Reproducibility of Host Cell Protein quantification with HCP PROFILER solution

Introduction

Biotherapeutics, molecules produced from biological systems, require rigorous purification steps to remove impurities and host cell proteins (HCP). Regulatory guidelines require pharmaceutical laboratories to monitor process-related impurities along the dow stream process purification workflow. ANAQUANT has developed an innovative standard dedicated to Host Cell Proteins identification and quantification (HCP analysis): the HCP PROFILER.  This solution is composed by a hydro-soluble bead (READYBEADS^TM technology) which, after being spiked directly into the sample, releases peptides at well controlled amount. The HCP PROFILER solution allows accurate and individual Host Cell Protein quantification assessment, in a single LC-MS analysis, with high reproducibility performances. It represents a technical solution for batches comparison (consistency) and downstream process performance studies regarding HCP removal. In this study we demonstrated the robustness of quantification results obtained with HCP PROFILER.

Results

The HCP profiler solution was thought to provide a calibration curve, with stability and reproducibility capabilities. Quantification reproducibility was tested over several months to demonstrate product reliability. The HCP quantification was performed in a viral vaccine intermediate of purification, known to be strongly stable over several months, and to be highly complex in the regards of protein number.

Briefly, sample was digested after that quality control was added (BSA from the NIST), then one HCPBEADS was added before a desalting step and MS analysis (high resolution MS analysis with Qexactive instrument). According to an easy and automatized data processing workflow, concentrations were obtained for each HCP. This analytical pipeline was performed at time 0 (production of the HCPBEADS), and after 7 and 9 months of storage (T7 and T9 respectively). First of all, the amount obtained for each individual protein was added to assess the total sample concentration. Results (table 1) demonstrated that, at the global level, the total protein concentration was highly reproducible, since the variation coefficient was found lower than 1% for the 3 samples. Also, the accuracy found for the BSA (QC) was always up to 80%.

Sample	T9mois	T7mois	T0	Variation coefficient
Total sample concentration [µg/ml]	395.83	388.13	390.96	0.99%
Total sample quantity [ng]	329.86	323.44	325.8	0.99%
Quality control Accuracy	84%	79%	94%

Table 1: Assessment of quantification reproducibility using HCP PROFILER solution by comparison of total Host Cell Proteins concentration in a same sample over 9 months. Assessment of quantification accuracy with BSA used as QC.

Then, a focus was done on the 20 most abundant proteins, which take part for approximatively 80% of the total concentration.

Figure 1: Assessment of quantification using HCP PROFILER standard by comparison of the twenty most abundant Host Cell Proteins quantity in a same sample over 9 months.

Results (Figure 1) demonstrated a good repeatability of proteins quantitation with HCP-PROFILER, indeed a large part of determined quantities are slightly similar between time 0 and time 7 or 9, since CVs between the 3 replicates never exceeded 22% for the 20 most abundant proteins. Thus, these results confirmed the reproducibility of HCP-PROFILER solution for identification and quantification purposes at large scale.

Conclusion

This product solves the reproducibility problems and biases user-induced while it ensures stability of standards over several months. It appears a valuable tool for biopharmaceuticals companies to ensure accuracy and reproducibility in Host cell protein analysis studies, particularly to monitor purification process performances and/or reproducibility.