Vasco Filipe - Academia.edu (original) (raw)
Papers by Vasco Filipe
Pharmaceutical Research, 2010
Purpose To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic l... more Purpose To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates. Methods Standard polystyrene beads of sizes ranging from 60 to 1,000 nm and physical mixtures thereof were analyzed with NTA and DLS. The influence of different ratios of particle populations was tested. Drug delivery nanoparticles and protein aggregates were analyzed by NTA and DLS. Live monitoring of heat-induced protein aggregation was performed with NTA. Results NTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples. Sample visualization and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000 nm) particles generally does not compromise the accuracy of NTA measurements, and a broad range of population ratios can easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS. Live monitoring of heat-induced protein aggregation provides information about aggregation kinetics and size of submicron aggregates. Conclusion NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates.
Pharmaceutical Research, 2011
Purpose To evaluate the potential of fluorescence single particle tracking (fSPT) for the charact... more Purpose To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA). Methods A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.1 μm (control) were diluted in buffer, serum and plasma, and their size distributions were analyzed by fSPT and nanoparticle tracking analysis (NTA). In a separate experiment, IgG and HSA, fluorescently labeled with different dyes, were mixed and subjected to heat stress. The stressed sample was analyzed by fSPT using a dual color mode and by NTA. Results The accuracy and precision of fSPT proved to be comparable to NTA. fSPT was able to successfully measure all the samples in buffer, serum and plasma. The average size of the cross-linked protein aggregates showed a slight increase in biological fluids. Moreover, fSPT analysis showed that a significant proportion of the aggregates formed by subjecting an IgG/HSA mixture to heat stress were composed of both proteins. Conclusion fSPT is a powerful technique for the characterization of submicron protein aggregates in biological fluids and complex formulations.
Pharmaceutical Research, 2010
Purpose The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly se... more Purpose The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly sensitive to viscosity, for the characterization of polysorbate-containing IgG formulations and compare them to the polarity-sensitive dyes ANS, Bis-ANS and Nile Red. Methods IgG formulations with polysorbate 20 or 80 were stressed below the aggregation temperature and analyzed by steady-state and time-resolved fluorescence and by HP-SEC with UV and fluorescent dye detection (Bis-ANS and CCVJ). Furthermore, commercial protein preparations of therapeutic proteins (Enbrel®50 mg, Humira®40 mg and MabThera®100 mg) were aggregated accordingly and analyzed with CCVJ fluorescence and HP-SEC. Results Contrarily to (Bis-)ANS and Nile Red, the molecular rotors DCVJ and CCVJ showed low background fluorescence in polysorbate-containing buffers. Time-resolved fluorescence experiments confirmed the steady-state fluorescence data. Both DCVJ and CCVJ showed enhanced fluorescence intensity for aggregated IgG formulations and were suitable for the characterization of polysorbate-containing IgG formulations in steady-state fluorescence and HP-SEC with dye detection (CCVJ). CCVJ was capable of detecting thermally induced aggregation in the commercial polysorbate-containing products Enbrel®50 mg, Humira®40 mg and MabThera®100 mg. Conclusion Fluorescent molecular rotors are suitable probes to detect aggregation in polysorbate-containing IgG formulations.
Pharmaceutical Research, 2010
Purpose The aim of this study was to develop a method to characterize intact soluble monoclonal I... more Purpose The aim of this study was to develop a method to characterize intact soluble monoclonal IgG1 antibody (IgG) oligomers by mass spectrometry. Methods IgG aggregates (dimers, trimers, tetramers and high-molecular-weight oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatography (HP-SEC), dialyzed against ammonium acetate pH 6.0 (a mass spectrometry-compatible volatile buffer), and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). Results Monomeric and aggregated IgG fractions in the stressed IgG formulation were successfully isolated by HP-SEC. ESI-TOF MS analysis enabled us to determine the molecular weight of the monomeric IgG as well as the aggregates, including dimers, trimers and tetramers. HP-SEC separation and sample preparation proved to be necessary for good quality signal in ESI-TOF MS. Both the HP-SEC protocol and the ESI-TOF mass spectrometric technique were shown to leave the IgG oligomers largely intact. Conclusions ESI-TOF MS is a useful tool complementary to HP-SEC to identify and characterize small oligomeric protein aggregates.
Pharmaceutical Research, 2010
Purpose To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic l... more Purpose To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates. Methods Standard polystyrene beads of sizes ranging from 60 to 1,000 nm and physical mixtures thereof were analyzed with NTA and DLS. The influence of different ratios of particle populations was tested. Drug delivery nanoparticles and protein aggregates were analyzed by NTA and DLS. Live monitoring of heat-induced protein aggregation was performed with NTA. Results NTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples. Sample visualization and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000 nm) particles generally does not compromise the accuracy of NTA measurements, and a broad range of population ratios can easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS. Live monitoring of heat-induced protein aggregation provides information about aggregation kinetics and size of submicron aggregates. Conclusion NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates.
Pharmaceutical Research, 2011
Purpose To evaluate the potential of fluorescence single particle tracking (fSPT) for the charact... more Purpose To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA). Methods A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.1 μm (control) were diluted in buffer, serum and plasma, and their size distributions were analyzed by fSPT and nanoparticle tracking analysis (NTA). In a separate experiment, IgG and HSA, fluorescently labeled with different dyes, were mixed and subjected to heat stress. The stressed sample was analyzed by fSPT using a dual color mode and by NTA. Results The accuracy and precision of fSPT proved to be comparable to NTA. fSPT was able to successfully measure all the samples in buffer, serum and plasma. The average size of the cross-linked protein aggregates showed a slight increase in biological fluids. Moreover, fSPT analysis showed that a significant proportion of the aggregates formed by subjecting an IgG/HSA mixture to heat stress were composed of both proteins. Conclusion fSPT is a powerful technique for the characterization of submicron protein aggregates in biological fluids and complex formulations.
Pharmaceutical Research, 2010
Purpose The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly se... more Purpose The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly sensitive to viscosity, for the characterization of polysorbate-containing IgG formulations and compare them to the polarity-sensitive dyes ANS, Bis-ANS and Nile Red. Methods IgG formulations with polysorbate 20 or 80 were stressed below the aggregation temperature and analyzed by steady-state and time-resolved fluorescence and by HP-SEC with UV and fluorescent dye detection (Bis-ANS and CCVJ). Furthermore, commercial protein preparations of therapeutic proteins (Enbrel®50 mg, Humira®40 mg and MabThera®100 mg) were aggregated accordingly and analyzed with CCVJ fluorescence and HP-SEC. Results Contrarily to (Bis-)ANS and Nile Red, the molecular rotors DCVJ and CCVJ showed low background fluorescence in polysorbate-containing buffers. Time-resolved fluorescence experiments confirmed the steady-state fluorescence data. Both DCVJ and CCVJ showed enhanced fluorescence intensity for aggregated IgG formulations and were suitable for the characterization of polysorbate-containing IgG formulations in steady-state fluorescence and HP-SEC with dye detection (CCVJ). CCVJ was capable of detecting thermally induced aggregation in the commercial polysorbate-containing products Enbrel®50 mg, Humira®40 mg and MabThera®100 mg. Conclusion Fluorescent molecular rotors are suitable probes to detect aggregation in polysorbate-containing IgG formulations.
Pharmaceutical Research, 2010
Purpose The aim of this study was to develop a method to characterize intact soluble monoclonal I... more Purpose The aim of this study was to develop a method to characterize intact soluble monoclonal IgG1 antibody (IgG) oligomers by mass spectrometry. Methods IgG aggregates (dimers, trimers, tetramers and high-molecular-weight oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatography (HP-SEC), dialyzed against ammonium acetate pH 6.0 (a mass spectrometry-compatible volatile buffer), and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). Results Monomeric and aggregated IgG fractions in the stressed IgG formulation were successfully isolated by HP-SEC. ESI-TOF MS analysis enabled us to determine the molecular weight of the monomeric IgG as well as the aggregates, including dimers, trimers and tetramers. HP-SEC separation and sample preparation proved to be necessary for good quality signal in ESI-TOF MS. Both the HP-SEC protocol and the ESI-TOF mass spectrometric technique were shown to leave the IgG oligomers largely intact. Conclusions ESI-TOF MS is a useful tool complementary to HP-SEC to identify and characterize small oligomeric protein aggregates.
Pharmaceutical Research, 2010
Purpose To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic l... more Purpose To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates. Methods Standard polystyrene beads of sizes ranging from 60 to 1,000 nm and physical mixtures thereof were analyzed with NTA and DLS. The influence of different ratios of particle populations was tested. Drug delivery nanoparticles and protein aggregates were analyzed by NTA and DLS. Live monitoring of heat-induced protein aggregation was performed with NTA. Results NTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples. Sample visualization and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000 nm) particles generally does not compromise the accuracy of NTA measurements, and a broad range of population ratios can easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS. Live monitoring of heat-induced protein aggregation provides information about aggregation kinetics and size of submicron aggregates. Conclusion NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates.
Pharmaceutical Research, 2011
Purpose To evaluate the potential of fluorescence single particle tracking (fSPT) for the charact... more Purpose To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA). Methods A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.1 μm (control) were diluted in buffer, serum and plasma, and their size distributions were analyzed by fSPT and nanoparticle tracking analysis (NTA). In a separate experiment, IgG and HSA, fluorescently labeled with different dyes, were mixed and subjected to heat stress. The stressed sample was analyzed by fSPT using a dual color mode and by NTA. Results The accuracy and precision of fSPT proved to be comparable to NTA. fSPT was able to successfully measure all the samples in buffer, serum and plasma. The average size of the cross-linked protein aggregates showed a slight increase in biological fluids. Moreover, fSPT analysis showed that a significant proportion of the aggregates formed by subjecting an IgG/HSA mixture to heat stress were composed of both proteins. Conclusion fSPT is a powerful technique for the characterization of submicron protein aggregates in biological fluids and complex formulations.
Pharmaceutical Research, 2010
Purpose The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly se... more Purpose The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly sensitive to viscosity, for the characterization of polysorbate-containing IgG formulations and compare them to the polarity-sensitive dyes ANS, Bis-ANS and Nile Red. Methods IgG formulations with polysorbate 20 or 80 were stressed below the aggregation temperature and analyzed by steady-state and time-resolved fluorescence and by HP-SEC with UV and fluorescent dye detection (Bis-ANS and CCVJ). Furthermore, commercial protein preparations of therapeutic proteins (Enbrel®50 mg, Humira®40 mg and MabThera®100 mg) were aggregated accordingly and analyzed with CCVJ fluorescence and HP-SEC. Results Contrarily to (Bis-)ANS and Nile Red, the molecular rotors DCVJ and CCVJ showed low background fluorescence in polysorbate-containing buffers. Time-resolved fluorescence experiments confirmed the steady-state fluorescence data. Both DCVJ and CCVJ showed enhanced fluorescence intensity for aggregated IgG formulations and were suitable for the characterization of polysorbate-containing IgG formulations in steady-state fluorescence and HP-SEC with dye detection (CCVJ). CCVJ was capable of detecting thermally induced aggregation in the commercial polysorbate-containing products Enbrel®50 mg, Humira®40 mg and MabThera®100 mg. Conclusion Fluorescent molecular rotors are suitable probes to detect aggregation in polysorbate-containing IgG formulations.
Pharmaceutical Research, 2010
Purpose The aim of this study was to develop a method to characterize intact soluble monoclonal I... more Purpose The aim of this study was to develop a method to characterize intact soluble monoclonal IgG1 antibody (IgG) oligomers by mass spectrometry. Methods IgG aggregates (dimers, trimers, tetramers and high-molecular-weight oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatography (HP-SEC), dialyzed against ammonium acetate pH 6.0 (a mass spectrometry-compatible volatile buffer), and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). Results Monomeric and aggregated IgG fractions in the stressed IgG formulation were successfully isolated by HP-SEC. ESI-TOF MS analysis enabled us to determine the molecular weight of the monomeric IgG as well as the aggregates, including dimers, trimers and tetramers. HP-SEC separation and sample preparation proved to be necessary for good quality signal in ESI-TOF MS. Both the HP-SEC protocol and the ESI-TOF mass spectrometric technique were shown to leave the IgG oligomers largely intact. Conclusions ESI-TOF MS is a useful tool complementary to HP-SEC to identify and characterize small oligomeric protein aggregates.
Pharmaceutical Research, 2010
Purpose To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic l... more Purpose To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates. Methods Standard polystyrene beads of sizes ranging from 60 to 1,000 nm and physical mixtures thereof were analyzed with NTA and DLS. The influence of different ratios of particle populations was tested. Drug delivery nanoparticles and protein aggregates were analyzed by NTA and DLS. Live monitoring of heat-induced protein aggregation was performed with NTA. Results NTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples. Sample visualization and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000 nm) particles generally does not compromise the accuracy of NTA measurements, and a broad range of population ratios can easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS. Live monitoring of heat-induced protein aggregation provides information about aggregation kinetics and size of submicron aggregates. Conclusion NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates.
Pharmaceutical Research, 2011
Purpose To evaluate the potential of fluorescence single particle tracking (fSPT) for the charact... more Purpose To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA). Methods A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.1 μm (control) were diluted in buffer, serum and plasma, and their size distributions were analyzed by fSPT and nanoparticle tracking analysis (NTA). In a separate experiment, IgG and HSA, fluorescently labeled with different dyes, were mixed and subjected to heat stress. The stressed sample was analyzed by fSPT using a dual color mode and by NTA. Results The accuracy and precision of fSPT proved to be comparable to NTA. fSPT was able to successfully measure all the samples in buffer, serum and plasma. The average size of the cross-linked protein aggregates showed a slight increase in biological fluids. Moreover, fSPT analysis showed that a significant proportion of the aggregates formed by subjecting an IgG/HSA mixture to heat stress were composed of both proteins. Conclusion fSPT is a powerful technique for the characterization of submicron protein aggregates in biological fluids and complex formulations.
Pharmaceutical Research, 2010
Purpose The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly se... more Purpose The aim was to evaluate fluorescent molecular rotors (DCVJ and CCVJ), which are mainly sensitive to viscosity, for the characterization of polysorbate-containing IgG formulations and compare them to the polarity-sensitive dyes ANS, Bis-ANS and Nile Red. Methods IgG formulations with polysorbate 20 or 80 were stressed below the aggregation temperature and analyzed by steady-state and time-resolved fluorescence and by HP-SEC with UV and fluorescent dye detection (Bis-ANS and CCVJ). Furthermore, commercial protein preparations of therapeutic proteins (Enbrel®50 mg, Humira®40 mg and MabThera®100 mg) were aggregated accordingly and analyzed with CCVJ fluorescence and HP-SEC. Results Contrarily to (Bis-)ANS and Nile Red, the molecular rotors DCVJ and CCVJ showed low background fluorescence in polysorbate-containing buffers. Time-resolved fluorescence experiments confirmed the steady-state fluorescence data. Both DCVJ and CCVJ showed enhanced fluorescence intensity for aggregated IgG formulations and were suitable for the characterization of polysorbate-containing IgG formulations in steady-state fluorescence and HP-SEC with dye detection (CCVJ). CCVJ was capable of detecting thermally induced aggregation in the commercial polysorbate-containing products Enbrel®50 mg, Humira®40 mg and MabThera®100 mg. Conclusion Fluorescent molecular rotors are suitable probes to detect aggregation in polysorbate-containing IgG formulations.
Pharmaceutical Research, 2010
Purpose The aim of this study was to develop a method to characterize intact soluble monoclonal I... more Purpose The aim of this study was to develop a method to characterize intact soluble monoclonal IgG1 antibody (IgG) oligomers by mass spectrometry. Methods IgG aggregates (dimers, trimers, tetramers and high-molecular-weight oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatography (HP-SEC), dialyzed against ammonium acetate pH 6.0 (a mass spectrometry-compatible volatile buffer), and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). Results Monomeric and aggregated IgG fractions in the stressed IgG formulation were successfully isolated by HP-SEC. ESI-TOF MS analysis enabled us to determine the molecular weight of the monomeric IgG as well as the aggregates, including dimers, trimers and tetramers. HP-SEC separation and sample preparation proved to be necessary for good quality signal in ESI-TOF MS. Both the HP-SEC protocol and the ESI-TOF mass spectrometric technique were shown to leave the IgG oligomers largely intact. Conclusions ESI-TOF MS is a useful tool complementary to HP-SEC to identify and characterize small oligomeric protein aggregates.