caa a22 4500 445313447 CHVBK 20180317142638.0 cr unu---uuuuu 170323e20110401xx s 000 0 eng 10.1007/s11095-010-0343-z doi (NATIONALLICENCE)springer-10.1007/s11095-010-0343-z Frozen State Storage Instability of a Monoclonal Antibody: Aggregation as a Consequence of Trehalose Crystallization and Protein Unfolding [Elektronische Daten] [Satish Singh, Parag Kolhe, Anjali Mehta, Steven Chico, Alanta Lary, Min Huang] ABSTRACT: Purpose: 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. Springer Science+Business Media, LLC, 2011 aggregation nationallicence cryoprotectant nationallicence glass transition temperature nationallicence monoclonal antibody nationallicence trehalose nationallicence Singh Satish BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut Kolhe Parag BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut Mehta Anjali BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut Chico Steven BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut Lary Alanta BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut Huang Min BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut Pharmaceutical Research Springer US; http://www.springer-ny.com 28/4(2011-04-01), 873-885 0724-8741 28:4<873 2011 28 11095 https://doi.org/10.1007/s11095-010-0343-z text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11095-010-0343-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Singh Satish BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut NATIONALLICENCE 700 1- Kolhe Parag BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut NATIONALLICENCE 700 1- Mehta Anjali BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut NATIONALLICENCE 700 1- Chico Steven BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut NATIONALLICENCE 700 1- Lary Alanta BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut NATIONALLICENCE 700 1- Huang Min BioTherapeutics Pharmaceutical Sciences, Pfizer Inc., 63017, Chesterfield, Missouri, USA aut NATIONALLICENCE 773 0- Pharmaceutical Research Springer US; http://www.springer-ny.com 28/4(2011-04-01), 873-885 0724-8741 28:4<873 2011 28 11095 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer