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Sandeep Yadav - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of Super Refined™ Polysorbate 20 With Respect to Polysorbate Degradation, Particle Formation and Protein Stability.
Journal of pharmaceutical sciences, 2020Co-Authors: Nidhi Doshi, Raphael Fish, Karina Padilla, Sandeep YadavAbstract:Abstract Super Refined™ and Tween™ 20 HP Polysorbate 20 (PS20) are two commercially available compendial grades of PS20 frequently used in biopharmaceutical formulations as protein stabilizing surfactants. PS20 degradation has been a major concern recently for potentially limiting drug product shelf life due to free fatty acid particle formation. This work is a side-by-side comparison of SR and HP PS20 in terms of PS20 degradation, particle formation and protein stability. The use of SR grade PS20 results in higher levels of oxidative PS20 degradation, protein oxidation, peroxides and protein aggregation, and therefore requires addition of methionine as an antioxidant to mitigate these issues. No clear root cause was identified as to why SR PS20 is more prone to oxidative degradation. This work also suggests that SR PS20 is less prone to particle formation than HP PS20 when there is preferential degradation of mono-esters of PS20, while more susceptible to particle formation when there is preferential degradation of higher order esters of PS20. Overall, this publication summarizes the potential risks and benefits of SR PS20 compared to HP PS20 to enable a formulator to make an informed decision when choosing between the two surfactant grades in their drug product formulations.
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Dual Effect of Histidine on Polysorbate 20 Stability: Mechanistic Studies
Pharmaceutical Research, 2018Co-Authors: Lin Zhang, Sandeep Yadav, Y. John Wang, Olivier Mozziconacci, Christian SchӧneichAbstract:Purpose L-Histidine (L-His) and Polysorbate 20 (PS20) are two excipients frequently included in parenteral products to stabilize biotherapeutics. The objective of the current work was to investigate the impact of L-His on PS20 stability in aqueous solutions when subjected to forced oxidation and accelerated stability testing. Methods The stability of PS20 in L-His buffer was compared with that in acetate buffer. Forced oxidation of PS20 in these two buffer systems was initiated by a free radical generator, 2,2′-azobis (2-amidinopropane) hydrochloride (AAPH), while accelerated stability tests were carried out at 40°C. Ultra-performance liquid chromatography mass spectrometry was utilized to monitor intact PS20 and to analyze degradation products. Results Our results demonstrate a dual effect of L-His on PS20 stability. During exposure to AAPH, L-His protects PS20 from oxidation. Stable isotope labeling of L-His with ^13C was employed for mechanistic investigations. The protection of L-His was abrogated when acetate was added to L-His buffer, implying that the anti-oxidative activity of L-His may be compromised by specific counter ions. The replacement of L-His by various His derivatives led to significant changes in the protection of PS20 against AAPH-induced degradation. In contrast to forced degradation, the addition of L-His promoted oxidative PS20 degradation during accelerated storage at 40°C in solution, generating mainly short chain POE-laurates. Conclusion L-His exhibits a dual effect on the stability profile of PS20, protecting against AAPH-induced oxidation but promoting oxidative degradation during accelerated stability testing.
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Dual Effect of Histidine on Polysorbate 20 Stability: Mechanistic Studies.
Pharmaceutical research, 2018Co-Authors: Lin Zhang, Y. John Wang, Sandeep Yadav, Olivier Mozziconacci, Christian SchӧneichAbstract:L-Histidine (L-His) and Polysorbate 20 (PS20) are two excipients frequently included in parenteral products to stabilize biotherapeutics. The objective of the current work was to investigate the impact of L-His on PS20 stability in aqueous solutions when subjected to forced oxidation and accelerated stability testing. The stability of PS20 in L-His buffer was compared with that in acetate buffer. Forced oxidation of PS20 in these two buffer systems was initiated by a free radical generator, 2,2′-azobis (2-amidinopropane) hydrochloride (AAPH), while accelerated stability tests were carried out at 40°C. Ultra-performance liquid chromatography mass spectrometry was utilized to monitor intact PS20 and to analyze degradation products. Our results demonstrate a dual effect of L-His on PS20 stability. During exposure to AAPH, L-His protects PS20 from oxidation. Stable isotope labeling of L-His with 13C was employed for mechanistic investigations. The protection of L-His was abrogated when acetate was added to L-His buffer, implying that the anti-oxidative activity of L-His may be compromised by specific counter ions. The replacement of L-His by various His derivatives led to significant changes in the protection of PS20 against AAPH-induced degradation. In contrast to forced degradation, the addition of L-His promoted oxidative PS20 degradation during accelerated storage at 40°C in solution, generating mainly short chain POE-laurates. L-His exhibits a dual effect on the stability profile of PS20, protecting against AAPH-induced oxidation but promoting oxidative degradation during accelerated stability testing.
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Degradation Mechanisms of Polysorbate 20 Differentiated by ^18O-labeling and Mass Spectrometry
Pharmaceutical Research, 2017Co-Authors: Lin Zhang, Y. John Wang, Sandeep Yadav, Olivier Mozziconacci, Barthélemy Demeule, Christian SchӧneichAbstract:Purpose To investigate the mechanisms of Polysorbate (PS) degradation with the added objective of differentiating the hydrolysis and oxidation pathways. Methods Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was utilized to characterize all-laurate Polysorbate 20 (PS20) and its degradants. ^18O stable isotope labeling was implemented to produce ^18O-labeled degradation products of all-laurate PS20 in H_2 ^18O, with subsequent UPLC-MS analysis for location of the cleavage site on the fatty acid-containing side chain of PS20. Results The analysis reveals that hydrolysis of all-laurate PS20 leads to a breakdown of the ester linkage to liberate free lauric acid, showing a distinct dependence on pH. Using a hydrophilic free radical initiator, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) to study the oxidative degradation of all-laurate PS20, we demonstrate that free lauric acid and polyoxyethylene (POE) laurate are two major decomposition products. Measurement of ^18O incorporation into free lauric acid indicated that hydrolysis primarily led to ^18O incorporation into free lauric acid via “acyl-cleavage” of the fatty acid ester bond. In contrast, AAPH-exposure of all-laurate PS20 produced free lauric acid without ^18O-incorporation. Conclusions The ^18O-labeling technique and unique degradant patterns of all-laurate PS20 described here provide a direct approach to differentiate the types of PS degradation.
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understanding particle formation solubility of free fatty acids as Polysorbate 20 degradation byproducts in therapeutic monoclonal antibody formulations
Molecular Pharmaceutics, 2015Co-Authors: Nidhi Doshi, Barthélemy Demeule, Sandeep YadavAbstract:The purpose of this work was to determine the aqueous solubilities at 2–8 °C of the major free fatty acids (FFAs) formed by Polysorbate 20 (PS20) degradation and identify possible ways to predict, delay, or mitigate subsequent particle formation in monoclonal antibody (mAb) formulations. The FFA solubility limits at 2–8 °C were determined by titrating known amounts of FFA in monoclonal antibody formulations and identifying the FFA concentration leading to visible and subvisible particle formation. The solubility limits of lauric, myristic, and palmitic acids at 2–8 °C were 17 ± 1 μg/mL, 3 ± 1 μg/mL, and 1.5 ± 0.5 μg/mL in a formulation containing 0.04% (w/v) PS20 at pH 5.4 and >22 μg/mL, 3 ± 1 μg/mL, and 0.75 ± 0.25 μg/mL in a formulation containing 0.02% (w/v) PS20 at pH 6.0. For the first time, a 3D correlation between FFA solubility, PS20 concentration, and pH has been reported providing a rational approach for the formulator to balance these with regard to potential particle formation. The results sug...
Weishan Li - One of the best experts on this subject based on the ideXlab platform.
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synergistic effect of polyethylene glycol 600 and Polysorbate 20 on corrosion inhibition of zinc anode in alkaline batteries
Journal of Applied Electrochemistry, 2011Co-Authors: Man Liang, Hebing Zhou, Qiming Huang, Shejun Hu, Weishan LiAbstract:Polyethylene glycol 600 (PEG 600) and Polysorbate 20 (Tween 20) were used as a composite corrosion inhibitor of zinc in alkaline solution for the first time. The effects of the composite and individual inhibitors on corrosion inhibition of zinc were evaluated by weight-loss analysis and electrochemical methods including potentiodynamic, potentiostatic, and electrochemical impedance spectroscopic measurements. It was found that there was a synergistic effect between PEG 600 and Tween 20 on corrosion inhibition of zinc. The corrosion inhibition efficiency of the composite inhibitor, 500 ppm PEG 600 + 500 ppm Tween 20, was 89%, much higher than that of the individual inhibitor, 1000 ppm Tween 20 (71%) or 1000 ppm PEG 600 (55%). The battery (Zn/MnO2) discharge performance tests showed that the composite inhibitor reduced the self-discharge of zinc anode more effectively than the individual inhibitor. The synergistic mechanism between PEG 600 and Tween 20 was discussed.
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synergistic effect of polyethylene glycol 600 and Polysorbate 20 on corrosion inhibition of zinc anode in alkaline batteries
Journal of Applied Electrochemistry, 2011Co-Authors: Man Liang, Hebing Zhou, Qiming Huang, Shejun Hu, Weishan LiAbstract:Polyethylene glycol 600 (PEG 600) and Polysorbate 20 (Tween 20) were used as a composite corrosion inhibitor of zinc in alkaline solution for the first time. The effects of the composite and individual inhibitors on corrosion inhibition of zinc were evaluated by weight-loss analysis and electrochemical methods including potentiodynamic, potentiostatic, and electrochemical impedance spectroscopic measurements. It was found that there was a synergistic effect between PEG 600 and Tween 20 on corrosion inhibition of zinc. The corrosion inhibition efficiency of the composite inhibitor, 500 ppm PEG 600 + 500 ppm Tween 20, was 89%, much higher than that of the individual inhibitor, 1000 ppm Tween 20 (71%) or 1000 ppm PEG 600 (55%). The battery (Zn/MnO2) discharge performance tests showed that the composite inhibitor reduced the self-discharge of zinc anode more effectively than the individual inhibitor. The synergistic mechanism between PEG 600 and Tween 20 was discussed.
Barthélemy Demeule - One of the best experts on this subject based on the ideXlab platform.
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a rapid high sensitivity reversed phase ultra high performance liquid chromatography mass spectrometry method for assessing Polysorbate 20 degradation in protein therapeutics
Journal of Pharmaceutical Sciences, 2019Co-Authors: Ying Cheng, Barthélemy Demeule, Anthony Tomlinson, Camellia Zamiri, Toshiro Carcelen, Yinges Yigzaw, Matt KaloAbstract:Polysorbate 20 (PS20), a widely used surfactant in protein therapeutics, has been reported to undergo hydrolytic degradation during product storage, causing the release of free fatty acids. The accumulation of free fatty acids in protein therapeutics was found to result in the formation of particles due to their limited aqueous solubility at 2°C-8°C. Quantitation of free fatty acids originating from PS20 degradation is thus important during bioprocess optimization and stability testing in formulation development to ensure optimum PS20 stability as well as product and process consistency in final drug products. This work reports the development of a simple and robust, high-throughput, reversed-phase ultra high performance liquid chromatography mass spectrometry method for high-sensitivity quantitation of lauric acid and myristic acid by using isotope-labeled fatty acid internal standards. The high sensitivity (<100 ng/mL for lauric acid) and suitable precision (intermediate precision relative standard deviation of 11%) of this method enable accurate detection of lauric acid produced from the degradation of less than 1% of PS20 in a 0.2-mg/mL formulation. Using accelerated thermal stability testing, this method identifies processes that exhibit fast PS20 degradation within only days and consequently allows faster iterative optimization of the process.
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A Rapid High-Sensitivity Reversed–Phase Ultra High Performance Liquid Chromatography Mass Spectrometry Method for Assessing Polysorbate 20 Degradation in Protein Therapeutics
Journal of pharmaceutical sciences, 2019Co-Authors: Ying Cheng, Barthélemy Demeule, Anthony Tomlinson, Camellia Zamiri, Toshiro Carcelen, Yinges Yigzaw, Matt KaloAbstract:Polysorbate 20 (PS20), a widely used surfactant in protein therapeutics, has been reported to undergo hydrolytic degradation during product storage, causing the release of free fatty acids. The accumulation of free fatty acids in protein therapeutics was found to result in the formation of particles due to their limited aqueous solubility at 2°C-8°C. Quantitation of free fatty acids originating from PS20 degradation is thus important during bioprocess optimization and stability testing in formulation development to ensure optimum PS20 stability as well as product and process consistency in final drug products. This work reports the development of a simple and robust, high-throughput, reversed-phase ultra high performance liquid chromatography mass spectrometry method for high-sensitivity quantitation of lauric acid and myristic acid by using isotope-labeled fatty acid internal standards. The high sensitivity (
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Degradation Mechanisms of Polysorbate 20 Differentiated by ^18O-labeling and Mass Spectrometry
Pharmaceutical Research, 2017Co-Authors: Lin Zhang, Y. John Wang, Sandeep Yadav, Olivier Mozziconacci, Barthélemy Demeule, Christian SchӧneichAbstract:Purpose To investigate the mechanisms of Polysorbate (PS) degradation with the added objective of differentiating the hydrolysis and oxidation pathways. Methods Ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was utilized to characterize all-laurate Polysorbate 20 (PS20) and its degradants. ^18O stable isotope labeling was implemented to produce ^18O-labeled degradation products of all-laurate PS20 in H_2 ^18O, with subsequent UPLC-MS analysis for location of the cleavage site on the fatty acid-containing side chain of PS20. Results The analysis reveals that hydrolysis of all-laurate PS20 leads to a breakdown of the ester linkage to liberate free lauric acid, showing a distinct dependence on pH. Using a hydrophilic free radical initiator, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) to study the oxidative degradation of all-laurate PS20, we demonstrate that free lauric acid and polyoxyethylene (POE) laurate are two major decomposition products. Measurement of ^18O incorporation into free lauric acid indicated that hydrolysis primarily led to ^18O incorporation into free lauric acid via “acyl-cleavage” of the fatty acid ester bond. In contrast, AAPH-exposure of all-laurate PS20 produced free lauric acid without ^18O-incorporation. Conclusions The ^18O-labeling technique and unique degradant patterns of all-laurate PS20 described here provide a direct approach to differentiate the types of PS degradation.
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understanding particle formation solubility of free fatty acids as Polysorbate 20 degradation byproducts in therapeutic monoclonal antibody formulations
Molecular Pharmaceutics, 2015Co-Authors: Nidhi Doshi, Barthélemy Demeule, Sandeep YadavAbstract:The purpose of this work was to determine the aqueous solubilities at 2–8 °C of the major free fatty acids (FFAs) formed by Polysorbate 20 (PS20) degradation and identify possible ways to predict, delay, or mitigate subsequent particle formation in monoclonal antibody (mAb) formulations. The FFA solubility limits at 2–8 °C were determined by titrating known amounts of FFA in monoclonal antibody formulations and identifying the FFA concentration leading to visible and subvisible particle formation. The solubility limits of lauric, myristic, and palmitic acids at 2–8 °C were 17 ± 1 μg/mL, 3 ± 1 μg/mL, and 1.5 ± 0.5 μg/mL in a formulation containing 0.04% (w/v) PS20 at pH 5.4 and >22 μg/mL, 3 ± 1 μg/mL, and 0.75 ± 0.25 μg/mL in a formulation containing 0.02% (w/v) PS20 at pH 6.0. For the first time, a 3D correlation between FFA solubility, PS20 concentration, and pH has been reported providing a rational approach for the formulator to balance these with regard to potential particle formation. The results sug...
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Polysorbate 20 degradation in biopharmaceutical formulations quantification of free fatty acids characterization of particulates and insights into the degradation mechanism
Molecular Pharmaceutics, 2015Co-Authors: Anthony Tomlinson, Barthélemy Demeule, Sandeep YadavAbstract:Polysorbate 20 (PS20), a commonly used surfactant in biopharmaceuticals, showed degradation upon long-term (∼18–36 months) storage of two monoclonal antibody (mAb, mAb-A, and mAb-B) drug products at 2–8 °C. The PS20 degradation resulted in the accumulation of free fatty acids (FFA), which ultimately precipitated to form particles upon long-term storage. This study documents the development, qualification, and application of a method for FFA quantification in soluble and insoluble fraction of protein formulation. The method was applied to the quantification of capric acid, lauric acid, myristic acid, palmitic/oleic acid, and stearic acid in placebo as well as active protein formulations on stability. Quantification of FFA in both the soluble and insoluble fraction of mAb-A and mAb-B provided a better mechanistic understanding of PS20 degradation and the dynamics of subsequent fatty acid particle formation. Additionally, the use of this method for monitoring and quantitation of the FFA on real time storage ...
Alfred Blume - One of the best experts on this subject based on the ideXlab platform.
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Surface Tension and Self-association Properties of Aqueous Polysorbate 20 HP and 80 HP Solutions: Insights into Protein Stabilisation Mechanisms
Journal of Pharmaceutical Innovation, 2020Co-Authors: Patrick Garidel, Julia Buske, Michaela Blech, Alfred BlumeAbstract:Objective Polysorbates 20 and 80 are the most used surfactants for the development of parenteral protein formulations, because of their beneficial safety and stabilisation profile. Although, showing excellent stabilisation properties, the stabilisation mechanism(s) of these surfactants for aqueous protein formulations are still unclear. Different stabilisation models have been discussed in the literature, among them the possible formation of protein-surfactant micelle complexes. Methods This study focusses on the determination of the self-association properties of compendial grade Polysorbate 20 HP (PS 20 HP) and Polysorbate 80 HP (PS 80 HP) with regard to the formed micelle size (by dynamic light scattering, DLS), the concentration upon which the surfactant molecules self-associate (cmr, critical micelle concentration range) to form micelles, and the related surface tension. Surface tension and micelle size were determined as a function of temperature, as well as composition of the formulation (presence of a buffer salt and influence of ionic strength). Results The critical micelle concentration range values (cmr) at 25 °C are between 15–75 μM for PS 20 HP, but considerably lower for PS 80 HP with 7–16 μM, depending only slightly on the formulation composition. With increasing temperature, the cmr decreases slightly. PS 80 HP forms larger micelles (R_h = 4.5 nm) compared with PS 20 HP (R_h = 3.5 nm) (25 °C). The temperature dependency of the micelle size is more pronounced for PS 80 HP. Conclusions Based on these results, the suggested stabilisation mechanism, especially for antibody formulations, by the formation of antibody-Polysorbate micelle complexes, is critically discussed, and the current study shows that this stabilisation mechanism is not likely, for commonly used monoclonal antibody formulations.
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Surface Tension and Self-association Properties of Aqueous Polysorbate 20 HP and 80 HP Solutions: Insights into Protein Stabilisation Mechanisms
Journal of Pharmaceutical Innovation, 2020Co-Authors: Patrick Garidel, Julia Buske, Michaela Blech, Alfred BlumeAbstract:Polysorbates 20 and 80 are the most used surfactants for the development of parenteral protein formulations, because of their beneficial safety and stabilisation profile. Although, showing excellent stabilisation properties, the stabilisation mechanism(s) of these surfactants for aqueous protein formulations are still unclear. Different stabilisation models have been discussed in the literature, among them the possible formation of protein-surfactant micelle complexes. This study focusses on the determination of the self-association properties of compendial grade Polysorbate 20 HP (PS 20 HP) and Polysorbate 80 HP (PS 80 HP) with regard to the formed micelle size (by dynamic light scattering, DLS), the concentration upon which the surfactant molecules self-associate (cmr, critical micelle concentration range) to form micelles, and the related surface tension. Surface tension and micelle size were determined as a function of temperature, as well as composition of the formulation (presence of a buffer salt and influence of ionic strength). The critical micelle concentration range values (cmr) at 25 °C are between 15–75 μM for PS 20 HP, but considerably lower for PS 80 HP with 7–16 μM, depending only slightly on the formulation composition. With increasing temperature, the cmr decreases slightly. PS 80 HP forms larger micelles (Rh = 4.5 nm) compared with PS 20 HP (Rh = 3.5 nm) (25 °C). The temperature dependency of the micelle size is more pronounced for PS 80 HP. Based on these results, the suggested stabilisation mechanism, especially for antibody formulations, by the formation of antibody-Polysorbate micelle complexes, is critically discussed, and the current study shows that this stabilisation mechanism is not likely, for commonly used monoclonal antibody formulations.
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a thermodynamic analysis of the binding interaction between Polysorbate 20 and 80 with human serum albumins and immunoglobulins a contribution to understand colloidal protein stabilisation
Biophysical Chemistry, 2009Co-Authors: Patrick Garidel, Claudia Hoffmann, Alfred BlumeAbstract:Abstract The development of liquid therapeutic protein drugs imposes the presence of specific stabilisation agents to prevent protein degradation in order to reach shelf-lives of at least 2 years for drugs stored at 2–8 °C. Non-ionic detergents are used to avoid protein adsorption and the formation of protein aggregates. Depending on the protein and excipient (detergent) used the stabilisation effect is quite different and cannot be predicted up to now. One reason for this is the inadequate understanding of the principles that govern the stabilisation of proteins in the presence of detergents. One stabilisation mechanism discussed implicates a direct binding of detergent molecules to the hydrophobic surface area(s) of the protein in order to minimise protein–protein interactions and thus protein aggregation. Therefore, the presented study considers the interaction and binding of Polysorbate 20 and 80 to various human serum albumins and immunoglobulins of different subtypes. The interaction is analysed by means of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). From ITC the binding constant is derived as well as the thermodynamic parameters. The thermal protein stability is obtained from DSC. The results show that binding of the two detergents to human serum albumin is observed with binding constants of approximately ≈ 10 3 M − 1 , with 1–3 detergent molecules binding to the albumins. The exact Polysorbate–albumin ratio depends on the used albumin fraction. The interaction of the detergent is also obvious from the DSC results, showing an increase of the denaturation temperature. However, the binding of the detergent to the three investigated immunoglobulins is quite low and negligible, thus showing that for immunoglobulins a direct and strong Polysorbate binding to the protein is not the reason for the colloidal stabilisation effect of immunoglobulins in solution in the presence of Polysorbate 20 or 80.
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a thermodynamic analysis of the binding interaction between Polysorbate 20 and 80 with human serum albumins and immunoglobulins a contribution to understand colloidal protein stabilisation
Biophysical Chemistry, 2009Co-Authors: Patrick Garidel, Claudia Hoffmann, Alfred BlumeAbstract:The development of liquid therapeutic protein drugs imposes the presence of specific stabilisation agents to prevent protein degradation in order to reach shelf-lives of at least 2 years for drugs stored at 2-8 degrees C. Non-ionic detergents are used to avoid protein adsorption and the formation of protein aggregates. Depending on the protein and excipient (detergent) used the stabilisation effect is quite different and cannot be predicted up to now. One reason for this is the inadequate understanding of the principles that govern the stabilisation of proteins in the presence of detergents. One stabilisation mechanism discussed implicates a direct binding of detergent molecules to the hydrophobic surface area(s) of the protein in order to minimise protein-protein interactions and thus protein aggregation. Therefore, the presented study considers the interaction and binding of Polysorbate 20 and 80 to various human serum albumins and immunoglobulins of different subtypes. The interaction is analysed by means of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). From ITC the binding constant is derived as well as the thermodynamic parameters. The thermal protein stability is obtained from DSC. The results show that binding of the two detergents to human serum albumin is observed with binding constants of approximately approximately 10(3) M(-1), with 1-3 detergent molecules binding to the albumins. The exact Polysorbate-albumin ratio depends on the used albumin fraction. The interaction of the detergent is also obvious from the DSC results, showing an increase of the denaturation temperature. However, the binding of the detergent to the three investigated immunoglobulins is quite low and negligible, thus showing that for immunoglobulins a direct and strong Polysorbate binding to the protein is not the reason for the colloidal stabilisation effect of immunoglobulins in solution in the presence of Polysorbate 20 or 80.
Patrick Garidel - One of the best experts on this subject based on the ideXlab platform.
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An in-depth examination of fatty acid solubility limits in biotherapeutic protein formulations containing Polysorbate 20 and Polysorbate 80.
International journal of pharmaceutics, 2020Co-Authors: Nils Glücklich, Julia Buske, Mridula Dwivedi, Stefan Carle, Karsten Mäder, Patrick GaridelAbstract:Abstract Two of the most widely used surfactants to stabilize biologicals against e.g. interfacial stress are Polysorbate 20 (PS20) and Polysorbate 80 (PS80). In recent years, Polysorbate degradation in biopharmaceutical formulations has been observed. Polysorbate (PS) is mainly composed of sorbitan and isosorbide fatty acid (FA) esters, varying in their FA composition. Especially hydrolysis, which can be induced chemically as well as enzymatically, leads to the release of FAs from PS. These FAs are poorly soluble in aqueous buffer systems due to their hydrophobic nature and therefore prone to precipitation and particle formation. Since the emergence of particles in liquid formulations has to be avoided, it is important to prevent their formation. This study evaluates the solubility limits of selected FAs, which are likely to be released during the degradation of PS20 and PS80 in the presence of defined PS concentrations. Our results show that the solubility is highly dependent on the pH, the temperature, the used PS concentration and the aliphatic chain of respective FAs. Solubility of FAs, such as palmitic and oleic acid under the conditions determined in this study, are in the range of 3 to 130 µg·ml-1 (12 to 460 µM). Furthermore, the results allow making an estimation to which extent PS may degrade before particle formation in the drug product may be expected.
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Surface Tension and Self-association Properties of Aqueous Polysorbate 20 HP and 80 HP Solutions: Insights into Protein Stabilisation Mechanisms
Journal of Pharmaceutical Innovation, 2020Co-Authors: Patrick Garidel, Julia Buske, Michaela Blech, Alfred BlumeAbstract:Objective Polysorbates 20 and 80 are the most used surfactants for the development of parenteral protein formulations, because of their beneficial safety and stabilisation profile. Although, showing excellent stabilisation properties, the stabilisation mechanism(s) of these surfactants for aqueous protein formulations are still unclear. Different stabilisation models have been discussed in the literature, among them the possible formation of protein-surfactant micelle complexes. Methods This study focusses on the determination of the self-association properties of compendial grade Polysorbate 20 HP (PS 20 HP) and Polysorbate 80 HP (PS 80 HP) with regard to the formed micelle size (by dynamic light scattering, DLS), the concentration upon which the surfactant molecules self-associate (cmr, critical micelle concentration range) to form micelles, and the related surface tension. Surface tension and micelle size were determined as a function of temperature, as well as composition of the formulation (presence of a buffer salt and influence of ionic strength). Results The critical micelle concentration range values (cmr) at 25 °C are between 15–75 μM for PS 20 HP, but considerably lower for PS 80 HP with 7–16 μM, depending only slightly on the formulation composition. With increasing temperature, the cmr decreases slightly. PS 80 HP forms larger micelles (R_h = 4.5 nm) compared with PS 20 HP (R_h = 3.5 nm) (25 °C). The temperature dependency of the micelle size is more pronounced for PS 80 HP. Conclusions Based on these results, the suggested stabilisation mechanism, especially for antibody formulations, by the formation of antibody-Polysorbate micelle complexes, is critically discussed, and the current study shows that this stabilisation mechanism is not likely, for commonly used monoclonal antibody formulations.
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Surface Tension and Self-association Properties of Aqueous Polysorbate 20 HP and 80 HP Solutions: Insights into Protein Stabilisation Mechanisms
Journal of Pharmaceutical Innovation, 2020Co-Authors: Patrick Garidel, Julia Buske, Michaela Blech, Alfred BlumeAbstract:Polysorbates 20 and 80 are the most used surfactants for the development of parenteral protein formulations, because of their beneficial safety and stabilisation profile. Although, showing excellent stabilisation properties, the stabilisation mechanism(s) of these surfactants for aqueous protein formulations are still unclear. Different stabilisation models have been discussed in the literature, among them the possible formation of protein-surfactant micelle complexes. This study focusses on the determination of the self-association properties of compendial grade Polysorbate 20 HP (PS 20 HP) and Polysorbate 80 HP (PS 80 HP) with regard to the formed micelle size (by dynamic light scattering, DLS), the concentration upon which the surfactant molecules self-associate (cmr, critical micelle concentration range) to form micelles, and the related surface tension. Surface tension and micelle size were determined as a function of temperature, as well as composition of the formulation (presence of a buffer salt and influence of ionic strength). The critical micelle concentration range values (cmr) at 25 °C are between 15–75 μM for PS 20 HP, but considerably lower for PS 80 HP with 7–16 μM, depending only slightly on the formulation composition. With increasing temperature, the cmr decreases slightly. PS 80 HP forms larger micelles (Rh = 4.5 nm) compared with PS 20 HP (Rh = 3.5 nm) (25 °C). The temperature dependency of the micelle size is more pronounced for PS 80 HP. Based on these results, the suggested stabilisation mechanism, especially for antibody formulations, by the formation of antibody-Polysorbate micelle complexes, is critically discussed, and the current study shows that this stabilisation mechanism is not likely, for commonly used monoclonal antibody formulations.
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Development and validation of a selective marker-based quantification of Polysorbate 20 in biopharmaceutical formulations using UPLC QDa detection.
Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2020Co-Authors: Dirk Heinrich Evers, Torsten Schultz-fademrecht, Patrick Garidel, Julia BuskeAbstract:Abstract Polysorbates are widely used as non-ionic surfactant in biopharmaceutical formulations. Recently, the degradation of Polysorbate moved into the focus of attention, because in several published studies it was described, that stability issues in Polysorbate containing formulations were observed leading to the formation and appearance of sub-visible and visible particles. For this reason, monitoring of Polysorbate and its degradation products is of importance throughout the development of parenterals. The aim of the study was to develop a method for the selective marker-based quantification of adequate Polysorbate 20 components of interest without the need to apply derivatization or complex detection techniques. A single quadrupole mass (QDa) detector was used coupled to an ultra-high performance liquid chromatography (UHPLC) system. Method development was based on a developed reversed phase-high performance liquid chromatography assay coupled to a charge aerosol detector (RP-HPLC CAD). Instead of a charge aerosol detector (CAD) a QDa detector was used in order to significantly improve the selectivity. The focus of this study is the development of the QDa based method for the analysis of Polysorbate 20. Modifications of the mobile phase and the type of chromatography column allowed the separation of several components of Polysorbate 20 from polar non-esterified to apolar higher order species. In addition, a multitude of components could be quantified by their individual m/z values. The peak assignment identified 676 compounds which originated from Polysorbate 20. Some of these were selected and defined as marker components. It was shown that the developed method is capable to determine Polysorbate 20 in different biopharmaceutical formulations. The proposed assay is based on an artful sample preparation as well as a unique calibration procedure that make the determination of several selected components achievable. Furthermore, it was successfully demonstrated that the analytical procedure is valid to reliably quantify several Polysorbate 20 components at its 100% level (corresponds to 0.4 mg/mL intact Polysorbate 20) and even at lower concentrations that occur e.g. in case of Polysorbate 20 degradation. In conclusion, the method is beneficial to determine selected Polysorbate 20 species during formulation development of biopharmaceuticals as well as during stability testing and trouble shooting.
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a thermodynamic analysis of the binding interaction between Polysorbate 20 and 80 with human serum albumins and immunoglobulins a contribution to understand colloidal protein stabilisation
Biophysical Chemistry, 2009Co-Authors: Patrick Garidel, Claudia Hoffmann, Alfred BlumeAbstract:Abstract The development of liquid therapeutic protein drugs imposes the presence of specific stabilisation agents to prevent protein degradation in order to reach shelf-lives of at least 2 years for drugs stored at 2–8 °C. Non-ionic detergents are used to avoid protein adsorption and the formation of protein aggregates. Depending on the protein and excipient (detergent) used the stabilisation effect is quite different and cannot be predicted up to now. One reason for this is the inadequate understanding of the principles that govern the stabilisation of proteins in the presence of detergents. One stabilisation mechanism discussed implicates a direct binding of detergent molecules to the hydrophobic surface area(s) of the protein in order to minimise protein–protein interactions and thus protein aggregation. Therefore, the presented study considers the interaction and binding of Polysorbate 20 and 80 to various human serum albumins and immunoglobulins of different subtypes. The interaction is analysed by means of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). From ITC the binding constant is derived as well as the thermodynamic parameters. The thermal protein stability is obtained from DSC. The results show that binding of the two detergents to human serum albumin is observed with binding constants of approximately ≈ 10 3 M − 1 , with 1–3 detergent molecules binding to the albumins. The exact Polysorbate–albumin ratio depends on the used albumin fraction. The interaction of the detergent is also obvious from the DSC results, showing an increase of the denaturation temperature. However, the binding of the detergent to the three investigated immunoglobulins is quite low and negligible, thus showing that for immunoglobulins a direct and strong Polysorbate binding to the protein is not the reason for the colloidal stabilisation effect of immunoglobulins in solution in the presence of Polysorbate 20 or 80.