Capto S, 10 L

Column	Maximum operation pressure (bar)
HiScale	20
XK 16	5
XK 26	5
XK 50	3
BPG 100	8
BPG 140	6
BPG 200	6
BPG 300	4
BPG 450	2.5
BioProcess LPLC	6
BioProcess MPLC	20
Chromaflow 400	3
Chromaflow 600	3
Chromaflow 800	3
Chromeflow 1000	3
FineLINE Pilot 35	20
FineLINE 70	20
FineLINE 100	20
FineLINE 200	20
FineLINE 350	20

 

Suction packing technique. 

Please read the Application Note: Methods for packing Capto S and Capto Q in production-scale columns.

The same packing method is valid for Capto DEAE, Capto Q and Capto S.

Packing Capto S in XK 16/20 and 16/40 columns


Materials needed
Capto S
Plastic spoon or spatula
Measuring cylinder
20% ethanol
Lab stand
Equilibrate all materials to room temperature

Preparing the packing slurry
Capto S is delivered in 0.2 M sodium acetate in 20% ethanol with a slurry concentration of approximately 75%. To pack XK columns, the slurry can be poured directly from the medium container.

Equipment needed
ÄKTAdesign 100 systems or a stand-alone pump such as Pump P-900 with a pressure monitor can be used for packing.The columns should be packed without using a slurry reservoir, as it is important to lock the medium bed quickly at the end of the packing after the flow has been stopped.

XK 16/20 and XK 16/40 packing procedure
To pack the column, use 20% ethanol and proceed as follows:

1. Assemble the column. Ensure that all parts of the column are clean and intact.

2. Mount the bottom piece/adapter to the column tube.

3. Wet the bottom piece/adapter filter with 20% ethanol with the aid of a syringe mounted on the outlet tubing. When filter is wet, mount an end plug on the outlet.

4. Wet the top adapter filter with 20% ethanol by connecting the inlet tubing to the pump and starting a flow. Make sure no air is trapped underneath the filters on both adapter(s) and bottom piece.

5. Mount the column vertically on a lab stand.

6. Pour an appropriate amount of gel slurry into the column. Avoid formation of air bubbles in the gel by pouring it along a thin capillary.

7. If necessary, add packing solution to the upper edge of the glass tube.

8. Mount the top adapter without trapping any air beneath the filter. Tighten the sealing ring. Do not let the medium to sediment.

9. Pack the medium for 15 min at 900 cm/h (30 ml/min). Make sure the back pressure does not exceed 4 bar. If necessary, reduce the flow.

10. Stop the flow and mount an end plug on the outlet.

11. Disconnect the top adapter tubing from the pump. Quickly adjust the adapter down to the bed surface.

12. Connect the top adapter tubing to the pump. Remove the end plug from the outlet tubing.

13. Pack the medium for an additional 5 min at 900 cm/h.

14. Stop the flow and disconnect the top adapter tubing from the pump. Mount an end plug on the outlet.

15. Quickly adjust the adapter down to the bed surface and then a further 1 to 2 mm into the medium bed. Lock the adapter at this level.

Testing the packed column

See packing evaluation protocol.

Packing buffer: Water, ethanol or sodium chloride. Because of the increased influence of the wall support, salt solutions is required in columns with small diameters, e.g. BPG 100.

Please read the Application Note: Methods for packing Capto S and Capto Q in production-scale columns in Related Documents.

The same packing method is valid for Capto DEAE, Capto Q and Capto S.

Constant flow packing. 

Packing buffer: 10 mM NaCl

BPG 100
23 µm nets
Adaptor net Code no: 18-1103-08
End piece net Code no: 18-9252-01
BPG 140
23 µm nets
Adaptor net Code no: 18-1113-01
End piece net Code no: 18-1113-00
BPG 200
23 µm nets
Adaptor net Code no: 18-9253-01
End piece net Code no: 18-9254-01
BPG 300
23 µm nets
Adaptor net Code no: 18-1012-54
End piece net Code no: 18-1012-34

 

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)
XK 16	1.1	50 - 60	20 - 30	1200 - 900
XK 16	1.1	50 - 60	30 - 40	900 - 600
XK 16	1.1	50 - 60	40 - 45	600 - 450
XK 26	1.1	50 - 60	20 - 30	1200 - 900
XK 26	1.1	50 - 60	30 - 40	900 - 600
XK 26	1.1	50 - 60	40 - 45	600 - 450
XK 50	1.1	50 - 60	20 - 30	1200 - 900
XK 50	1.1	50 - 60	30 - 40	900 - 600
XK 50	1.1	50 - 60	40 - 45	600 - 450

 

Recommended net porosity for Capto adhere, Capto DEAE, Capto MMC, Capto Q, Capto Viral Q and Capto S: 20 µm

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.

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 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 400-1000	1.1	50 - 60	20 - 45	Initial step 425 Compression step 1500-600

Column evaluation
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.

HETP calculation
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
where,
L = Bed height (cm)
N = Number of theoretical plates.

N is defined by the equation:
N = 5.54 (Ve /Wh)2
where,
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
Acetone	18800	900	2417	4203	0.024

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
where,
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.

20 µm net
Stainless steel or plastic

For code no and detailed information see the spare part section.

XK 16/40
10 µm nets (5 pieces/pack) Code no: 18-8761-01
XK 26/40
10 µm nets (5 pieces/pack) Code no: 18-8760-01
XK 50/30
10 µm nets (5 pieces/pack) Code no: 18-8759-01

 

Column	Compression factor*	Packin factor**	Slurry concentration (%)	Bed height (cm)	Flow velocity (cm/h)
BPG 100-300	1.1	1.10 (water) 1.10 (20% EtOH) 1.17 (10mM NaCl) 1.20 (0.2 M sodium acetate, 20% EtOH)	50 - 60	20 - 45	30 to settle the bed

* Compression factor = Gravity settled bed height / Packed bed height
** Packing factor = Consolidated bed height at 30-60 cm/h / Packed bed height