The Role of Protein Structure in ADRs

During the last thirty years, the global market for biopharmaceuticals has prospered, achieving sales of more than $176 billion in 2015 ^[1]. While effective, biotherapeutics are expensive and exert substantial financial stress upon patients and the overall healthcare system ^[2]. The desire to reduce health care costs combined with the patent expirations for many of the world’s largest biotherapeutics has led to the development of generic versions commonly referred to as biosimilars ^{[3, 4]}.  Unlike conventional pharmaceuticals, biosimilars are not exact replicates of originals, as they are complex, heterogeneous mixtures of three-dimensional biomolecules ^[3]. The structure and functional activity of biopharmaceuticals are dependent on various aspects of their production and environment. The presence of proteins having improper higher order structure (HOS) has been linked to adverse drug reactions (ADR) ^{[5, 6]}.  The range of ADR’s extends from simple irritation to patient death. The morbidity and mortality of reported ADR’s have alerted the biopharmaceutical industry to the critical role that protein structure plays in the safety and function of biotherapeutics. Scientists and regulators have become increasingly aware of the importance of HOS to ensure stability, safety, and biological function of bio-therapeutics.  Although a variety of HOS analytics exists today, their inadequacies to reliably predict efficacy and safety has been brought into question, establishing the unmet need for new and improved HOS analytics ^[7].

SAS Footprints as Predictors of Efficacy and Toxicity

An emerging HOS analysis technique addressing the challenges for improved biotherapeutic safety and efficacy is hydroxyl radical protein footprinting (HRPF). HRPF has confidently detected defects in biopharmaceutical HOS and function ^[8]; detected defects in monoclonal antibody production (Mab) ^[9-11]; as well as detected biosimilar failure and storage induced defects for innovator products ^[12], with impressive fidelity. HRPF irreversibly labels a protein’s exterior surface by covalent reaction with hydroxyl (OH) radicals photolytically derived from hydrogen peroxide (H₂O₂), and when followed with subsequent mass spectrometric analysis, HRPF identifies the Solvent Accessible Surface Area (SASA) of a protein by its increased mass ^[13-15]. Biotherapeutic SAS is in direct contact with the surrounding aqueous environment and constitutes the “business end” of molecular interaction.  As such, SAS footprints are good predictors of biotherapeutic efficacy and/or toxicity.

Limitations of Traditional Analysis Methods

The most widely used method for generating OH radicals from H₂O₂ employs a quick burst of ultraviolet (UV) light and is appropriately called fast photochemical oxidation of proteins (FPOP) ^[16-22]. Typically, a complicated, expensive, and hazardous Class IV UV laser is used. While academic laboratories have demonstrated the utility of FPOP HRPF for HOS analysis, adoption in pharma has been minuscule at best, predominantly in laboratories that have hired researchers from or closely collaborate with FPOP academic labs.

GenNext has identified several technical, safety, ease-of-use, and cost barriers that have limited adoption of HRPF in the biopharmaceutical industry. These impediments include:

1. The use of dangerous, expensive lasers that demand substantial expertise and safety measures ^[23];
2. the irreproducibility of HRPF caused by uncontrolled or unexpected background scavenging of OH radicals that complicates reproducibility and limits comparative studies ^[20]; and
3. the absence of intuitive, efficient, and easy-to-use data processing tools to streamline and facilitate HRPF data analysis. As such, there are no commercial solutions for HRPF analysis, despite the demonstrated need for its HOS analytical power.

HRPF Breakthroughs Enabled by GenNext Technologies

By introducing the Fox^® Protein Footprinting platform, GenNext obsoleted costly, difficult, and dangerous lasers while dramatically improving HRFP reproducibility and streamlining data analysis. The platform includes simple, safe, and integrated components along with data analysis software to accelerate your HRPF research. Our novel products and services for Hydroxyl Radical Protein Footprinting will accelerate your biopharmaceutical development and reduce manufacturing costs, while improving therapeutic efficacy and safety.