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Characterization of protein aggregation: the case of a therapeutic immunoglobulin

Demeule, Barthélemy
Lawrence, M Jayne
Drake, Alex F.
Published in Biochimica et biophysica acta. 2007, vol. 1774, no. 1, p. 146-53
Abstract In this paper, a therapeutic immunoglobulin (Antibody A) has been characterized in two solutions: (1) 0.1% acetic acid containing 50 mM magnesium chloride, a solution in which the immunoglobulin is stable, and (2) 10 mM sodium phosphate buffer pH approximately 7. The protein solutions were characterized by microscopy, asymmetrical flow field-flow fractionation (FFF), light scattering, circular dichroism, fluorescence and fluorescence lifetime spectroscopy. The results show that Antibody A dissolved in 0.1% acetic acid containing 50 mM magnesium chloride exists as 88% monomer, 2% low molecular weight aggregates and 10% high molecular weight aggregates (>1 million Dalton). In phosphate buffer, Antibody A formed micrometre-sized aggregates that were best characterized by fluorescence microscopy. The aggregation of Antibody A in phosphate buffer was shown to be concomitant with conformational changes in amino acid residue side chains. The aggregates formed in phosphate buffer were easily disrupted during FFF analysis, indicating that they are formed by weak interactions. The combination of microscopy, asymmetrical flow field-flow fractionation (FFF) and spectroscopy allowed a reliable assessment of protein self association and aggregation.
Keywords Acetic AcidAntibodies, Monoclonal/chemistryBuffersCircular DichroismFluorescence PolarizationFractionation, Field FlowLightMagnesium ChlorideMicroscopy, FluorescencePhosphatesProtein Structure, QuaternaryScattering, RadiationSpectrometry, Fluorescence
PMID: 17142116
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DEMEULE, Barthélemy et al. Characterization of protein aggregation: the case of a therapeutic immunoglobulin. In: Biochimica et biophysica acta, 2007, vol. 1774, n° 1, p. 146-53. doi: 10.1016/j.bbapap.2006.10.010 https://archive-ouverte.unige.ch/unige:13246

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Deposited on : 2011-01-24

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