Separation and Purification
Accelerating and simplifying bioprocessing
Ultrafiltration preserves biological activity and saves time - Protein purification technology has progressed from methods as diverse as chemical precipitation for sample concentration or dialysis for buffer exchange towards pressure-driven purification cross flow systems utilizing ultrafiltration membranes. Ultrafiltration (UF) techniques rely on the use of polymeric membranes with highly defined pore sizes to separate molecules according to size. Simply put, UF procedures rely on the use of fluid pressure to drive the migration of the smaller molecules through a UF membrane with the simultaneous retention of larger molecules.
While chemical precipitation can be used to concentrate a protein sample, separation with ultrafiltration is based on mechanical rather than chemical interactions allowing a researcher to perform sample concentration without the addition of denaturing solvents or salts. Buffer exchange using dialysis technologies use large volumes of buffer and since the only force acting upon the solution is diffusion, the process can take several days. Pre-assembled and simple to use ultrafiltration devices can rapidly perform either concentration or buffer exchange procedures without the extensive handling required for many other techniques.
Ultrafiltration can be performed in one of two operational modes: Direct Flow Filtration (DFF), or Tangential Flow Filtration (TFF, Figure 1). DFF works well for small volumes (up to 30 mL) using centrifugal devices, however, DFF technologies can fall prey to problems with membrane fouling. To reduce the formation of a gel layer, cross flow can be generated on the upstream side of the membrane using a floating stir bar configuration (stirred cell) or by creating a controlled laminar flow. While stirred cell operations tend to improve UF performance, they are still limited to achieving the optimal performance since the velocity and subsequent level of agitation is dependent on the sweep of the bar that varies along the radius of the sweep.
Tangential flow filtration (TFF) is a rapid and efficient method for separation and purification of biomolecules. It can be applied to a wide range of biological fields such as immunology, protein chemistry, molecular biology, biochemistry, and microbiology. TFF can be used to concentrate and desalt sample solutions ranging in volume from 10 mL to thousands of liters. It can be used to fractionate large from small biomolecules, harvest cell suspensions, and clarify fermentation broths and cell lysates.
Why Use Tangential Flow Filtration?
- Easy to set up and use – Simply connect the TFF device to a pump and pressure gauge(s) with tubing and a few fittings, add your sample to the reservoir, and begin filtration.
- Fast and efficient – It is easier to set up and much faster than dialysis. Higher concentrations can be achieved in less time than when using centrifugal devices or stirred cells.
- Perform two steps with one system – Concentrate and diafilter a sample on the same system, saving time and avoiding product loss.
- Can be scaled up or scaled down – Materials of construction and cassette path length allow conditions established during pilot-scale trials to be applied to process scale applications. TFF devices that can process sample volumes as small as 10 mL or as large as thousands of liters are available.
- Economical – TFF devices and cassettes can be cleaned and reused, or disposed of after single use. A simple integrity test can be performed to confirm that membrane and seals are intact.
Consider the Biomolecule of Interest
Your biomolecule of interest, or product, can be retained and separated from the low molecular weight contaminants, or it can be passed and purified from higher molecular weight contaminants and particles.
In general, a membrane with a molecular weight cut-off (MWCO) should be selected that is three to six times smaller than the molecular weight of the protein to be retained. Other factors can also impact the selection of the appropriate MWCO. For example, if flow rate (or processing time) is a major consideration, selection of a membrane with an MWCO toward the lower end of this range (3x) will yield higher flow rates. If recovery is the primary concern, selection of a tighter membrane (6x) will yield maximum recovery (with a slower flow rate). These values should be used as a general guide, as solute retention and selectivity can vary depending on many factors, such as transmembrane pressure, molecular shape or structure, solute concentration, presence of other solutes, and ionic conditions.
Our membranes are highly selective and typically achieve recoveries in the range of 95 to 99%. The narrow pore size distribution of these membranes results in minimal molecule retention of molecular weights below the MWCO of the membrane.
Consider Fluid Characteristics
Sample concentration and viscosity determine the type of channel that is required for the process run. Our lab scale TFF devices are available in screen or suspended screen configurations. Typically, screen channel configuration is used for clarified, dilute solutions free of particulate or aggregates. Suspended screen channel TFF cassettes provide better performance with highly viscous or particulate laden solutions.
Consider the Sample Volume and Processing Time
Choosing the appropriate cassette or device size depends on the total sample volume, the required process time, and the desired final sample volume.
Our Minimate™ system works with the Minimate capsules to easily process sample volumes up to 1000 mL. For process development and scale-up applications, Pall Life Sciences offers an extensive line of TFF holders and cassettes. With these products, a complete TFF system for full production can be optimized using the volumes typically generated in the development or discovery lab.
Key Applications for TFF
The primary applications for TFF are concentration, diafiltration (desalting and buffer exchange), and fractionation of large from small biomolecules. In addition, it can be used for clarification and removal of cells, as well as cellular debris from fermentation or cell culture broths.
Concentration is a simple process that involves removing fluid from a solution while retaining the solute molecules. The concentration of the solute increases in direct proportion to the decrease in solution volume (i.e., halving the volume effectively doubles the concentration). To concentrate a sample, choose an ultrafiltration (UF) membrane with an MWCO that is substantially lower than the molecular weight of the molecules to be retained. This is important in order to assure complete retention and high recovery of the target molecule.
Diafiltration is the fractionation process that washes smaller molecules through a membrane and leaves larger molecules in the retentate without ultimately changing concentration. It can be used to remove salts or exchange buffers. It can remove ethanol or other small solvents or additives.
There are several ways to perform diafiltration. In continuous diafiltration, the diafiltration solution (water or buffer) is added to the sample feed reservoir at the same rate as filtrate is generated. In this way, the volume in the sample reservoir remains constant, but the small molecules (e.g., salts) that can freely permeate through the membrane are washed away. Using salt removal as an example, each additional diafiltration volume (DV) reduces the salt concentration further. (Adding a volume of water or buffer to the feed reservoir equal to the volume of product in the system, then concentrating back to the starting volume constitutes one diafiltration volume. For example, if you have a 500 mL sample to start, 1 DV = 500 mL.) Using 5 DV will reduce the ionic strength by ~99% with continuous diafiltration.
In discontinuous diafiltration, the solution is first diluted and then concentrated back to the starting volume. This process is then repeated until the required concentration of small molecules (e.g., salts) remaining in the reservoir is reached. Each additional DV reduces the salt concentration further. Using 5 DV will reduce the ionic strength by ~96% with discontinuous diafiltration. Continuous diafiltration requires less filtrate volume to achieve the same degree of salt reduction as discontinuous diafiltration, as illustrated in the table on the right. By first concentrating a sample, the amount of diafiltration solution required to achieve a specified ionic strength can be substantially reduced. To reduce the ionic strength of a 1 liter sample by 96% using discontinuous diafiltration requires 5 DV or, in this case, 5 liters. If the sample is first concentrated tenfold to 100 mL, then 5 DV is now only 500 mL. This represents a substantial savings in buffer and time.