Figures & data
Table 1. Cross-industry studies demonstrating the application LC-MS/MS methods in support of process development.
Table 2. Recent studies and trend of LC-MS based analysis in HCP characterization.
Table 3. Comparison of different LC and MS methods with their advantages and limitations for HCP analysis.
Figure 1. General workflow for HCP characterization by LC-MS/MS analysis.
Decision tree for a workflow supporting process development with HCP identification and quantitation. If a specific HCP is identified in in-process pools or UF/DF pools above certain LOD, a quantitative method is developed to confirm the protein level. More characterization may be needed based on the process development.
Table 4. Our commonly used HCP identification methods by LC-MS/MS analysis.
Figure 2. Workflow for HCP quantitation by SWATH and targeted (MRM/PRM) MS analysis.
Workflow for HCP relative and absolute quantitation. For relative quantitation by SWATH MS analysis, we spike recombinant reference proteins that are not from the host (e.g., CHO, or E. coli). The quantity of proteins of interest is determined by comparison with the top 3 peaks from reference proteins at a known concentration. To get a more accurate quantity of a specific protein for UF/DF pool samples, we can perform absolute quantitation with MRM analysis by measuring the target protein based on an external calibration curve made from spiking the recombinant protein-protein of interest into matrix samples at various known concentrations. The matrix is a blank sample that is similar to the test articles but with no or least amount of HCP of interest present. Samples are the test articles. In each sample, stable isotope-labeled peptides are spiked at a consistent level as internal standards.
