Understanding the conformational stability of protein therapeutics using Raman spectroscopy
White Paper Jul 10, 2014
The combination of Dynamic Light Scattering (DLS) with Raman Spectroscopy provides the ability to extract a wealth of chemical, structural, and physical information about biotherapeutic proteins under formulation conditions.
Raman spectroscopy simultaneously derives protein secondary structure (Amide I and III) and tertiary structure markers (aromatic side chains, disulfide bonds, hydrogen bonding, local hydrophobicity). These higher order structural determinations are performed at actual formulation concentrations (50 mg/mL or greater for mAbs), rather than at the lower concentrations required by conventional methods, i.e. less than a few mg/mL for Circular Dichroism (CD).
By use of Raman spectroscopy, protein secondary and tertiary structure perturbation/ unfolding, melting temperature, onset temperature of aggregation, and van 't Hoff enthalpy values can all be derived, leading to improved understanding of competing pathways of unfolding/structure change and aggregation, and ultimately, unique insights into the mechanism(s) of aggregation to help improve formulation stability.
The unique coupling of Raman spectroscopy and DLS provides the ability to simultaneously correlate protein structure with colloidal parameters to enhance the understanding of protein therapeutic formulations under a variety of stress conditions, e.g., thermal, formulation, chemical degradation, extrinsic particulates.