38 results on '"Koulov A"'
Search Results
2. Identification of an Oxidizing Leachable from a Clinical Syringe Rubber Stopper
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Sonja Peter, Atanas Koulov, Andreas Zerr, Ombeline Danton, Hanns-Christian Mahler, Michael Jahn, Ariane Schröter, Pascal Chalus, and Jörg Huwyler
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Pharmaceutical Science ,02 engineering and technology ,030226 pharmacology & pharmacy ,Chemical reaction ,Ferrous ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Natural rubber ,Oxidizing agent ,Sample preparation ,Drug Packaging ,Dichloromethane ,chemistry.chemical_classification ,Chromatography ,Syringes ,Polymer ,021001 nanoscience & nanotechnology ,chemistry ,Leaching (chemistry) ,visual_art ,visual_art.visual_art_medium ,Rubber ,Drug Contamination ,0210 nano-technology ,Oxidation-Reduction - Abstract
Leaching of toxic or reactive chemicals from polymeric materials can adversely affect the quality and safety of biopharmaceuticals. It was therefore the aim of the present study to analyze leachables from a disposable clinical administration syringe using a polysorbate-containing surrogate solution and to assess their chemical reactivity. Analytical methods did include (headspace) GC-MS, Fourier-transform-infrared spectroscopy, a ferrous oxidation-xylenol orange assay, and nuclear magnetic resonance analysis. In the syringe leachables solution, the carcinogenic 1,1,2,2-tetrachloroethane (TCE) was detected in concentrations above the ICH M7-derived analytical evaluation threshold. TCE was shown to be an oxidation product of dichloromethane used during sample preparation. Since TCE was only isolated from incubations with the contained rubber stopper, we hypothesized that a stopper-derived leachable acted as a reactive oxidant promoting this chemical reaction. Subsequently, the leachable was identified to be the polymerization initiator Luperox® 101. Combining different analytical approaches led to the structural elucidation of a chemical reactive oxidant, which has the potential to interact and alter drug products. We conclude that chemically reactive compounds, such as the newly identified rubber stopper leachable Luperox® 101, may be of concern and therefore should be routinely considered if a prolonged exposure of polymers with drug products can be anticipated.
- Published
- 2021
3. 4-Hydroxynonenal – A Toxic Leachable from Clinically Used Administration Materials
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Ariane Schröter, Hanns-Christian Mahler, Michael Jahn, Atanas Koulov, Jörg Huwyler, and Nadia Ben Sayed
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Aldehydes ,Chromatography ,Polymers ,Pharmaceutical Science ,02 engineering and technology ,021001 nanoscience & nanotechnology ,030226 pharmacology & pharmacy ,4-Hydroxynonenal ,Kinetics ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,Drug product ,0210 nano-technology ,Derivatization - Abstract
Introduction: The migration of chemicals from processing materials into biopharmaceuticals can lead to various problems. Leachables from administration materials, with no possibility of further clearance, are of particular concern. Released chemicals can be toxic or react with formulation components, thereby impacting product safety. Therapeutic proteins, which are susceptible to chemical modifications, have highest risk to be affected. Aim: The aim of this study was to identify a previously unknown leachable compound from clinical administration sets, which was present above the applied generic safety threshold. Methods: Extracts of commonly used clinical administration sets were analyzed using a recently established specific assay allowing the identification and quantification of the α,β-unsaturated aldehyde 4-hydroxynonenal (HNE) in a drug product surrogate solution. HNE was quantified after derivatization with 2,4-dinitrophenylhydrazine (DNPH) and liquid extraction of the formed hydrazone by LC-MRM analysis. Results: Potentially genotoxic HNE was a leachable compound from all tested administration sets, in parts exceeding safety thresholds for genotoxicants. The HNE-releasing polymer was identified as PVC. Conclusion: Clinical administration sets should be, like manufacturing materials and container closure systems, in the focus of routine leachables studies. Manufacturers of clinical administration sets should show responsibility to avoid the presence of safety concerning chemicals, like HNE.
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- 2021
4. Tracking the physical stability of fluorescent-labeled mAbs under physiologic in vitro conditions in human serum and PBS
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Andrew J. Racher, Roman Mathaes, Joerg Huwyler, Atanas Koulov, Joachim Schuster, Pascal Detampel, Hanns-Christian Mahler, and Susanne Joerg
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Serum ,medicine.drug_class ,Pharmaceutical Science ,02 engineering and technology ,Monoclonal antibody ,030226 pharmacology & pharmacy ,Phosphates ,Flow cytometry ,03 medical and health sciences ,0302 clinical medicine ,In vivo ,Fluorescence microscope ,medicine ,Humans ,Particle Size ,Fragmentation (cell biology) ,Alexa Fluor ,medicine.diagnostic_test ,Chemistry ,Antibodies, Monoclonal ,General Medicine ,Flow Cytometry ,021001 nanoscience & nanotechnology ,In vitro ,Isoelectric point ,Microscopy, Fluorescence ,Biochemistry ,Chromatography, Gel ,Saline Solution ,0210 nano-technology ,Hydrophobic and Hydrophilic Interactions ,Biotechnology - Abstract
In recent years, the stability of biotherapeutics in vivo has received increasing attention. Assessing the stability of biotherapeutics in serum may support the selection of adequate molecule candidates. In our study, we compared the physical stability of 8 different monoclonal antibodies (mAbs) in phosphate-buffered saline (PBS) and human serum. mAbs were Alexa Fluor 488-labeled and characterized with respect to fragmentation, aggregation, and proteinaceous particle formation. Samples were analyzed using size-exclusion chromatography, light obscuration, and flow imaging. In addition, novel methods such as flow cytometry and fluorescence microscopy were applied. mAbs were selected based on their hydrophobicity and isoelectric point. All mAbs studied were inherently less stable in human serum as compared to PBS. Particle size and particle counts increased in serum over time. Interestingly, certain mAbs showed significant levels of fragmentation in serum but not in PBS. We conclude that PBS cannot replicate the physical stability measured in serum. The stability of labeled mAbs in human serum did not correlate with their hydrophobicity and isoelectric point . Serum stability significantly differed amongst the tested mAbs.
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- 2020
5. Particle Analysis of Biotherapeutics in Human Serum Using Machine Learning
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Roman Mathaes, Hanns-Christian Mahler, Pascal Detampel, Joerg Huwyler, Joachim Schuster, Susanne Joerg, Atanas Koulov, and Kai D. Schleicher
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Serum ,Pharmaceutical Science ,02 engineering and technology ,Machine learning ,computer.software_genre ,030226 pharmacology & pharmacy ,Machine Learning ,03 medical and health sciences ,0302 clinical medicine ,Microscopy ,Therapeutic Immunoglobulin ,Fluorescence microscope ,Biological fluids ,Humans ,Particle Size ,Particle analysis ,Chemistry ,business.industry ,Proteins ,021001 nanoscience & nanotechnology ,Microscopy, Fluorescence ,Particle ,Particle size ,Artificial intelligence ,0210 nano-technology ,business ,computer ,Light obscuration - Abstract
In recent years, an increasing number of studies assessed the stability of biotherapeutics in biological fluids. Such studies aim to simulate the conditions encountered in the human body and investigate the in vivo stability under in vitro conditions. However, on account of complexity of biological fluids, standard pharmaceutical methods are poorly suited to assess the stability of biotherapeutics. In this study, a fluorescent-labeled therapeutic immunoglobulin G (IgG) was analyzed for proteinaceous particles after mixing with human serum and after incubation at 37°C for 5 days. Samples were analyzed using standard pharmaceutical methods (light obscuration and dynamic imaging). Moreover, we developed a fluorescence microscopy method allowing to semiquantitatively detect IgG particles in serum. Several hundred IgG particles were detected after exposure to serum. Moreover, particle counts and particle size increased in serum over time. The results showed that an IgG may form particles on mixing with serum and novel methods such as fluorescence microscopy are required to gain insight on the stability of biotherapeutics in biological fluids. Furthermore, we showed distinct advantages of machine learning over traditional threshold-based methods by analyzing microscopy images. Machine learning allowed simplifying particles in regards to count, size, and shape.
- Published
- 2020
6. 4-Hydroxynonenal is An Oxidative Degradation Product of Polysorbate 80
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Hanns-Christian Mahler, Ariane Schröter, Michael Jahn, Atanas Koulov, and Jörg Huwyler
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Antioxidant ,medicine.medical_treatment ,Linoleic acid ,Polysorbates ,Pharmaceutical Science ,02 engineering and technology ,medicine.disease_cause ,030226 pharmacology & pharmacy ,4-Hydroxynonenal ,Adduct ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,Derivatization ,chemistry.chemical_classification ,Aldehydes ,Chromatography ,021001 nanoscience & nanotechnology ,Amino acid ,Oxidative Stress ,chemistry ,0210 nano-technology ,Oxidation-Reduction ,Oxidative stress ,Polyunsaturated fatty acid - Abstract
Introduction Polysorbates (PS) are used in biopharmaceuticals to stabilize therapeutic proteins. Oxidative degradation of (poly)unsaturated fatty acids (PUFAs) contained in PS was shown to lead to α,β-unsaturated carbonyls. Aim The n-6-PUFA linoleic acid accounts for up to 18% of all FAs contained in multi-compendial grade PS80. 4-Hydroxynonenal (HNE) is highly reactive towards nucleophilic amino acids, potentially leading to covalent protein modifications. This study tests whether HNE may be a pharmaceutically relevant PS80 peroxidation product. Methods Since HNE was not directly detectable in the PS80 matrix by UV and MS, a new quantification method was established. After derivatization with 2,4-dinitrophenyl hydrazine (DNPH) and extraction of the formed hydrazone with a salting-out assisted liquid-liquid extraction, the HNE-DNPH adduct was analyzed by multiple reaction monitoring. Kinetic oxidation studies were conducted incubating PS80 in presence and absence of the antioxidant butylhydroxytoluene (BHT). Results HNE was confirmed as PS80 degradant in oxidatively stressed samples. BHT was shown to prevent its formation. Conclusion HNE is a detectable PS80 degradation product raising questions about the potential impact on critical quality attributes of biopharmaceuticals formulated with PS80. Addition of BHT prevented HNE formation under oxidative stress. Consequently, BHT might be a valuable additive in PS used in biopharmaceuticals.
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- 2021
7. Characterization of Polymeric Syringes Used for Intravitreal Injection
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Hanns-Christian Mahler, Dhananjay Jere, Atanas Koulov, Roman Mathaes, Ahmad S. Sediq, Susanne Joerg, Martin Vogt, Anja Matter, Sarah S. Peláez, Maximilian Zaeh, and Pascal Chalus
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Potential impact ,Materials science ,Hold time ,Syringes ,Pharmaceutical Science ,02 engineering and technology ,021001 nanoscience & nanotechnology ,030226 pharmacology & pharmacy ,Vial ,Dead volume ,Silicone oil ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,Pharmaceutical Preparations ,Needles ,NEEDLE GAUGE ,Intravitreal Injections ,Silicone Oils ,0210 nano-technology ,Protein concentration ,Syringe ,Biomedical engineering - Abstract
Intravitreal (IVT) injection is currently the state of the art for drug delivery to the back of the eye. Drug Products (DP) intended for IVT injections usually pose challenges such as a very low injection volume (e.g. 50 μL) and high injection forces. DPs in vials are typically transferred and injected using disposable polymer syringes, which can feature a silicone oil (SO) coating. In our syringe in-use study, we compared dead volume, total SO content and SO layer distributions of three IVT transfer injection syringes. We assessed multiple potential impact factors such as protein concentration, needle gauge, injection speed, surfactant type and the impact of the in-use hold time on sub-visible particle (SvP) formation and injection forces. Pronounced differences were observed between the syringes regarding SvP generation. Siliconized syringes showed higher SvP counts as compared to non-siliconized syringes. In some cases injection forces exceeded 20 N, which caused needles to burst off during injection. The syringes also showed relevant differences in total SO content and dead volume. In conclusion, specific consideration in the selection of an adequate transfer injection syringe are required. This includes extensive testing and characterization under intended and potential in-use conditions and the development of in-use handling procedures.
- Published
- 2020
8. Measuring Lipolytic Activity to Support Process Improvements to Manage Lipase-Mediated Polysorbate Degradation
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Hanns-Christian Mahler, Filip Maciej Fedorowicz, Andreas Zerr, Finn Brigger, Michael Jahn, and Atanas Koulov
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Swine ,Lipolysis ,Pharmaceutical Science ,Polysorbates ,02 engineering and technology ,030226 pharmacology & pharmacy ,03 medical and health sciences ,chemistry.chemical_compound ,Hydrolysis ,0302 clinical medicine ,medicine ,Animals ,Pharmacology (medical) ,Lipase ,Pharmacology ,Polysorbate ,Chromatography ,biology ,Organic Chemistry ,Substrate (chemistry) ,021001 nanoscience & nanotechnology ,Solvent ,Orlistat ,Biopharmaceutical ,chemistry ,biology.protein ,Molecular Medicine ,0210 nano-technology ,Biotechnology ,medicine.drug - Abstract
Polysorbates are critical stabilizers in biopharmaceutical protein formulations. However, they may degrade in drug substance (DS) or drug product (DP) during storage. Degradation catalyzed by lipases present in host cell proteins (HCPs) is one suspected root cause. The purpose of this study was to develop an assay to detect lipolytic activity in biopharmaceutical DS and DP formulations. The assay is based on the hydrolysis of the lipase substrate 4-methylumbelliferyl oleate to yield the fluorescent product 4-methylumbelliferone. First, the assay components and their concentrations (buffer salts and pH, solvent and inhibitor Orlistat) were established and optimized using a model lipase (Porcine pancreatic lipase) and cell culture harvest fluid that exhibited lipolytic activity. The assay was then successfully applied and thereby qualified in protein formulations and at lipase concentrations possibly encountered in actual biopharmaceutical DS and DP formulations. The lipase assay can be used to detect lipolytic activity in intermediate and final DS, for example during process optimization in downstream purification, to better and specifically reduce the level, or deplete, lipases from HCPs. The assay is also suitable to be applied during root cause investigations related to polysorbate degradation in biopharmaceutical DP.
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- 2020
9. In Vivo Stability of Therapeutic Proteins
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Hanns-Christian Mahler, Pascal Detampel, Roman Mathaes, Joachim Schuster, Satish K. Singh, Atanas Koulov, and Joerg Huwyler
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Drug ,Chemistry, Pharmaceutical ,media_common.quotation_subject ,Pharmaceutical Science ,02 engineering and technology ,Pharmacology ,030226 pharmacology & pharmacy ,Excipients ,03 medical and health sciences ,Route of administration ,0302 clinical medicine ,Drug Stability ,Pharmacokinetics ,In vivo ,Animals ,Humans ,Pharmacology (medical) ,media_common ,Chemistry ,Immunogenicity ,Organic Chemistry ,Proteins ,Therapeutic protein ,021001 nanoscience & nanotechnology ,Pharmaceutical Preparations ,Drug development ,Pharmacodynamics ,Molecular Medicine ,0210 nano-technology ,Biotechnology - Abstract
Significant efforts are made to characterize molecular liabilities and degradation of the drug substance (DS) and drug product (DP) during various product life-cycle stages. The in vivo fate of a therapeutic protein is usually only considered in terms of pharmacokinetics (PKs) and pharmacodynamics (PDs). However, the environment in the human body differs substantially from that of the matrix (formulation) of the DP and may impact on the stability of an injected therapeutic protein. Stabilizing excipients used in protein formulations are expected to undergo more rapid distribution and dissociation in vivo, compared to a protein as a highly charged macromolecule. Thus, in vivo stability may significantly differ from shelf-life stability. In vivo degradation of the therapeutic protein may alter efficacy and/or safety characteristics such as immunogenicity. Studying the stability of a therapeutic protein in the intended body compartment can de-risk drug development in early stages of development by improving the selection of better clinical lead molecules. This review assesses the considerations when aiming to evaluate the in vivo fate of a therapeutic protein by comparing the physiology of relevant human body compartments and assessing their potential implications on the stability of a therapeutic protein. Moreover, we discuss the limitations of current experimental approaches mimicking physiologic conditions, depending on the desired route of administration, such as intravenous (IV), subcutaneous (SC), intravitreal (IVT), or intrathecal (IT) administration(s). New models more closely mimicking the relevant physiologic environment and updated analytical methods are required to understand the in vivo fate of therapeutic proteins.
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- 2020
10. Functional amyloid--from bacteria to humans
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Fowler, Douglas M., Koulov, Atanas V., Balch, William E., and Kelly, Jeffery W.
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Glycoproteins -- Research ,Tissue engineering -- Research ,Biological sciences ,Chemistry - Abstract
Amyloid--a fibrillar, cross [beta]-sheet quaternary structure--was first discovered in the context of human disease and tissue damage, and was thought to always be detrimental to the host. Recent studies have identified amyloid fibers in bacteria, fungi, insects, invertebrates and humans that are functional. For example, human Pmel17 has important roles in the biosynthesis of the pigment melanin, and the factor XII protein of the hemostatic system is activated by amyloid. Functional amyloidogenesis in these systems requires tight regulation to avoid toxicity. A greater understanding of the diverse physiological applications of this fold has the potential to provide a fresh perspective for the treatment of amyloid diseases.
- Published
- 2007
11. Stabilizing Polysorbate 20 and 80 Against Oxidative Degradation
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Atanas Koulov, Hanns-Christian Mahler, Ariane Schmidt, Michael Jahn, and Jörg Huwyler
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Polysorbate ,Chromatography ,Antioxidant ,Chemical structure ,medicine.medical_treatment ,Hydrolysis ,Pharmaceutical Science ,Polysorbates ,02 engineering and technology ,021001 nanoscience & nanotechnology ,030226 pharmacology & pharmacy ,Peroxide ,Micelle ,Gas Chromatography-Mass Spectrometry ,Ferrous ,03 medical and health sciences ,chemistry.chemical_compound ,Oxidative Stress ,0302 clinical medicine ,chemistry ,medicine ,Polysorbate 20 ,0210 nano-technology ,Oxidation-Reduction - Abstract
Polysorbates are stabilizers typically required in therapeutic protein formulations. On account of their chemical structure, polysorbates are prone to degradation, which can render a pharmaceutical product instable or incompliant. The purpose of this study was to investigate if the addition of butylhydroxytoluene (BHT) and butylhydroxyanisole (BHA) protects polysorbate 20 (PS20) and polysorbate 80 (PS80) against oxidative degradation. PS20 and PS80 solutions containing BHA, BHT—or as control without an antioxidant—were stressed by exposure to air at 40°C for 7 weeks. The following assays were performed: ferrous oxidation-xylenol orange assay, liquid chromatography coupled to UV and mass spectrometry (MS), pH measurement, liquid chromatography fluorescence micelle assay, headspace–gas chromatography coupled with MS. PS20 and PS80 solutions containing an antioxidant were found to be more stable, as demonstrated by lower peroxide levels, lower free fatty acid contents, stable pH, intact polysorbate micelle structure/composition, and less volatile degradants. PS20 and PS80 solutions containing BHT or BHA are more stable against oxidative degradation compared to nonstabilized solutions. It might be beneficial to formulate bulk polysorbate with the antioxidant(s) to ensure stabilization during all process steps.
- Published
- 2019
12. Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs
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Philippe Ringler, Atanas Koulov, Volker Schnaible, Gerald Gellermann, Christof Finkler, Melissa A. Graewert, Henning Stahlberg, Jan Olaf Stracke, Friederike Plath, Arne C. Rufer, Dmitri I. Svergun, Alexandra Graff-Meyer, and Matthias E. Lauer
- Subjects
0301 basic medicine ,Chemistry ,medicine.drug_class ,Stereochemistry ,Dimer ,Immunology ,Antibodies, Monoclonal ,Protein aggregation ,Monoclonal antibody ,030226 pharmacology & pharmacy ,03 medical and health sciences ,Crystallography ,chemistry.chemical_compound ,030104 developmental biology ,0302 clinical medicine ,Antibodies monoclonal ,Covalent bond ,Report ,Immunoglobulin G ,medicine ,Humans ,Immunology and Allergy ,Hinge region ,Dimerization - Abstract
The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.
- Published
- 2016
13. Considerations for the Use of Polysorbates in Biopharmaceuticals
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Atanas Koulov, Maloney Kevin, Wei Liu, Alexander M. Harmon, Hanns-Christian Mahler, Dilbir S. Bindra, Ping Y Yeh, Kapil Gupta, Sandeep Yadav, Vincent Corvari, R. Matthew Fesinmeyer, Michael T. Jones, John Wang, Satish K. Singh, and Kenneth D Hinds
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Computer science ,Drug Compounding ,Pharmacology toxicology ,Polysorbates ,Pharmaceutical Science ,02 engineering and technology ,030226 pharmacology & pharmacy ,Excipients ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Drug Stability ,Animals ,Humans ,Pharmacology (medical) ,Particle Size ,Pharmacology ,Polysorbate ,Biological Products ,Protein Stability ,Hydrolysis ,Organic Chemistry ,Proteins ,021001 nanoscience & nanotechnology ,Biopharmaceutical ,chemistry ,Molecular Medicine ,Polysorbate 20 ,Biochemical engineering ,0210 nano-technology ,Oxidation-Reduction ,Biotechnology - Abstract
Polysorbates are commonly added to protein formulations and serve an important function as stabilizers. This paper reviews recent literature detailing some of the issues seen with the use of polysorbate 80 and polysorbate 20 in protein formulations. Based on this knowledge, a development strategy is proposed that leads to a control strategy for polysorbates in protein formulations. A consortium of Biopharmaceutical scientists working in the area of protein formulations, shared experiences with polysorbates as stabilizers in their formulations. Based on the authors experiences and recent published literature, a recommendation is put forth for a development strategy which will lead into the appropriate control strategy for these excipients. An appropriate control strategy may comprise one or more elements of raw material, in-process and manufacturing controls. Additionally, understanding the role, if any, polysorbates play during stability will require knowledge of the criticality of the excipient, based upon its impact on CQAs due to variations in concentration and degradation level.
- Published
- 2018
14. Factors Governing the Precision of Subvisible Particle Measurement Methods – A Case Study with a Low-Concentration Therapeutic Protein Product in a Prefilled Syringe
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Atanas Koulov, Adeline Boillon, Christof Finkler, Roland Schmidt, Michael Adler, Jens Lamerz, Hanns-Christian Mahler, Thierry Da Cunha, Joerg Huwyler, and Anacelia Ríos Quiroz
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Pharmacology toxicology ,Pharmaceutical Science ,Nanotechnology ,02 engineering and technology ,030226 pharmacology & pharmacy ,Chemistry Techniques, Analytical ,Protein Aggregates ,03 medical and health sciences ,0302 clinical medicine ,Protein particles ,Pharmacology (medical) ,Particle Size ,Process engineering ,Prefilled Syringe ,Volume concentration ,Pharmacology ,Measurement method ,Chemistry ,business.industry ,Syringes ,Organic Chemistry ,Proteins ,Therapeutic protein ,021001 nanoscience & nanotechnology ,Molecular Medicine ,Particle ,Sources of error ,0210 nano-technology ,business ,Biotechnology - Abstract
The current study was performed to assess the precision of the principal subvisible particle measurement methods available today. Special attention was given to identifying the sources of error and the factors governing analytical performance.The performance of individual techniques was evaluated using a commercial biologic drug product in a prefilled syringe container. In control experiments, latex spheres were used as standards and instrument calibration suspensions.The results reported in this manuscript clearly demonstrated that the particle measurement techniques operating in the submicrometer range have much lower precision than the micrometer size-range methods. It was established that the main factor governing the relatively poor precision of submicrometer methods in general and inherently, is their low sampling volume and the corresponding large extrapolation factors for calculating final results.The variety of new methods for submicrometer particle analysis may in the future support product characterization; however, the performance of the existing methods does not yet allow for their use in routine practice and quality control.
- Published
- 2015
15. Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule
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Emilien Folzer, Katrin Bomans, Patrick Bulau, Christof Finkler, Hanns-Christian Mahler, Jörg Huwyler, Roland Schmidt, Katharina Diepold, and Atanas Koulov
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chemistry.chemical_classification ,Methionine ,Ultraviolet Rays ,Tryptophan ,Antibodies, Monoclonal ,Pharmaceutical Science ,Biological activity ,Hydrogen Peroxide ,Protein oxidation ,Amino acid ,chemistry.chemical_compound ,Biochemistry ,chemistry ,Metals ,Immunoglobulin G ,Reagent ,Forced degradation ,Indicators and Reagents ,Hydrogen peroxide ,Oxidation-Reduction - Abstract
Oxidation of methionine and tryptophan are common degradation pathways for monoclonal antibodies and present major analytical challenges in biotechnology. Generally, protein oxidation is detectable in stability and/or stressed samples (e.g., exposed to hydrogen peroxide, UV light, or metal ions). The induced chemical modifications may impact the biological activity of antibodies and may have biological consequences. However, these effects and the contribution of individual protein modifications are difficult to delineate as different amino acids are often oxidized simultaneously and accompanied by other degradants such as aggregates, especially in forced degradation studies. Here, we report a new method to obtain selective oxidation of methionine or tryptophan by using oxidation reagents combined with large excess of free tryptophan or methionine, correspondingly. More specifically, using hydrogen peroxide or tert-butyl hydroperoxide in combination with addition of free tryptophan allowed for selective oxidation of methionine. Conversely, the use of 2,2-azobis(2-amidinopropane) dihydrochloride in combination with free methionine resulted in selective tryptophan oxidation, whereas methionine oxidation was not significantly altered. This novel stress model system may prove to be valuable tool in future mechanistic studies of oxidative degradation of protein therapeutics.
- Published
- 2015
16. Extensive Chemical Modifications in the Primary Protein Structure of IgG1 Subvisible Particles Are Necessary for Breaking Immune Tolerance
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Antonio Iglesias, Roland Schmidt, Björn Boll, Atanas Koulov, Juliana Bessa, Christof Finkler, Patrick Bulau, Anacelia Ríos Quiroz, Emilien Folzer, Hanns-Christian Mahler, and Jörg Huwyler
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0301 basic medicine ,Genetically modified mouse ,Pharmaceutical Science ,Mice, Transgenic ,030226 pharmacology & pharmacy ,Immune tolerance ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Protein structure ,Immune system ,Drug Discovery ,Immune Tolerance ,Size fractions ,Animals ,Humans ,Amino Acid Sequence ,Particle Size ,Chemistry ,Immunogenicity ,Antibodies, Monoclonal ,Virology ,Mice, Inbred C57BL ,030104 developmental biology ,Immunoglobulin G ,Antibody Formation ,Biophysics ,Molecular Medicine ,Particle ,Stress conditions - Abstract
A current concern with the use of therapeutic proteins is the likely presence of aggregates and submicrometer, subvisible, and visible particles. It has been proposed that aggregates and particles may lead to unwanted increases in the immune response with a possible impact on safety or efficacy. The aim of this study was thus to evaluate the ability of subvisible particles of a therapeutic antibody to break immune tolerance in an IgG1 transgenic mouse model and to understand the particle attributes that might play a role in this process. We investigated the immunogenic properties of subvisible particles (unfractionated, mixed populations, and well-defined particle size fractions) using a transgenic mouse model expressing a mini-repertoire of human IgG1 (hIgG1 tg). Immunization with proteinaceous subvisible particles generated by artificial stress conditions demonstrated that only subvisible particles bearing very extensive chemical modifications within the primary amino acid structure could break immune tolerance in the hIgG1 transgenic mouse model. Protein particles exhibiting low levels of chemical modification were not immunogenic in this model.
- Published
- 2017
17. Size Fractionation of Microscopic Protein Aggregates Using a Preparative Fluorescence-Activated Cell Sorter
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Anja Matter, Kamal Egodage, Cyril Allard, Markus Bluemel, Verena Rombach-Riegraf, Friedrich Raulf, Rene Strehl, Atanas V. Koulov, Margit Jeschke, Bahman Ossuli, and Eline Angevaare
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Chromatography ,Light ,medicine.diagnostic_test ,Chemistry ,Pharmaceutical Science ,Fractionation ,Protein aggregation ,Cell sorting ,Flow Cytometry ,Fluorescence activated cell sorter ,Flow cytometry ,Immunoglobulin G ,Biological property ,medicine ,Biophysics ,Humans ,Scattering, Radiation ,Particle size ,Particle Size ,Degradative Pathway - Abstract
Protein aggregation, which takes place both in vivo and in vitro, is an important degradative pathway for all proteins. Protein aggregates have distinct physicochemical and biological properties that are important to study and characterize from the perspective of both fundamental and applied sciences. The size of protein aggregates varies across a huge range, spanning several orders of magnitude. Currently, protein aggregates larger than hundreds of nanometers in diameter are impossible to physically fractionate. Here, we present a new method to fractionate microscopic proteinaceous particles using preparative fluorescence-activated cell sorting technology. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2128–2135, 2013
- Published
- 2013
18. Structure-Activity Relationships in Cholapod Anion Carriers: Enhanced Transmembrane Chloride Transport through Substituent Tuning
- Author
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Anthony P. Davis, Germinal Magro, Jean-Baptiste Joos, Timothy N. Lambert, Bradley D. Smith, Atanas V. Koulov, Beth A. McNally, John P. Clare, Valentina Sgarlata, Adam L. Sisson, and Luke W. Judd
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Anions ,Models, Molecular ,Molecular Structure ,Stereochemistry ,Vesicle ,medicine.medical_treatment ,Organic Chemistry ,Transporter ,General Chemistry ,Chloride ,Article ,Catalysis ,Transmembrane protein ,Steroid ,Structure-Activity Relationship ,chemistry.chemical_compound ,Membrane ,Chlorides ,chemistry ,Lipophilicity ,medicine ,Steroids ,Lucigenin ,medicine.drug - Abstract
Chloride transport by a series of steroid-based “cholapod” receptors/carriers was studied in vesicles. The principal method involved preincorporation of the cholapods in the vesicle membranes, and the use of lucigenin fluorescence quenching to detect inward-transported Cl−. The results showed a partial correlation between anion affinity and transport activity, in that changes at the steroidal 7 and 12 positions affected both properties in concert. However, changes at the steroidal 3-position yielded irregular effects. Among the new steroids investigated the bis-p-nitrophenylthiourea 3 showed unprecedented activity, giving measurable transport through membranes with a transporter/lipid ratio of 1:250 000 (an average of
- Published
- 2008
19. Functional amyloid – from bacteria to humans
- Author
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Jeffery W. Kelly, Douglas M. Fowler, William E. Balch, and Atanas V. Koulov
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Melanins ,Amyloid ,Hemostasis ,Factor XII ,Bacteria ,biology ,Protein Conformation ,Fungi ,Microbial metabolism ,biology.organism_classification ,Models, Biological ,Biochemistry ,Melanin ,chemistry.chemical_compound ,Amyloid disease ,Protein structure ,Biosynthesis ,chemistry ,Animals ,Humans ,Protein quaternary structure ,Molecular Biology - Abstract
Amyloid--a fibrillar, cross beta-sheet quaternary structure--was first discovered in the context of human disease and tissue damage, and was thought to always be detrimental to the host. Recent studies have identified amyloid fibers in bacteria, fungi, insects, invertebrates and humans that are functional. For example, human Pmel17 has important roles in the biosynthesis of the pigment melanin, and the factor XII protein of the hemostatic system is activated by amyloid. Functional amyloidogenesis in these systems requires tight regulation to avoid toxicity. A greater understanding of the diverse physiological applications of this fold has the potential to provide a fresh perspective for the treatment of amyloid diseases.
- Published
- 2007
20. Comparative Evaluation of Two Methods for Preparative Fractionation of Proteinaceous Subvisible Particles--Differential Centrifugation and FACS
- Author
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Atanas Koulov, Roland Schmidt, Emilien Folzer, Hanns-Christian Mahler, Christof Finkler, Björn Boll, and Jörg Huwyler
- Subjects
Pharmacology ,Differential centrifugation ,Future studies ,Chromatography ,Chemistry ,Organic Chemistry ,Pharmaceutical Science ,Antibodies, Monoclonal ,Fractionation ,Flow Cytometry ,Mass measurement ,Comparative evaluation ,Protein Aggregates ,Immunoglobulin G ,Centrifugation, Density Gradient ,Molecular Medicine ,Pharmacology (medical) ,Critical assessment ,Particle size ,Particle Size ,Light obscuration ,Biotechnology - Abstract
The goal of this study was to compare and evaluate two preparative techniques for fractionation of proteinaceous subvisible particles. This work enables future studies to address the potential biological consequences of proteinaceous subvisible particles in protein therapeutic products. Particles were generated by heat stress and separated by size using differential centrifugation and FACS (Fluorescence-activated cell sorter). Resulting fractions were characterized by size-exclusion chromatography, light obscuration, flow imaging microscopy and resonant mass measurement. Here we report the optimization and comprehensive evaluation of two methods for preparative fractionation of subvisible proteinaceous particles into distinct size fractions in the range between 0.25 and 100 μm: differential centrifugation and FACS. Using these methods, well-defined size fractions were prepared and characterized in detail. Critical assessment and comparison of the two techniques demonstrated their complementarity and for the first time—their relative advantages and drawbacks. FACS and differential centrifugation are valuable tools to prepare well-defined size-fractions of subvisible proteinaceous particles. Both techniques possess unique and advantageous attributes and will likely find complementary application in future research on the biological consequences of proteinaceous subvisible particles.
- Published
- 2015
21. Synthesis and supramolecular properties of conformationally restricted and flexible phospholipids
- Author
-
Vares, Lauri, Koulov, Atanas V., and Smith, Bradley D.
- Subjects
Phospholipids -- Research ,Biological sciences ,Chemistry - Abstract
Short synthetic routes are described order to produce structurally related phospholipids that are either conformationally restricted or flexible. The supramolecular properties of structurally related phospholipids are investigated.
- Published
- 2003
22. Biophysical studies of a synthetic mimic of the apoptosis-detecting protein annexin v
- Author
-
Bradley D. Smith, Roger G. Hanshaw, Atanas V. Koulov, C. Lakshmi, and Kenneth A. Stucker
- Subjects
chemistry.chemical_compound ,Quenching (fluorescence) ,Biochemistry ,Dipicolylamine ,Chemistry ,Annexin ,Phosphatidylcholine ,Vesicle ,Fluorescence microscope ,Biophysics ,General Chemistry ,Phosphatidic acid ,Phosphatidylserine - Abstract
A Zn 2+ -dipicolylamine coordination compound is shown to sense the presence of anionic phospholipids in a membrane bilayer. The sensor contains two dipicolylamine subunits attached to an anthracene scaffold, which exhibits a maxi- mum absorbance at 380 nm, and undergoes an enhancement in fluorescence inten- sity when exposed to membranes enriched in phosphatidylserine. For these reasons, the compound is referred to as PSS-380 (Phosphatidylserine Sensor, 380 nm). The fluorescence emission of PSS-380 is enhanced up to tenfold by the presence of vesicles containing the anionic phospholipids phosphatidylserine, phosphati- dylglycerol, or phosphatidic acid. No enhancement in fluorescence is observed upon exposure to vesicles containing only zwitterionic phosphatidylcholine, or exposure to monodispersed (non-aggregated) anionic phospholipids. The sensing effect is cooperative; not only does association to the vesicles increase if the vesicles have raised levels of anionic phospholipid, but the maximum fluorescence at sensor saturation is also enhanced. It appears that sensing is triggered by the three-compo- nent self-assembly of sensor, Zn 2+ , and the anionic membrane surface, which leads to diminished photo-induced electron transfer (PET) quenching. The utility of PSS- 380 in flow cytometry and fluorescence microscopy is demonstrated by using the molecule to detect the appearance of phosphatidylserine on the plasma membrane surface of various cell lines. Thus, PSS-380 can identify apoptotic cells in the same way as the commonly used protein reagent annexin V.
- Published
- 2005
23. The immunogenicity of antibody aggregates in a novel transgenic mouse model
- Author
-
Sonja Schlicht, Björn Boll, Sabine Boeckle, Anna Kiialainen, Martin Ebeling, Thomas Singer, Hermann Beck, Thomas Buckel, Thomas Weiser, Antonius G. Rolink, Atanas Koulov, Antonio Iglesias, and Juliana Bessa
- Subjects
Genetically modified mouse ,medicine.drug_class ,Transgene ,Molecular Sequence Data ,Pharmaceutical Science ,Mice, Transgenic ,Protein aggregation ,Monoclonal antibody ,complex mixtures ,Flow cytometry ,Immune tolerance ,Protein Aggregates ,medicine ,Immune Tolerance ,Animals ,Humans ,Pharmacology (medical) ,Transgenes ,Pharmacology ,biology ,medicine.diagnostic_test ,Base Sequence ,Chemistry ,Immunogenicity ,Organic Chemistry ,Antibodies, Monoclonal ,Flow Cytometry ,Molecular biology ,Immunoglobulin G ,Antibody Formation ,biology.protein ,Molecular Medicine ,Immunoglobulin Light Chains ,Antibody ,Immunoglobulin Heavy Chains ,Stress, Psychological ,Biotechnology - Abstract
Protein aggregates have been discussed as a potential risk factor related to immunogenicity. Here we developed a novel human IgG transgenic (tg) mouse system expressing a mini-repertoire of human IgG1 antibodies (Abs) for the assessment of immunogenic properties of human mAb preparations.Transgenic mice were generated using germline versions of the human Ig heavy chain γ1 (IgH-γ1), and the human Ig light chain (IgL) κ and λ genes. Only the soluble form of human IgH-γ1 was used to avoid expression of the membrane Ig-H chain and concomitant allelic exclusion of endogenous murine Ig genes. IgG1 aggregates were generated by different stress conditions such as process-related, low pH and exposure to artificial light.The expression of human Ig proteins induced immunological tolerance to a broad range of human IgG1 molecules in the tg mice. Immunization with IgG1 aggregates demonstrated that soluble oligomers induced by significant light-exposure and carrying neo-epitopes induced a strong immune response in tg mice. In contrast, Ab aggregates alone and monomers with neo-epitopes were not immunogenic.This mouse model is able to recognize immunogenic modifications of human IgG1. While the degree of stress-induced aggregation varies for different mAbs, our findings using a particular mAb (mAb1) demonstrate that non-covalently modified aggregates do not break tolerance, contrary to widely held opinion. The immunogenic potential of soluble aggregates of human IgG strongly depends on the presence of neo-epitopes resulting from harsh stress conditions, i.e. extensive exposure to artificial light.
- Published
- 2014
24. Subvisible (2-100 μm) Particle Analysis During Biotherapeutic Drug Product Development: Part 1, Considerations and Strategy
- Author
-
Irene Cecchini, Paolo Mangiagalli, Robert Simler, Hanns-Christian Mahler, Douglas P. Nesta, Patrick Garidel, Dean C. Ripple, Atanas Koulov, Mara Rossi, Bernardo Perez-Ramirez, Satish K. Singh, Thomas Spitznagel, Alla Polozova, Andrew Weiskopf, Nataliya Afonina, Linda O. Narhi, Andrea Herre, Roland Schmidt, Tony Lubiniecki, Vincent Corvari, Michael Rosario Defelippis, and Klaus Wuchner
- Subjects
Microscopy ,Light ,Chemistry ,business.industry ,Protein Stability ,Drug Compounding ,Pharmaceutical Science ,Proteins ,Nanotechnology ,Protein Aggregates ,New product development ,Drug Discovery ,Drug product ,Animals ,Humans ,Scattering, Radiation ,Physical stability ,Biochemical engineering ,Particle size ,Particle analysis ,Particle Size ,business - Abstract
Measurement and characterization of subvisible particles (defined here as those ranging in size from 2 to 100 μm), including proteinaceous and nonproteinaceous particles, is an important part of every stage of protein therapeutic development. The tools used and the ways in which the information generated is applied depends on the particular product development stage, the amount of material, and the time available for the analysis. In order to compare results across laboratories and products, it is important to harmonize nomenclature, experimental protocols, data analysis, and interpretation. In this manuscript on perspectives on subvisible particles in protein therapeutic drug products, we focus on the tools available for detection, characterization, and quantification of these species and the strategy around their application.
- Published
- 2014
25. Detection of apoptotic cells using a synthetic fluorescent sensor for membrane surfaces that contain phosphatidylserine
- Author
-
Kenneth A. Stucker, J P Robinson, Bradley D. Smith, C. Lakshmi, and Atanas V. Koulov
- Subjects
Dose-Response Relationship, Drug ,Cell Membrane ,Apoptosis ,Phosphatidylserines ,Cell Biology ,Phosphatidylserine ,Fluorescence ,Jurkat cells ,Cell biology ,Jurkat Cells ,chemistry.chemical_compound ,Membrane ,Microscopy, Fluorescence ,chemistry ,Humans ,Annexin A5 ,Coloring Agents ,Molecular Biology - Abstract
Detection of apoptotic cells using a synthetic fluorescent sensor for membrane surfaces that contain phosphatidylserine
- Published
- 2003
26. Characterization of Aggregates and Particles Using Emerging Techniques
- Author
-
Manuel Diez, Markus Bluemel, Hui Zhao, Mariola Bozova, Kurt Forrer, and Atanas V. Koulov
- Subjects
Chemistry ,Nanotechnology ,Characterization (materials science) - Published
- 2012
27. Facilitated transport of sodium or potassium chloride across vesicle membranes using a ditopic salt-binding macrobicycle
- Author
-
Joseph M. Mahoney, Atanas V. Koulov, and Bradley D. Smith
- Subjects
Magnetic Resonance Spectroscopy ,Time Factors ,Potassium ,Sodium ,Inorganic chemistry ,chemistry.chemical_element ,Salt (chemistry) ,Sodium Chloride ,Biochemistry ,Chloride ,Potassium Chloride ,Chlorides ,medicine ,Physical and Theoretical Chemistry ,Ions ,chemistry.chemical_classification ,Facilitated diffusion ,Vesicle ,Organic Chemistry ,Biological Transport ,Membranes, Artificial ,Electrophysiology ,Membrane ,Models, Chemical ,chemistry ,Phosphatidylcholines ,Salts ,Efflux ,medicine.drug - Abstract
A synthetic receptor, with an ability to bind sodium or potassium chloride as a contact ion-pair, is shown to effectively transport either salt across vesicle membranes. Significant transport is observed even when the transporter ∶ phospholipid ratio is as low as 1 ∶ 2500. Chloride efflux from unilamellar vesicles is monitored using a chloride selective electrode. Mechanistic studies indicate that the facilitated efflux is due to the uncomplexed transporter diffusing into the vesicle and the transporter–salt complex diffusing out. Vesicle influx experiments are also reported, where the facilitated influx of chloride and sodium ions into vesicles is observed directly by 35Cl and 23Na NMR, respectively.
- Published
- 2002
28. Discrimination between silicone oil droplets and protein aggregates in biopharmaceuticals: a novel multiparametric image filter for sub-visible particles in microflow imaging analysis
- Author
-
Margit Jeschke, Verena Rombach-Riegraf, Markus Bluemel, Atanas V. Koulov, Kamal Egodage, Rene Strehl, and Manuel Diez
- Subjects
Pharmacology ,Microscopy ,Materials science ,Organic Chemistry ,Pharmaceutical Science ,Proteins ,Nanotechnology ,Filter (signal processing) ,Protein aggregation ,Composite image filter ,Silicone oil ,Imaging analysis ,chemistry.chemical_compound ,chemistry ,Microscopic image ,Image Processing, Computer-Assisted ,Molecular Medicine ,Protein particles ,Particle ,Silicone Oils ,Pharmacology (medical) ,Biological system ,Biotechnology - Abstract
Accurate monitoring of the sub-visible particle load in protein biopharmaceuticals is increasingly important to drug development. Manufacturers are expected to characterize and control sub-visible protein particles in their products due to their potential immunogenicity. Light obscuration, the most commonly used analytical tool to count microscopic particles, does not allow discrimination between potentially harmful protein aggregates and harmless pharmaceutical components, e.g. silicone oil, commonly present in drug products. Microscopic image analysis in flow-microscopy techniques allows not only counting, but also classification of sub-visible particles based on morphology. We present a novel approach to define software filters for analysis of particle morphology in flow-microscopic images enhancing the capabilities of flow-microscopy. Image morphology analysis was applied to analyze flow-microscopy data from experimental test sets of protein aggregates and silicone oil suspensions. A combination of four image morphology parameters was found to provide a reliable basis for automatic distinction between silicone oil droplets and protein aggregates in protein biopharmaceuticals resulting in low misclassification errors. A novel, custom-made software filter for discrimination between proteinaceous particles and silicone oil droplets in flow-microscopy imaging analysis was successfully developed.
- Published
- 2011
29. Auto-ionizing 3s to np resonances in neutral potassium and calcium
- Author
-
Natalia O. Vasetskaya, Michael A. Koulov, and Vadim K. Ivanov
- Subjects
Chemical species ,Chemistry ,Potassium ,Krypton ,chemistry.chemical_element ,Resonance ,Photoionization ,Electron ,Atomic physics ,Excitation ,Ion - Abstract
Results of many electron calculations of 3p-subshell photoionization cross section for neutral potassium and calcium are presented. Resonance structure associated with excitation of autoionizing 3s3p64s1,2n p states has been studied. The dynamic polarization and screening contribution to resonance parameters has been taken into account. A crucial role of double-electron excitations has been shown.© (2004) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.
- Published
- 2004
30. Synthesis and supramolecular properties of conformationally restricted and flexible phospholipids
- Author
-
and Atanas V. Koulov, Bradley D. Smith, and Lauri Vares
- Subjects
Cyclopropanes ,Magnetic Resonance Spectroscopy ,Calorimetry, Differential Scanning ,Stereochemistry ,Surface Properties ,Bilayer ,Vesicle ,Organic Chemistry ,Supramolecular chemistry ,Phospholipid ,Molecular Conformation ,Phosphonate ,Fluorescence ,chemistry.chemical_compound ,Membrane ,chemistry ,Liposomes ,Phosphatidylcholines ,Pressure ,Phosphorus-31 NMR spectroscopy ,Fluorescence anisotropy ,Phospholipids ,Fluorescent Dyes ,Ovum - Abstract
Short synthetic routes are described to produce structurally related phospholipids that are either conformationally restricted or flexible. The conformationally restricted structures have a cyclopropyl ring in the interfacial region of the phospholipid. The key synthetic step is a cyclopropanation reaction between 2(5H)-furanone and a stabilized chloroallylic phosphonate anion. The synthetic routes enable the incorporation of different polar headgroups as well as nonpolar tails at late stages in the sequence. The phosphoethanolamine derivatives 1b and 2b readily form encapsulating vesicles, however, dye leakage from vesicles composed of the restricted phospholipid 1b is significantly slower than from vesicles composed of flexible analogue 2b. Physicochemical analyses using 31P NMR, differential scanning calorimetry, fluorescence anisotropy, and Langmuir-Blodgett films suggest that the decreased permeability of membranes composed of conformationally restricted 1b is due to its ability to pack more closely in a bilayer assembly.
- Published
- 2003
31. Double-electron excitation effects in the 4s4p<formula><roman>6</roman></formula>np auto-ionizing resonances in Kr and its isoelectronic ions sequence
- Author
-
Vadim K. Ivanov and Michael A. Koulov
- Subjects
Chemical species ,Electron excitation ,Chemistry ,Krypton ,chemistry.chemical_element ,Resonance ,Electron ,Photoionization ,Atomic physics ,Excitation ,Ion - Abstract
New results of many electron calculations of 4p-subshell photoionization cross section for Kr, Rb+, Sr2+, and Y are presented. The resonance structure associated with excitation of autoionizing 4s4p6np states has been studied. The dynamic polarization and screening effects are taken into account for calculations of resonance parameters. Double-electron excitations is shown to play a crucial role in describing the resonance shapes and widths.© (2003) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.
- Published
- 2003
32. Highly impermeable vesicles composed of conformationally restricted phosphatidylethanolamine
- Author
-
Lauri Vares, Atanas V. Koulov, and Bradley D. Smith
- Subjects
Phosphatidylethanolamine ,Cyclopropanes ,Stereochemistry ,Chemistry ,Vesicle ,Phosphatidylethanolamines ,Lipid Bilayers ,Metals and Alloys ,Molecular Conformation ,Membranes, Artificial ,General Chemistry ,Catalysis ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,chemistry.chemical_compound ,Liposomes ,Materials Chemistry ,Ceramics and Composites ,Organic chemistry ,Anisotropy ,Derivative (chemistry) ,Fluorescent Dyes - Abstract
Vesicles composed of a phosphatidylethanolamine derivative with a cyclopropyl-containing interfacial region are twenty-seven times less permeable than vesicles composed of a closely related analogue.
- Published
- 2002
33. Cationic triple-chain amphiphiles facilitate vesicle fusion compared to double-chain or single-chain analogues
- Author
-
Mahim Jain, Lauri Vares, Bradley D. Smith, and Atanas V. Koulov
- Subjects
Vesicle fusion ,Lipid Bilayers ,Biophysics ,Molecular Conformation ,Phosphatidic Acids ,Membrane curvature ,Calorimetry ,Biochemistry ,Membrane Fusion ,Drug Delivery Systems ,Cations ,Amphiphile ,Organic chemistry ,Cationic liposome ,Lipid bilayer ,Fluorescent Dyes ,Calorimetry, Differential Scanning ,Chemistry ,Rhodamines ,Vesicle ,Phosphatidylethanolamines ,Cationic polymerization ,Gene Transfer Techniques ,Temperature ,Lipid bilayer fusion ,Cell Biology ,Hydrogen-Ion Concentration ,Quaternary Ammonium Compounds ,4-Chloro-7-nitrobenzofurazan ,Drug delivery ,Phosphatidylcholines ,Molecular shape - Abstract
Cationic, triple-chain amphiphiles promote vesicle fusion more than structurally related double-chain or single-chain analogues. Two types of vesicle fusion experiments were conducted, mixing of oppositely charged vesicles and acid-triggered self-fusion of vesicles composed of cationic amphiphile and anionic cholesteryl hemisuccinate (CHEMS). Vesicle fusion was monitored by standard fluorescence assays for intermembrane lipid mixing, aqueous contents mixing and leakage. Differential scanning calorimetry was used to show that triple-chain amphiphiles lower the lamellar–inverse hexagonal (Lα–HII) phase transition temperature for dipalmitoleoylphosphatidylethanolamine. The triple-chain amphiphiles may enhance vesicle fusion because they can stabilize the inversely curved membrane surfaces of the fusion intermediates, however, other factors such as extended conformation, packing defects, chain motion, or surface dehydration may also contribute. From the perspective of drug delivery, the results suggest that vesicles containing cationic, triple-chain amphiphiles (and cationic, cone-shaped amphiphiles in general) may be effective as fusogenic delivery capsules.
- Published
- 2002
34. Photoionization cross section of 4s24p6shells in the vicinities of the 4s4p6np autoionizing resonances in Kr isoelectronic sequence
- Author
-
Michael A. Koulov and Vadim K. Ivanov
- Subjects
Chemistry ,Ionization ,Krypton ,Physics::Atomic and Molecular Clusters ,Photoionization mode ,Resonance ,chemistry.chemical_element ,Physics::Atomic Physics ,Photoionization ,Electron ,Atomic physics ,Excitation ,Ion - Abstract
The results of many electron calculations of photoionization cross section in Kr isoelectronic sequence are presented. Theresonance structure associated with excitation of autoionizing 4s4p6np states has been studied with account for the dy-narnic many electron correlations. Double-electron excitations are shown to play a crucial role in describing of resonanceshapes.Keywords: autoionizing resonance, double-electron excitation, photoionization, positive ion.
- Published
- 2002
35. A Chaperone Trap Contributes to the Onset of Cystic Fibrosis
- Author
-
Atanas V. Koulov, Abbas Razvi, John R. Yates, Sandra Pankow, Judith A. Coppinger, William E. Balch, and Darren M. Hutt
- Subjects
Proteomics ,Protein Folding ,congenital, hereditary, and neonatal diseases and abnormalities ,Cystic Fibrosis ,Biophysics ,Cystic Fibrosis Transmembrane Conductance Regulator ,lcsh:Medicine ,Biochemistry ,Mass Spectrometry ,Ion Channels ,03 medical and health sciences ,0302 clinical medicine ,Autosomal Recessive ,Heat shock protein ,Genetics ,Humans ,HSP90 Heat-Shock Proteins ,Heat shock ,Protein Interactions ,lcsh:Science ,Biology ,030304 developmental biology ,Clinical Genetics ,0303 health sciences ,Spectrometric Identification of Proteins ,Multidisciplinary ,biology ,Chemistry ,Physics ,lcsh:R ,HSC70 Heat-Shock Proteins ,Proteins ,Human Genetics ,Hsp90 ,Transmembrane protein ,Cystic fibrosis transmembrane conductance regulator ,Chaperone Proteins ,Cell biology ,Proteostasis ,Chaperone (protein) ,biology.protein ,Medicine ,lcsh:Q ,Protein folding ,030217 neurology & neurosurgery ,Research Article - Abstract
Protein folding is the primary role of proteostasis network (PN) where chaperone interactions with client proteins determine the success or failure of the folding reaction in the cell. We now address how the Phe508 deletion in the NBD1 domain of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein responsible for cystic fibrosis (CF) impacts the binding of CFTR with cellular chaperones. We applied single ion reaction monitoring mass spectrometry (SRM-MS) to quantitatively characterize the stoichiometry of the heat shock proteins (Hsps) in CFTR folding intermediates in vivo and mapped the sites of interaction of the NBD1 domain of CFTR with Hsp90 in vitro. Unlike folding of WT-CFTR, we now demonstrate the presence of ΔF508-CFTR in a stalled folding intermediate in stoichiometric association with the core Hsps 40, 70 and 90, referred to as a ‘chaperone trap’. Culturing cells at 30 C resulted in correction of ΔF508-CFTR trafficking and function, restoring the sub-stoichiometric association of core Hsps observed for WT-CFTR. These results support the interpretation that ΔF508-CFTR is restricted to a chaperone-bound folding intermediate, a state that may contribute to its loss of trafficking and increased targeting for degradation. We propose that stalled folding intermediates could define a critical proteostasis pathway branch-point(s) responsible for the loss of function in misfolding diseases as observed in CF.
- Published
- 2012
36. A fluorescent assay for chloride transport; identification of a synthetic anionophore with improved activity
- Author
-
Jean-Baptiste Joos, Bradley D. Smith, Atanas V. Koulov, Anthony P. Davis, and Beth A. McNally
- Subjects
Ion Transport ,Luminescent Agents ,Chromatography ,Molecular Structure ,Chemistry ,Vesicle ,Metals and Alloys ,General Chemistry ,Fluorescence ,Chloride ,Catalysis ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,chemistry.chemical_compound ,Spectrometry, Fluorescence ,Chlorides ,Biochemistry ,Materials Chemistry ,Ceramics and Composites ,medicine ,Acridines ,Lucigenin ,Ion transporter ,medicine.drug - Abstract
A fluorescent assay based on the chloride-sensitive probe, lucigenin, is developed for monitoring chloride transport into vesicles, and used to compare the effectiveness of three steroid-derived transporters.
- Published
- 2005
37. ChemInform Abstract: SEKUNDAERE ALPHA-HYDROHEXAFLUORISOBUTTERSAEUREAMIDE
- Author
-
N. P. Aktaev, G. A. Sokol'skii, and A. N. Koulov
- Subjects
Chemistry ,Radiochemistry ,General Medicine - Published
- 1974
38. ChemInform Abstract: ZUR NATUR DER STICKSTOFF-PHOSPHORBINDUNG IN BICYCLISCHEN UND ALICYCLISCHEN IMINOPHOSPHATEN
- Author
-
V. G. Yurchenko, A. I. Sedlov, E. S. Koulov, and I. N. Zhmurova
- Subjects
Chemistry ,General Medicine ,Medicinal chemistry - Published
- 1975
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