Most antibody manufacturers currently use a three column platform comprising Protein A affinity chromatography
for product capture, followed by anion exchange (AEX) chromatography in flow-through mode to extract negatively charged contaminants, and then cation exchange (CEX) chromatography or hydrophobic interaction chromatography (HIC) in retention mode to remove positively charged contaminant species. This article presents a new process that uses membrane adsorbers for both flow-through and retention steps in antibody polishing. The process shows that a membrane adsorber in retention mode can be used efficiently in a commercial antibody manufacturing process.
Antibodies have been at the forefront of the biopharmaceuticals market for most of the last decade, and judging by the hundreds of antibody-based products at all stages of the clinical pipeline, these will continue to dominate the market for the foreseeable future.1 About half of all biopharmaceutical process trains have been designed for the production of monoclonal antibodies, and the industry has focused on a small number of platform technologies optimized to squeeze every last milligram of product from the feedstream.2 The vast majority of companies currently producing monoclonal antibodies (MAbs) use a three-column platform approach comprising Protein A affinity chromatography for product capture, followed by anion exchange (AEX) chromatography in flow-through mode to extract negatively charged contaminants such as host cell protein (HCP), endotoxins, host DNA, and leached Protein A, and then cation exchange (CEX) chromatography or hydrophobic interaction chromatography (HIC) in retention mode to remove positively charged contaminant species including residual HCP and product aggregates.
One disadvantage of this approach is that as cell culture titers increase, the downstream process becomes a bottleneck and costs increase in line with production scale, i.e., there is no economy of scale. The industry has been evaluating strategies to address such capacity and cost issues and has investigated different approaches, some at the low-technology end of the spectrum and others at the high-technology end. Among the latter, membrane adsorbers offer a flexible and cost-effective solution to some of the bottlenecks in antibody manufacture and are gaining acceptance as an alternative to traditional resin-based chromatography, particularly because they eliminate cleaning and validation costs, provide flexibility in production train design and scale-up, and can be used in viral clearance steps.3–5 Membrane adsorbers are thin, synthetic, microporous or macroporous membranes that are derivatized with functional groups akin to those on the equivalent resins. The membranes are stacked 10–15 layers deep in a comparatively small cartridge, generating a much smaller footprint than columns with a similar output. This reduces buffer consumption but increases the flow rate even though the bed height is much lower and there is a reduced pressure drop. Despite the increased flow rate, adsorption is efficient because the transport of solutes to their binding sites in a membrane adsorber occurs mainly by convection, while pore diffusion (the predominant mechanism in resins) is minimal. These benefits reduce process times to less than 10% of those associated with traditional stainless-steel columns.6 Another important advantage is the linear scale-up for parameters such as frontal surface area, bed volume, flow rate, and static binding capacity, while normalized dynamic binding capacity remains fairly constant at 10% or complete breakthrough.7 Scaling up process-scale operations therefore is a lot less troublesome than would be the case for standard resins, making the processing of up to 100-kg batches of antibodies a realistic proposal.8
SARTORIUS STEDIM BIOTECH GMBH
Currently, the benefits of membrane adsorbers are most apparent in flow-through applications because capacity constraints in retention mode, particularly at high loading rates, make resin chromatography a more attractive option for capture steps. Packed-bed chromatography certainly remains the preferred operation for capturing molecules of <200 kDa, especially when peak cutting and gradients are required for the separation of closely related species.8 However, for larger molecules (including most of the anticipated contaminants in antibody manufacture), membranes offer higher capacity and faster processing. For example, flow-through AEX for antibody polishing with a membrane adsorber can be conducted with a bed height of 4 mm at flow rates of more than 600 cm/h, providing a much higher frontal surface area to bed height ratio than is possible with columns. Even though the flow rate is much greater than would be possible with a column, there is sufficient retention time to reduce DNA, most HCP, and many viruses by up to four log reduction values, allowing membrane adsorbers to be used not only to separate the product from inert impurities but also as an integrated viral clearance step.9,10
This article presents a new process developed at Philogen that uses membrane adsorbers for both flow-through and retention steps in antibody polishing. The set-up was used successfully with Teleukin, a new MAb fusion protein in Phase 1–2 clinical development at Philogen.11 After an initial capture step using a traditional Protein A column, the feed is loaded onto a Sartobind Q AEX membrane adsorber (Sartorius Stedim Biotech, Göttingen, Germany), which elutes directly into a Sartobind S CEX adsorber (Sartorius Stedim). The AEX adsorber is operated in flow-through mode to retain HCP, DNA, and leachate, while the CEX adsorber operates in retention mode, allowing the pure antibody to be separated from positively charged impurities. Because the eluate from the first adsorber is loaded directly and automatically onto the second, this can be regarded as a single, integrated polishing step that achieves up to 90% recovery and 99.9% purity as determined by size exclusion chromatography, cation exchange HPLC, and SDS–PAGE.