Protection Mechanisms of Excipients on Lactate Dehydrogenase during Freeze-Thawing and Lyophilization
type: CreativeWork
description: Problem - The primary objective of this dissertation was to elucidate the protective effects and mechanisms of excipients on a labile model protein, lactate dehydrogenase (LDH), during freeze-thawing and lyophilization. Cryoprotectants are excipients that protect labile molecules during freezing. Some investigators believe that preferential exclusion is the predominant mechanism to explain the cryoprotection of labile proteins by the polyethylene glycols (PEG's). They assume that the hydration shell around a labile protein remains in tact during freezing, as in solution. But freezing is a separation process, that extracts much of the water into a crystalline ice matrix that is distinctly different from pockets containing the solid amorphous protein. Additionally, proponents of the preferential exclusion theory have not presented direct evidence to support integrity of the hydration shell at temperatures beneath the glass transition temperature. Another possible stabilizing mechanism could be that some excipients preferentially bind to a labile protein to stabilize the macromolecule. A preferential binding mechanism to induce protein stabilization in the frozen state would in direct contrast to the proposed Timasheff preferential exclusion mechanism of protein stabilization. Different hyotheses have been proposed to explain the mechanism/s of protection during the drying portion of the lyophilization cycle. Currently, there are two main hypotheses to explain the mechanisms of labile protein lyoprotection (i.e., stabilization during drying) with excipients. The first is the "single amorphous immobilization hypothesis". This theory proposes that amorphous protein stabilization can only be achieved if another amorphous compound provides immobilization and spatial separation in a glassy, solid matrix during dehydration. The second hypothesis is the "water replacement hypothesis". According to this theory, sugars stabilize proteins by hydrogen bonding and other weak force…
abstract: Problem - The primary objective of this dissertation was to elucidate the protective effects and mechanisms of excipients on a labile model protein, lactate dehydrogenase (LDH), during freeze-thawing and lyophilization. Cryoprotectants are excipients that protect labile molecules during freezing. Some investigators believe that preferential exclusion is the predominant mechanism to explain the cryoprotection of labile proteins by the polyethylene glycols (PEG's). They assume that the hydration shell around a labile protein remains in tact during freezing, as in solution. But freezing is a separation process, that extracts much of the water into a crystalline ice matrix that is distinctly different from pockets containing the solid amorphous protein. Additionally, proponents of the preferential exclusion theory have not presented direct evidence to support integrity of the hydration shell at temperatures beneath the glass transition temperature. Another possible stabilizing mechanism could be that some excipients preferentially bind to a labile protein to stabilize the macromolecule. A preferential binding mechanism to induce protein stabilization in the frozen state would in direct contrast to the proposed Timasheff preferential exclusion mechanism of protein stabilization. Different hyotheses have been proposed to explain the mechanism/s of protection during the drying portion of the lyophilization cycle. Currently, there are two main hypotheses to explain the mechanisms of labile protein lyoprotection (i.e., stabilization during drying) with excipients. The first is the "single amorphous immobilization hypothesis". This theory proposes that amorphous protein stabilization can only be achieved if another amorphous compound provides immobilization and spatial separation in a glassy, solid matrix during dehydration. The second hypothesis is the "water replacement hypothesis". According to this theory, sugars stabilize proteins by hydrogen bonding and other weak force…
author: Mi, Yanli
keywords: LDH, PEG, cryoprotection, lyoprotection, stabilization, freeze-thawing, lyophilization, non-specific binding, CD, SEC-HPLC, FTIR, Freeze-dry cryostage microscope, Medicinal and Pharmaceutical Chemistry, Medicine and Health Sciences, Pharmacy and Pharmaceutical Sciences
date published: 2002-12-01T08:00:00Z
date modified: 2026-07-24T05:00:11Z
provider: University of Tennessee Health Science Center
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Tennessee Health Science Center