Host Cell Proteins (HCPs) in protein biopharmaceuticals can be analyzed by mass spectrometry – for both identification and quantification of the individual HCPs. The analysis requires optimized sample preparation protocols for enzymatic protein cleavage into peptides. It also requires highly advanced liquid chromatography mass spectrometry (LC-MS) techniques.
The major analytical challenges are:
- Measurement of low level HCPs (nanogram amount) in the presence of mg amounts of drug substance protein.
- Reproducibility of the MS techniques to identify and quantify each low-level HCP.
The solutions is simple; SWATH LC-MS can solve these challenges. This makes it an ideal tool in HCP analysis for bioprocess optimization.
You will find a description of the principles of LC-MS/MS of peptides and advantages of SWATH MS for HCP analysis below:
For LC-MS/MS of peptides several terms are important to understand
In the figure above, the term MS (or MS1) describes the analysis where the MS-instrument measures the intact mass of a peptide. The term MS/MS (or MS2) describes the analysis where you isolate a specific peptide mass (m/z) and fragment it into specific fragment ions. You can use the masses of a peptide and its fragment ions to identify the peptide. Also, you may use the intensity of the MS signals to quantify the peptide.
Mass spectrometers can perform MS/MS analysis in Data Dependent Acquisition mode (DDA). DDA is also sometimes referred to as Information Dependent Acquisition mode (IDA), as illustrated in the figure above.
In DDA mode, the mass spectrometer selects the most intense peptide ions (top 10-15 peaks) in MS1. It then sends them one-by-one into the fragmentation analysis (MS2).
Experience shows that DDA analysis of a complex peptide sample generates different protein identification results for identical technical replicates. In proteomics research you commonly see reports of protein identification results when 2 out of 3 technical replicates observe a given protein. In DDA mode of a complex sample the instrument does not have enough time to select all peptides for MS/MS analysis. Instead, you only analyze the top 10-15 peptides. The instrument decides on-the-fly which peptide to select – which creates variation from run to run.
Advanced mass spectrometers can also perform Data Independent Acquisition (DIA). Then they fragment all peptides within a certain mass range in MS/MS. This instrument is not selecting individual peptides, and therefore the data acquisition in DIA-mode is more reproducible.
What is SWATH? SWATH is a special type of DIA. In HCP SWATH mode, the Sciex mass spectrometer divides the mass range into small mass windows. Eventually, there can be up to hundreds of windows. The instrument performs MS/MS analysis on all peptides in each window. With dynamic size mass windows you will have small windows in areas with many peptides. – While larger windows in areas with few peptides.
“The beauty of SWATH is that you can acquire MS/MS data for all peptides without knowing what to look for”
Interpretation of a complex HCP SWATH mass spectrum with fragmentation of multiple peptides requires comparison to an ion library with MS/MS data on known peptides. The ion library contains information about each peptide, including HPLC retention time, MS/MS data and amino acid sequence. The ion library can be build using DDA analysis of the samples. Furthermore, you can expand the library with new data on more samples.
Key advantages of HCP SWATH analysis by LC-MS:
- Low interference from the high amount of the drug substance protein. Because your aquire the data on low level HCP peptides in data-independent mode in small mass windows.
- Highly reproducible HCP identification and quantification because data are acquired in Data Independent Acquisition mode.
- All recorded SWATH data can be re-analyzed – when the ion library is updated with more known compounds and their fragmentation pattern.
See examples of HCP SWATH analysis for biopharmaceutical proteins and bioprocess sample >>
Download our application note for a description of Alphalyse’s HCP analysis based on SWATH LC-MS >>