Theoretical approximations and experimental extinction coefficients of biopharmaceuticals.
Anal Bioanal Chem
; 408(5): 1523-30, 2016 Feb.
Article
em En
| MEDLINE
| ID: mdl-26715251
UV spectrophotometric measurement is a widely accepted and standardized routine analysis for quantitation of highly purified proteins; however, the reliability of the results strictly depends on the accuracy of the employed extinction coefficients. In this work, an experimental estimation of the differential refractive index (dn/dc), based on dry weight measurements, was performed in order to determine accurate extinction coefficients for four biotherapeutic proteins and one synthetic copolymer after separation in a size-exclusion ultra-performance liquid chromatograph coupled to an ultraviolet, multiangle light scattering and refractive index (SE-UPLC-UV-MALS-RI) multidetection system. The results showed small deviations with respect to theoretical values, calculated from the specific amino acid sequences, for all the studied immunoglobulins. Nevertheless, for proteins like etanercept and glatiramer acetate, several considerations, such as glycan content, partial specific volume, polarizability, and higher order structure, should be considered to properly calculate theoretical extinction coefficient values. Herein, these values were assessed with simple approximations. The precision of the experimentally obtained extinction coefficients, and its convergence towards the theoretical values, makes them useful for characterization and comparability exercises. Also, these values provide insight into the absorbance and scattering properties of the evaluated proteins. Overall, this methodology is capable of providing accurate extinction coefficients useful for development studies.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Refratometria
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Acetato de Glatiramer
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Trastuzumab
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Rituximab
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Infliximab
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Etanercepte
/
Modelos Teóricos
Limite:
Humans
Idioma:
En
Revista:
Anal Bioanal Chem
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
México
País de publicação:
Alemanha