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A sensitive and robust HPLC method to quantify recombinant antibody fragments in E. coli crude cell lysate
Journal of Chromatography B ( IF 2.8 ) Pub Date : 2018-03-02 , DOI: 10.1016/j.jchromb.2018.02.044
Thomas Gundinger , Alexander Pansy , Oliver Spadiut

Antibody fragments (Fabs) represent a highly interesting class of biopharmaceuticals with a broad range of medical applications. They bind antigens comparable to full length monoclonal antibodies, but are smaller in size allowing increased tissue penetration, and lack the glycosylated Fc domain, which is why they can be produced in the prokaryotic expression host E. coli. Due to the presence of disulfide bonds Fabs are usually produced in the oxidative environment of the E. coli periplasm. Even though recombinant production in E. coli is cheaper and easier to realize than in mammalian cells, this intracellular production of the Fab in E. coli entails difficulties in subsequent product analytics. Whereas Fabs are produced extracellularly by mammalian cells and thus can be analyzed in the environment of a defined medium, recombinantly produced Fabs in E. coli have to be analyzed in crude cell lysate containing a lot of host cell proteins, host cell DNA and lipids. Thus, robust and sensitive HPLC analytics for Fab quantification is still scarce today and recombinant Fabs from E. coli are conventionally quantified by expensive and cumbersome immunoassays. In this study we developed a sensitive and robust affinity-based HPLC method for the quantification of recombinant Fabs in E. coli crude cell lysates with a limit of quantification down to 46 μg/mL and high reproducibility. This method will definitely be of key importance for strain generation as well as for early steps in upstream process development for recombinant E. coli producing Fabs.



中文翻译:

一种灵敏和鲁棒HPLC法在定量重组抗体片段ë大肠杆菌粗细胞裂解液

抗体片段(Fabs)代表了非常有趣的一类生物药物,具有广泛的医学应用。它们结合与全长单克隆抗体相当的抗原,但是尺寸较小,允许增加的组织渗透,并且缺少糖基化的Fc结构域,这就是为什么可以在原核表达宿主E中产生它们的原因。大肠杆菌。由于二硫键的存在,通常在E的氧化环境中产生Fab 。大肠杆菌周质。即使在重组生产ë大肠杆菌是便宜并且比在哺乳动物细胞中更容易实现,这胞内产生所述Fab在ë大肠杆菌在后续的产品分析中会遇到困难。Fab是由哺乳动物细胞在细胞外产生的,因此可以在特定培养基的环境中进行分析,而重组产生的Fab在E中大肠杆菌必须在含有大量宿主细胞蛋白,宿主细胞DNA和脂质的粗细胞裂解物中进行分析。因此,匮乏的今天和从重组的Fab的Fab量化健壮和敏感HPLC分析仍然是Ë大肠杆菌通常通过昂贵且繁琐的免疫测定来定量。在这项研究中,我们开发了一种灵敏且稳健的基于亲和力的HPLC方法,用于定量E中的重组Fab 。大肠杆菌粗细胞裂解物的定量限低至46μg/ mL,并且具有很高的重现性。此方法肯定会在上游工艺开发用于重组早期步骤的应变产生关键的重要性以及ë。产生大肠杆菌的Fab。

更新日期:2018-03-02
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