Biopharmaceutical manufacturing is divided into two areas: upstream fermentation or cell culture and downstream purification processes. Each area contains multiple unit operations. A unit operation is defined as a step in processing using a particular type of equipment. Here, we focus on downstream process development, which must reliably produce a highly purified drug substance (often >99%). Downstream processing includes recovery, capturing, and polishing steps.

The primary downstream unit operation is chromatography because of its simplicity and high resolving power (1). Most process trains involve at least two distinct, orthogonal chromatography steps chosen from the number of chromatography supports available with various ligands attached to solid matrix. Affinity, cation-exchange (CEX), anion-exchange (AEX), ceramic hydroxyapatite (CHT), and hydrophobic-interaction chromatography (HIC) are the main types of chromatography modes used in large-scale bioseparations. The mode used for polishing depends mainly on the nature of the impurities (2). Chromatography can be operated in bind-and-elute (e.g., during the initial capture on protein A resin in antibody manufacturing) or flow-through mode (e.g., during the contaminant removal with AEX membrane adsorbers or resins) (3, 4). For monoclonal antibody purification, AEX and HIC are often operated using flow-through mode, in which the product does not bind to the chromatography support but specific contaminants are retained (2).

In current biomanufacturing processes, which emphasize speed-to-market for business success, a standardized platform approach to downstream process development is applied to reduce time to clinic and improve process economics (2, 5, 6). Downstream costs account for nearly 50% of total manufacturing costs (6). Therefore, during process development, techniques are evaluated that reduce process complexity, buffer consumption, labor, and time — while still producing the required high-quality product. Furthermore, higher upstream productivity has prompted discussion on increasing throughput in downstream unit operations (7, 8).

Gottschalk, Thoemmes, and Etzel evaluated alternative formats for recovery and purification unit operations with nonchromatographic formats and membrane chromatography (7, 8). Given these requirements, a novel membrane adsorber was developed for large-scale purification of biomolecules based on HIC principles. A macroporous membrane structure was designed for high flow rates and binding capacities. Using the advantages of membrane chromatography technology, the novel Sartobind phenyl membrane can be considered as an alternative to resins for applications in which throughput and processing time need improvement.

Advantages of Membrane Chromatography Technology

Membrane chromatography is amenable to large-scale purification of biotherapeutic products and has been part of a commercial, FDA-approved process for DNA and virus clearance since 2001 (9). The performance of disposable membrane chromatography makes it suitable as a platform technology in large-scale biomanufacturing. A significant functional advantage of membranes over resins is that the transport of molecules to their binding sites takes place mainly by convection with minimal pore diffusion (Figure 1) (10), which results in a binding capacity more or less independent of the flow rate (11). The flow-rate limitation associated with HIC column chromatography can increase the risk of protein degradation because of long contact time on the hydrophobic surface. Moreover, because of their hydrodynamic benefits, flow-through processes based on membrane chromatography involve much smaller devices than columns with a similar throughput. This consequently reduces buffer consumption, processing time, and space requirements. Flow-through membrane chromatography can reduce ≤95% of buffer consumption and 66% of processing time (4). The advantages and performance of membrane chromatography have been well described (12,13,14,15).

Figure 1: (Click to enlarge)

Membrane adsorbers come in reusable form as well as a fully disposable technology that eliminates the need for reuse validation at large scales (10, 16). Our company has developed a Sartobind HIC membrane adsorber as detailed below.

Hydrophobic-Interaction Membrane Chromatography

HIC is used to purify molecules based on differences in their hydrophobicity. It is an efficient mode to remove dimers and high—molecular-weight aggregates in a monoclonal antibody purification process as a polishing step (17). HIC has evolved into one of the most powerful methods in preparative biochemistry (18,19,20,21). It has also been described as a method of clearing multiple product-related impurities in Fc fusion protein purification (22).