Do you want to identify host cell proteins in your pre-clinical tox batch or GMP phase I/II batches?
We have developed sensitive mass spectrometry and gel-based methods for detection, vizualization and relative quantification of host cell proteins down to low ppm concentrations. FDA and EMA are increasingly aware of mass spectrometry (MS) as an orthogonal method to ELISA.
Currently, we offer three orthogonal methods for comparison of batch-to-batch HCP profiles between pre-clinical and clinical batches:
- HCP analysis by in-solution digestion and SWATH LC MS analysis
- GeLC-MS/MS by 1D SDS PAGE, in-gel digestion and nanoLC MS protein identification
- 2D PAGE MALDI MS by 2D electrophoresis, in-gel digestion and MALDI MS protein identification
The SWATH LC-MS method provides identification and relative quantification of HCPs down to low ppm concentrations. We digest the drug substance in-solution and analyse the complex peptide mixture by LC-MS/MS. Our scientists perform this analysis on a Sciex TrippleTOF 6600 instrument using data-independent SWATH data acquisition and ion library searching. The analysis is under development, promissing to deliver low-to-sub-ppm detection limits and highly reproducible quantification of each HCP.
The GeLC-MS/MS method is for identification of host cell proteins down to ~50 ppm. We separate the sample by 1D SDS PAGE; each lane is completely excised into 8 gel consecutive pieces and in-gel digested with trypsin. Afterwards, we analyze the peptides by high sensitivity nanoLC-MS/MS and search the data against a customized database containing host cell proteins from your specific cell line and the drug substance sequence. The MS-data is searched against this specifically designed database for HCP identification.
The GeLC-MS/MS analysis provides you with a comprehensive list of observed HCPs, amino acids sequences, theoretical pI/MW, database accession number, etc. The sensitivity of the analysis is in the low-to-sub nanogram range demonstrated on protein standards.
Read more about HCP analysis using GeLC-MS/MS in our poster Combining platform ELISA and orthogonal GeLC-MS/MS for HCP detection, and in the application note Detection of small HCPs by GeLC-MS/MS.
The 2D PAGE with MALDI MS method is for visualization and identification of proteins separated by 2D PAGE according to pI and Mw. We stain the proteins by coomassie blue or fluorescense, then cut out the visible spots for in-gel trypsin digestion and MALDI MS/MS peptide analysis of each spot. The HCP protein identification is obtained by a MASCOT search in customized HCP protein database. Typically it identifies >95% of visible protein spots. The sensitivity of the analysis is demonstrated on known protein standards.
We also develop host cell protein analysis for up- and downstream bioprocess optimization