Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

Sisi Zhang; Hui Xiao; Ning Li

doi:10.3791/65544

JoVE Journal
Chemistry

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JoVE Journal Chemistry

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

エンリッチメントビーズと限定消化を組み合わせた宿主細胞タンパク質分析(英語)

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09:45 min

January 19, 2024

DOI: 10.3791/65544-v

Sisi Zhang¹, Hui Xiao¹, Ning Li¹

¹Analytical Chemistry,Regeneron Pharmaceuticals Inc.

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Overview

This article presents a sensitive method for enriching and detecting low-abundance host cell proteins (HCPs) from drug products using proteome enrichment beads. The approach is exemplified with a monoclonal antibody drug substance, providing a reference for evaluating various analytical methods.

Key Study Components

Area of Science

Biotechnology
Pharmaceutical Analysis
Proteomics

Background

Host cell proteins (HCPs) can interfere with drug product efficacy and safety.
Detecting low-abundance HCPs is crucial for quality control in biopharmaceuticals.
Traditional methods often struggle with the dynamic range between antibodies and HCPs.
Improving detection limits is essential for accurate HCP analysis.

Purpose of Study

To develop a method for enriching and detecting HCPs in drug products.
To identify challenges in HCP analysis and propose solutions.
To evaluate the performance of the method using a well-characterized monoclonal antibody.

Methods Used

Protein enrichment beads for HCP isolation.
Liquid chromatography coupled with mass spectrometry (LC-MS).
Targeted methods including immunoprecipitation and molecular weight cutoff.
Multiple reaction monitoring for enhanced sensitivity.

Main Results

The method successfully enriched HCPs from the drug product.
Improved detection limits were achieved compared to traditional methods.
The approach effectively reduced the dynamic range issues in LC-MS analysis.
Demonstrated applicability with a monoclonal antibody reference material.

Conclusions

The developed method enhances the detection of low-abundance HCPs.
It addresses significant challenges in HCP analysis for biopharmaceuticals.
This protocol can serve as a benchmark for future studies in HCP detection.

Frequently Asked Questions

What are host cell proteins?

Host cell proteins (HCPs) are proteins derived from the cells used to produce biopharmaceuticals, which can affect drug safety and efficacy.

Why is detecting HCPs important?

Detecting HCPs is crucial for ensuring the quality and safety of biopharmaceutical products.

What challenges exist in HCP analysis?

The main challenge is the dynamic range between the abundant therapeutic antibodies and the low-abundance HCPs.

How does the new method improve HCP detection?

The method enhances sensitivity and reduces the dynamic range issues by using protein enrichment beads and targeted detection techniques.

What techniques are used in this study?

Techniques include protein enrichment, liquid chromatography, and mass spectrometry.

Can this method be applied to other drug products?

Yes, the method can be adapted for various biopharmaceuticals beyond monoclonal antibodies.

医薬品(DP)から宿主細胞タンパク質(HCP)を濃縮し、プロテオーム濃縮ビーズを使用してペプチドを検出するためのプロトコルが示されています。この分析法は、自社製のモノクローナル抗体(mAb)原薬(DS)を使用して実証されており、これは、性能の観点からさまざまな分析法を評価および比較するための十分に特性評価された標準物質です。

私たちは、プロテオーム濃縮ビーズを用いて、医薬品由来の低存在量の宿主細胞タンパク質を検出する高感度法を開発しました。このアプローチは、医薬品に関連する根本原因とリスクを見つけるのに役立ちます。プロテインAの枯渇、消化の制限、分子量カットオフ、免疫沈降など、ノンターゲット法であるHCP分析の検出限界を改善する方法は複数あります。

液体クロマトグラフィー、多重反応モニタリング、非液体クロマトグラフィー並列反応モニタリングなど、宿主細胞タンパク質を検出するための標的を絞った方法。これらのHCP分析に関連する主な課題は、抗体薬物と宿主細胞タンパク質の間の大きなダイナミックレンジです。そのため、ほとんどの方法は、抗体ペプチドとHCPペプチドの間のダイナミックレンジを狭めるために、LCMS分析の前に抗体の量を減らし、HCPを濃縮することを目的としています。

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