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J Chromatogr A. 2018 Oct 12;1571:55-64. doi: 10.1016/j.chroma.2018.08.014. Epub 2018 Aug 7.

A linear epitope coupled to DsRed provides an affinity ligand for the capture of monoclonal antibodies.

Author information

1
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074 Aachen, Germany. Electronic address: clemens.ruehl@gmail.com.
2
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074 Aachen, Germany. Electronic address: johannes.buyel@rwth-aachen.de.
3
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074 Aachen, Germany. Electronic address: patrick.opdensteinen@ime.fraunhofer.de.
4
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074 Aachen, Germany; Institute for Molecular Biotechnology, Worringerweg 1, RWTH Aachen University, 52074 Aachen, Germany. Electronic address: johannes.buyel@ime.fraunhofer.de.

Abstract

Monoclonal antibodies (mAbs) dominate the market for biopharmaceutical proteins because they provide active and passive immunotherapies for many different diseases. However, for most mAbs, two expensive manufacturing platforms are required. These are mammalian cell cultures for upstream production and Protein A chromatography for product capture during downstream processing. Here we describe a novel affinity ligand based on the fluorescent protein DsRed as a carrier for the linear epitope ELDKWA, which can capture the HIV-neutralizing antibody 2F5. We produced the DsRed-2F5-Epitope (DFE) in transgenic tobacco (Nicotiana tabacum) plants and purified it using a combination of heat treatment and immobilized metal-ion affinity chromatography, resulting in a yield of 24 mg kg-1 at 90% purity. Using a design-of-experiments approach, we coupled up to 15 mg DFE per mL Sepharose. The resulting affinity resin was able to capture 2F5 from the clarified extract of N. benthamiana plants, achieving a purity of 97%, a recovery of >95% and an initial dynamic binding capacity at 10% product breakthrough of 4 mg mL-1 after a contact time of 2 min. The resin capacity declined to 15% of the starting value within 25 cycles when 1.25 M magnesium chloride was used for elution. We confirmed the binding activity of the 2F5 product by surface plasmon resonance spectroscopy. DFE is not yet optimized, and a cost analysis revealed that boosting DFE expression and increasing its capacity by fourfold will make the resin cost-competitive with some Protein A counterparts. The affinity resin can also be exploited to purify idiotype-specific mAbs.

KEYWORDS:

Affinity chromatography; Design of experiments; Fluorescent protein carrier; HIV-neutralizing monoclonal antibody; Plant molecular farming; Transient protein production

PMID:
30104060
DOI:
10.1016/j.chroma.2018.08.014
[Indexed for MEDLINE]
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