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.

Protein adsorption to biomaterial surfaces is important for the function of such materials with anchorage-dependent cell adhesion requiring the presence of adsorbed proteins. The current study evaluated five solid surfaces with poly(acrylic acid) (PAA) grafted from the surface of a poly(tetrafluoroethylene) membrane with respect to the adsorption of serum albumin (SA), lactoferrin (Lf), and lysozyme (Lys) from a phosphate buffer and NaCl solution or water for specific combinations. With the use of x-ray photoelectron spectroscopy, the relative amounts and protein layer thickness were evaluated. SA adsorption was governed by ionic repulsive forces and hydrophobic interactions as evidenced from an increase in the protein adsorption at lower pH (6.5 compared to 7.4) and a correlation with surface coverage when water (pH 6.5) was used as the medium. The adsorption of Lf and Lys followed similar trends for all samples. In general, ionic attractive forces dominated and a strong correlation of increasing protein adsorption with the PAA chain length was evident. This study concluded that all surfaces appear suitable for use in biomaterial applications where tissue ingrowth is desired and that the enhanced protein adsorption in a medium with high ionic strength (e.g., biological fluid) correlates with the PAA chain length rather than the surface coverage.

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.

Protein biologics are prone to conformational changes during formulation development. Limited methods are available for conformational analysis of proteins in solid state and in the presences of formulation excipients. The aim of this study was to investigate the secondary structures of proteins encased in solid lipid matrices as a novel indicator of their stability upon in vitro release. Model proteins namely catalase and lysozyme were incorporated into lipid namely Precirol® AT05 (glycerol palmitostearate, melting point 58°C) at 30% w / w loading using melting and mixing and wet granulation methods. Attenuated total reflectance (ATR-FTIR) spectroscopy, size-exclusion chromatography (SEC) and biological activity analyses were performed. The information about secondary structure was acquired using second derivative analysis of amide-I band (1600–1700 cm −1 ). ATR analysis demonstrated interference of lipid spectrum with protein amide-I band which was subsequently subtracted to allow the analysis of the secondary structure. ATR spectra amide-I bands showed shifts peak band positions compared to native protein for matrices prepared using wet granulation. SEC analysis gave evidence of protein aggregation for catalase which was increased using wet granulation. The biological activity of catalase was statistically different from that of control and was affected by the incorporation method and was found to be in alignment with ATR spectral changes and extent of aggregation. In conclusion, ATR spectroscopy could analyze protein secondary structure in lipid matrices provided lipid interference was minimized. The ATR spectral changes and formation of aggregates can indicate the loss in biological activity of protein released from solid lipid matrices.

Protein drugs may encounter conformational perturbations during the formulation processing of lipid-based solid dosage forms. In aqueous protein solutions, attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy can investigate these conformational changes following the subtraction of spectral interference of solvent with protein amide I bands. However, in solid dosage forms, the possible spectral contribution of lipid carriers to protein amide I band may be an obstacle to determine conformational alterations. The objective of this study was to develop an ATR FT-IR spectroscopic method for the analysis of protein secondary structure embedded in solid lipid matrices. Bovine serum albumin (BSA) was chosen as a model protein, while Precirol AT05 (glycerol palmitostearate, melting point 58 ℃) was employed as the model lipid matrix. Bovine serum albumin was incorporated into lipid using physical mixing, melting and mixing, or wet granulation mixing methods. Attenuated total reflection FT-IR spectroscopy and size exclusion chromatography (SEC) were performed for the analysis of BSA secondary structure and its dissolution in aqueous media, respectively. The results showed significant interference of Precirol ATO5 with BSA amide I band which was subtracted up to 90% w/w lipid content to analyze BSA secondary structure. In addition, ATR FT-IR spectroscopy also detected thermally denatured BSA solid alone and in the presence of lipid matrix indicating its suitability for the detection of denatured protein solids in lipid matrices. Despite being in the solid state, conformational changes occurred to BSA upon incorporation into solid lipid matrices. However, the extent of these conformational alterations was found to be dependent on the mixing method employed as indicated by area overlap calculations. For instance, the melting and mixing method imparted negligible effect on BSA secondary structure, whereas the wet granulation mixing method promoted more changes. Size exclusion chromatography analysis depicted the complete dissolution of BSA in the aqueous media employed in the wet granulation method. In conclusion, an ATR FT-IR spectroscopic method was successfully developed to investigate BSA secondary structure in solid lipid matrices following the subtraction of lipid spectral interference. The ATR FT-IR spectroscopy could further be applied to investigate the secondary structure perturbations of therapeutic proteins during their formulation development.

The demand for highly efficient macromolecular drugs, used in the treatment of many severe diseases, is continuously increasing. However, the hydrophilic character and large molecular size of these drugs significantly limit their ability to permeate across cellular membranes and thus impede the drugs in reaching their target sites in the body. Cell-penetrating peptides (CPP) have gained attention as promising drug excipients, since they can facilitate drug permeation across cell membranes constituting a major biological barrier. Fluorophores are frequently covalently conjugated to CPPs to improve detection, however, the ensuing change in physico-chemical properties of the CPPs may alter their biological properties. With complementary biophysical techniques, we show that the mode of biomembrane interaction may change considerably upon labeling of the CPP penetratin (PEN) with a fluorophore. Fluorophore-PEN conjugates display altered modes of membrane interaction with increased insertion into the core of model cell membranes thereby exerting membrane-thinning effects. This is in contrast to PEN, which localizes along the head groups of the lipid bilayer, without affecting the thickness of the lipid tails. Particularly high membrane disturbance is observed for the two most hydrophobic PEN conjugates; rhodamine B or 1-pyrene butyric acid, as compared to the four other tested fluorophore-PEN conjugates.

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.

Ethnopharmacological relevance: Plants used in the traditional medicine of Europe to treat memory dysfunction and/or to enhance memory were investigated for activity against the underlying mechanisms of Alzheimer's disease.Aim of the study: To investigate 35 ethanolic extracts of plants, selected using an ethnopharmacological approach, for anti-amyloidogenic activity as well as an ability to inhibit the enzymatic activity of acetylcholinesterase.Materials and methods: The anti-amyloidogenic activity of the extracts against amyloid beta was investigated by Thioflavin T fibrillation assays and the ability to inhibit the enzymatic activity of acetylcholinesterase was evaluated monitoring the hydrolysis of acetylthiocholineResults: Under the experimental conditions investigated, extracts of two plants, Carum carpi and Olea sylvestris, inhibited amyloid beta fibrillation considerably, eight plant extracts inhibited amyloid beta fibrillation to some extent, 16 plant extracts had no effect on amyloid beta fibrillation and nine extracts accelerated fibrillation of amyloid beta. Furthermore, five plant extracts from Corydalis species inhibited the enzymatic activity of acetylcholinesterase considerably, one plant extract inhibited the enzymatic activity of acetylcholinesterase to some extent and 29 plant extract had no effect on the enzymatic activity of acetylcholinesterase.Conclusions: An optimal extract in this study would possess acetylcholinesterase inhibitory activity as well as anti-amyloidogenic activity in order to address multiple facets of Alzheimer's disease, until the molecular origin of the disease is unraveled. Unfortunately no such extract was found.

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.

The presence of Lewy bodies and Lewy neurites is a major pathological hallmark of Parkinson's disease and is hypothesized to be linked to disease development, although this is not yet conclusive. Lewy bodies and Lewy neurites primarily consist of fibrillated α-Synuclein; yet, there is no treatment available targeting stabilization of α-Synuclein in its native state. The aim of the present study was to investigate the inhibitory activity of an ethanolic extract of Geum urbanum against α-Synuclein fibrillation and examine the structural changes of α-Synuclein in the presence of the extract. The anti-fibrillation and anti-aggregation activities of the plant extract were monitored by thioflavin T fibrillation assays and size exclusion chromatography, while structural changes were followed by circular dichroism, Fourier transform infrared spectroscopy, intrinsic fluorescence, small angle X-ray scattering and electron microscopy. Since the extract is a complex mixture, structure-function relationships could not be determined. Under the experimental conditions investigated, Geum urbanum was found to inhibit α-Synuclein fibrillation in a concentration dependent way, and to partly disintegrate preformed α-Synuclein fibrils. Based on the structural changes of α-Synuclein in the presence of extract, we propose that Geum urbanum delays α-Synuclein fibrillation either by reducing the fibrillation ability of one or more of the aggregation prone intermediates or by directing α-Synuclein aggregation towards a non-fibrillar state. However, whether these alterations of the fibrillation pathway lead to less pathogenic species is yet to be determined.

Lipidation of proteins is used in the pharmaceutical field to increase the therapeutic efficacy of proteins. In this study, we investigate the effect of a 14-carbon fatty acid modification on the adsorption behavior of human insulin to a hydrophobic solid surface and the subsequent fibrillation development under highly acidic conditions and elevated temperature by comparing to the fibrillation of human insulin. At these stressed conditions, the lipid modification accelerates the rate of fibrillation in bulk solution. With the use of several complementary surface-sensitive techniques, including quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), and neutron reflectivity (NR), we show that there are two levels of structurally different protein organization at a hydrophobic surface for both human insulin and the lipidated analogue: a dense protein layer formed within minutes on the surface and a diffuse outer layer of fibrillar structures which took hours to form. The two layers may only be weakly connected, and proteins from both layers are able to desorb from the surface. The lipid modification increases the protein surface coverage and the thickness of both layer organizations. Upon lipidation not only the fibrillation extent but also the morphology of the fibrillar structures changes from fibril clusters on the surface to a more homogeneous network of fibrils covering the entire hydrophobic surface.

In the present study, the stabilizing effect of excipients present in recombinant Human Serum Albumin (rHSA) solutions are studied using rheological analysis. The study was conducted using Design of Experiment (DoE) principles and multivariate analysis approach. It is observed that the excipients octanoate and polysorbate 80 used in the Recombumin Alpha® formulation both increase the stability of the protein reflected by an observed decrease in the complex viscosity interpreted as a decreasing degree of self-association. Octanoate seems to be efficient in protecting the protein at higher temperatures, whereas polysorbate protects against self-association at lower temperatures. Additionally, a synergistic effect of octanoate and polysorbate 80 was observed in a non-linear fashion. This effect would be difficult to assess in a study using univariate analysis of these complex formulations, however the multivariate approach using DoE and Principal Component Analysis (PCA) efficiently illustrated its usefulness in the current study.

Protein fibrillation is the pathological hallmark of several neurodegenerative diseases and also complicates the manufacturing and use of protein drugs. As a case study, the inhibitory activity of the natural compound aloin against insulin fibrillation was investigated. Based on Thioflavin T assays, high-performance liquid chromatography and transmission electron microscopy it was found that a degradation product of aloin, formed over weeks of storage, was able to significantly inhibit insulin fibrillation. The activity of the stored aloin was significantly reduced in the presence of small amounts of sodium azide or ascorbic acid, suggesting the active compound to be an oxidation product. A high-performance liquid chromatography method and a liquid chromatography-mass spectrometry method were developed to investigate the degradation products in the aged aloin solution. We found that the major compounds in the solution were aloin A and aloin B. In addition, 10-hydroxy aloin and elgonica dimers were detected in smaller amounts. The identified compounds were isolated and tested for activity by means of Thioflavin T assays, but no activity was observed. Thus, the actual fibrillation inhibitor is an as yet unidentified and potentially metastable degradation product of aloin. These results suggest that degradation products, and in particular oxidation products, are to be considered thoroughly when natural products are investigated for activity against protein fibrillation.

In the original version of this article, there is an error in the Early Event in Competition Studies section on page 598. The correct text is given below. An intuitive approach to explain this is by the different diffusion coefficients of PS80 (1.8×10−6 cm2/s for the monomer, 1.8×10−7 cm2/s for the micelle)1.Amani A. York P. de Waard H. Anwar J. Molecular dynamics simulation of a polysorbate 80 micelle in water.Soft Matter. 2011; 7: 2900-2908Crossref Scopus (75) Google Scholar and IgG1 (~4×10−7 cm2/s).2.Saltzman W.M. Radomsky M.L. Whaley K.J. Cone R.A. Antibody diffusion in human cervical mucus.Biophys J. 1994; 66: 508-515Abstract Full Text PDF PubMed Scopus (320) Google Scholar, 3.Tyn M.T. Gusek T.W. Prediction of diffusion coefficients of proteins.Biotechnol Bioeng. 1990; 35: 327-338Crossref PubMed Scopus (389) Google Scholar The diffusion coefficient of the mAb and the PS80 monomer (the competing species), differ by only a factor ~4. Hence, there could be a limited degree of competition for the surface in the initial phase, where a fraction of the mAb would adsorb and be resistant to PS80, while another but smaller fraction initially adsorbs and is then removed by the surfactant due to insufficient contact points. However, the time scale of the event (in terms of height and width of the spike) remained constant regardless of surfactant concentrations being below or above CMC (Supplementary Figure 5), which would argue against this explanation. This same observation negates a potential transient PS80 depletion from the bulk in the early adsorption phase as a result of adsorption to tubing, since also in that case the height and width should be reduced upon increasing PS80 concentration. Competitive Adsorption of Monoclonal Antibodies and Nonionic Surfactants at Solid Hydrophobic SurfacesJournal of Pharmaceutical SciencesVol. 104Issue 2PreviewTwo monoclonal antibodies from the IgG subclasses one and two were compared in their adsorption behavior with hydrophobic surfaces upon dilution to 10 mg/mL with 0.9% NaCl. These conditions simulate handling of the compounds at hospital pharmacies and surfaces encountered after preparation, such as infusion bags and i.v. lines. Total internal reflection fluorescence and quartz crystal microbalance with dissipation monitoring were used to follow and quantify this. Furthermore, the influence of the nonionic surfactant polysorbate 80 (PS80) on the adsorption process of these two antibodies was investigated. Full-Text PDF

Purpose: This study describes how protein release from polymer matrices correlate with simple measurements on the intrinsic viscosity of the polymer solutions used for casting the matrices and calculations of the solubility parameters of polymers and solvents used.Method: Matrices of poly(DL-lactide-co-glycolide) (PLGA) were cast with bovine serum albumin (BSA) as a model drug using different solvents (acetone, dichloromethane, ethanol and water). The amount of released protein from the different matrices was correlated with the Hildebrand and Hansen solubility parameters of the solvents, and the intrinsic viscosity of the polymer solutions. Matrix microstructure was investigated by transmission and scanning electron microscopy (TEM and SEM). Polycaprolactone (PCL) matrices were used in a similar way to support the results for PLGA matrices.Results: The maximum amount of BSA released and the release profile from PLGA matrices varied depending on the solvent used for casting. The maximum amount of released BSA decreased with higher intrinsic viscosity, and increased with solubility parameter difference between the solvent and polymer used. The solvent used also had an effect on the matrix Microstructure as determined by TEM and SEM. Similar results were obtained for the PCL polymer systems.Conclusions: The smaller the difference in the solubility parameter between the polymer and the solvent used for casting a polymer matrix, the lower will be the maximum protein release. This is because of the presence of smaller pore sizes in the cast matrix if a solvent with a solubility parameter close to the one of the polymer is used. Likewise, the intrinsic viscosity of the polymer solution increases as solubility parameter differences decrease, thus, simple measurements of intrinsic viscosity and solubility parameter difference, allow the prediction of protein release profiles. (C) 2015 Elsevier B.V. All rights reserved.

Two monoclonal antibodies from the IgG subclasses one and two were compared in their adsorption behavior with hydrophobic surfaces upon dilution to 10 mg/mL with 0.9% NaCl. These conditions simulate handling of the compounds at hospital pharmacies and surfaces encountered after preparation, such as infusion bags and i.v. lines. Total internal reflection fluorescence and quartz crystal microbalance with dissipation monitoring were used to follow and quantify this. Furthermore, the influence of the nonionic surfactant polysorbate 80 (PS80) on the adsorption process of these two antibodies was investigated. Despite belonging to two different IgG subclasses, both antibodies displayed comparable adsorption behavior. Both antibodies readily adsorbed in the absence of PS80, whereas adsorption was reduced in the presence of 30 mg/L surfactant. The sequence of exposure of the surfactant and protein to the surface was found to have a major influence on the extent of protein adsorption. Although only a fraction of adsorbed protein could be removed by rinsing with 30 mg/L surfactant solution, adsorption was entirely prevented when surfaces were pre-exposed to PS80.

Future Science Book SeriesAdvances in the Discovery and Development of Peptide Therapeutics Advances in peptide formulationsLene JørgensenLene JørgensenPublished Online:17 Nov 2015https://doi.org/10.4155/fseb2013.14.5AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit View chapterReferences1. Jorgensen L , Hostrup S , Moeller EH et al. Recent trends in stabilising peptides and proteins in pharmaceutical formulation – considerations in the choice of excipients . Expert Opin. Drug Deliv. 6 ( 11 ), 1219 – 1230 ( 2009 ). Crossref, Medline, CAS, Google Scholar2. Manning M , Chou D , Murphy B et al. Stability of protein pharmaceuticals: an update . Pharm. Res. 27 ( 4 ), 544 – 575 ( 2010 ). Crossref, Medline, Google Scholar3. Hawe A , Wigggenhorn M , van de Weert M et al. Forced degradation of therapeutic proteins . J. Pharm. Sci. 101 ( 3 ), 895 – 913 ( 2013 ). Crossref, Google Scholar4. 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Complex coacervates of hyaluronic acid and lysozyme, a model protein, were formed by ionic interaction using bulk mixing and were characterized in terms of binding stoichiometry and protein structure and stability. The complexes were formed at pH 7.2 at low ionic strength (6 mM) and the binding stoichiometry was determined using solution depletion and isothermal titration calorimetry. The binding stoichiometry of lysozyme to hyaluronic acid (870 kDa) determined by solution depletion was found to be 225.9 ± 6.6 mol, or 0.1 bound lysozyme molecules per hyaluronic acid monomer. This corresponded well with that obtained by isothermal titration calorimetry of 0.09 bound lysozyme molecules per hyaluronic acid monomer. The complexation did not alter the secondary structure of lysozyme measured by Fourier-transform infrared spectroscopy overlap analysis and had no significant impact on the Tm of lysozyme determined by differential scanning calorimetry. Furthermore, the protein stability of lysozyme was found to be improved upon complexation during a 12-weeks storage study at room temperature, as shown by a significant increase in recovered protein when complexed (94 ± 2% and 102 ± 5% depending on the polymer-protein weight to weight ratio) compared to 89 ± 2% recovery for uncomplexed protein. This study shows the potential of hyaluronic acid to be used in combination with complex coacervation to increase the physical stability of pharmaceutical protein formulations.