Polishing with membrane chromatography (MC) has achieved acceptance as state-of- the-art technology for charged impurities. Traditionally, anion-exchange (AEX) and cation-exchange (CEX) membrane chromatography have been used to remove charged contaminants such as host-cell proteins (HCPs), recombinant DNA, protein A, endotoxins, and viruses. In monoclonal antibody (MAb) processes, polishing steps usually follow a protein A affinity column step. In some cases, CEX capture is applied, either with at least one AEX or a combined AEX and CEX step. The latter may be replaced by a hydrophobic-interaction chromatography (HIC) step. Ceramic hydroxyapatite is also used, though less frequently.

Hydrophobic antibody aggregates formed during MAb manufacturing are frequent process-related impurities that must be removed during downstream processing because they can cause loss of activity as well as toxicity and immunogenicity. Because of their toxic potential, such aggregates can cause an unwanted response or even overreaction of a patient's immune system (anaphylaxis).

Typically, product aggregate levels are monitored using size-exclusion chromatography (SEC). Removal of aggregates from a protein solution, however, is typically performed using HIC because monomeric proteins display less hydrophobicity than aggregates do. Because they form at lower concentrations, flow-through mode is most favorable for modern MC, which is primarily driven by volume rather than mass capacity. This is reasonable because a flow-through approach significantly reduces buffer consumption and allows application of disposable devices. Until recently, however, HIC has been applied only in a bead/column format and bind-and-elute mode. Trace contaminants can be efficiently removed, particularly HCPs, recombinant DNA, leached protein A, and product-related impurities such as soluble aggregates.

PRODUCT FOCUS: PROTEINS (ANTIBODIES)



PROCESS FOCUS: DOWNSTREAM PROCESSING



WHO SHOULD READ: PROCESS DEVELOPMENT ENGINEERS, ANALYSTS



KEYWORDS: HYDROPHOBIC-INTERACTION CHROMATOGRAPHY, POLISHING, DISPOSABLES, LABORATORY SCALE



LEVEL: INTERMEDIATE

To make use of membrane capabilities for high flow rates and convective flow, Sartorius Stedim Biotech addressed the limitation of conventional beads and developed a hydrophobic membrane adsorber carrying a phenyl ligand to efficiently remove product aggregates (1). The novel phenyl membrane adsorber has proven useful for aggregate removal in a MAb purification process.

(Click to enlarge)

Development of the HIC Membrane

Flow rate and diffusion limitations with packed-bed resins can lengthen process times, which may increase the risk of protein unfolding and denaturation, leading to product loss (2). The developer's intention was to create a hydrophobic adsorber that shows hydrophobic interaction at high salt concentrations but keeps mass transfer limitation as small as possible. That would circumvent a number of disadvantages seen with traditional resins.

The new macroporous phenyl membrane adsorber has a pore size of >3 µm with a recommended flow rate of five bed volumes per minute. Binding sites for proteins are accessible by convection rather than diffusion. That minimizes the effect of decreased binding capacity at high flow rates (3). The mechanism for capturing hydrophobic target molecules is defined by interactions between the hydrophobic surfaces of proteins and the adsorber. A number of hydrophobic spots on each protein are open for interaction with the hydrophobic matrix at high salt concentrations.

Membrane Matrix: A second-generation membrane was developed that displays a porous structure to enhance surface accessibility. Structure and pore size of the base membrane drives permeability, accessibility, and binding capacity of this membrane (4). To exclude grafting processes (as known from traditional adsorbers), the HIC ligand was directly attached to cross-linked and reinforced cellulose. Binding capacity at high salt concentrations was almost equal to that of conventional beads, to which selectivity is similar when the membrane is loaded with protein mixtures (3).

Ligand: HIC separates and purifies biomolecules based on differences in their hydrophobicity. Half of a protein surface may be accessible for hydrophobic interactions. In this case, the strength of interaction depends on a sufficient number of exposed hydrophobic groups and on membrane ligand type and density. Sample properties, temperature, type, and pH influence the binding process, as do concentrations of salt and additives. The main development reason for choosing the phenyl ligand in this membrane adsorber was its capability to remove product-derived hydrophobic impurities and contaminants during MAb production. The ligand also displayed high selectivity and ≤20 mg MAb/mL dynamic binding capacity, making it a good compromise for polishing IgG in bind-and-elute operations (3).