Analysis of Host Cell Proteins (HCPs): Methods, Challenges, and an Efficient MWCO-Based Characterization Technique

The presence of host cell proteins (HCPs) in therapeutic protein drugs can have adverse effects on the production process and product performance. To effectively assess and control the risks associated with HCPs in therapeutic protein products, it is crucial to develop methods that can identify and monitor all HCP components.

Traditional Methods and their Challenges

The most commonly used method for HCP assay in therapeutic protein production is the enzyme-linked immunosorbent assay (ELISA). While ELISA allows for quantitative analysis of the overall abundance of HCPs using polyclonal antibodies, it lacks the ability to rapidly quantify individual HCP fractions and may not detect less immunogenic or non-immunogenic HCPs.

Complementary assays such as 1D/2D-PAGE, mass spectrometry (MS)-based techniques, and others have been developed for monitoring HCPs. Among these, liquid chromatography-tandem mass spectrometry (LC-MS/MS) stands out as an orthogonal complementary analysis method to ELISA, capable of simultaneous identification and quantitative analysis of HCP impurities.

However, the major challenge with MS-based methods is the large concentration difference between HCPs (usually in low ppm levels) and the target protein (high concentration). Mass spectrometers often struggle to identify and detect low concentrations of HCPs amidst high concentrations of target proteins. To address this challenge, two general strategies are available: the addition of separation methods such as 2D-LC and/or ion mobility to remove interfering peptides and improve separation efficiency, or focusing on sample preparation by enriching HCPs through affinity purification or molecular weight cut-off (MWCO) ultrafiltration. The latter strategy has shown great efficiency in enriching HCPs for subsequent identification and analysis.

Efficient MWCO-Based HCP Characterization Technique

The MWCO-based HCP characterization technique involves the following steps: first, the sample is treated with surfactant to release the interaction between the target protein and HCPs. Subsequently, most of the target protein is removed through MWCO ultrafiltration, while the HCPs are enriched, reducing the concentration difference between HCPs and the target protein. This strategy effectively narrows the dynamic concentration range, significantly improving the sensitivity of detecting low-abundance HCPs.

Sample Preparation and Protein Digestion

In the MWCO-based technique, target protein samples containing HCPs are dried and dissolved in Tris HCl dissociation buffer containing surfactants. Ultrafiltration is then performed to remove the target protein, followed by reduction, alkylation, and trypsin digestion of the remaining protein mixture. The resulting peptides are acidified, and a biphasic system is formed by mixing with ethyl acetate. The aqueous phase, containing the peptides, is collected, dried, and resuspended for further analysis.

LC-MS/MS Analysis

The MWCO-desalted peptide mixture is resuspended in formic acid solution and injected into an HPLC system coupled to a mass spectrometer. Peptides are separated on a C18 column using a linear gradient elution. The mass spectrometer operates in a data-dependent acquisition mode, generating data for subsequent proteomic analysis.

PRM Proteomic Analysis

Parallel reaction monitoring (PRM) is a targeted proteomics technology that enables the selective detection of specific proteins and peptides for absolute quantification. PRM analysis using high-resolution, high-precision mass spectrometry facilitates accurate identification and quantification of target proteins/peptides.

Data Analysis

Proteomic data analysis involves searching against the UniprotKB murine protein database, considering various modifications, cleavage deletions, and false discovery rate settings. PRM data are manually curated using targeted proteomics software for HCP identification, requiring at least two specific peptides.

Conclusion

Characterization and monitoring of HCPs are crucial in biopharmaceutical processes utilizing cell culture. The presence of numerous HCPs, varying in molecular weights and abundances, poses challenges for traditional detection methods. The MWCO-based technique, combined with Shotgun proteomics analysis, offers a simple and efficient approach to identify HCPs in therapeutic protein drug products. By removing interference from the target protein and enriching HCPs, this method significantly improves the detection of low-abundance HCPs, achieving sensitivity down to 1 ppm. However, the detection range for HCPs is influenced by the MWCO filter's pore size, necessitating the use of multiple orthogonal and complementary approaches for comprehensive HCP characterization.

As HCP characterization technology continues to advance, aided by proteomic databases, genomic information, and algorithms, the future holds promise for more accurate HCP detection. Predicting the immunogenicity, biological activity, safety, and interactions of HCPs with the target protein based on such data will enable better guidance for standardized production, quantitative risk assessment, process control space design, assessment of process change/improvement risks, comparability studies, and process optimization throughout the product life cycle, ultimately enhancing the quality and stability of the final product.