Laboratory Scale Production and Purification of a Therapeutic Antibody

Summary

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

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

The success of therapeutic antibodies continues to drive substantial investment into antibody development as a wave of next generation therapeutics begins. The antibody market is expected to be reshaped by antibody fragments ¹, antibody-drug conjugates ², bispecific antibodies ³ and engineered antibodies with favorable properties ⁴. Another class gaining pharmaceutical interest are biosimilars. Biosimilar antibodies are 'highly similar' replicate products of a therapeutic antibody that has already received regulatory approval. A proposed biosimilar must be comparabl....

Protocol

NOTE: A suitable mammalian expression vector must be used for this protocol. Here, a single construct containing two expression cassettes is used (i.e. heavy and light chain expression is driven by separate promoters). Trastuzumab heavy and light chains were previously cloned into the vector. This vector was a gift from Andrew Beavil, obtained through a not-for-profit plasmid repository ¹³.

1. Recovery and Scale-up of Vector DNA

NOTE: Vector DNA was received as a soft agar stab culture in Escherichia coli XL-1 Blue strain; vector carries hygromycin resistance.

1. Insert a sterile inoculation stab or loop....

Results

Stable production of trastuzumab by transfected HEK-293 cells was confirmed using bio-layer interferometry (BLI) as presented in Figure 1. An IgG standard curve was generated by measuring the binding rate between an IgG antibody standard and Protein A biosensor (Figure 1A). The crude supernatant sample was similarly measured, then its concentration interpolated from the standard curve (Figure 1B). The supernatant concentration was measure.......

Discussion

This protocol details the transfection, stable expression and purification of a therapeutic antibody in HEK-293 cells. Stable expression of antibody genes is the first step in generating an antibody-producing cell line for the development and manufacture of a therapeutic antibody. While Chinese hamster ovary (CHO) cells remain the expression platform of choice for therapeutic proteins, the HEK-293 cell line is gaining prominence with the realization that proteins produced in these cells are a closer match to naturally oc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pFUSE vector series	InvivoGen	N/A	Heavy and light antibody genes expressed in separate vectors that require co-transfection.
mAbXpress vector series	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	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	Addgene	61883	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	Jomar Life Research	fas-hg-s	Used to prepare low salt LB agar containing 75 µg/ml hygromycin.
Fast-Media Hygro TB	Jomar Life Research	fas-hg-l	Used to prepare low salt TB broth containing 75 µg/ml hygromycin.
Glycerol, BioXtra, ≥99%	Sigma-Aldrich	G6279	Prepare to 80% with water and autoclave. Store at room temperature.
Jestar 2.0/LFU Plasmid Maxi Kit	Astral Scientific	G221020	Plasmid Maxi Prep Kit; elute or resuspend DNA in water (pH 7.0-8.5).
FreeStyle 293-F Cells	Life Technologies	R790-07	HEK-293 cell line adapted to suspension culture in serum-free media.
FreeStyle 293 Expression Medium	Life Technologies	12338-018	Serum-free media specially formulated for maintaing 293-F cell line and high protein expression.
Kolliphor P188	Sigma-Aldrich	K4894	Non-ionic surfactant; pluronic F-68; prepare to 10% in water and filter-sterilize using 0.22 μm filter. Store at 4 °C.
DMEM, high glucose	Life Technologies	11995-065
Heat-Inactivated Foetal Bovine Serum	Life Technologies	10082-147
Polyethylenimine, Linear, MW 25,000	Polysciences, Inc.	23966	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 -80 °C until use.
OptiPro SFM	Life Technologies	12309-050	Transfection formulated serum-free media
Hygromycin B Solution	Jomar Life Research	ant-hg-1
Dimethylsulphoxide (DMSO)	Thermo Fisher Scientific	AJA2225
Tryptone (casein peptone)	Thermo Fisher Scientific	LP0042B	Prepare to 20% in PBS and filter-sterilize using 0.22 μm filter. Store at 4 °C.
Phosphate Buffered Saline (PBS) Tablets, pH 7.4, 100 ml	Astral Scientific	09-2051-100
HiTrap Protein A High Performance, 1 x 5 ml column	Sigma-Aldrich	GE17-0403-01
AKTApurifier 100	GE Healthcare	28406266	Automated FPLC system, which can include a P-960 sample pump and Frac-920 fraction collector.
Glycine-HCl	Sigma-Aldrich	G2879
Citric Acid, monohydrate	Astral Scientific	BIOC2123
Sodium Citrate, trisodium salt dihydrate	Astral Scientific	BIOCB0035
1 M Tris-HCl solution pH 9.0	Astral Scientific	BIOSD8146
Amicon Ultra Centrifugal Filters (30 MWCO)	Merck Millipore	UFC803008/UFC903008	Used to buffer exchange and concentrate purified protein.
Pierce Bicinchoninic Acid (BCA) Assay Kit	Thermo Fisher Scientific	23227
BLItz System	fortéBIO	45-5000	Instrument used for bio-layer interferometry (BLI) measurements.
Protein A biosensors	fortéBIO	18-5010
Acrylamide/Bisacrylamide (37.5:1), 40% solution	Astral Scientific	786-502
Ammonium Persulfate (APS)	Astral Scientific	AM0486
TEMED	Astral Scientific	AM0761
Coomassie Brilliant Blue R-250	Astral Scientific	786-498
Precision Plus Dual-Color Protein Standard	Bio-Rad	1610374