A new multimodal membrane adsorber for monoclonal antibody purifications

Abstract

This contribution describes research on the use of a newly developed multimodal membrane (MMM) adsorber that can be used as a chromatographic stationary phase in bioseparation processes. Compared with commercial cationic multimodal adsorbers, this MMM has superior static binding capacity (SBC = 180 mg IgG/ml), dynamic binding capacity (DBC 10% = 60 mg IgG/ml), and load productivity (>10 mg/ml/min). Furthermore, the incorporation of functional groups that provide orthogonal modes of interactions increases the range of ionic strength for operation of the MMM relative to conventional ion-exchange and hydrophobic interaction chromatography media. The effects of different salt types (kosmotropic, neutral, chaotropic salts) and ionic strength on IgG binding were investigated. To further understand the protein adsorption on the MMM, a thermodynamic model was employed to describe IgG adsorption isotherms on the MMM by providing a unique set of physically meaningful parameters for each salt type. The model was also a precise predictor of the adsorption isotherms under non-test conditions. A breakthrough analysis was used to determine dynamic binding capacities. The MMM maintained 70% DBC as ionic strength increased from 0 to 300 mM

Antibody purification from CHO cell supernatant using new multimodal membranes

“…The difference between DBC and equilibrium binding capacity is much more significant for the MMM than for Protein A HP resin. Previously we found that the binding reaction for this MMM is relatively slow and might possibly originate from the hydrophobicity of the ligands, which affects both the polymer conformation and the number of easily accessible binding sites . It appears that over time as protein binds within the polymer layer, it swells and provides access to additional binding sites, resulting in high equilibrium capacities.…”

“…The volumetric flow rate was 1 CV/min (1 min residence time). Much shorter residence times could be used for the membrane column, as we have demonstrated . A 1 min residence time was selected for comparison of the columns so that the Protein A column would have sufficient binding capacity, as its capacity decreases with increasing flow rate.…”

“…Figures and show results of the purification strategy to capture hIgG 1 from CHO cell supernatant using a size‐exclusion desalting column followed by MMM chromatography. The desalting operation was used to remove chaotropic salts reported in the cell culture medium, since our previous study has shown that chaotropes interrupt protein binding to the MMM. From a protein sample load volume study with the desalting column, it was found that a 5 mL sample CHO cell supernatant volume gave sufficiently high peak resolution.…”

“…At the same time, hydrophobic domains on the protein molecules also interact with hydrophobic groups on the MMMs, which helps to maintain the high binding capacity even under conditions of 150 mM NaCl used in the desalting column. Previously we showed that 150 mM NaCl has only a minor effect on the MMM binding capacity . In Figure , data collection was started when the hIgG 1 solution was injected.…”

“…The MMMs were developed and prepared in our laboratory, as described previously . The membranes comprise a macroporous regenerated cellulose membrane with a coating of poly(glycidyl methacrylate) that is modified by reaction with 4‐mercaptobenzoic acid.…”