Column Packing Protocol for Amsphere A3
Posted on January 27,2017
This packing protocol is for small columns (1-20 mL, ID=0.5-1 cm). The recommended packing method is by flow.
Preparation of the slurry
The slurry of Amsphere A3 is provided in a storage buffer solution containing 16% ethanol in 20 mM sodium phosphate, pH 7.5. Decant the storage solution once the resin has settled, and add 0.1 M sodium chloride to obtain a slurry concentration of 50% (v/v) (buffers other than 0.1 M sodium chloride can be used but could influence the packing quality).
Caution: Do not mix or re-slurry with a magnetic stir bar.
Note: Column packing with water or 1 M sodium chloride is possible but not recommended.
The volume of the slurry needed to pack the bed can be calculated using the following formula:
Volume slurry = volume packed bed Å~ (100/slurry (%)) Å~ compression factor
Compression factor of Amsphere A3: 1.10 (Å}0.02)
Amsphere A3 is a new protein A resin designed with a surface modified base bead and alkali-resistant optimized ligand.
Protein A ligand
• High DBC via controlled conformation and orientation
• High alkaline stability from protein engineering Surface modification
• Low HCP levels by surface hydrophilization Base bead formulation
• High DBC at high flow rate
• Excellent pressure and flow properties via rigid crosslinking
TABLE 1: RECOMMENDED PACKING CONDITIONS (FOR 20 cm BED HEIGHT)
Degassing of all buffers is recommended prior to use.
- Assemble the column according to instructions. If the column does not allow for 50% slurry, or 2X of the packing buffer compared to the settled bed volume, please attach a packing reservoir tube.
- Add 0.5 mL of the packing buffer to the empty column and then pour slurry into the column (avoid air bubbles).
- Pack the column by a downward flow. Flow rate should be 120-200% of the operating flow rate or refer to the recommended packing condition.
- Let the flow run until bed has consolidated.
- Measure the bed height.
- Stop the flow and disconnect the packing reservoir if used from the column. Add the top adapter according to instructions and lower the adapter until it touches the resin bed. Make sure that no air is trapped or that the pressure does not increase.Do not exceed the pressure specifications of the media or the column.
- Flow the column according to your maximum operational velocity for 2-5 CV to condition the bed.
Before evaluation: Flush 2-5 CV of equilibration buffer, e.g., 20 mM sodium phosphate, 150 mM sodium chloride, 7.5 pH at 120-200% of the operating flow rate.
Caution: Sometimes replacing buffer causes the bed height to reduce slightly. When this occurs, adjust the adapter to the new bed height.
Before use: Wash the column with 5 CV of elution buffer. Then flush the column with equilibration buffer until the pH and conductivity become stable.
Evaluation of Column Packing Efficiency
Packing evaluation can be represented with the number of theoretical plates (N) and peak asymmetry (As).
To evaluate As, use (i) 1 M sodium chloride as the test marker for conductivity and (ii) acetone (or other substance with low molecular
weight that is not retained in the media) as the test marker for UV (absorbance at 280 nm). Equilibrate the column with 150 mM sodium
chloride before evaluation. The injection volume of the test marker should be 1-3% of column volume.
Note: The acceptable value range of As is usually between 0.7-1.5, but the criteria is application dependent.
Example of Column Packing
Below is an example of column packing using the protocol on the previous page.
Materials and Method
Column Tricorn 5/200 (4 mL)
Packing solution 0.1 M sodium chloride
Equilibration buffer 20 mM sodium phosphate, 150 mM sodium chloride, 7.5 pH
Packing flow rate 3.9 mL/min (1200 cm/hr)
Equipment AKTA system
Loading Equilibration buffer
Inject solution 1% acetone in 1 M sodium chloride
Inject solution 100 μL
Flow rate 1.0 mL/min (300 cm/hr)
Chromatogram and evaluation results can be seen below.
Bed height for the column after packing was 20.6 cm and volume was 4.03 mL.
Asymmetry and plates per meter were within specification (see below).
Good packing was observed.
For storing a column packed with Amsphere A3, equilibrate the column with 16 – 20% ethanol, and store between 2 – 8 ÅãC. Instead of 16 – 20% ethanol, 2% benzyl alcohol can be used. After long-term storage, it is recommended to do a blank run including CIP before use. Please refer to General Instructions for details.
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