Frozen State Storage Instability of a Monoclonal Antibody: Aggregation as a Consequence of Trehalose Crystallization and Protein Unfolding
- 1.3k Downloads
To investigate the cause of unexpected and erratic increase in aggregation during long-term storage of an IgG2 monoclonal antibody in a trehalose formulation at −20°C.
Frozen matrix was sampled, stored frozen at various temperatures and analyzed by SEC over time.
Aggregation increased with time at −20°C but not at −40°C or −10°C. The cause of the instability was the crystallization of freeze-concentrated trehalose from the frozen solute when the storage temperature exceeds the glass transition temperature of the matrix (−29°C). Crystallization at −20°C deprives the protein of the cryoprotectant, leading to a slow increase in aggregation. Storage at −10°C also leads to crystallization of trehalose but no increase in aggregation. It is hypothesized that significantly higher mobility in the matrix at −10°C allows protein molecules that are unfolded at the ice interface on freezing to refold back before significant aggregation can occur. In contrast, lack of mobility at −40°C prevents crystallization, refolding, and aggregation.
Aggregation in the frozen state when stored above the glass transition temperature is a consequence of balance between rate of crystallization leading to loss of cryoprotectant, rate of aggregation of the unfolded protein molecules, and rate of refolding that prevents aggregation.
KEY WORDSaggregation cryoprotectant glass transition temperature monoclonal antibody trehalose
- 1.Singh SK. Storage consideration as part of the formulation development program for biologics. Am Pharm Rev. 2007;10(3):26–33.Google Scholar
- 3.Singh SK, Kolhe P, Wang W, Nema S. Freezing of biologics—A practitioner’s review. Part II: Practical advice. Bioprocess Int. 2009;7(10):34–42.Google Scholar
- 4.Singh SK, Kolhe P, Wang W, Nema S. Freezing of biologics—A practitioner’s review. Part I: Fundamental aspects. Bioprocess Int. 2009;7(9):32–44.Google Scholar
- 8.Franks F. Biophysics and biochemistry at low temperatures. Cambridge: Cambridge University Press; 1985. p. 86–9.Google Scholar
- 10.Wisniewski R, Wu V. Large-scale freezing and thawing of biopharmaceutical products. In: Avis KE, Wu VL, editors. Biotechnology and biopharmaceutical manufacturing, processing, and preservation. Buffalo Grove: Interpharm Press; 1996. p. 7–59.Google Scholar
- 11.Kolhe P, Holding E, Lary A, Chico S, Singh SK. Large-scale freezing of biologics—Part 2. Understanding protein and solute concentration changes in a cryovessel in the frozen state. BioPharm Int. 2010;23(7):40–9.Google Scholar
- 14.Kolhe P, Holding E, Lary A, Chico S, Singh SK. Large-scale freezing of biologics—Part 1. Understanding protein and solute concentration changes in a cryovessel during freezing. BioPharm Int. 2010;23(6):53–60.Google Scholar
- 17.Roos Y, Karel M. Amorphous state and delayed ice formation in sucrose solutions. Int J Food Sci Technol. 1991;26:553–66.Google Scholar
- 27.Izutsu K, Yoshioka S, Kojima S. Effect of cryoprotectants on the eutectic crystallization of NaCl in frozen solutions studied by differential scanning calorimetry (DSC) and broad-line pulsed NMR. Chem Pharm Bull. 1995;43:1804–6.Google Scholar