A plethora of device configurations and membranes make it difficult to determine a method for a specific sample. Choosing the appropriate membrane to fit the macromolecule involves an understanding of how ultrafiltration works.

In the mid-1960s, Alan Michaels, PhD, and his colleagues at Amicon Inc. perfected a method developed by Loeb and Sourirajan to produce asymmetric ultrafiltration (UF) membranes. Rapidly, Amicon (acquired by Millipore in 1997) became the pioneer in UF device development (Figure 1). Laboratory-scale ultrafiltration has evolved significantly since that time. Originally developed as a technique to concentrate and desalt protein samples, the scope of the technology expanded as new device platforms have been engineered.

Numerous device configurations and membrane choices can make it difficult to determine an appropriate UF device for a specific sample. Choosing the appropriate membrane for a specific macromolecule involves an understanding of how UF membranes separate molecules on the basis of molecular size.

Before the development of UF, several classical methods were used to concentrate and desalt proteins, primarily dialysis, protein precipitation, and lyophilization. Dialysis

click the image to enlarge   Figure 1. Ultrafiltration devices timeline. (All images courtesy of Millipore Corp.)

is a gentle, passive method where solutes pass through a thin membrane with an associated molecular weight cut-off (MWCO). It is a slow diffusive process with the biological solution and the exchange solvent on opposing sides of the dialysis membrane. Dialysis is used mostly for desalting and buffer exchange, but not for protein concentration. Desalting by dialysis is time-consuming and relatively inefficient for highly diluted proteins. Pressure differentials can cause the dialysis membrane to stretch, changing the MWCO rating of the pores [S. S. Kuwahara, J. H. Chuan, "Process Validation of Separation Systems," in Separation Technology, W. P. Olson, Ed. (Interpharm Press Inc., Buffalo Grove, Ill., 1995), p. 448].

Protein precipitation is a method that uses solvents (e.g., acetone or aceto-nitrile) or salts (e.g., ammonium sulfate) to precipitate proteins out of solution. Proteins are then collected by centrifugation and resolubilized. The method is harsh and often results in irreversible protein denaturation and decreased solubility. Lyophilization is a freeze-drying method in which the protein solution is first frozen and then the solvent is reduced in a drying process involving sublimation and desorption. Lyophilization requires expensive equipment, is complex and time-consuming, and does not work well at low volumes.

Compared with these methods, UF is a convenient and gentle alternative. Unlike precipitation and lyophilization, there is no phase change during concentration. Proteins and other macromolecules can be concentrated safely and easily or desalted. It is a fast and efficient method which can be performed at room temperature or in a cold room with less of an effect on processing time than with dialysis. Unlike dialysis, UF can be used for both buffer exchange and concentration. Protein recovery after UF is typically greater than 90% when the appropriate device and membrane are used.

UF membranes can separate small particles and dissolved macromolecules from fluids. The membranes typically have a mean pore size between 10 Å and 500 Å (or 1.0 nm and 50 nm), which is significantly smaller than the range of microfiltration (MF) membranes (0.025 to 10 mm). Ultrafiltration effectively separates molecules that differ by at least an order of magnitude in size. Macromolecules ranging in size from 1 to 1,000 kDa are retained by an appropriately sized UF membrane, while salts and water pass through. Typical applications for UF are buffer exchange, protein concentration, and desalting. UF also is used to separate free from protein bound ligands, to remove unincorporated labels from labeled proteins and nucleic acids, and in the removal and exchange of sugars.

UF membranes are not absolute in their pore size ratings. Separation occurs as a result of differences in the filtration rate of different components across the membrane in response to a given pressure. Unlike UF membranes, microporous membranes have a precisely controlled pore size that ensures quantitative retention of particles, biomolecules, and microorganisms greater than the pore size of the membrane.

Rating ultrafilter membranes
Microfiltration membrane pore size can be characterized conveniently using the membrane's "bubble point." Each pore size of a specific membrane polymer has a range of pressure where bubbles will form when gas is applied to the wet membrane. Conversely, it is not as straightforward to characterize a UF membrane. Two methods are used for rating and differentiating UF membranes: protein markers and dextran challenge. Retention of proteins such as cytochrome C (MW=12,400 daltons) for membranes ≤10,000 nominal molecular weight limit (NMWL); BSA (MW=67,000 daltons) for membranes >10,000 and ≤50,000 NMWL; and IgG (MW=156,000 daltons) for 100,000 NMWL membranes is relatively standard across the industry. The percent retention of these proteins by membranes with the same rating can differ, depending on materials of composition, manufacturing quality, and reproducibility. For example, a regenerated cellulose membrane typically will exhibit protein retention of 90 to 95% for a globular solute at that molecular weight limit, but a polyethersulfone membrane, which has a more open structure and higher flow characteristics, will show 80 to 85% retention for the same protein. For example, a regenerated cellulose membrane typically will have protein retention of 90 to 95% for a globular solute at that molecular weight limit, but a polyethersulfone membrane, which has a more open structure and higher flow characteristics, has an 80 to 85% retention for the same protein.

Characterization of UF membranes also can be achieved using a polydispersed mixture of dextrans to determine retention behavior. The sigmoidal curve in Figure 2 demonstrates the percent retention of different molecular weight dextrans with a 100,000 NMWL membrane. At the membrane's rated size, it retains solutes of 100,000 dalton size at 90% efficiency, but it also retains solutes of 30,000 dalton size at 50% efficiency. This is the reason solutes must differ in size by an order of magnitude to be efficiently separated by UF.

Another UF application is purification and concentration of nucleic acids. Because nucleic acids have a rod-like structure, the appropriate membrane pore size for a given DNA or RNA is related to the length (in nucleotides) rather than the molecular weight. Therefore, a nucleotide cutoff (NCO) guideline is used instead. Other factors affecting

click the image to enlarge   Figure 2. Mixed dextran rejection profile for a 100,000 NMWL Ultracel regenerated cellulose membrane.

isolation of nucleic acids include the strandedness of the DNA or RNA molecule; whether the DNA is relaxed, linear, or supercoiled; the ionic strength of the solvent; and the centrifugal force applied to the membrane. Ideally, nucleic acid recovery is achieved in low-salt buffers run under conditions of relatively low velocity. Removal of unincorporated primers and salts from polymerase chain reaction (PCR) is commonly performed using either multiwell plates or centrifugal devices incorporating UF membranes.

Appropriate membranes
To assist researchers in choosing the best membrane for their globular protein application, a rule has been developed to rapidly calculate the appropriate membrane pore size (NMWL). It is a simple calculation based on the molecular weight of the protein to be concentrated or removed on the upstream side of the membrane. Used for regenerated cellulose membranes, the "rule of two" requires a membrane cutoff two times smaller than the proteins' molecular weight. The "rule of three" is applied to polyethersulfone membranes.

Using the example of a 65,000 MW protein, a 30,000 NMWL regenerated cellulose membrane would be appropriate because it is less than half of 65,000. For polyethersulfone, the 10,000 NMWL membrane is more than three times smaller than 65,000. Employing these rules results in >90% recovery of the solute of interest for a wide range of protein solutes. Other factors to consider when determining an optimal membrane include flow rate, also known as flux, solute concentration, solute composition, and temperature.

Ultrafiltration devices
Figure 1 tracks the introduction of UF devices developed over the past 40 years. Cones and holders introduced by Amicon were the first centrifugal UF devices available to researchers and were widely used for protein concentration and deproteination applications. In the 1980s, prefabricated devices were developed, eliminating the need to assemble the membranes and holders. Since the introduction of the Centricon device, many disc-based devices, which process samples from <1 mL up to 100 mL, have been commercialized. Invert spin technology is essential for maximum recovery of concentrated samples <0.1 mL.

Recently, improvements have been made to disc-based devices that process larger volumes of sample. The traditional technology employed normal flow filtration (NFF) by which fluid is convected directly toward the membrane. NFF often results in the accumulation of retained solute on the upstream side of the filter, a phenomenon known as concentration polarization. This layer of solute acts like an additional filter and slows or even stops filtration.

Newer designs employ a more efficient form of filtration known as tangential flow filtration (TFF), where solution flows tangentially along the surface of the membrane and

click the image to enlarge   Figure 3. The removal of salts from retained solutes using diafiltration. The sodium chloride concentration is reduced by dilution.

retained components do not build up on the surface. When used in a swinging bucket rotor, the centrifugal or g-force is parallel to the membrane, thus improving filtration efficiency. The vertical panel design accommodates a larger filter than traditional disc designs, so filtration is faster and more efficient. A dead-stop design feature prevents spinning samples to dryness, therefore maximizing sample recovery.

Another UF platform is a classical method developed prior to centrifugal devices—the stirred cell system. A stirring bar mixes the fluid above the UF membrane as air pressure drives filtration through the membrane. Stirred cell systems are capable of processing from a few milliliters to 2,000 mL, and more closely resemble an NFF than a TFF system. The system's flexibility can take advantage of the large selection of UF discs available in various materials of construction and NMWL. The time-consuming setup, cleaning, and maintenance are one drawback. Stirred cells also have a significant amount of dead volume (areas of the device that cannot be accessed), which presents recovery challenges. Recent developments in larger volume centrifugal devices have dramatically improved recovery and convenience for the 20 to 500 mL volume segment formerly serviced by stirred cell systems.

Ultrafiltration applications
The main protein applications for UF devices are concentration of chromatography fractions in protein purification protocols, desalting, and buffer exchange. The key benefit of using centrifugal devices instead of dialysis tubing is that proteins can be concentrated and buffer can be exchanged in the same device. The process of exchanging buffer using UF is termed diafiltration (Figure 3). During diafiltration, buffer is introduced into the upstream side of the membrane while filtrate is removed downstream. Diafiltration washes components out of the product pool into the filtrate, thereby exchanging buffers and reducing the concentration of undesirable species (salts, unincorporated labels, etc.).

Diafiltration is a valuable technique for antibodies and enzymes, since salt concentration, pH, and other buffer components used during purification can inactivate the proteins, and buffer exchange is typically necessary. Figure 4 illustrates the 20-fold difference in the activity of human RNA-dependent protein kinase (PKR) with and without buffer exchange using ultrafiltration [P. A. Lemaire, J. Cole, "Concentration of PKR and Buffer Exchange" in Ultrafiltration Handbook (Millipore Corp., Billerica, Mass., 2004), pp. 18-19]. This data also verifies that ultrafiltration is a gentle process which preserves activity.

Centrifugal UF devices are convenient alternatives to gel filtration for removal of free from bound species. Examples include removal of unincorporated labels (e.g., fluorescent, radioactive) in labeling applications and separation of free from protein-bound drug in drug discovery. UF simultaneously removes unincorporated ribonucleotides and salts from transcripts and concentrates RNA, a method which is much more efficient than gel purification or LiCl precipitation.

For low-throughput drug discovery applications and amino acid analysis, centrifugal devices are a convenient deproteinization method for serum, plasma, urine, and other

Figure 4. Comparison of human protein kinase autophosphorylation with buffer exchange by ultrafiltration and without buffer exchange.

biological samples. A 10,000 NMWL low-binding membrane will remove 95% of proteins with high recovery of small molecular weight solutes (for example, drugs, amino acids, and peptides) in the filtrate [E. Chernokalskaya et al., "Ultrafiltration for Proteomic Sample Preparation," Electrophoresis, vol. 25, pp. 2461-2468 (2004)]. Rapid PCR clean-up has been achieved for many years by using centrifugal UF devices. Concentrating extracted DNA samples improved the success rate of short tandem repeat (STR) amplification and yields a full STR allele profile, even with minute blood or semen stains ["Preparing Samples for Forensics Identification Analysis with Microcon Centrifugal Filters" in Ultrafiltration Handbook (Millipore Corp., Billerica, Mass., 2004), p. 39]. Recovery of amplified DNA is dependent on the total mass of PCR fragment and its size. Typically, recovery ranges from 60% for smaller fragments to near quantitative recoveries for fragments greater than about 500 bp.

Genomic DNA can be isolated and concentrated using centrifugal UF, a tool that often is employed in forensic analysis. Ultrafiltration is a fast, efficient and high-recovery method of preparation for a variety of biological samples. Recent improvements in membrane consistency and device design have made ultrafiltration devices the method of choice for concentration and purification of proteins and nucleic acids. Ultrafiltration is finding its way into many other applications including PCR purification, drug binding studies, and biomarker analysis.

The development of more accurate molecular weight cut-off membranes and devices can now accommodate larger membrane surface area in optimized device architecture. Next-generation devices could be designed to improve recovery and purity of macromolecules and potentially separate species that are closer in molecular weight.

About the Author
Kavonian is group product manager for protein research products, and Chernokalskaya is the director of technology development, Bioscience Division, Millipore Corp., Danvers, Mass.

This article was published in G & P magazine: Vol. 6, No. z, January/February, 2006, pp. 24-29.