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1.
Anal Chem ; 85(24): 12055-61, 2013 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-24224685

RESUMO

Native mass spectrometry and ion mobility spectrometry were used to investigate the gas-phase structures of selected cations and anions of proteins and protein complexes with masses ranging from 6 to 468 kDa. Under the same solution conditions, the average charge states observed for all native-like anions were less than those for the corresponding cations. Using an rf-confining drift cell, similar collision cross sections were measured in positive and negative ion mode suggesting that anions and cations have very similar structures. This result suggests that for protein and protein complex ions within this mass range, there is no inherent benefit to selecting a specific polarity for capturing a more native-like structure. For peptides and low-mass proteins, polarity and charge-state dependent structural changes may be more significant. The charged-residue model is most often used to explain the ionization of large macromolecules based on the Rayleigh limit, which defines the upper limit of charge that a droplet can hold. Because ions of both polarities have similar structures and the Rayleigh limit does not depend on polarity, these results cannot be explained by the charged-residue model alone. Rather, the observed charge-state distributions are most consistent with charge-carrier emissions during the final stages of analyte desolvation, with lower charge-carrier emission energies for anions than the corresponding cations. These results suggest that the observed charge-state distributions in most native mass spectrometry experiments are determined by charge-carrier emission processes; although the Rayleigh limit may determine the gas-phase charge states of larger species, e.g., virus capsids.


Assuntos
Gases/química , Espectrometria de Massas/métodos , Proteínas/química , Modelos Moleculares , Peso Molecular , Conformação Proteica
2.
Int J Nanomedicine ; 13: 3689-3711, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29983563

RESUMO

BACKGROUND: Adjuvants have the potential to increase the efficacy of protein-based vaccines but need to be maintained within specific temperature and storage conditions. Lyophilization can be used to increase the thermostability of protein pharmaceuticals; however, no marketed vaccine that contains an adjuvant is currently lyophilized, and lyophilization of oil-in-water nanoemulsion adjuvants presents a specific challenge. We have previously demonstrated the feasibility of lyophilizing a candidate adjuvanted protein vaccine against Mycobacterium tuberculosis (Mtb), ID93 + GLA-SE, and the subsequent improvement of thermostability; however, further development is required to prevent physicochemical changes and degradation of the TLR4 agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water nanoemulsion (SE). MATERIALS AND METHODS: In this study, we took a systematic approach to the development of a thermostable product by first identifying compatible solution conditions and stabilizing excipients for both antigen and adjuvant. Next, we applied a design-of-experiments approach to identify stable lyophilized drug product formulations. RESULTS: We identified specific formulations that contain disaccharide or a combination of disaccharide and mannitol that can achieve substantially improved thermostability and maintain immunogenicity in a mouse model when tested in accelerated and real-time stability studies. CONCLUSION: These efforts will aid in the development of a platform formulation for use with other similar vaccines.


Assuntos
Adjuvantes Imunológicos/farmacologia , Emulsões/química , Nanopartículas/química , Temperatura , Vacinas contra a Tuberculose/imunologia , Animais , Formação de Anticorpos , Química Farmacêutica , Difusão Dinâmica da Luz , Excipientes , Feminino , Liofilização , Concentração de Íons de Hidrogênio , Imunidade Celular , Lipídeos/química , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/imunologia , Nefelometria e Turbidimetria , Tamanho da Partícula , Tuberculose/imunologia , Tuberculose/patologia
3.
Methods Mol Biol ; 1494: 273-283, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27718201

RESUMO

Adjuvants in modern vaccines boost and shape immune responses and allow for antigen dose-sparing. Analysis of protein antigens in the presence of adjuvants can prove challenging, especially if the adjuvant interferes with visualization of the protein band on an SDS-PAGE gel. In this chapter, a variety of different techniques are presented to mitigate the interference of a nanoemulsion adjuvant, GLA-SE, with different recombinant proteins of varying molecular weight by addressing sample preparation and staining methods.


Assuntos
Adjuvantes Imunológicos/química , Eletroforese em Gel de Poliacrilamida/métodos , Coloração e Rotulagem/métodos , Vacinas/química , Emulsões , Proteínas Recombinantes/química
4.
J Pharm Sci ; 104(2): 768-74, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25242027

RESUMO

Aluminum salts have a long history as safe and effective vaccine adjuvants. In addition, aluminum salts have high adsorptive capacities for vaccine antigens and adjuvant molecules, for example, Toll-like receptor 4 (TLR4) agonists. However, the physicochemical properties of aluminum salts make direct quantitation of adsorbed molecules challenging. Typical methods for quantifying adsorbed molecules require advanced instrumentation, extreme sample processing, often destroy the sample, or rely on an indirect measurement. A simple, direct, and quantitative method for analysis of adsorbed adjuvant molecules is needed. This report presents a method utilizing Fourier transform infrared spectroscopy with a ZnSe-attenuated total reflectance attachment to directly measure low levels (<30 µg/mL) of TLR4 agonists adsorbed on aluminum salts with minimal sample preparation.


Assuntos
Hidróxido de Alumínio/análise , Glucosídeos/análise , Lipídeo A/análise , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Receptor 4 Toll-Like/agonistas , Adsorção , Hidróxido de Alumínio/metabolismo , Glucosídeos/metabolismo , Lipídeo A/metabolismo
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