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1.
Chonnam Med J ; 60(1): 1-12, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38304124

RESUMO

Extracellular vesicles (EVs) function as potent mediators of intercellular communication for many in vivo processes, contributing to both health and disease related conditions. Given their biological origins and diverse functionality from correspondingly unique "cargo" compositions, both endogenous and modified EVs are garnering attention as promising therapeutic modalities and vehicles for targeted therapeutic delivery applications. Their diversity in composition, however, has revealed a significant need for more comprehensive analytical-based characterization methods, and manufacturing processes that are consistent and scalable. In this review, we explore the dynamic landscape of EV research and development efforts, ranging from novel isolation approaches, to their analytical assessment through novel characterization techniques, and to their production by industrial-scale manufacturing process considerations. Expanding the horizon of these topics to EVs for in-human applications, we underscore the need for stringent development and adherence to Good Manufacturing Practice (GMP) guidelines. Wherein, the intricate interplay of raw materials, production in bioreactors, and isolation practices, along with analytical assessments compliant with the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines, in conjunction with reference standard materials, collectively pave the way for standardized and consistent GMP production processes.

2.
Adv Sci (Weinh) ; 9(15): e2103222, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35332686

RESUMO

Exosomes are extracellular vesicles that share components of their parent cells and are attractive in biotechnology and biomedical research as potential disease biomarkers as well as therapeutic agents. Crucial to realizing this potential is the ability to manufacture high-quality exosomes; however, unlike biologics such as proteins, exosomes lack standardized Good Manufacturing Practices for their processing and characterization. Furthermore, there is a lack of well-characterized reference exosome materials to aid in selection of methods for exosome isolation, purification, and analysis. This review informs exosome research and technology development by comparing exosome processing and characterization methods and recommending exosome workflows. This review also provides a detailed introduction to exosomes, including their physical and chemical properties, roles in normal biological processes and in disease progression, and summarizes some of the on-going clinical trials.


Assuntos
Exossomos , Vesículas Extracelulares , Exossomos/química , Exossomos/metabolismo , Desenvolvimento Industrial , Proteínas/metabolismo
3.
AAPS J ; 21(3): 44, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30915582

RESUMO

Biologic products encounter various types of interfacial stress during development, manufacturing, and clinical administration. When proteins come in contact with vapor-liquid, solid-liquid, and liquid-liquid surfaces, these interfaces can significantly impact the protein drug product quality attributes, including formation of visible particles, subvisible particles, or soluble aggregates, or changes in target protein concentration due to adsorption of the molecule to various interfaces. Protein aggregation at interfaces is often accompanied by changes in conformation, as proteins modify their higher order structure in response to interfacial stresses such as hydrophobicity, charge, and mechanical stress. Formation of aggregates may elicit immunogenicity concerns; therefore, it is important to minimize opportunities for aggregation by performing a systematic evaluation of interfacial stress throughout the product development cycle and to develop appropriate mitigation strategies. The purpose of this white paper is to provide an understanding of protein interfacial stability, explore methods to understand interfacial behavior of proteins, then describe current industry approaches to address interfacial stability concerns. Specifically, we will discuss interfacial stresses to which proteins are exposed from drug substance manufacture through clinical administration, as well as the analytical techniques used to evaluate the resulting impact on the stability of the protein. A high-level mechanistic understanding of the relationship between interfacial stress and aggregation will be introduced, as well as some novel techniques for measuring and better understanding the interfacial behavior of proteins. Finally, some best practices in the evaluation and minimization of interfacial stress will be recommended.


Assuntos
Produtos Biológicos/química , Desenvolvimento de Medicamentos , Produtos Biológicos/administração & dosagem , Química Farmacêutica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Transição de Fase , Agregados Proteicos , Estabilidade Proteica , Propriedades de Superfície , Água/química
4.
Anal Chem ; 88(21): 10404-10410, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27686335

RESUMO

Magnetic microbeads exhibit rapid separation characteristics and are widely employed for biomolecule and cell isolations in research laboratories, clinical diagnostics assays, and cell therapy manufacturing. However, micrometer particle diameters compromise biomarker recognition, which leads to long incubation times and significant reagent demands. Here, a stimuli-responsive binary reagent system is presented that combines the nanoscale benefits of efficient biomarker recognition and the microscale benefits of rapid magnetic separation. This system comprises magnetic nanoparticles and polymer-antibody (Ab) conjugates that transition from hydrophilic nanoscale reagents to microscale aggregates in response to temperature stimuli. The binary reagent system was benchmarked against Ab-labeled Dynabeads in terms of biomarker isolation kinetics, assay speed, and reagent needs. Surface plasmon resonance (SPR) measurements showed that polymer conjugation did not significantly alter the Ab's binding affinity or kinetics. ELISA analysis showed that the unconjugated Ab, polymer-Ab conjugates, and Ab-labeled Dynabeads exhibited similar equilibrium dissociation constants (Kd), ∼2 nM. However, the binary reagent system isolated HIV p24 antigen from spiked serum specimens (150 pg/mL) much more quickly than Dynabeads, which resulted in shorter binding times by tens of minutes, or about 30-50% shorter overall assay times. The binary reagent system showed improved performance because the Ab molecules were not conjugated to large, solid microparticle surfaces. This stimuli-responsive binary reagent system illustrates the potential advantages of nanoscale reagents in molecule and cell isolations for both research and clinical applications.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Proteína do Núcleo p24 do HIV/sangue , Imunoconjugados/química , Nanopartículas de Magnetita/química , Polímeros/química , Ressonância de Plasmônio de Superfície/métodos , Biomarcadores/sangue , HIV/isolamento & purificação , Proteína do Núcleo p24 do HIV/análise , Infecções por HIV/sangue , Humanos , Interações Hidrofóbicas e Hidrofílicas , Indicadores e Reagentes , Multimerização Proteica , Temperatura
5.
Nanomedicine ; 12(7): 2031-2041, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27184097

RESUMO

Carbohydrate receptors on alveolar macrophages are attractive targets for receptor-mediated delivery of nanostructured therapeutics. In this study, we employed reversible addition fragmentation chain transfer polymerization to synthesize neoglycopolymers, consisting of mannose- and galactose methacrylate-based monomers copolymerized with cholesterol methacrylate for use in functional liposome studies. Glycopolymer-functional liposomes were employed to elucidate macrophage mannose receptor (CD206) and macrophage galactose-type lectin (CD301) targeting in both primary macrophage and immortal macrophage cell lines. Expression of CD206 and CD301 was observed to vary significantly between cell lines (murine alveolar macrophage, murine bone marrow-derived macrophage, RAW264.7, and MH-S), which has significant implications in in vitro targeting and uptake studies. Synthetic glycopolymers and glycopolymer augmented liposomes demonstrated specific receptor-mediated uptake in a manner dependent on carbohydrate receptor expression. These results establish a platform capable of probing endogenous carbohydrate receptor-mediated targeting via glycofunctional nanomaterials.


Assuntos
Metabolismo dos Carboidratos , Lipossomos , Macrófagos Alveolares , Animais , Linhagem Celular , Portadores de Fármacos , Humanos , Lectinas , Macrófagos , Manose , Camundongos
6.
Methods Enzymol ; 562: 501-27, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26412665

RESUMO

This chapter illustrates how analytical ultracentrifugation methods, coupled with the fluorescence detection system, are an excellent approach to characterizing and comparing protein-binding interactions in dilute solution and concentrated, crowded solutions like serum. We show that in serum, the binding and assembly states for a pair of endogenous protein ligands and an antibody inhibitor are dramatically different than those observed in dilute, simple buffers. This type of analysis approach may be helpful in research efforts intent at discerning the underpinnings to a therapeutic's activity and pharmacokinetic properties in vivo.


Assuntos
Ligante RANK/isolamento & purificação , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Soluções Tampão , Humanos , Osteoprotegerina/química , Osteoprotegerina/isolamento & purificação , Ligação Proteica , Multimerização Proteica , Ligante RANK/química , Soro/química , Albumina Sérica/química , Albumina Sérica/isolamento & purificação , Soluções , Ultracentrifugação/métodos
7.
Biochim Biophys Acta ; 1848(10 Pt A): 1974-80, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26074010

RESUMO

Integral membrane proteins (IMPs) are of therapeutic interest and are targeted by a majority of approved drugs. It's difficult to express, purify, and maintain the functional conformation of IMPs. Nanodisc presents a reliable method to solubilize and stabilize IMPs in detergent-free condition. In this study, we demonstrate the assembly and purification of a chimeric ion channel, KcsA-Kv1.3 Nanodisc. We further detail biophysical analysis of the assembled Nanodisc using analytical ultracentrifugation (AUC), surface plasmon resonance (SPR), and back scattering interferometry (BSI). AUC is employed to determine the molecular composition of the empty and KcsA-Kv1.3 Nanodisc. Combination of SPR and BSI overcomes each other's limitation and provides insight of equilibrium binding properties of peptide and small molecule ligands to KcsA-Kv1.3.


Assuntos
Proteínas de Bactérias/química , Canal de Potássio Kv1.3/química , Nanopartículas/química , Nanopartículas/ultraestrutura , Bloqueadores dos Canais de Potássio/química , Canais de Potássio/química , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Sítios de Ligação , Canal de Potássio Kv1.3/antagonistas & inibidores , Dados de Sequência Molecular , Complexos Multiproteicos/síntese química , Complexos Multiproteicos/ultraestrutura , Ligação Proteica
8.
J Pharm Sci ; 103(9): 2605-2614, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24867196

RESUMO

Processing protein solutions into the solid state is a common approach for generating stable amorphous protein mixtures that are suitable for long-term storage. Great care is typically given to protecting the protein native structure during the various drying steps that render it into the amorphous solid state. However, many studies illustrate that chemical and physical degradations still occur in spite of this amorphous material having good glassy properties and it being stored at temperatures below its glass transition temperature (Tg). Because of these persistent issues and recent biophysical studies that have refined the debate ascribing meaning to the molecular dynamical transition temperature and Tg of protein molecules, we provide an updated discussion on the impact of assessing and managing localized, individual protein molecule nondiffusive motions in the context of proteins being prepared into bulk amorphous mixtures. Our aim is to bridge the pharmaceutical studies addressing bulk amorphous preparations and their glassy behavior, with the biophysical studies historically focused on the nondiffusive internal protein dynamics and a protein's activity, along with their combined efforts in assessing the impact of solvent hydrogen-bonding networks on local stability. We also provide recommendations for future research efforts in solid-state formulation approaches.


Assuntos
Química Farmacêutica/métodos , Simulação de Dinâmica Molecular , Proteínas/química , Estabilidade de Medicamentos , Armazenamento de Medicamentos/métodos , Liofilização/métodos , Soluções/química , Temperatura de Transição
9.
Comb Chem High Throughput Screen ; 13(2): 170-87, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20053162

RESUMO

This review describes methods for quantifying the binding of small molecule drug candidates to plasma proteins and the application of these methods in drug discovery and development. Particular attention is devoted to methods amenable to medium-to-high throughput analysis and those well suited for measurement of compounds that are highly protein bound. The methods reviewed herein include the conventional techniques of equilibrium dialysis, ultrafiltration and ultracentrifugation, as well as some more novel approaches utilizing micropartitioning and biosensor-based analysis. Additional concepts that are discussed include plasma protein structure, enantioselective protein binding, drug displacement, the effect of patient demographics and disease states on free (unbound) drug levels, and the influence of protein binding on drug candidate pharmacokinetics and pharmacodynamics. Practical considerations pertaining to the evaluation of highly protein bound drug candidates are also highlighted.


Assuntos
Proteínas Sanguíneas/metabolismo , Descoberta de Drogas/métodos , Preparações Farmacêuticas/metabolismo , Técnicas Biossensoriais/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ligação Proteica
10.
Biochemistry ; 45(20): 6341-53, 2006 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-16700545

RESUMO

The heme-containing cytochrome P450s (CYPs) are a major enzymatic determinant of drug clearance and drug-drug interactions. The CYP3A4 isoform is inhibited by antifungal imidazoles or triazoles, which form low-spin heme iron complexes via formation of a nitrogen-ferric iron coordinate bond. However, CYP3A4 also slowly oxidizes the antifungal itraconazole (ITZ) at a site that is approximately 25 A from the triazole nitrogens, suggesting that large antifungal azoles can adopt multiple orientations within the CYP3A4 active site. Here, we report a surface plasmon resonance (SPR) analysis with kinetic resolution of two binding modes of ITZ, and the related drug ketoconazole (KTZ). SPR reveals a very slow off-rate for one binding orientation. Multiphasic binding kinetics are observed, and one of the two binding components resolved by curve fitting exhibits "equilibrium overshoot". Preloading of CYP3A4 with the heme ligand imidazole abolishes this component of the antifungal azole binding trajectories, and it eliminates the conspicuously slow off-rate. The fractional populations of CYP3A4 complexes corresponding to different drug orientations can be manipulated by altering the duration of the pulse of drug exposure. UV-vis difference absorbance titrations yield low-spin spectra and K(D) values that are consistent with the high-affinity complex resolved by SPR. These results demonstrate that ITZ and KTZ bind in multiple orientations, including a catalytically productive mode and a slowly dissociating inhibitory mode. Most importantly, they provide the first example of a SPR-based method for the kinetic characterization of binding of a drug to any human CYP, including mechanistic insight not available from other methods.


Assuntos
Antifúngicos/química , Citocromo P-450 CYP3A/química , Itraconazol/química , Cetoconazol/química , Ressonância de Plasmônio de Superfície/métodos , Antifúngicos/metabolismo , Sítios de Ligação , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Imidazóis/metabolismo , Itraconazol/metabolismo , Cetoconazol/metabolismo , Cinética , Luz , Conformação Proteica/efeitos dos fármacos , Espectrofotometria Ultravioleta , Estereoisomerismo
11.
J Pharm Sci ; 94(8): 1636-67, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15965985

RESUMO

The internal, dynamical fluctuations of protein molecules exhibit many of the features typical of polymeric and bulk small molecule glass forming systems. The response of a protein's internal molecular mobility to temperature changes is similar to that of other amorphous systems, in that different types of motions freeze out at different temperatures, suggesting they exhibit the alpha-beta-modes of motion typical of polymeric glass formers. These modes of motion are attributed to the dynamic regimes that afford proteins the flexibility for function but that also develop into the large-scale collective motions that lead to unfolding. The protein dynamical transition, T(d), which has the same meaning as the T(g) value of other amorphous systems, is attributed to the temperature where protein activity is lost and the unfolding process is inhibited. This review describes how modulation of T(d) by hydration and lyoprotectants can determine the stability of protein molecules that have been processed as bulk, amorphous materials. It also examines the thermodynamic, dynamic, and molecular factors involved in stabilizing folded proteins, and the effects typical pharmaceutical processes can have on native protein structure in going from the solution state to the solid state.


Assuntos
Estabilidade de Medicamentos , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Água/química , Biofarmácia , Biopolímeros/química , Crioprotetores/química , Cristalização , Dessecação , Armazenamento de Medicamentos , Liofilização , Soluções , Temperatura , Termodinâmica , Temperatura de Transição
12.
Biochemistry ; 42(35): 10418-28, 2003 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-12950168

RESUMO

Exploiting the principle of bivalent binding, we have designed symmetrical, bifunctional inhibitors to simultaneously occupy both active sites of cytosolic glutathione S-transferase, with enhanced specificity for the P1-1 isoform. We have prepared two series of compounds that differ in their binding domains-the first is a series of bis-glutathione conjugates, and the second is a series of compounds each possessing two equivalents of Uniblue A, an analogue of Cibacron Blue. For each series, a monofunctional reference compound was also prepared to determine the relative advantage of the bivalent inhibitors. Within each series, the most potent inhibitors exhibited IC(50) values 2 orders of magnitude lower than the relevant reference compounds. Moreover, within the bis-glutathionyl series, a 10-fold increase in selectivity was achieved for GST P1-1 over the A1-1 isoform. Isothermal titration calorimetry with a representative bis-glutathione conjugate and a monofunctional reference compound indicates that the bivalent inhibitor exhibits the expected increase in intrinsic affinity and decrease in stoichiometry relative to the monofunctional compound, supporting the overall design strategy.


Assuntos
Inibidores Enzimáticos/metabolismo , Glutationa Transferase/antagonistas & inibidores , Glutationa/química , Isoenzimas/antagonistas & inibidores , Animais , Sítios de Ligação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/classificação , Glutationa/metabolismo , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Triazinas/química , Triazinas/metabolismo
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