Protein Aggregation in Frozen Trehalose Formulations: Effects of Composition, Cooling Rate, and Storage Temperature.

This study was designed to assess the effects of cooling rate, storage temperature, and formulation composition on trehalose phase distribution and protein stability in frozen solutions. The data demonstrate that faster cooling rates (>100°C/min) result in trehalose crystallization and protein aggregation as determined by Fourier Transform Near-Infrared (FT-NIR) spectroscopy and size-exclusion chromatography, respectively. Conversely, at slower cooling rates (≤1°C/min), trehalose remains predominantly amorphous and there is no effect on protein stability. Evaluation of storage temperatures demonstrates that aggregation increases more rapidly at -14°C compared with higher (-8°C) and lower (-20°C) storage temperatures; however, a relatively higher amount of cumulative aggregation was observed at lower (-20°C) temperature compared with higher storage temperatures (-14°C and -8°C). Further evaluation of the effects of formulation composition suggests that the phase distribution of amorphous and crystallized trehalose dihydrate in frozen solutions depends on the ratio of trehalose to mAb. The results identify an optimal range of trehalose-mAb (w/w) ratio, 0.2-2.4, capable of physically stabilizing mAb formulations during long-term frozen storage-even for fast cooled (>100°C/min) formulations.

[1]  F. Franks,et al.  Protein destabilization at low temperatures. , 1995, Advances in protein chemistry.

[2]  Yrjö H. Roos,et al.  Amorphous state and delayed ice formation in sucrose solutions , 2007 .

[3]  R. Suryanarayanan,et al.  Trehalose Crystallization During Freeze-Drying: Implications On Lyoprotection , 2010 .

[4]  F Franks,et al.  The thermodynamics of protein stability. Cold destabilization as a general phenomenon. , 1988, Biophysical chemistry.

[5]  Hanns-Christian Mahler,et al.  Glycation during storage and administration of monoclonal antibody formulations. , 2008, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[6]  A. Mcauley,et al.  Sorbitol Crystallization Can Lead to Protein Aggregation in Frozen Protein Formulations , 2006, Pharmaceutical Research.

[7]  R. Suryanarayanan,et al.  Predicting the crystallization propensity of carboxylic acid buffers in frozen systems--relevance to freeze-drying. , 2011, Journal of pharmaceutical sciences.

[8]  Chung C. Hsu,et al.  Surface Denaturation at Solid-Void Interface—A Possible Pathway by Which Opalescent Participates Form During the Storage of Lyophilized Tissue-Type Plasminogen Activator at High Temperatures , 2004, Pharmaceutical Research.

[9]  Raj Suryanarayanan,et al.  Crystallization of Mannitol below Tg′ during Freeze-Drying in Binary and Ternary Aqueous Systems , 2004, Pharmaceutical Research.

[10]  J. D. de Pablo,et al.  Thermophysical Properties of Trehalose and Its Concentrated Aqueous Solutions , 1997, Pharmaceutical Research.

[11]  M. Pikal,et al.  Study of the individual contributions of ice formation and freeze-concentration on isothermal stability of lactate dehydrogenase during freezing. , 2008, Journal of Pharmacy and Science.

[12]  Min Huang,et al.  Frozen State Storage Instability of a Monoclonal Antibody: Aggregation as a Consequence of Trehalose Crystallization and Protein Unfolding , 2011, Pharmaceutical Research.

[13]  J. Bischof,et al.  Freezing-induced phase separation and spatial microheterogeneity in protein solutions. , 2009, The journal of physical chemistry. B.

[14]  Y. J. Wang,et al.  Vibrational spectroscopy and chemometrics to characterize and quantitate trehalose crystallization. , 2010, Analytical biochemistry.

[15]  J H Crowe,et al.  Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying , 1995, Applied and environmental microbiology.

[16]  G. Strambini,et al.  Protein stability in ice. , 2007, Biophysical journal.

[17]  Margaret Ricci,et al.  The LC/MS analysis of glycation of IgG molecules in sucrose containing formulations. , 2007, Journal of pharmaceutical sciences.

[18]  S. Yoshioka,et al.  Effect of mannitol crystallinity on the stabilization of enzymes during freeze-drying. , 1994, Chemical & pharmaceutical bulletin.

[19]  R. Suryanarayanan,et al.  Crystallization Behavior of Mannitol in Frozen Aqueous Solutions , 2004, Pharmaceutical Research.

[20]  K. Avis,et al.  Cryopreservation : Applications in Pharmaceuticals and Biotechnology , 1999 .

[21]  Ana D. Lopez,et al.  Trehalose: A Cryoprotectant That Enhances Recovery and Preserves Function of Human Pancreatic Islets After Long-Term Storage , 1997, Diabetes.

[22]  T. Anchordoquy,et al.  Effect of glycine on pH changes and protein stability during freeze-thawing in phosphate buffer systems. , 2002, Journal of pharmaceutical sciences.

[23]  S. Yoshioka,et al.  Decreased Protein-Stabilizing Effects of Cryoprotectants Due to Crystallization , 1993, Pharmaceutical Research.

[24]  S. Singh,et al.  Frozen-state storage stability of a monoclonal antibody: aggregation is impacted by freezing rate and solute distribution. , 2013, Journal of pharmaceutical sciences.

[25]  C. Angell,et al.  Phase relations and vitrification in saccharide-water solutions and the trehalose anomaly , 1989 .

[26]  R. Suryanarayanan,et al.  Crystallization of Trehalose in Frozen Solutions and its Phase Behavior during Drying , 2010, Pharmaceutical Research.

[27]  N. Warne,et al.  Impact of sucrose level on storage stability of proteins in freeze-dried solids: I. Correlation of protein-sugar interaction with native structure preservation. , 2009, Journal of pharmaceutical sciences.

[28]  F. Franks,et al.  Stability of proteins at subzero temperatures: thermodynamics and some ecological consequences , 1991 .

[29]  M. Descamps,et al.  Vitrification and Polymorphism of Trehalose Induced by Dehydration of Trehalose Dihydrate , 2002 .

[30]  R. Suryanarayanan,et al.  Influence of the Active Pharmaceutical Ingredient Concentration on the Physical State of Mannitol—Implications in Freeze-Drying , 2005, Pharmaceutical Research.

[31]  U. T. Lashmar,et al.  Bulk Freeze – Thawing of Macromolecules Effects of Cryoconcentration on Their Formulation and Stability , 2007 .

[32]  M. Pikal,et al.  Measurement of the Kinetics of Protein Unfolding in Viscous Systems and Implications for Protein Stability in Freeze-Drying , 2005, Pharmaceutical Research.

[33]  Raj Suryanarayanan,et al.  Influence of Crystallizing and Non-crystallizing Cosolutes on Trehalose Crystallization During Freeze-Drying , 2010, Pharmaceutical Research.

[34]  Michael J. Pikal,et al.  Protein Stability During Freezing: Separation of Stresses and Mechanisms of Protein Stabilization , 2007, Pharmaceutical development and technology.

[35]  T. Bewley,et al.  Effect of Freezing on Aggregation of Human Growth Hormone , 1991, Pharmaceutical Research.

[36]  J. Carpenter,et al.  An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. , 1989, Biochemistry.

[37]  S J Prestrelski,et al.  Factors affecting short-term and long-term stabilities of proteins. , 2001, Advanced drug delivery reviews.

[38]  E. Gabellieri,et al.  Proteins in frozen solutions: evidence of ice-induced partial unfolding. , 1996, Biophysical journal.

[39]  A. Rosenberg,et al.  Effects of protein aggregates: An immunologic perspective , 2006, The AAPS Journal.

[40]  Michael C. Williams,et al.  Volumetric interpretation of viscosity for concentrated and dilute sugar solutions , 1981 .

[41]  Enhong Cao,et al.  Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. , 2003, Biotechnology and bioengineering.

[42]  Serguei Tchessalov,et al.  Impact of sucrose level on storage stability of proteins in freeze-dried solids: II. Correlation of aggregation rate with protein structure and molecular mobility. , 2009, Journal of pharmaceutical sciences.

[43]  D S Reid,et al.  Is trehalose special for preserving dry biomaterials? , 1996, Biophysical journal.

[44]  D. Kalonia,et al.  Effect of vacuum drying on protein-mannitol interactions: The physical state of mannitol and protein structure in the dried state , 2004, AAPS PharmSciTech.

[45]  S. Yalkowsky,et al.  Handbook of aqueous solubility data , 2003 .

[46]  J L Cleland,et al.  A specific molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody. , 2001, Journal of pharmaceutical sciences.