title: Capillary isoelectric focusing method development and validation for investigation of recombinant therapeutic monoclonal antibody.
abstract: Capillary isoelectric focusing (cIEF) is a basic and highly accurate routine analytical tool to prove identity of protein drugs in quality control (QC) and release tests in biopharmaceutical industries. However there are some "out-of-the-box" applications commercially available which provide easy and rapid isoelectric focusing solutions for investigating monoclonal antibody drug proteins. However use of these kits in routine testings requires high costs. A capillary isoelectric focusing method was developed and validated for identification testing of monoclonal antibody drug products with isoelectric point between 7.0 and 9.0. A method was developed providing good pH gradient for internal calibration (R(2)>0.99) and good resolution between all of the isoform peaks (R=2), minimizing the time and complexity of sample preparation (no urea or salt used). The method is highly reproducible and it is suitable for validation and method transfer to any QC laboratories. Another advantage of the method is that it operates with commercially available chemicals which can be purchased from any suppliers. The interaction with capillary walls (avoid precipitation and adsorption as far as possible) was minimized and synthetic isoelectric small molecular markers were used instead of peptide or protein based markers. The developed method was validated according to the recent ICH guideline (Q2(R1)). Relative standard deviation results were below 0.2% for isoelectric points and below 4% according to the normalized migration times. The method is robust to buffer components with different lot numbers and neutral capillaries with different type of inner coatings. The fluoro-carbon coated column was chosen because of costs-effectivity aspects.
authors.affiliation: Chemical Works of Gedeon Richter Plc, Gyömrői út 19.-21., 1103 Budapest, Hungary. Electronic address: d.suba@richter.hu.
authors.forename: Dávid
authors.initials: D
authors.lastname: Suba
authors.affiliation: Chemical Works of Gedeon Richter Plc, Gyömrői út 19.-21., 1103 Budapest, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Urbányi
authors.affiliation: Budapest University of Technology and Economics, Műegyetem rakpart 3., 1111 Budapest, Hungary.
authors.forename: András
authors.initials: A
authors.lastname: Salgó
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002021:Buffers; D002138:Calibration; D004566:Electrodes; D004573:Electrolytes; D019075:Electrophoresis, Capillary / Q000379:methods; D004867:Equipment Design; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry*; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D020033:Protein Isoforms; D011786:Quality Control; D011994:Recombinant Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012492:Salts / Q000737:chemistry; D014508:Urea / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D002021:Buffers; D004573:Electrolytes; D007074:Immunoglobulin G; D020033:Protein Isoforms; D011994:Recombinant Proteins; D012492:Salts; D014508:Urea
keywords: Capillary electrophoresis; Isoelectric focusing; Method development; Monoclonal antibody; Validation