title: Charge profiling and stability testing of biosimilar by capillary isoelectric focusing.
abstract: CIEF was developed for the rapid analysis of charge heterogeneity of trastuzumab biosimilar using commercially available fluorocarbon-coated capillary. The CIEF master mix was composed of 0.30% w/v methyl cellulose, 2.3 M urea, 56.8 mM l-arginine, 1.52 mM iminodiacetic acid, 4.5% v/v carrier ampholytes (broad-range pI 3-10 and narrow-range pI 8-10.5 with ratio of 3:1), and 0.45% v/v 10.0, 9.5, 7.0, 5.5, 4.1 pI markers. To get a robust method to analyze charge heterogeneity, some separation parameters, including focusing time and separation temperature, were investigated and optimized. The optimized method gave good precision in estimated pI values of charge variants with RSDs of not more than 0.16% intraday analysis (n = 6) and < 0.18% interday analysis (n = 9). In addition, the applications of this method including purity, stability, lot consistency, peptide N-glycosidase F digest, and C-terminal lysine variants characterization were also investigated.
authors.affiliation: School of Pharmacy, Yantai University, Yantai, P. R. China.
authors.forename: Junzi
authors.initials: J
authors.lastname: Cao
authors.forename: Wen
authors.initials: W
authors.lastname: Sun
authors.forename: Feifei
authors.initials: F
authors.lastname: Gong
authors.forename: Wanhui
authors.initials: W
authors.lastname: Liu
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000032:analysis; D001120:Arginine / Q000032:analysis; D002021:Buffers; D006031:Glycosylation; D007525:Isoelectric Focusing / Q000379:methods*; D008747:Methylcellulose / Q000032:analysis; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D015203:Reproducibility of Results; D000068878:Trastuzumab; D014508:Urea / Q000032:analysis
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D002021:Buffers; D014508:Urea; D008747:Methylcellulose; D001120:Arginine; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000068878:Trastuzumab
keywords: CIEF; Charge heterogenity; Monoclonal antibody; Trastuzumab biosimilar