Effect of pH and Excipients on Structure, Dynamics, and Long-Term Stability of a Model IgG1 Monoclonal Antibody upon Freeze-Drying
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To investigate the mechanism of IgG1 mAb stabilization after freeze-drying and the interdependence of protein structural preservation in the solid state, glassy state dynamics and long-term storage stability under different formulation conditions.
IgG1 mAb was formulated with mannitol at pH 3.0, 5.0, and 7.0 in the presence and absence of sucrose and stability was monitored over 1 year at different temperatures. Physical and covalent degradation of lyophilized formulation was monitored using SEC, CEX, and light obscuration technique. Secondary and tertiary structure of the protein in the solid state was characterized using FTIR and fluorescence spectroscopy respectively. Raman spectroscopy was also used to monitor changes in secondary and tertiary structure, while SS-NMR 1H relaxation was used to monitor glassy state dynamics.
IgG1 mAb underwent significant secondary structural perturbations at pH 3.0 and conditions without sucrose, while pH 5.0 condition with sucrose showed the least structural change over time. The structural changes correlated with long-term stability with respect to protein aggregate formation and SbVP counts. SS-NMR data showed reduced relaxation time at conditions that were more stable.
Native state protein structural preservation and optimal solid-state dynamics correlate with improved long-term stability of the mAb in the different lyophilized formulations.
KEY WORDSIgG1 antibody lyophilization protein formulation protein structure solid state NMR solid state stability
fourier transform infrared spectroscopy
high molecular weight species
high performance liquid chromatography
low molecular weight species
residual water content
solid state nuclear magnetic resonance.
glass transition temperature
Acknowledgments and Disclosures
Thanks to Twinkle Christian and Chris Woods for helping with FTIR analysis. Special thanks to Dr. Margaret Ricci, Dr. Ping Yeh, Prof. David Volkin and Dr. David Brems for their technical input and support.
- 5.Chang L, Shepherd D, Sun J, Ouellette D, Grant KL, Tang X, et al. Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and/or immobilization in a glassy matrix? J Pharm Sci. 2005;94(7):1427–44.PubMedCrossRefGoogle Scholar
- 31.USP General Chapters: <788> Particulate matter in injections. Pharmacopeial Forum. 2009; (28):1930, USP32–NF27.Google Scholar
- 38.Koenig JL. Raman spectroscopy of biological molecules: a review. J Polymer Sci. 1972;6(1):59–177.Google Scholar
- 45.Brunner H, Holz M. Raman studies of the conformation of the basic pancreatic trypsin inhibitor. Biochimica et Biophysica Acta (BBA)-Protein. Structure. 1975;379(2):408–17.Google Scholar