David Rubin

Ultrafiltration (UF) is a low-pressure purification process that uses membranes to separate fine substances based on size and ionic charge. First developed during the late 1960s, UF technology has evolved through the years. As a result of improved membrane materials and re-engineered manufacturing techniques, current robust UF processes now serve a critical role in many biopharmaceutical applications (Table I).	

Normal flow filtration (NFF) and tangential flow filtration (TFF) are two widely adopted ultrafiltration platforms that are related, but differ in fluid stream flow mechanics. In NFF processes, the fluid stream is introduced perpendicular to the membrane surface (Figure 1). Substances smaller than the membrane pores become trapped either on the membrane's surface or within the membrane matrix, whereas the filtrate passes through the membrane. Sometimes referred to as "dead-end" or "depth" filtration, NFF is commonly used in applications such as clarification, prefiltration, sterile filtration and virus removal.	

In TFF processes, the fluid stream is introduced parallel to the membrane surface, resulting in a continuous sweeping of the filtration medium. Under low pressure, substances smaller than the membrane's pores escape as filtrate or permeate, and larger particles are retained as retentate. Because of TFF's inherent sweeping action and cross-flowing process stream, TFF-based platforms run more cleanly than NFF processes, in which separated particles can accumulate either on or in the membrane.	

Table I:  Biopharmaceutical applications for TFF-UF systems.	

TFF systems exhibit highly predictable performance characteristics, scalability, ease of use, speed and reliability - all of which have contributed to establishing this platform as the preferred separation method for many biopharmaceutical applications. TFF systems are frequently used in applications in which small molecules (1-1000 kD) in solution need to be separated (Table II). 	

The proper selection of UF membranes is critical to obtaining the desired separation results, and early users of membranes had few options.  Today, a wide variety of membranes is available, allowing users to be very discriminating in their selection.	

Figure 1:  Fluid stream flow dynamics for NFF and TFF systems.				

Membrane selection is based on several process parameters and, of course, the separation objective. The primary parameters are:	

Separation performance (retention). Perhaps the most critical parameter, separation performance is determined by NMWL (normal molecular weight limit). Membranes are available in numerous NMWLs, offering process engineers choices that allow fairly precise separations of proteins and other similarly sized substances.

Processing rate (flux). Membrane flux, as measured by flow rate per unit area, determines how much membrane is required to process the feed stream in the desired time period. Higher flux results when the actual pore size closely resembles the desired NMWL cut-off.  Maximizing membrane pore size allows process fluids to pass more quickly, thus increasing flux.

Mechanical integrity. A strong, mechanically intact membrane will perform consistently for a long period of time. Being quite fragile, the membrane is cast on a substrate to add strength and rigidity. Substrate composition and structure is as important to mechanical integrity as the membrane itself. Common membrane substrate materials include polyethersulfone (PES) and composite regenerated cellulose (RC).

The biopharmaceutical industry benefits from continuous improvements to both membranes and substrates. High performance composite membranes constructed of both RC and PES are now available in void-free structures (Figures 2A, 2B). The consistent internal matrix of composite membranes offers a more robust performance when compared with conventional membranes, in which cavernous voids (Figure 3) beneath a thin, dense skin can result in surface defects and reduced membrane strength.		

Table II:  Membrane classification by size.	

As a result of their unique structure and mechanical integrity, void-free membranes are highly stable, resist fouling and provide extremely high flux with consistent performance (Figures 4A, 4B). High performance membranes are manufactured with a more open average pore that allows 3040% greater permeability with high flux, compared with conventional membranes (Figure 5). Therefore, production systems employing void-free membranes provide high retention in a smaller, more efficient footprint. 	

Today's high-performance RC membranes are highly hydrophilic and, therefore, resistant to both fouling and protein absorption. RC membranes are also compatible with organic solvents and cleaning solutions with pH levels of 2-13. These membranes range in molecular weight cut-offs from 1-1000 kD. 	

Figure 2 A: Composite regenerated cellulose membrane. B: Composite polyethersulfone membrane. (In the UF membranes shown, the void structure has been eliminated.)	

Unlike conventional PES membranes that tend to absorb proteins and other biological compounds, Biomax PES membranes (Millipore Corporation, Bedford, Massachusetts, USA) are hydrophilically modified to be more resistant to fouling. These membranes operate across the entire range of pH values and can withstand exposure to oxidizing 	

Figure 3:  Conventional UF membranes exhibit cavernous voids beneath a thin, dense skin.	

chemicals, making them suitable for applications in which harsh cleaning agents are used or when sanitization is required. Similar to high performance RC membranes, these advanced PES membranes are available in NMWL cut-offs that range between 1-1000 kD.	

The first step in deciding upon a UF membrane format is to fully characterize both the process stream and the separation objective. Considerations that need to be addressed include the following:				

Will the product be contained in the retentate (such as protein concentration) or in the permeate?

What is the viscosity at the operating temperature? 

Will feedstream components have a tendency to bind to a membrane?

Once these questions have been answered, a membrane material can then be selected. High performance membranes are available in both PES and RC composite (Table III) and offer the best combination of performance variables.		

Figure 4A:  Void-free membranes significantly reduce the incidence of microdefects.

Membranes can be incorporated into a variety of configurations, including cassettes, spiral wound and hollow fibre (Figure 6), and each style has successfully been applied in a variety of applications for the biopharmaceutical industry. Determining the ideal UF membrane configuration depends on a number of factors such as cost, feed stream component characteristics, production volume and scaling needs.	

Spiral wound modules are considered an economical option for very large process volumes. These devices incorporate alternate layers of membranes and separator screens wrapped around a central core. The feed is introduced at one end of the module and flows down the cartridge's axis, while the filtrate (permeate) spirals to the core, where it is removed.				

Figure 4B:  Void-free membranes demonstrate consistent return of water permeability after cleaning.	

Hollow fibre modules consist of a bundle of membrane tubes that range in diameter from 0.1-2.0 mm. Feed is introduced inside the tube from which permeate passes through the tube wall and is collected in a shell. Hollow fibre modules enjoy the advantage of low shear and thus are better suited to specific applications. In general, however, hollow fibre modules are less efficient than cassettes for processing protein-rich feeds. Larger systems require high pumping capacities to achieve fluxes that are comparable with other membrane configurations, at the expense of yields and recovery.	

Cassette (or flat plate) configurations are the most common and versatile membranes in biopharmaceutical applications. Here, alternating layers of membrane and spacer screens are stacked together and then sealed. Cassettes accommodate numerous membrane and screen types and have high packing densities. Most importantly, cassettes containing high performance membranes allow linear scaling, which, for many research and development applications, makes them the obvious choice. Any benchtop or pilot plant work performed with a scalable membrane will greatly reduce the potential for surprises later in the development cycle.				

Figure 5:  High flux membrane versus conventional polyethersulfone UF membrane.	

Once a membrane material and configuration is determined, the actual membrane can be selected, using NMWL data as a guide. It is important to select a membrane with sufficient retention to meet yield goals. For most applications, the membrane's NMWL rating should be 20-30% of the molecular weight of the product to be retained. The morphology of proteins and molecules is usually complex - rarely do these non-spherical particles align optimally with a membrane pore. Additionally, proteins can assume different shapes when the pH changes. Therefore, changing a buffer or cleaning solution can affect membrane performance. Membranes also retain finer components from highly fouling feedstocks than they retain from cleaner feeds. For all these reasons, membrane ratings are nominal as they typically separate 80-90% of species at the rated molecular weight. Select at least two, preferably more, membranes for testing based on this criterion.				

The initial testing process can begin once the membrane type, NMWL rating and the device configuration are determined. Several key process parameters must be set, including cross-flow rate, transmembrane pressure, filtrate control, membrane area and diafiltration design (if any). Once these variables are established, testing of the selected membrane filtration devices can begin. Tests of each membrane should be conducted across the entire range of potential process flow rates and concentrations. Additionally, this battery of tests should be repeated for at least five different transmembrane pressures for each concentration.			

Table III: Void-free membane properties.								

Today's high performance, void-free membranes offer high-purity, high-yield and fast processing in a scalable cassette format. The membranes are available in both PES and composite RC in numerous NMWL ratings. These UF membrane devices enjoy widespread usage throughout the biopharmaceutical industry in which quality, reliability and performance are essential.			

Figure 6: UF devices are available in a variety of configurations.	

Articles

Ultrafiltration is a pressure-driven membrane filtering process used to separate and/or purify dissolved or suspended particles from water and other liquids. Recent advances in materials and membrane manufacturing techniques have led to ultrafiltration playing a pivotal role in a number of biopharmaceutical processes, including protein concentration and blood for actionation. This article examines the criteria that should be considered when selecting membranes for such applications.