Journal of Chromatography & Separation Techniques
Open Access

Development and validation of analytical methods based on RP-HPLC: Quantifying HER1 extracellular domain in culture supernatant and peptide mapping of a monoclonal antibody

3^rd International Conference and Exhibition on Advances in Chromatography & HPLC Techniques

July 13-14, 2017 Berlin, Germany

Yadira S Prieto Curbelo

Center for Molecular Immunology, Cuba

Posters & Accepted Abstracts: J Chromatogr Sep Tech

Abstract :

Techniques based on high-resolution liquid chromatography are currently widely used to quantify recombinant proteins from culture supernatants, as well as their characterization. Such assays can be easily and rapidly developed, in case of reverse phase chromatography. In this study, we describe the development and validation of an analytical technique for quantifying HER1 extracellular domain (HER1 ECD) in bioreactor supernatant using reversed-phase chromatography with a C8 column. On the other hand, we validated the methodology for the peptide mapping of monoclonal antibody using C4 column. For both study cases, the proteins were analyzed by monitoring the absorbance of the sample at 214 nm. The resulting analytical methodology was found to provide precise and accurate results for a wide range of concentrations (10â��120 �¼g/mL) of HER1 ECD. The accuracy of the method varied from 86 to 109%, while the repeatability and the day-to-day intermediate precision were less than 7.25 and 7.85%, respectively. In the case of peptide mapping of mAb, the methodology provides a range of 35-40 well resolved peaks. As a criterion is set, RTâ�¤0.5 min and the % peak height relative to the reference material must be 70% to 130%. These methodologies constitute a useful tool that can be applied during the production of the HER1 ECD vaccine and in the identification of modifications on the primary structure of the mAb due to changes in biomanufacturing process.

Biography :

Email: yadirap@cim.sld.cu