laboratory scale columns for performance screening tricorn ...7b8b445b-01c7-403d-b221-5c916… ·...

1.0 Introduction

It is of critical importance for biopharmaceutical production that the purification process is robust and reproducible lot to lot. It is therefore, vital that chromatography columns are properly and efficiently packed and able to be qualified in a reasonable time frame.

An efficiently packed column is critical to achieve maximum efficiency, high product yield and purity. Irregularities in packing can create uneven flow distribution through the column that will result in peak broadening, poor resolution and lower yields as a result of a drop in capacity. All of this will have a detrimental impact in overall product quality.

At process scale the column designs are robust, and commercially available resins come with well-designed packing methods to achieve the most efficiently packed columns for process purification. Alternatively, pre-packed formats can be purchased from most resin manufacturer’s, negating the impact of irregularities in packing for the end user.

However, at the process development stage on laboratory scale, the design of the column used and packing approach can significantly impact the results of screening evaluations. A resin cannot be truly evaluated if not packed efficiently.

In this short investigation we have evaluated two of the most common formats used to pack columns at small scale; GE Healthcare Tricorn and Kinesis Omnifit columns.

Laboratory Scale Columns for Performance ScreeningTricorn versus Omnifit

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1.3 Results

The 10% breakthrough capacity using Tricorn columns packed using the method described in Appendix A, showed poor reproducibility and a high level of variability between different packings and operators.

The 10% breakthrough capacity using Omnifit columns packed using the method described in Appendix B, showed excellent reproducibility and low levels of variability between different packings and operators.

Each result presented in Figure 1 is an average of 3 experiments per analyst.

Protocol Buffer Column volumes

Equilibration Phosphate buffered saline, pH 7.2 5 CV

Sample Application Load up to 40 CV’s of 5 mg/ml hlgG - set cut-off at 80% of 100% signal N/A Column Wash Phosphate buffered saline, pH 7.2 5 CV Elution 0.1 M acetic acid 5 CV Equlibration Phosphate buffered saline, pH 7.2 8 CV

Table 1. Dynamic binding capacity determination protocol for Protein A resins

1.2 Experiment

MabSelect™ SuRe™ LX was packed per the manufacturer’s instructions and Praesto® AP was packed into Tricorn columns using the method described in Appendix A.

MabSelect™ SuRe™ LX and Praesto® AP were packed into Omnifit columns using the method described in Appendix B. Dynamic binding capacities on the laboratory scale columns were performed using polyclonal immunoglobin G and the following protocol;



Binding capacity hlgG, 10% breakthrough (mg/ml) - 6 mins RT

Column Tricorn Omnifit

Analyst 1 49.6 62.4

Analyst 2 50.6 62.8

Analyst 3 47.7 62.2

RSD (%) 2.97 0.51

The use of Omnifit columns and the packing method in Appendix A reproducibly produces capacity results that closely mirror capacity data generated for the in-house reference standard, a Repligen Minichrom 8-20 column. The reference column is machine packed, producing identical packing performance on each column of a batch and as a result is used as a quality check.

The results in Figure 2 and Table 3 show that up to a 25% drop in capacity can be seen by a poorly packed tricorn column. This could have a dramatic effect on resin screening evaluations.

Whereas, the design of the Omnifit column along with an efficient packing method from the resin vendors allow the user to gain the maximum capacity at laboratory scale.

Figure 1. Dynamic binding capacities - 10% breakthrough hlgG for MabSelect™ SuRe™ LX packed in Tricorn and Omnifit columns.

Table 2. Dynamic binding capacities - 10% breakthrough hlgG for MabSelect™ SuRe™ LX packed in Tricorn and Omnifit columns.



Figure 2. Dynamic binding capacities - 10% breakthrough hlgG for Praesto® AP packed in Tricorn and Omnifit columns.

Table 3. Dynamic binding capacities - 10% breakthrough hlgG for Praesto® AP packed in Tricorn and Omnifit columns.

Binding capacity hlgG, 10% breakthrough (mg/ml) - 6 mins RT

Column Tricorn Omnifit Pre-packed - Minichrom

Residence time (mins)

3 31.8 43.1 43.3

4.8 42.2 54.0 55.6

6 47.6 57.5 59.0

10 54.6 64.3 66.3



1.4 Conclusions

GE’s Tricorn and Kinesis Omnifit empty laboratory scale columns are both versatile small scale columns that give the user great flexibility when packing multiple different resin types.

However, the adaptor design of the Tricorn column (Figure 3) does not distribute the flow evenly over the circumference of the top of the column bed. As a result, the column will appear efficiently packed or even over packed when it is in fact under packed. This under packing explains the loss in overall capacity.

Kinesis’s Omnifit column is similar in design apart from the adaptor and plunger mechanism. The flow is evenly distributed over the circumference of the adaptor before the frit (Figure 4), allowing for an evenly distributed flow during packing and experimental runs. The same would apply for GE’s XK or HiScale columns and YMC columns that have designs which allow for a more even flow distribution from the adaptor.

Figure 3. Schematic representation of Tricorn empty high performance column.

Figure 4. Schematic representation of Omnifit laboratory scale columns.

2.0 Appendices

Appendix A - Tricorn Column Packing Method



Tricorn 5/50: Materials and Equipment

• Praesto® AC, APc or AP • Tricorn 5/50 packing equipment • Tricorn 5/50 column• Glass filter funnel• Plastic beaker• Plastic spatula• Measuring cylinder• Purified Water or 100 mM NaCl solution (Packing Solution)• A Chromatography system, such as a BIO-RAD NGC

or an AKTA system. Alternatively, a stand-alone pump (depending on the flow rate required) can be used for packing.

Operation

Set up a filter funnel over an appropriate collection vessel. Pour the medium onto the filter funnel, and wash the medium by pouring 500 ml of purified water or 100 mM NaCl solution. This will remove the 20% ethanol storage solution.Remove the medium from the filter funnel, add to an appropriate size falcon tube and add H2O or 100 mM NaCl. Then centrifuge at 1800 rpm for 10 minutes.Calculate the slurry % and add or remove packing solution to obtain a 50 % slurry.Calculate the required slurry volume for a 5 cm packed bed.

1. Determine the slurry volume for column packing.2. Determine the desired packed bed height. 3. Calculate the column volume (CV) of a packed column by the following equation; a. Cross-sectional area of the column (CSA) × bed height (Bh) b. Multiply the column volume by a compression factor (C.F) (CV×C.F) (C.F = 1.2) c. Divide by the slurry concentration. d. Example calculation;

Column: Tricorn 5/50Desired bed height: 5 cm

Slurry concentration: 50 %Compression Factor (Praesto® AC, APc or AP): 1.2

(CSA ×Bh ×C.F)/ (Slurry Concentration)((O.25)² × π) ×5 ×1.2)/0.5 = 2.36 ml

Required slurry volume for a 5 cm packed bed = 2.4 ml



Operation

Unpack a Tricorn 5/50 column, assemble and connect Tricorn 5/50 packing equipment as per the manufacturer’s instructions (GE Healthcare).

Stir the column media gently to ensure homogeneity, fill the column with the calculated slurry and top up with packing solution.

Insert top filter at a 45° angle to prevent air bubbles forming at the top of the column and screw the top cap of the packing column.

Start a flow rate of 0.5 ml/min of packing solution through column switching valve of the selected system. Once a flow is established, connect tubing from the column switching valve to the top of the packing column.

Remove the stop plug from the bottom of the column and replace with the outlet column tubing connected to the column switching valve. Disconnect the outlet tubing from the system and place in a collection vessel.

Start a packing flow at a linear velocity of 600 cm/h (2ml/min) and leave to pack for 10 minutes or 10 column volumes.

Turn the flow off and attach a stop plug to the bottom of the column. Dismount the packing tube and remove excess resin using a pipette.

Top up the column with packing solution and attach an adaptor with a filter in place. Screw down the adaptor to 1 to 2 mm above the packed bed. Turning the adaptor down will expel any air in the adaptor tubing.

Reconnect the column to the system following the steps described in 2.1.7 and 2.1.8.

Start a packing flow at 600 cm/h (2ml/min) and leave to run for 10 minutes or 10 column volumes.

Mark the bed height after 10 minutes and stop the pump.

Turn the adaptor down to the mark point, and then give the adaptor an extra 1/3 turn.

Start a conditioning flow of 600 cm/h (2ml/min) through the column and allow to run for 10 column volumes.

Note: If a gap has formed between the bed and the adaptor during flow conditioning, turn the adaptor down to close the gap and restart the conditioning flow.

Note: Check for any pressure spikes during the packing procedure. Any rapid increase in pressure without stabilisation would indicate a filter blockage.

The column is now ready to be tested.



Appendix B - Omnifit Column Packing Method

Omnifit 10/100 (3 cm bed height): Materials and Equipment

• Praesto® AC, APc or AP • Omnifit -10/100 column• Glass filter funnel• Plastic beaker• Plastic spatula• Measuring cylinder• Purified Water or 100 mM NaCl solution (Packing Solution)• A Chromatography system, such as a BIO-RAD NGC or an AKTA

system. Alternatively, a stand-alone pump (depending on the

flow rate required) can be used for packing.

Operation

Set up a filter funnel over an appropriate collection vessel. Pour the medium onto the filter funnel, and wash the medium by pouring 500 ml of purified water or 100 mM NaCl solution. This will remove the 20% ethanol storage solution.Remove the medium from the filter funnel, add to an appropriate size falcon tube and add H2O or 100 mM NaCl. Then centrifuge at 1800 rpm for 10 minutes.Calculate the slurry after the last centrifugation and top up with either purified H2O or 100 mM NaCl to obtain a 50% slurry.

The volume of slurry required to pack a given bed height can be estimated using the following formula:Volume slurry = volume packed bed x (100 / slurry %) x 1.2 (compression factor)Volume slurry = 2.36 x 2 x 1.15 = 5.66 mlBed height (50% slurry) = 7.2 cm

Assemble the bottom end piece to the column and mark the given bed height (7.2 cm) on the column tube.

Assemble the top adaptor and connect it to the designated system. Start the flow at 3 ml/min, allow to run for 5 minutes to allow any air to pass through the top adaptor.

Stir the slurry to ensure homogeneity and add the required volume to the column. (Up to the 7.2 cm marked point).

Fill the column with packing buffer and insert the top adaptor at a 45° angle to prevent air bubbles forming. Stop the flow and bring the adaptor to approximately 1 mm above the bed formation. Restart the flow and connect the bottom tubing to the system.

Bring the adaptor down until the gap above the bed is closed. No further compression is needed.

Condition the packed column at 2.6 ml/min (200 cm/h) by flowing 3 column volumes of packing buffer upflow, followed by 3 column volumes downflow. Repeat this step 3 times.

(check the pressure; usually it is less than 3 bar = 0.3 MPa).

Note: If a gap has formed, compress further and repeat the conditioning step.The column is now ready to be tested



Ordering InformationTo place your order, simply contact us via email, or telephone using the information at the base of this sheet.

If you wish to discuss your purification challenges with a specialist, we have dedicated experts on-hand, across the globe to provide knowledgeable, same day technical assistance.

Contact UsPurolite LtdLlantrisant Business ParkLlantrisantWales, UKCF72 8LF

T. +44 1443 222336F. +44 1443 227073E. [email protected]

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