Effect of hydration on the secondary structure of lyophilized proteins as measured by fourier transform infrared (FTIR) spectroscopy

Luthra, Sumit; Kalonia, Devendra S.; Pikal, Michael J.

doi:10.1002/jps.20890

Cited by 29 publications

(26 citation statements)

References 19 publications

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“…Although structure appeared well preserved for our model proteins, studies have demonstrated the risk of structure alterations for e.g. freeze-dried samples, 42,43 which indicates that secondary structure perturbations upon drying cannot generally be discounted. Addition of saccharides to protect native secondary structure could be considered in such situations although the additive must allow the formation of transparent protein films.…”

Section: Drying Has Limited Effect On Protein Structurementioning

confidence: 92%

Characterization of dry globular proteins and protein fibrils by synchrotron radiation vacuum UV circular dichroism

et al. 2008

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Circular dichroism using synchrotron radiation (SRCD) can extend the spectral range down to approximately 130 nm for dry proteins, potentially providing new structural information. Using a selection of dried model proteins, including alpha-helical, beta-sheet, and mixed-structure proteins, we observe a low-wavelength band in the range 130-160 nm, whose intensity and peak position is sensitive to the secondary structure of the protein and may also reflect changes in super-secondary structure. This band has previously been observed for peptides but not for globular proteins, and is compatible with previously published theoretical calculations related to pi-orbital transitions. We also show that drying does not lead to large changes in the secondary structure and does not induce orientational artifacts. In combination with principal component analysis, our SRCD data allow us to distinguish between two different types of protein fibrils, highlighting that bona fide fibrils formed by lysozyme are structurally more similar to the nonclassical fibrillar aggregates formed by the SerADan peptide than with the amyloid formed by alpha-synuclein. Thus, despite the lack of direct structural conclusions, a comprehensive SRCD-based database of dried protein spectra may provide a useful method to differentiate between various types of supersecondary structure and aggregated protein species.

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Section: Drying Has Limited Effect On Protein Structurementioning

confidence: 92%

Characterization of dry globular proteins and protein fibrils by synchrotron radiation vacuum UV circular dichroism

et al. 2008

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“…Freeze-drying of the protein from the buffer systems resulted in a varied extent of the lyophilization-induced structural perturbation as observed in the broad amide I spectra and reduced a-helix band (1656 cm Ϫ1 ) intensity. 2,24,[28][29][30] Larger structural changes were suggested in freeze-drying of the protein from sodium phosphate and sodium succinate buffer solutions. Figure 5 shows area-normalized second-derivative amide I spectra of BSA freeze-dried with the organic acids and their salts (50 mM).…”

Section: Resultsmentioning

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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“…Originally, sample preparation prior to FTIR measurements in transmission mode involved grinding of the lyophile with potassium bromide and subsequent compression into a pellet under vacuum . This procedure had been claimed not to cause significant alterations in the protein–disaccharide interactions and to produce only minor, if any, effects on protein secondary structures . However, this meant that only a putatively representative sample portion could be measured.…”

Section: Methodsmentioning

confidence: 99%

“…Furthermore, the results presented as “bottom” and “top” are assumed to refer to a sample portion, not exclusively to the outermost layer of the sample. Such an assumption is reasonable and takes into account the FTIR‐ATR beam penetration depth and the porosity of freeze‐dried samples, potentially causing intermediate layers to fill the voids of the outermost layer during fixation.…”

Section: Methodsmentioning

confidence: 99%

Impact of Microscale and Pilot-Scale Freeze-Drying on Protein Secondary Structures: Sucrose Formulations of Lysozyme and Catalase

Peters

Leskinen

Molnár

et al. 2015

Journal of Pharmaceutical Sciences

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Effect of hydration on the secondary structure of lyophilized proteins as measured by fourier transform infrared (FTIR) spectroscopy

Cited by 29 publications

References 19 publications

Characterization of dry globular proteins and protein fibrils by synchrotron radiation vacuum UV circular dichroism

Characterization of dry globular proteins and protein fibrils by synchrotron radiation vacuum UV circular dichroism

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

Impact of Microscale and Pilot-Scale Freeze-Drying on Protein Secondary Structures: Sucrose Formulations of Lysozyme and Catalase

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