Phase Separation of Excipients during Lyophilization: Effects on Protein Stability

Raman spectroscopy and multivariate analysis for the rapid discrimination between native-like and non-native states in freeze-dried protein formulations

“…It is well known that co-lyophilized mixtures of sucrose and polymers (e.g. proteins) interact at a molecular level through hydrogen bonding, forming miscible amorphous molecular dispersions [40][41][42]. As there is evidence that the C=O stretching vibration is strongly influenced by the polymer-sucrose hydrogen bond [40], it is expected that this will notably influence the amide I band, typically dominated by signals of the backbone C=O stretching vibrations.…”

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

“…Therefore, glassy (amorphous) formulations should not be subjected to of water with the protein during drying and thus stabilize the protein conformation (Carpenter 59 and Crowe, 1989). These two theories are not mutually exclusive; both mechanisms play a 60 role in protein stabilization (Grasmeijer et al, 2013;Randolph, 1997 …”

Effect of Sodium Tetraborate (Borax) on the Thermal Properties of Frozen Aqueous Sugar and Polyol Solutions

“…5,6) Freezing an aqueous solution concentrates the solutes into a supercooled solution surrounded by ice crystals. 7,8) The sugars and polyols protect the proteins and cell membranes against dehydration-induced structural perturbation by substituting water molecules through direct molecular interactions (e.g., hydrogen bonding). Decreased molecular mobility in the polyol-based glass-state amorphous phase reduces chemical changes of the components.…”

“…Decreased molecular mobility in the polyol-based glass-state amorphous phase reduces chemical changes of the components. 3,7) Controlling the physical properties of the freeze-concentrates and subsequently freeze-dried solids should provide the key to production of stable protein and other biopolymer formulations. 1,2,7) Thermal analysis of frozen aqueous carbohydrate solutions often shows several transitions of the amorphous supercooled phase including the "real" glass transition (T g ) and the glass transition of maximally freeze-concentrated solutes (T g Ј) at temperatures depending on the solute compositions.…”

“…3,7) Controlling the physical properties of the freeze-concentrates and subsequently freeze-dried solids should provide the key to production of stable protein and other biopolymer formulations. 1,2,7) Thermal analysis of frozen aqueous carbohydrate solutions often shows several transitions of the amorphous supercooled phase including the "real" glass transition (T g ) and the glass transition of maximally freeze-concentrated solutes (T g Ј) at temperatures depending on the solute compositions. 9,10) The terming and implications of these thermal transitions are still under some debate, whereas it has been established that the changes in the solute and surrounding water mobility at the transition temperatures have significant impact on the physical and chemical stability of the components.…”