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. !

Public Health Relevance

Monoclonal antibodes (mAb) make up an important and fast growing drug class and they are also imperative tools for the research and development of new drugs and diagnostics, but their commercial production is expensive and time consuming. The first phase in the purification process is a capture step via Protein A chromatography, which requires expensive resin and is difficult to execute at the industrial scale. Our technology will provide an innovative, chromatography-free alternative to Protein A chromatography: by fusing an antibody-binding domain to a stimulus-responsive biopolymer, the mAb can undergo phase separation from contaminants with a small amount of heat or salt. Because this technology uses a batch-type process and just three simple pieces of equipment, it has the potential to make mAb purification faster, easier, and more affordable.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZRG1)
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Wu, Mary Ann
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Isolere Bio, Inc.
United States
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