SBIR-STTR Award

The biological function and physicochemical properties of protein-­based therapeutics are determined by their higher order structures (HOS). Therefore, it is imperative to analyze the three-­dimensional higher order structure of protein therapeutics at several stages of the drug development process to ensure both safety and efficacy of the drug. Biopharmaceutical manufacturers are required to demonstrate the consistency of the protein HOS conformation to the regulatory agencies. Moreover, Identifying the binding site of a protein to its corresponding antigen (known as epitope mapping) is critical for the development of new therapeutics, vaccines and diagnostics. Food and Drug Administration (FDA) guidelines require specific binding site information between a drug and its target for the regulatory filing. Current techniques for protein HOS characterization and epitope mapping are slow, expensive and difficult to perform. We have developed a technology called Plasma Induced Modification to Biomolecules that addresses the need of the industry for routine structural, mass spectrometry-­based protein HOS analysis. PLIMB generates sub microsecond bursts of hydroxyl (OH) radicals from water to label proteins in solution. The OH radicals covalently labels the solvent accessible regions of the protein and subsequent mass spectrometric analysis reveals single amino acid level structural information. A way to quickly and efficiently analyze higher order structures of proteins on a benchtop scale, PLIMB will enable faster development of protein therapeutics, throughout the drug discovery process. In SBIR Phase I, we will prove the feasibility of a hydroxyl radical detection system that will tightly control the OH radical dose generated by PLIMB to yield highly reproducible HOS data. After completion of the SBIR Phase I project, we will continue the development and validation of the PLIMB system for commercial use. Ultimately, PLIMB will enable fast, high resolution structural analysis of proteins, a capability which is highly sought after in the pharmaceutical industry.

Public Health Relevance Statement:
Project Narrative The goal of this SBIR is to develop a highly reproducible, benchtop instrument to perform Hydroxyl Radical Protein Footprinting (HRF) for analysis of three-dimensional structures in protein therapeutics. This instrument will enable structural characterization of proteins at a much higher throughput than traditional techniques to better drive the discovery and development of protein therapeutics.

Project Terms:
absorption; Address; Adopted; Amino Acids; antigen antibody binding; Antigens; Autoimmune Diseases; base; Binding Sites; Biological Process; Biological Products; Biotechnology; Chemicals; commercial application; Computer software; Consumption; Data; design; Detection; detector; Development; Diagnostic; Disease; Dose; drug development; drug discovery; drug efficacy; Drug Industry; Ensure; Epidermal Growth Factor Receptor; Epitope Mapping; Equilibrium; Feedback; Generations; Goals; Guidelines; Health; high dimensionality; Higher Order Chromatin Structure; Human; Hydroxyl Radical; improved; Industry; Industry Standard; instrument; Label; Legal patent; Life; Malignant Neoplasms; Manufacturer Name; Mass Spectrum Analysis; Measurement; Measures; Medicine; Methods; Modification; Molecular Conformation; Monitor; novel therapeutics; Nuclear Magnetic Resonance; off-patent; Outcome; Oxides; Peptides; Peroxides; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Plasma; Preclinical Drug Development; Process; Property; Protein Analysis; Protein Engineering; Protein Footprinting; Protein Region; protein structure; Proteins; Quality Control; rapid growth; Reproducibility; Resolution; Safety; Sampling; Small Business Innovation Research Grant; Solvents; Spectrum Analysis; Speed; Structural Protein; Structure; Surface; Synchrotrons; System; Techniques; Technology; Therapeutic; therapeutic protein; three dimensional structure; Three-dimensional analysis; Time; United States Food and Drug Administration; Universities; Vaccines; Validation; Water; Wisconsin; Work; X-Ray Crystallography

Immuto Scientific Inc. is developing a robust, automated benchtop instrument to perform high-throughput structural characterization of protein biotherapeutics in a fraction of time of traditional structural characterization techniques such as x-ray crystallography and cryo-EM. Protein-based biotherapeutics (such as antibodies, insulin, recombinant vaccines, etc.) are one of the most effective classes of modern medicines for the treatment of a wide variety of diseases including cancers, autoimmunity/inflammation, genetic disorders, and infectious diseases such as COVID-19. The biological function and physicochemical properties of biotherapeutics are determined by their higher order structures (HOS)-the folding and three-dimensional conformation that largely dictates function and stability. Therefore, it is imperative to analyze the three- dimensional higher order structure of protein therapeutics at several stages of the drug development process to ensure both safety and efficacy of the drug. Biopharmaceutical manufacturers are required to demonstrate the consistency of the protein HOS conformation to the regulatory agencies. Moreover, identifying the binding site of a therapeutic protein (such as a monoclonal antibody) to its corresponding antigen (known as epitope mapping) is critical for the development of new antibody therapeutics, vaccines and diagnostics. Food and Drug Administration (FDA) guidelines require specific binding site information between a drug and its target for the regulatory filing. Current techniques for protein HOS characterization and epitope mapping are complex, resource intensive and can take up to 6-12 months to perform. Based on feedback received from over 300 customer pharmaceutical customers, we have developed a technology called Plasma Induced Modification to Biomolecules (PLIMB) that addresses the need of the industry for routine structural, mass spectrometry-based protein HOS analysis. PLIMB generates sub microsecond bursts of hydroxyl (OH) radicals from water to label proteins in solution. The OH radicals covalently label the solvent accessible regions of the protein and subsequent mass spectrometric analysis reveals single amino acid level structural information. With PLIMB, HOS analysis and epitope mapping can be performed in under 48 hours where current techniques such as x- ray crystallography and Cryo-EM takes several months to perform. In Phase II, we will first incorporate the hydroxyl radical detection system that was designed in Phase I into a fully automated, manufacturable PLIMB instrument, and then validate the PLIMB system for commercial use. Ultimately, PLIMB will be a revolutionary new tool for pharmaceutical researchers that will provide new capabilities to better engineer highly effective protein biotherapeutics and accelerate the drug discovery timeline.

Public Health Relevance Statement:
Project Narrative The goal of this SBIR Phase II project is to develop a robust, high throughput, automated benchtop instrument to perform Hydroxyl Radical Protein Footprinting (HRF) for analysis of three-dimensional structures in protein therapeutics. This instrument will enable structural characterization of proteins at a much higher throughput than traditional techniques to better drive the discovery and development of protein therapeutics.

Project Terms:
Amino Acids; aminoacid; Amplifiers; Antibodies; Monoclonal Antibodies; Clinical Treatment Moab; mAbs; Antigens; immunogen; Autoimmune Diseases; autoimmune condition; autoimmune disorder; Autoimmunity; Autoimmune Status; Binding Sites; Combining Site; Reactive Site; Biological Products; Biologic Products; Biological Agent; biologics; biopharmaceutical; biotherapeutic agent; Biological Response Modifier Therapy; Biological Therapy; biological therapeutic; biological treatment; biologically based therapeutics; biotherapeutics; biotherapy; Biotechnology; Biotech; Malignant Neoplasms; Cancers; Malignant Tumor; malignancy; neoplasm/cancer; Communicable Diseases; Infectious Disease Pathway; Infectious Diseases; Infectious Disorder; Crystallization; Data Analyses; Data Analysis; data interpretation; Disease; Disorder; Double-Blind Method; Double-Blind Study; Double-Blinded; Double-Masked Method; Double-Masked Study; Drug Industry; Pharmaceutic Industry; Pharmaceutical Industry; Pharmaceutical Preparations; Drugs; Medication; Pharmaceutic Preparations; drug/agent; Engineering; Equilibrium; balance; balance function; Feedback; Fiber Optics; Goals; Health; Human; Modern Man; Industry; Inflammation; Insulin; Humulin R; Novolin R; Regular Insulin; Lasers; Laser Electromagnetic; Laser Radiation; Medicine; Methionine; Methods; Molecular Conformation; Molecular Configuration; Molecular Stereochemistry; conformation; conformational state; oxidation; Peroxides; Blood Plasma; Plasma Serum; Reticuloendothelial System, Serum, Plasma; Plasma; genetic protein engineering; protein design; Protein Engineering; Proteins; Quality Control; Recombinant Vaccines; Investigators; Researchers; Research Personnel; Research Resources; Resources; Safety; Cell Communication and Signaling; Cell Signaling; Intracellular Communication and Signaling; Signal Transduction Systems; Signaling; biological signal transduction; Signal Transduction; Software; Computer software; Solvents; Mass Photometry/Spectrum Analysis; Mass Spectrometry; Mass Spectroscopy; Mass Spectrum; Mass Spectrum Analyses; Mass Spectrum Analysis; Technology; Testing; Time; United States Food and Drug Administration; Food and Drug Administration; USFDA; Universities; Vaccines; Water; Hydrogen Oxide; Wisconsin; Hydroxyl Radical; Hydroxyl; methionine sulfoxide; Measures; TimeLine; Guidelines; Tube; base; Label; improved; Surface; Phase; Single Crystal Diffraction; X Ray Crystallographies; X-Ray Diffraction Crystallography; X-Ray/Neutron Crystallography; Xray Crystallography; X-Ray Crystallography; Ensure; Modern Medicine; Measurement; Epitope Mapping; Biological Function; Biological Process; Collaborations; Exposure to; tool; instrument; Diagnostic; Knowledge; Life; Adopted; Hour; Complex; Source; Techniques; System; 3-D; 3D; three dimensional; 3-Dimensional; Performance; photomultiplier; Cryo-electron Microscopy; Electron Cryomicroscopy; cryo-EM; cryoEM; Cryoelectron Microscopy; antigen antibody binding; drug efficacy; voltage; Speed; Structure; Sampling; performance tests; Property; response; drug development; native protein drug; pharmaceutical protein; protein drug agent; protein-based drug; therapeutic protein; Protein Footprinting; drug discovery; Manufacturer Name; Manufacturer; Pharmaceutical Agent; Pharmaceuticals; Pharmacological Substance; Pharmacologic Substance; Address; Dose; protein structures; proteins structure; protein structure; Data; Higher Order Chromatin Folding; Higher Order Structure; Higher Order Chromatin Structure; Protein Region; Reproducibility; Resolution; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Preparation; Process; Modification; Development; developmental; rapid growth; design; designing; Three-dimensional analysis; 3-D analysis; 3-dimensional analysis; 3D analysis; Outcome; three dimensional structure; 3-D structure; 3-dimensional structure; 3D structure; Consumption; Coupling; Therapeutic antibodies; commercial application; prototype; phase 1 study; Phase I Study; Industry Standard; Genetic Diseases; genetic condition; genetic disorder; COVID-19; COVID19; CV-19; CV19; corona virus disease 2019; coronavirus disease 2019; coronavirus disease-19; coronavirus infectious disease-19; detection platform; detection system