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Debottlenecking downstream mAb purification.
With ever increasing titers, column chromatography has become a bottleneck in the downstream processing of monoclonal antibodies (mAbs) and a major barrier in the development of a truly disposable single-use mAb manufacturing facility. Recent developments in high- capacity membrane chromatography have shown the potential to provide disposable chromatographic solutions. There are several reasons to consider single-use purification. Emerging trends in the biopharmaceutical industry are for smaller, flexible, and multiproduct facilities that lead to lower manufacturing costs. These trends have emerged because of increased bioreactor titers, smaller market size for new biopharmaceuticals, and the high cost of a dedicated facility. Single-use purification allows an organization to capitalize on these emerging trends.
Protein A as a capture step
Protein A has traditionally been the most widely used capture step in the purification of mAbs. It yields a high purity material (> 99.5%) that only needs polishing steps to remove aggregates, residual host-cell protein (HCP), DNA, virus, and leached protein A. Its disadvantages are that it is not single-use ($ 9,000–12,000 per liter), needs an enzyme-linked immunosorbent assay (i.e., protein A) release assay, and requires a fireproof facility. In an effort to overcome protein A's disadvantages, several companies have introduced cation-exchange chromatography (i.e., chromatography beads) as a substitute capture step with good results.
Researchers at the biopharmaceutical company Percivia have demonstrated efficient capture (> 90 mg mAb/mL of resin), yield (> 95%) and purity (95% reduction in HCP) with the use of a cation-exchange resin, GigaCap S 650-M (Tosoh Bioscience) (1). Abbott Laboratories has used a cation-exchange resin for a capture step for its drug Humira (adalimumab), and the biopharmaceutical company Medarex also has used a cation exchange resin for capture of a mAb (1–2). In an optimization study, Genentech (now part of Roche) used cation-exchange capture (SP-Sepharose FF, Pharmacia) followed by hydrophobic interaction chromatography (HIC) and strong anion-exchange chromatography to reduce HCP to traditional levels achieved with protein A (3).
Single-use high-capacity membrane chromatography
Two recent studies have used single-use high-capacity cation (weak C) exchange membranes as a capture step for mAbs. The advantages of these membranes are high dynamic binding capacities, short processing times, low cost per membrane volume, and single use. Lawton has optimized the capture step for a high-capacity cation exchange membrane (Advective Flow Chromatography "C," Natrix Separations) and obtained greater than 75 mg mAb/mL membrane (10% dynamic capacity) with > 95% purity and removal of aggregates (4). Kuczewski et al. have described a complete single-use purification process using the same high capacity cation exchange membrane as Lawton (Advective Flow Chromatography "C," Natrix Separations) (4, 5). In its process, Percivia obtained bindings of 55 mg/mL membrane, yields > 95%, HCP reductions of > 96%, and some removal of aggregates. Further membrane polishing steps, consisting of anion-exchange flow-through (Chromasorb, Millipore) and HIC flow-through (Sartobind Phenyl, Sartorious Stedim Biotech) reduced HCP to less than 50 ppm and aggregates to less than 0.5%, which was in an acceptable range.
Recent results using high-capacity membrane chromatography sets the stage for single-use purification of mAbs. Combined with single-use bioreactors, flexible multiproduct facilities can be built for the low-cost manufacture of the next generation of mAbs.
Carl W. Lawton, PhD, is director of the Massachusetts Biomanufacturing Center at the University of Massachusetts, Lowell, Carl_Lawton@uml.edu
1. B Lain, M. Cacciuttolo, and G. Zarbis-Papastoitsis, Bioprocess Int. 7 (5), 26–34 (2009).
2. G. Ferreira et al., BioPharm. Int. 20 (5) 32–43 (2007).
3. D. Follman and R. Fahrner, J. Chromatogr. A, 1024 (1–2), 79–85 (2004).
4. C. Lawton, presentation at the BioProcess International Conference (Long Beach, CA, Nov. 2011).
5. M. Kuczewski et al., Biotechnol. J. 6 (1), 56–65 (2011).