Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

Sisi Zhang; Hui Xiao; Ning Li

doi:10.3791/65544

JoVE Journal
Chemistry

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Chemistry

Host Cell Protein Analysis using Enrichment Beads Coupled with Limited Digestion

제한된 분해와 결합된 농축 비드를 사용한 숙주 세포 단백질 분석

Full Text

2,905 Views

09:45 min

January 19, 2024

DOI: 10.3791/65544-v

Sisi Zhang¹, Hui Xiao¹, Ning Li¹

¹Analytical Chemistry,Regeneron Pharmaceuticals Inc.

Please note that some of the translations on this page are AI generated. Click here for the English version.

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.

의약품(DP)에서 숙주 세포 단백질(HCP)을 농축하고 단백질체 농축 비드를 사용하여 펩타이드를 검출하기 위한 프로토콜이 제시됩니다. 이 방법은 자체 제조한 단클론 항체(mAb) 원료의약품(DS)을 사용하여 시연되며, 이는 성능 측면에서 다양한 방법을 평가하고 비교하기 위한 잘 특성화된 표준 물질입니다.

당사는 단백질체 농축 비드를 사용하여 의약품에서 저농도 숙주 세포 단백질을 검출하는 고감도 방법을 개발했습니다. 이 접근 방식은 의약품과 관련된 근본 원인과 위험을 찾는 데 도움이 됩니다. HCP 분석의 검출 한계를 개선하는 방법에는 여러 가지가 있으며, 단백질 A 고갈, 제한된 소화, 분자량 컷오프, 면역침전을 포함하되 이에 국한되지 않는 비표적 분석법이 있습니다.

액체 크로마토그래피, 다중 반응 모니터링 및 비액체 크로마토그래피 병렬 반응 모니터링을 포함한 숙주 세포 단백질을 검출하는 표적 방법. 이러한 HCP 분석과 관련된 주요 과제는 항체 약물과 숙주 세포 단백질 사이의 유의미한 동적 범위입니다. 따라서 대부분의 방법은 항체 펩타이드와 HCP 펩타이드 사이의 동적 범위를 줄이기 위해 LCMS 분석 전에 항체의 양을 줄이고 HCP를 농축하는 것을 목표로 합니다.

View the full transcript and gain access to thousands of scientific videos