Raman Marker Bands for Secondary Structure Changes of Frozen Therapeutic Monoclonal Antibody Formulations During Thawing
Astrid Hauptmann, Georg Hoelzl, Martin J. Mueller, Karoline Bechtold-Peters, Thomas Loerting
Abstract
In this work we use Raman spectroscopy for protein characterization in the frozen state. We investigate the behavior of frozen therapeutic monoclonal antibody IgG1 formulation upon thawing by Raman spectroscopy. Secondary and tertiary structure of the protein in three different mab formulations in the frozen state are followed through observation of marker bands for a-helix, b-sheet and random coil. We identify the tyrosine intensity ratio I 856 /I 830 as a marker for mab aggregation. Upon fast cooling (40 C/min) to -80 C we observe a significant increase of random coil and a -helical structures, while this is not the case for slower cooling (20 C/min) to -80 C. Most changes in the protein's secondary structure are observed in the course of thawing in the range up to -20 C, when passing through the glass transitions and cold-crystallization of the two types of freeze-concentrated solutions formed through macro-and microcryoconcentration. An increase of protein concentration and the addition of mannitol suppress secondary structural changes but do no impact on aggregation.