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采用体积排阻色谱法对完整的人源单克隆抗体聚集体进行质谱分析。

Mass spectrometric analysis of intact human monoclonal antibody aggregates fractionated by size-exclusion chromatography.

机构信息

Division of Drug Delivery Technology Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.

出版信息

Pharm Res. 2010 Oct;27(10):2197-204. doi: 10.1007/s11095-010-0224-5. Epub 2010 Aug 3.

DOI:10.1007/s11095-010-0224-5
PMID:20680668
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2939344/
Abstract

PURPOSE

The aim of this study was to develop a method to characterize intact soluble monoclonal IgG1 antibody (IgG) oligomers by mass spectrometry.

METHODS

IgG aggregates (dimers, trimers, tetramers and high-molecular-weight oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatography (HP-SEC), dialyzed against ammonium acetate pH 6.0 (a mass spectrometry-compatible volatile buffer), and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS).

RESULTS

Monomeric and aggregated IgG fractions in the stressed IgG formulation were successfully isolated by HP-SEC. ESI-TOF MS analysis enabled us to determine the molecular weight of the monomeric IgG as well as the aggregates, including dimers, trimers and tetramers. HP-SEC separation and sample preparation proved to be necessary for good quality signal in ESI-TOF MS. Both the HP-SEC protocol and the ESI-TOF mass spectrometric technique were shown to leave the IgG oligomers largely intact.

CONCLUSIONS

ESI-TOF MS is a useful tool complementary to HP-SEC to identify and characterize small oligomeric protein aggregates.

摘要

目的

本研究旨在开发一种通过质谱法对完整的可溶性单克隆 IgG1 抗体（IgG）寡聚物进行特征分析的方法。

方法

通过使 IgG 制剂经历多次 pH 跳跃来产生 IgG 聚集物（二聚体、三聚体、四聚体和高分子量寡聚物）。通过高效体积排阻色谱（HP-SEC）分离蛋白质寡聚物级分，用乙酸铵 pH 6.0（质谱兼容的挥发性缓冲液）透析，并通过天然电喷雾电离飞行时间质谱（ESI-TOF MS）进行分析。

结果

通过 HP-SEC 成功分离了应激 IgG 制剂中的单体和聚集 IgG 级分。ESI-TOF MS 分析使我们能够确定单体 IgG 以及包括二聚体、三聚体和四聚体在内的聚集物的分子量。HP-SEC 分离和样品制备被证明对于 ESI-TOF MS 中的良好质量信号是必要的。HP-SEC 方案和 ESI-TOF 质谱技术均表明 IgG 寡聚物基本保持完整。

结论

ESI-TOF MS 是一种与 HP-SEC 互补的有用工具，可用于鉴定和表征小的寡聚蛋白聚集物。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/4ca3a58629eb/11095_2010_224_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/c9b4e26f9349/11095_2010_224_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/d6a353e91749/11095_2010_224_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/ea18903f9a69/11095_2010_224_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/92542bac25e5/11095_2010_224_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/7cfc11c2b4d4/11095_2010_224_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/4ca3a58629eb/11095_2010_224_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/c9b4e26f9349/11095_2010_224_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/d6a353e91749/11095_2010_224_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/ea18903f9a69/11095_2010_224_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/92542bac25e5/11095_2010_224_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/7cfc11c2b4d4/11095_2010_224_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fc58/2939344/4ca3a58629eb/11095_2010_224_Fig6_HTML.jpg

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