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Characterization of a Protein Conjugate Using an Asymmetrical-Flow Field-Flow Fractionation and a Size-Exclusion Chromatography with Multi-Detection System

In this study we present detailed characterization of a protein-PEG conjugate using two separation techniques, that is, asymmetrical-flow field-flow fractionation (AF4) and size-exclusion chromatography (SEC), which were online coupled to a series of successively connected detectors: an ultraviolet, a multiangle light-scattering, a quasi-elastic light-scattering, and a refractive-index detector (UV-MALS(QELS)-RI). Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a complementary characterization technique. The results of AF4 as well as SEC on two columns connected in series, with both separation techniques coupled to a multidetection system, indicate the uniform molar mass and chemical composition of the conjugate, that is, the molar ratio of protein to PEG is 1/1, the presence of minute amounts of residual unreacted protein and the aggregates with the same chemical composition as that of the conjugate. Since the portion of aggregated species is smalle...

Authors:   Katja Rebolj; David Pahovnik; Ema Žagar
Journal:   Analytical Chemistry
Year:   2012
DOI:   10.1021/ac3010378
Publication date:   17-Aug-2012
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