TY - JOUR
T1 - Theoretical approximations and experimental extinction coefficients of biopharmaceuticals
AU - Miranda-Hernández, Mariana P.
AU - Valle-González, Elba R.
AU - Ferreira-Gómez, David
AU - Pérez, Néstor O.
AU - Flores-Ortiz, Luis F.
AU - Medina-Rivero, Emilio
N1 - Publisher Copyright:
© 2015 Springer-Verlag Berlin Heidelberg.
PY - 2016/2/1
Y1 - 2016/2/1
N2 - 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.
AB - 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.
KW - Biopharmaceuticals
KW - Dry weight
KW - Extinction coefficient
KW - Protein quantitation
KW - Refractive index increment
UR - http://www.scopus.com/inward/record.url?scp=84955673568&partnerID=8YFLogxK
U2 - 10.1007/s00216-015-9261-6
DO - 10.1007/s00216-015-9261-6
M3 - Artículo
SN - 1618-2642
VL - 408
SP - 1523
EP - 1530
JO - Analytical and Bioanalytical Chemistry
JF - Analytical and Bioanalytical Chemistry
IS - 5
ER -