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

Monoclonal antibodies (mAbs) dominate themarketfor biopharmaceutical proteins because they provide active and passive immunotherapies for many different diseases. However, for most mAbs,two expensive manufacturing platforms are required.

Journal of Chromatography A, 1571 (2018) 55–64 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma A linear epitope coupled to DsRed provides an affinity ligand for the capture of monoclonal antibodies C Rühl a , M Knödler a , P Opdensteinen a , J.F Buyel a,b,∗ a b 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 a r t i c l e i n f o Article history: Received June 2018 Received in revised form 19 July 2018 Accepted August 2018 Available online August 2018 Keywords: Affinity chromatography Design of experiments Fluorescent protein carrier HIV-neutralizing monoclonal antibody Plant molecular farming Transient protein production a b s t r a c t 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-ofexperiments 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 mg mL−1 after a contact time of 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 © 2018 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Introduction Antibodies dominate the biopharmaceutical market, with more than 50 approved products and more than 300 candidates in the development pipeline [1] The total sales volume was more than D 40 billion in 2013, which is about 33% of all biopharmaceutical protein sales Most products are monoclonal antibodies (mAbs) that are typically produced in mammalian cells, such as Chinese hamster ovary (CHO) cells, with titers regularly exceeding ∼5 g L−1 in the culture supernatant [2] Despite these high product titers, upstream Abbreviations: CV, column volume; DoE, design of experiments; IMAC, immobilized metal-ion affinity chromatography; SPR, surface plasmon resonance; TSP, total soluble protein ∗ Corresponding author at: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074 Aachen, Germany E-mail addresses: clemens.ruehl@gmail.com (C Rühl), johannes.buyel@rwth-aachen.de, matthias.knoedler@ime.fraunhofer.de (M Knödler), patrick.opdensteinen@ime.fraunhofer.de (P Opdensteinen), johannes.buyel@ime.fraunhofer.de (J.F Buyel) production in mammalian cells is expensive due to the cost of media and the need for sterile conditions Alternative expression systems are therefore being investigated, including yeast such as Pichia pastoris [3] and plants, the latter offering a scalable and safe production platform [4] Plant-derived mAbs have already been tested in clinical trials, including the HIV-neutralizing mAb 2G12 [5] Regardless of the expression host, another major cost driver for mAb manufacturing is the reliance of most processes on a Protein A capture step, which has become the gold standard for initial purification [6] Although the production of this protein-based affinity ligand in bacterial systems is cost-effective, the resin is nevertheless expensive given the need for qualification before its use in processes that comply with good manufacturing practices (GMP) and also the substantial margin which reflects the lack suitable alternatives Depending on the production scale, the costs for the resin alone can amount to 10 million euros (assuming × 15,000-L bioreactors, and a 10-ton output of mAb product per year) [7] This corresponds to more than 25% of the total process costs [8] The impact of the Protein A resin on the cost of goods is one reason for the high market prices, often exceeding 2000 euros per g purified https://doi.org/10.1016/j.chroma.2018.08.014 0021-9673/© 2018 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/ 4.0/) 56 C Rühl et al / J Chromatogr A 1571 (2018) 55–64 mAb [9] Such prices are a major burden for healthcare systems and can be prohibitive in developing countries, especially if large doses of product are required For example, up to 12 g of mAb per patient is required for a lymphoma therapy [10], and up to g annually per person for a prophylactic anti-HIV treatment [5] Therefore, several inexpensive non-protein ligands have been developed that could in principle replace Protein A [11] Many of them preferentially target the constant regions of mAbs, e.g the MEP ligand binds to the CH2 domain [12], facilitating rapid process development due to the uniform elution conditions [13] However, the performance of such alternative resins in terms of recovery and purity has been inconsistent compared to Protein A, e.g both high and low purities have been reported following mAb elution from MEP [14–16], whereas >95% purity is typically achieved when using Protein A [17,18] Here we have developed an alternative approach for the affinity purification of mAbs based on the use of linear epitopes, in this case ELDKWA (one-letter amino acid code) for the HIV-neutralizing antibody 2F5 [19,20] We fused this epitope to the fluorescent protein DsRed [21] as a carrier, generating the fusion protein DsRed-2F5-Epitope (DFE) We then produced DFE in transgenic tobacco (Nicotiana tabacum) plants and purified it by singlestep immobilized metal-ion affinity chromatography (IMAC) We optimized the coupling of DFE to a Sepharose resin using a designof-experiments (DoE) approach, resulting in a novel affinity resin which we used to purify mAb 2F5 (transiently expressed in N benthamiana) from clarified leaf extracts We discuss the optimization of elution conditions and provide an initial cost evaluation, compared with a Protein A-based process counterpart construct using either the vacuum infiltration method [29] or manual injection into leaves [30] Whole plants or leaf sections were infiltrated with A tumefaciens (OD600nm = 1.0) in infiltration buffer (0.5 g L−1 Fertilizer MEGA (Planta Düngemittel GmbH, Regenstauf, Germany), 200 ␮M acetosyringone, pH 5.6) and cultivated for a further days before harvesting [30] 2.4 Protein extraction and clarification Proteins were extracted from plants by blade-based homogenization in mL extraction buffer (50 mM sodium phosphate, 500 mM sodium chloride, 10 mM sodium bisulfite, pH 8.0) per gram wet biomass, followed by clarification using a sequence of bag, depth and sterile filters [31] Tobacco extracts containing DFE were heat treated before clarification [28] 2.5 Immobilized metal-ion affinity chromatography DFE was purified by immobilized metal-ion affinity chromatography (IMAC) on an ÄKTApure system (GE Healthcare, Little Chalfont, UK) using an XK-26 column containing 53 mL of chelating Sepharose fast flow IMAC resin loaded with nickel ions After loading the clarified extract onto a conditioned column (extraction buffer without sodium bisulfite), the resin was washed with 10 column volumes (CVs) of buffer without imidazole followed by elution in buffer containing 300 mM imidazole at a flow rate of 50 cm h−1 The concentrations of protein and nucleic acid were monitored at 280 and 260 nm, respectively Materials and methods 2.1 Design of experiments 2.6 Coupling DFE to Sepharose resin Design Expert v10 (Stat-Ease, Minneapolis, MN, USA) was used to set up and evaluate all experimental designs The factors and levels are presented in the supplementary data (Table S1), and the detailed DoE method is discussed elsewhere [22] The purified DFE affinity ligand was immobilized on HiTrap NHS-activated [32] Sepharose HP columns (GE Healthcare) with a bed volume of mL Before coupling, the columns were washed with mL ice-cold mM hydrochloric acid at a flow rate of