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1.
Pharm Res ; 40(10): 2355-2370, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37131104

RESUMO

BACKGROUND: Spray-drying is considered a promising alternative drying method to lyophilization (freeze-drying) for therapeutic proteins. Particle counts in reconstituted solutions of dried solid dosage forms of biologic drug products are closely monitored to ensure product quality. We found that high levels of particles formed after reconstitution of protein powders that had been spray-dried under suboptimal conditions. METHODS: Visible and subvisible particles were evaluated. Soluble proteins in solution before spray-drying and in the reconstituted solution of spray-dried powder were analyzed for their monomer content levels and melting temperatures. Insoluble particles were collected and analyzed by Fourier transform infrared microscopy (FTIR), and further analyzed with hydrogen-deuterium exchange (HDX). RESULTS: Particles observed after reconstitution were shown not to be undissolved excipients. FTIR confirmed their identity as proteinaceous in nature. These particles were therefore considered to be insoluble protein aggregates, and HDX was applied to investigate the mechanism underlying aggregate formation. Heavy-chain complementarity-determining region 1 (CDR-1) in the aggregates showed significant protection by HDX, suggesting CDR-1 was critical for aggregate formation. In contrast, various regions became more conformationally dynamic globally, suggesting the aggregates have lost protein structural integrity and partially unfolded after spray-drying. DISCUSSION: The spray-drying process could have disrupted the higher-order structure of proteins and exposed the hydrophobic residues in CDR-1 of the heavy chain, contributing to the formation of aggregate through hydrophobic interactions upon reconstitution of spray-dried powder. These results can contribute to efforts to design spray-dry resilient protein constructs and improve the robustness of the spray-drying process.


Assuntos
Microscopia , Proteínas , Pós/química , Liofilização , Tamanho da Partícula
2.
MAbs ; 14(1): 2095701, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35799328

RESUMO

Although monoclonal antibodies have greatly improved cancer therapy, they can trigger side effects due to on-target, off-tumor toxicity. Over the past decade, strategies have emerged to successfully mask the antigen-binding site of antibodies, such that they are only activated at the relevant site, for example, after proteolytic cleavage. However, the methods for designing an ideal affinity-based mask and what parameters are important are not yet well understood. Here, we undertook mechanistic studies using three masks with different properties and identified four critical factors: binding site and affinity, as well as association and dissociation rate constants, which also played an important role. HDX-MS was used to identify the location of binding sites on the antibody, which were subsequently validated by obtaining a high-resolution crystal structure for one of the mask-antibody complexes. These findings will inform future designs of optimal affinity-based masks for antibodies and other therapeutic proteins.


Assuntos
Anticorpos Monoclonais , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Sítios de Ligação
3.
J Pharm Sci ; 111(3): 699-709, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34808214

RESUMO

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a polydisperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.


Assuntos
Laboratórios , Software , Tamanho da Partícula
4.
Biochemistry ; 54(3): 807-19, 2015 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-25521717

RESUMO

The man o' war (mow) phenotype in zebrafish is characterized by severe craniofacial defects due to a missense mutation in UDP-α-d-xylose synthase (UXS), an essential enzyme in proteoglycan biosynthesis. The mow mutation is located in the UXS dimer interface ∼16 Å away from the active site, suggesting an indirect effect on the enzyme mechanism. We have examined the structural and catalytic consequences of the mow mutation (R236H) in the soluble fragment of human UXS (hUXS), which shares 93% sequence identity with the zebrafish enzyme. In solution, hUXS dimers undergo a concentration-dependent association to form a tetramer. Sedimentation velocity studies show that the R236H substitution induces the formation of a new hexameric species. Using two new crystal structures of the hexamer, we show that R236H and R236A substitutions cause a local unfolding of the active site that allows for a rotation of the dimer interface necessary to form the hexamer. The disordered active sites in the R236H and R236A mutant constructs displace Y231, the essential acid/base catalyst in the UXS reaction mechanism. The loss of Y231 favors an abortive catalytic cycle in which the reaction intermediate, UDP-α-d-4-keto-xylose, is not reduced to the final product, UDP-α-d-xylose. Surprisingly, the mow-induced hexamer is almost identical to the hexamers formed by the deeply divergent UXS homologues from Staphylococcus aureus and Helicobacter pylori (21% and 16% sequence identity, respectively). The persistence of a latent hexamer-building interface in the human enzyme suggests that the ancestral UXS may have been a hexamer.


Assuntos
Biocatálise , Carboxiliases/genética , Carboxiliases/metabolismo , Mutação/genética , Multimerização Proteica , Animais , Carboxiliases/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Proteínas Mutantes/química , Nucleotídeos/metabolismo , Fenótipo , Dobramento de Proteína , Estrutura Quaternária de Proteína , Proteoglicanas/biossíntese , Soluções , Uridina Difosfato Xilose/química , Uridina Difosfato Xilose/metabolismo , Peixe-Zebra
5.
Cell ; 157(4): 795-807, 2014 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-24813607

RESUMO

It is widely believed that perinatal cardiomyocyte terminal differentiation blocks cytokinesis, thereby causing binucleation and limiting regenerative repair after injury. This suggests that heart growth should occur entirely by cardiomyocyte hypertrophy during preadolescence when, in mice, cardiac mass increases many-fold over a few weeks. Here, we show that a thyroid hormone surge activates the IGF-1/IGF-1-R/Akt pathway on postnatal day 15 and initiates a brief but intense proliferative burst of predominantly binuclear cardiomyocytes. This proliferation increases cardiomyocyte numbers by ~40%, causing a major disparity between heart and cardiomyocyte growth. Also, the response to cardiac injury at postnatal day 15 is intermediate between that observed at postnatal days 2 and 21, further suggesting persistence of cardiomyocyte proliferative capacity beyond the perinatal period. If replicated in humans, this may allow novel regenerative therapies for heart diseases.


Assuntos
Diferenciação Celular , Proliferação de Células , Coração/crescimento & desenvolvimento , Miócitos Cardíacos/citologia , Animais , Separação Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/fisiologia , Tri-Iodotironina/metabolismo
6.
J Dent Hyg ; 85(3): 220-8, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21888779

RESUMO

PURPOSE: The purpose of this study was to determine the impact of a 4 hour continuing education (CE) program on the oral health knowledge and behaviors of dentists, dental hygienists and dental assistants in providing oral health services to young children in West Virginia general dental practices. METHODS: A free CE program was provided for 92 general dentists, 123 dental hygienists and 37 dental assistants (n=252) at 4 sites across West Virginia. Participants completed a pre- and post-test on topics including the timing of the first dental exam, fluorides, xylitol, Alternative Restorative Technique (ART) and their practice pattern of caring for children under 3 years old. A 6 month follow-up questionnaire was mailed to participants to assess outcomes. RESULTS: Participants showed a 22% increase in knowledge from the pre-test to the post-test (p<0.001) for all questions except for ART, which showed no change. The majority of dentists and hygienists (89%) increased their comfort in providing services for children under 3 years old. Participants (80%) stated they would increase the number of children under 3 years old they examine, yet 6 months after the program only 42% responded affirmatively (p<0.001). At the time of the program, 62% reported that they currently examine children at 1 year of age, and there was no significant change 6 months later. While 54% responded that they would contact their local physicians about early oral health care, only 27% followed through. CONCLUSION: This program significantly increased the participants' knowledge and comfort level for providing infant and toddler oral health care. However, it did not motivate most to alter their practice behaviors to conform to national best practice guidelines.


Assuntos
Assistência Odontológica para Crianças/normas , Cárie Dentária/prevenção & controle , Recursos Humanos em Odontologia/educação , Odontólogos/estatística & dados numéricos , Educação Continuada em Odontologia/métodos , Fatores Etários , Pré-Escolar , Assistentes de Odontologia/educação , Assistentes de Odontologia/estatística & dados numéricos , Higienistas Dentários/educação , Higienistas Dentários/estatística & dados numéricos , Recursos Humanos em Odontologia/estatística & dados numéricos , Educação Continuada em Odontologia/estatística & dados numéricos , Humanos , Lactente , Saúde Bucal , West Virginia
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