Packing large-scale chromatography columns with Capto™ S ImpAct resin

Calculating the packing factor, compression factor, and column volume

The height of a gravity-settled bed differs from the height of a bed settled under low flow (consolidated) due to the properties and the behavior of the resin under different conditions. You should therefore separate the compression factor (CF) from the packing factor (PF). For example, for a 20 cm bed in 150 mM NaCl when consolidated at 60 cm/h, the PF is 1.20 and the CF is 1.23 for Capto™ S ImpAct resin. This information is helpful when the amount of gravity-settled resin needed for a specific column size is calculated (the amount of gravity-settled resin needed to obtain a bed with a specific PF [e.g., 1.20] is calculated as column volume [Vc] × CF [e.g., 1.23]).

Equations to calculate CF, PF, and column volume (CV) are shown in Equation 1 to 3, respectively:

Eq 1.

Compression factor, CF = L_settled / L_packed

Eq 2.

Packing factor, PF = L_cons / L_packed

where

L_settled = bed height measured after settling by gravity

L_cons = consolidated bed height, that is, bed height measured after settling the resin at a given flow velocity

L_packed = packed bed height

Eq 3.

CV = L_packed × A_C

where

A_C = cross-sectional area of the column

When packing BPG and AxiChrom™ columns, PF is used in the packing procedure to calculate the final packed bed height after the consolidation step. CF is used in the chromatography resin preparation step to calculate the resin volume needed to pack a desired bed height. Because Chromaflow™ columns are pack-in-place columns, they have no registered consolidated bed heights, and the CF is used throughout the packing process.

Properties of Capto™ S ImpAct in various packing solutions

Capto™ S ImpAct resin settles quickly both in water and in 20% ethanol. When using these solutions, remember to rinse tubing and nozzles directly after packing to prevent clogging of the flow path. Adding salt to packing solutions slows the settling of the resin particles and allows them to settle less tightly.

You should add salt (150 mM NaCl) to the packing solution to maximize the flow properties of Capto™ S ImpAct resin.

The packing factor should be chosen based on the target bed height (Fig 1).

Fig 1. Bed height and PF curve for AxiChrom™ columns up to 1000 mm, illustrating the decreasing linearity of PF for different bed heights. For each bed height, there is an acceptable interval for the PF. Packing factors are shown are for packing with 150 mM NaCl.

Slurry preparation

To achieve the correct amount of chromatography resin for packing to target bed height or compression, you must measure the slurry concentration correctly. To get an accurate slurry concentration, use the slurry concentration kit.

When calculating the slurry volume (V), use CF = 1.24, which is valid when the resin is in storage solution. When you prepare the slurry, calculate the chromatography resin volume (V) needed to pack the desired bed height.

Chromatography slurry volume, 
V = A_C × L_packed × CF

C_slurry

where

C_slurry= concentration of the slurry

You can prepare the resin to form a slurry manually by shaking or stirring, or by using a mixer such as the Media Wand™ slurry mixing and transfer tool. Shaking gives good results but is often not practical for larger volumes. When stirring, use soft stirrers without sharp edges. Media Wand™ suspends the resin directly in the container and transfers the slurry to the BioProcess™ resin mixer in one operation, which makes it suitable for large-scale packing.

Capto™ S ImpAct resin is supplied in 20% ethanol with 0.2 M sodium acetate. Before packing, transfer the resin to the packing solution as described in the packing instructions for the relevant column.

AxiChrom™ columns are low-pressure, mechanical axial compression chromatography columns designed for process development and biopharmaceutical manufacturing. Mechanical axial compression enables accurate and reproducible control of the packing, even with large-diameter columns. The columns are available in different configurations and materials.

The columns are scalable and will give you predictable results over the entire range of scales by enabling a uniform plug flow through the bed, irrespective of column size. The columns feature Intelligent packing with preprogrammed methods that support all column sizes. For more information and general considerations for automated packing in AxiChrom™ columns, see our article on packing large-scale columns with Capto™ ImpRes ion exchange chromatography resins.

Intelligent packing makes column operation straightforward and gives you very high packing success rates. The packing methods we describe here apply to bed heights of up to 40 cm depending on column diameter.

Packing variables and recommended packing buffers are given in Table 2.

Table 2. Packing variables for packing of Capto™ S ImpAct resin in AxiChrom™ columns

BPG columns are glass columns for process development and manufacturing. The single-screw adapter allows easy and efficient packing and running. The columns have diameters from 100 to 450 mm. Capto™ S ImpAct resin can be packed in BPG 100 to 300 mm diameter columns. The bed height interval recommended for BPG 100 to 300 columns is 10 to 40 cm.

Capto™ S ImpAct resin is packed with 150 mM NaCl in BPG 100 and BPG 300 columns.

The packing factor for packing of Capto™ S ImpAct to various bed heights in BPG columns is 1.14.

Packing variables and recommended packing buffers for BPG columns are given in Table 3.

Table 3. Packing variables for packing Capto™ S ImpAct in BPG columns

Resin preparation

You can equilibrate the resin to the packing solution by using the BPG column as a “filter”. Pour the resin into the column (for calculation of amount, see Slurry preparation earlier) Mount the adapter, tighten the adapter O-ring, move the adapter down, and compress the bed slightly. The pump should then be connected and the resin washed with at least three column volumes (CV) of the packing solution.

Unpack and resuspend the slurry and pack according to the method below.

Note! Equilibration is critical for Capto™ S ImpAct in BPG columns as the delivery solution contains 0.2 M sodium acetate in 20% ethanol, and the recommended packing solution is 150 mM NaCl.

Column and system preparation

A detailed description of column preparation is available in the BPG operating instructions. The packing pump should be as pulsation-free as possible. Screw and rotary lobe pumps are the most suitable for this task, but multiheaded diaphragm pumps are also satisfactory.

Caution: Ensure that the column has no visible scratches in the glass tube and that the adapter moves smoothly in both upward and downward directions before packing. In addition, you should not find tightening the adapter O-ring to the column inner wall difficult.

1. Place a new 10 μm net on both the adapter and the bottom end pieces.
2. Level the column with the aid of a spirit level.
3. A pressure relief valve should be used for safety, especially against pressure spikes. Position this valve on the pump outlet and add a pressure gauge to the adapter.
4. Mount one 4-port, 2-way valve on the bottom inlet and one on top of the pressure gauge, i.d. 10 mm for BPG 300 and i.d. 6 mm for BPG 100 columns, respectively.

Packing

1. Set the pressure alarm or pressure relief valve according to the pressure rating of the column. Purge the system and tubing of air.
2. Purge the end piece net of trapped air by draining some packing solution through the column outlet. Leave about 2 cm of solution in the column and close the bottom valve. If air is still trapped under the end piece net, add more packing solution and connect tubing to the suction side of a pump. Start the pump and place the tubing on the bottom net and extract any remaining air.
3. Add the slurry to the column and, if needed, additional packing solution to about 40 cm. Mix the resin and the packing solution to a homogeneous slurry.
   Note! The available height to allow insertion of the adapter into a 50 cm column tube (for filling slurry) is 40 cm. Use a longer column tube when packing beds higher than 20 cm: 75 cm and 95 cm tubes are available.
4. Rinse the wall from particles and let the resin settle until there is about 1 cm of clear liquid on top of the slurry. This reduces the risk of particles sticking between the O-ring and the column wall, which can otherwise cause leakage.
5. Insert the top adapter and secure it to the column top flange. Lower the adapter to the surface of the slurry and allow some clear liquid to pass the O-ring. Tighten the adapter O-ring.
6. Make sure that the column top valve is open. Slowly move the adapter down until no air bubbles can be seen leaving the top valve.
7. Start the pump and adjust the settling velocity to 60 cm/h (4.7 L/h for BPG 100 and 42.4 L/h for BPG 300). Shift the top valve into the column and immediately open the bottom valve and lead the liquid to waste.
8. Run the settling flow until the bed is completely consolidated. Note the consolidated bed height and calculate the packed bed height. For BPG 100 - 300, PF = 1.14 in 150 mM NaCl for bed heights up to 40 cm. The packed bed height is the ratio between the consolidated bed height and the PF. Use a marker pen to indicate the packed bed height on the column.
9. Stop the flow and close the bottom valve. Loosen the O-ring and lower the adapter down to 1 cm above the settled bed and seal the adapter O-ring. Shift the top valve to waste and use the adapter to mechanically compress the bed to the mark on the column (step 8). Excessive packing solution is removed through the adapter tube.
   Note! Compressing Capto™ S ImpAct in BPG columns, especially the larger BPG 300, is physically demanding. Do not use extension rods on the adapter height adjuster to compress the resin. To increase the performance and stability of the bed, flow condition the column downwards for 5 CV and upward for 5 CV at 110 cm/h with packing solution.
10. Connect the pump to the top of the column. Purge the system and tubing by running the mobile phase to waste by bypassing the column inlet with the 4-port valve. Start at a low flow velocity (approximately 60 cm/h).
11. Shift the top valve to direct the flow to the column and immediately open the bottom mobile phase to waste or connect it to the buffer tank for recirculation.
12. Increase the flow to 110 cm/h and run the column at this flow for 5 CV.
13. Slowly decrease the flow for 2 min to avoid disturbance of the conditioned bed.
14. Exchange the mobile phase connections. Connect the tubing from the pump to the bottom valve and open the top valve to waste or to the buffer tank for recirculation.
15. Repeat steps 10 through 13 with upward flow to complete the conditioning procedure.
16. Test the packing at the optimal test velocity (20 to 30 cm/h). Packing variables and recommended packing buffers are given in Table 3.

These columns are acrylic or steel, pack-in-place columns for GMP manufacturing. The columns have diameters ranging from 400 to 2000 mm. The packing method we describe applies to Chromaflow™ columns up to 600 mm with a standard configuration.

The method we describe in this section is only verified for packing the resin in Chromaflow™ 600 to a bed height of 20 cm. Lower beds will not be compressed sufficiently because the extreme velocities needed to efficiently pack a shorter bed using standard equipment.

To pack Capto™ S ImpAct resin in Chromaflow™ 600, you introduce the slurry from the top nozzle using Chromaflow™ packing station pack 100.

Note! The recommended operational flow velocity for Capto™ S ImpAct in Chromaflow™ 600 at 20 cm bed height is 150 cm/h.

Note! The flow capacity of Chromaflow™ packing station pack 100 is required for packing Capto™ S ImpAct resin in your Chromaflow™ 600 column.

Resin preparation

Use 10 mM NaCl as packing solution for Chromaflow™ columns. This packing solution is used throughout the whole procedure.

To avoid trapping air in the column when packing, you will need additional 5% to 10% slurry for the extra volumes in the tank and tubing. Add the slurry to a mixer such as the BioProcess™ resin mixer and stir the resin. Dilute the suspension to a slurry concentration of about 50%.

Column and system preparation

For a more detailed description of the column and packing station preparation, see Chromaflow™ columns operating instructions and Chromaflow™ packing stations instructions for use. In this application note, we use standard Chromaflow™ equipment and connections on the column and the packing station.

Note: Ensure that the air supply flow rate follows the specification for Chromaflow™ packing station pack 100 (100 L/min) and that the air pressure in the packing station is 6.0 to 7.0 bar (87.0 to 101.5 psi, 0.6 to 0.7 MPa).

1. Set up the column according to the Chromaflow™ column and Chromaflow™ packing station instructions for use.
2. A pressure relief valve should be used for safety reasons. Position this on the slurry inlet top (SIT). Place a pressure gauge on the mobile phase top (MPT) to record the pressure during packing. Mount a 3-port, 2-way valve on top of the pressure gauge and the mobile phase bottom (MPB). The top valve should lead in two directions: one side into the system and one to the waste for purging the tubing. On the bottom valve, one side leads to the system and a 1.5” to 2” .i.d. tubing leads to the waste (for packing). Part of the MPB waste tubing should be placed above the outlet valve to eliminate air entering through the MPB.
3. Connect appropriate tubing (1” or 1.5” i.d.) and tanks to the column and packing station. If a flow meter is used, place it between the SIT and the packing station.
4. Level the adapter to the desired bed height. Remember to loosen the nuts on the adapter rods to allow the adapter to be raised or lowered. Flush the adapter rods with 20% ethanol as lubrication.

Packing

Note: Packing Chromaflow™ columns is a rapid procedure compared with other packing procedures and it is therefore important to thoroughly read the packing instructions and go through the packing steps in advance of the packing.

1. Set both nozzles in run position to prime the tubing with slurry. Lead the slurry outlet top (SOT) tubing back to the slurry tank, and secure it. Stir the slurry to keep it homogeneous, select slurry and SIT on the packing station, open the slurry tank and start the packing pump.
2. The initial flow velocity should be at least 1400 cm/h, corresponding to an air pressure on the packing station of at least 2.5 bar (36.3 psi, 0.25 MPa) for packed bed heights of 20 and 30 cm.
3. When the tubing is primed and the flow velocity set, set the SIT/slurry inlet bottom (SIB) to the position between SIT and SIB. This blocks the flow during step 4 while maintaining the correct flow rate for the next step.
4. Move the top nozzle down into the packing position.
5. Two operators should simultaneously open the bottom mobile phase valve to waste and turn the SIT/SIB valve to SIT on the packing station. The column starts to fill with slurry and the bed builds up slowly from the bottom as excess liquid exits via the MPB.
   Note: Column pressure must not exceed the operating pressure limit of the column (i.e., 3.0 bar [43.5 psi, 0.3 MPa]). If this pressure is reached, gently decrease the packing flow so that the pressure remains just below 3.0 bar (43.5 psi, 0.3 MPa). Typically, the final pressure in the column is 2.0 to 3.0 bar (29 to 43.5 psi, 0.2 to 0.3 MPa) depending on the viscosity of the packing solution, column diameter, bed height, and so forth.
6. Stop the pump when the building bed is 5 mm from the top bed support by setting the SIT/SIB to the position between SIT and SIB, as described in step 3. Once the flow is stopped, the bed will expand to meet the adapter.
   Note: If you are using a nontransparent column tube, stop the packing flow when the calculated volume of slurry has been introduced into the column. Check the volume in the slurry tank or use a volume totalizator.
7. Immediately move the top nozzle back to the run position.
8. Close the MPB valve when the pressure in the column is between 0.3 bar (4.35 psi, 0.03 MPa) and 0.1 bar (1.45 psi, 0.01 MPa).
9. Use packing solution to rinse residual resin from the tubing and the top nozzle. Pump the packing solution through the top nozzle back into the slurry tank.
10. Close the slurry tank and empty the tubing between the tank and the packing station.
11. Pump liquid upwards through the column until the air is expelled.

Figure 2 shows the increase in pressure and decrease in flow velocity when packing a 20 cm bed height in Chromaflow™ 600 column.

Fig 2. Column pressure and flow velocity during packing of a 20 cm bed height in Chromaflow™ 600 using Packing Station Pack 100.

Process-scale, packed columns must perform with a high degree of efficiency over many processing cycles (i.e., display very high stability). The efficiency of a packed column can be expressed in terms of height equivalent to a theoretical plate (HETP) and asymmetry factor (A_S). This test should be repeated regularly to monitor the state of the packed bed throughout the working life of the column. If the test results are to be comparable over time, conditions such as, flow velocity (cm/h), liquid pathway, sample composition, and elution buffer should be kept constant.

Set the requirements of the test under the test conditions and the goal of your purification. This is further described in  this application note. To compare the performance of columns packed with chromatography resin of different particle diameters, the reduced plate height (h = HETP/average bead diameter [dp]) is typically used. As a guideline, a value of h < 3 is very good at optimal test conditions.

Test conditions used in this study

Sample: 2% v/v acetone

0.8 M NaCl for AxiChrom™ 50, 100, and 200 columns

Sample volume: 1% of the bed volume (V_C)>

Test flow velocity: 30 cm/h for AxiChrom and BPG columns, 20 cm/h for Chromaflow™ columns

Eluent: 0.15 M NaCl or 0.4 M NaCl for AxiChrom™ 50, 100, and 200 columns

The results for Capto™ S ImpAct in column sizes ranging from 100 to 1000 mm were very similar, showing that the verified packing methods available in Intelligent packing give equivalent results, independent of column size and bed height.

The stability test showed that the resin particles were stable when running in water at velocities given in Table 4.

Table 4. Column efficiency data for Capto S ImpAct resin packed in AxiChrom™ columns

Pressure-flow curves provide you with a simple, yet effective illustration of column performance in terms of the maximum operating flow velocity at which the purification process can be run. These curves also show the magnitude of the back pressure in the system at a certain flow velocity and for different bed heights.

Pressure-flow curves for Capto™ S ImpAct packed in AxiChrom™ 300, 600, and 1000 at different bed heights are shown in Figures 3–5.

NOTE! All pressure/flow values are normalized to 20°C and respective bed height (i.e., 10.0, 20.0 cm etc) and empty column pressure is removed. The last measuring point (the gap flow point) is removed from all flow data.

Fig 3. Pressure-flow curves for Capto™ S ImpAct in 150 mM NaCl packed in AxiChrom™ 300 column to different bed heights using different packing factors.

Fig 4. Pressure-flow curves for Capto™ S ImpAct in 150 mM NaCl packed in AxiChrom™ 300, 600, and 1000 columns at 20 cm bed height using the same PF.

Fig 5. Pressure-flow curves for Capto™ S ImpAct in 150 mM NaCl packed in AxiChrom 600 column at different bed heights and PF.

The packing method we describe works well for Capto™ S ImpAct in different sizes of BPG columns at different bed heights.

The stability test showed that the bed was stable in water at the flow velocities given in Table 5.

Table 5. Column efficiency data for Capto™ S ImpAct in BPG columns

The pressure-flow curves for Capto™ S ImpAct at 40 cm bed height in BPG 100 and BPG 300 are shown in Figure 6. Capto™ S ImpAct shows linear behavior in these columns.

Fig 6. Pressure-flow curves for Capto™ S ImpAct in 150 mM NaCl packed to 40 cm bed height (PF = 1.14) in BPG 100 and BPG 300. System and tubing pressure is excluded.

A bed height of 20 cm gives a good plate number and asymmetry factor. In addition, the stability test shows that the bed is stable when running in water at the velocity given in Table 6.

Table 6. Column efficiency data for Capto™ S ImpAct in Chromaflow™ columns

The pressure-flow curves are shown in Figure 7. As the optimal compression factor is difficult to achieve in standard pack-in-place columns, the maximum flow velocity that can be run through the packed bed is limited.

The highest operating flow velocity recommended for Capto™ S ImpAct in the Chromaflow™ 600 column is 150 cm/h. Note that bed efficiency and bed stability are very good, provided this 150 cm/h guideline is met.

Fig 7. Pressure-flow curve for Capto™ S ImpAct in Chromaflow™ 600 column, 20 cm bed height.

AxiChrom™ 300-1600 chromatography columns

BioProcess™ resin mixer

BPG chromatography columns

Capto™ S ImpAct cation exchange chromatography resin

Chromaflow™ chromatography columns

Media Wand™ slurry mixing and transfer tool

Application note: Packing large-scale columns with Capto™ ImpRes ion exchange chromatography resins

Application note: How to pack Capto™ HIC resins using verified packing methods

Application note: How to determine resin slurry concentration

Application note: Column efficiency testing