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

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

RWTH Aachen University

Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.

Downstream processing (DSP) is a major cost factor during the production of biopharmaceutical proteins. Clarification can account for ∼40% of these costs, especially when a large amount of dispersed particulate material is generated, such as during the extraction of intracellular proteins from plants. Filter capacity can be increased (and DSP costs reduced) by using flocculants. Here we show that cellulose-based filter aids can enhance the positive effect of flocculants by improving depth filter capacity even further. A design-of-experiments (DoE) approach was used to identify the optimal size and concentration of filter aids, at different values of pH and conductivity, for the clarification of tobacco leaf extracts during the production of a monoclonal antibody and a fluorescent protein. Filter aids ∼28 or ∼100 μm in length at concentrations of ∼10 and ∼5 g L(-1) respectively were most efficient in combination with a strong cationic flocculant, but were ineffective without the flocculant. The filter aids increased depth filter capacity by 35-fold compared to an additive-free extract reaching ∼1000 L m(-2) without affecting the target proteins. Thus, filter aids can be used to reduce production costs of plant-derived biopharmaceuticals while the DoE approach enabled the identification of robust process conditions.

Cellulose-based filter aids increase the capacity of depth filters during the downstream processing of plant-derived biopharmaceutical proteins.

The 2013-2016 Ebola outbreak highlighted the limited treatment options and lack of rapid response strategies for emerging pathogen outbreaks. Here, we propose an efficient development cycle using glycoengineered Pichia pastoris to produce monoclonal antibody cocktails against pathogens. To enable rapid genetic engineering of P. pastoris, we introduced a genomic landing pad for reliable recombinase-mediated DNA integration. We then created strains expressing each of the three monoclonal antibodies that comprise the ZMapp cocktail, and demonstrated that the secreted antibodies bind to the Ebola virus glycoprotein by immunofluorescence assay. We anticipate that this approach could accelerate the production of therapeutics against future pathogen outbreaks.

Production of Functional Anti-Ebola Antibodies in Pichia pastoris.

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.

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

The production of biopharmaceutical proteins in plants offers many advantages over traditional expression platforms, including improved safety, greater scalability and lower upstream production costs. However, most products are retained within plant cells or the apoplastic space instead of being secreted into a liquid medium, so downstream processing necessarily involves tissue and cell disruption followed by the removal of abundant particles and host cell proteins (HCPs). We investigated whether ultrafiltration/diafiltration (UF/DF) can simplify the purification of the model recombinant protein cyanovirin-N (CVN), an ~ 11 kDa HIV-neutralizing lectin, from tobacco extracts prior to chromatography. We compared different membrane types and process conditions, and found that at pH 8.0 and 50 mS cm an UF step using a 100 kDa regenerated cellulose membrane removed more than 80% of the ~ 0.75 mg mL total soluble protein present in the clarified plant extract. We recovered ~ 70% of the CVN and the product purity increased ~ 3-fold in the permeate. The underlying effects of tobacco HCP retention during the UF/DF step were investigated by measuring the zeta potential and particle size distribution in the 2-10,000 nm range. Combined with a subsequent 10 kDa DF step, this approach simultaneously reduced the process volume, conditioned the process intermediate, and facilitated early, chromatography-free purification. Due to the generic, size-based nature of the method, it is likely to be compatible with most products smaller than ~50 kDa.

A Combined Ultrafiltration/Diafiltration Step Facilitates the Purification of Cyanovirin-N From Transgenic Tobacco Extracts.

Cancer is the leading cause of death in industrialized countries. Cancer therapy often involves monoclonal antibodies or small-molecule drugs, but carbohydrate-binding lectins such as mistletoe (Viscum album) viscumin offer a potential alternative treatment strategy. Viscumin is toxic in mammalian cells, ruling them out as an efficient production system, and it forms inclusion bodies in Escherichia coli such that purification requires complex and lengthy refolding steps. We therefore investigated the transient expression of viscumin in intact Nicotiana benthamiana plants and Nicotiana tabacum Bright Yellow 2 plant-cell packs (PCPs), comparing a full-length viscumin gene construct to separate constructs for the A and B chains. As determined by capillary electrophoresis the maximum yield of purified heterodimeric viscumin in N. benthamiana was ~7 mg/kg fresh biomass with the full-length construct. The yield was about 50% higher in PCPs but reduced 10-fold when coexpressing A and B chains as individual polypeptides. Using a single-step lactosyl-Sepharose affinity resin, we purified viscumin to ~54%. The absence of refolding steps resulted in estimated cost savings of more than 80% when transient expression in tobacco was compared with E. coli. Furthermore, the plant-derived product was ~3-fold more toxic than the bacterially produced counterpart. We conclude that plants offer a suitable alternative for the production of complex biopharmaceutical proteins that are toxic to mammalian cells and that form inclusion bodies in bacteria.

Comparison of microbial and transient expression (tobacco plants and plant-cell packs) for the production and purification of the anticancer mistletoe lectin viscumin.

Patrick Opdensteinen

Institute for Molecular Biotechnology

RWTH Aachen University

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

Patrick Opdensteinen

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RWTH Aachen University

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

Institute for Molecular Biotechnology