Host cell protein (HCP) analysis is essential to detect and quantify low-level proteins coproduced in the cell culture from the host cells used in manufacturing therapeutic proteins. These contaminants must be minimized to ensure product safety and efficacy and meet regulatory requirements. Detection and analysis of low-level contaminants can be challenging, requiring advanced technology with high sensitivity and specificity to deliver reliable and reproducible results. Agilent has a complete portfolio of samples-to-answers solutions for HCP analysis including automation, LC, LC/MS, and software solutions.
HCPs can be confidently identified using MS1 profiling, iterative-directed MS and 2D-LC/MS fractionation strategies.
An automated HCP analysis workflow including LC–MS/MS to improve protein identification and data analysis software to confirm identification.
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Sample preparation for host cell protein analysis can be time consuming and tedious. The Agilent AssayMAP Bravo platform is a powerful protein and peptide sample preparation solution. Automation increases workflow reproducibility by reducing sample preparation variability and frees up scientists for more value-added work.
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Discover a method for sub-ppm host cell protein quantification in a monoclonal antibody therapeutic. The workflow featured an Agilent AssayMAP Bravo automated protein sample preparation platform, an 1290 Infinity III LC System, and an Agilent 6495 Triple Quadrupole LC/MS.
To detect low-level host cell proteins by LC/MS, it is often necessary to inject large amounts of a therapeutic, and sometimes more than 100 µg is required. Here, a balance between loading amount and host cell protein detection levels is explored. As expected, the largest injection amount of the sample resulted in detection of the largest number of proteins and obtained the highest sensitivity. The smaller injection was still able to detect proteins with a concentration under 1 ppm. Alternatively, the sample could be injected twice with an iterative MS/MS run. A workflow showing high analytical sensitivity using the Agilent AdvanceBio Peptide Plus column, Agilent AdvanceBio 6545XT LC/Q-TOF, and Protein Metrics Byos demonstrate high analytical sensitivity for sample-limited applications such as AAV HCP analysis.
The AssayMAP Bravo automated protein and peptide sample preparation platform delivered reproducibility, scalability, flexibility, and ease-of-use for host cell protein sample preparation. Iterative MS/MS with the 6545XT AdvanceBio LC/Q-TOF improves protein identification coverage. Using LC-MS/MS coupled with iterative MS/MS acquisition identified all spiked-in standard proteins above 8 ppm with high confidence. By adding HPRP fractionation using the AssayMAP Bravo, all the spiked-in proteins above 2 ppm were identified.
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