The Use of Disaccharides in Inhibiting Enzymatic Activity Loss and Secondary Structure Changes in Freeze-Dried β-Galactosidase during Storage

ABSTRACTPurposeThe purpose of this study is to show how disaccharides differ in their ability to protect lyophilized β-galactosidase from enzymatic activity loss and secondary structure changes during storage.Methodsβ-galactosidase was lyophilized with trehalose, sucrose, cellobiose or melibiose at 2:1, 20:1 and 40:1 excipient/protein weight ratios, and stored up to 90 days at 45°C. Protein enzymatic activity was studied using o-nitrophenyl-β-d-galactopyranoside cleavage test, and its secondary structure in lyophilizates analyzed using Fourier transform infrared spectroscopy. The crystallization tendencies, glass transition temperatures and water contents of lyophilizates were evaluated using x-ray powder diffractometry, differential scanning calorimetry and thermogravimetry, respectively.ResultsThe enzymatic activity of β-galactosidase decreased more slowly in lyophilizates containing trehalose or melibiose at 2:1 excipient/protein weight ratio when compared to those containing sucrose or cellobiose. Similar behavior was observed when analyzing the protein’s secondary structure in lyophilizates. In 20:1 and 40:1 excipient/protein weight ratio lyophilizates the decrease of enzymatic activity was less dependent on the excipient, but activity was always amongst the highest in melibiose lyophilizates.ConclusionsMelibiose was shown to be effective in protecting lyophilized β-galactosidase during storage. The protein secondary structure was shown to change at comparable rate in lyophilizates as its enzymatic activity after rehydration.

[1]  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.

[2]  W. Caughey,et al.  Protein secondary structures in water from second-derivative amide I infrared spectra. , 1990, Biochemistry.

[3]  M. Davenport,et al.  Intestinal Permeability and Absorptive Capacity in Children with Portal Hypertension , 2002, Scandinavian journal of gastroenterology.

[4]  M. Pikal,et al.  Mass and heat transfer in vial freeze-drying of pharmaceuticals: role of the vial. , 1984, Journal of pharmaceutical sciences.

[5]  M. Pikal,et al.  Solid state chemistry of proteins: II. The correlation of storage stability of freeze-dried human growth hormone (hGH) with structure and dynamics in the glassy solid. , 2008, Journal of pharmaceutical sciences.

[6]  Michael J Pikal,et al.  Effect of sorbitol and residual moisture on the stability of lyophilized antibodies: Implications for the mechanism of protein stabilization in the solid state. , 2005, Journal of pharmaceutical sciences.

[7]  T. Arakawa,et al.  Dehydration-induced conformational transitions in proteins and their inhibition by stabilizers. , 1993, Biophysical journal.

[8]  J. May,et al.  Determination of residual moisture in freeze-dried viral vaccines: Karl Fischer gravimetric and thermogravimetric methodologies. , 1982, Journal of biological standardization.

[9]  Daan J.A. Crommelin,et al.  Physical Methods to Characterize Pharmaceutical Proteins , 1995, Pharmaceutical Biotechnology.

[10]  K. Knutson,et al.  Fourier transform infrared spectroscopy investigations of protein structure. , 1995, Pharmaceutical biotechnology.

[11]  Yrjö H. Roos,et al.  Frozen state transitions in relation to freeze drying , 1997 .

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

[13]  H. Mantsch,et al.  Determination of protein secondary structure by Fourier transform infrared spectroscopy: a critical assessment. , 1993, Biochemistry.

[14]  Bruno C. Hancock,et al.  The Relationship Between the Glass Transition Temperature and the Water Content of Amorphous Pharmaceutical Solids , 1994, Pharmaceutical Research.

[15]  M. Řezáčová,et al.  Increased melibiose/rhamnose ratio in bile of rats with acute cholestasis , 2008, Journal of gastroenterology and hepatology.

[16]  M. Manning,et al.  Quantitation of the area of overlap between second-derivative amide I infrared spectra to determine the structural similarity of a protein in different states. , 1996, Journal of pharmaceutical sciences.

[17]  M. Davenport,et al.  Intestinal absorption and permeability in paediatric short-bowel syndrome: a pilot study. , 1999, Journal of pediatric gastroenterology and nutrition.

[18]  R. Parker,et al.  Aspects of the glass transition behaviour of mixtures of carbohydrates of low molecular weight. , 1990, Carbohydrate research.

[19]  David Ouellette,et al.  Mechanism of protein stabilization by sugars during freeze-drying and storage: native structure preservation, specific interaction, and/or immobilization in a glassy matrix? , 2005, Journal of pharmaceutical sciences.

[20]  J. Carpenter,et al.  Application of infrared spectroscopy to development of stable lyophilized protein formulations. , 1998, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[21]  S J Prestrelski,et al.  Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. I. Enzyme activity and calorimetric studies. , 1993, Archives of biochemistry and biophysics.

[22]  G. Zografi,et al.  Sugar-polymer hydrogen bond interactions in lyophilized amorphous mixtures. , 1998, Journal of pharmaceutical sciences.

[23]  S. Wold,et al.  PLS-regression: a basic tool of chemometrics , 2001 .

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

[25]  J. Hofrichter,et al.  Protein reaction kinetics in a room-temperature glass , 1995, Science.

[26]  M. Pikal,et al.  Effect of hydration on the secondary structure of lyophilized proteins as measured by fourier transform infrared (FTIR) spectroscopy. , 2007, Journal of pharmaceutical sciences.

[27]  A. Muga,et al.  Structural and Functional Studies on the Interaction of Sodium Dodecyl Sulfate with β-Galactosidase , 1993 .

[28]  Bruno C. Hancock,et al.  CHARACTERIZATION OF THE TIME SCALES OF MOLECULAR MOTION IN PHARMACEUTICALLY IMPORTANT GLASSES , 1999 .

[29]  A. Muga,et al.  An infrared spectroscopic study of β‐galactosidase structure in aqueous solutions , 1989 .

[30]  W. Jones,et al.  The glass transition temperatures of amorphous trehalose-water mixtures and the mobility of water: an experimental and in silico study. , 2007, Carbohydrate research.

[31]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[32]  H. Frijlink,et al.  Inulin is a promising cryo- and lyoprotectant for PEGylated lipoplexes. , 2005, Journal of controlled release : official journal of the Controlled Release Society.

[33]  S. Yoshioka,et al.  Significance of Local Mobility in Aggregation of β-Galactosidase Lyophilized with Trehalose, Sucrose or Stachyose , 2007, Pharmaceutical Research.

[34]  Y. Inoue,et al.  Infrared spectroscopic study on the properties of the anhydrous form II of trehalose. Implications for the functional mechanism of trehalose as a biostabilizer. , 2001, Carbohydrate research.

[35]  W. Wang,et al.  Lyophilization and development of solid protein pharmaceuticals. , 2000, International journal of pharmaceutics.

[36]  K. Griebenow,et al.  Effect of excipients on the stability and structure of lyophilized recombinant human growth hormone. , 1998, Journal of pharmaceutical sciences.

[37]  J. Rantanen,et al.  In-line monitoring of solid-state transitions during fluidisation , 2007 .

[38]  S. Allison,et al.  Counteracting effects of thiocyanate and sucrose on chymotrypsinogen secondary structure and aggregation during freezing, drying, and rehydration. , 1996, Biophysical journal.

[39]  S J Prestrelski,et al.  Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. II. Structural studies using infrared spectroscopy. , 1993, Archives of biochemistry and biophysics.

[40]  B. Chang,et al.  Physical factors affecting the storage stability of freeze-dried interleukin-1 receptor antagonist: glass transition and protein conformation. , 1996, Archives of biochemistry and biophysics.

[41]  O. Antikainen,et al.  Determination of the crushing strength of intact tablets using Raman spectroscopy. , 2008, International journal of pharmaceutics.

[42]  F. Franks.  Freeze-drying: from empiricism to predictability. The significance of glass transitions. , 1992, Developments in biological standardization.