Separation of Freezing- and Drying-Induced Denaturation of Lyophilized Proteins Using Stress-Specific Stabilization

Carpenter, John F.; Prestrelski, Steven J.; Arakawa, Tsutomu

doi:10.1006/abbi.1993.1309

Cited by 271 publications

(209 citation statements)

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“…A Perkin-Elmer differential scanning calorimeter 7 (DSC 7, Perkin Elmer Ltd., Beaconsfield, Bucks) examined DNase I and T able1-T hespr a y-drying conditions applied to lysozyme, deoxyribonuclease I (DNase I) and lactate dehydrogenase (LDH) Carpenter et al, 1993). Empty pans with vented lids were used as references.…”

Section: Differential Scanning Calorimetry (Dsc)mentioning

confidence: 99%

Study of protein conformational stability and integrity using calorimetry and FT-Raman spectroscopy correlated with enzymatic activity

Elkordy

Forbes²,

Barry³

2008

European Journal of Pharmaceutical Sciences

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Enzymatic assay after cooling of thermally denatured protein solutions from HSDSC determined if thermal transition reversibility was related to biological activity. HSDSC data showed that molecules from lyophilised lysozyme were able to refold better than the spraydried form. This was confirmed by enzymatic assay. Moreover, enzymatic assay results revealed that lysozyme folding reversibility was related to the native structure of the protein that is essential for the biological activity.Thermal denaturation of DNase I and LDH samples in HSDSC was not reversible upon cooling of thermally denatured proteins (in contrast to lysozyme). Hence, it was decided to identify the effect of protein initial structures on its propensity to thermal denaturation via FT-Raman spectroscopy. In other words, proteins may denature with structural alterations due to stresses such as heat and the protein loses its enzymatic activity. Consequently, FTRaman investigated the effects of spray drying and heating of solid DNase I and LDH samples, from differential scanning calorimetry, on protein conformational integrities. Lyophilised and spray-dried DNase I and LDH solid samples were heated to two temperatures, one before the apparent denaturation temperatures (T m ) and the other after the T m . Samples heated below their T m showed some alterations of the secondary structure and some enzy- IntroductionProteins fold in three dimensions. Protein structure is organised from primary structure to quaternary structure. primary structure or the amino acid sequence of the peptide chains is the ultimate determinant for the protein native form. The protein secondary structure is the assignment of ␣-helices and ␤-sheets along the main peptide chain; whilst the ter-0928-0987/$ -see front matter

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Section: Differential Scanning Calorimetry (Dsc)mentioning

confidence: 99%

Study of protein conformational stability and integrity using calorimetry and FT-Raman spectroscopy correlated with enzymatic activity

Elkordy

Forbes²,

Barry³

2008

European Journal of Pharmaceutical Sciences

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“…Trehalose has also been found to be very effective in the stabilization of labile proteins during lyophilization (15,16) and exposure to high temperatures in solution (2,8,9). Sugars in general protect proteins against dehydration by hydrogen bonding to the dried protein by serving as water substitute (15,17).…”

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confidence: 99%

“…Sugars in general protect proteins against dehydration by hydrogen bonding to the dried protein by serving as water substitute (15,17). Several studies carried out by Timasheff and coworkers (9,18) show that sugars and polyols stabilize the folded structure of proteins in solution as a result of greater preferential hydration of the unfolded state compared with the native state.…”

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confidence: 99%

Why Is Trehalose an Exceptional Protein Stabilizer?

Kaushik¹,

Bhat

2003

Journal of Biological Chemistry

547

246

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Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 M trehalose was observed to raise the transition temperature, T m of RNase A by as much as 18°C and Gibbs free energy by 4.8 kcal mol ؊1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (⌬C p ) in trehalose solutions for all the studied proteins. An increase in the ⌬G and a decrease in the ⌬C p values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (⌬G versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 M concentration 4 -7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.Sugars have been known to protect proteins against loss of activity (1, 2), chemical (3, 4), and thermal denaturation (5-9). Among several sugars, ␣,␣-trehalose (␣-D-glucopyranosyl(131)-␣-D-glucopyranoside) has been known to be a superior stabilizer in providing protection to biological materials against dehydration and desiccation (10, 11). It is a compatible osmolyte that gets accumulated in organisms under stress conditions (12, 13). Because of this unique property, tremendous interest has been generated in understanding the molecular basis of stress management through induction of trehalose biosynthesis (13, 14).Trehalose has also been found to be very effective in the stabilization of labile proteins during lyophilization (15, 16) and exposure to high temperatures in solution (2,8,9). Sugars in general protect proteins against dehydration by hydrogen bonding to the dried protein by serving as water substitute (15, 17). Several studies carried out by Timasheff and coworkers (9,18) show that sugars and polyols stabilize the folded structure of proteins in solution as a result of greater preferential hydration of the unfolded state compared with the native state. The mechanism is fundamentally different from stabil...

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confidence: 99%

“…19) Sucrose and trehalose protect proteins by substituting surrounding water molecules through hydrogen bonds during the freeze-drying process. 4,[20][21][22] Limited molecular mobility in glass-state lyophilized disaccharide solids also protects embedded proteins from chemical degradation (e.g., deamidation) during storage. 23) The present study assesses the physical properties and protein-stabilizing effects of carboxylic acid buffer systems (e.g., sodium citrate, sodium L-tartrate, sodium succinate) and their constituting salts against lyophilization-induced protein secondary structure changes.…”

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confidence: 99%

Stabilization of Protein Structure in Freeze-Dried Amorphous Organic Acid Buffer Salts

Izutsu¹,

Kadoya

Yomota³

et al. 2009

Chem. Pharm. Bull.

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The development of protein pharmaceuticals requires rational formulation design to ensure appropriate storage stability, because the degradation of such pharmaceuticals through various chemical and physical pathways not only reduces their therapeutic effects but also increases the risk of product immunogenicity. [1][2][3][4][5] Freeze-drying is a popular method of conferring long-term stability of therapeutic proteins that is not achievable in aqueous solutions. Removal of the surrounding water molecules during the freeze-drying process, however, often perturbs the protein structure, leading to irreversible aggregation in the reconstituted solutions. The structurally altered protein molecules are also prone to chemical degradation during storage.1) Maintaining the protein conformation by process and ingredient (e.g., stabilizer, pH buffer, isotonic agents) optimization thus improves both the physical and chemical stability of protein formulations.Choosing the solution pH and buffer system appropriate to a particular protein is a simple but significant element in the formulation design because the chemical and physical integrity of proteins in the aqueous solutions and freeze-dried solids depend largely on the pH. 6) Some buffer components also favorably or adversely affect the protein stability through direct interactions and/or through changing the local environments in the dried state. For example, freezing of certain buffer systems (e.g., sodium phosphate) often induces crystallization of a component salt and resulting shift of the local pH surrounding the proteins.7-11) Freeze-drying from some buffer systems (e.g., L-histidine, citrate, or Tris) often leads to higher activity retention of proteins (e.g., coagulation factor VIII, recombinant human interleukin-1 receptor antagonist) relative to those from other buffers.12-15) Conformation of the proteins lyophilized in these buffer systems is of particular interest.Reported properties of some carboxylic acid salts, including stabilization of native protein conformation in aqueous solutions (e.g., antithrombin III) 16,17) and their propensity to form glass-state amorphous solids upon lyophilization, 18) suggest their ability to protect protein conformation against dehydration stress through mechanisms similar to disaccharides. Non-reducing disaccharides (e.g., sucrose, trehalose) are popular stabilizers in solution and freeze-dried protein formulations. Various saccharides and polyols thermodynamically favor native protein structures over denatured states in aqueous solutions by a "preferential exclusion" mechanism. 19) Sucrose and trehalose protect proteins by substituting surrounding water molecules through hydrogen bonds during the freeze-drying process. 4,[20][21][22] Limited molecular mobility in glass-state lyophilized disaccharide solids also protects embedded proteins from chemical degradation (e.g., deamidation) during storage. 23)The present study assesses the physical properties and protein-stabilizing effects of carboxylic acid buffer systems (e.g.,...

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Separation of Freezing- and Drying-Induced Denaturation of Lyophilized Proteins Using Stress-Specific Stabilization

Cited by 271 publications

References 0 publications

Study of protein conformational stability and integrity using calorimetry and FT-Raman spectroscopy correlated with enzymatic activity

Study of protein conformational stability and integrity using calorimetry and FT-Raman spectroscopy correlated with enzymatic activity

Why Is Trehalose an Exceptional Protein Stabilizer?

Stabilization of Protein Structure in Freeze-Dried Amorphous Organic Acid Buffer Salts

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