Maximize Biomolecule Separation With Tangential Flow Filtration

Introduction to tangential flow filtration What is tangential flow filtration Ultrafiltration fundamentals Applications TFF system selection considerations Capsules, cassettes, and systems Frequently asked questions Page  | Home  Page  | Home Contents 2 Introduction 02 01 03 04 05 06 07 08 What is tangential flow filtration? Introduction Applications pg 4 pg 3 pg 22 pg 9 pg 26 pg 30 pg 35 pg 13 TFF system selection considerations Ultrafiltration fundamentals Applications Concentration Diafiltration Fractionation TFF capsules, cassettes, and systems Frequently asked questions Glossary 01 Tangential flow filtration (TFF), also known as cross flow filtration, is an efficient method for separation and purification of biomolecules. The process fluid passes tangentially across the surface of a filter membrane, and as a pressure differential is applied to the system, constituents in the sample that are small enough to travel through the pore structure of the membrane will pass in to the filtrate. Larger constituents will be retained and recirculate around the flow path of the system. The method can be applied to many biological fields such as immunology, protein chemistry, molecular biology, biochemistry, and microbiology. TFF can be used to concentrate and desalt sample solutions ranging in volumes from 10 mL to thousands of liters. It can be used to fractionate large and small biomolecules, harvest cell suspensions, and clarify fermentation broths and cell lysates.  Page  | Home Introduction 3 Introduction 02 What is tangential flow filtration 4 What is Tangential flow filtration  Page  | Home Membrane filtration is a separation technique widely used in the life science laboratory. Depending on membrane porosity, it can be classified as a microfiltration or ultrafiltration (UF) process. Microfiltration membranes with pore sizes between 0.1 μm and 10 μm are used for clarification, sterilization, and removal of microparticulates or for cell harvesting. Ultrafiltration membranes, have much smaller pore sizes between, 0.001 μm and 0.1 μm, and are used for concentrating and desalting dissolved molecules, exchanging buffers, and gross fractionation. Ultrafiltration membranes are typically classified by molecular weight cut-off (MWCO) rather than pore size. There are two main membrane filtration modes that are used for microfiltration or ultrafiltration membranes: Direct flow filtration (DFF): Also known as “dead-end” filtration, applies the feed stream perpendicular to the membrane face and attempts to pass 100% of the fluid through the membrane. The resulting filtrate is then collected. What is tangential flow filtration Tangential flow filtration (TFF): Also known as crossflow filtration, where the feed stream passes parallel to the membrane face. Sample constituents that are small enough to travel through the pore structure of the membrane will pass, this is called the permeate or filtrate. Larger constituents are retained and recirculate around the system, this is called the retentate. Both the filtrate and retentate may be collected. In DFF, the small and large particulate mixture is forced toward the filter. Some smaller particles pass through but larger particles form a layer on the surface of the filter. This prevents small particles at the top of the mixture from reaching the filter (Fig 1). In contrast, operating in a TFF mode prevents the formation of a restrictive layer by recirculating the mixture. The turbulences remove the large particles that can block the holes in the flter, allowing the small particles at the top of the mixture to pass through (Fig 1). (A) (B) Fig 1. (A) Applying direct pressure to the mixture allows small particles to move through the filter A layer of larger particles quickly builds up and prevents further smaller particles from passing through the sieve. (B) Recirculating the sample breaks up the aggregated layer of large particles, allowing for the smaller particles to continue to pass through. 5 What is Tangential flow filtration  Page  | Home Fig 2. Direct flow filtration process. The feed is directed into the membrane. Molecules larger than the pores accumulate at the membrane surface blocking the flow of liquid and smaller molecules that could pass through. This layer of large molecules is referred to as a “gel layer”. As the volume filtered increases, fouling increases and the flux rate decreases rapidly, resulting in the membrane blocking and the end of the filtration. Sample solution flows through the feed channel and tangentially along the surface of the membrane. The crossflow prevents at the surface of the membrane. This disruption of the gel layer allows for the flow of molecules that are small enough to pass through the pores of the membrane to continue into the filtrate. The TFF process prevents the rapid decline in flux rate seen in direct flow filtration allowing a greater volume to be processed per unit area of membrane surface. Fig 3. Tangential flow filtration process. When processing a solution the differences between DFF and TFF can be visualized by plotting graphs of filtrate flux rate against volume filtered. The resulting plots can be seen in Figures 2 and 3. Volume filtration Flitrate flux rate Volume filtration Flitrate flux rate 6 What is Tangential flow filtration  Page  | Home What is Tangential flow filtration  Page  | Home Tangential flow filtration system configurations TFF systems typically require a TFF membrane device, a pump, tubing, valves or clamps, one or more pressure gauges, and a sample reservoir. Pressure gauges are typically installed at the feed and retentate ports in development and process TFF systems. While it is possible to run a TFF system without pressure gauges, the use of at least one pressure gauge between pump and TFF unit is strongly recommended. Pressure is an important variable in the TFF process. The ability to monitor and control the pressure leads to more consistent results, and can be very helpful for troubleshooting system problems. Fig 4. Flow path through a TFF system. Two of the important variables involved in all TFF devices are transmembrane pressure (TMP) and crossflow velocity (CF). 1. The transmembrane pressure is the force that drives fluid through the membrane, carrying along the permeable molecules. 2. The crossflow velocity is the rate of the solution flow through the feed channel and across the membrane. It provides the force that sweeps away molecules that can foul the membrane and restrict filtrate flow. Sample reservoir Pump Membrane Feed Retentate port Retentate valve Rententate Filtrate Filtrate Feed Port 7 What is Tangential flow filtration  Page  | Home Tangential flow filtration system operation When operating a TFF system, the fluid is pumped from the sample reservoir into the feed port, across the membrane surface, out the retentate port and back into the sample reservoir (Fig 4). The crossflow sweeps away larger molecules and aggregates that are retained on the surface of the membrane, preventing gel polarization. Liquid flowing through the narrow feed channel creates a pressure difference between the feed and retentate ports. This pressure, which is applied to the membrane, can be further increased by increasing the crossflow rate or by restricting the tubing at the retentate port by tightening the retentate valve. The resulting TMP is the force that drives liquid through the membrane. Liquid that flows through the membrane, called filtrate or permeate, carries molecules smaller than the membrane pores through the filter. Larger molecules that are unable to pass through the pores of the membrane will remain in the retentate and will continue to circulate around the flow path of the TFF system. Effectively regulating both the TMP and crossflow rate will prevent membrane fouling, thus allowing a greater volume of product to be processed in the least amount of time and improve the operational efficiency of the TFF system. Operation of a TFF system consists of the following steps: 1. Rinse the TFF device before use to remove any storage agent. 2. Establish the normalized water permeability (NWP) of the membrane to establish a baseline for the device performance, note - this step is not necessary but strongly recommended if the device will be cleaned and reused. 3. Condition the system with a sample buffer, conditioning helps remove air from the system, adjusts the systems temperature, and prevents possible precipitation or denaturation of biomolecules resulting from contact with flushing solution. 4. Process the sample (concentration and/or diafiltration, or fractionation). 5. Clean the system and TFF device and determine cleaning efficiency. 6. Store TFF device. Comprehensive and optimal systems operation including; capsule and cassette conditioning, NWP protocols, recommeded retentate flow rates, cleaning and storage protocols are supplied in Cytiva’s TFF Instructions for Use Guide. 8 03 Ultrafiltration fundamentals 9 Ultrafiltration fundamentals  Page  | Home Ultrafiltration (UF) is a membrane separation technique used to separate extremely small particles and dissolved molecules in fluids. The primary basis for separation is molecular size, although other factors such as molecular shape and charge can also play a role. Molecules larger than the membrane pores will be retained at the surface of the membrane and concentrated during the ultrafiltration process. Compared to non-membrane processes ultrafiltration: • Is gentler to the molecules being processed • Does not require an organic extraction which may denature labile proteins • Maintains the ionic and pH environment • Is fast and relatively inexpensive • May be performed at low temperatures (like a cold room) • Is very efficient and can simultaneously concentrate and purify molecules The retention properties of ultrafiltration membranes are expressed as molecular weight cut-off (MWCO). This value refers to the approximate molecular weight (MW) of a dilute globular solute, i.e., a typical protein, which is > 90% retained by the membrane. However, a molecule’s shape can have a direct effect on its retention by a membrane. Ultrafiltration fundamentals For example, linear molecules like DNA may find their way through pores that will retain a globular species of the same molecular weight. Our Omega™ membranes are highly selective. The curves in Fig. 5 illustrate the selectivity of these membranes. The narrow pore size distribution results in minimal retention of molecules whose molecular weights fall below the MWCO of the membrane. There are three generic applications for ultrafiltration: Concentration: Ultrafiltration is a very convenient method for the concentration of dilute protein or DNA/RNA samples. It is gentle and efficient. Desalting and buffer exchange (diafiltration): Ultrafiltration provides a very convenient and efficient way to remove or exchange salts, remove detergents, separate free from bound molecules, remove low molecular weight materials, or rapidly change the ionic or pH environment. Fractionation: Ultrafiltration will not accomplish a sharp separation of two molecules with similar molecular weights. However, ultrafiltration can be used to perform gross fractionation. Fig 5. Selectivity of ultrafiltration membranes. Ultrafiltration fundamentals  Page  | Home The molecules to be separated should differ by at least one order of magnitude in size for effective separation. Fractionation using ultrafiltration is effective in applications such as the preparation of protein-free filtrates or the separation of unbound and bound constituents. Average retention (%) 102 103 104 105 106 1.0 μm Molecular weight (kDa) 1K MWCO 3K MWCO 5K MWCO 10K MWCO 30K MWCO 50K MWCO 100K MWCO 300K MWCO 1000K MWCO l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l 10 Ultrafiltration fundamentals  Page  | Home Importance of selection correct MWCO Table 1 provides retention characteristics of different MWCO membranes for various solutes. For proteins, it is recommended that a MWCO be selected that is 3 to 6 times smaller than the molecular weight of the solute being retained. If flow rate is a consideration, choose a membrane with a MWCO at the lower end of this range; if the main concern is retention, choose a tighter membrane. It is important to recognize that retention of a molecule by a UF membrane is determined by a variety of factors, among which its molecular weight serves only as a general indicator. Therefore, choosing the appropriate MWCO for a specific application requires the consideration of a number of factors including molecular shape, electrical charge, sample concentration, sample composition, and operating conditions. Common variables that increase molecular passage: • Sample concentration less than 1 mg/mL • Linear versus globular molecules • High transmembrane pressure • Buffer composition that favors breakup of molecules • pH and ionic conditions that change the molecule Omega membrane Cytiva’s Omega polyethersulfone (PES) membranes offer high flux and selectivities. They have been specifically modified to minimize protein binding to the surface and interstitial structure of the membrane. This polymeric membrane is stable against biological and physical degradation due to the unique chemical properties of PES. Omega membranes are cast on a highly porous, nonwoven polyolefin support. They have an anisotropic structure, a thin-skinned like top layer with a highly porous underlying support. The structure of the skin determines the porosity and permeability characteristics of the membrane and can typically be cleaned quicker and easier than membranes with a uniform, sub-micron depth structure. This membrane is compatible with acids, bases and a variety of other cleaning agents. Omega membranes are available in a wide range of nominal MWCOs. Common variables that decrease molecular passage: • Sample concentration higher than 10 mg/mL • Buffer conditions that permit molecules to aggregate • Presence of other molecules that increase sample concentration • Lower transmembrane pressure • Adsorption to the membrane or device • Low temperature (4°C versus 24°C) Because different manufacturers use different molecules to define the MWCO of their membranes, it is important to perform pilot experiments to verify the membrane performance for a particular application. Cytiva centrifugal UF devices containing the Omega membrane can aid in offering an indication of membrane performance before scaling up to a larger Cytiva TFF system. Centrifugal devices allow the testing of small volumes of processing solution in a very easy to use and cost-effective way. 11 Ultrafiltration fundamentals  Page  | Home Table 1. Retention characteristics of Omega membrane MWCO selection of protein application MWCO (K) Membrane nominal pore size* (nm) Biomolecular size (nm) Biomolecule molecular weight (K) 1K - - 3K - 10K 3K - - 10K - 20K 5K - - 15K - 30K 10K - - 30K - 90K 30K - - 90K - 180K 50K 5 nm 15 - 30 nm 150K - 300K 70K - - 210K - 420K 100K 10 nm 30 - 90 nm 300K - 900K 300K 35 nm 90 -200 nm 900K - 1800K 500K - - 1500K - 3000K 1000K 100 nm 00 - 600 nm > 3000K MWCO selection for nucleic acid applications MWCO (K) Base pairs (DS) (BP) Bases (SS) (Bs) 1K 5 - 16 Bp 9 - 32 Bs 3K 16 - 32 Bp 32 - 65 Bs 5K 25 - 50 Bp 50 - 95 Bs 10K 50 - 145 Bp 90 - 285 Bs 30K 145 - 285 Bp 285 - 570 Bs 50K 240 - 475 Bp 475 - 950 Bs 100K 475 - 1450 Bp 950 - 2900 Bs 300K 1450 - 2900 Bp 2900 - 5700 Bs 1000K 4800 - 9500 Bp > 9500 Bs MWCO selection for virus applications MWCO (K) Membrane nominal pore size* (nm) Virus of particle diameter (nm) 100K 10 nm 30 - 90 nm 300K 35 nm 90 - 200 nm * Nominal pore size as measured by electron microscopy 12 04 Applications 13 Applications  Page  | Home The primary applications for TFF are concentration, diafiltration, and fractionation of large from small biomolecules. In addition, TFF can be used for clarification and removal of cells as well as cellular debris from fermentation or cell culture broths. Concentration 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 a UF membrane with a MWCO that is substantially lower than the MW of the molecules to be retained. This is important in order to assure complete retention and high recovery of the target molecule. A good general rule is to select a membrane with a MWCO that is 3 to 6 times lower than the MW of the molecules to be retained. If the sample will only be concentrated, then 3 times lower is sufficient. If significant diafiltration will also be applied to the sample, then an even lower MWCO may be advisable. The membrane is installed, or a disposable TFF capsule selected, and the TFF system is initialized. The sample is then added, a crossflow is established, feed and retentate pressures are set, then filtrate is collected. When the desired concentration is reached, the process is stopped, and sample recovered. Applications (A) (B) Fig 6. TFF concentration. When recovering the concentrate, it is important to note that a significant portion of the concentrated product could be on the membrane in the form of a gel layer. This will need to be recovered back into the solution before the system is drained. We supply detailed procedures on how to maximize concentrate recovery in the TFF Instructions for Use Guide. Applications  Page  | Home Retentate or concentrate Filtrate 14 Applications  Page  | Home Diafiltration Diafiltration is a technique that uses ultrafiltration membranes to completely remove, replace, or lower the concentration of salts or solvents from solutions containing proteins, peptides, nucleic acids, and other biomolecules. benefits of diafiltration Conventional techniques used for salt removal or buffer exchange such as membrane dialysis and column based gel filtration are effective but have limitations. Dialysis procedures can take up to several days, require large volumes of water for equilibration, and risk product loss through manual manipulation of the dialysis bags. Gel filtration results in a dilution of the sample and often requires an additional ultrafiltration step to concentrate it back. Adding steps to a process can risk sample loss or possible contamination. With diafiltration, salt or solvent removal and buffer exchange can be performed quickly and conveniently.The sample is concentrated on the same system, minimizing the risk of sample loss or contamination. There are several ways to perform diafiltration. While the end result may be the same, the time and volume required to complete the process may vary considerably. It is important to understand the differences in the methods used and when to choose one over the other. Continuous diafiltration The technique of continuous diafiltration involves washing out the original buffer salts or other low MW species in the retentate by adding water or a new buffer at the same rate as filtrate is being generated. Figure 7 shows a typical continuous diafiltration system setup using a Minimate™ EVO TFF system. The retentate volume and product concentration does not change during the diafiltration process. If water is used for diafiltering, the salts will be washed out and the conductivity lowered. If a buffer is used for diafiltering, the new buffer salt concentration will increase at a rate inversely proportional to that of the species being removed. The amount of salt removed is related to the filtrate volume generated, relative to the retentate volume. The filtrate volume generated is usually referred to in terms of diafiltration volumes (DV). A single DV is the volume of retentate when diafiltration is started. For continuous diafiltration, liquid is added at the same rate as filtrate is generated. When the volume of filtrate collected equals the starting retentate volume, 1 DV has been processed. Using continuous diafiltration, greater than 99.5% of a 100% permeable solute can be removed by washing through 6 retentate volumes with the buffer of choice. Molecules that are larger than salts and solvents, but are smaller than the pores in the membrane, can also be washed out. The permeability of these molecules may be less than 100%. In such cases, it will take more liquid, i.e., more DV’s, to completely wash a partially permeable molecule through the membrane. Typically, the larger the molecule, the lower the permeability and the greater the wash volume required. The permeability of a molecule through a specific membrane can be determined by measuring the concentration of the molecule in the filtrate compared to the concentration in the retentate under specified conditions. % permeability = (Conc.FILTRATE/Conc.RETENTATE) x 100 Permeability is often described in terms of rejection coefficient of the membrane, i.e. the membrane’s ability to hold back or reject a given molecule from passing through. Rejection coefficient = 1 – (Conc.FILTRATE/Conc.RETENTATE) A rejection coefficient of 1 equals 0% permeability A rejection coefficient of 0 equals 100% permeability Permeability will be affected by such factors as transmembrane pressure, crossflow rate, retentate concentration, pH, and ionic strength, and gel layer formation. Therefore, the permeability may change during the process. 15 Applications  Page  | Home A reservoir lid seals tightly allowing for diafiltration solution to be drawn directly into the reservoir without the need of a transfer pump. Vacuum is created as filtrate is generated through the TFF device allowing continuous diafiltration to be performed. Table 2 shows the relationship between permeability through a membrane and the number of diafiltration volumes required for removal of permeating species. As noted earlier, a greater volume of buffer is required to remove a molecule that is partially retained. To remove 99.9% of a molecule that is 75% permeable to the membrane requires 9 DVs, while for a 100% permeable species, only 7 DVs are required. Fig 7. Minimate EVO TFF system. Table 2. Continuous (constant volume) diafiltration Diafiltration volumes MWCO Permeability 100% Rejection Coefficient = 0 (%) Permeability 75% Rejection Coefficient = 0.25 (%) 1 63% 53% 2 86% 77% 3 95% 89% 4 98.2% 95% 5 99.3% 97.6% 6 99.7% 98.9% 7 99.9% 99.4% 8 99.7% 9 99.9% 0% - Salts, solvents, buffers, etc. 25% - Molecules lower in MW than MWCO of membrane but bigger than salts 16 Applications  Page  | Home Discontinuous diafiltration: Sequential dilution Discontinuous diafiltration by sequential dilution involves diluting the sample with water or replacement buffer to a predetermined volume. The diluted sample is then concentrated back to its original volume by ultrafiltration. This process is repeated until the unwanted salts, solvents, or smaller molecules are removed. Each subsequent dilution removes more of the small molecules. As shown in Figure 8, the sample is generally diluted with an equal volume of buffer (1 DV). Alternatively, multiple volumes can be added at once, provided the process tank is large enough to hold the entire volume. Diluting the sample usually lowers the viscosity, which may increase the filtrate flux rate. Start 1 DV=50% 2 DV=75% 3 DV=88% 2X Dilution 2X Dilution 2X Dilution 2X Conc. 2X Conc. 2X Conc. Large molecules: bigger than pores in membrane Small molecules: salts or solvent Large molecules: bigger than pores in membrane Small molecules: salts or solvent 1 DV=50% 2 DV=75% 3 DV=88% 2X Conc. 2X Conc. 2X Conc. 2X Dilution 2X Dilution 2X Dilution % Removal exchange % Removal exchange 5 8 5 4 5 2 8 12 14 5 5 16 16 5 8 5 4 5 5 2 8 5 4 5 2 8 12 14 5 16 5 8 5 4 Fig 8. Discontinuous diafiltration: Sequential dilution. Fig 9. Discontinuous diafiltration with volume reduction. Start 1 DV=50% 2 DV=75% 3 DV=88% 2X Dilution 2X Dilution 2X Dilution 2X Conc. 2X Conc. 2X Conc. Large molecules: bigger than pores in membrane Small molecules: salts or solvent Large molecules: bigger than pores in membrane Small molecules: salts or solvent 1 DV=50% 2 DV=75% 3 DV=88% 2X Conc. 2X Conc. 2X Conc. 2X Dilution 2X Dilution 2X Dilution % Removal exchange % Removal exchange 5 8 5 4 5 2 8 12 14 5 5 16 16 5 8 5 4 5 5 2 8 5 4 5 2 8 12 14 5 16 5 8 5 4 17 Applications  Page  | Home Discontinuous diafiltration: Volume reduction Discontinuous diafiltration by volume reduction reverses this procedure. The sample is first concentrated to a predetermined volume, and then diluted back to its original volume with water or replacement buffer. This is repeated until the unwanted salts, solvents, or smaller molecules are removed. Each subsequent concentration and dilution removes more of the small molecule (Fig 9). Following diafiltration, the sample may be concentrated or the next purification step performed. The final product, after diafiltration by either method is at the same volume and concentration as when diafiltration started. The salt concentration has been equally reduced in both examples. However, the volume of diafiltration buffer used by the volume reduction method was half that used in sequential dilution. This is because the initial concentration step reduced the volume in half. A diafiltration volume is equal to the volume where dilution occurs. Therefore, half the volume was required. This being the case, it would seem that concentrating before diafiltration, by either discontinuous sequential dilution or constant volume diafiltration, should reduce the required diafiltration buffer volume and save time. And in most cases this is true. The factor we have not accounted for is filtrate flux rate, which equates to process time. As the product becomes concentrated, viscosity increases and the filtrate flux rate decreases. The filtrate flux rate varies inversely as the log of the concentration factor. J = k ln(CG/CB) Where: J = Filtrate flux rate k = constant CG = gel layer concentration CB = retentate concentration This becomes very significant as the product concentration (CB) increases above a few percent and is dependent on the characteristics of the specific molecules that make up the sample. So, although it might take significantly less volume to diafilter a concentrated sample, it could take considerably more time compared to a less concentrated sample. Simple protocols are available to find optimum conditions to maximize productivity. Continuous or discontnuous diafiltration: Which technique should be used? When deciding which technique to use and where in the process to perform diafiltration, consider the following factors: • Initial sample volume, concentration, and viscosity • Required final sample concentration • Stability of sample at various concentrations • Volume of buffer required for diafiltration • Total processing time • Reservoir size available • Economics The choice of which method to use must be based on several criteria. Scale is an important consideration. What we will do at laboratory scale may be very different than at process scale, especially if the process is automated. At lab scale discontinuous diafiltration is often used for simplicity. Continuous diafiltration requires a pump or equipment to add the diafiltration solution at a constant rate. Both techniques can be automated for process applications. If we eliminate the equipment issue and focus on the process, we can compare the differences. 18 Applications  Page  | Home Table 3. Salt reduction from sample using reduction of constant volume diafiltration 2X Volume reduction Continuous diafiltration (constant volume) Diafiltration volumes 100% Permeable 0% Retention* (%) 75% Permeable 25% Retention* (%) 100% Permeable 0% Retention*(%) 75% Permeable 25% Retention*(%) 1 50% 41% 63% 53% 2 75% 65% 86% 77% 3 88% 79% 95% 89% 4 94% 88% 98.2% 95% 5 96.9% 93% 99.3% 97.6% 6 98.4% 95.6% 99.7% 98.9% 7 99.2% 97.4% 99.9% 99.4% 8 99.6% 98.4% 99.7% 9 99.8% 99.0% 99.9% 10 99.9% 99.4% * Retention of smaller molecules 0% - Salts, solvents, buffers, etc. 25% - Molecules lower in MW than MWCO of membrane but bigger than salts The ionic strength, buffer composition, and stabilizer concentration can affect stability of the sample. Diafiltration may remove salts or stabilizing molecules, resulting in protein product denaturation and aggregation. The process of concentrating and diluting a protein solution can also affect molecular interactions resulting in denaturation or aggregation as well as subsequent precipitation and product loss. It is necessary to evaluate the effect of concentration on the product to determine where diafiltration is best performed relative to concentration effects. Continuous diafiltration offers an advantage over discontinuous diafiltration in that the retentate concentration remains constant. It is often seen as a gentler process relative to the stability of the product. When to perform diafiltration: Before or after concentration? We have already seen that concentrating a sample first can significantly reduce the volume of diafiltration solution required. We have also seen that continuous diafiltration takes less volume than discontinuous diafiltration with sequential dilution. Therefore, if the sample is first concentrated to the final concentration required and then continuous diafiltration performed, acceptable results should be obtained. However, above a certain concentration, filtrate flux rates may become prohibitively slow. It may actually take longer to diafilter the concentrated sample than it would if the sample were first diluted to reduce the concentration. In this situation, even though continuous diafiltration of the diluted sample requires a greater diafiltration volume, the total processing time would be less due to the faster filtrate flux rate. (Process Time = Filtrate Flow Rate x Volume) In general, the optimum retentate concentration for performing (continuous) diafiltration is at: ln (CG/CR) = 1 or CR(optimum) = CG/e = 0.37CG Where: CG = gel layer concentration CR = retentate concentration. CR(optimum) = highest retentate concentration where diafiltration should be performed The CG value for a sample can be determined from experimentation by concentrating a sample on a membrane and recording and plotting data for filtrate flux rate vs. log concentration (or concentration factor). The curve can then be extrapolated to filtrate flux rate = “0”. The CG value will be the same for this product regardless of the starting concentration or filtrate flux rate. 19 Applications  Page  | Home Diafiltration summary Diafiltration is a fast and effective technique for desalting or buffer exchange of solutions. It can be performed in a continuous or discontinuous mode. Continuous diafiltration usually takes less volume to achieve the same degree of salt reduction and can be easier to perform. Continuous diafiltration is also perceived as a kinder and gentler process on active biomolecules. On the other hand, discontinuous diafiltration with volume reduction takes less volume than continuous diafiltration. Concentrating the sample before diafiltration usually reduces the required filtrate volume and saves time. However, if the sample viscosity becomes too great, the filtrate flux rate decreases and the process time can increase substantially. Finding the CG for the sample can help dermine. At what concentration diafiltration should be performed Product concentration Concentration factor 70 60 50 40 30 20 10 0 Filtrate flux rate 1 10 100 Fig 10. Determine the CG value of a product. In this example the CG value is a concentration factor of approximately 33X. Therefore the optimal concentration to perform diafiltration would be 0.37 CG = 12.2X. If the starting product concentration is 5 mg/mL, then diafiltration should be performed when the concentration reaches 61 mg/mL. If the final concentration will be less than 61 mg/mL, then diafiltration should be performed after concentration, unless it is necessary to remove a specific molecule prior to concentration. The ultrafiltration product selected may dictate choice of continuous or discontinuous diafiltration. Stirred cells and centrifugal devices are best suited for discontinuous diafiltration because of their mode of operation. Tangential flow devices have the advantage of being useful for either diafiltration technique. 20 Applications  Page  | Home Fractionation Ultrafiltration can be used to separate molecules based on their size. The molecules to be separated should differ by at least one order of magnitude in size for effective separation. When performing fractionation, the larger molecular fraction will remain in the retentate and continue to circulate around the flow path of the system, the smaller molecular fraction will travel through the membrane filter into the filtrate. The larger molecular fraction will slowly start to concentrate as the buffer solution passes into the filtrate. To avoid concentration of the larger molecular fraction it is possible to add new buffer to the sample feed reservoir at the same rate as filtrate is being generated. Fractionation using ultrafiltration is effective in applications such as the preparation of protein-free filtrates or the separation of unbound and bound constituents. If it’s not possible to accomplish a sharp separation of two molecules with similar molecular weights using ultrafiltration, more expensive chromatographical methods would have to be used. (A) (B) Fig 11. TFF fractionation. When recovering the filtrate, it is important to note that some solution may remain in the vent ports and tubing of the system. Cytiva supplies detailed procedures on how to maximize both concentrate and filtrate recovery in the TFF Instructions for user guide. Retentate or concentrate Filtrate 21 05 TFF system selection considerations 22 TFF system selection considerations  Page  | Home TFF system selection considerations  Page  | Home Step 1: Define the purpose of TFF process The biomolecule of interest in your sample is called a product. Separation can occur by choosing a membrane that retains the product while passing any low molecular weight contaminants. Alternatively, a membrane can be chosen that passes the product while retaining higher molecular weight components in the sample. It is also possible to combine both separations in a two-stage process that will fractionate out the product from both higher and lower molecular weight components. In the first stage, a membrane is chosen that passes the product and retains the higher molecular weight components. The filtrate from the first stage then becomes the sample for the second stage. For the second stage, the membrane is chosen to concentrate the product and remove lower molecular weight substances. You will need to define your separation goals – concentration, diafiltration, or fractionation. You must also consider the process volumes that you have to work with and any future scale up requirements. It is important to know the concentration factor or the level of salt reduction required in order to choose the most appropriate membrane and system for the process. In order to choose the best TFF system for your requirements review and consider the following steps: TFF system selection considerations Step 2: Choose the membrane MWCO The MWCO of a membrane is defined by its ability to retain a given percent of a molecule in solution, typically > 90% retention. As discussed earlier, to retain a product, select a membrane with a MWCO that is 3 to 6 times lower than the MW of the target protein. For fractionation, select a membrane MWCO that is lower than the MW of the molecule to be retained but higher than the MW of the molecule you are trying to pass. Please refer to Page 10 to review the retention characteristics of the Omega membrane. Step 3: Determine the required membrane area for the application Choosing an appropriate device, capsule, or cassette depends on the total sample volume, required process time, and desired final sample volume. Different TFF devices contain different membrane surface areas. Use the following equation to calculate the membrane area required for processing a sample in a specified time: A= V J × T Where: A = Membrane area (m2 ) V = Volume of filtrate generated (L) J = Filtrate flux rate [L/m2 /h (LMH)] T = Process time (h) Examples Example 1: What TFF system should I use to concentrate 10 L to 200 mL in 2.5 h? Assume the average filtrate flux rate of 50 L/m2 /h, LMH. Volumetric throughput (volume of filtrate) = 10 L – 0.2 L = 9.8 L A= 9.8 = 9.8 = 0.08 m2 50 L/m²/h x 2.5 h 125 Recommended System: Centramate™ holder with 1 membrane cassette, area of 0.093 m². Example 2: You have 50 mL of sample (MW = 54KD) collected from a Mustang™ Q membrane chromatography module that was eluted in a buffer solution (0.05 M Tris, 0.5 M NaCl). 23 TFF system selection considerations  Page  | Home You need to reduce the salt concentration below 0.05 M and then concentrate to 10 mL. Using a Minimate TFF capsule with a 10KD membrane on a Minimate EVO TFF system, how long will the process time be if the average filtrate flux rate is 40 LMH and 3 diafiltration volumes (constant volume diafiltration) are required to get the salt concentration below 0.05 M? Minimate area = 50 cm² = 0.005 m² Sample volume = 50 mL Diafiltration volume (1DV) = 50 mL Average filtrate flux rate = 40 LMH Total filtrate volume (VT )= VD+ VC Where: VD = Filtrate volume from diafiltration step VC = Filtrate volume from concentration step VT = VD + VC = (3 DV x 50 mL) + (50 mL – 10 mL) VT = VD + VC = 150 mL + 40 mL = 190 mL = 0.19 L A = V/(JxT) Rewrite equation to solve for T T = V/(JxA) T = 0.19 L / (40 L/m²/h x 0.005 m²) T = 0.19 / 0.2 = 1 h When diafiltration is performed, the total volumetric throughput (filtrate volume) equals the initial sample volume multiplied by the number of diafiltration volumes. To save on buffer volume and processing time, very often sample is first concentrated and then subjected to diafiltration. Example 3: You have a 1 L sample (0.1 mg/mL) that you need to concentrate 10 times and diafilter to remove at least 99% of the salts. Using a Centramate cassette holder with one cassette 0.093 m2 (1 ft²) how much time will it take to process your sample? The average filtrate flux rate for the process if you concentrate first and then diafilter is 40 LMH. If you do the diafiltration first and then concentrate, the average flux rate is 50 LMH. Scenario A: The sample is first concentrated 10X (from 1.0 L to 0.1 L) followed by continuous diafiltration for 6 DV’s to remove salt. Total filtrate volume (VT ) = VC + VD Where: VC = Filtrate volume in concentration step VD = Total diafiltration volume (1 DV = 0.1 L) VT = (1.0 - 0.1) + (6 x 0.1) = 0.9 + 0.6 = 1.5 L Average filtrate flux rate = 40 LMH Area (A) = Filtrate volume (V) Average filtrate flux (J) x Process time (T) Rewrite to solve for T T = V J x A T = 1.5 L = 0.4 h 40 L/m²/h x 0.093 m² Scenario B: The sample is diafiltered first by continuous diafiltration for 6 DV’s to remove salt and then concentrated 10X, from 1.0 L to 0.1 L. Total filtrate volume (VT ) = VD + VC Where: VD = Total diafiltration volume (1 DV = 1 L) VC = Filtrate volume in concentration step VT = (6 x 1.0) + (1.0 - 0.1) = 6 + 0.9 = 6.9 L Average filtrate flux rate = 50 LMH T = 6.9 L = 1.48 h 50 L/m²/h x 0.093 m² In this example, concentrating the sample first followed by diafiltration takes 0.4 h. Reversing the process and doing diafiltration first takes 1.5 h. Therefore, concentrating first has saved about 1 h of process time. If the sample had been fairly concentrated to start, the results may have been very different. In designing a process it is important to look at the total process and evaluate how filtrate flux rate changes may affect the process requirements. 24 TFF system selection considerations  Page  | Home Table 4. General product selection based on starting sample volume TFF capsule or cassette1 Membrane area Typical filtrate flow rate2 at 50 LMH (20°C) Recomended retentate flow rate (Screen channel) Starting sample volume range Minimum concentrated volume3 Lab scale and scale up devices Minimate 50 cm2 – 30 - 80 mL/min 25 - 500 mL 15 mL LV Centramate 0.01 m2 (0.1 ft2 ) 8 mL/min 60 - 80 mL/min 40 - 2000 mL 10 mL LV Centramate 0.02 m2 (0.2 ft2 ) 15 mL/min 120 -160 mL/min 60 - 4000 mL 15 mL Process development and small scale production Centramate 0.093 m2 (1.0 ft2 ) 4.6 L/hr 600 - 800 mL/min 0.2 - 20 L 100 mL 1 Data is per unit or cassette. Centramate holder can hold 5 cassettes. Other column data can be calculated by multiplying table values by the number of cassettes installed in the holder. 2 Typical filtrate flow rate is based on an average filtrate flow rate of 50 LMH and a process time of about 4 h. Actual value may be higher or lower depending on the MWCO of membrane, sample composition and viscosity, operating conditions i.e. TMP, cross flow rate, temperature, etc. 3 Minimum concentrate volume depends on system hold-up volume, reservoir design and pump type and speed. Smaller volumes can be achieved by minimizing tubing lengths and use of properly sized components, tubing, fittings, etc. 25 06 Capsules, cassettes, and systems 26 Capsules, cassettes, and systems  Page  | Home Capsules, cassettes, and systems  Page  | Home Choosing the appropriate cassette or device size depends on the total sample volume, the required process time, and the desired final sample volume. Cytiva supplies an extensive line of TFF holders and cassettes from laboratory friendly plug-in devices to fully scalable systems for process development and small scale production: Minimate tangential flow filtration capsules TDisposable TFF device for bioprocessing applications accelerates and • Contains our Omega polyethersulfone (PES) membranes that offer high flux and have been specifically modified to minimize protein binding. This polymeric membrane is stable against biological and physical degradation due to the unique chemical properties of PES. Omega membranes are available in a wide range of molecular weight cut-offs. • The cost-effective plastic construction of the Minimate TFF capsule and chemical compatibility of the Omega PES ultrafiltration membrane facilitate cleaning and reuse. • Each Minimate capsule is 100% integrity tested during manufacture to ensure reliable performance. For critical applications, users can re-test the integrity after initial use. A certificate of quality is included with each capsule. • Features an effective filtration area of 50 cm2 . Capsules, cassettes, and systems Minimate EVO tangential flow filtration system Versatile benchtop TFF system designed for highly reliable buffer exchange or concentration of samples up to 1 L. • Designed to work with Minimate TFF capsules. • A low system working volume achieved through the use of a conical bottom reservoir and compact design enables high concentration factors from up to 1 L or more of sample to be achieved. Concentrate your sample down to as little as 15 mL. • Roller head peristaltic pumps provide gentle processing and are the choice for critical applications such as fragile biomolecules. • All wetted components are made from low protein binding, chemically resistant materials. • The reservoir design allows for either continuous or discontinuous diafiltration to be performed in the same system without sample transfer. • Includes the addition of a downstream pressure gauge to enable accurate TMP differential calculations, allowing greater user control and easier validation. Minimate tangential flow filtration capsules Minimate EVO tangential flow filtration system 27 Capsules, cassettes, and systems  Page  | Home Centramate T-Series TFF cassettes with Omega membrane Available with 0.02 m2 or 0.1 m2 effective filtration areas and feature Omega PES membranes. Feature a durable and stable construction that exhibits very low extractables and offer broad chemical compatibility. The cassettes are designed to deliver optimal mass transfer to improve your process economics. Omega PES membrane provides superior performance and is stable against biological and physical degradation, offering high flux, high selectivity, and low protein binding. Omega membranes are available in a wide range of MWCOs. Centramate cassettes offer easy scale-up for robust purification processes, they feature the same materials of construction from development to production-scale processes. All materials of construction in the T-Series cassettes have been tested and meet requirements for United States Pharmacopeia (USP) Biological Reactivity Test, In Vivo at 70°C (158ºF). LV Centramate lab tangential flow filtration holder Designed for maximum product recovery for lab-scale or scale-up process volumes up to 4 L. • Stainless steel holder designed for use with 0.02 m2 Centramate cassettes. • Designed for a low hold-up volume allowing for high concentration factors to be achieved from small starting volumes. • Features easy connections through luer lock fitted ports with polished 316L stainless steel to ensure the same compatibility characteristics as productionscale holders. • Can be used with the Minimate EVO tangential flow filtration system Centramate T-Series TFF cassette LV Centramate lab tangential flow filtration holder 28 Capsules, cassettes, and systems  Page  | Home Centramate and Centramate PE lab tangential flow filtration holders and systems Suitable for process development and small-scale production of 1 to 125 L. • Holders designed for use with Centramate cassettes. • Holders are available with Type 316L stainless steel Centramate holder or economically priced, extremely durable, ultra-high molecular weight polyethylene Centramate PE holder. • Filtration area is easily expanded by adding additional membrane cassettes. • Identical fluid path lengths provide precise linear scale-up to larger process systems. • Fittings kits containing clamps, tubing and gauges can be purchased separately or as part of complete Centramate or Centramate PE systems. Centramate SS system Centramate PE system 29 07 Frequently asked questions 30 Frequently asked questions  Page  | Home Frequently asked questions  Page  | Home In a recent interview, Joshua Smith, Global Product Manager - Capsules and Vents, answered a number of frequently asked questions about TFF and the use of TFF systems. Centrifugal devices are often used in research to perform ultrafiltration applications, but when and why would researchers choose to move to using TFF? UF centrifugal devices are ideal for the processing of small volumes of samples, however, centrifugal devices are dead-end filters, meaning that the membrane will suffer from fouling and will eventually block. As users scale up to processing larger volumes of solution then TFF systems can offer greater efficiencies, the tangential flow of fluid across the surface of the membrane in TFF causes a sweeping action which reduces the potential for membrane fouling and therefore gives far more efficient processing. TFF can also be scaled up from research to pilot and then to process scale, operating with volumes in excess of 10,000 L. TFF systems can offer more versatility than centrifugal devices. For example, proteins can be extremely sensitive to solution conditions, they tend to aggregate at high concentrations. TFF offers more control over processing parameters, so it’s possible to accurately achieve desired concentration factors without proteins crashing out of solution. The versatility of TFF also allows for multiple processing steps to be performed in one system, this prevents excess sample handling and potential sample loss. For example, it’s very simple to perform continuous diafiltration followed by a concentration step all within the same TFF system. Frequently asked questions Centrifugal devices can actually be very useful when wanting to work with TFF systems from Cytiva. We offer a range of centrifugal devices, these devices contain the Omega (modified polyethersulfone) membrane that is found in the larger TFF devices. So when trying to select the best MWCO membrane our centrifugal devices can offer a cost-effective way to predict membrane performance, and help you maximize protein concentration and recovery. Obviously, it should be noted that this type of test will only offer an indication of performance as proteins can behave differently based on the volume of solution they are in, pH conditions and concentration factors. Centrifugal filters 31 Use of the Minimate capsules significantly reduces processing times A 2 mg/mL BSA solution was concentrated ten-fold (1000 to 100 mL) in either a 350 mL stirred cell device or Minimate capsule. The Minimate contains a pre-assembled Omega 10K membrane. The crossflow, set at 50 mL/min with retentate loop backpressure applied to create an initial filtrate flow of about 15 mL/min. The stirred cell devices used PES or regenerated cellulose (RC) disks and were pressurized with filtered air at 55 psi giving a starting filtrate flow of about 6 mL/min. Error bars indicate standard error for five independent runs. Measuring the absorbance at 260 nm for both the filtrate and retentate fractions provided verification of protein concentration process. Using stirred cell devices it was observed that BSA leaked through the 10K PES membrane during processing indicating a failure in integrity. Subsequently, in order to ensure a statistically significant comparison between configurations additional experiments were performed until a total of five successful stirred-cell runs were completed. Frequently asked questions  Page  | Home The pre-assembled Minimate eliminates integrity failures Sample concentration was performed as described in figure 12A. Aliquots of the starting material, final retentate, and final filtrate pools were analyzed for protein concentration at 280 nm. Average concentrations are plotted with error bars indicating standard error for (5 - Minimate), (10 - SC PES), and (5 - SC RC) runs respectively. For simplicity in the graphical representation, the retentate values are plotted on the Y2 axis to accommodate the ten-fold concentration that occurred in the processing. By monitoring the filtrate for protein bypass, it was observed that the competitive PES membrane in stirred cell operation suffered significant integrity failures. The Minimate TFF and regenerated cellulose stirred cell devices were all integral, however, the competitive PES membranes failed in 5 out of 10 runs each at different stages in the processing. Time (Hr) 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Minimate Comp PES Comp RC Start/filtrate BSA (mg/mL) 2.5 2.0 1.5 1.0 0.5 0.0 Minimate SC PES SC RC Start Retentate Filtrate 25 20 15 10 5 0 Retentate BSA (mg/mL) Time (Hr) 6.0 5.0 4.0 3.0 2.0 1.0 0.0 Minimate Comp PES Comp RC Start/filtrate BSA (mg/mL) 2.5 2.0 1.5 1.0 0.5 0.0 Minimate SC PES SC RC Start Retentate Filtrate 25 20 15 10 5 0 Retentate BSA (mg/mL) Fig 12A. Fig 12B. What advantages does TFF have over older ultrafiltration methods using stirred cells? Stirred cells are simply devices for performing ultrafiltration applications, however, as with centrifugal devices they operate under direct flow filtration, also known as “dead-end” filtration, which can fall prey to problems with membrane fouling. To try and reduce the formation of a gel layer, stirred cell devices utilize a floating stir bar on the upside of the membrane that generates turbulence, however, they are limited in performance since the velocity and subsequent level of agitation is dependant on the sweep of the bar, and that varies along the radius of the sweep. In contrast TFF operation allows for a uniform and gentle recirculation of sample over the entire membrane surface resulting in improved flux rates, significantly reducing processing times and increasing productivity. There are a number of major differences between the operation of stirred cell devices and TFF systems. Firstly, many of the commercially available stirred cell devices require researchers to assemble the units themselves, manoeuvring UF membrane disks into the housing and screwing in place. This manual handing may create leaks or fluid bypass and could even damage the structure of the membrane. Major leaks can be easily identified when visualizing abnormally high filtrate flow rates, more critical however, is that a partial bypass can look like a normal run, but the loss of molecule would not be detected until it was too late or even at the end of a run. Cytiva has undertaken studies and generated data comparing the processing times and device integrity of laboratory scale stirred cell devices and the Minimate TFF capsule (Fig 12A and 12B). Finally, one of the main differences between the operation of stirred cell devices and TFF systems is the need for a pressure vessel or a nitrogen tank which creates the desired pressure to run a stirred cell device. This can require more specialist handling for stirred cell systems and raise laboratory safety concerns. 32 What are the advantages of using a bespoke system, such as the Minimate EVO TFF system over putting a basic system together? While it is possible to put together a simple TFF system using a pump, tubing and gauges, a bespoke system such as the Minimate EVO TFF system offers numerous advantages. The Minimate EVO TFF system is delivered with all the components needed to set up the system, Cytiva supplies a quick start guide that takes a researcher through the simple steps to assemble the system. The system is designed to be plugand-play using luer connections, making installation rapid and easy. The Minimate EVO TFF system features an optimized flow path design, we guide users as to the tubing lengths to use on it, this helps provide higher product recoveries and the ability to operate at low working volumes, achieve low volumes of concentrate and ensure low hold-up volumes within the system. Included with the system is a variable-speed, roller-head peristaltic pump for gentle processing, two pressure gauges, valves, tubing, and 500 mL reservoir with a magnetic stir bar and stir plate, all assembled on a compact drip tray. All wetted components are made from chemically resistant materials. How many times can a typical TFF cassette be used? There are no specific guidelines for the total number of times a TFF capsule or cassette can be reused. The lifespan of a device is very much dependent on usage frequency, particle load of process fluids, storage conditions and execution of clean-in-place (CIP) procedures. We recommend that a normalized water permeability (NWP) value is taken on new Minimate capsules and Centramate cassettes prior to use. Then after each use and following a CIP procedure the NWP can be re-evaluated. 33 Frequently asked questions  Page  | Home Typically, after a CIP procedure the NWP of capsule or cassette should be greater than 75% of the original NWP value, if not then we would recommend performing the CIP procedure again. Once the NWP value on a used device drops below 50% of the original NWP value then we would suggest using a new capsule or cassette. Our comprehensive instructions for use documents contain information on how to correctly handle our TFF devices, how to perform both CIP and NWP procedures, and how to store devices when not in use. What is scale up and how can it help researchers? Scalability is the ability to scale up or even scale down products and systems based on the volume and processing requirements of a solution. Scalability can be viewed in different ways, firstly from a proof-of-concept perspective, or secondly where devices and systems can be scaled in a truly linear way. Scale up can help remove bottlenecks, reducing evaluation times, freeing up resources to focus on process optimization. Proof-of-concept testing can be very valuable when performing ultrafiltration applications. A good example is ultilizing a Cytiva UF centrifugal device to gain an indication of MWCO performance before moving to a TFF capsule or cassette. Minimate capsules have the same path length and materials of construction as the larger Centramate cassette product range that can be used in pilot and production plants. While the Minimate capsule may not be linear scalable to the Centramate cassette product range it offers the ability for super fast set up and easy plug-and-play lab scale processing or proof-of-concept TFF testing, offering predictable performance, which saves time when scaling up a process. It is also possible to scale up the Minimate system itself. By connecting several Minimate capsules in parallel you will achieve a greater membrane surface area, allowing for an increase in initial process volume or an increase in the speed of processing time. The LV Centramate and Centramate cassette range feature identical fluid path lengths providing precise linear scalability. What are the tangential flow filtration systems options to suit different lab needs? The Minimate TFF system is perfect for small to medium-scale research and development applications. It’s an easy-to-use manual system suitable for lower volume processing. In contrast, the ÄKTA flux™ tangential flow filtration system is an automated, advanced solution for medium to large-scale applications. The ÄKTA flux system also integrates with other systems, offering high-throughput capabilities for larger volumes and more complex applications. For more information on ÄKTA flux and the different models visit cytiva.com ÄKTA flux tangential flow filtration system 34 Frequently asked questions  Page  | Home Can I attach an LV Centramate holder and cassette to the Minimate EVO TFF system, and what would I need to do so? Yes, you can use a LV Centramate with the Minimate EVO TFF system. We recommend ordering the Minimate EVO TFF fittings kit, part number 97014, this will provide the additional 3-way stop cocks, hose barb adapters and tubing needed to properly connect the LV Centramate holder (Fig 13). How would you summarize the benefits of tangential flow filtration? It’s easy to set up and use, especially if you are using a purpose-built system such as the Minimate EVO TFF system. TFF is fast and efficient, the tangential flow of solution across the surface of membrane reduces membrane fouling allowing for efficient and gentle sample processing. Multiple processing steps can be performed in one system without the need to transfer samples. You can perform diafiltraion and concentration of a sample in the same system, saving time and avoiding product loss. TFF can be scaled up or scaled down, either for proof-of-concept or in the case of the LV Centramate and Centramate cassette range by precise linear scalability. This allows TFF to be performed at lab, pilot or process scale. Finally, TFF is very economical, devices and cassettes can be cleaned and reused. Fig 13. Minimate EVO TFF system with LV Centramate. 08 Glossary 35 Glossary  Page  | Home Concentration polarization: The accumulation of the retained molecules (gel layer) on the surface of the membrane caused by a combination of the following factors: transmembrane pressure, crossflow velocity, sample viscosity, and solute concentration. Continuous diafiltration: The technique of continuous diafiltration, also referred to as constant volume diafiltration, involves washing out the original buffer salts or other low molecular weight species in the retentate (sample) by adding water or a new buffer to the retentate at the same rate as filtrate is being generated. Crossflow rate (CF): The retentate flow rate; units in L/min. Provides the “sweeping” effect to reduce concentration polarization. The pressure drop, P (PFEED – RRETENTATE), is directly related to the CF. Crossflow flux rate (CFR): Crossflow rate per unit area of membrane, units or L/min/m2 . Diafiltration: The fractionation process that washes smaller molecules through a membrane and leaves larger molecules in the retentate (concentrate). It can be used to remove salts or exchange buffers, remove ethanol or other small molecules such as detergents, small peptides or nucleic acids. Discontinuous diafiltration-sequential dilution: Discontinuous diafiltration by sequential dilution involves first diluting the sample to a predetermined volume, then concentrating the sample back to its original volume with water or replacement buffer. This is repeated until the unwanted salts, solvents, or smaller molecules are removed. Each subsequent dilution removes more of the small molecules. Glossary Discontinuous diafiltration-volume reduction: Discontinuous diafiltration by volume reduction involves first concentrating the sample to a predetermined volume, then diluting the sample back to its original volume with water or replacement buffer. This is repeated until the unwanted salts, solvents, or smaller molecules are removed. Each subsequent concentration and dilution removes more of the small molecule. Concentration polarization: The accumulation of the retained molecules (gel layer) on the surface of the membrane caused by a combination of the following factors: transmembrane pressure, crossflow velocity, sample viscosity, and solute concentration. Filtrate: The solution that passes through the membrane. Filtrate flux rate: Filtrate flow rate per unit area, unit L/m2/h (LMH). Filtrate flux rate is affected by crossflow rate, TMP and viscosity. Gel layer: The microscopically thin layer of molecules that forms on the top of the membrane. It causes a reduction in the filtrate flow rate and may increase the retention of molecules that would normally cross into the filtrate. Hold-up volume: The volume of retentate fluid remaining in the filter and system tubing after sample recovery. Microfiltration (MF): Microfiltration refers to filtration using filter media with pore sizes typically between 0.1 μm and 10 μm. Generally used for clarification, sterilization, and removal of microparticulates or for cell harvesting in life science applications. Minimal operating volume: The minimal volume of process fluid that can be handled effectively by the TFF system. 36 Glossary  Page  | Home Molecular weight cut off (MWCO): The molecular weight cut-off of a membrane sometimes called Nominal Molecular Weight Limit (NMWL) is defined by its ability to retain a given percent of a molecule in solution (typically 90% retention). Normalized water permeability (NWP): Membrane water permeability corrected to a temperature of 20ºC. Membrane water permeability: Filtrate flux rate for water per unit of applied TMP. The water permeability is related to the membrane hydraulic resistance. It is significantly affected by temperature. Membrane recovery: Measure of the water permeability of the membrane after processing and cleaning compared to the water permeability of the “original” membrane. % Membrane recovery = (NWPCLEAN/NWPORIGINAL) x 100% Product recovery: The amount of product (mass or activity) recovered after processing compared to the amount in the starting sample. Usually expressed as a percentage of starting material. Retentate: The sample that passes through the feed channel (does not pass through the membrane). Also known as the concentrate. Tangential flow filtration (TFF) or crossflow filtration: A process where the feed stream flows parallel to the membrane face. Applied pressure causes one portion of the flow stream to pass through the membrane (filtrate) while the remainder (retentate) is recirculated back to the feed reservoir. Transmembrane pressure (TMP): It is the driving force for liquid transport through the ultrafiltration membrane. Calculated as the average pressure applied to the membrane minus any filtrate pressure. TMP = (PFEED - PRETENTATE)/2 - PPERMEATE In most cases, pressure at filtrate port equals zero. Ultrafiltration (UF): A process that separates solutes based on their molecular weight or size. Ultrafiltration membranes have pore sizes between 0.001 and 0.1 μm, and are typically used for concentrating and desalting dissolved molecules (protein, peptides, nucleic acids, carbohydrates, and other biomolecules), exchanging buffers, and gross fractionation. Ultrafiltration membranes are typically classified by MWCO rather than pore size. Related products available from Cytiva 1. AcroPrep™ 24, AcroPrep Advance 96 and 384-well filter plates are an excellent platform for a wide variety of molecular biology, analytical, and high throughput sample preparation and detection applications.) 2. ÄKTA flux tangential flow filtration system 37 Glossary  Page  | Home cytiva.com Cytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corp. or an affiliate doing business as Cytiva. Centramate, Minimate, Mustang, Omega, Centramate and ÄKTA flux are trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva. Any other trademarks are the property of their respective owners. If a third-party trademark is used in a promotional manner, reach out to your Cytiva contact for guidance. The Danaher trademark is a proprietary mark of Danaher Corporation. © 2025 Cytiva For local office contact information, visit cytiva.com/contact CY46090-29Jan25-PO  Page  | Home