IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Charge isoform distribution for a monoclonal antibody by capillary isoelectrofocusing
Autor/es:
WEPFER, J; COLLAZO, D; VIZIOLI, NM
Reunión:
Simposio; 23rd Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical, and Industrial Applications of Capillary Electrophoresis and Microchip Technology; 2017
Resumen:
Capillary isoelectrofocusing (cIEF) is the capillary version of isoelectric focusing gelelectrophoresis, which separates proteins on the basis of overall charge, or isoelectric point (pI).cIEF separation forcharacterization of therapeutic proteins has beenincreasingly adopted inrecent years. Determination of pI adds a critical dimension to establishing identity, purity, posttranslationalmodification and stability oftherapeutic protein preparations.In this work, the distribution of charge isoforms of a monoclonal antibody (mAb) was studied.The neutral capillary (50 μm x 45 cm), the gel (polymer solution) and the five pI peptidemarkers were from Beckman Coulter. The analytical procedure was carried out on a P/ACEMDQ capillary electrophoresis system (Beckman Coulter). Ampholytes covered a pH range of3-10 and detection was performed at 280 nm. Before analysis, the mAb samples were treatedwith carboxypeptidase B to remove C-terminal lysine and eliminate ambiguities introduced bythe presence of multiple C-terminal variants for each charged species.Under the experimental conditions, four mayor peaks were observed. The pI of the main peakwas 8.9. Results were compared with those obtained on a commercially available imaging cIEFanalyzer. Using the cIEF analysis kit from Beckman Coulter it was possible to detect anadditional charge isoform in the studied mAb. The optimization of focusing step was easy andbrief. cIEF analysis method required no hard analyst training period which made this more attractive.