title: A novel reagent significantly improved assay robustness in imaged capillary isoelectric focusing.
abstract: Imaged Capillary Isoelectric Focusing (icIEF) has been used as primary method for charge variants analysis of therapeutic antibodies and proteins [1], [9]. Proteins tend to precipitate around their pI values during focusing [14], which directly affects the reproducibility of their charge profiles. Protein concentration, focusing time and various supplementing additives are key parameters to minimize the protein precipitation and aggregation. Urea and sucrose are common additives to reduce protein aggregation, solubilize proteins in sample matrix and therefore improve assay repeatability [15]. However some proteins and antibodies are exceptions, we found urea and sucrose are not sufficient for a typical fusion protein (Fusion protein A) in icIEF assay and high variability is observed. We report a novel reagent, formamide, significantly improved reproducibility of protein charge profiles. Our results show formamide is a good supplementary reagent to reduce aggregation and stabilize proteins in isoelectric focusing. We further confirmed the method robustness, linearity, accuracy and precision after introducing the new reagent; extremely tight pI values, significantly improved method precision and sample on-board stability are achieved by formamide. Formamide is also proven to be equally functional to multiple antibodies as urea, which makes it an extra tool in icIEF method development.
authors.affiliation: Department of Biological Process Development, Bristol-Myers Squibb, USA. Electronic address: Xin.Zhang4@bms.com.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Biological Process Development, Bristol-Myers Squibb, USA.
authors.forename: Sergey
authors.initials: S
authors.lastname: Voronov
authors.affiliation: Department of Biological Process Development, Bristol-Myers Squibb, USA.
authors.forename: Nesredin
authors.initials: N
authors.lastname: Mussa
authors.affiliation: Department of Biological Process Development, Bristol-Myers Squibb, USA.
authors.forename: Zhengjian
authors.initials: Z
authors.lastname: Li
mesh_terms: D001681:Biological Assay / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005559:Formamides / Q000737:chemistry*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D007753:Laboratories / Q000592:standards*; D057054:Molecular Imaging / Q000379:methods*; D020033:Protein Isoforms; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: D005559:Formamides; D020033:Protein Isoforms; D011506:Proteins; C031066:formamide
keywords: Charge profile; Formamide; Robustness; Urea; icIEF