Optimized DIA-MS workflow for Host Cell Proteins (HCP) characterization and quantification in bioreactors and top-down mass spectrometry analysis for monoclonal antibody production

Biopharmaceutical proteins have revolutionized treatment of diseases and are one of the fastest growing segments of the pharmaceutical industry. Close monitoring of Critical Quality attributes (CQAs), - attributes of the product or impurities known to influence efficacy, safety or stability of the final product, - is necessary.

Host cell proteins (HCPs) are especially challenging to monitor and to deplete trough purification. The industrial standard to monitor HCP are enzyme-linked immunosorbent assays (ELISA). These assays are only able to detect immunoreactive HCP species and only give a total quantity. In contrast mass spectrometry (MS) is a non-targeted approach and allows unbiased identification and quantification of HCPs population down to single HCPs.

Here, we present an optimized workflow to monitor and quantify HCP using a DIA bottom-up MS approach. The developed method is capable of quantifying more than 1000 HCPs from harvest samples including 20 high risk proteins identified as being especially problematic for biopharmaceuticals, while having improved throughput due to reduction in processing and acquisition time through the implementation of a high temperature digest in combination with a micro-flow LC system and untargeted DIA acquisition on a SCIEX ZenoTOF 7600. The full workflow as well as unit operations were evaluated for robustness, sensitivity, repeatability and influences on the quantification results.

We will also show top-down mass spectrometry analysis of the monoclonal antibody product and good fragmentation by electron activated dissociation (EAD)in order to look at post translational modifications in a shorter timeframe than with a bottom-up approach.