Advancements in Characterizing Therapeutic Protein Higher-Order Structure

Electrospray-ionization mass spectrometry (ESI-MS) is a well-established tool for biotherapeutic analysis. It draws intact proteins or peptide ions into the vacuum of a mass spectrometer, where the ion mass is measured. Electrospray ion-mobility mass spectrometry (ESI-IMS) introduces ions into a low-pressure gas, where the effects of aerodynamic drag reveal their shape. This technique is just emerging as a valuable tool for characterizing intact proteins, even though for a decade it’s been the basis of a commercially available medical diagnostic test that measures the size distributions of lipoproteins for cardiovascular risk assessment.

A growing body of evidence shows that ion mobility provides critical information about intact antibodies, protein–protein interactions, and protein aggregation. It leverages some concepts behind ESI-MS (e.g., sensitivity, dynamic range, and reproducibility) but differs in that ESI-IMS measures protein shape directly and rapidly detects subtle changes in higher-order structure. It’s becoming a sensitive probe for protein conformation and can be used to characterize intact biotherapeutics, drug-conjugates, and nanoparticle–drug assemblies.

Recently, IonDx has made key advances in the application of electrospray ion mobility to biotherapeutics. Our new protein characterization platform for biologics is based on improvements to several ion-mobility technologies. Our patented technology combines electrospray ionization with improved ion optics and detection, eliminates charge distortion typically encountered with highly-charged electrospray ions, and introduces a new way to quantify variations in the shape of biotherapeutic molecules.

One promising application for ESI-IMS is to generate biophysical “fingerprints” for comparing biosimilars with innovator drugs. As more of the latter fall off patent, the former continue to increase in number. Regulators require biosimilars to exhibit biophysical fingerprints that are comparable to the innovator drugs they are meant to replace. With increasing emphasis on analytical data for biosimilar characterization, John Jenkins (director of the Office of New Drugs at the US FDA) suggests that applicants develop new and innovative approaches for studying their products. Fingerprints help establish comparability, according to recently released guidances (2, 3). Interchangeability also can be demonstrated with fingerprints, but some are better than others.

Key to generating a biophysical fingerprint of higher-order structure using ESI-IMS is to analyze singly charged ions rather than the typical highly charged antibodies measured by native MS. To do so, we reduce the net charge on electrosprayed droplets to a single charge. Eliminating high charge states prevents them from stretching a protein in the gas phase and simplifies data interpretation by eliminating the need to deconvolute charge states. We have overcome these challenges by developing a standalone ion-mobility spectrometer with patented detector technology suitable for measuring the ion mobility of singly charged macromolecular ions.

Historically, a protein’s three-dimensional structure has been determined with methodologies developed by the X-ray crystallography community. Indeed, their techniques serve as the gold standard against which other techniques are contrasted. But X-ray crystallography captures only a single protein structure. If a protein has more than one structure, or its uncrystallized form varies, those variations are not revealed. Thus, less-precise but faster techniques for determining protein structure are relied upon increasingly — even if they provide only an indirect measurement of protein structure. Our technology measures ion shape both directly and rapidly.

Mike Bogan is director of development, and Henry Benner is founder and CEO of IonDx, Inc., 8 Harris Court C5-6, Monterey CA 93940; www.iondx.com.