Screening of novel excipients for freeze-dried protein formulations.

The typical excipients used as bulking agents and lyoprotectants for freeze-drying are usually limited to only a few selected substances, such as sucrose and mannitol. Considering the sheer diversity amongst proteins, it is doubtful that this limited choice should, in every case, provide the best possible option in order to achieve the most stable product. In this work, a screening of 12 proteins with 64 excipients was conducted in order to increase the knowledge space of potential excipients. Three critical quality attributes (CQAs) of the freeze-dried products, namely the solid state, the cake appearance and the protein integrity based on changes in tryptophan fluorescence were investigated by high throughput X-ray powder diffraction, image analysis and intrinsic fluorescence spectroscopy, respectively. It was found, that in some cases the excipient had a dominating influence on the CQAs, whilst in other cases the CQAs were primarily protein dependent, or that the CQAs were dependent on the combination of both. In the course of this investigation, a general view of potentially relevant excipients, and their interplay with various proteins, was obtained, thereby furthermore paving the way for the use of novel freeze-drying excipients.

Exploring the chemical space for freeze-drying excipients.

Commonly, a limited number of generally accepted bulking agents and lyoprotectants are used for freeze-drying; predominantly mannitol, glycine, sucrose and trehalose. The purpose of this study was to combine a theoretical approach using molecular descriptors with a large scale experimental screening to evaluate the suitability of a broad range of excipients for freeze-drying. A large selection of sugars, polyols and amino acids was characterized by modulated differential scanning calorimetry (mDSC) and X-ray powder diffraction (XRPD) after well-plate based freeze-drying. The calculated molecular descriptors were investigated with both hierarchical cluster analysis and principal component analysis. A clear clustering of the excipients according to the size-related and weight-related descriptors was observed; however other relevant descriptors could also be identified. From a practical perspective, a trend was observed with regard to a higher likelihood for amorphisation and a higher glass transition temperature of the maximally freeze-concentrated solution with increasing molecular size. A translation of the molecular descriptors on pharmaceutical performance was more successful for lyoprotectants than for bulking agents. Additionally, in the course of the experimental screening, several new potential bulking agents and lyoprotectants were identified.

Using dextran of different molecular weights to achieve faster freeze-drying and improved storage stability of lactate dehydrogenase.

Freeze-drying of protein formulations is frequently used to maintain protein activity during storage. The freeze-drying process usually requires long primary drying times because the highest acceptable drying temperature to obtain acceptable products is dependent on the glass transition temperature of the maximally freeze-concentrated solution (Tg'). On the other hand, retaining protein activity during storage is related to the glass transition temperature (Tg) of the final freeze-dried product. In this study, dextrans with different molecular weight (1 and 40 kDa) and mixtures thereof at the ratio 3:1, 1:1, and 1:3 (w/w) were used as cryo-/lyoprotectant and their impact on the stability of the model protein lactate dehydrogenase (LDH) was investigated at elevated temperatures (40 °C and 60 °C). The dextran formulations were then compared to formulations containing sucrose as cryo-/lyoprotectant. Because of the higher Tg' values of the dextrans, the primary drying times could be reduced compared to freeze-drying with sucrose. Similarly, the higher Tg and Tg' of dextrans relative to sucrose led to benefits during storage which was shown through improved protection of LDH activity.

The use of molecular descriptors in the development of co-amorphous formulations.

Co-amorphous systems consisting of a drug and an amino acid have been investigated extensively for the enhancement of drug solubility and amorphous stability. The purpose of this study is to investigate which molecular descriptors are important for predicting the likelihood of a successful co-amorphisation between amino acid and drug. The predictions are thought to be used in an early screening phase to identify potential drug-amino acid combinations for further studies. A large variety of molecular descriptors was calculated for six drugs (carvedilol, mebendazole, carbamazepine, furosemide, indomethacin and simvastatin) and the twenty naturally occurring amino acids. The descriptor differences for all drug-amino acid combinations were calculated and used as input in the X-matrix of a Partial Least Square Discriminant Analysis (PLS-DA). The Y-matrix of the PLS-DA consisted of the X-ray powder diffraction response ("co-amorphous" or "not co-amorphous") obtained by ball milling all combinations for 60 min. The PLS-DA model showed a clear separation of the not co-amorphous and the co-amorphous samples and was successfully predicting the class membership of 19 out of the 20 completely left out drug-amino acid combinations of mebendazole. The approach seems to be promising for predicting the ability of new drug-amino acids combinations to become co-amorphous.

Correlation between calculated molecular descriptors of excipient amino acids and experimentally observed thermal stability of lysozyme.

A quantitative structure-property relationship (QSPR) between protein stability and the physicochemical properties of excipients was investigated to enable a more rational choice of stabilizing excipients than prior knowledge. The thermal transition temperature and aggregation time were determined for lysozyme in combination with 13 different amino acids using high throughput fluorescence spectroscopy and kinetic static light scattering measurements. On the theoretical side, around 200 2D and 3D molecular descriptors were calculated based on the amino acids' chemical structure. Multivariate data analysis was applied to correlate the descriptors with the experimental results. It was possible to identify descriptors, i.e. amino acids properties, with a positive influence on either transition temperature or aggregation onset time, or both. A high number of hydrogen bond acceptor moieties was the most prominent stabilizing factor for both responses, whereas hydrophilic surface properties and high molecular mass density mostly had a positive influence on the unfolding temperature. A high partition coefficient (logP(o/w)) was identified as the most prominent destabilizing factor for both responses. The QSPR shows good correlation between calculated molecular descriptors and the measured stabilizing effect of amino acids on lysozyme.

Surface area, volume and shape descriptors as a novel tool for polymer lead design and discovery.

In recent years, the demand and interest for functionalized polymers have increased for drug delivery purposes. Because of the increased interest, methods that can be used to predict physical and chemical properties of polymers prior to synthesis would be of high value for the design and development of novel polymer structures. Through use of molecular descriptors and Principal Component Analysis, this study explores the possibilities of using in silico methods for polymer design and characterization for property prediction. The results presented in this paper suggest that it is possible to produce a model, which can successfully distinguish between a set of both structurally similar and different polymers based on their surface properties.