|Column||Compression factor||Slurry concentration (%)||Bed height (cm)|
|BPG 100||1.15||50 - 70||10 - 30|
|BPG 140||1.15||50 - 70||10 - 30|
|BPG 200||1.15||50 - 70||10 - 30|
|BPG 300||1.15||50 - 70||10 - 30|
|Column||Maximum operation pressure (bar)|
|FineLINE Pilot 35||20|
Packing in place technique .
Packing buffer: water
For more information please contact your local Cytiva representative.
|10 µm nets (5 pieces/pack) Code No. 18-8761-01|
|10 µm nets (5 pieces/pack) Code No. 18-8760-01|
|23 µm nets|
|Adaptor net Code no: 18-1103-08|
|End piece net Code no: 18-9252-01|
|23 µm nets|
|Adaptor net Code no: 18-1113-01|
|End piece net Code no: 18-1113-00|
|23 µm nets|
|Adaptor net Code no: 18-9253-01|
|End piece net Code no: 18-9254-01|
|23 µm nets|
|Adaptor net Code no: 18-1012-54|
|End piece net Code no: 18-1012-34|
FineLINE Pilot 35. Approximative values for compression factor, slurry concentration and recommended bed heights
|Column||Compression factor||Slurry concentration (%)||Bed height (cm)|
|FineLINE Pilot 35||1.15||50 - 70||10 - 301|
1 Run FineLINE Pilot 35 in series
Constant flow packing technology.
Packing buffer: 20 % EtOH
For more information, please read Packing MabSelect and MabSelect SuRe in a XK 16/40 column.
Packing MabSelect and MabSelect SuRe in a XK 16/40 column
Constant flow packing
Below is an example of packing the chromatography medium in a XK 16/40 column.
Preparing the suspension
The chromatography medium is supplied as a suspension in 20% ethanol. Decant the 20% ethanol solution and replace it with packing buffer before use.
45 ml chromatography medium
Glass filter funnel
20% EtOH + 0.25 M NaCl
Washing the medium
Equilibrate all material to room temperature. Mount the glass filter funnel onto the filtering flask. Pour the medium into the funnel and wash according to following instructions.
1. 100 ml 20% EtOH.
2. 200 ml 20% EtOH + 0.25 M NaCl.
Preparation of the slurry
1. Move the sedimented medium from the funnel into a beaker.
2. Add 50 ml of 20% EtOH + 0.25 M NaCl.
Packing the column
Packing buffer: 20% EtOH
Column XK 16/40
Pump P 900 (ÄKTA)
Two AK 16 adaptors
Assembling the column
1. Details of the column parts and packing equipment can be found in the instructions supplied. Before packing ensure that all parts, particularly the nets, net fasteners and glass tube, are clean and intact.
2. Attach the packing reservoir to the column.
3. Inject 20% ethanol into the column bottom adaptor with a syringe. Ensure that there are no air bubbles trapped under the net. Close the tubing with a stopper.
4. Flush the column and reservoir with 20% EtOH, leaving a few ml at the bottom. Mount the column vertically on a laboratory stand.
Packing the column
1. Pour the slurry into the column in one continuous motion. Pouring down a glass rod held against the wall of the column prevents the introduction of air bubbles. Immediately fill the remainder of the column and reservoir with 0.25 M NaCl in 20% EtOH. Mount the lid on the packing reservoir and connect it to the pump.
2. Open the column outlet and start the packing by pumping 20% EtOH through the column at a flow rate of 25 ml/min (750 cm/h) until the bed height stabilizes (approx. 5 min).
Note: Make sure that the backpressure does not exceed the pressure limits of the column during packing.
3. Switch off and disconnect the pump. Close the column outlet.
4. Take the column from the stand and remove the packing reservoir over a sink. Remount the column vertically and fill to the top with 20% EtOH.
5 Wet the column adaptor by submerging the plunger end in 20% ethanol, and drawing through with a syringe. Ensure that all bubbles have been removed.
6. Insert the adaptor into the top of the column, taking care not to trap air under the net.
7. With the adaptor outlet open, push the adaptor into the column and down onto the medium bed, allowing the 20% EtOH to displace any air remaining in the tubing.
8. Lock the adaptor in position, connect it to the pump, open the column outlet and continue packing at a flow rate of 25 ml/min for a further 3 min.
9. Mark the position of the bed surface on the column. Stop the pump, close the column outlet and reposition the adaptor to approximately 2 mm below the marked position.
Packing small-scale AxiChrom columns
AxiChrom™ 50, 70, 100, 140, and 200 columns are designed to simplify column handling and maintenance during process development.
- Preprogrammed, verified packing methods that include Capto™, MabSelect™ media
- Reproducible, predictable and scalable results.
- Rotating pivot stand for easy emptying, maintenance and safer operation
- Proven sanitary design with construction materials recognized for use in GMP environments.
- Operates with ÄKTAexplorer™ and ÄKTApilot™ with UNICORN™ v 5.01 (and later).
AxiChrom columns are designed to simplify column handling and maintenance. Packing is facilitated by the Intelligent Packing methodology in the UNICORN software. The preprogrammed, verified packing methods shorten start-up time, eliminate errors and pack the column with optimal bed compression.
The pivot design allows the column tube to be tilted for convenient manual emptying as well as easy access to parts for maintenance.
Scalable and predictable performance over the entire AxiChrom platform is assured through the use of computational fluid dynamics in designing the liquid distribution system.
The AxiChrom column is sanitizable, and materials of construction are suitable for use in regulated environments. Comprehensive documentation, including IQ/OQ documents are available for all column sizes.
Packing large-scale AxiChrom columns
Large-scale AxiChrom™ columns are supported with Intelligent Packing and intuitive handling via the AxiChrom Master.The novel, swing-out-design facilitates maintenance and provides a safer working enivironment.
- Preprogrammed verified packing methods that include Capto™ and MabSelect™ media
- Reproducible and predictable results
- Swing-out, hoist-free design for easy maintenance and safer operation
- Proven, sanitary design meets stringent cGMP production standards
- Key steps guided by AxiChrom Master
Intelligent Packing provides preprogrammed, verified axial compression packing methods that promote accurate and reproducible results. Intuitive handling supports preparation and maintenance procedures. Both are controlled by the AxiChrom Master, a separate unit with a touch-screen interface that guides the operator through critical steps for priming, packing, unpacking, and maintenance.
Packing large-scale AxiChrom columns (Video below)
Easy access to all column parts for maintenance in-situ is facilitated by the swing-out, hoist-free, column tube design.
The distribution system design has been developed using analytical methods and modern computational fluid dynamic modeling tools to ensure reproducible and predictable results over the entire range of scales.
Materials of construction meet the ASME Bioprocessing Equipment Standard for biopharmaceutical manufacture.
AxiChrom columns are supplied with comprehensive documentation to facilitate set-up, installation, and operation.
The efficiency of a column depends on how well it is packed. A poorly packed column gives rise to uneven flow, resulting in zone broadening and reduced resolution. It is thus important to have a method by which the column can be tested before it is put into operation. Such a method should be simple, quantitative and should not introduce contaminating materials. It is also an advantage if the same method can be used to monitor column performance over its working life, so that it is easy to determine when the medium should be re-packed or replaced.
Avoid methods that use colored compounds such as Blue Dextran. They do not meet the above criteria and cannot be used with ion exchange and affinity chromatography media.
Experience has shown that the best method of expressing the efficiency of a column is in terms of the height equivalent to a theoretical plate, HETP, reduced plate number, h, and the peak asymmetry factor, As. These values can be determined easily by applying a NaCl or acetone solution, to the column (see below).
It is important that the column is properly equilibrated ( >2 column volumes) before evaluating the packing. Ideally, run three test runs to see whether the values are stable. If an initially poor result improves during a later test, the reason can be that the column was not properly equilibrated. To check that the bed is stable, run the column at 70% packing pressure for 20 hours and test it again.
Note that pressure spikes may cause poor packing (cracking). If this happens, fit an air trap and a pressure relief valve between the pump and column. Locate the pressure relief valve between the air trap and the column.
Choice of test sample for columns
The most appropriate material for column testing is, of course, the sample that is to be run in the application, but this is not always practical or economical. As an alternative, a solution of either NaCl or acetone will give a good indication of the column packing quality. The eluate is monitored by measuring conductivity or UV absorption, and the resulting elution profile is used to calculate the HETP value.
The advantages of using NaCl are that it is readily available and can be used safely to test all columns. One disadvantage is that NaCl may interact with the medium matrix, especially ion exchanger matrices, and thus give erroneous results.
Acetone, in contrast, does not interact with the matrix and is detected by UV absorption at 280 nm. Alternatively, you can increase the running buffer concentration 10-fold and use it as test solution.
The figure below shows a UV trace for acetone in a typical BPG column application and gives calculated HETP and As values.
The sample volume should be approximately 1% of the total bed volume and the concentration 1.0% v/v NaCl, or equivalent when using stronger buffer. Alternatively, use 1.0% v/v acetone. The flow velocity should be between 10 and 30 cm/h depending on the bead size of the chromatography medium. The high flow velocity could be used for small beads whereas large beads only allow low flow velocity. To avoid diluting the sample, apply it as close to the column inlet as possible. If an airtrap is included in the system, by-pass it during sample application to avoid back-mixing. Calculate the HETP value from the conductivity (or UV) curve as follows:
HETP, in its simplest terms, is expressed as:
HETP = L/N
L = Bed height (cm)
N = Number of theoretical plates.
N is defined by the equation:
N = 5.54 (Ve /Wh)2
Ve = Elution volume (ml)
Wh = Peak width at half height (ml)
Ve is measured as the volume passed through the column to the peak maximum.
Wh is measured as the peak width at half-peak height.
From the example in the figure, the HETP value can be calculated from the chromatogram as follows:
|Ve (ml)||Wh (ml)||N||N/m||HETP cm|
Well-packed columns have low HETP values. However, it is only possible to compare columns that have been packed with the same type of media and that have been tested under identical conditions.
As a general rule-of-thumb, a good HETP value is approximately two to four times the mean bead diameter of the medium in question, provided that the sample does not interact with the medium.
In practice, the correlation between HETP and column performance can only be assessed by the column operator. Once this has been established, a standard can be set to judge the acceptability of a column packing.
For example, the column operator may know from experience that a column packed with Sephadex G-25 gel filtration medium with HETP values above 0.05 cm does not give the required separation. Consequently, the operator will set this value as the maximum permissible i.e. the minimum acceptable quality.
Reduced plate number
Definition of reduced plate number: h = HETP/dp
h = reduced plate number.
HETP = above described height equivalent to a theoretical plate.
dp = mean particle diameter of the chromatography medium beads.
The reduced plate number should be in the range of 2-4 times the mean particle diameter of the chromatography medium beads.
Peak asymmetry factor calculation
The peak asymmetry factor should be as close as possible to 1, and the shape of the peak should be as symmetrical as possible. This is usually the case for gel filtration media, but for certain ion exchange and affinity media, the shape may be asymmetrical due to interaction with the media. A change in peak shape is usually the first indication of column deterioration.
The peak asymmetry factor, As, is calculated from the graph above:
As = b/a
a = distance from peak apex to 10% of the peak height on the ascending side of the peak
b = distance from peak apex to 10% of the peak height on the descending part of the peak
Note: Measuring HETP, h and As values is the best way to judge the condition of the packed column. A packed column can look good, but still need repacking for optimal performance. Always check the column after packing and regularly between runs to ensure best performance.
Chromaflow. Approximative values for compression factor, slurry concentration and packing flow velocity
Recommended flow velocity intervals in the table below are for packing the column. The intervals are given in reverse order to indicate that the high flow velocity can be used for shorter beds whereas higher beds only allow lower flow velocity, due to the higher pressure drop across the bed.
|Column||Compression factor||Slurry concentration (%)||Bed height (cm)||Flow velocity (cm/h)|
|Chromaflow 400||1.15||50 - 70||10 - 30||Initial flow velocity 3000 - 1000|
|Chromaflow 600||1.15||50 - 70||10 - 30||Initial flow velocity 3000 - 1000|
|Chromaflow 800||1.15||50 - 70||10 - 30||Initial flow velocity 3000 - 1000|
|Chromaflow 1000||1.15||50 - 70||15 - 30||Initial flow velocity 3000 - 1000|
Constant flow packing followed by axial compression.
Packing buffer: 20% ethanol
20 µm net
Stainless steel or plastic
For code no and detailed information see the spare part section.
Recommended flow velocities in the table below are for packing the column.
|Column||Compression factor||Slurry concentration (%)||Bed height||Flow velocity (cm/h)|
|XK 16/40||1.15||50 - 70||10 - 30||750|
|XK 26/40||1.15||50 - 70||10 - 30||710|