# Plasma-Generated Hydroxyl Radicals for Analysis of Three-Dimensional Structures in Protein Therapeutics

> **NIH NIH R44** · IMMUTO SCIENTIFIC, INC. · 2022 · $1,037,792

## Abstract

Abstract
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.

## Key facts

- **NIH application ID:** 10547178
- **Project number:** 2R44GM134849-02
- **Recipient organization:** IMMUTO SCIENTIFIC, INC.
- **Principal Investigator:** Faraz Choudhury
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $1,037,792
- **Award type:** 2
- **Project period:** 2019-09-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10547178

## Citation

> US National Institutes of Health, RePORTER application 10547178, Plasma-Generated Hydroxyl Radicals for Analysis of Three-Dimensional Structures in Protein Therapeutics (2R44GM134849-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10547178. Licensed CC0.

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