Anaquant HCP analysis I Protein characterisation I Protein analysis
Introduction & strategy
In the production of biotherapeutics, therapeutic proteins are often expressed in host cells such as CHO (Chinese Hamster Ovary) cells. However, during the downstream process (DSP), Host Cell Proteins (HCPs) can be co-purified with the final biotherapeutic product. Some HCPs pose risks to drug safety and efficacy due to their immunogenic properties or their ability to degrade the therapeutic substance. For these reasons, it is critical to monitor and quantify specific HCPs to ensure the quality of the drug substance (DS) and adherence to regulatory standards.
The CHO HCPtargeted: LCMS Kit has been designed to address this challenge by providing a targeted and robust method for the quantification of 74 critical HCPs. Using liquid chromatography-mass spectrometry (LC-MS), a superior orthogonal method to ELISA, this kit offers highly sensitive and specific quantification, ensuring greater control over HCPs at all stages of the DSP.
The Power of Targeted Mass Spectrometry
Broad approaches such as Data Dependent Acquisition (DDA) and Data Independent Acquisition (DIA) are commonly used to obtain detailed HCP profiles. However, to produce robust and reproducible results, these methods need to be validated with more targeted techniques. The CHO HCPtargeted: LCMS Kit employs selected/multiple reaction monitoring (SRM/MRM) or parallel reaction monitoring (PRM), enabling highly precise and reproducible quantification of key HCPs.
This targeted approach is crucial for regulatory filings, as it allows for the reliable detection and quantification of critical HCPs in the DS as well as during various DSP steps. The CHO HCPtargeted: LCMS Kit is particularly useful for batch-to-batch comparisons and comprehensive downstream process evaluations, enabling manufacturers to make informed decisions about purification steps and overall product quality.
Method Overview
The CHO HCPtargeted: LCMS Kit is built on ANAQUANT’s proprietary READYBEADS® technology, which integrates seamlessly with SCOUT-MRM methods. In this application, we targeted 74 critical HCPs extracted from an internal HCP database and validated through literature, including well-known immunogenic proteins such as PLBL2 lipase, HTRA1 protease, and BIP proteins.
To perform the analysis, 82 peptides were screened using the SCOUT SRM method. The corresponding heavy internal standard peptides were adsorbed onto READYBEADS®, following a patented ANAQUANT process. This workflow ensures highly reproducible results, with normalized signals across different sample types, making it an ideal tool for comparing HCP levels between samples at various DSP stages.
Results
Analytical strategy was performed on therapeutic mAb harvest sample, on its post-protein A purified version and on the DS sample obtained after whole purification process. The ultimate objective was to screen all 74 well-known HCPs and to validate the best downstream process to use after post protein A purification.
Figure 2: Extracted chromatogram of PBPL2 (blue), Pyruvate kinase (grey), Protein chaperon BIP (Red) in harvest mAb sample, post prot A mAb sample, and DS mAb sample
Among all 74 well described HCPs screened, 50 were identified in harvest sample, 15 remain present after proteinA purification step, while no risky HCPs were identified in corresponding DS sample (Table).
| Risky/well known HCPs screened | HCPs identified in Harvest | HCPs identified in post protA | HCPs identified in DS1 |
| 74 | 50 | 15 | 0 |
Concluding remark
he CHO HCPtargeted: LCMS Kit offers a powerful, easy-to-implement solution for the targeted quantification of 74 critical HCPs across different samples. By combining ANAQUANT’s innovative SCOUT-MRM method with READYBEADS® technology, this kit ensures high sensitivity, specificity, and reproducibility for routine HCP monitoring in biopharmaceutical production.
This robust analytical tool can significantly enhance DSP validation and regulatory compliance, ensuring product safety and quality. It is designed to be both scalable and user-friendly, making it an indispensable part of any biotherapeutic manufacturing workflow.
For further details or to learn more about how the CHO HCPtargeted: LCMS Kit can improve your HCP monitoring, contact ANAQUANT today.