title: Frozen state storage instability of a monoclonal antibody: aggregation as a consequence of trehalose crystallization and protein unfolding.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  Frozen matrix was sampled, stored frozen at various temperatures and analyzed by SEC over time.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., Chesterfield, Missouri 63017, USA. satish.singh@pfizer.com
authors.forename: Satish K
authors.initials: SK
authors.lastname: Singh
authors.forename: Parag
authors.initials: P
authors.lastname: Kolhe
authors.forename: Anjali P
authors.initials: AP
authors.lastname: Mehta
authors.forename: Steven C
authors.initials: SC
authors.lastname: Chico
authors.forename: Alanta L
authors.initials: AL
authors.lastname: Lary
authors.forename: Min
authors.initials: M
authors.lastname: Huang
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D003460:Crystallization; D004355:Drug Stability; D004356:Drug Storage; D005615:Freezing; D007074:Immunoglobulin G / Q000737:chemistry*; D017510:Protein Folding; D055550:Protein Stability; D058767:Protein Unfolding; D013816:Thermodynamics; D013997:Time Factors; D014199:Trehalose / Q000737:chemistry*; D014783:Viscosity
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D014199:Trehalose