Use of the Minimate TFF Capsule with Chromatography Systems

Scientific & Technical Report PN33339

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

Introduction protocols include a variety of separation techniques that may include general filtration/ Tangential Flow Filtration clarification, size exclusion chromatography, Tangential flow filtration (TFF) is an effective affinity chromatography, ion exchange method for both diafiltration and concentration chromatography, ultrafiltration, and possibly operations in purification strategies for bio - a sterilization step. TFF is frequently used in molecules. 1,2 The TFF process recirculates a process before chromatographic steps to sample flow over the surface of a membrane, desalt and buffer exchange (diafiltration) the controlling gel layer formation and effectively sample into a suitable state for binding or to reducing fouling. The operator develops an adjust the sample concentration. It may also effective and reproducible process by be applied as a bridge between chromato - optimizing flow rates and controlling trans- graphic steps when the elution buffer at the membrane pressure. This results in improved end of one process is incompatible with the flux rates and productivity. 3 A TFF process binding conditions of a subsequent step. developed on lab-scale devices can be At the end of a process, it may be used to readily scaled to larger process volumes. adjust the final concentration and ionic Traditionally, TFF separations have used strength of the product as required. peristaltic pumps and flexible tubing to Few protein chemists are without access provide the needed pressure and flow. to an LC pump system that has pumping Although standard lab pump systems are capacities approaching 100 mL/min. These sufficient for most TFF applications, the use systems can serve as robust alternatives to of high-performance liquid chromatography the use of a peristaltic pump for TFF protocols (LC) systems can offer some additional with significant additional benefits. Here we advantages with respect to process report the use of an ÄKTA Explorer N liquid development and control. This paper chromatography system (GE Healthcare) describes the novel use of an LC system to operate the Minimate TFF capsule. as the control platform for performing TFF Specific advantages of using an LC pump using a Minimate TFF capsule. system to drive TFF are: Liquid Chromatography and TFF • Use of chromatography equipment already Protein purification is rarely a simple process in place saves equipment cost, bench - and almost never a single step. Instead, it space, and training time. requires an ordered sequence of purification • Precise control of pressure and flow steps assembled into a process compatible rate assures uniform development of with the biochemistry of the target protein the gel layer to improve reproducibility to achieve the required purity. Purification between runs. • Ability to monitor and record process parameters in steps. An optimized fluid path minimizes hold-up and real time (pH, conductivity, absorbance, tempera - working volumes and allows easy scale up to larger ture, feed flow rate, valve positions, system pressure) TFF systems like Centramate™ and Centrasette™ gives a researcher an auditable trail for process systems. The Minimate TFF capsule and larger devices validation. utilize consistent materials of construction to assist in • Reduction in system working volume with narrow process validation. Finally, each pharmaceutical grade bore PEEK N and Tefzel N tubing allows greater Minimate TFF capsule is 100% integrity tested during concentration factors. manufacture to ensure reliable performance. • Operation of system valves to multiplex purification Technical Data procedures using the same pumping system Effective Filtration Area 50 cm 2 (0.05 ft 2) (sequential operations on multiple cartridges and Recommended Crossflow Rate 30-80 mL/min columns). TFF devices can be sanitized in place (0.6-1.6 L/min/ft 2) while other column operations are underway. 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 mL The Ultrareservoir container is designed to hold up to 500 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, which allows high concentration factors to be achieved. The reservoir lid seals tightly, allowing additional sample volume or diafiltration solution to be drawn into the reservoir by vacuum that is created as filtrate is gener - ated through the TFF device. This allows continuous diafiltration to be performed without the need for a transfer pump.

Minimate TFF Capsule with LC Pump System Equipment and Reagents 1. ÄKTA Explorer Chromatography Workstation with Unicorn N 4.0 Software (GE Healthcare) Materials and Methods 2. Digital Pressure Gauges (Cole Parmer, Model 1200) Minimate TFF Capsule 3. 1XPBS (phosphate buffered saline) Solution Minimate TFF capsules contain Omega™ polyether - (Invitrogen, Carlsbad, CA) sulfone membrane with an effective filtration area of 50 cm 2. The Omega ultrafiltration (UF) membrane, 4. 1M NaCl in PBS available in a wide range of molecular weight cut-offs, 5. Bovine Serum Albumin (BSA) solution: 1 mg/mL offers low adsorption characteristics resulting in high dissolved in PBS with 1M NaCl product recoveries. Sample batch sizes of up to 1 liter 6. 0.5M sodium hydroxide in water can be desalted and subsequently concentrated to volumes as low as 5 mL with little user intervention. 7. 0.1M sodium hydroxide in water The reusable/disposable capsule can perform single or sequential concentration/diafiltration steps using the same device in a closed loop connection, saving time and avoiding product loss associated with transfer

2 Step-by-Step Procedures (P1) and then connect P1 to one of the “Feed/ Retentate” ports of the Minimate TFF capsule. Table 1 Connect top column valve to a digital pressure Parts List for Connecting the Minimate TFF Capsule to gauge (P2) and then connect P2 to the other ÄKTA Explorer FPLC System “Feed/Retentate” port of the capsule. Orient the capsule in a vertical position with the end con - Description Qty nected to the top valve facing up. Connect the Tefzel tubing, 1/8" (use the minimal amount of tubing) 3 ft upper “Permeate/Drain” port of the capsule to a PEEK tubing (green), 0.030 x 1/16" (use the minimal 3 ft digital gauge (P3). Connect a length of tubing to amount of tubing) the outlet from the pressure gauge and put the Te fzel tubing, 1/16" (use the minimal amount of tubing) 3 ft other end in a waste container. Connect the Adapter assembly, 1/8" NPT to 5/16, 24 flat 3 each other “Permeate/Drain” port to a three-way valve bottom female connector. Use the valve to close off the port. Tubing connector, 1/8" male 3 each 4. Put separate buffer valve lines from the ÄKTA into Ferrule for 1/8" tubing (use the minimal amount 3 each the following solutions: of tubing) A13: PBS Union, 5/16" female 1/16" male 1 each A14: 0.5M sodium hydroxide Fingertight connector, 1/16" 12 each A12: 0.1M sodium hydroxide A11: BSA solution Pressure gauge TEE 3 each Digital pressure gauge, 0-300 psi range 3 each Membrane Equilibration and Line Flushing 10-32 female to male luer assembly 2 each 1. Using manual instruction, set the flow path to Micro-metering valve, 1/16" 2 each the following: 10-32 female to female luer assembly 2 each Column Position: 4 Outlet Valve: Waste F1 Buffer Valve: A13 Set-up Flow Direction: Up Flow 1. Connect an outlet valve line from the ÄKTA 2. Start the pump by setting a flow rate of 20 mL/min. (e.g., F8) to the “From Device” outlet of the Increase flow rate until the feed pressure reaches Ultrareservoir container. 2.7 bar (40 psi). 2. Connect a buffer valve line from the ÄKTA 3. Inspect the line connection to make sure there (e.g., A18) to the “To Pump” outlet of the is no leak. Ultrareservoir container. 4. Run for at least 5 minutes or until the pH, conduc - 3. Choose a column valve position from ÄKTA (e.g., tivity, and absorbance signals flatten out. position 4) for the Minimate capsule. Connect bottom column valve to a digital pressure gauge 5. Stop pump.

Diagram 1 Partnership of TFF with Column Chromatography

A Minimate TFF capsule is installed between the top and bottom column valves. Pump flow is directed into the TFF feed port, and the retentate flow goes through the analysis sensors and back to the Ultrareservoir container. Pressure sensors or gauges upstream and downstream of the capsule monitor pressure, and a third gauge is placed along the filtrate path to monitor backpressure. In diafiltration mode, the Ultrareservoir container is connected to the diafiltration buffer vessel. In concentration mode, the Ultrareservoir container is left open to the atmosphere.

www.pall.com/lab 3 Buffer Exchange 1. Leave the diafiltration buffer feed line open to air. A buffer exchange protocol was developed using 1M 2. Using manual instruction, set the flow path to the NaCl in PBS as the starting sample. This solution was following: diafiltered with PBS to remove salt and then back to Column Position: 4 high salt with a solution of 1M NaCl in PBS. Once the Outlet Valve: F8 process conditions were developed, the process was Buffer Valve: A18 repeated using a starting solution of 1 mg/mL BSA, Flow Direction: Up Flow 1M NaCl in PBS. 3. Start the pump at 20 mL/min. Adjust the flow rate 1. Equilibrate the Minimate TFF capsule according to so that the feed pressure (P1) is just under 2.7 bar the procedure described in the preceding section. (40 psi). 2. AutoZero the UV absorbance monitor of the ÄKTA 4. Collect the permeate in a graduated cylinder. (This and set the column position to “Bypass.” will allow the permeate volume to be measured and 3. Set the buffer valve line to A11 and pump flow rate concentration factor to be determined.) to 50 mL/min. Fill the Ultrareservoir container with 5. Stop the pump once the desired concentration 200 mL BSA solution (1 mg/mL dissolved in PBS is reached. with 1M NaCl). 4. Put the diafiltration buffer feed line (connected Washing, Regenerating, and Storing to the top part of the Ultrareservoir) into a flask the Minimate Capsule containing PBS. 1. Using manual instruction, set the flow path to the following: 5. Using manual instruction, set the flow path to Column Position: 4 the following: Outlet Valve: Waste F1 Column Position: 4 Buffer Valve: A13 (PBS) Outlet Valve: F8 Flow Direction: Up Flow Buffer Valve: A18 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 6. Start the pump at 20 mL/min. Adjust the flow rate 2.7 bar (40 psi). so that the feed pressure (P1) is just under 2.7 bar (40 psi). 3. Wash the system until the UV, pH, and conductivity signals stabilize. Note: Flow will start from the permeate tube. Diafiltration solution will start to be drawn into the Ultrareservoir con - 4. Switch the buffer valve line to A14 (0.5M sodium tainer in a few minutes. If this does not occur, check hydroxide). that the reservoir lid is completely inserted and sealed. 5. Wash the system until the UV, pH, and conductivity 7. Stop the pump once the conductivity signal reaches signals stabilize. a plateau. 6. Stop the pump. Let the Minimate capsule soak 8. Put the diafiltration buffer feed line into a flask in 0.5M sodium hydroxide for 1 hour. containing 1M NaCl in PBS solution. 7. Switch the buffer valve line to A12 (0.1M sodium 9. Repeat steps 5-7. hydroxide). 8. Wash the system until the UV, pH, and conductivity Sample Concentration signals flatten out. After the buffer exchange is complete, a sample can be concentrated by opening the diafiltration buffer 9. Stop the pump. The Minimate capsule can be feed line to air. This will break the vacuum generated stored in 0.1M sodium hydroxide at 4-20 °C. under the diafiltration mode.

4 I

Results and Discussion B Decreasing Salt 100 Continuous Diafiltration for Process Development 450 90 To develop a process protocol, a mock buffer- 80 C ) ) U exchange protocol was created where a buffer-only 350 m c

A 70 Conductivity / m S sample (1N NaCL in PBS) was first diafiltered with (

60 e (

PBS to reduce the salt concentration (Figures 1A, 250 y n t i

a 50 v i b t

1B). In-line monitoring allowed the documentation r c o u

s 40 d of salt concentration changes in real-time using the b 150 n A 30 o

conductivity sensor and data acquisition program C built into the ÄKTA Explorer. Consistent with previous 50 20 observations, 1 the passage of four volumes of A280 10 020406080100120140 diafiltration buffer was adequate to achieve > 98% -50 0 buffer exchange. Time (min) A sequential buffer exchange back to the high salt buffer was possible without disconnecting the device D C Increasing Salt from the system by replacing the low salt (PBS) 450 100 diafiltration buffer with a high salt buffer (PBS, 400 90 1M NaCl). Again, successful buffer exchange was Conductivity 350 80 ) detected in-line with the exchange of four buffer 70 ) U 300 m c A volumes (Figures 1C, 1D). Absorbance at 280 nm /

m 60 S (

250 m

was monitored to act as a sample process control e (

c 50 y n t

200 i

to determine if the absorbance readings are deflected a

v i b 40 t

c by the changing salt concentrations. The ability to o 150 u s 30 d b n monitor and document the process “live” allows the A 100 20 o researcher to save time and buffer, as well as provides C 50 10 a basis for future scale up decisions. A280 0 0 01020304050607080 Figure 1 -50 -10 Development of Buffer Exc hange Parameters Time (min) with In-line Monitoring A Decreasing Salt D Increasing Sa lt 500 100 100 90 450 90 400 80 Conductivity 80 ) ) ) ) 350

U U m Conductivity 70 m 70 c A c A

/ 300 / m m S S

( (

60 60

m m

e e ( (

c c 250

y y n n t t

i 50 200 50 i a a v v i i b b t t r r c c o 40 o 40 u u s s 150 d d b b n

100 n A 30 A o

30 o C C A280 20 20 0 50 01020304050607080 10 A280 10 0102030405060 -100 0 -50 0 Time (min) Time (mi n) D Diafiltration (buffer exchange) processing was documented from high salt to low salt then back to high salt. Repeated runs using a 10K Minimate capsule were processed at 20 (graphs A and C) and 25 (graphs B and D) mL/min recirculation rates. Salt and protein (A280) concentrations were monitored using in-line sensors.

www.pall.com/lab 5

Buffer Exchange at Constant C Increasing Salt Protein Concentration 300 100

Once a simple buffer-exchange protocol was devel - 90 250 oped, a second protocol was used to demonstrate 80 Conductivity ) diafiltration of a protein solution (1 mg/mL BSA) going ) 200 70 m U c / A first from high salt to low salt buffer (Figures 2A, 2B) S

m 60

( m

150 (

e and then back to a high salt buffer (Figures 2C, 2D). I n y c t

50 i n

v i a

A280 addition to monitoring protein and salt concentrations, t

b 100

c r 40 u o d in-line sensors documented the A600 values to ensure s n b 30 o

A 50

that the protein solution had not precipitated during C 20 processing. A precipitated protein solution would 0 A600 produce turbidity and cause a reading at A600. 0 10 20 30 40 50 60 70 80 90 100 10 The use of in-line monitors allows the researcher -50 0 Time (min) to immediately trace the incident back to the salt concentration where prote in precipitation first occurred, I thus saving valuable time in troubleshooting. D Increasing Salt 450 100 Figure 2 C Validation of Diafiltration Parameters in the 400 90 Conductivity Presence of Protein 350 80 ) ) m

300 70

c Decreasing U Salt

A I / A

120 S

m 250 60 m (

(

180 e y

t c

200 50 i n 100 v i a t i b A280 c r 150 40 u ) ) o C d A280 s U m

130 n b

80 c A 100 30 / o A m S C (

e 50 20

(

60 y

n t

i A600 a 80

v 0 10 i b t r Conductivity 020406080100120140160180 c o u s 40 -50 0 d b n A

o Time (min)

30 C 20 A600 Diafiltration (buffer exchange) processing was documented from 020406080100120140160180200 high salt to low salt then back to high salt with a protein solution -20 0 (1 mg/mL BSA). Repeated runs using a 10K Minimate capsule Time (min) 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. D B Decreasing Salt 140 100 Precise Control of Sample Concentration 120 90 In concentration mode, the Ultrareservoir container 80 100 A280 is left open to atmosphere. The concentration of the ) )

U 70 m retained solute increases as permeate is removed, c A 80 / m

S 60 (

decreasing the process volume. The in-line monitors

e (

60 50 y n

t allow the researcher to track the progress of the

C i a

Conductivity v i b t r 40

c concentration of the protein sample. While the salt

o 40 u s d b

30 n concentration (which is 100% permeable to the A 20 o A600 20 C membrane) remains constant, the protein solution 0 concentration increases. The use of sensors allows 010203040506070809010010 -20 0 the researcher to stop the process once the desired

Time (min) concentration is achieved. D ocumenting turbidity

6 (A600) provides a good diagnostic to determine Figure 4 experimentally the sample concentration limit prior to Sequential Operations for Diafiltration and Concentration protein precipitation. The 1 mg/mL BSA solution could 100 be concentrated over four-fold within 40 minutes with - 1100 90 out the formation of turbidity (Figure 3). While these 900 80 ) m )

conditions were chosen for this demonstration, in c U

70 / A reality, much higher concentration factors for BSA can 700 S m 60 m ( (

Conductivity y e be achieved. Other protein solutions may precipitate t i c 50 v

n 500 i t a

more easily depending on solubility. c

b 40 r

A280 u o d

s 300 30 n b o

Figure 3 A 20 C Precise Control of Protein Concentration 100 A600 10 250 25 80 100120140160180200220240260 -100 0 Time (min) 200 20

Conductivity ) Sequential diafiltration followed by concentration was documented ) m U c

150 / for a single run using a 1 mg/mL BSA solution in 1X PBS, 1M NaCl A S

m 15 starting solution. The run using a Minimate 10K capsule was ( m

(

A280 e processed at 25 mL/min recirculation rate with the buffer exchange y c t

100 i n

v from high salt to low salt. The diafiltration buffer feed line was then i a t b c r 10 opened to air, allowing the concentration of the sample. Salt, protein u o d s

50 n (A280), and turbidity (A600) were monitored using in-line sensors. b o A C A600 5 0 Scalability 510152025303540 TFF process development can be performed on a -50 0 Minimate TFF capsule with a simple lab-scale system Time (min) incorporating a peristaltic pump or with an LC pump The concentration of a 1 mg/mL BSA solution was accomplished and in-line monitoring system as described above. by leaving the diafiltration feed line open to air. The run using a Both allow development of procedures that are Minimate 10K capsule was processed at 25 mL/min recirculation rate. Salt, protein (A280), and turbidity (A600) were monitored using ultimately scalable. The advantage of the LC system in-line sensors. is greater control and documentation. The automation and documentation aspects simplify development of Sequential Buffer Exchange and Concentration validated protocols and provide an audit trail for labs An advantage of processing using in-line sensors was under Good Lab Practices (GLP). This is extremely demonstrated by performing sequential processing useful in the scale-up process. operations. Diafiltration of the protein sample (1 mg/mL BSA) reduced the salt concentration by While the Minimate TFF capsule is a valuable tool for > 98% with about four volumes of buffer. As soon as small process volumes (typically under 1 liter), larger the conductivity leveled off, indicating that desalting volumes can be processed by plumbing several was complete, sample concentration was started Minimate TFF capsules in parallel. The Minimate TFF by simply disconnecting the buffer line to the Ultra - capsule was designed with the same flow path length reservoir container. Concentration of retained solute as larger TFF devices (Centramate and Centrasette increased as permeate was removed, decreasing the systems) allowing predictable performance and saving process volume. This process is facilitated using the valuable optimization time when scaling beyond lab features of an LC system, such as the multiport valves scale to volumes used in pilot and production plants. and in-line sensors. The ability to monitor “live” allows greater control over the process during the run and provides documentation that can be used to develop and validate the scale-up protocol.

www.pall.com/lab 7 References Literature Resources

1. L. Schwartz, 2003. Diafiltration: A Fast, Efficient Method • Product Data, Minimate Tangential Flow Filtration System for Desalting or Buffer Exchange of Biological Samples. and Minimate TFF Capsule, PN 33366 Pall Scientific & Technical Report. • Technical Article, Desalting and Buffer Exchange by 2. L. Schwartz, 2003. Desalting and Buffer Exchange by Dialysis, Gel Filtration, or Diafiltration, available online Dialysis, Gel Filtration or Diafiltration. Pall Scientific & at www.pall.com/lab Technical Report. • Technical Article, Diafiltration: A Fast, Efficient Method 3. L. Schwartz and K. Seeley, 2002. Introduction for Desalting or Buffer Exchange of Bio logical Samples, to Tangential Flow Filtration for Laboratory and available online at www.pall.com/lab Process Development Applications. Pall Scientific • Technical Article, Increased Productivity Using Minimate & Technical Report, PN 33213. Capsules to Replace Stirred Cell Systems, PN 33342 • Technical Article, Introduction to Tangential Flow Filtration for Laboratory and Process Development Applications, PN 33213

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