Characterization of protein aggregation: the case of a therapeutic immunoglobulin.
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.
Demeule
Barthélemy
B
Department of Pharmaceutics and Biopharmaceutics, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.
Lawrence
M Jayne
MJ
Drake
Alex F
AF
Gurny
Robert
R
Arvinte
Tudor
T
D019342:Acetic Acid; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002021:Buffers; D002942:Circular Dichroism; D005454:Fluorescence Polarization; D031521:Fractionation, Field Flow; D008027:Light; D015636:Magnesium Chloride; D008856:Microscopy, Fluorescence; D010710:Phosphates; D020836:Protein Structure, Quaternary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D002021:Buffers; D010710:Phosphates; D015636:Magnesium Chloride; D019342:Acetic Acid; C018279:sodium phosphate