Higher Order Structure 2021

Complex formulations that include excipients, ligands, surfactants, and preservatives provide a challenging environment to measure protein secondary structure and detect change. Traditional spectroscopic methodologies such as Fourier Transform Infrared, Circular Dichroism, and Nuclear Magnetic Resonance have limitations when these common environments are encountered due to interfering absorbances and concentration requirements.
In this symposium, RedShiftBio will introduce the AQS3pro, an innovative technology designed for accurate measurement of protein secondary structure in complex formulation conditions. Powered by Microfluidic Modulation Spectroscopy (MMS), the AQS3pro utilizes infrared spectroscopy to provide high sensitivity measurements across a wide dynamic concentration range. In combination with the AQS3delta Data Analysis package, this novel technology facilitates a detailed look into the effect of the formulation environment on higher order structure. This presentation will include case studies examining the measured effects of complex formulation conditions, including ligand binding and purification methodologies, on protein structure and stability.

Small changes in structure can lead to loss of activity and/or an increase in aggregation. Structure-activity relationships are not isolated to the epitope either – both proximal and distal changes can have deleterious impacts on a biologics’ function. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) enables investigation of these localized or global changes through the exchange of labile protons with deuterium ions. The rate and magnitude of the exchange provides information regarding protein dynamics, conformation and interactions. This talk will focus on the increased resolution of HDX-MS via hardware improvements and how ion mobility spectroscopy aides in deconvolution and simplifies complex spectra.

In the half century since the first crystallography derived structure of a protein; structural biology has blossomed into a mature discipline exploring the correlation between structure, dynamics and function of proteins. The latest frontier in structural biology is the study of higher order structures (HOS) of proteins and their complexes. This is of particular relevance in biopharma where the quantification of different HOS species is directly correlated to determining the dosage and efficacy of the therapeutic protein.

Analytical ultracentrifugation (AUC) is one of the most versatile biophysical tools used today for the characterization of biological samples ranging from small drug molecules to intact viruses, vesicles and microparticles. AUC works with biological samples in the native state and does not depend on a matrix, reporter species or custom-coated substrates. AUC separates biomolecules based upon both size, molecular mass and anisotropy and can also be used to quantify interactions between different species. In this talk, we will discuss the principles of AUC and discuss the workflow to characterize the presence of HOS/aggregate species in an antibody sample. We will also touch upon other centrifugation instruments used in a typical protein purification and characterization workflow.

Magnetic Resonance (MR) is a proven technology that provides detailed information on protein aggregation and conformational states, stability monitoring and structural changes and chemical modifications at atomic resolution.1 Information rich data and ease of use in the study of the Higher Order Structure (HOS) of biotherapeutic proteins make this technique a direct measure of their Critical Quality Attributes (CQA). Recently, new developments in acquisition and data analysis have emerged to establish MR as a powerful metric for validating the HOS and monitoring the self-association of biotherapeutics, utilizing both high resolution NMR and Relaxometry methods.2,3 This presentation describes examples of the technologies offered by Bruker BioSpin Corp. to validate the CQAs of biotherapeutics required for assessment by regulatory agencies and development by the pharmaceutical industry.