Monoclonal Antibody (mAb)- based products are one of the most prominent biopharmaceutical products in recent years. mAb-based products were responsible for $38 billion in revenue for the 2009 global biopharmaceuticals market. Among genuinely new biopharmaceuticals, half (13 of 25) coming online in 2009 were mAb-based products; in particular, five of the ten top-selling products are mAb-based, confirming the preeminence of this product group in the biopharma sector [4]. The total global market for protein-based products is projected to grow at between 7% and 15% annually over the next several years and protein-based products are also predicted to represent 4 of the 5 top-selling drugs globally. Due to the emerging importance and potential of antibody products for therapeutic treatments, we have conducted a literature search in order to examine the current industry cost of producing antibody products.

It is clear that downstream processing of recombinantly produced antibodies is the limiting factor in large-scale production. Upstream processing, which typically involves the fermentation of mammalian or bacterial cell cultures to produce recombinant antibodies, has improved in recent years so that the focus is now on downstream processing [6]. Downstream processing relies heavily on several expensive filtration and chromatographic techniques, which can constitute up to 80% of the total cost of production [4]. Virus filters have a price tag of about $25,000 per run, and due to their single-use nature they have a significant impact to process costs [4]. Protein A chromatography, an industry standard for purification, is also expensive – up to $10,000 per liter (around $1.5 million for one column’s worth [7]). Although Protein A is recyclable, the regeneration process is expensive and time consuming [7].

Nearly every top mAb manufacturer is looking for alternatives to Protein A capture [7]. We surveyed various methods to circumvent the problem of current downstream processing techniques. Pressures to drive down manufacturing costs have encouraged the search for alternative production platforms, such as the use of transgenic expression systems in E. coli for antibody fragments [1]. Although most biopharmaceutical products are produced from mammalian Chinese hamster ovary (CHO) lines, E. coli is responsible for 17 of the 58 products produced from 2006-20104. Exploring E. coli as a production system for proteins that require an oxidizing environment has been shown to be difficult due to the necessary renaturation steps [3]. Improperly folded proteins can elicit an immune response in humans, however secretion could help avoid this problem. In addition, use of E. coli as a production system is beneficial due to the lack of post-translational modifications (PTM’s) that could increase blood half-life for some drugs [3].

Our project could provide an advantage over traditional antibody production methods by having a modular secretion system in place for single chain variable fragments (scFv) using E. coli as a host platform. scFv’s are favorable for a variety of reasons. Due to the absence of the Q2 on the constant region, scFv’s do not require N-glycosylation [5]. scFvs are also the smallest antibody formats that still retain antigen-binding properties. Their small size allows them to facilitate tissue penetration, and compared to full-size antibodies they demonstrate a short in vivo half-life, which is advantageous in medical imaging [5]. However, the formation of two intramolecular disulfide bonds is essential for correct scFv assembly. Our system secretes proteins into the periplasm and media via fusion to hyperosmotically inducible protein Y (OsmY), allowing oxidation of sulfhydryl groups and formation of disulfide bonds. Systems that enable secretion of the antibodies are also favored because it facilitates downstream processing [5]. Recent advances in scFv expression from the fungal model system Ustilago maydis, demonstrate optimization of their secretion system by use of a strong constitutive promoter and elimination of extracellular proteases [5]. This prevents bottlenecks in levels of mRNA and degradation due to extracellular proteases. By applying these optimization techniques to our system, we could further improve it in order to make a large impact on industrial antibody production.

References: [1] Farid, S. S. Process economics of industrial monoclonal antibody manufacture. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 848, 8–18 (2007).
[2] Novais, J., Titchener‐Hooker, N. & Hoare, M. Economic comparison between conventional and disposables‐based technology for the production of biopharmaceuticals. Biotechnology and Bioengineering 75, 143–153 (2001).
[3] Datar, R., Cartwright, T. & Rosen, C.-G. Process Economics of Animal Cell and Bacterial Fermentations: A Case Study Analysis of Tissue Plasminogen Activator. Bio/Technology 11, 349–357 (1993)
[4] Walsh, G. Biopharmaceutical benchmarks 2010. Nature Biotechnology (2010).
[5] Sarkari, P. et al. Improved expression of single-chain antibodies in Ustilago maydis.Journal of biotechnology (2014).
[6] Shukla, A. & Thömmes, J. Recent advances in large-scale production of monoclonal antibodies and related proteins. Trends in Biotechnology (2010).
[7] DePalma, Angelo. "Removing Impediments in Downstream Processing." Gen: Genetic Engineering & Biotechnology. 15 Apr. 2009. Web. 17 Oct. 2014.