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

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 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 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.

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

“…The design of the DFE fusion protein and the generation of transgenic plants are described elsewhere 5,17 .…”

“…The favored expression platform for mAbs are Chinese hamster ovary cell lines, which typically produce high mAb titers of up to 10 g•L -1 in the culture supernatant 3,4 . However, the production of mAbs in mammalian cell cultures is expensive due to the high cost of the medium and the need for sterile fermentation 5 . Alternative expression platforms such as plants potentially offer a faster, simpler, less expensive and more scalable approach for industrial manufacturing 6,7 .…”

“…Such alternatives may also circumvent the disadvantages of Protein A chromatography, including the harsh elution conditions at low pH (typically <3.5) that can potentially cause mAbs to undergo conformational changes that promote aggregation 13 . Importantly, Protein A is selective only for the Fc region of certain IgG subclasses, so non-functional molecules with truncated binding domains may co-purify with the intact product 5 , whereas mAb derivaties such as single-chain variable fragments do not bind to Protein A at all.…”

“…Here, we describe an alternative affinity chromatography resin for the capture of the HIV-neutralizing mAb 2F5 using its linear epitope ELDKWA (one letter amino acid code) 5,14 . The purification strategy for mAb 2F5 was evaluated in terms of antibody purity, yield and ligand stability.…”

“…This performance was comparable to Protein A resins 25,26 . The equilibrium dissociation constant (K D ) of DFE-purified 2F5 antibody and the synthetic ligand Fuzeon was 791 pM whereas that of a Protein A-purified counterpart was 763 pM 5 . Furthermore, no substantial color loss was observed in the resin over a total of 25 bind-and-elute cycles.…”