Application note Polishing mAb derived host cell proteins with MiMode PuraBead® HL4 mixed-mode adsorbent Protein A adsorbents are commonly used as the first chromatography step in downstream purification to capture monoclonal antibodies (mAbs) and fragments containing an Fc region. Intermediate and polishing steps are incorporated after the capture step to achieve the final purity required for the intended application. These chromatographic steps remove residual impurities, including host cell proteins (HCPs), host cell DNA, aggregates, leached Protein A, endotoxin, and a variety of viruses. This application note describes the optimal conditions for the use of MiMode PuraBead® HL4 mixed-mode adsorbent for the polishing of mAb derived host cell proteins.
The primary capture of mAbs and fragments within this application note, use the GORE® Protein Capture Device, a composite PTFE membrane with a native-Protein A ligand. Use of intermediate washes with high salt, low pH, or arginine were evaluated for optimizing HCP removal on the GORE® Protein Capture Device. Post-affinity mAbs and fragments were then polished using MiMode PuraBead® HL4 , a novel adsorbent consisting of a proprietary mixed-mode ligand on beaded agarose.
MiMode PuraBead® HL4 : A unique mixed-mode adsorbent MiMode PuraBead® HL4 primarily utilizes hydrogen bonding and hydrophobic interaction chromatography to bind host cell impurities while the target protein remains unbound. Figure 1 visualizes the chemical diversity of mixed-mode adsorbers, including MiMode PuraBead® HL4 resin, by mapping ligand structures based on their functional group similarities. Each point represents a different resin, with its position determined by the properties of its ligand groups rather than a simple characteristic such as charge. Clusters in the middle of the graph indicate resins with similar ligand structures, suggesting comparable binding behaviors. In contrast, MiMode PuraBead® HL4 resin is positioned distinctly to the right, highlighting its unique ligand composition and different mode of action. The distinct ligand chemistry of MiMode PuraBead® HL4 resin, as shown in Figure 1, offers unique purification advantages compared to other resins with similar structures that may behave alike. This underscores the importance of selecting appropriate adsorbents for specific purification needs. This reinforces the value of a toolbox approach, allowing for resin selection based on specific process needs rather than a onesize-fits-all solution.
Figure 1: Chemical space diagram of commercial mixed-mode adsorbents.
Exploring the pH wash regime for Protein A capture Clarified immunoglobulin G (IgG) produced in Chinese hamster ovary (CHO) cells was loaded onto a 1 mL GORE® Protein Capture Device with Protein A equilibrated with PhosphateBuffered Saline (PBS). The device was washed with PBS, followed by a high-salt wash of PBS and 1.8 M NaCl. The salt was washed off with PBS before being eluted with 100 mM sodium citrate pH 3.4. An identical run was performed with a 100 mM sodium citrate pH 5 wash added prior to elution. IgG concentration was measured by UV, while aggregates, HCP levels, and HC DNA were assessed using SEC HPLC, ELISA, and PicoGreen, respectively. Table 1 summarizes the analytical results, showing that the run with the pH 5 wash had a similar impurity profile but a lower IgG yield.