المؤلفون: Shifeng Yu, Yongjia Wu, Shuyu Wang, Michael Siedler, Peter M. Ihnat, Dana I. Filoti, Ming Lu, Lei Zuo

المصدر: Biosensors, Vol 12, Iss 6, p 422 (2022)

مصطلحات موضوعية: thermal stability, antibody, differential scanning calorimeter, MEMS, Biotechnology, TP248.13-248.65

الوصف: Calorimeters, which can be used for rapid thermal characterization of biomolecules, are getting intense attention in drug development. This paper presents a novel MEMS-based differential scanning calorimeter (DSC) for direct thermal characterization of protein samples. The DSC consisted of a pair of temperature sensors made by vanadium oxide (VOx) film with a temperature coefficient of resistivity of −0.025/K at 300 K, a microfluidic device with high thermal insulation (2.8 K/mW), and a Peltier heater for linear temperature scanning. The DSC exhibited high sensitivity (6.1 µV/µW), low noise (0.4 µW), high scanning rate (45 K/min), and low sample consumption volume (0.63 µL). The MEMS DSC was verified by measuring the temperature-induced denaturation of lysozyme at different pH, and then used to study the thermal stability of a monoclonal antibody (mAb), an antigen-binding fragment (Fab), and a dual variable domain immunoglobulin (DVD-Ig) at pH = 6. The results showed that lysozyme is a stable protein in the pH range of 4.0–8.0. The protein stability study revealed that the transition temperatures of the intact Fab fragment, mAb, and DVD proteins were comparable with conformational stability results obtained using conventional commercial DSC. These studies demonstrated that the MEMS DSC is an effective tool for directly understanding the thermal stability of antibodies in a high-throughput and low-cost manner compared to conventional calorimeters.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/2079-6374/12/6/422Test; https://doaj.org/toc/2079-6374Test

الوصول الحر: https://doaj.org/article/653ca2d886614184b153e9f5a89956acTest

المؤلفون: Joshua R Laber, Thomas M Laue, Dana I Filoti

المصدر: Antibody Therapeutics. 5:211-215

مصطلحات موضوعية: Immunology, Immunology and Allergy

الوصف: The diffusion interaction parameter (kD) has been demonstrated to be a high-throughput technique for characterizing interactions between proteins in solution. kD reflects both attractive and repulsive interactions, including long-ranged electrostatic repulsions. Here, we plot the mutual diffusion coefficient (Dm) as a function of the experimentally determined Debye–Hückel–Henry surface charge (ZDHH) for seven human monoclonal antibodies (mAbs) in 15 mM histidine at pH 6. We find that graphs of Dm versus ZDHH intersect at ZDHH, ~ 2.6, independent of protein concentration. The same data plotted as kD versus ZDHH show a transition from net attractive to net repulsive interactions in the same region of the ZDHH intersection point. These data suggest that there is a minimum surface charge necessary on these mAbs needed to overcome attractive interactions.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::575125721035aefc5eedbc2e8c7309fbTest
https://doi.org/10.1093/abt/tbac018Test

المؤلفون: Daniel D. Vallejo, Chae Kyung Jeon, Kristine F. Parson, Hayley R. Herderschee, Joseph D. Eschweiler, Dana I. Filoti, Brandon T. Ruotolo

المصدر: Analytical Chemistry. 94:6745-6753

مصطلحات موضوعية: Ion Mobility Spectrometry, Antibodies, Monoclonal, Mass Spectrometry, Analytical Chemistry

الوصف: Stability is a key critical quality attribute monitored throughout the development of monoclonal antibody (mAb) therapeutics. Minor changes in their higher order structure (HOS) caused by stress or environment may alter mAb aggregation, immunogenicity, and efficacy. In addition, the structures of the resulting mAb aggregates are largely unknown, as are their dependencies on conditions under which they are created. In this report, we investigate the HOS of mAb monomers and dimers under a variety of forced degradation conditions with ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) technologies. We evaluate two model IgG1 antibodies that differ significantly only in their complementarity-determinant regions: IgG1α and IgG1β. Our data covering both heat- and pH-based forced degradation conditions, aquired on two different IM-MS platforms, show that these mAbs undergo global HOS changes at both monomer and dimer levels upon degradation, but shifts in collision cross section (CCS) differ under pH or heat degradation conditions. In addition, the level of CCS change detected is different between IgG1α and IgG1β, suggesting that differences in the CDR drive differential responses to degradation that influence the antibody HOS. Dramatically different CIU fingerprints are obtained for IgG1α and IgG1β monomers and dimers for both degradation conditions. Finally, we constructed a series of computational models of mAb dimers for comparison with experimental CCS values and found evidence for a compact, overlapped dimer structure under native and heat degradation conditions, possibly adopting an inverted or nonoverlapped quaternary structure when produced through pH degredation. We conclude by discussing the potential impact of our findings on ongoing biotherapeutic discovery and development efforts.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::d30baf8cbb571aa86626d2ebb16cd28dTest
https://doi.org/10.1021/acs.analchem.2c00160Test

المؤلفون: Shifeng Yu, Yongjia Wu, Shuyu Wang, Michael Siedler, Peter M. Ihnat, Dana I. Filoti, Ming Lu, Lei Zuo

المصدر: Biosensors; Volume 12; Issue 6; Pages: 422

مصطلحات موضوعية: Calorimetry, Differential Scanning, Clinical Biochemistry, Biomedical Engineering, Temperature, Antibodies, Monoclonal, General Medicine, Micro-Electrical-Mechanical Systems, Analytical Chemistry, Immunoglobulin Fab Fragments, thermal stability, antibody, differential scanning calorimeter, MEMS, Muramidase, Instrumentation, Engineering (miscellaneous), Biotechnology

الوصف: Calorimeters, which can be used for rapid thermal characterization of biomolecules, are getting intense attention in drug development. This paper presents a novel MEMS-based differential scanning calorimeter (DSC) for direct thermal characterization of protein samples. The DSC consisted of a pair of temperature sensors made by vanadium oxide (VOx) film with a temperature coefficient of resistivity of −0.025/K at 300 K, a microfluidic device with high thermal insulation (2.8 K/mW), and a Peltier heater for linear temperature scanning. The DSC exhibited high sensitivity (6.1 µV/µW), low noise (0.4 µW), high scanning rate (45 K/min), and low sample consumption volume (0.63 µL). The MEMS DSC was verified by measuring the temperature-induced denaturation of lysozyme at different pH, and then used to study the thermal stability of a monoclonal antibody (mAb), an antigen-binding fragment (Fab), and a dual variable domain immunoglobulin (DVD-Ig) at pH = 6. The results showed that lysozyme is a stable protein in the pH range of 4.0–8.0. The protein stability study revealed that the transition temperatures of the intact Fab fragment, mAb, and DVD proteins were comparable with conformational stability results obtained using conventional commercial DSC. These studies demonstrated that the MEMS DSC is an effective tool for directly understanding the thermal stability of antibodies in a high-throughput and low-cost manner compared to conventional calorimeters. Published version

وصف الملف: application/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::76d138bae9c7af3e1b7f4575da6671b7Test
https://hdl.handle.net/10919/110905Test

المؤلفون: Tariq Ghayur, W. Blaine Stine, Dana I. Filoti

المصدر: Development of Biopharmaceutical Drug-Device Products ISBN: 9783030314149

مصطلحات موضوعية: Computer science, Variable domain, Computational biology, DUAL (cognitive architecture), Design for manufacturability

الوصف: The successful delivery of any novel biotherapeutic to patients requires the translation of discovery concepts to clinical enablement. Based on our experience in the development of mAb therapeutics and in evaluating 1000s of DVD-Ig molecules, in this chapter we will briefly discuss (i) critical aspects considered in developing the DVD-Ig platform and (ii) the lessons learned in selecting therapeutic grade DVD-Ig molecules, specifically with regard to how various structural components impact (a) structural-functional properties, (b) biophysical and biochemical properties, (c) PK profiles, and (d) manufacturability (i.e., expression, formulation, etc.).

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::a2cc5f407a25a2c2285c77dab42eb296Test
https://doi.org/10.1007/978-3-030-31415-6_5Test

المؤلفون: Dana I. Filoti, Ming Lu, Peter M. Ihnat, Michael S. Siedler, Shuyu Wang, Lei Zuo, Shifeng Yu

المصدر: Sensors and Actuators B: Chemical. 256:946-952

مصطلحات موضوعية: Materials science, Polydimethylsiloxane, 010401 analytical chemistry, Thermistor, Metals and Alloys, Analytical chemistry, 02 engineering and technology, 021001 nanoscience & nanotechnology, Condensed Matter Physics, 01 natural sciences, Vanadium oxide, 0104 chemical sciences, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, Step response, chemistry.chemical_compound, Differential scanning calorimetry, chemistry, Materials Chemistry, Sensitivity (control systems), Electrical and Electronic Engineering, 0210 nano-technology, Adiabatic process, Instrumentation, Polyimide

الوصف: This paper presented a power compensated MEMS differential scanning calorimeter (DSC) for protein stability characterization. In this microfabricated sensor, PDMS (Polydimethylsiloxane) and polyimide were used to construct the adiabatic chamber (1 μL) and temperature sensitive vanadium oxide was used as the thermistor material. A power compensation system was implemented to maintain the sample and reference at the same temperature. The resolution study and step response characterization indicated the high sensitivity (6 V/W) and low noise level (60 μk) of the device. The test with IgG1 antibody (mAb1) samples showed clear phase transitions and the data was confirmed to be reasonable by comparing it with the results of commercial DSC’s test. This device used ∼1uL sample amount and could complete the scanning process in 4 min, significantly increasing the throughput of the bimolecular thermodynamics study like drug formulation process.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::77d7bd290050c6f0faa5b68b0ad9f1e6Test
https://doi.org/10.1016/j.snb.2017.10.034Test

المؤلفون: Thomas M. Laue, Peter B. Crowley, Ciara Kyne, Dana I. Filoti, Kiara Jordon

المصدر: Protein Science. 26:258-267

مصطلحات موضوعية: 0301 basic medicine, Gel electrophoresis, Chromatography, 030102 biochemistry & molecular biology, biology, Chemistry, Cytochrome c, Size-exclusion chromatography, Protein primary structure, medicine.disease_cause, Biochemistry, Sulfate binding, 03 medical and health sciences, Electrophoresis, 030104 developmental biology, medicine, biology.protein, Biophysics, Solubility, Molecular Biology, Escherichia coli

الوصف: Decades of dilute-solution studies have revealed the influence of charged residues on protein stability, solubility and stickiness. Similar characterizations are now required in physiological solutions to understand the effect of charge on protein behavior under native conditions. Toward this end, we used free boundary and native gel electrophoresis to explore the charge of cytochrome c in buffer and in Escherichia coli extracts. We find that the charge of cytochrome c was ∼2-fold lower than predicted from primary structure analysis. Cytochrome c charge was tuned by sulfate binding and was rendered anionic in E. coli extracts due to interactions with macroanions. Mutants in which three or four cationic residues were replaced with glutamate were charge-neutral and “inert” in extracts. A comparison of the interaction propensities of cytochrome c and the mutants emphasizes the role of negative charge in stabilizing physiological environments. Charge–charge repulsion and preferential hydration appear to prevent aggregation. The implications for molecular organization in vivo are discussed.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::677105403fbb18d8d29b2ef35b18e9d8Test
https://doi.org/10.1002/pro.3077Test

المؤلفون: Harmen B. Steele, Dana I. Filoti, J.B. Alexander Ross, Stephen G. Sligar, Thomas M. Laue, Cheng Her, Mark A. McLean

المصدر: Biophysical Journal. 111:989-998

مصطلحات موضوعية: Electrophoresis, 0301 basic medicine, Lipid Bilayers, Biophysics, Phospholipid, Analytical chemistry, Phase Transition, 03 medical and health sciences, chemistry.chemical_compound, Phosphatidylcholine, Escherichia coli, Magnesium, Lipid bilayer, POPC, Phospholipids, Ions, Phosphatidylethanolamine, Membranes, 030102 biochemistry & molecular biology, Temperature, Phosphatidic acid, Hydrogen-Ion Concentration, Polyelectrolyte, Nanostructures, Crystallography, 030104 developmental biology, Membrane, chemistry, Calcium, lipids (amino acids, peptides, and proteins)

الوصف: Phospholipids (PLs) are a major, diverse constituent of cell membranes. PL diversity arises from the nature of the fatty acid chains, as well as the headgroup structure. The headgroup charge is thought to contribute to both the strength and specificity of protein-membrane interactions. Because it has been difficult to measure membrane charge, ascertaining the role charge plays in these interactions has been challenging. Presented here are charge measurements on lipid Nanodiscs at 20°C in 100 mM NaCl, 50 mM Tris, at pH 7.4. Values are also reported for measurements made in the presence of Ca(2+) and Mg(2+) as a function of NaCl concentration, pH, and temperature, and in solvents containing other types of cations and anions. Measurements were made for neutral (phosphatidylcholine and phosphatidylethanolamine) and anionic (phosphatidylserine, phosphatidic acid, cardiolipin, and phosphatidylinositol 4,5-bisphosphate (PIP2)) PLs containing palmitoyl-oleoyl and dimyristoyl fatty acid chains. In addition, charge measurements were made on Nanodiscs containing an Escherichia coli lipid extract. The data collected reveal that 1) POPE is anionic and not neutral at pH 7.4; 2) high-anionic-content Nanodiscs exhibit polyelectrolyte behavior; 3) 3 mM Ca(2+) neutralizes a constant fraction of the charge, but not a constant amount of charge, for POPS and POPC Nanodiscs; 4) in contrast to some previous work, POPC only interacts weakly with Ca(2+); 5) divalent cations interact with lipids in a lipid- and ion-specific manner for POPA and PIP2 lipids; and 6) the monovalent anion type has little influence on the lipid charge. These results should help eliminate inconsistencies among data obtained using different techniques, membrane systems, and experimental conditions, and they provide foundational data for developing an accurate view of membranes and membrane-protein interactions.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::847e783401a08f176e3a3b07b4f4eed8Test
https://doi.org/10.1016/j.bpj.2016.06.041Test

المؤلفون: Anhdao T Darcy, Leslie Alessandri, Lucia Eaton, Sahana Bose, David Banach, Silvino Sousa, Gregory M. Preston, Jeremy Freeman, David Winarta, Dana I. Filoti, Reema Raghavendra, Nathan Brown, David Ouellette, Ivan Correia, Alexander V. Ivanov, Ramkrishna Sadhukhan

المصدر: Cogent Biology, Vol 4, Iss 1 (2018)

مصطلحات موضوعية: musculoskeletal diseases, mab, Necrosis, medicine.drug_class, polymer, tnfα, elastin, Inflammation, Monoclonal antibody, tem, medicine, fusion protein, elastomeric, lcsh:QH301-705.5, biology, Chemistry, General Engineering, Fusion protein, lcsh:Biology (General), anti-tnfα, biology.protein, Cancer research, Tumor necrosis factor alpha, Antibody, medicine.symptom, Elastin

الوصف: Injecting anti-tumor necrosis factor (TNF)α antibodies into patient joints at the site of inflammation, inter-articular (IA) delivery, has demonstrated modest success for treatment of Spondyloarthritis (SpA), Rheumatoid Arthritis (RA), and osteoarthritis. However, IA delivery is not the treatment route of choice due to rapid clearance from the site of administration. Elastin-like polypeptides (ELPs) are reported to undergo phase transition; forming reversible aggregates above their transition temperature, which when injected into IA space have a 25-fold longer half-life compared to the soluble form. Here, we fused an ELP repeat to the C-terminus of each heavy chain of an anti-TNFα monoclonal antibody (mAb) and provide detailed characterization of the fusion IgG molecule. Expression and purification yielded homogenous protein confirmed by gels, hydrophobic-interaction chromatography, Capilary Electrophoresis (CE), Mass Spectrometry (MS), and analytical ultracentrifugation. The ELPs altered hydrophobicity and pI of the parent mAb and new elastic properties were imparted to the molecule; forming large stable complexes with a hydrodynamic radius of 40 nm above 39°C that dissociated into soluble, active monomer below 37°C. The fusion mAb retained its affinity and ability to neutralize TNFα as determined by surface plasmon resonance and cell-based assay, respectively, with equal potency to unmodified anti-TNFα mAb. Differential-scanning calorimetry studies show stabilization of adjacent CH2 and CH3 domains in the fusion molecule and the aggregated molecule was found to be fully functional after 7 days at 37°C. For the first time, we reveal architecture of an ELP-fusion mAb and binding to antigen using single-particle-transmission-electron microscopy. Unstructured ELP was visualized at the C-terminus and binding to antigen was shown at the N-terminus. Collectively, these studies indicate that it is possible to impart elastic properties to a monoclonal antibody while retaining purity, stability, and ability to effectively bind and neutralize antigen.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b5d542796cbe7fb2a928260250736277Test
https://doaj.org/article/045686369d4c466580ae1d9a6b5be47dTest

المؤلفون: Sandeep Yadav, Dana I. Filoti, Thomas M. Laue, Steven J. Shire

المصدر: Journal of Pharmaceutical Sciences. 104:2123-2131

مصطلحات موضوعية: Electrophoresis, Free-flow electrophoresis, Chromatography, biology, Chemistry, Osmolar Concentration, Temperature, Analytical chemistry, Proteins, Pharmaceutical Science, Charge (physics), Hydrogen-Ion Concentration, Human serum albumin, Effective nuclear charge, Capillary electrophoresis, Models, Chemical, biology.protein, medicine, Animals, Humans, Electrophoretic light scattering, Bovine serum albumin, Serum Albumin, medicine.drug

الوصف: As interest in high-concentration protein formulations has increased, it has become apparent that routine, accurate protein charge measurements are necessary. There are several techniques for charge measurement, and a comparison of the methods is needed. The electrophoretic mobility, effective charge, and Debye-Hückel-Henry charge have been determined for bovine serum albumin, and human serum albumin. Three different electrophoretic methods were used to measure the electrophoretic mobility: capillary electrophoresis, electrophoretic light scattering, and membrane confined electrophoresis. In addition, the effective charge was measured directly using steady-state electrophoresis. Measurements made at different NaCl concentrations, pH, and temperatures allow comparison with previous charge estimates based on electrophoresis, Donnan equilibrium, and pH titration. Similar charge estimates are obtained by all of the methods. The strengths and limitations of each technique are discussed, as are some general considerations about protein charge and charge determination.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::91ccd31fa97e96a82e40fe68f07e7a43Test
https://doi.org/10.1002/jps.24454Test