Protein Concentration and Diafiltration by Tangential Flow Filtration

An Overview In Normal Flow Filtration (NFF), fluid is convected directly toward the Table of Contents PURPOSE membrane under an applied pressure. Purpose 1 Membrane-based Tangential Flow Particulates that are too large to pass What is TFF? 1 Filtration (TFF) unit operations are used through the pores of the membrane TFF Basics 3 for clarifying, concentrating, and accumulate at the membrane surface or in the depth of the filtration media, Define Your Process Goals 5 purifying proteins. This technical brief is a practical introduction to protein while smaller molecules pass through Choosing the Right Equipment 5 processing using tangential flow to the downstream side. This type of Optimization of Key Process filtration. It is intended to help scientists process is often called dead-end Parameters 9 and engineers achieve their protein filtration. However, the term “normal” indicates that the fluid flow occurs in Characterizatiom of Performance 13 processing objectives by discussing how the choice of key components the direction normal to the membrane Putting the Process Together 15 and operating parameters will affect surface, so NFF is a more descriptive System Considerations 17 process performance. and preferred name. NFF can be used for sterile filtration of clean Major Process Considerations 19 What is TFF? streams, clarifying prefiltration, and Glossary of Terms 23 Filtration is a pressure driven virus/protein separations and will not Technical References 23 separation process that uses be discussed in this document. membranes to separate components in In Tangential Flow Filtration (TFF), a liquid solution or suspension based the fluid is pumped tangentially along on their size and charge differences. the surface of the membrane. An Filtration can be broken down into applied pressure serves to force a two different operational modes – portion of the fluid through the Normal Flow Filtration and Tangential membrane to the filtrate side. As in Flow Filtration. The difference in fluid NFF, particulates and macromolecules flow between these two modes is that are too large to pass through the membrane pores are retained on the TECHNICAL BRIEF illustrated in figure 1.

Normal Flow Filtration Tangential Flow Filtration

Feed Flow Pressure Pressure

Feed Flow

Membrane Membrane

Filtrate Filtrate

Figure 1. Comparison of NFF and TFF upstream side. However, in this case weights lower than the NMWL will This process type is used to separate the retained components do not build be retained by the membrane. A virus particles from proteins or from up at the surface of the membrane. component’s shape, its ability to smaller media components, as either a Instead, they are swept along by the deform, and its interaction with other virus reduction step or a virus harvest tangential flow. This feature of TFF components in the solution all affect its step. HPTFF is a high resolution makes it an ideal process for finer retention. Different membrane process where protein-protein sized-based separations. TFF is also manufacturers use different criteria to separations can be carried out on the commonly called cross-flow filtration. assign the NMWL ratings to a family basis of both size and charge, resulting However, the term “tangential” is of membranes. The technical in product yields and purification descriptive of the direction of fluid references at the end of this document factors similar to chromatography. flow relative to the membrane, so it is provide more detail on membrane Membrane NMWLs used for HPTFF the preferred name. retention determination as well as are in the range of 10 kD to 300 kD. additional information on other related Reverse Osmosis (RO) and How is TFF Used in Protein topics. Nanofiltration (NF) are types of TFF Processing? Microfiltration (MF) is usually used where very tight membranes are used TFF can be further subdivided into upstream in a recovery process to to separate salts and small molecules categories based on the size of separate intact cells and some cell with molecular masses typically lower components being separated. For debris/lysates from the rest of the than 1500 Daltons from water or protein processing, these can range components in the feed stream. Either other solvents. Membranes with from the size of intact cells to buffer the retained cells or the clarified filtrate NMWLs of 1 kD and lower are used. salts. Figure 2 details typical can be the product stream. Membrane Finally, Diafiltration (DF) is a TFF components that would be retained by pore size cutoffs used for this type of process that can be performed in a membrane and that would pass separation are typically in the range combination with any of the other through a membrane for each of the of 0.05 µm to 1.0 µm. categories of separation to enhance subdivisions. In addition, it shows the Ultrafiltration (UF) is one of the most either product yield or purity. During range of membrane pore size ratings widely used forms of TFF and is used DF, buffer is introduced into the or nominal molecular weight limits to separate proteins from buffer recycle tank while filtrate is removed (NMWL) that generally fall into each components for buffer exchange, from the unit operation. In processes category. desalting, or concentration. where the product is in the retentate, A membrane pore size rating, Depending on the protein to be diafiltration washes components out typically given as a micron value, retained, membrane NMWLs in the of the product pool into the filtrate, indicates that particles larger than the range of 1 kD to 1000 kD are used. thereby exchanging buffers and rating will be retained by the Two types of UF are Virus filtration reducing the concentration of membrane. A NMWL, on the other (VF) and High Performance tangential undesirable species. When the hand, is an indication that most flow filtration (HPTFF). For VF, product is in the filtrate, diafiltration dissolved macromolecules with membrane NMWL ratings range from washes it through the membrane into molecular weights higher than the 100 kD to 500 kD, or up to 0.05 µm. a collection vessel. NMWL and some with molecular

Microfiltration Virus High-Performance Ultrafiltration Nanofiltration/ Filtration Filtration TFF Reverse Osmosis

Components retained Antibiotics by membrane Intact cells Sugars Cell debris Viruses Proteins Proteins Salts

membrane membrane membrane membrane membrane membrane membrane membrane membrane membrane membrane membrane membrane membrane

Colloidal material Proteins Proteins Small Peptides (Salts) Components passed Viruses Salts Salts Salts Water through membrane Proteins Salts Approximate membrane 0.05 µm – 1 µm 100 kD – 0.05 µm 10 kD – 300 kD 1 kD – 1000 kD <1 kD cutoff range

Figure 2. Subdivisions of tangential flow filtration processes

2 The remainder of this document will Diafiltration Retentate Valve to focus on the development of Buffer Return Apply Pressure concentration and diafiltration steps for protein processing. Retentate Pressure TFF Basics Feed Tank Feed In a TFF unit operation, a pump is Pressure used to generate flow of the feed stream through the channel between two membrane surfaces. A schematic Filtration Filtrate of a simple TFF system is shown in Module Stream figure 3. During each pass of fluid Feed Pump over the surface of the membrane, the applied pressure forces a portion of the fluid through the membrane and Figure 3. Schematic of a simple TFF system into the filtrate stream. The result is a gradient in the feedstock concentration from the bulk conditions at the center of the channel to the more Membrane concentrated wall conditions at the membrane surface. There is also a concentration gradient along the length of the feed channel from the Q Q inlet to the outlet (retentate) as F Cb R progressively more fluid passes to the filtrate side. Figure 4 illustrates the Cw flows and forces described above with the parameters defined as: TMP Qf, Cf -1 Membrane QF: feed flow rate [L h ] -1 QR: retentate flow rate [L h ] -1 Figure 4. Flows and forces in a TFF channel Qf: filtrate flow rate [L h ]

Cb: component concentration in the bulk solution [g L-1]

Cw: component concentration at the membrane surface [g L-1]

Cf: component concentration in the filtrate stream [g L-1] Feed Pressure, PF TMP: applied pressure across the membrane [bar]

The flow of feedstock along the length of the membrane causes a pressure drop from the feed to the retentate end of the channel. The flow Pressure on the filtrate side of the membrane is Average TMP Retentate Pressure, PR typically low and there is little restriction, so the pressure along the length of the membrane on the filtrate side is approximately constant. A standard pressure profile in a TFF Filtrate Pressure, PF channel is shown in figure 5. InletFeed Channel Length Outlet

Figure 5. Pressure profile in a TFF channel

3 Definitions Transmembrane Pressure (TMP) is the average applied pressure from the feed to the filtrate side of the membrane.

TMP [bar] = [(PF + PR)/2] – Pf

Pressure Drop (∆P) is the difference in pressure along the feed channel of the membrane from the inlet to the outlet.

∆P [bar] = PF – PR

Conversion Ratio (CR) is the fraction of the feed side flow that passes through the membrane to the filtrate.

CR [-] = Qf/QF

Apparent Sieving (Sapp) is the fraction of a particular protein that passes through the membrane to the filtrate stream based on the measurable protein concentrations in the feed and filtrate streams. A sieving coefficient can be calculated for each protein in a feedstock.

Sapp [-] = (concentration in filtrate, Cf)/(concentration in feed, Cb)

Intrinsic Sieving (Si) is also the fraction of a particular protein that passes through the membrane to the filtrate stream. However, it is based on the protein concentration at the membrane surface. Although it cannot be directly measured, it gives a better understanding of the membrane's inherent separation characteristics.

Si [-] = (concentration in filtrate, Cf)/(concentration at membrane wall, Cw)

Retention (R) is the fraction of a particular protein that is retained by the membrane. It can also be calculated as either apparent or intrinsic retention. Retention is often also called rejection.

Rapp [-] = 1 – Sapp or Ri = 1 - Si

2 Filtrate Flux (Jf) is the filtrate flow rate normalized for the area of membrane [m ] through which it is passing.

-2 -1 Jf [L m h ] = Qf/area

Mass Flux (Jm) is the mass flow of protein through the membrane normalized for the area of membrane [m2] through which it is passing.

-2 -1 Jm [g m h ] = Qf x Cf/area

Volume Concentration Factor (VCF or X) is the amount that the feed stream has been reduced in volume from the initial volume. For instance, if 20 L of feedstock are processed by ultrafiltration until 18 L have passed through to the filtrate and 2 L are left in the retentate, a ten-fold concentration has been performed so the Volume Concentration Factor is 10. In a Fed-Batch concentration process, where the bulk feedstock is held in an external tank and added to the TFF operation continuously as filtrate is removed, VCF should be calculated based only on the volume that has been added to the TFF operation. VCF or X [-] = Total starting feed volume added to the operation/current retentate volume

Concentration Factor (CF) is the amount that the product has been concentrated in the feed stream. This depends on both the volume concentration factor and the retention. As with the VCF, for a Fed-Batch concentration process, calculate CF based only on the volume of feedstock added to the TFF operation.

R CF [-] = Final product concentration/Initial product concentration = VCF( app)

A Diavolume (DV or N) is a measure of the extent of washing that has been performed during a diafiltration step. It is based on the volume of diafiltration buffer introduced into the unit operation compared to the retentate volume. If a constant-volume diafiltration is being performed, where the retentate volume is held constant and diafiltration buffer enters at the same rate that filtrate leaves, a diavolume is calculated as: DV or N [-] = Total buffer volume introduced to the operation during diafiltration/retentate volume

4 Define Your Process Goals Choosing the Right Equipment All regenerated cellulose The first step of TFF process The primary components of a TFF membranes are very hydrophilic, development is to define what the process are the membrane material exhibiting low fouling and ultra-low process must achieve and what goals and the membrane module format. protein adsorption. They are more must be met. A good understanding of Choosing the most appropriate compatible with organic solvents than these objectives will enable the components early in the development are the polyethersulfone-based successful selection of an appropriate process, with consideration for the membranes, but are less tolerant to unit operation and operating requirements of the process, increases extreme pH’s. Ultracel membranes parameters. Important process the success and robustness of the final are recommended for use in all objectives to define are: step. applications where harsh pH • Final product concentration conditions are not needed and Membranes especially when protein loading is • Feed volume reduction 2 Millipore provides ultrafiltration low (<20 g/m ) or the feedstock is • Extent of buffer exchange membranes in several different highly fouling. • Contaminant removal specification materials to suit a wide range of Traditional polyethersulfone applications. The different membrane membranes tend to adsorb protein as Next, identify and quantify any materials offer alternatives in fouling well as other biological components, criteria by which the success of the characteristics and chemical leading to membrane fouling and operation will be measured. The compatibility. Each of the membrane lowered flux. Millipore’s Biomax® primary goals for a successful protein materials is available in a number of membrane is polyethersulfone-based, processing are: NMWLs. Two of the most common but has been hydrophilically modified • High product yield materials for ultrafiltration membranes to be more resistant to fouling. The • High product quality (or activity) are regenerated cellulose and Biomax membrane line ranges in polyethersulfone. NMWLs from 5 to 1000 kD. Biomax • High product purity Millipore's Ultracel® membrane is membranes operate over a wide • Controlled bioburden and endotoxin regenerated cellulose. The Ultracel PL temperature range and are highly In addition, the process should family, standard regenerated stable at pH’s from 1 to 14, but have scale up accurately, enable cellulose, is available in NMWLs from limited solvent compatibility. The use straightforward validation, and be 1 to 300 kD. The Ultracel PLC of Biomax membrane is recommended robust during continued use at composite regenerated cellulose for applications where very harsh pH industrial scale. Finally, the economic ranges in NMWLs from 5 to 1000 kD. conditions are required for processing objectives for the process must be met Ultracel PLC membranes are cast on a or cleaning. In order to minimize and any constraints such as process microporous polyethylene substrate adsorption losses maintain moderately time, unit operation size, or buffer use and have superior resistance to high protein loading ( >20 g/m2). must be observed. Discussion on how reverse pressure versus the PL series. the process design impacts yield, quality, bioburden, scalability, robustness, and economics begins on page 19.

10 kD Biomax® Traditional PES 10 10 kD Ultracel® PLC

Scanning electron micrograph images of cross sections of different membranes. 5 Table 1 shows the magnitude of For a batch UF and constant- membrane, the amount of diafiltration protein losses due to adsorption on volume DF process, where retention and/or volume concentration has to several UF membrane materials. These remains constant throughout the be reduced. For example, if the losses were measured at Millipore process, this loss is calculated as: number of diavolumes is reduced to with model protein feedstocks. In 4.3, the value of the term (ln VCF + N) addition, the percentage yield loss Product Loss (%) = 100* (1 – e (R-1)[lnVCF + N]) is now 7.3 and the amount of product due to adsorption is shown for two lost to the filtrate is 7.0%, as indicated theoretical processes. The “Low Protein by point “B”. However, the extent of The relationship is plotted in figure 6 Case” is a process in which 1000 L buffer exchange is drastically reduced. for processes in which the product is of 0.1 g/L solution is concentrated to To reduce the product loss without in the retentate. To illustrate how to use 2 g/L on a 10 m2 unit. The “High changing the process, a membrane figure 6, consider a process where the Protein Case” is a process in which with higher retention of the product goals are to perform a 20-fold volume 1000 L of 10 g/L solution is concen- must be chosen. If the retention is concentration factor (VCF), a 7 diavolume trated to 200 g/L on a 20 m2 unit. increased to 0.999 while the value of buffer exchange, and lose less than The unique construction of both the (ln VCF + N) remains at 10, product 7% of the product to the filtrate. For Biomax and Ultracel PLC product lines loss to the filtrate drops to only 1.0%, this example, the natural log (ln) of the makes these membranes free of voids as indicated by point “C”. In many VCF is 3 and N is 7, so the value of and defects and well-attached to the cases, product retention is different the term (ln VCF + N) is 10. If a substrate. The membranes are rugged, during the UF and DF sections of a membrane is chosen that has a have very high integrity, and have process. It is important to check this for retention of 0.99 for the product, the excellent retention characteristics. A each process. When retention product loss to the filtrate will be membrane from either the Biomax or changes, product loss to the filtrate is 9.5%, as indicated by point “A” on Ultracel PLC family should be the first determined separately for each section the graph. Therefore, the yield loss choice when developing a process. by following the appropriate retention goal is not met. In order to reduce the Since NMWLs for UF membranes curve and summing the two results. do not indicate absolute product loss while still using the same retention/sieving ratings, some rules of thumb are useful in determining Membrane Material Protein Adsorption Low Protein Case High Protein Case what membrane rating is applicable [-] [g m-2] [% Yield Loss] [% Yield Loss] for a particular process. As a rule, choose a membrane that has a Polyethersulfone 0.5 5.0 0.10 NMWL one-third to one-fifth of the Biomax (polyethersulfone) 0.2 2.0 0.04 molecular weight of a product that is to be retained. Also, a minimum size Regenerated cellulose 0.1 1.0 0.02 difference of approximately five-fold between components that are being Table 1. Typical protein adsorption onto UF membranes separated is optimal. Highly fouling feedstocks tend to (R-1)*[In VCF + N] have higher retention of like-sized Product Loss (%) = 100 * (1 - e^ ) 50 proteins than cleaner feedstocks. In R = 0.8 addition, a process operating at very 45 R = 0.9 high TMPs has lower retentions due to 40 an increased protein concentration at 35 the membrane surface. Since each 30 protein feedstock and process is 25 unique, two or more membranes may need to be tested before choosing an 20 optimal one. 15 R = 0.99 10 A Choose a membrane that has B sufficiently high retention to meet Product Loss to the Filtrate (%) 5 R = 0.999 your yield goal. Product loss to the C 0 filtrate due to incomplete retention is 0 2 46810121416 cumulative for the concentration and LN (VCF) + N diafiltration sections of a process. Figure 6. The effect of product retention on product yield during a batch ultrafiltration/ constant-volume diafiltration process where the product is in the retentate and the retention is constant throughout the process. 6 Modules Screens are often inserted into the Flat Plate Membranes are fabricated into feed and/or filtrate channels in spiral (Often referred to as Cassettes) modules in several formats. The most wound and flat plate modules to In a flat plate membrane module, common formats used for tangential increase turbulence in the channels layers of membrane either with or flow filtration are: and reduce concentration without alternating layers of separator • Flat plate polarization. This is not an option with screen are stacked together and then hollow fiber modules. The turbulence- sealed into a package. Feed fluid is • Spiral wound promoted channels have higher mass pumped into alternating channels at • Hollow fiber transfer coefficients at lower crossflow one end of the stack and the filtrate The basic flowpaths for each of rates, meaning that higher fluxes are passes through the membrane into the these modules is shown in figure 7. achieved with lower pumping filtrate channels. Flat plate modules requirements. Turbulence-promoted generally have high packing densities feed channels are, therefore, more (area of membrane per area of floor efficient than open channels. Using a space), allow linear scaling, and suspended screen in a flat plate some offer the choice of open or module gives some of the benefits of turbulence promoted channels. both open and turbulence-promoted channels. Figure 8 illustrates the different types of channel configurations.

Feed Feed Filtrate Filtrate Filtrate Retentate Hollow, thin-walled membrane tube

Membrane sheet Membrane

Filtrate Retentate Feed Filtrate Filtrate

Hollow Fiber Spiral-Wound Flat Plate

Figure 7. Fluid flowpaths through different TFF modules

Open Suspended Coarse Fine

Figure 8. Open and turbulence-promoted feed channels in TFF module types

7 Spiral Wound Hollow Fiber available in several sizes of linearly In a spiral wound module, alternating Hollow fiber modules are comprised scalable modules to process volumes layers of membrane and separator of a bundle of membrane tubes with from 20 mL to 10,000 L. These screen are wound around a hollow narrow diameters, typically in the modules incorporate all of the central core. The feed stream is range of 0.1 to 2.0 mm. In a hollow membrane materials discussed above pumped into one end and flows down fiber module, the feed stream is and Millipore offers the choice of the axis of the cartridge. Filtrate pumped into the lumen (inside) of the several different feed channel screens passes through the membrane and tube and filtrate passes through the to optimize the turbulence-promotion spirals to the core, where it is membrane to the shell side, where it is for a particular process. removed. The separator screens removed. Because of the very open Spiral wound Helicon modules are increase turbulence in the flowpath, feed flowpath, low shear is generated useful for lower cost processing of very leading to a higher efficiency module even with moderate crossflow rates. large volumes. Modules with standard than hollow fibers. One drawback to While this may be useful for highly polyethersulfone and regenerated spiral wound modules is that they are shear-sensitive products, in general it cellulose membranes are available. not linearly scaleable because either reduces the efficiency of the module Spiral wound Prep/Scale modules are the feed flowpath length (cartridge by requiring very high pumping easy to use for processing smaller length) or the filtrate flowpath length capacity to achieve competitive fluxes. volumes when a spiral-wound format (cartridge width) must be changed Millipore offers two premier TFF is preferred. However, flat plate within scales. However, spiral wound modules for ultrafiltration/diafiltration Pellicon XL modules offer superior low- modules are a good low cost option protein processing. These are the volume processing. for very large area unit operations, as flatplate Pellicon® 2 cassette modules, required for food and beverage and the spiral-wound Helicon™ applications. modules. Pellicon cassettes are

Prep/Scale filter modules Pellicon cassettes

8 Optimization of Key Process membrane resistance. The second, Filtrate Control Parameters level part of the curve is the pressure Many TFF applications apply a Before implementing a TFF step for independent regime. In this section, crossflow and pressure setpoint and protein processing, the parameters at the concentration of protein at the the filtrate flows uncontrolled and which the step will operate must be membrane surface is high and a unrestricted out of the module. This is defined. Key parameters to determine significant portion of the applied the simplest type of operation and are: pressure is working against the protein most concentration and/or diafiltration • Crossflow rate osmotic pressure. As protein processes where the target product is concentration increases or feed flow in the retentate operate in this manner. • Transmembrane pressure rate decreases, the TMP at which the For other applications, however, it is • Filtrate control flux plateaus decreases. A typical helpful to use some type of filtrate • Membrane area trend of flux with increasing TMP, control beyond that achieved by protein concentration, and feed flow simply adjusting the pressure with the • Diafiltration design rate is shown in figure 9. retentate valve. These parameters are typically If the process is run with a TMP When using very open UF arrived at through a combination of setpoint in the pressure independent membranes, the membrane rules of thumb, experimentation, and regime, maximum flux is achieved, permeability is so high that nearly all consideration of process requirements and this minimizes the required of the crossflow is converted to filtrate and limitations. membrane area. However, the protein with very little applied TMP. Although wall concentration is high and could Crossflow Rate this results in high fluxes, it is similar to The crossflow rate greatly depends on exceed a solubility limitation, leading operating in an NFF mode and the which module and feed channel to yield losses. On the other hand, if a benefits of the tangential flow are lost. turbulence promoter are chosen. TMP setpoint is chosen in the pressure Often, very high wall concentrations Millipore provides recommendations dependent regime, fluxes are lower and high membrane fouling occur, for crossflow rates for each feed and more membrane area is required. especially during the startup of the channel type in the Maintenance Therefore, for a standard UF/DF process. To reduce the filtrate rate and Procedures manuals. In general, a process, the optimum TMP at which to enable the TMP to be controlled at the higher crossflow rate gives higher flux run a process is at the knee of the low values required for robust TFF at equal TMP. It increases the curve, where nearly the highest flux is operations the filtrate flow must be sweeping action across the membrane achieved without exerting excessive controlled. and reduces the concentration gradient pressure or reaching exceedingly high In a controlled flow filtrate towards the membrane surface. This protein wall concentrations. For HPTFF operation, a pump or valve on the also tends to slightly increase the processes, where two similarly-sized filtrate line restricts filtrate flow to a set observed retention of most components. components are being separated, the value, as shown in Figure 10. In However, higher crossflow rates cause optimum operating point is determined addition to reducing the filtrate flow to the product to experience more passes differently. maintain adequate tangential flow, it through the pump in a given amount creates pressure in the filtrate line to of time. This can lead to degradation reduce the TMP while the feed and of product quality. Also, higher retentate pressures remain fixed. crossflow rates require the use of larger pumps and larger diameter Low Protein Concentration piping, which increase the system or High Feed Flow holdup volume and could increase product losses due to unrecoverable holdup. Therefore, balance the ) Optimum -1 increase in flux with the increase in h Operating Point -2 pump passes and holdup volume when choosing a crossflow rate.

Flux(L m High Protein Concentration TMP or Low Feed Flow In a TFF unit operation, filtrate flux increases with increasing TMP up to a point and then it levels off. The first part of the curve, where the flux increases with pressure, is the pressure Transmembrane Pressure (bar) dependent regime. Here, the primary factor limiting flux is the fouled Figure 9. A typical trend of flux versus transmembrane pressure for a TFF process 9 Diafiltration Retentate Valve to Buffer Return Apply Pressure Retentate Pressure Feed Tank Feed Filtrate Pressure Pump

Filtration Filtrate Module Stream Feed Pump

Figure 10. Schematic of a TFF system using a pump for a filtrate control

Membrane Area After determining the process flux and the total volume to be processed, the membrane area required for the final unit operation can be determined. However, since flux is filtrate flow rate divided by both area and time, the membrane area is also a function of the total process time. Choosing a longer process time leads to lower membrane area requirements. This is beneficial because membrane and capital costs are reduced. In addition, unrecoverable holdup volume is lower in smaller unit operations, minimizing yield losses. However, excessively long process times put the product at risk for quality degradation and/or bioburden contamination. Interestingly, product pump passes do not change significantly because a low-area operation has a low crossflow rate but a high process time while a high-area operation has a high crossflow rate and a short process time. Calculate the membrane area requirements as:

Membrane area [m2] = Filtrate volume [L] /Flux [L m-2 h-1] * Process time [h]

Flux typically drops as protein concentration increases, so choose an average flux for the above calculation. Alternatively, some processes exhibit several distinct sections with different fluxes. For example, a concentration followed by a diafiltration followed by another concentration will generally show a decreasing flux followed by a constant flux during DF followed by another section of decreasing flux. In this case, break out each section as:

2 Membrane area [m ] = [Filtrate volume1/Flux1 + Filtrate volume2/Flux2 + . . . ] /Process time

For a robust scaleup, always incorporate a safety margin into the membrane area requirements to account for lot to lot variability in membrane permeability, feedstock characteristics, and batch volumes. Typically, a safety margin of at least 20% extra membrane area is used, but this could increase or decrease depending on the expected variability in the process. Diafiltration Design If the process includes a diafiltration step, first choose the mode of diafiltration control. The two most common modes of diafiltration control are batch and constant-volume. In a batch DF process, a large volume of diafiltration buffer is added to the recycle tank and then the retentate is concentrated. When a certain retentate volume is reached, another volume of buffer is added. This cycle is continued until the desired total volume of DF buffer has been added. The benefit of this mode of diafiltration is that no level control is required. However, the buffer exchange is not as efficient as in a constant volume DF 10 2.5 process and the product concentration Constant Volume DF at which diafiltration is performed Batch DF cannot be optimized because protein 2 concentration changes as the buffer is added and then concentrated. Constant-volume diafiltration is the 1.5 more commonly used control mode. To perform a constant-volume DF, buffer is added to the recycle tank at the same rate that filtrate is removed. 1 The total volume of retentate remains constant throughout the process. This mode of operation requires some Retention Volume (Fraction of Original) Retention Volume 0.5 method of level control that will meter 0 2 46810the addition of DF buffer to keep the Diavolumes (-) retentate volume constant. The effect of the two modes of operation on 100 retentate volume and buffer exchange Constant Volume DF is illustrated in figure 11. The Batch DF remaining diafiltration discussion and 10 calculations will focus on constant- volume diafiltration processes, since they are more efficient and more 1 commonly used. A third mode of diafiltration control 0.1 is known as the optimum diafiltration

(% of Original) strategy. It is primarily used when a component that is partially retained is 0.01 being removed by diafiltration. Here, both the volume and concentration of Contaminant Remaining in Retentate product are changed along a 0.001 0 2 46810controlled path throughout the process to simultaneously optimize buffer use, Diavolumes (-) product yield, and buffer exchange. Please contact Millipore Technical Figure 11. Retentate volume and buffer exchange during batch and Services for more information constant-volume diafiltration on this control scheme. After choosing a control mode, determine the placement of the diafiltration step within the process. Starting Buffer Diafiltration Buffer For processes where the target protein is retained, flux typically drops as a ]

-1 protein is concentrated. Diafiltration at h

-2 lower protein concentrations then maximizes flux. However, at low protein concentration, the total volume of product to diafilter is high, increasing the membrane area and buffer volume required. Therefore, Filtrate Flux [L m there is an optimum protein concentration at which to perform diafiltration where the tradeoff between flux and volume is balanced 0.1 1 10 100 1000 and the minimum membrane area or Product Concentration [g L-1] process time is needed.

Figure 12. Typical trend of flux versus protein concentration in different buffers 11 In the past, the optimum Starting Buffer concentration for diafiltration has been Diafiltration Buffer determined by finding the concentration Larger Retentate Volume Higher Buffer Usage at which flux drops to zero (historically Higher Membrane Area called cg) and then dividing this Optimum Cb concentration by the constant e (e = for Diafiltration 2.718). However, this approach only gives an approximation of the optimum Smaller Retentate Volume point for processes where the flux Lower Buffer Usage Higher Membrane Area decay follows a well-defined standard curve. For standard pressure-controlled DF Optimization Parameter UF/DF processes, a more accurate and generally applicable approach for determining the optimum point at 0 20 40 60 80 100 120 which to diafilter is to first plot flux Product Concentration (g L-1) versus the log of protein concentration. It is important to plot this data with the Figure 13. Determination of the optimum protein concentration for diafiltration for a protein in both the initial and final standard TFF process buffers, since flux can often change significantly with different buffers. A typical trend is shown in figure 12. required, it may not always be diafiltration buffer required at the Next, choose several protein practical. Product volume at this expense of adding more membrane concentrations along each curve that concentration may be below the area or processing time. span the range from initial to final minimum recirculation volume of the Finally, the goal of a diafiltration concentrations expected in the process unit operation or the product may not step is to reduce buffer or contaminant and calculate the value of the DF be stable at this concentration. In species from a product in the retentate. Optimization Parameter at each point these cases, choose a lower Since the number of diavolumes that using the following equation: concentration at the expense of using are performed directly impacts both more diafiltration buffer and more yield and extent of purification, it must DF Optimization Parameter = C * Jf membrane area or longer processing be determined with the goal in mind. time. Alternately, choose a Figure 6 illustrates the relationship where: concentration higher than the optimum between product retention and C = product concentration in if the goal is to minimize the volume of product yield as a function of volume feedstock at data point [g L-1]

Jf = filtrate flux at data point Remaining Contaminant (%) = 100* e(R-1)*N -2 -1 [L m h ] 100 Finally, plot the optimization parameter versus protein concentration for each buffer, as shown in figure 13, 10 to find the product concentration where the value of the optimization 1 parameter is maximized. This is the optimum concentration at which to diafilter to minimize membrane area 0.1 R = 0.4 requirements. If the optimum is very (% of Original) different for the two buffers, it is most R = 0 0.01 conservative to choose the optimum R = 0.2 based on the buffer curve that results Contaminant Remaining in Retentate in the lower value. The actual value 0.001 will be between the two curves, since 0 5101520 throughout the diafiltration the product Diavolumes (-) will be gradually exchanged from the starting to the final buffer. Although operating at this concen- Figure 14. Removal of a contaminant during a constant-volume diafiltration process tration minimizes the membrane area where the product is in the retentate and the contaminant is in the filtrate

12 concentration factor and diavolumes. Characterization of Then, apply new conditions and Buffer exchange and contaminant Performance repeat the procedure. removal are easier to view in terms of Although the above discussion gives The method of startup and the order percent removal versus diavolumes, as general guidelines on how to choose of conditions tested can impact the shown in figure 14. an appropriate module and operating results, so take care to always begin There are several common reasons parameters, the performance of the with the least fouling conditions and why actual contaminant removal can process must be tested on the actual move towards more fouling be lower than the theoretical removal feedstock. One of the most important conditions. During startup of the shown in figure 14. For example, experiments for characterizing operation, first slowly ramp the feed retention of the contaminant can performance is to generate flux versus rate (and co-flow rate, if applicable) change throughout a diafiltration as its TMP curves at several crossflow rates without any applied pressure. When concentration and the buffer (or pressure drops) and several protein the feed rate setpoint is reached, composition change. The contaminant concentrations and to determine ramp the applied pressure to its can bind to the product of interest. The product retention at each point. In setpoint. Finally, if filtrate control is formation of surfactant micelles can addition, if the process contains a being used, ramp the filtrate to its change retention or cause partitioning diafiltration, it is important to generate setpoint. Shutdown of the operation of the contaminant into the micelle. these flux versus TMP curves in both should occur in reverse order from the The Donnan effect can increase the starting and final buffers, since flux startup. retention when low ionic strength and retention can change significantly When testing different flow, solutions are used. Finally, deadlegs with buffer conditions. If required, the concentration, and pressure points, in the system piping can result in small effects of processing at different conditions that are least fouling are volumes of solution that are not fully temperatures can also be those at low protein concentrations, washed throughout the diafiltration. incorporated. With a small volume of low TMPs, and high feed rates. A Since contaminants or residuals often feedstock and a single day’s work, good approach is to start with the must be removed from the product to this experiment generates a wealth of highest feed rate and lowest protein very low levels, incorporate a safety information about the process. The concentration and TMP to be tested. factor of at least two extra diavolumes experiment will be briefly described At constant feed rate and protein and test the process to ensure that here. concentration, increase the TMP until it actual residual levels are acceptable. Typically, determine TFF begins to level off. At this point, the performance at approximately three membrane is operating in the pressure different crossflow rates that span the independent regime (see figure 9) and range of manufacturer recommended higher TMPs will cause excessively rates for the module being used. high protein concentrations within the Likewise, approximately three different module without the benefit of protein concentrations should be increased flux. Maintaining the protein tested that span the range from initial concentration constant, repeat the protein concentration in the feedstock TMP excursion (low to high TMP) at to the highest concentration expected each feed rate to be tested, moving in the process. Investigate at least five from high to low feed rates. Then, transmembrane pressures for each increase the protein concentration and crossflow and protein concentration. repeat the entire procedure. TMPs will vary depending on the A sample sheet for data collection membrane module and the feedstock, is illustrated in figure 15. For each test but will typically be in the range of 5 point, calculate flux, TMP, and to 50 psid. retention. Then, generate graphs Perform the experiment by starting showing flux versus TMP at different ProFlux® M12 Benchtop TFF system with up the module in a total recycle mode, crossflow rates and protein spiral wound modules where both the retentate and the concentrations, and retention versus filtrate lines are directed back to the TMP at different crossflow rates and recycle tank. Set specific flow, protein concentrations. From the pressure, concentration, and retention data, calculate the predicted temperature conditions. After the yield losses as described by the module has equilibrated at the equation shown in figure 6. The conditions, record the flows and collection of this data enables the pressures and collect small samples of choice of successful and robust the feed and filtrate streams for operating conditions. analysis of protein concentration. 13 Experiment Title: Sample Experiment Lab book Reference: 10739-25 Date: 04/25/99 Operator: JCT Objective: Determine operating parameters Feedstock Product and pool: Protein Y IEX pool Feedstock lot #: 10739-18 Membrane Material, MWCO: PLCGC Membrane Area [m2]: 0.1 Device Holder: Pellicon-mini Device lot #:

Pre-Use Cleaning and Equilibration Step Time Feed Rate Pfeed Pret Pfilt Filt. Rate Temp DP TMP Flux [hh:mm] [L/min] [psig] [psig] [psig] [L/min] [°C] [psid] [psid] [L/m2 h] 5 L DI water flush 9:10 0.5 24 10 0 0.17 22 14 17 102 1 L 0.1N NaOH total recycle 9:20 0.5 24 10 0 0.17 22 14 17 102 Normalized Flux testing 9:50 0.5 24 10 0 0.17 22 14 17 102 Integrity testing 10:00 30 0.001 1 L Equilibration buffer recycle 10:10 0.5 25 10 0 0.15 22 15 17.5 90

Process: Starting Feedstock Volume = 4 L at 2 g/L Step Time Run Time Feed Rate Pfeed Pret Pfilt Filt. Rate Filt Vol. Temp DP TMP Flux [hh:mm] [min] [L/min] [psig] [psig] [psig] [L/min] [L] [°C] [psid] [psid] [L/m2 h] J v T, Q1, C =2 g/L 10:20 0.7 33 7 0 0.108 22 26 20 65 10:30 0.7 43 17 0 0.152 22 26 30 91 10:40 0.7 63 37 0 0.196 22 26 50 118 J v T, Q2, C = 2 g/L 10:50 0.5 17 3 0 0.042 22 14 10 25 11:00 0.5 27 13 0 0.087 22 14 20 52 11:10 0.5 37 23 0 0.125 22 14 30 75 11:20 0.5 47 33 0 0.147 22 14 40 88 11:30 0.5 57 43 0 0.155 22 14 50 93 J v T, Q3, C = 2 g/L 11:40 0.3 13 7 0 0.032 22 6 10 19 11:50 0.3 23 17 0 0.065 22 6 20 39 12:00 0.3 33 27 0 0.092 22 6 30 55 Conc to C = 20 g/L 12:10 0 0.5 37 23 0 0.125 0 22 14 30 75 12:27 17 0.5 37 23 0 0.110 2 22 14 30 66 12:43 33 0.5 38 23 0 0.088 3.6 22 15 30.5 53 12:47 37 0.5 40 23 0 0.073 3.2 22 17 31.5 44 J v T, Q2, C = 20 g/L 12:57 0.5 17 3 0 0.025 22 14 10 15 1:07 0.5 27 13 0 0.052 22 14 20 31 1:17 0.5 37 23 0 0.073 22 14 30 44 1:27 0.5 47 33 0 0.080 22 14 40 48

Post-Use Cleaning Step Time Feed Rate Pfeed Pret Pfilt Filt. Rate Temp DP TMP Flux [hh:mm] [L/min] [psig] [psig] [psig] [L/min] [°C] [psid] [psid] [L/m2 h] 2 L 0.1N NaOH flush 1:45 0.5 24 10 0 0.16 22 14 17 96 1 L 0.1N NaOH total recycle 1:50 0.5 24 10 0 0.16 22 14 17 96 Normalized Flux testing 2:20 0.5 24 10 0 0.16 22 14 17 96 Integrity testing 2:10 30 0.001 1 L Storage solution recycle 2:20 0.5 24 10 0 0.16 22 14 17 96

Figure 15. Example of data collection sheet for a TFF performance characterization experiment

14 Test the Process! Set up System After choosing the membrane, Install Membranes module, and all operating parameters, run the entire process to ensure that performance meets all criteria for Clean Membranes acceptability. During the process, and System monitor flows and pressures. Collect samples of all initial and final streams. Calculate process time to ensure that it Test Integrity and Permeability is within the expected range. Test the quality of the final product with reliable assays, ideally the assays that Equilibrate with will be used during actual processing Process Buffer for qualifying product release. In order to understand where the product is going during a process, it is Concentrate important to calculate not only yield, Labscale™ Benchtop TFF System with Diafilter but also mass balance. Determine the Pellicon XL module. Complete, linear total protein in each of the retentate, scalable solution for small-volume the filtrate, and the unrecoverable processing. Remove Product holdup volume. Ideally, these amounts from System sum to the total amount that was put into the unit operation. If they fall process is in place. In addition, it will short, there were likely some help to ensure that the process Clean Membranes adsorption and/or solubility losses parameters were not determined and System during the process. However, if the based on a best-case run that is not amount of protein unaccounted for is a reproducible. large percent of the total, either the Test Integrity process is not operating correctly or Putting the Process Together and Permeability some operating parameters need to Once a protein processing procedure be changed to reduce the losses. The has been developed, it must be yield and mass balance follow the integrated into a complete process. Store law of conservation of mass where: The typical sequence of steps in an ultrafiltration/diafiltration process are Figure 16. Typical sequence of steps in a V * C = V * C + V * C + V * C outlined in figure 16. o o r r f f h h TFF process Set Up and Pre-Use Cleaning The subscripts o, r, f, and h refer to Before installation of membranes into original, retentate, filtrate, and holdup, a new TFF holder, thoroughly clean appropriate Millipore Maintenance respectively. The percent yield in any and flush all components of the holder Procedures for recommended one of the streams can be calculated and system to remove potential cleaning, sanitization, and depyro- by dividing the amount protein in that contaminants that were introduced genation solutions, recirculation times, stream by the total amount in the during manufacture and assembly. and temperatures. feedstock. For instance, the yield in Scrubbing exposed surfaces with a the retentate is calculated as: soap solution and recirculating the Integrity and Permeability solution through all piping with the use Testing Yield [%] = 100* Vr * Cr / Vo* Co of special cleaning gaskets, followed In order to ensure that the installed by extensive flushing with high quality membranes have not sustained any Finally, to understand how robust a water removes residual dirt and oils. damage during storage and handling, process is to feedstock variability and After new membranes have been Millipore recommends integrity testing multiple cycles, it is very helpful to run installed, and before their first use on all TFF assemblies prior to startup and the process several times. Although product, clean, sanitize, after each post-use cleaning. An air this is not always possible, especially depyrogenate, and flush the assembly diffusion test identifies problems such when feedstock is extremely limited, it to remove membrane preservatives as macroscopic holes in the membrane, can help guard against unexpected and any contaminants introduced cracks in the seals, or improperly performance degradation once the during installation. Please refer to the seated modules.

15 When air is applied to the retentate side of the membranes at a controlled pressure, it diffuses through the water in the pores at a predictable rate. If there are defects, the air will be able to flow through them at a much higher rate than the background diffusion, giving a failing test value. To obtain accurate test results, fully wet the membranes with water and then completely drain the modules. In the Maintenance Procedures manuals, Millipore provides instructions on performing integrity tests and lists test pressures and diffusion specifications for all of its membranes. Prior to the first use on protein and after each post-use cleaning, measure the clean membrane permeability to establish a baseline for flows and pressures. This value, also called the normalized flux, is determined by recording crossflow and filtrate flow rates, feed, retentate, and filtrate pressures, and temperature

Normalized Flux [L m-2 h-1 psig-1] = (flux x temperature correction factor)/TMP

during recirculation of a solution. Then, calculate: For a given TFF setup, always take the measurement at similar operating conditions, preferably at a low TMP, using the same solution. Refer to the Millipore Maintenance Procedures for specific instructions and temperature correction factors.

Pre-Use Equilibration and Protein Processing Since the membranes are typically in storage solution just before processing, drain the TFF system and then flush it with high quality water to reduce the level of residual storage components to acceptably low levels. It is good practice to then equilibrate the system in a buffer solution before introduction of protein. This prevents any solubility or product quality changes that could occur if the protein solution was suddenly exposed to very different pH or ionic strength conditions. Also, it minimizes the exposure of the protein to air/liquid interfaces that result from using the protein solution to fill the empty recycle tank or start the recirculation with empty piping. Perform the equilibration step at the same flow and pressure conditions at which the protein will be processed. Once the piping is filled with buffer and the recycle tank is filled to a level that, at minimum, submerges the retentate return pipe, introduce the protein feedstock into the tank and process according to the determined parameters.

Product Recovery Product recovery is the process of removing the product from the TFF system into a vessel appropriate for storage or further processing. Devise a procedure to remove the product as completely as possible from the system in order to maximize yield. The bulk of the product, which is typically in the recycle tank, is pumped out using the feed pump. However, some liquid remains held up in the piping and the modules. A well-designed system has minimal deadlegs in the piping and is sloped to a collection port at the lowest point in the piping to improve draining. Beyond simply draining the system, however, one of the following methods can be used to increase product recovery: • Low-pressure air blowdown • Buffer displacement • Buffer flush • Buffer recirculation

Air Blowdown Using air pressure to enhance volumetric recovery from a system, introduce the pressurized line at a high point in the piping and collect the product from the lowest point. Take care to avoid bubbling into the product, since this causes

16 foaming and potential product collected along with the product, buffer flush or recycle will reduce the degradation. In addition, in order to diluting the final product, so use the remaining volumes even further. maximize recovery the blowdown minimum amount required for good Post-Use Cleaning and Testing should occur gradually, since once an recovery. This dilution needs to be After each membrane use and product air path through the piping is formed, compensated for during any recovery, clean the TFF assembly using further blowdown will not increase the concentration steps of the process to the same cleaning, sanitization, and volumetric recovery. ensure that the final diluted product depyrogenation protocol that was concentration meets any specification. Buffer Displacement performed before use. In addition, Since over-concentrating a product is Alternatively, use buffer to push out the perform integrity and permeability often impractical because of solubility remaining liquid. If this is done while testing. Comparing the post-use or minimum volume reasons, a buffer the piping is still filled with product, it cleaned membrane permeability with flush is not always feasible. is called a buffer displacement; if it is the original value indicates the done after the piping has been emptied, Buffer Recirculation effectiveness of the cleaning. The trend it is called a buffer flush. A buffer A final method for increasing the of the value with repeated cycles can displacement can be performed recovery of product from the be used to gauge expected membrane without significantly diluting the final membrane modules and piping is a lifetime and to set a limit on maximum product if the volume of buffer used is buffer recycle. For this procedure, add number of cycles for one set of only slightly larger than the volume of buffer to the drained system, membranes. piping to be cleared. Since the buffer recirculate, and then recover it in the Storage is being used like a plug to push the same manner that the product was If another protein processing run product through the piping, in theory recovered. The buffer dilutes out any doesn’t immediately follow the no buffer should be collected in the residual product in the system. The cleaning, recirculate an appropriate product tank until the product has recirculated buffer is added to the storage solution through the TFF been completely replaced by buffer in product, so this procedure has the assembly to prevent organism growth. the piping. The effectiveness of a dis- same issues of product dilution as The membrane modules must remain placement step, however, is reduced if a mentioned above. The amount of filled with storage solution until the lot of mixing occurs between the buffer liquid remaining in different types of next run to prevent drying of the displacer and the held up product. filter modules after gravity draining as membranes. At this point, the well as after draining followed by Buffer Flush membranes and holder can be low-pressure air blowdown compared During a buffer flush procedure, the isolated and removed to allow the to the volume in the modules when full buffer rinses the system of residual tank, piping, and instrumentation to be is shown in table 2. A well-executed product. The entire amount of buffer used for other processes. added to the system for the flush is System Considerations To implement a complete TFF process, Module Liquid Holdup Liquid Holdup after Liquid Holdup after when Full Gravity Drain Drain and Blowdown the piping and equipment associated with the membrane modules must be [mL m-2] [mL m-2] [mL m-2] selected and a method for controlling Pellicon 2 180 140 10 the process parameters at their setpoints must be chosen. Helicon 400 20 15 Equipment Options Table 2. Liquid holdup in UF filter modules In addition to the membrane modules and holder, at minimum a working TFF Item Sanitary Consideration operation requires a recycle vessel, a feed pump, a retentate control valve, Piping, Fittings Stainless Steel, 316 L or better, 20 Ra ID finish or better and pressure sensors for the feed and Connections Tri-Clamp® style retentate lines. Many systems also include feed and filtrate flow meters, a Elastomers EPDM, silicone filtrate pressure sensor, and sensors for Valves Diaphragm preferred temperature, pH, conductivity, or UV absorbance. Pumps Circumferential piston displacement, rotary lobe, centrifugal Most TFF systems used for protein Instrumentation Sanitary fluid flow path (materials and design) processing are operated in a sanitary manner. Table 3 lists the primary

Table 3. Acceptable components and materials for sanitary systems 17 sanitary design considerations for different system components. Finally, consider the process requirements for volume reduction, buffer exchange, and product recovery when choosing equipment design and layout. In a typical ultrafiltration process, the maximum practical VCF is approximately 50 – 100 before the limitation of minimum recirculation volume in a single tank becomes a significant problem, even when more novel tank designs are used. Examples of tank features that can reduce the minimum recirculation volume are a conical bottom, a reduced-diameter lower section, and a low side-entry retentate return port. Likewise, diafiltration of non-retained species is typically limit- Fully automated 80 m2 Pellicon system for concentration and diafiltration. ed to a maximum of approximately 14 diavolumes, since beyond this any incomplete mixing or deadlegs in the Constant Crossflow Rate Constant Retentate Pressure system will significantly reduce the To control the tangential flow based The simplest way to control the effectiveness of further buffer exchange on crossflow rate, install a flow meter applied pressure is to set a constant or contaminant removal. on the unit operation downstream of retentate pressure by adjusting a valve Postprocessing recovery of a retentate the feed pump and prior to the mem- on the retentate line. For unit product is enhanced when a system brane modules. The benefit of this type operations where the tangential flow is has minimal deadlegs, minimal piping of control is that the crossflow rate is controlled based on a crossflow rate, length, and piping that is sloped to a known to be constant even if the the TMP changes slightly throughout recovery port at the lowest point. resistance to flow through the feed the process. For unit operations where channels changes. Constant crossflow the tangential flow is controlled based Process Control Options control is especially useful when on a pressure drop, the TMP remains Throughout a TFF process, as protein processing solutions that experience constant. is concentrated or exchanged into viscosity changes during processing Constant TMP different buffers, the process and to facilitate accurate pump sizing Alternatively, set a constant TMP for parameters need to be adjusted so during scale-up. that they remain at their setpoints. crossflow rate controlled operations by Several methods of process control are Constant Pressure Drop changing the retentate pressure used to accomplish this. The tangential Alternatively, control the tangential setpoint throughout the process as the flow can be controlled to maintain flow by setting a constant feed feed pressure changes. This is slightly either pressure or pressure drop. A flowmeter more complicated and there is usually is not required for this type of control, no significant benefit. • Constant crossflow rate only pressure gauges are needed, so Constant Flux • Constant pressure drop the instrumentation is simpler and less Change the retentate pressure expensive. However, pressure drop The applied pressure can be throughout a process in response to often changes throughout a process controlled to maintain a constant changes in the filtrate rate to maintain due to changes in solution viscosity or, • Retentate pressure a constant flux setpoint. This type of occasionally, restriction of feed control is useful for realizing some of • TMP channels by foulants. In addition, the benefits of constant Cwall variability in membranes and feed- • Flux processing without requiring a fully stock cause lot to lot pressure drop • Cwall automated system. A constant flux changes. When choosing a method setpoint can also be achieved through • Mixed mode control of tangential flow control, consider the the use of a pump or a control valve characteristics of the process fluid as placed on the filtrate line, instead of well as the precision required to using the retentate control valve. This achieve process objectives. control scheme is very common on 18 open UF (>100kD) and MF applications and was described in more detail on page 9. Constant Cwall An alternate method of process control, called Cwall process control, maintains a constant protein concentration at the membrane surface throughout a process. The retentate pressure is modified to maintain a flux setpoint that changes according to an algorithm that takes into account both the protein mass transfer coefficient in the specific buffer and the instantaneous protein concentration. One of the benefits of Cwall process control is that it allows operation at the optimum TMP throughout a process even as that TMP changes. Therefore, yield and membrane area are both optimized in the same process. The drawback to this method of process control is that it is more complicated than the other Large-scale spiral wound UF/DF system schemes and requires the use of an automated control system. Product Yield show measurable filtrate loss when Mixed Control There are four contributors to product they are significantly concentrated or To prevent the unit operation from loss during a TFF step: diafiltered. In addition, because of exceeding certain undesirable • Retention losses charge effects, retention of a molecule operating conditions regardless of can change if the pH and ionic fluid changes throughout the process, • Adsorption losses strength of the solution changes. use a mixed mode approach to • Solubility losses Adsorption Losses process control. For example, in • Unrecoverable holdup volume Adsorption losses occur when product addition to a constant filtrate flow losses binds to a membrane and cannot be setpoint, set a maximum TMP control In addition, if protein quality is desorbed in an active form prior to setpoint that overrides the filtrate compromised during processing, the recovery. For applications in which control. In many processes, retention yield of usable protein will be product concentration is high in changes with TMP, so the overriding reduced. With an optimally designed comparison to the membrane area TMP setpoint keeps the process from process, yield loss can be minimized used to process it, adsorption operating at conditions where in each of these areas. Table 4 shows probably won’t be a significant mode retention is unacceptable. the relative magnitudes of product loss of yield loss. However, if product concentrations are very low and/or a Major Process Considerations that can be attributed to each of the different sources noted above. In very large membrane area is required As previously noted, it is important to addition, the process choices that for processing, this loss mechanism define and prioritize the goals and affect each of the loss mechanisms are should not be ignored. The membrane requirements of a TFF operation during listed. material that is chosen will affect how the development phase so that the much protein is adsorbed for a given operating parameters and system Retention Losses area. In general, hydrophilic options that are chosen will result in a Choosing a membrane with membranes will exhibit lower protein successful process. This section will appropriate retention characteristics is binding than membranes that are discuss in more detail the key critical to ensuring high product yield. more hydrophobic. Adsorption losses considerations of product yield, If a product in the retentate is being will also be affected by other product quality, bioburden control, concentrated or desalted, low components in the feed stream that scalability, robustness, and economics retention results in product being lost may interact with both the membrane and will define how each is affected through the membrane to the filtrate. and the product. by the process design. Even highly retained products can

19 Source of Loss Retention Adsorption Hold-Up Solubility/Quality

Magnitude of Loss [%] 0.4 – 10 0.02 – 2 0.2 – 10 0.1 – 20 Choices affecting loss Membrane NMWL Membrane material System design Operating parameters Operating parameters System materials Recovery method System components Buffer selection Protein susceptibility Buffer selection

Table 4. Typical product yield losses during a TFF process

Solubility Losses Product Quality Finally, filling the system with buffer Solubility losses are a third mechanism During the course of a TFF process, before introducing protein solution will of product loss during protein pro- the quality of a protein could be minimize any air entrainment during cessing. The final bulk concentration compromised due to aggregation or startup. of a product may not be beyond its denaturation caused by High Protein Concentration solubility limit. However, polarization • Micro-cavitation As described in the previous section, at the membrane surface and feed • Air/liquid interfaces there is the potential for highly stream volume reduction along the concentrated areas to exist within the • High protein concentrations channel result in areas of higher TFF unit. Since protein aggregation is concentration throughout the TFF unit. • Temperature effects a result of protein-protein interactions In addition, inadequate mixing will that are concentration-dependent, increase concentration differences. The potential for this damage depends, in part, on the susceptibility higher concentrations could result in Product concentration in these areas more aggregated protein. The same could potentially exceed a solubility of the particular protein being processed. However, even for considerations for process control limitation. Since higher fluxes lead to should be made as mentioned above. higher localized concentrations, delicate products, damage can be reducing flux is one way to minimize minimized or eliminated by designing Temperature Effects solubility losses if they are significant a robust process and system. Lowering the process temperature at in a particular process. Alternatively, Micro-Cavitation which a TFF process is run is a method using a process control scheme where To prevent cavitation, which occurs often used to attempt to minimize the concentration of product at the when a pump pulls a vacuum at its product quality degradation. membrane surface is controlled can inlet and fluid subsequently degasses, However, it can exacerbate any maximize flux without exceeding the feed pump should always be run solubility problems, since proteins are solubility limitations. with a minimum inlet pressure equal to typically less soluble at lower temperatures. In addition, filtrate flux is Holdup Volume Losses the manufacturer’s recommendation. However, micro-cavitation will still reduced at lower temperatures Finally, unrecoverable holdup volume because of the corresponding viscosity in a unit operation leads to product occur to some extent when the protein feedstock makes multiple passes increase and mass transfer coefficient loss. After processing, a certain decrease. Flux decreases amount of liquid remains in both the through the feed pump and/or through a partially closed pressure approximately 2 – 3% per degree filter modules and the system piping. Celsius of temperature reduction. In cases where the final product control valve. The selection of appropriate pumps and valves can Therefore, for equal membrane area, concentration is high and/or when the process time will be longer at a lower final product volume is small, these help prevent the protein denaturation that microcavitation can cause. temperature. Since protein losses could be significant. Careful degradation can be a kinetic design of the piping, optimization of Air/Liquid Interfaces phenomenon, a longer process time total membrane area, and Other air/liquid interfaces can occur may eliminate the benefit of the lower development of an efficient product in several places throughout a TFF temperature. Alternatively, if the recovery step will help to minimize the system. In the recycle tank, the membrane area was increased to product loss incurred due to retentate stream should always be compensate for the flux decrease, a unrecoverable holdup. returned below the liquid surface to higher crossflow rate would be prevent foaming, and vortex formation needed, which would expose the should be avoided by using an off protein to more passes through the center drain or baffles in the tank. pump.

20 Bioburden Control The ability to reliably control bioburden levels in a process stream is very important in protein processing. An outbreak of bioburden in an upstream process step may raise quality concerns but be tolerable, while in a downstream or final process step it may cause failure of the entire batch. While the risk of bioburden contamination is not necessarily greater in a TFF step than in other processing steps, there are ways that the risk can be minimized during TFF processing. The method used to clean and sanitize a TFF unit operation between uses is obviously important in controlling bioburden (as well as ensuring removal of endotoxin). The chemicals that are chosen must be not only effective for cleaning and Pellicon 2 Industrial Scale sanitization, but also compatible with the membrane and piping materials and able to be rinsed out to The simplest way to ensure acceptably low levels before further accurate and predictable translation of processing. Typical chemicals used to product yield and purity from bench to clean TFF systems include sodium industrial scale is to use linear scale hydroxide and sodium hypochlorite. techniques. To linearly scale a TFF The Millipore Maintenance Procedures process, all fluid dynamic and provide appropriate cleaning membrane module parameters must be concentrations, times, and kept constant between scales of temperatures for specific membrane operation. Fluid dynamic parameters, systems. which are set by the user, include the The total cycle time for a TFF ratio of feed volume to membrane process can affect bioburden loads. area (at constant feed concentration), Since TFF processes are typically run feed rate per membrane area, filtrate under sanitized but non-sterile flux, and retentate and filtrate Pellicon 2 Mini Holder conditions, extended processing times pressures. Membrane module increase the potential for higher parameters are inherent in the specific bioburden loads. filters that are chosen, and include troubleshoot or develop improvements to membrane material and pore size, a process once it has been implemen- Scaleup and Scaledown turbulence promoter, channel height, ted into a manufacturing line. Once a process has been developed channel length, and feed and filtrate at lab bench scale, it must be flow geometries. Robustness translated to industrial scale and Linear scaling is also used to Once implemented at large scale, a validated, and this can present reduce the scale of a process. This process must be robust if it is to be unanticipated surprises and can be very useful for process successful. A robust TFF process will challenges. Often, there is little validation, where it is sometimes not perform well within the lot-to-lot opportunity to perform intermediate- economically feasible to perform all of feedstock and membrane variations scale runs due to time and material the required validation at full operating that it encounters. While developing a constraints. In addition, material from scale. Validation performed at small process, it can be very useful to test the initial industrial-scale runs is usually scale will only be acceptable if it can performance at the extremes of these required for time-sensitive clinical or be shown to produce results that are variations, if possible. For instance, marketed supply. Therefore, accurate equivalent to industrial scale. In determining flux of the most fouling lot and dependable scaleup is critical for addition, linear scaledown is used to of feedstock using membranes having the success of a process. the lowest acceptable permeability

21 would allow the membrane area to be Typical capital costs include The number of times a set of sized such that the maximum process membrane holders, plant floor space membranes will be used before time was not unacceptable. requirements, utilities, pumps, valves, installing new membranes affects the Alternatively, determining retention of instrumentation, piping, tanks, and labor and materials costs. For single the product in the least fouling lot of process automation. A unit operation use membranes, the membrane feedstock using membranes having the with the smallest acceptable expense is high. However, labor, highest acceptable permeability would membrane area minimizes the cost power consumption, and ensure that the chosen membrane associated with the holders and water/chemical costs associated with cutoff would not lead to significant piping. An efficient unit operation will cleaning are minimized. In addition, product loss. A robust TFF unit have a high packing density of the cost for validating the acceptability operation should also be able to membranes, requiring minimal plant of reuse is avoided. The labor withstand some variations in operation floor space for a large membrane required for installing a new set of without catastrophic failure. area. Manually operated systems membranes before each run could be Consideration of membrane and have lower instrumentation and high, depending on the membrane module characteristics such as automation costs associated with them area and module type. On the other susceptibility to reverse transmembrane than fully automated systems. hand, using membranes for multiple pressure, chemical compatibility, or Optimizing the diafiltration step and runs lowers the per-run membrane cost stability at high pressures help to minimizing the number of different at the expense of higher power, guide the choice of a unit operation buffer or cleaning solutions will water, chemical, and validations that will operate robustly. minimize tank costs. Finally, a system costs. However, if a single set of that processes multiple products will membranes is used for an excessive Process Economics be able to split capital expenses over number of runs, the value gained by The economics driving the success or a larger profit base. not installing a new set of membranes failure of TFF process steps are case Typical materials costs include is negated if membrane performance specific, since different applications membrane modules, buffer and water begins to degrade. This approach have very different economic goals. usage, cleaning chemical usage, also greatly increases both validation For some very high value products, the power consumption, and product loss. costs and risk to the product. product cost is relatively insensitive to As with the capital costs, consumable the economics of a single process costs are minimized if as low a step, while for other products every membrane area as acceptable is cost must be minimized for the product installed. Choosing membranes that to be competitive. The costs are easy to clean helps to reduce the associated with TFF steps break down water and chemicals used after each into four categories – capital, materials, run. Labor costs are reduced by using labor, and overhead. an automated unit operation, but this will increase capital expenditures.

22 Glossary of Terms Technical References Cb: Component concentration in the bulk solution [g L-1] Cheryan, M. 1986. Ultrafiltration Handbook. Technomic Publishing Co., Cf : Component concentration in the filtrate stream [g L-1] Inc., Pennsylvania. CF: Protein concentration factor [-] Koros, W.J.; Ma, Y.H.; Shimidzu, T. CR: Conversion ratio [-] 1996. Terminology for Membranes Crossflow: The flow of fluid through the and Membrane Processes (IUPAC feed channels of the membrane modules Recommendations 1996). Pure & created by a pump Appl. Chem. 68: 1479 – 1489. Cw: Component concentration at the membrane surface [g L-1] Ng, P.; Lundblad, J.; Mitra, G. 1976. Optimization of Solute Separation by DF: Diafiltration Diafiltration. Separation Science 2: DV: Diavolume [-] 499 – 502. Feed: The fluid that flows from the recycle tank into the feed channels of the Tkacik, G.; Michaels, S. 1991. A membrane modules Rejection Profile Test for Ultrafiltration Filtrate: The fluid that passes through the Membranes and Devices. Bio/Technol. membranes, also commonly called 9: 941 – 946. permeate HPTFF: High performance tangential flow van Reis, R.; Gadam, S.; Frautschy, filtration L.N.; Orlando, S.; Goodrich, E.M.; -2 -1 Saksena, S.; Kuriyel, R.; Simpson, Jf: Filtrate flux [L m h ] C.M.; Pearl, S.; Zydney, A.L. 1997. J : Mass flux [g m-2 h-1] m High Performance Tangential Flow kD: Kilodalton Filtration. Biotech. Bioeng. 56: Mass balance: The amount of the target 71 – 82. product in all pools compared to the total amount put into the unit operation [%] van Reis, R.; Goodrich, E.M.; Yson, MF: Microfiltration C.L.; Frautschy, L.N.; Dzegeleski, S.; NFF: Normal flow filtration Lutz, H. 1997. Linear Scale NMWL: nominal molecular weight limit Ultrafiltration. Biotechnol. Bioeng. 55: 737 – 746. PF: Feed pressure [bar] Pf : Filtrate pressure [bar] van Reis, R.; Goodrich, E.M.; Yson, Pool: A general term denoting a total C.L.; Frautschy, L.N.; Whiteley, R.; volume of fluid Zydney, A.L. 1997. Constant Cwall

PR: Retentate pressure [bar] Ultrafiltration Process Control. -1 J. Membrane Sci. 130: 123 – 140. QF: Feed flow rate [L h ] -1 Qf : Filtrate flow rate [L h ] van Reis, R.; Zydney, A.L. 1999. -1 QR: Retentate flow rate [L h ] Protein Ultrafiltration. In M.C. Flikinger, Rapp : Apparent or observed retention [-] S.W. Drew (eds.) Encyclopedia of Recirculation: The flow of fluid through the Bioprocess Technology. John Wiley channels of the membrane modules and Sons, Inc. New York. created by a pump Zeman, L.J.; Zydney, A.L. 1996. Retentate: The fluid that flows out of the Microfiltration and Ultrafiltration: feed channels of the membrane modules back into the recycle tank Principles and Applications. Marcel Dekker, New York. Ri: Intrinsic retention [-] RO: Reverse osmosis

Sapp: Apparent or observed sieving [-] Si: Intrinsic sieving [-] TFF: Tangential flow filtration TMP: Transmembrane pressure [bar] UF: Ultrafiltration VCF: Volume concentration factor [-] VF: Virus filtration Yield: The amount of the target product in a pool compared to the total amount put into the unit operation [%] ∆P: Pressure drop [bar] 23 For More Information If you would like to learn more about Tangential Flow Filtration processing, as well as other protein purification operations, Millipore offers a variety of information and assistance. Training Millipore offers several classes on TFF processing for customers. These classes blend lecture time with hands on time in the lab to help you become proficient in both the theory and operation of tangential flow filtration. Please visit our website at www.millipore.com for information on the variety of training courses available. Expert Help Millipore’s applications specialists have extensive knowledge of the biotechnology and pharmaceutical fields. The AccessSM Services group works with you to optimize the use of TFF in your facility, and get your process up and running quickly and easily.

Discover the More in Millipore™ In every application, every step and every scale, count on Millipore to be everywhere for you—from monoclonals to vaccines, from clinical through pilot to full-scale manufacturing. Our technologies are used by most of the world’s major biopharmaceutical companies. But we deliver more than advanced separation, purification, sterilization and quality control products. With Millipore, you get services to optimize and validate your processes, comprehensive resources to streamline and enhance your operation, unmatched know how forged from nearly 50 years’ experience—and solutions that integrate it all. For higher yields, improved process economics and faster speed to market, discover the more in Millipore.

To Place an Order or Receive Technical Assistance For additional information call your nearest Millipore office: In the U.S. and Canada, call toll-free 1-800-MILLIPORE (1-800-645-5476) In the U.S., Canada and Puerto Rico, fax orders to 1-800-MILLIFX (1-800-645-5439) Outside of North America contact your local office. To find the office nearest you visit www.millipore.com/offices. Internet: www.millipore.com Technical Service: www.millipore.com/techservice

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