title: Analysis of recombinant monoclonal antibodies by RPLC: toward a generic method development approach.
abstract: Monoclonal antibodies (mAbs) are an emerging class of therapeutic agents that have recently gained importance. To attain acceptable kinetic performance with mAbs in reversed phase liquid chromatography, there is a need to work with the last generation of wide-pore sub-2 μm fully porous or core-shell particles stationary phases. In addition, temperature in the range 60-90 °C was found to be mandatory to limit adsorption phenomenon of mAbs and their fragments. A generic method development strategy was proposed to account for the selectivity, efficiency, recovery, and the possible thermal degradation. This study also demonstrated that the gradient steepness and temperature cannot be optimized using van't Hoff type linear models. Similarly, the common linear solvent strength model also generated some error in predicting the retention times. In contrast, when quadratic models were employed, the prediction accuracy of retention times was found to be excellent (relative error between 0.5 and 1%) using a reasonable number of experiments (9 or 6 experiments for optimization of gradient time and temperature, which requires between 6 and 8 h). Two separations of mAbs fragments were performed to demonstrate the reliability of the quadratic approach.
authors: School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland. szfekete@mail.bme.hu
authors: Szabolcs
authors: S
authors: Fekete
authors: Serge
authors: S
authors: Rudaz
authors: Jenő
authors: J
authors: Fekete
authors: Davy
authors: D
authors: Guillarme
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation; D056148:Chromatography, Reverse-Phase / Q000295:instrumentation; D004867:Equipment Design; D007128:Immunoglobulin Fragments / Q000032:analysis*; D016014:Linear Models; D008956:Models, Chemical; D016062:Porosity; D011489:Protein Denaturation; D055550:Protein Stability; D015203:Reproducibility of Results; D012984:Software; D012997:Solvents / Q000737:chemistry; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D012997:Solvents