1. Analysis of therapeutic proteins and peptides using multiangle light scattering coupled to ultra high performance liquid chromatography.
- Author
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Espinosa‐de la Garza, Carlos E., Miranda‐Hernández, Mariana P., Acosta‐Flores, Lilia, Pérez, Néstor O., Flores‐Ortiz, Luis F., and Medina‐Rivero, Emilio
- Subjects
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THERAPEUTIC use of proteins , *PEPTIDES , *HIGH performance liquid chromatography , *LIGHT scattering , *COPOLYMERS , *REFRACTIVE index , *MOLAR mass - Abstract
Analysis of the physical properties of biotherapeutic proteins is crucial throughout all the stages of their lifecycle. Herein, we used size-exclusion ultra high performance liquid chromatography coupled to multiangle light scattering and refractive index detection systems to determine the molar mass, mass-average molar mass, molar-mass dispersity and hydrodynamic radius of two monoclonal antibodies (rituximab and trastuzumab), a fusion protein (etanercept), and a synthetic copolymer (glatiramer acetate) employed as models. A customized instrument configuration was set to diminish band-broadening effects and enhance sensitivity throughout detectors. The customized configuration showed a performance improvement with respect to the high-performance liquid chromatography standard configuration, as observed by a 3 h column conditioning and a higher resolution analysis in 20 min. Analysis of the two monoclonal antibodies showed averaged values of 148.0 kDa for mass-average molar mass and 5.4 nm for hydrodynamic radius, whereas for etanercept these values were 124.2 kDa and 6.9 nm, respectively. Molar-mass dispersity was 1.000 on average for these proteins. Regarding glatiramer acetate, a molar mass range from 3 to 45 kDa and a molar-mass dispersity of 1.304 were consistent with its intrinsic peptide diversity, and its mass-average molar mass was 10.4 kDa. Overall, this method demonstrated an accurate determination of molar mass, overcoming the difficulties of size-exclusion chromatography. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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