Development and validation of analytical methods based on RPâ€‘HPLC: Quantifying HER1 extracellular domain in culture supernatant and peptide mapping of a monoclonal antibody | Yadira S Prieto Curbelo | Center for Molecular Immunology, Cuba |

Center for Molecular Immunology, Cuba

Techniques based on high-resolution liquid chromatography are currently used to quantify recombinant proteins from

culture supernatants, as well as their characterization. Such assays can be easily and rapidly developed, this is the 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 validate 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. Th e resulting

analytical methodology was found to provide precise and accurate results for a wide range of concentrations (10–120 μg/mL)

of HER1 ECD. Th e 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 RT ≤0.5 min and the percentage peak height relative to the reference

material must be 70% to 130%. Th ese methodologies constitute a useful tool that can be applied during the production of the

HER1 ECD vaccine and in the identifi cation of modifi cations on the primary structure of the mAb due to changes in biomanufacturing

process.

Chromatography