Chirascan systems provide invaluable information for the characterization of protein biotherapeutics.
Whether these are recombinant enzymes or regulatory proteins, or monoclonal antibodies (mAbs) and constructs derived thereof, including bispecifics and antibody-drug conjugates—Circular Dichroism spectroscopy is an essential orthogonal technique and indispensable in any toolset for biophysical characterization.
To assist with the complex task of characterizing protein-based biotherapuetics such as mAbs, the National Institute of Standards and Technology (NIST) in the USA undertook a holistic study using a humanized mAb, the NISTmAb, in 2016.
The study utilized the biophysical techniques most commonly employed during biotherapeutic development, and the NISTmAb is now available to biopharmaceutical companies as a representative reference molecule.
Applied Photophysics Ltd. had been approached by the NIST to contribute CD data to this project. A primary standard of the NISTmAb, PS 8670, was compared with the NISTmAb reference material, RM 8671.
As typical for mAbs, the far-UV spectrum is characteristic of a protein with high β-sheet content. However, the far-UV spectra contain information beyond secondary structure elements—signals of lower amplitude around and above 230 nm arise from disulfide bonds.
The profile of the near-UV spectra is much more complex as it is composed of overlapping contributions by aromatic amino acid side chains and disulfide linkages.
Although the near-UV spectra appear to be superimposable by visual inspection, statistical analysis reveals that the tertiary structure of RM 8671 is significantly different to that of PS 8670, while this is not the case for the secondary structure.
Such small spectral differences in the near-UV can only be found with state-of-the-art CD instruments that provide the required sensitivity. Moreover, the automation offered by a Chirascan Q100 enabled the reproducible generation of a sufficient number of replicates as required for robust statistical analysis.
Use the Chirascan Q100 for characterization of your own protein therapeutics!