Freeze-Dry Microscopy of Protein/Sugar Mixtures: Drying Behavior, Interpretation of Collapse Temperatures and a Comparison to Corresponding Glass Transition Data

doi:10.1002/jps.21586

Journal of Pharmaceutical Sciences

Volume 98, Issue 9, September 2009, Pages 3072-3087

https://doi.org/10.1002/jps.21586 Get rights and content

ABSTRACT

The purpose of this study is to investigate the change in collapse appearance and temperature of protein/sugar mixtures as a function of nucleation temperature (T_n), sublimation velocity (V_sub) and the sugar/protein mole ratio when performing freeze-dry microscopy experiments. BSA and HSA were used as sample proteins and mixed with either sucrose or trehalose. Differential scanning calorimetry was used to determine the corresponding glass transition temperatures (T′_g). To allow a more representative comparison between these analytical methods, a collapse midpoint temperature (T_c-50) was introduced. While there was no distinct correlation between T_n and the onset of collapse (T_oc) for either mixture, V_sub was found to correlate with the measured collapse temperature which is important for comparability of experiments. Furthermore, V_sub could be used to qualitatively investigate the product resistance to water vapor flow. A dramatic change in the appearance of collapse was found for high sugar/protein mole ratios (≥ 362:1) which needs to be considered to avoid a misinterpretation of T_oc data. At low protein concentrations midpoint T′_g data showed good agreement with T_oc values but were found significantly lower at higher protein concentrations. Application of the Gordon–Taylor equation failed to predict the critical temperature for any of the protein/sugar mixtures studied

Section snippets

INTRODUCTION

Freeze-drying is still the method of choice to achieve improved stability of biopharmaceuticals when the product is not sufficiently stable in a liquid formulation. A well designed freeze-drying cycle forms a glassy solid which can minimize degradation reactions of complex structures such as proteins or peptides and may provide acceptable shelf life for worldwide shipping and storage.1, 2 It was estimated that about 200 new antibody products are currently in development, many of them in a

Material

BSA, HSA, sucrose and trehalose, potassium chloride (KCl), sodium chloride (NaCl) and magnesium chloride (MgCl2) were purchased from Sigma Chemical Company (St. Louis, MO). All excipients used were of highest available analytical grade. Water was double distilled from an all-glass apparatus and filtered through a 0.22 μL membrane before used (Millipore®, Billerica, MA). Liquid nitrogen was obtained from Linde (Nuremberg, Germany).

Experimental Procedure

Solutions of pure BSA, HSA, sucrose and trehalose, as well as

Nucleation Temperature and Corresponding Collapse Temperature of the Sample

Nucleation temperatures (T_n) for both the pure protein solutions and the binary mixtures with sucrose and trehalose were observed in a temperature range from approximately –9 to –20 °C (cf. Fig. 1) which is in good agreement with the degree of supercooling found during laboratory scale freeze-drying experiments.1, 2 Performing replicate experiments under identical conditions (i.e., same solution but new preparation of experiment) clearly showed that T_n cannot be controlled during FDM

ACKNOWLEDGMENTS

The authors would like to thank Dr. Michael J. Pikal for his active participation in discussions for bringing understanding to the results obtained during this study.

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Freeze-Dry Microscopy of Protein/Sugar Mixtures: Drying Behavior, Interpretation of Collapse Temperatures and a Comparison to Corresponding Glass Transition Data

ABSTRACT

Section snippets

INTRODUCTION

Material

Experimental Procedure

Nucleation Temperature and Corresponding Collapse Temperature of the Sample

ACKNOWLEDGMENTS

Eur J Pharm Biopharm

J Pharm Sci

J Pharm Sci

J Pharm Sci

Eur J Pharm Bio-pharm

Int J Pharm

Arch Biochem Biophys

J Pharm Sci

Thermochim Acta

J Pharm Sci

J Pharm Sci

J Pharm Sci

Cryobiology

J Pharm Sci

Int J Pharm

J Pharm Sci

Cryobiology

Freeze Drying

Lyophilization of biopharmaceuticals

Antibodies and genetically engineered related molecules: Production and purification

Biotechnol Prog

Rational design of stable lyophilized protein formulations: Some practical advice

Pharm Res

Freeze-drying of proteins. Part II: Formulation selection

Bio Pharm

Protein stability during freezing: Separation of stresses and mechanisms of protein stabilization

Pharm Dev Technol

Lyophilization and development of solid protein pharmaceuticals

Int J Pharm

Practical formulation and process development of freeze-dried products

Pharm Dev Technol