The ice-water interface and protein stability: a review

The ice-water interface is commonly encountered in our life, and comes into play in a wide number of natural phenomena. Here, attention will be focused on its effects on protein stability, with specific reference to the case of pharmaceutical proteins. This field represents a fascinating, and not yet fully understood, subject of investigation. Some background information on the ice-water phase diagram, as well as to the mechanisms of nucleation and crystal growth, will be provided. We will eventually discuss the effect of ice on protein activity, reviewing the mechanisms of ice-induced denaturation that have been proposed so far and discussing the strategies that may help prevent, or minimize, undesired loss of therapeutic activity

A model-based approach for the rational design of the freeze-thawing of a protein-based formulation

Proteins are unstable molecules that may be severely injured by stresses encountered during freeze-thawing. Despite this, the selection of freeze-thaw conditions is currently empirical, and this results in reduced process control. Here we propose a mathematical model that takes into account the leading causes of protein instability during freeze-thawing, i.e. cold denaturation and surface-induced unfolding, and may guide the selection of optimal operating conditions. It is observed that a high cooling rate is beneficial for molecules that are extremely sensitive to cold denaturation, while the opposite is true when ice-induced unfolding is dominant. In all cases, a fast thawing rate is observed to be beneficial. The simulation outputs are confirmed by experimental data for myoglobin and lactate dehydrogenase, suggesting that the proposed modeling approach can reproduce the main features of protein behavior during freeze-thawing. This approach can therefore guide the selection of optimal conditions for protein-based formulations that are stored in a frozen or freeze-dried state

Image segmentation applied to the investigation of craquelure domains in paintings

Old paintings are generally characterized by the presence of cracks patterns, formed during the drying process. This network of cracks, known as craquelure, represents a key feature for the authentication of artworks and the discovery of forgery. In this work, an image segmentation approach is used for a quantitative study of cracks patterns in paintings. We will show how the size distribution and orientation of the craquelure domains can be evaluated using this technique

The role of cyclodextrins against interface-induced denaturation in pharmaceutical formulations: a molecular dynamics approach

Protein-based pharmaceutical products are subject to a variety of environmental stressors, during both production and shelf-life. In order to preserve their structure, and, therefore, functionality, it is necessary to use excipients as stabilizing agents. Among the eligible stabilizers, cyclodextrins (CDs) have recently gained interest in the scientific community thanks to their properties. Here, a computational approach is proposed to clarify the role of β-cyclodextrin (βCD) and 2-hydroxypropyl-β-cyclodextrin (HPβCD) against granulocyte colony-stimulating (GCSF) factor denaturation at the air-water and ice-water interfaces, and also in bulk water at 300 or 260 K. Both traditional molecular dynamics (MD) simulations and enhanced sampling techniques (metadynamics, MetaD) are used to shed light on the underlying molecular mechanisms. Bulk simulations revealed that CDs were preferentially included within the surface hydration layer of GCSF, and even included some peptide residues in their hydrophobic cavity. HPβCD was able to stabilize the protein against surface-induced denaturation in proximity of the air-water interface, while βCD had a destabilizing effect. No remarkable conformational changes of GCSF, or noticeable effect of the CDs, were instead observed at the ice surface. GCSF seemed less stable at low temperature (260 K), which may be attributed to cold-denaturation effects. In this case, CDs did not significantly improve conformational stability. In general, the conformationally altered regions of GCSF seemed not to depend on the presence of excipients that only modulated the extent of destabilization with either a positive or a negative effect

Surface treatment of glass vials for lyophilization: Implications for vacuum-induced surface freezing

Freeze-drying is commonly used to increase the shelf-life of pharmaceuticals and biopharmaceuticals. Freezing represents a crucial phase in the freeze-drying process, as it determines both cycle efficiency and product quality. For this reason, different strategies have been developed to allow for a better control of freezing, among them, the so-called vacuum-induced surface freezing (VISF), which makes it possible to trigger nucleation at the same time in all the vials being processed. We studied the effect of different vial types, characterized by the presence of hydrophilic (sulfate treatment) or hydrophobic (siliconization and TopLyo Si–O–C–H layer) inner coatings, on the application of VISF. We observed that hydrophobic coatings promoted boiling and blow-up phenomena, resulting in unacceptable aesthetic defects in the final product. In contrast, hydrophilic coatings increased the risk of fogging (i.e., the undesired creeping of the product upward along the inner vial surface). We also found that the addition of a surfactant (Tween 80) to the formulation suppressed boiling in hydrophobic-coated vials, but it enhanced the formation of bubbles. This undesired bubbling events induced by the surfactant could, however, be eliminated by a degassing step prior to the application of VISF. Overall, the combination of degasification and surfactant addition seems to be a promising strategy for the successful induction of nucleation by VISF in hydrophobic vials

ADD force field for sugars and polyols: predicting the additivity of protein-osmolyte interaction

The protein-osmolyte interaction has been shown experimentally to follow an additive construct, where the individual osmolyte-backbone and osmolyte-side-chain interactions contribute to the overall conformational stability of proteins. Here, we computationally reconstruct this additive relation using molecular dynamics simulations, focusing on sugars and polyols, including sucrose and sorbitol, as model osmolytes. A new set of parameters (ADD) is developed for this purpose, using the individual Kirkwood-Buff integrals for sugar-backbone and sugar-side-chain interactions as target experimental data. We show that the ADD parameters can reproduce the additivity of protein-sugar interactions and correctly predict sucrose and sorbitol self-association and their interaction with water. The accurate description of the separate osmolyte-backbone and osmolyte-side-chain contributions also automatically translates into a good prediction of preferential exclusion from the surface of ribonuclease A and α-chymotrypsinogen A. The description of sugar polarity is improved compared to previous force fields, resulting in closer agreement with the experimental data and better compatibility with charged groups, such as the guanidinium moiety. The ADD parameters are developed in combination with the CHARMM36m force field for proteins, but good compatibility is also observed with the AMBER 99SB-ILDN and the OPLS-AA force fields. Overall, exploiting the additivity of protein-osmolyte interactions is a promising approach for the development of new force fields

Impact of controlled vacuum induced surface freezing on the freeze drying of human plasma

During the freezing step of a typical freeze drying process, the temperature at which nucleation is induced is generally stochastically distributed, resulting in undesired within-batch heterogeneity. Controlled nucleation techniques have been developed to address this problem; these make it possible to trigger the formation of ice crystals at the same time and temperature in all the batch. Here, the controlled nucleation technique known as vacuum induced surface freezing is compared to spontaneous freezing for the freeze drying of human plasma, a highly concentrated system commonly stored in a dried state. The potency of Factor VIII (FVIII), a sensitive, labile protein present in plasma, and the reconstitution time of the dried cakes are evaluated immediately after freeze drying, and after 1, 3, 6 or 9 months storage at different degradation temperatures. We show that the application of controlled nucleation significantly reduces the reconstitution time and in addition helps to improve FVIII stability

Vacuum-Induced Surface Freezing for the Freeze-Drying of the Human Growth Hormone: How Does Nucleation Control Affect Protein Stability?

Abstract In the present work, the effect of controlled nucleation on the stability of human growth hormone (hGH) during freeze-drying has been investigated. More specifically, the vacuum-induced surface freezing technique has been compared to conventional freezing, both with and without an annealing step. Size exclusion chromatography and cell-based potency assays have been used to characterize the formation of soluble aggregates and the biological activity of hGH, respectively. The results obtained indicate that controlled nucleation has a positive effect on both cycle performance and product homogeneity because of the formation of bigger ice crystals, and characterized by a narrower dimensional distribution. From the point of view of hGH stability, we observed that vacuum-induced surface freezing is not detrimental to the biological activity of the protein, or aggregate formation. In addition, the effect of 2 different formulations, including trehalose or cellobiose, on protein preservation was also considered for this study

Image segmentation and 3D reconstruction for improved prediction of the sublimation rate during freeze drying

In a freeze drying process, the freezing step determines the pore size distribution within the product, which, in turn, affects the sublimation rate. Traditionally, pore analysis is carried out on SEM images by means of a manual, time-consuming approach. Here, an image segmentation technique was used to automatize this process and improve its reliability. A 3D structure of the cake was then reconstructed from the distribution of the super-pixels. We show that the approach herein proposed can remarkably improve prediction of the sublimation rate with respect to traditional methods

Water entrapment and structure ordering as protection mechanisms for protein structural preservation

In this paper, Molecular Dynamics is used to further gain insight into the mechanisms by which typical pharmaceutical excipients preserve the protein structure. More specifically, the water entrapment scenario will be analyzed, which states that excipients form a cage around the protein, entrapping and slowing water molecules. Human growth hormone will be used as model protein, but the results obtained are generally-applicable. We will show that water entrapment, as well as the other mechanisms of protein stabilization in the dried state proposed so far, may be related to the formation of a dense hydrogen bonding network between excipient molecules. We will also present a simple phenomenological model capable of explaining the behavior and stabilizing effect provided by typical cryo- and lyo-protectants. This model uses, as input data, molecular properties which can be easily evaluated. We will finally show that the model predictions compare fairly well with experimental data