Marco van de Weert | University of Copenhagen (original) (raw)

Papers by Marco van de Weert

Research paper thumbnail of Correlation between calculated molecular descriptors of excipient amino acids and experimentally observed thermal stability of lysozyme

International journal of pharmaceutics, Jan 15, 2017

A quantitative structure-property relationship (QSPR) between protein stability and the physicoch... more 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 prope...

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Research paper thumbnail of Rapid Conformational Analysis of Protein Drugs in Formulation by Hydrogen/Deuterium Exchange Mass Spectrometry

Journal of Pharmaceutical Sciences, 2016

Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) has become an established metho... more Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) has become an established method for analysis of protein higher order structure. Here, we use HDX-MS methodology based on manual solid-phase extraction (SPE) to allow fast and simplified conformational analysis of proteins under pharmaceutically relevant formulation conditions. Of significant practical utility, the methodology allows global HDX-MS analyses to be performed without refrigeration or external cooling of the setup. In mode 1, we used dimethyl sulphoxide-containing solvents for SPE, allowing the HDX-MS analysis to be performed at acceptable back-exchange levels (<30%) without the need for cooling any components of the setup. In mode 2, SPE and chromatography were performed using fast isocratic elution at 0°C resulting in a back-exchange of 10%-30%. Real-world applicability was demonstrated by HDX-MS analyses of interferon-β-1a in formulation, using an internal HDX reference peptide (P7I) to control for any sample-to-sample variations in back-exchange. Advantages of the methodology include low sample use, optimized excipient removal using multiple solvents, and fast data acquisition. Our results indicate that HDX-MS can provide a reliable approach for fast conformation analysis of proteins in their intended formulations, which could facilitate an increased use of the technique in pharmaceutical development research.

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Research paper thumbnail of Mechanistic study of the inhibitory activity of Geum urbanum extract against α-Synuclein fibrillation

Biochimica et biophysica acta, 2016

The presence of Lewy bodies and Lewy neurites is a major pathological hallmark of Parkinson's... more 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-funct...

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Research paper thumbnail of Effect of ethanol as a co-solvent on the aerosol performance and stability of spray-dried lysozyme

International journal of pharmaceutics, Jan 9, 2016

In the spray drying process, organic solvents can be added to facilitate drying, accommodate cert... more In the spray drying process, organic solvents can be added to facilitate drying, accommodate certain functional excipients, and modify the final particle characteristics. In this study, lysozyme was used as a model pharmaceutical protein to study the effect of ethanol as a co-solvent on the stability and aerosol performance of spray-dried protein. Lysozyme was dissolved in solutions with various ratios of ethanol and water, and subsequently spray-dried. A change from spherical particles into wrinkled and folded particles was observed upon increasing the ratio of ethanol in the feed. The aerosol performance of the spray-dried lysozyme from ethanol-water solution was improved compared to that from pure water. The conformation of lysozyme in the ethanol-water solution and spray dried powder was altered, but the native structure of lysozyme was restored upon reconstitution in water after the spray drying process. The enzymatic activities of the spray-dried lysozyme showed no significant...

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Research paper thumbnail of Effect of the Freezing Step in the Stability and Bioactivity of Protein-Loaded PLGA Nanoparticles Upon Lyophilization

Pharmaceutical research, Jan 21, 2016

The freezing step in lyophilization is the most determinant for the quality of biopharmaceutics. ... more The freezing step in lyophilization is the most determinant for the quality of biopharmaceutics. Using insulin as model of therapeutic protein, our aim was to evaluate the freezing effect in the stability and bioactivity of insulin-loaded PLGA nanoparticles. The performance of trehalose, sucrose and sorbitol as cryoprotectants was evaluated. Cryoprotectants were co-encapsulated with insulin into PLGA nanoparticles and lyophilized using an optimized cycle with freezing at -80°C, in liquid nitrogen, or ramped cooling at -40°C. Upon lyophilization, the stability of protein structure and in vivo bioactivity were assessed. Insulin was co-encapsulated with cryoprotectants resulting in particles of 243-394 nm, zeta potential of -32 to -35 mV, and an association efficiency above 90%. The cryoprotectants were crucial to mitigate the freezing stresses and better stabilize the protein. The insulin structure maintenance was evident and close to 90%. Trehalose co-encapsulated insulin-loaded PLGA...

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Research paper thumbnail of Ligand Binding and Thermostability of Different Allosteric States of the Insulin Zinc-Hexamer

Biochemistry Usa, Mar 1, 2006

The influence of ligand binding and conformation state on the thermostability of hexameric zinc-i... more The influence of ligand binding and conformation state on the thermostability of hexameric zinc-insulin was studied by differential scanning calorimetry (DSC). The insulin hexamer exists in equilibrium between the forms T6, T3R3, and R6. Phenolic ligands induce and stabilize the T3R3- and R6-states which are further stabilized by binding of certain anions that do not stabilize the T6-state. It was shown that the thermostability of the resorcinol-stabilized R6-state was significantly higher than that of the T6-state. Further analysis showed that phenol- and m-cresol-stabilized R6-hexamer loses three ligands before reaching the unfolding temperature and hence unfolds from the T3R3-state. The relative affinity of the four tested anionic ligands was found, by DSC, to be thiocyanate > or = 4-hydroxy-3-nitrobenzoate > p-aminobenzoate > chloride. The results correlate with other methods and demonstrate that DSC provides a general and useful method of evaluation of both phenolic and anionic ligand binding to insulin without the use of probes or other alterations of the system of interest. However, it is a prerequisite that the binding is strong enough to saturate the binding sites at temperatures around the unfolding transition.

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Research paper thumbnail of Chemical and Thermal Stability of Insulin: Effects of Zinc and Ligand Binding to the Insulin Zinc-Hexamer

Pharmaceutical Research, Sep 13, 2006

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Research paper thumbnail of The Molecular Chaperone a-Crystallin as an Excipient in an Insulin Formulation

Pharmaceut Res, 2010

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Research paper thumbnail of Lipidation effect on the associated fibrillation of the model protein insulin at hydrophobic surfaces

Langmuir, 2016

Lipidation of proteins is used in the pharmaceutical field to increase the therapeutic efficacy o... more 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.

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Research paper thumbnail of Protein pharmaceuticals

Drug Discovery Today: Technologies, 2008

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Research paper thumbnail of The Chaperone-like Protein a-Crystallin Dissociates Insulin Dimers and Hexamers

Biochemistry Usa, 2009

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Research paper thumbnail of The Chaperone-like Protein α-Crystallin Dissociates Insulin Dimers and Hexamers

Biochemistry, 2009

The protein α-crystallin, a member of the small heat shock protein family, has the ability to pre... more The protein α-crystallin, a member of the small heat shock protein family, has the ability to prevent aggregation of partially denatured proteins, an effect demonstrated both in vivo and in vitro. In this work, we have probed the apparent thermal destabilization of bovine insulin by α-...

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Research paper thumbnail of Large-scale Biophysical Evaluation of Protein PEGylation Effects: in vitro Properties of 61 Protein Entities

Molecular Pharmaceutics, 2016

PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but su... more PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but surprisingly limited public data is available on the biophysical effects of protein PEGylation. Here we report the first large-scale study, with site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total using a common set of analytical methods. Predictions of molecular size were typically accurate in comparison with actual size determined by size-exclusion chromatography (SEC) or dynamic light scattering (DLS). In contrast, there was no universal trend regarding the effect of PEGylation on the thermal stability of a protein based on data generated by circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorimetry (DSF). In addition, DSF was validated as a fast and inexpensive screening method for thermal unfolding studies of PEGylated proteins. Multivariate data analysis revealed clear trends in biophysical properties upon PEGylation for a subset of proteins, although no universal trends were found. Taken together, these findings are important in the consideration of biophysical methods and evaluation of second-generation biopharmaceutical drug candidates.

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Research paper thumbnail of Study on the binding of Thioflavin T to �-sheet-rich and non-�-sheet cavities

J Struct Biol, 2007

Amyloid fibril formation plays a role in more than 20 diseases including Alzheimer’s disease. In ... more Amyloid fibril formation plays a role in more than 20 diseases including Alzheimer’s disease. In vitro detection of these fibrils is often performed using Thioflavin T (ThT), though the ThT binding mode is largely unknown. In the present study, spectral properties of ThT in binding environments representing β-sheet-rich and non-β-sheet cavities were examined. Acetylcholinesterase and γ-cyclodextrin induced a characteristic ThT fluorescence similar to that with amyloid fibrils, whereas β-cyclodextrin and the β-sheet-rich transthyretin did not. The cavities of acetylcholinesterase and γ-cyclodextrin were of similar diameter and only these cavities could accommodate two ThT ions according to molecular modelling. Binding stoichiometry studies also showed a possible binding of two ThT ions. Thus, the characteristic ThT fluorescence is induced in cavities with a diameter of 8–9 Å and a length able to accommodate the entire length of the ThT ion. The importance of a cavity diameter capable of binding two ThT ions, among others, indicates that an excimer formation is a plausible mechanism for the characteristic fluorescence. We propose a similar ThT binding mode in amyloid fibrils, where cavities of an appropriate size running parallel to the fibril axis have previously been proposed in several amyloid fibril models.

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Research paper thumbnail of Preparing and evaluating delivery systems for proteins

Eur J Pharm Sci, 2006

From a formulation perspective proteins are complex and therefore challenging molecules to develo... more From a formulation perspective proteins are complex and therefore challenging molecules to develop drug delivery systems for. The success of a formulation depends on the ability of the protein to maintain the native structure and activity during preparation and delivery as well as during shipping and long-term storage of the formulation. Therefore, the development and evaluation of successful and promising drug delivery systems is essential. In the present review, some of the particulate drug delivery systems for parenteral delivery of protein are presented and discussed. The challenge for incorporation of protein in particulate delivery systems is exemplified by water-in-oil emulsions.

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Research paper thumbnail of The Inhibitory Effect of Natural Products on Protein Fibrillation May Be Caused by Degradation Products – A Study Using Aloin and Insulin

PLOS ONE, 2016

Protein fibrillation is the pathological hallmark of several neurodegenerative diseases and also ... more 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.

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Research paper thumbnail of The Molecular Chaperone α-Crystallin as an Excipient in an Insulin Formulation

Pharmaceutical Research, Mar 1, 2010

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Research paper thumbnail of Co-encapsulation of lyoprotectants improves the stability of protein-loaded PLGA nanoparticles upon lyophilization

International journal of pharmaceutics, Jan 21, 2015

The purpose of this work was to evaluate the influence of the co-encapsulation of lyoprotectants ... more The purpose of this work was to evaluate the influence of the co-encapsulation of lyoprotectants with insulin into PLGA nanoparticles, on the stability of the protein and nanoparticles upon lyophilization. Different lyoprotectants were used, namely trehalose, glucose, sucrose, fructose and sorbitol at 10% (w/v). Insulin-loaded PLGA nanoparticles with co-encapsulated lyoprotectants achieved a mean particle size of 386-466nm, and a zeta potential ranging between -34 and -38mV, dependent on the lyoprotectant used. Formulations had association efficiencies and loading capacities of 85-91% and 10-12%, respectively. The lyophilization process increased the colloidal stability of nanoparticles, and maintained their spherical shape and smooth surface, particularly in presence of lyoprotectants. XRPD revealed that the lyophilizates of nanoparticles with co-encapsulated lyoprotectants were amorphous, whereas formulations with externally added lyoprotectants, except trehalose, showed crystalli...

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Research paper thumbnail of Influence of Tableting on the Conformation and Thermal Stability of Trypsin as a Model Protein

Journal of Pharmaceutical Sciences, 2015

The objective of this study was to investigate the influence of compaction on the conformation of... more The objective of this study was to investigate the influence of compaction on the conformation of trypsin, its transition temperature (Tm ) of unfolding, and its folding reversibility after thermal denaturation. Plain trypsin was compacted at 40-382 MPa. Pressure-induced changes in the trypsin conformation and the extent of their reversibility were determined using solid- and liquid-state IR spectroscopy together with principal component analysis and an area overlap approach. Trypsin enzymatic activity was determined by a photometric assay. Liquid-state differential scanning calorimetry was performed to determine the Tm as well as the folding reversibility after thermal denaturation of the reconstituted samples. It was found that compacted samples showed reduced activity accompanied by an altered secondary structure. Conformational changes that occur in the solid state were partially reversible upon tablet reconstitution. Aqueous-state IR spectroscopy combined with partial least squares was shown to be a powerful tool to follow irreversible structural changes and evaluate sample bioactivity. Besides its conformation, the thermal stability of trypsin was altered as a result of the applied compaction pressure, indicated by a reduced folding reversibility. In conclusion, this study reveals that tableting can have a negative impact on the biological quality of protein APIs. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.

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Research paper thumbnail of Quantitative release of active lysozyme from dextran based hydrogels

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Research paper thumbnail of Correlation between calculated molecular descriptors of excipient amino acids and experimentally observed thermal stability of lysozyme

International journal of pharmaceutics, Jan 15, 2017

A quantitative structure-property relationship (QSPR) between protein stability and the physicoch... more 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 prope...

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Research paper thumbnail of Rapid Conformational Analysis of Protein Drugs in Formulation by Hydrogen/Deuterium Exchange Mass Spectrometry

Journal of Pharmaceutical Sciences, 2016

Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) has become an established metho... more Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) has become an established method for analysis of protein higher order structure. Here, we use HDX-MS methodology based on manual solid-phase extraction (SPE) to allow fast and simplified conformational analysis of proteins under pharmaceutically relevant formulation conditions. Of significant practical utility, the methodology allows global HDX-MS analyses to be performed without refrigeration or external cooling of the setup. In mode 1, we used dimethyl sulphoxide-containing solvents for SPE, allowing the HDX-MS analysis to be performed at acceptable back-exchange levels (<30%) without the need for cooling any components of the setup. In mode 2, SPE and chromatography were performed using fast isocratic elution at 0°C resulting in a back-exchange of 10%-30%. Real-world applicability was demonstrated by HDX-MS analyses of interferon-β-1a in formulation, using an internal HDX reference peptide (P7I) to control for any sample-to-sample variations in back-exchange. Advantages of the methodology include low sample use, optimized excipient removal using multiple solvents, and fast data acquisition. Our results indicate that HDX-MS can provide a reliable approach for fast conformation analysis of proteins in their intended formulations, which could facilitate an increased use of the technique in pharmaceutical development research.

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Research paper thumbnail of Mechanistic study of the inhibitory activity of Geum urbanum extract against α-Synuclein fibrillation

Biochimica et biophysica acta, 2016

The presence of Lewy bodies and Lewy neurites is a major pathological hallmark of Parkinson's... more 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-funct...

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Research paper thumbnail of Effect of ethanol as a co-solvent on the aerosol performance and stability of spray-dried lysozyme

International journal of pharmaceutics, Jan 9, 2016

In the spray drying process, organic solvents can be added to facilitate drying, accommodate cert... more In the spray drying process, organic solvents can be added to facilitate drying, accommodate certain functional excipients, and modify the final particle characteristics. In this study, lysozyme was used as a model pharmaceutical protein to study the effect of ethanol as a co-solvent on the stability and aerosol performance of spray-dried protein. Lysozyme was dissolved in solutions with various ratios of ethanol and water, and subsequently spray-dried. A change from spherical particles into wrinkled and folded particles was observed upon increasing the ratio of ethanol in the feed. The aerosol performance of the spray-dried lysozyme from ethanol-water solution was improved compared to that from pure water. The conformation of lysozyme in the ethanol-water solution and spray dried powder was altered, but the native structure of lysozyme was restored upon reconstitution in water after the spray drying process. The enzymatic activities of the spray-dried lysozyme showed no significant...

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Research paper thumbnail of Effect of the Freezing Step in the Stability and Bioactivity of Protein-Loaded PLGA Nanoparticles Upon Lyophilization

Pharmaceutical research, Jan 21, 2016

The freezing step in lyophilization is the most determinant for the quality of biopharmaceutics. ... more The freezing step in lyophilization is the most determinant for the quality of biopharmaceutics. Using insulin as model of therapeutic protein, our aim was to evaluate the freezing effect in the stability and bioactivity of insulin-loaded PLGA nanoparticles. The performance of trehalose, sucrose and sorbitol as cryoprotectants was evaluated. Cryoprotectants were co-encapsulated with insulin into PLGA nanoparticles and lyophilized using an optimized cycle with freezing at -80°C, in liquid nitrogen, or ramped cooling at -40°C. Upon lyophilization, the stability of protein structure and in vivo bioactivity were assessed. Insulin was co-encapsulated with cryoprotectants resulting in particles of 243-394 nm, zeta potential of -32 to -35 mV, and an association efficiency above 90%. The cryoprotectants were crucial to mitigate the freezing stresses and better stabilize the protein. The insulin structure maintenance was evident and close to 90%. Trehalose co-encapsulated insulin-loaded PLGA...

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Research paper thumbnail of Ligand Binding and Thermostability of Different Allosteric States of the Insulin Zinc-Hexamer

Biochemistry Usa, Mar 1, 2006

The influence of ligand binding and conformation state on the thermostability of hexameric zinc-i... more The influence of ligand binding and conformation state on the thermostability of hexameric zinc-insulin was studied by differential scanning calorimetry (DSC). The insulin hexamer exists in equilibrium between the forms T6, T3R3, and R6. Phenolic ligands induce and stabilize the T3R3- and R6-states which are further stabilized by binding of certain anions that do not stabilize the T6-state. It was shown that the thermostability of the resorcinol-stabilized R6-state was significantly higher than that of the T6-state. Further analysis showed that phenol- and m-cresol-stabilized R6-hexamer loses three ligands before reaching the unfolding temperature and hence unfolds from the T3R3-state. The relative affinity of the four tested anionic ligands was found, by DSC, to be thiocyanate > or = 4-hydroxy-3-nitrobenzoate > p-aminobenzoate > chloride. The results correlate with other methods and demonstrate that DSC provides a general and useful method of evaluation of both phenolic and anionic ligand binding to insulin without the use of probes or other alterations of the system of interest. However, it is a prerequisite that the binding is strong enough to saturate the binding sites at temperatures around the unfolding transition.

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Research paper thumbnail of Chemical and Thermal Stability of Insulin: Effects of Zinc and Ligand Binding to the Insulin Zinc-Hexamer

Pharmaceutical Research, Sep 13, 2006

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Research paper thumbnail of The Molecular Chaperone a-Crystallin as an Excipient in an Insulin Formulation

Pharmaceut Res, 2010

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Research paper thumbnail of Lipidation effect on the associated fibrillation of the model protein insulin at hydrophobic surfaces

Langmuir, 2016

Lipidation of proteins is used in the pharmaceutical field to increase the therapeutic efficacy o... more 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.

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Research paper thumbnail of Protein pharmaceuticals

Drug Discovery Today: Technologies, 2008

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Research paper thumbnail of The Chaperone-like Protein a-Crystallin Dissociates Insulin Dimers and Hexamers

Biochemistry Usa, 2009

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Research paper thumbnail of The Chaperone-like Protein α-Crystallin Dissociates Insulin Dimers and Hexamers

Biochemistry, 2009

The protein α-crystallin, a member of the small heat shock protein family, has the ability to pre... more The protein α-crystallin, a member of the small heat shock protein family, has the ability to prevent aggregation of partially denatured proteins, an effect demonstrated both in vivo and in vitro. In this work, we have probed the apparent thermal destabilization of bovine insulin by α-...

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Research paper thumbnail of Large-scale Biophysical Evaluation of Protein PEGylation Effects: in vitro Properties of 61 Protein Entities

Molecular Pharmaceutics, 2016

PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but su... more PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but surprisingly limited public data is available on the biophysical effects of protein PEGylation. Here we report the first large-scale study, with site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total using a common set of analytical methods. Predictions of molecular size were typically accurate in comparison with actual size determined by size-exclusion chromatography (SEC) or dynamic light scattering (DLS). In contrast, there was no universal trend regarding the effect of PEGylation on the thermal stability of a protein based on data generated by circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorimetry (DSF). In addition, DSF was validated as a fast and inexpensive screening method for thermal unfolding studies of PEGylated proteins. Multivariate data analysis revealed clear trends in biophysical properties upon PEGylation for a subset of proteins, although no universal trends were found. Taken together, these findings are important in the consideration of biophysical methods and evaluation of second-generation biopharmaceutical drug candidates.

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Research paper thumbnail of Study on the binding of Thioflavin T to �-sheet-rich and non-�-sheet cavities

J Struct Biol, 2007

Amyloid fibril formation plays a role in more than 20 diseases including Alzheimer’s disease. In ... more Amyloid fibril formation plays a role in more than 20 diseases including Alzheimer’s disease. In vitro detection of these fibrils is often performed using Thioflavin T (ThT), though the ThT binding mode is largely unknown. In the present study, spectral properties of ThT in binding environments representing β-sheet-rich and non-β-sheet cavities were examined. Acetylcholinesterase and γ-cyclodextrin induced a characteristic ThT fluorescence similar to that with amyloid fibrils, whereas β-cyclodextrin and the β-sheet-rich transthyretin did not. The cavities of acetylcholinesterase and γ-cyclodextrin were of similar diameter and only these cavities could accommodate two ThT ions according to molecular modelling. Binding stoichiometry studies also showed a possible binding of two ThT ions. Thus, the characteristic ThT fluorescence is induced in cavities with a diameter of 8–9 Å and a length able to accommodate the entire length of the ThT ion. The importance of a cavity diameter capable of binding two ThT ions, among others, indicates that an excimer formation is a plausible mechanism for the characteristic fluorescence. We propose a similar ThT binding mode in amyloid fibrils, where cavities of an appropriate size running parallel to the fibril axis have previously been proposed in several amyloid fibril models.

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Research paper thumbnail of Preparing and evaluating delivery systems for proteins

Eur J Pharm Sci, 2006

From a formulation perspective proteins are complex and therefore challenging molecules to develo... more From a formulation perspective proteins are complex and therefore challenging molecules to develop drug delivery systems for. The success of a formulation depends on the ability of the protein to maintain the native structure and activity during preparation and delivery as well as during shipping and long-term storage of the formulation. Therefore, the development and evaluation of successful and promising drug delivery systems is essential. In the present review, some of the particulate drug delivery systems for parenteral delivery of protein are presented and discussed. The challenge for incorporation of protein in particulate delivery systems is exemplified by water-in-oil emulsions.

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Research paper thumbnail of The Inhibitory Effect of Natural Products on Protein Fibrillation May Be Caused by Degradation Products – A Study Using Aloin and Insulin

PLOS ONE, 2016

Protein fibrillation is the pathological hallmark of several neurodegenerative diseases and also ... more 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.

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Research paper thumbnail of The Molecular Chaperone α-Crystallin as an Excipient in an Insulin Formulation

Pharmaceutical Research, Mar 1, 2010

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Research paper thumbnail of Co-encapsulation of lyoprotectants improves the stability of protein-loaded PLGA nanoparticles upon lyophilization

International journal of pharmaceutics, Jan 21, 2015

The purpose of this work was to evaluate the influence of the co-encapsulation of lyoprotectants ... more The purpose of this work was to evaluate the influence of the co-encapsulation of lyoprotectants with insulin into PLGA nanoparticles, on the stability of the protein and nanoparticles upon lyophilization. Different lyoprotectants were used, namely trehalose, glucose, sucrose, fructose and sorbitol at 10% (w/v). Insulin-loaded PLGA nanoparticles with co-encapsulated lyoprotectants achieved a mean particle size of 386-466nm, and a zeta potential ranging between -34 and -38mV, dependent on the lyoprotectant used. Formulations had association efficiencies and loading capacities of 85-91% and 10-12%, respectively. The lyophilization process increased the colloidal stability of nanoparticles, and maintained their spherical shape and smooth surface, particularly in presence of lyoprotectants. XRPD revealed that the lyophilizates of nanoparticles with co-encapsulated lyoprotectants were amorphous, whereas formulations with externally added lyoprotectants, except trehalose, showed crystalli...

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Research paper thumbnail of Influence of Tableting on the Conformation and Thermal Stability of Trypsin as a Model Protein

Journal of Pharmaceutical Sciences, 2015

The objective of this study was to investigate the influence of compaction on the conformation of... more The objective of this study was to investigate the influence of compaction on the conformation of trypsin, its transition temperature (Tm ) of unfolding, and its folding reversibility after thermal denaturation. Plain trypsin was compacted at 40-382 MPa. Pressure-induced changes in the trypsin conformation and the extent of their reversibility were determined using solid- and liquid-state IR spectroscopy together with principal component analysis and an area overlap approach. Trypsin enzymatic activity was determined by a photometric assay. Liquid-state differential scanning calorimetry was performed to determine the Tm as well as the folding reversibility after thermal denaturation of the reconstituted samples. It was found that compacted samples showed reduced activity accompanied by an altered secondary structure. Conformational changes that occur in the solid state were partially reversible upon tablet reconstitution. Aqueous-state IR spectroscopy combined with partial least squares was shown to be a powerful tool to follow irreversible structural changes and evaluate sample bioactivity. Besides its conformation, the thermal stability of trypsin was altered as a result of the applied compaction pressure, indicated by a reduced folding reversibility. In conclusion, this study reveals that tableting can have a negative impact on the biological quality of protein APIs. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci.

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Research paper thumbnail of Quantitative release of active lysozyme from dextran based hydrogels

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