High DBC at fast flow rates increases process productivity. With that higher alkaline stability and higher DBC, a bioprocess that includes Toyopearl AF-rProtein L-650F resin will require five times less resin to purify an amount of target fragment when that same process uses Capto L resin. A typical NaOH-contact time of 15 minutes allows running 40 cycles when using the Toyopearl AF-rProtein L-650F before dropping below 90% SBC, but only 19 cycles when using Capto L resin. Toyopearl AF-rProtein L-650F resin shows a higher alkaline stability than Capto L resin. Moreover, a higher DBC enables the use of highly concentrated elution pools, reducing the costs of subsequent purification steps. From a process economics viewpoint, that means that a process using Toyopearl AF-rProtein L-650F will require 2.5 times less resin to purify the same amount of target fragment than a process using Capto L resin. Equilibration buffer: 0.1 M sodium phosphate, pH 7.0, elution buffer: 0.1 M glycine/HCl, pH 2.5.Ĭompared with the DBC of Capto L resin, the DBC of Toyopearl AF-rProtein L-650F resin is roughly 2.5 times higher at any given residence time. We compared the DBC for two different fragments at different flow rates using Toyopearl AF-rProtein L-650F resin and Capto L resin (from Cytiva) (Figure 1).įigure 1: Toyopearl AF-rProtein L-650F resin exhibits a 2.5 times higher dynamic binding capacity (DBC) for scFv and Fab compared with Capto L resin. Table 1 lists process conditions by which we achieved an HCP log reduction of 3.37, a DNA log reduction of 4.30, and a recovery of 95.3%. We developed a process that allows those two main contaminants to flow through a column bed while target molecules are bound and later eluted from the column at low pH. The goal of the protein L capture step is to reduce the levels of host-cell protein (HCP) and DNA contaminations while increasing concentrations of target fragments.
That improves the efficiency and reduces the cost of capture steps for antibody fragments. After linking that ligand to the well-established Toyopearl rigid methacrylic polymer backbone, we obtained a stable resin with high dynamic binding capacity (DBC) at fast flow rates. Our ligand is a genetically optimized recombinant protein L with high chemical stability. Tosoh Bioscience has developed Toyopearl AF-rProtein L-650F as the best-in-class affinity resin for antibody fragment capture. Protein L is a surface protein of Peptostreptococcus magnus, which binds the κ-light-chain of antibodies present in most common fragments. As antibody fragments do not have an Fc-region, chromatography experts cannot use protein A affinity and must select protein L as a ligand for fragment capture. Downstream processing, however, can be challenging. They also can be produced in prokaryotic cells (such as Escherichia coli), a cell line more economic than mammalian expression systems. Recombinant-based antibody fragments can be modified to meet specific needs of affinity, avidity, valence, and action mode.
Upstream processing for antibody fragments is easier than it is for standard antibodies. These drugs offer several therapeutic advantages over conventional monoclonal antibodies. Antibody fragments are either separate functional subunits of antibodies or recombinant molecules, which, just like antibodies, are composed of immunoglobulin domains. Some of the latest promising biopharmaceutical drug substances are antibody fragments. Tosoh Bioscience’s SkillPak 1-mL and 5-mL prepacked columns
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