Metal-oxide Photo-Oxidation of Proteins (MoPOP) for Biopharmaceutical Higher Order Structural Analysis

Gennext Technologies, Inc., Half Moon Bay CA

Investigators

Abstract

The GenNext submssion entitled ?Metal-oxide Photo-Oxidation of Proteins (MoPOP) for Biopharmaceutical Higher Order Structural Analysis? is responsive to the ackowledged need for new and improved tools for higher order structural analysis (HOS) of biopharmaceuticals. Biopharmaceuticals are complex, heterogeneous mixtures of 3- dimensional biomolecules, whose safety and efficacy is reliant upon proper HOS. The presence of proteins with improper HOS has been linked to severe adverse drug reactions (ADR), establishing the need for new and improved HOS analytics. An emerging HOS analysis technique is hydroxyl radical foot-printing (HRPF). HRPF involves the irreversible labeling of a protein?s exterior by reaction with hydroxyl radicals (?OH) generated from UV photolysis of hydrogen peroxide (H2O2) with subsequent MS analysis to identify the outer portions of the protein. Academic laboratories have demonstrated the utility of HRPF for HOS analysis; however adoption in pharma has been minuscule at best. Guided by the critiques of scientists at leading biopharmaceutical companies, we have identified barriers that have limited pharma?s adoption of FPOP HRPF. A substantial barrier is artifacts created by H2O2. While H2O2 is a fine source of ?OH, its potential for protein oxidation under ambient conditions is well described and represents a limitation for HRPF adoption. In our experience, biopharmaceutical exposure to H2O2 must be limited to less than 15 minutes to avoid labeling artifacts. Such temporal limitations constrain the number of samples that can be queued in microplates, placing H2O2 HRPF at odds with market desires of throughput and productivity. Moreover, clients have expressed the need for a rapid screening technique that could serve to triage their HOS studies. This proposal aims to replace photolysis of H2O2 with a novel metal-oxide photo-catalytic process that produces ?OH from water. Our proprietary, patent-pending process has been coined Metal-oxide Photo-Oxidation of Proteins (MoPOP?). MoPOP theoretical ?OH yields are predicted to support HRPF experimentation, eliminating the use of H2O2 and its associated complications. When irradiated with an economical continuous wave (CW) diode source, MoPOP provides a facile, inexpensive, and ?reagent-free? means to label biopharmaceuticals. In addition to LC MS analysis, MoPOP oxidative products can be detected and quantified using selective fluorescence dyes for carbonylated proteins, addressing the market need for HOS screening.