvest; Immunoglobulin Constant Region; improved; Industry; innovation; Liquid substance; Mammalian Cell; Methods; microbial; Modeling; Monoclonal Antibodies; Natural regeneration; novel; novel therapeutics; Outcome; Oxygen; Peptides; Pharmaceutical Preparations; Phase; Phase Transition; Plant Resins; Plasmids; Play; Polymers; polypeptide; Process; Production; Productivity; Protein Engineering; protein purification; Proteins; Protocols documentation; Quality Control; Reagent; Recombinant Fusion Proteins; Recombinants; Research; Residual state; Running; Sales; scale up; Schedule; Side; Small Business Innovation Research Grant; Sodium Chloride; Specificity; Staphylococcal Protein A; Staphylococcus aureus; Stimulus; Technology; Temperature; Therapeutic Human Experimentation; Time; time use; tool; United States National Institutes of Health; Virus; Vision