Summary

实验室规模的生产和治疗性抗体的纯化

Published: January 24, 2017
doi:

Summary

这个协议描述在哺乳动物表达系统中生产的治疗性抗体的。所描述的方法包括制备载体DNA,稳定转染和人胚肾293细胞系的无血清适应的,设置大规模培养和纯化使用亲和层析组成。

Abstract

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

Introduction

治疗性抗体的成功将继续推动大量投资进入的抗体开发为下一代疗法的浪潮开始。抗体市场预期由抗体片段1,抗体-药物偶联物2,双特异性抗体3和工程化抗体具有有利性质4重塑。另一类获得医药感兴趣的是生物仿制药。生物仿制药抗体是“高度相似”复制这种已经获得监管部门的批准治疗性抗体产品。拟议的生物仿制药必须是就其结构,功能,动物毒性,临床安全性和有效性,人体药代动力学(PK),药效学(PD)和免疫原性5,6鼻祖抗体相媲美。

批准ř生物仿制药的抗体的茨一直缓慢由于在产品的最终质量的严格限制。确切的制造过程,如通过对最终的处理步骤的特定细胞系和培养条件可以保持专有的。更重要的是,抗体的制造固有涉及程度可变性的可加至生产高度类似产品的挑战。全面理化和生物物理表征和比较是相当困难的,但许多研究表明生物仿制药的抗体的特性出现在文献7,8,9。

产生治疗性抗体便从哺乳动物宿主细胞用携带的基因的各抗体的载体的转染。载体设计,细胞系和培养条件是建立前的关键考虑因素PRESSION系统。

抗体的DNA序列可以从药物银行(www.drugbank.ca),IMGT(www.igmt.org)或研究的出版物,包括专利中采购。例如,曲妥单抗的序列,可通过药物银行(DB编号:DB00072)。可变区的氨基酸序列可经历基因设计和优化用于合成在所需宿主物种。它是没有修饰的氨基酸序列制成的生物仿制药抗体重要。一旦合成,抗体基因可亚克隆到所选择的合适的载体。

人IgG抗体由两条相同的重链和两个相同的轻链组成。两条链的紧密调节表达是用于在哺乳动物细胞10的异源抗体蛋白的最佳生产的关键。帧内以及链间二硫键必须形成和一些翻译后修饰的需要成为在蛋白质的生物合成过程中troduced。许多载体是已专为表达抗体基因(参考材料表)可用。这些特定抗体 – 载体通常表示为重链和轻链,以便仅在每个链的可变区所需要克隆的恒定区。

有两个独立的构建体(共转染)细胞的转染是用于输送重和轻链编码基因的最常见的方法。也就是说,每个基因通过其自身的启动子被组装的内质网前驱动和转录为单独的抗体链。另一方面,多顺反子载体具有掺入内部核糖体进入位点(IRES)元件,使多个基因作为与由mRNA 11的内部区域允许翻译单一的mRNA转录物的表达。在这种情况下,重链和轻链编码基因被连接在一个arrangEMENT同时实现抗体链10,12的共表达。

而瞬时转染细胞产生足够的蛋白来执行的实验的数量有限,已经经过选择基因组整合稳定转染的细胞系可以提供更高的产量。较高的蛋白质的量允许为涉及体外表征检测开发,并能提供在考虑抗体质量为下游应用的指示,如克隆细胞系和铅候选选择。

本文的目的是描述在哺乳动物表达系统中产生的治疗性抗体的稳定表达和纯化。事实上,这种方法可以适用于一生物仿制药抗体的表达。该方法可用于抗体的初始特性继续到关键的,尽管是费时STE之前确定一个理想的克隆用于大规模生产的PS。此外,这种方法可用于表达其它蛋白质,而不只是抗体。

下面的详细方案描述的治疗性抗体曲妥单抗的表达。这包括制备载体DNA随后在HEK-293细胞系和抗体蛋白质的纯化稳定转染通过自动化色谱法的。

Protocol

注:合适的哺乳动物表达载体,必须使用该协议。这里,使用含有两个表达盒的单个构建体( 即重链和轻链表达由单独的启动子驱动)。曲妥单抗重和轻链先前克隆到载体中。这个载体是从安德鲁Beavil的礼物,通过一个不以营利为目的质粒库13获得。 1.恢复和载体DNA的规模化发展注:载体DNA收到大肠杆菌 XL-1蓝色株软琼脂穿刺培养;载体携带潮霉素抗性。 </…

Representative Results

使用生物层干涉(BLI)转染HEK-293细胞的稳定生产单抗的结果确认, 如图1中。通过测量IgG抗体标准和蛋白质的生物传感器( 图1A)之间的结合率产生的IgG的标准曲线。粗制上清液样品类似地测量,那么其浓度从标准曲线( 图1B)内插。上清液的浓度测得为大约25微克/毫升双周(从传代培养收集50毫升取样)的无血清适应之后和之前为?…

Discussion

该协议细节的转染,稳定表达和治疗性抗体的纯化在HEK-293细胞。抗体基因的稳定表达是在产生用于治疗性抗体的开发和制造的抗体产生细胞系的第一步。而中国仓鼠卵巢(CHO)细胞仍然是治疗性蛋白质的选择的表达平台,HEK-293细胞系获得突出的实现,在这些细胞中产生的蛋白质是天然存在的人类蛋白质更匹配,在后方面-translational修改和功能14,15。

Disclosures

The authors have nothing to disclose.

Acknowledgements

The research was supported by the University of Sydney. pVITRO1-Trastuzumab-IgG1/κ was a gift from Andrew Beavil (Addgene plasmid # 61883). We thank Tihomir S. Dodev for useful discussions regarding pVITRO1-Trastuzumab-IgG1/κ.

Materials

pFUSE vector series N/A InvivoGen Heavy and light antibody genes expressed in separate vectors that require co-transfection.
mAbXpress vector series N/A ACYTE Biotech Pty Ltd. Heavy and light antibody genes expressed in separate vectors that require co-transfection. Refer to: Jones, M. L. et al. A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies. J Immunol Methods. 354 (1-2), 85-90, doi:10.1016/j.jim.2010.02.001, (2010).
pVITRO1 vector N/A N/A Heavy and light antibody genes are each driven by a separate promoter in a single vector.  Refer to: Dodev, T. S. et al. A tool kit for rapid cloning and expression of recombinant antibodies. Sci Rep. 4 5885, doi:10.1038/srep05885, (2014).
GS vector series N/A Lonza Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript.
Multi-cistronic vector series 1 N/A N/A Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Li, J. et al. A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies. J Immunol Methods. 318 (1-2), 113-124, doi:10.1016/j.jim.2006.10.010, (2007).
Multi-cistronic vector series 2 N/A N/A Multi-cistronic vector with heavy and light antibody genes co-expressed and translated as single transcript. Refer to: Ho, S. C. et al. IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol. 157 (1), 130-139, doi:10.1016/j.jbiotec.2011.09.023, (2012).
pVITRO1-Trastuzumab-IgG1/κ 61883 Addgene Mammalian expression vector containing trastuzumab antibody genes with hygromycin resistance gene; pVITRO1-Trastuzumab-IgG1/κ was a gift from Andrew Beavil.
Fast-Media Hygro Agar fas-hg-s Jomar Life Research Used to prepare low salt LB agar containing 75 µg/ml hygromycin.
Fast-Media Hygro TB fas-hg-l Jomar Life Research Used to prepare low salt TB broth containing 75 µg/ml hygromycin. 
Glycerol, BioXtra, ≥99% G6279 Sigma-Aldrich Prepare to 80% with water and autoclave. Store at room temperature.
Jestar 2.0/LFU Plasmid Maxi Kit G221020 Astral Scientific Plasmid Maxi Prep Kit; elute or resuspend DNA in water (pH 7.0-8.5).
FreeStyle 293-F Cells R790-07 Life Technologies HEK-293 cell line adapted to suspension culture in serum-free media.
FreeStyle 293 Expression Medium 12338-018 Life Technologies Serum-free media specially formulated for maintaing 293-F cell line and high protein expression.
Kolliphor P188 K4894 Sigma-Aldrich Non-ionic surfactant; pluronic F-68; prepare to 10% in water and filter-sterilize using 0.22 μm filter. Store at 4oC.
DMEM, high glucose  11995-065 Life Technologies
Heat-Inactivated Foetal Bovine Serum 10082-147 Life Technologies
Polyethylenimine, Linear, MW 25,000  23966 Polysciences, Inc. Prepare to 1 mg/ml in water. Adjust to pH 7.0 with 1 M HCl (solution becomes clear) and filter-sterilize using 0.22 μm filter. Store at -80oC until use.
OptiPro SFM 12309-050 Life Technologies Transfection formulated serum-free media
Hygromycin B Solution ant-hg-1 Jomar Life Research
Dimethylsulphoxide (DMSO) AJA2225 Thermo Fisher Scientific
Tryptone (casein peptone) LP0042B Thermo Fisher Scientific Prepare to 20% in PBS and filter-sterilize using 0.22 μm filter. Store at 4oC. 
Phosphate Buffered Saline (PBS) Tablets, pH 7.4, 100 ml 09-2051-100 Astral Scientific
HiTrap Protein A High Performance, 1 x 5 ml column GE17-0403-01 Sigma-Aldrich
AKTApurifier 100 28406266 GE Healthcare Automated FPLC system, which can include a P-960 sample pump and Frac-920 fraction collector.
Glycine-HCl G2879 Sigma-Aldrich
Citric Acid, monohydrate BIOC2123 Astral Scientific
Sodium Citrate, trisodium salt dihydrate  BIOCB0035 Astral Scientific
1 M Tris-HCl solution pH 9.0 BIOSD8146 Astral Scientific
Amicon Ultra Centrifugal Filters (30 MWCO) UFC803008/UFC903008 Merck Millipore Used to buffer exchange and concentrate purified protein.
Pierce Bicinchoninic Acid (BCA) Assay Kit 23227 Thermo Fisher Scientific
BLItz System 45-5000 fortéBIO Instrument used for bio-layer interferometry (BLI) measurements.
Protein A biosensors 18-5010 fortéBIO
Acrylamide/Bisacrylamide (37.5:1), 40% solution 786-502 Astral Scientific
Ammonium Persulfate (APS) AM0486 Astral Scientific
TEMED AM0761 Astral Scientific
Coomassie Brilliant Blue R-250 786-498 Astral Scientific
Precision Plus Dual-Color Protein Standard 1610374 Bio-Rad

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Cite This Article
Elgundi, Z., Sifniotis, V., Reslan, M., Cruz, E., Kayser, V. Laboratory Scale Production and Purification of a Therapeutic Antibody. J. Vis. Exp. (119), e55153, doi:10.3791/55153 (2017).

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