HPLC and tandem detection to monitor conformational properties of biopharmaceuticals

被引：24

作者：

Luykx, DMAM

Goerdayal, SS

Dingemanse, PJ

Jiskoot, W

Jongen, PMJM

机构：

[1] Natl Inst Publ Hlth & Environm, RIVM, Ctr Biol Med & Med Technol, NL-3720 BA Bilthoven, Netherlands

[2] Univ Utrecht, Dept Pharmaceut, UIPS, NL-3508 TB Utrecht, Netherlands

来源：

JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES | 2005年 / 821卷 / 01期

关键词：

(reversed-phase) HPLC; biopharmaceuticals; protein conformation; intrinsic fluorescence; circular dichroism; tandem-detection; interferon-alfa; human growth hormone; anti tetanus immunoglobulin; human serum albumin;

D O I：

10.1016/j.jchromb.2005.04.005

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

High-performance liquid chromatography (HPLC) with UV, circular dichroism (CD) and intrinsic fluorescence detection was applied to monitor conformational properties of recombinant human interferon alpha 2b when performing size exclusion chromatography (SEC) and reversed-phase HPLC (RP-HPLC). In this way native conditions during SEC and structural changes of the protein during RP-HPLC were demonstrated. These results were confirmed by stand-alone fluorescence and CD measurements. With respect to HPLC tandem detection, the fluorescence detector compared favourably to the UV and CD detector regarding linearity, sensitivity and selectivity. SEC combined with intrinsic fluorescence scanning detection permits conformational analysis of small amounts of aggregates in the presence of excess native monomeric protein. In conclusion, HPLC with on-line UV and intrinsic fluorescence detection provides a promising concept for analysing the amount and conformational properties of a biopharmaceutical and its impurities. (c) 2005 Elsevier B.V. All rights reserved.

引用

页码：45 / 52

页数：8

共 26 条

[1] Trichloroacetic acid and trifluoroacetic acid-induced unfolding of cytochrome c:: stabilization of a native-like folded intermediate [J].