PN33339Scientific & Technical Report

The Partnership of the Minimate™ TFF Capsule with Liquid Chromatography Systems Facilitates Lab-scale Purifications and ProcessDevelopment Through In-Line Monitoring

Introduction

Tangential Flow FiltrationTangential flow filtration (TFF) is an effectivemethod for both diafiltration and concentrationoperations in purification strategies for bio-molecules.1,2 The TFF process recirculatessample flow over the surface of a membrane,controlling gel layer formation and effectivelyreducing fouling. The operator develops aneffective and reproducible process by optimizing flow rates and controlling trans-membrane pressure. This results in improvedflux rates and productivity.3 A TFF processdeveloped on lab-scale devices can be readily scaled to larger process volumes.

Traditionally, TFF separations have used peristaltic pumps and flexible tubing to provide the needed pressure and flow. Although standard lab pump systems aresufficient for most TFF applications, the useof high-performance liquid chromatography(LC) systems can offer some additional advantages with respect to process development and control. This paper describes the novel use of an LC system as the control platform for performing TFFusing a Minimate TFF capsule.

Liquid Chromatography and TFFProtein purification is rarely a simple processand almost never a single step. Instead, it requires an ordered sequence of purificationsteps assembled into a process compatiblewith the biochemistry of the target protein to achieve the required purity. Purification

protocols include a variety of separationtechniques that may include general filtration/clarification, size exclusion chromatography,affinity chromatography, ion exchange chromatography, ultrafiltration, and possibly a sterilization step. TFF is frequently used in a process before chromatographic steps to desalt and buffer exchange (diafiltration) thesample into a suitable state for binding or toadjust the sample concentration. It may alsobe applied as a bridge between chromato-graphic steps when the elution buffer at theend of one process is incompatible with thebinding conditions of a subsequent step. At the end of a process, it may be used toadjust the final concentration and ionicstrength of the product as required.

Few protein chemists are without access to an LC pump system that has pumping capacities approaching 100 mL/min. Thesesystems can serve as robust alternatives tothe use of a peristaltic pump for TFF protocolswith significant additional benefits. Here wereport the use of an ÄKTA Explorer� liquidchromatography system (GE Healthcare) to operate the Minimate TFF capsule.

Specific advantages of using an LC pumpsystem to drive TFF are:

• Use of chromatography equipment alreadyin place saves equipment cost, bench-space, and training time.

• Precise control of pressure and flow rate assures uniform development of the gel layer to improve reproducibility between runs.

• Ability to monitor and record process parameters inreal time (pH, conductivity, absorbance, tempera-ture, feed flow rate, valve positions, system pressure)gives a researcher an auditable trail for process validation.

• Reduction in system working volume with narrowbore PEEK� and Tefzel� tubing allows greater concentration factors.

• Operation of system valves to multiplex purification procedures using the same pumping system (sequential operations on multiple cartridges andcolumns). TFF devices can be sanitized in placewhile other column operations are underway.

Minimate TFF Capsule with LC Pump System

Materials and Methods

Minimate TFF CapsuleMinimate TFF capsules contain Omega™ polyether-sulfone membrane with an effective filtration area of 50 cm2. The Omega ultrafiltration (UF) membrane,available in a wide range of molecular weight cut-offs,offers low adsorption characteristics resulting in highproduct recoveries. Sample batch sizes of up to 1 litercan be desalted and subsequently concentrated tovolumes as low as 5 mL with little user intervention.The reusable/disposable capsule can perform singleor sequential concentration/diafiltration steps using thesame device in a closed loop connection, saving timeand avoiding product loss associated with transfer

steps. An optimized fluid path minimizes hold-up andworking volumes and allows easy scale up to largerTFF systems like Centramate™ and Centrasette™systems. The Minimate TFF capsule and larger devicesutilize consistent materials of construction to assist inprocess validation. Finally, each pharmaceutical gradeMinimate TFF capsule is 100% integrity tested duringmanufacture to ensure reliable performance.

Technical Data

Effective Filtration Area 50 cm2 (0.05 ft2)

Recommended Crossflow Rate 30-80 mL/min (0.6-1.6 L/min/ft2)

Operating Temperature Range 5-50 °C (41-122 °F)

Maximum Operating Pressure 4 bar (400 kPa, 60 psi)at 20 ºC (68 ºF)

pH Limits 1-14

Product Hold-up Volume (feed/retentate) ~1.6 mL

Ultrareservoir™ Container, 500 mLThe Ultrareservoir container is designed to hold up to500 mL of sample and give efficient mixing of product.The sloped bottom and optimally located feed and return ports reduce the system hold-up volume, whichallows high concentration factors to be achieved. Thereservoir lid seals tightly, allowing additional samplevolume or diafiltration solution to be drawn into thereservoir by vacuum that is created as filtrate is gener-ated through the TFF device. This allows continuousdiafiltration to be performed without the need for atransfer pump.

Equipment and Reagents1. ÄKTA Explorer Chromatography Workstation with

Unicorn� 4.0 Software (GE Healthcare)

2. Digital Pressure Gauges (Cole Parmer, Model 1200)

3. 1XPBS (phosphate buffered saline) Solution (Invitrogen, Carlsbad, CA)

4. 1M NaCl in PBS

5. Bovine Serum Albumin (BSA) solution: 1 mg/mLdissolved in PBS with 1M NaCl

6. 0.5M sodium hydroxide in water

7. 0.1M sodium hydroxide in water

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Step-by-Step Procedures

Table 1Parts List for Connecting the Minimate TFF Capsule toÄKTA Explorer FPLC System

Description Qty

Tefzel tubing, 1/8" (use the minimal amount of tubing) 3 ft

PEEK tubing (green), 0.030 x 1/16" (use the minimal 3 ftamount of tubing)

Tefzel tubing, 1/16" (use the minimal amount of tubing) 3 ft

Adapter assembly, 1/8" NPT to 5/16, 24 flat 3 eachbottom female

Tubing connector, 1/8" male 3 each

Ferrule for 1/8" tubing (use the minimal amount 3 eachof tubing)

Union, 5/16" female 1/16" male 1 each

Fingertight connector, 1/16" 12 each

Pressure gauge TEE 3 each

Digital pressure gauge, 0-300 psi range 3 each

10-32 female to male luer assembly 2 each

Micro-metering valve, 1/16" 2 each

10-32 female to female luer assembly 2 each

Set-up1. Connect an outlet valve line from the ÄKTA

(e.g., F8) to the “From Device” outlet of the Ultrareservoir container.

2. Connect a buffer valve line from the ÄKTA (e.g., A18) to the “To Pump” outlet of the Ultrareservoir container.

3. Choose a column valve position from ÄKTA (e.g.,position 4) for the Minimate capsule. Connect bottom column valve to a digital pressure gauge

(P1) and then connect P1 to one of the “Feed/Retentate” ports of the Minimate TFF capsule. Connect top column valve to a digital pressuregauge (P2) and then connect P2 to the other“Feed/Retentate” port of the capsule. Orient thecapsule in a vertical position with the end con-nected to the top valve facing up. Connect theupper “Permeate/Drain” port of the capsule to adigital gauge (P3). Connect a length of tubing to the outlet from the pressure gauge and put theother end in a waste container. Connect the other “Permeate/Drain” port to a three-way valveconnector. Use the valve to close off the port.

4. Put separate buffer valve lines from the ÄKTA intothe following solutions:A13: PBSA14: 0.5M sodium hydroxideA12: 0.1M sodium hydroxideA11: BSA solution

Membrane Equilibration and Line Flushing1. Using manual instruction, set the flow path to

the following:Column Position: 4Outlet Valve: Waste F1Buffer Valve: A13Flow Direction: Up Flow

2. Start the pump by setting a flow rate of 20 mL/min. Increase flow rate until the feed pressure reaches2.7 bar (40 psi).

3. Inspect the line connection to make sure there is no leak.

4. Run for at least 5 minutes or until the pH, conduc-tivity, and absorbance signals flatten out.

5. Stop pump.

A Minimate TFF capsule is installedbetween the top and bottom column valves. Pump flow is directed into the TFF feed port, andthe retentate flow goes through theanalysis sensors and back to the Ultrareservoir container. Pressuresensors or gauges upstream anddownstream of the capsule monitorpressure, and a third gauge isplaced along the filtrate path tomonitor backpressure. In diafiltrationmode, the Ultrareservoir container isconnected to the diafiltration buffervessel. In concentration mode, the Ultrareservoir container is left open to the atmosphere.

Diagram 1Partnership of TFF with Column Chromatography

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Buffer Exchange A buffer exchange protocol was developed using 1MNaCl in PBS as the starting sample. This solution wasdiafiltered with PBS to remove salt and then back tohigh salt with a solution of 1M NaCl in PBS. Once theprocess conditions were developed, the process wasrepeated using a starting solution of 1 mg/mL BSA,1M NaCl in PBS.

1. Equilibrate the Minimate TFF capsule according tothe procedure described in the preceding section.

2. AutoZero the UV absorbance monitor of the ÄKTAand set the column position to “Bypass.”

3. Set the buffer valve line to A11 and pump flow rateto 50 mL/min. Fill the Ultrareservoir container with200 mL BSA solution (1 mg/mL dissolved in PBSwith 1M NaCl).

4. Put the diafiltration buffer feed line (connected to the top part of the Ultrareservoir) into a flask containing PBS.

5. Using manual instruction, set the flow path to the following:Column Position: 4Outlet Valve: F8Buffer Valve: A18Flow Direction: Up Flow

6. Start the pump at 20 mL/min. Adjust the flow rateso that the feed pressure (P1) is just under 2.7 bar(40 psi).

Note: Flow will start from the permeate tube. Diafiltrationsolution will start to be drawn into the Ultrareservoir con-tainer in a few minutes. If this does not occur, checkthat the reservoir lid is completely inserted and sealed.

7. Stop the pump once the conductivity signal reaches a plateau.

8. Put the diafiltration buffer feed line into a flask containing 1M NaCl in PBS solution.

9. Repeat steps 5-7.

Sample Concentration After the buffer exchange is complete, a sample canbe concentrated by opening the diafiltration bufferfeed line to air. This will break the vacuum generatedunder the diafiltration mode.

1. Leave the diafiltration buffer feed line open to air.

2. Using manual instruction, set the flow path to thefollowing:Column Position: 4Outlet Valve: F8Buffer Valve: A18Flow Direction: Up Flow

3. Start the pump at 20 mL/min. Adjust the flow rateso that the feed pressure (P1) is just under 2.7 bar(40 psi).

4. Collect the permeate in a graduated cylinder. (This will allow the permeate volume to be measured andconcentration factor to be determined.)

5. Stop the pump once the desired concentration is reached.

Washing, Regenerating, and Storing the Minimate Capsule1. Using manual instruction, set the flow path to

the following:Column Position: 4Outlet Valve: Waste F1Buffer Valve: A13 (PBS)Flow Direction: Up Flow

2. Start the pump by setting a flow rate of 20 mL/min. Increase flow rate until the feed pressure reaches 2.7 bar (40 psi).

3. Wash the system until the UV, pH, and conductivity signals stabilize.

4. Switch the buffer valve line to A14 (0.5M sodium hydroxide).

5. Wash the system until the UV, pH, and conductivity signals stabilize.

6. Stop the pump. Let the Minimate capsule soak in 0.5M sodium hydroxide for 1 hour.

7. Switch the buffer valve line to A12 (0.1M sodium hydroxide).

8. Wash the system until the UV, pH, and conductivity signals flatten out.

9. Stop the pump. The Minimate capsule can bestored in 0.1M sodium hydroxide at 4-20 °C.

Results and Discussion

Continuous Diafiltration for Process DevelopmentTo develop a process protocol, a mock buffer-exchange protocol was created where a buffer-onlysample (1N NaCL in PBS) was first diafiltered withPBS to reduce the salt concentration (Figures 1A, 1B). In-line monitoring allowed the documentation of salt concentration changes in real-time using the conductivity sensor and data acquisition program built into the ÄKTA Explorer. Consistent with previousobservations,1 the passage of four volumes of diafiltration buffer was adequate to achieve > 98%buffer exchange.

A sequential buffer exchange back to the high saltbuffer was possible without disconnecting the devicefrom the system by replacing the low salt (PBS) diafiltration buffer with a high salt buffer (PBS, 1M NaCl). Again, successful buffer exchange was detected in-line with the exchange of four buffer volumes (Figures 1C, 1D). Absorbance at 280 nm was monitored to act as a sample process control to determine if the absorbance readings are deflectedby the changing salt concentrations. The ability tomonitor and document the process “live” allows theresearcher to save time and buffer, as well as providesa basis for future scale up decisions.

Figure 1Development of Buffer Exchange Parameters with In-line Monitoring

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Diafiltration (buffer exchange) processing was documented fromhigh salt to low salt then back to high salt. Repeated runs using a10K Minimate capsule were processed at 20 (graphs A and C) and25 (graphs B and D) mL/min recirculation rates. Salt and protein(A280) concentrations were monitored using in-line sensors.

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Buffer Exchange at Constant Protein ConcentrationOnce a simple buffer-exchange protocol was devel-oped, a second protocol was used to demonstrate diafiltration of a protein solution (1 mg/mL BSA) goingfirst from high salt to low salt buffer (Figures 2A, 2B)and then back to a high salt buffer (Figures 2C, 2D). Inaddition to monitoring protein and salt concentrations,in-line sensors documented the A600 values to ensurethat the protein solution had not precipitated duringprocessing. A precipitated protein solution would produce turbidity and cause a reading at A600. The use of in-line monitors allows the researcher to immediately trace the incident back to the salt concentration where protein precipitation first occurred,thus saving valuable time in troubleshooting.

Figure 2Validation of Diafiltration Parameters in the Presence of Protein

Diafiltration (buffer exchange) processing was documented fromhigh salt to low salt then back to high salt with a protein solution (1 mg/mL BSA). Repeated runs using a 10K Minimate capsule were processed at 25 (graphs A and C) and 20 (graphs B and D)mL/min recirculation rates. Salt, protein (A280), and turbidity (A600)were monitored using in-line sensors.

Precise Control of Sample ConcentrationIn concentration mode, the Ultrareservoir container is left open to atmosphere. The concentration of theretained solute increases as permeate is removed, decreasing the process volume. The in-line monitorsallow the researcher to track the progress of the concentration of the protein sample. While the saltconcentration (which is 100% permeable to the membrane) remains constant, the protein solutionconcentration increases. The use of sensors allowsthe researcher to stop the process once the desiredconcentration is achieved. Documenting turbidity

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(A600) provides a good diagnostic to determineexperimentally the sample concentration limit prior toprotein precipitation. The 1 mg/mL BSA solution couldbe concentrated over four-fold within 40 minutes with-out the formation of turbidity (Figure 3). While theseconditions were chosen for this demonstration, in reality, much higher concentration factors for BSA canbe achieved. Other protein solutions may precipitatemore easily depending on solubility.

Figure 3Precise Control of Protein Concentration

The concentration of a 1 mg/mL BSA solution was accomplishedby leaving the diafiltration feed line open to air. The run using a Minimate 10K capsule was processed at 25 mL/min recirculationrate. Salt, protein (A280), and turbidity (A600) were monitored usingin-line sensors.

Sequential Buffer Exchange and ConcentrationAn advantage of processing using in-line sensors wasdemonstrated by performing sequential processingoperations. Diafiltration of the protein sample (1 mg/mL BSA) reduced the salt concentration by > 98% with about four volumes of buffer. As soon asthe conductivity leveled off, indicating that desaltingwas complete, sample concentration was started by simply disconnecting the buffer line to the Ultra-reservoir container. Concentration of retained soluteincreased as permeate was removed, decreasing theprocess volume. This process is facilitated using thefeatures of an LC system, such as the multiport valvesand in-line sensors. The ability to monitor “live” allowsgreater control over the process during the run andprovides documentation that can be used to developand validate the scale-up protocol.

Figure 4Sequential Operations for Diafiltration and Concentration

Sequential diafiltration followed by concentration was documentedfor a single run using a 1 mg/mL BSA solution in 1X PBS, 1M NaClstarting solution. The run using a Minimate 10K capsule wasprocessed at 25 mL/min recirculation rate with the buffer exchangefrom high salt to low salt. The diafiltration buffer feed line was thenopened to air, allowing the concentration of the sample. Salt, protein(A280), and turbidity (A600) were monitored using in-line sensors.

ScalabilityTFF process development can be performed on aMinimate TFF capsule with a simple lab-scale systemincorporating a peristaltic pump or with an LC pumpand in-line monitoring system as described above.Both allow development of procedures that are ultimately scalable. The advantage of the LC system is greater control and documentation. The automationand documentation aspects simplify development ofvalidated protocols and provide an audit trail for labsunder Good Lab Practices (GLP). This is extremelyuseful in the scale-up process.

While the Minimate TFF capsule is a valuable tool forsmall process volumes (typically under 1 liter), largervolumes can be processed by plumbing several Minimate TFF capsules in parallel. The Minimate TFFcapsule was designed with the same flow path lengthas larger TFF devices (Centramate and Centrasettesystems) allowing predictable performance and savingvaluable optimization time when scaling beyond labscale to volumes used in pilot and production plants.

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References

1. L. Schwartz, 2003. Diafiltration: A Fast, Efficient Methodfor Desalting or Buffer Exchange of Biological Samples.Pall Scientific & Technical Report.

2. L. Schwartz, 2003. Desalting and Buffer Exchange byDialysis, Gel Filtration or Diafiltration. Pall Scientific &Technical Report.

3. L. Schwartz and K. Seeley, 2002. Introduction to Tangential Flow Filtration for Laboratory and Process Development Applications. Pall Scientific & Technical Report, PN 33213.

Literature Resources

• Product Data, Minimate Tangential Flow Filtration Systemand Minimate TFF Capsule, PN 33366

• Technical Article, Desalting and Buffer Exchange by Dialysis, Gel Filtration, or Diafiltration, available online at www.pall.com/lab

• Technical Article, Diafiltration: A Fast, Efficient Method for Desalting or Buffer Exchange of Biological Samples,available online at www.pall.com/lab

• Technical Article, Increased Productivity Using MinimateCapsules to Replace Stirred Cell Systems, PN 33342

• Technical Article, Introduction to Tangential Flow Filtrationfor Laboratory and Process Development Applications,PN 33213

Visit us on the Web at www.pall.com/labE-mail us at LabCustomerSupport@pall.com

© 2010, Pall Corporation. Pall, , Minimate, Omega, Centramate, Centrasette and Ultrareservoir aretrademarks of Pall Corporation. ® indicates a trademark registered in the USA. Filtration.Separation.Solution.SM is a service mark of Pall Corporation. Cibacron is a trademark of CIBA-GEIGY LTD. *ÄKTA andUNICORN are trademarks of GE Healthcare. PEEK polymer is a trademark of Victrex plc. Tefzel is a registeredtrademark of DuPont.

4/10, 1.5k, AA GN09.3204 PN33339

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