title: In-line near infrared spectroscopy during freeze-drying as a tool to measure efficiency of hydrogen bond formation between protein and sugar, predictive of protein storage stability.
abstract: Sugars are often used as stabilizers of protein formulations during freeze-drying. However, not all sugars are equally suitable for this purpose. Using in-line near-infrared spectroscopy during freeze-drying, it is shown here that hydrogen bond formation during freeze-drying, under secondary drying conditions in particular, can be related to the preservation of the functionality and structure of proteins during storage. The disaccharide trehalose was best capable of forming hydrogen bonds with the model protein, lactate dehydrogenase, thereby stabilizing it, followed by the molecularly flexible oligosaccharide inulin 4kDa. The molecularly rigid oligo- and polysaccharides dextran 5kDa and 70kDa, respectively, formed the least amount of hydrogen bonds and provided least stabilization of the protein. It is concluded that smaller and molecularly more flexible sugars are less affected by steric hindrance, allowing them to form more hydrogen bonds with the protein, thereby stabilizing it better.
authors.affiliation: Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Maarten A
authors.initials: MA
authors.lastname: Mensink
authors.affiliation: Laboratory of Pharmaceutical Process Analytical Technology, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: Pieter-Jan
authors.initials: PJ
authors.lastname: Van Bockstal
authors.affiliation: Laboratory of Pharmaceutical Process Analytical Technology, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: Sigrid
authors.initials: S
authors.lastname: Pieters
authors.affiliation: Laboratory of Pharmaceutical Process Analytical Technology, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: Laurens
authors.initials: L
authors.lastname: De Meyer
authors.affiliation: Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
authors.forename: Henderik W
authors.initials: HW
authors.lastname: Frijlink
authors.affiliation: Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands; Process Technology, Corbion Purac, P.O. Box 21,4200 AA Gorinchem, The Netherlands.
authors.forename: Kees
authors.initials: K
authors.lastname: van der Voort Maarschalk
authors.affiliation: Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. Electronic address: w.l.j.hinrichs@rug.nl.
authors.forename: Wouter L J
authors.initials: WL
authors.lastname: Hinrichs
authors.affiliation: Laboratory of Pharmaceutical Process Analytical Technology, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: De Beer
mesh_terms: D002241:Carbohydrates / Q000737:chemistry*; D005612:Freeze Drying; D006860:Hydrogen Bonding; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D019265:Spectroscopy, Near-Infrared / Q000379:methods*
chemical_list: D002241:Carbohydrates; D011506:Proteins
keywords: Fourier transform infrared (FTIR) spectroscopy; Molecular flexibility; Near-infrared (NIR) spectroscopy; Solid-state stability; Vitrification; Water-replacement