16 results on '"Koulov A"'
Search Results
2. Discrimination Between Silicone Oil Droplets and Protein Aggregates in Biopharmaceuticals: A Novel Multiparametric Image Filter for Sub-visible Particles in Microflow Imaging Analysis
- Author
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Strehl, René, Rombach-Riegraf, Verena, Diez, Manuel, Egodage, Kamal, Bluemel, Markus, Jeschke, Margit, and Koulov, Atanas V.
- Published
- 2012
- Full Text
- View/download PDF
3. Impact of non-ideal analyte behavior on the separation of protein aggregates by asymmetric flow field-flow fractionation
- Author
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Lena Josse, Jörg Huwyler, Björn Boll, Atanas Koulov, Sophie Hochenauer, Anja Heubach, and Christof Finkler
- Subjects
Analyte ,Materials science ,Resolution (mass spectrometry) ,Osmolar Concentration ,010401 analytical chemistry ,Proteins ,Filtration and Separation ,Fractionation ,Hydrogen-Ion Concentration ,010501 environmental sciences ,Protein aggregation ,Separation principle ,01 natural sciences ,Fractionation, Field Flow ,0104 chemical sciences ,Analytical Chemistry ,Characterization (materials science) ,Physics::Fluid Dynamics ,Asymmetric flow field flow fractionation ,Protein Aggregates ,Ionic strength ,Biological system ,0105 earth and related environmental sciences - Abstract
Asymmetric flow field-flow fractionation is a valuable tool for the characterization of protein aggregates in biotechnology owing to its broad size range and unique separation principle. However, in practice asymmetric flow field-flow fractionation is non-trivial to use due to the major deviations from theory and the influence on separation by various factors that are not fully understood. Here, we report methods to assess the non-ideal effects that influence asymmetric flow field-flow fractionation separation and for the first time identify experimentally the main factors that impact it. Furthermore, we propose new approaches to minimize such non-ideal behavior, showing that by adjusting the mobile phase composition (pH and ionic strength) the resolution of asymmetric flow field-flow fractionation separation can be drastically improved. Additionally, we propose a best practice method for new proteins.
- Published
- 2018
4. Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs
- Author
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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
5. Particle Analysis of Biotherapeutics in Human Serum Using Machine Learning.
- Author
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Schuster, Joachim, Koulov, Atanas, Mahler, Hanns-Christian, Joerg, Susanne, Huwyler, Joerg, Schleicher, Kai, Detampel, Pascal, and Mathaes, Roman
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PARTICLE analysis , *MACHINE learning , *BIOCOMPLEXITY , *SERUM , *FLUORESCENCE microscopy , *IMMUNOGLOBULIN G - 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. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
6. Size Fractionation of Microscopic Protein Aggregates Using a Preparative Fluorescence-Activated Cell Sorter
- Author
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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
7. Variance Between Different Light Obscuration and Flow Imaging Microscopy Instruments and the Impact of Instrument Calibration.
- Author
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Matter, Anja, Koulov, Atanas, Singh, Satish, Mahler, Hanns-Christian, Reinisch, Helena, Langer, Carolin, Zucol, Benjamin, and Mathaes, Roman
- Subjects
- *
MICROSCOPY , *REFRACTIVE index , *CALIBRATION , *TEST systems , *OPTICAL properties , *PRODUCT attributes - Abstract
Subvisible particles (SVPs) are an obligatory critical quality attribute of the product, and yet, they are found in all biopharmaceutical products intended for infusion or injection. Light obscuration (LO) is the primary pharmacopeial method used to quantify SVPs. However, the method may not be equally sensitive toward all particles that can possibly occur. Calibration of LO instruments is usually performed using polystyrene beads suspended in water. In this study, the dependence of the sizing accuracy of LO analysis was evaluated by using a calibration suspension of lower refractive index beads made of silica suspended in sucrose solution. It was demonstrated that the sizing accuracy was strongly dependent on the reference material's properties used for calibration. It was also demonstrated that flow imaging microscopy suffered from the same artifact, albeit to a smaller extent. We further tested different LO sensors and instruments. Interestingly, our results show that the sizing accuracy varied from instrument to instrument, strongly depending on the properties of the sensor. To summarize, sizing and counting accuracies were dependent on the material used for calibration and its optical properties as well as the calibration curve, the sensor, and the instrument supplier. Closer match of optical properties between calibration system and test system seems to improve the sensitivity of the measurement. The results of this study raise the following major practical implications: (1) LO and flow imaging microscopy are not truly orthogonal analytical methods, (2) while matching optimal properties of material used for calibration and test items increased sensitivity, this is of poor practical applicability given that analytes contain multiple particles, and (3) setting product-specific limits for SVPs require special considerations with regard to the data sets used. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
8. The immunogenicity of antibody aggregates in a novel transgenic mouse model
- Author
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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
9. Impact of non‐ideal analyte behavior on the separation of protein aggregates by asymmetric flow field‐flow fractionation.
- Author
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Boll, Björn, Josse, Lena, Heubach, Anja, Hochenauer, Sophie, Finkler, Christof, Huwyler, Jörg, and Koulov, Atanas V.
- Subjects
PROTEIN fractionation ,ASYMMETRY (Chemistry) ,IONIC strength ,HYDROGEN-ion concentration ,IMMUNOGLOBULINS - Abstract
Abstract: Asymmetric flow field‐flow fractionation is a valuable tool for the characterization of protein aggregates in biotechnology owing to its broad size range and unique separation principle. However, in practice asymmetric flow field‐flow fractionation is non‐trivial to use due to the major deviations from theory and the influence on separation by various factors that are not fully understood. Here, we report methods to assess the non‐ideal effects that influence asymmetric flow field‐flow fractionation separation and for the first time identify experimentally the main factors that impact it. Furthermore, we propose new approaches to minimize such non‐ideal behavior, showing that by adjusting the mobile phase composition (pH and ionic strength) the resolution of asymmetric flow field‐flow fractionation separation can be drastically improved. Additionally, we propose a best practice method for new proteins. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
10. Discrimination between silicone oil droplets and protein aggregates in biopharmaceuticals: a novel multiparametric image filter for sub-visible particles in microflow imaging analysis
- Author
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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
11. Tracking the physical stability of fluorescent-labeled mAbs under physiologic in vitro conditions in human serum and PBS.
- Author
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Schuster, Joachim, Mahler, Hanns-Christian, Koulov, Atanas, Joerg, Susanne, Racher, Andy, Huwyler, Joerg, Detampel, Pascal, and Mathaes, Roman
- Subjects
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SERUM , *ISOELECTRIC point , *MONOCLONAL antibodies , *FLUORESCENCE microscopy , *FLOW cytometry - 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. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
12. Factors Governing the Accuracy of Subvisible Particle Counting Methods.
- Author
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Ríos Quiroz, Anacelia, Finkler, Christof, Huwyler, Joerg, Mahler, Hanns-Christian, Schmidt, Roland, and Koulov, Atanas V.
- Subjects
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BIOPHARMACEUTICS , *DRUG development , *NANOMEDICINE , *PARTICLE size distribution , *SOLUTION (Chemistry) - Abstract
A number of new techniques for subvisible particle characterization in biotechnological products have emerged in the last decade. Although the pharmaceutical community is actively using them, the current knowledge about the analytical performance of some of these tools is still inadequate to support their routine use in the development of biopharmaceuticals (especially in the case of submicron methods). With the aim of increasing this knowledge and our understanding of the most prominent techniques for subvisible particle characterization, this study reports the results of a systematic evaluation of their accuracy. Our results showed a marked overcounting effect especially for low concentrated samples and particles fragile in nature. Furthermore, we established the relative sample size distribution as the most important contributor to an instrument’s performance in accuracy counting. The smaller the representation of a particle size within a solution, the more difficulty the instruments had in providing an accurate count. These findings correlate with a recent study examining the principal factors influencing the precision of the subvisible particle measurements. A more thorough understanding of the capabilities of the different particle characterization methods provided here will help guide the application of these methods and the interpretation of results in subvisible particle characterization studies. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
13. Mouse Models for Assessing Protein Immunogenicity: Lessons and Challenges.
- Author
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Jiskoot, Wim, Kijanka, Grzegorz, Randolph, Theodore W., Carpenter, John F., Koulov, Atanas V., Mahler, Hanns-Christian, Joubert, Marisa K., Jawa, Vibha, and Narhi, Linda O.
- Subjects
- *
IMMUNOGENETICS , *LABORATORY mice , *THERAPEUTIC use of proteins , *IMMUNE response , *RESEARCH - Abstract
The success of clinical and commercial therapeutic proteins is rapidly increasing, but their potential immunogenicity is an ongoing concern. Most of the studies that have been conducted over the past few years to examine the importance of various product-related attributes (in particular several types of aggregates and particles) and treatment regimen (such as dose, dosing schedule, and route of administration) in the development of unwanted immune responses have utilized one of a variety of mouse models. In this review, we discuss the utility and drawbacks of different mouse models that have been used for this purpose. Moreover, we summarize the lessons these models have taught us and some of the challenges they present. Finally, we provide recommendations for future research utilizing mouse models to improve our understanding of critical factors that may contribute to protein immunogenicity. [ABSTRACT FROM AUTHOR]
- Published
- 2016
- Full Text
- View/download PDF
14. Determination of the Density of Protein Particles Using a Suspended Microchannel Resonator.
- Author
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FOLZER, EMILIEN, KHAN, TARIK A., SCHMIDT, ROLAND, FINKLER, CHRISTOF, HUWYLER, JÖRG, MAHLER, HANNS-CHRISTIAN, and KOULOV, ATANAS V.
- Subjects
- *
PROTEIN structure , *DENSITY , *MICROCHANNEL flow , *MONOCLONAL antibodies , *PRIONS , *NANOPARTICLES - Abstract
One of the analytical tools for characterization of subvisible particles, which gained popularity over the last years because of its unique capabilities, is the resonance mass measurement technique. However, a challenge that this technique presents is the need to know the exact density of the measured particles in order to obtain accurate size calculations. The density of proteinaceous subvisible particles has not been measured experimentally yet and to date researchers have been using estimated density values. In this paper, we report for a first-time experimental measurements of the density of protein particles (0.2-5 μm in size) using particles created by stressing three different proteins using four different types of stress conditions. Interestingly, the particle density values that were measured varied between 1.28 and 1.33 g/cm3 and were lower than previous estimates. Furthermore, it was found that although the density of proteinaceous particles was affected to a very low degree by the stress conditions used to generate them, there is relatively larger difference between particles originating from different classes of proteins (e.g., monoclonal antibody vs. bovine serum albumin). [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
15. Subvisible (2-100 μm) Particle Analysis During Biotherapeutic Drug Product Development: Part 1, Considerations and Strategy.
- Author
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Narhi, Linda O., Corvari, Vincent, Ripple, Dean C., Afonina, Nataliya, Cecchini, Irene, Defelippis, Michael R., Garidel, Patrick, Herre, Andrea, Koulov, Atanas V., Lubiniecki, Tony, Mahler, Hanns-Christian, Mangiagalli, Paolo, Nesta, Douglas, Perez-Ramirez, Bernardo, Polozova, Alla, Rossi, Mara, Schmidt, Roland, Simler, Robert, Singh, Satish, and Spitznagel, Thomas M.
- Subjects
- *
DRUG development , *PARTICLE size determination , *PRIONS , *INFORMATION theory , *DATA analysis - 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. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:1899-1908, 2015 [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
16. Size fractionation of microscopic protein aggregates using a preparative fluorescence-activated cell sorter.
- Author
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Rombach-Riegraf, Verena, Allard, Cyril, Angevaare, Eline, Matter, Anja, Ossuli, Bahman, Strehl, Rene, Raulf, Friedrich, Bluemel, Markus, Egodage, Kamal, Jeschke, Margit, and Koulov, Atanas V.
- Subjects
- *
PROTEIN fractionation , *MICROSCOPY , *IMMOBILIZED proteins , *FLUORESCENCE , *CELL separation , *PROTEOLYSIS , *NANOSTRUCTURED materials , *FLOW cytometry - 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 [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
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