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users
guide (PDF) |
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For rapid
protein desalting buffer exchange
and removal of alcohol and detergents |
|
| Cellufine GH-25 desalting media is
based on porous, spherical, highly crosslinked cellulose particles.
The sharp 3kD exclusion limit allows proteins to pass through
the column in the void volume while retarding smaller molecular
weight solutes in the internal pores. Outstanding mechanical strength
allows operation at high flow rates even in large diameter process
scale columns, thus minimizing run times. |
Features
• Mechanical robust spherical particle
• Efficient salt removal
• Hydrophilic
• Pre-swollen
• pH stable 1 – 14 (0.1M HCl, 0.5M NaOH)
• Resistant to organic solvents
• Autoclavable (121 °C, 30 min) |
Benefits
• Enables high flow rates and short run times
• Permits large sample loads (typical: 5 – 30 minute
run times in columns from 1 ml to 100 liters, with loads up to
35 % bed volume)
• Low non-specific adsorption, high recovery
• Easy packing
• Easy cleaning and depyrogenation
• Permits use with all commonly used solvents and buffers
without shrinkage or swelling
• Sterilizable |
|
|
|
Matrix |
Crosslinked cellulose |
| Particle
Size |
44 – 105 µm |
| Gel
Exclusion Limit |
3kD |
| Efficiency |
98 % to 100 % recovery.
No deterioration after 250 days, 1000 cycles. |
| Autoclavable |
121 °C, 30 min. |
| Pressure
Resistance |
No collapse at up to 870 ml/h/cm2 flow
in large columns
|
| pH
Stability |
pH 1 – 14 |
| Chemical
Resistance |
Resistance to detergents
and dissociating agents.
No change after 30 days in 0.1M HCl or 0.1M NaOH. |
| Supplied |
Suspension in 20
% ethanol |
|
|
Flow Properties
Due to its rigidity, Cellufine
GH-25 delivers nearly twice
the flow rate of an equivalent sized Dextran gel. |
|
The specific selection curve of Cellufine GH-25
1: Glycine 75 2: (Gly)2 132
3: (Gly)3 189 4: (Gly)4 246
5: Calcium pantothenate 477 6: Vitamin B12
1355
7: insulin B chain 3495 |
|
Applications
• Desalting before lyophilization or concentration
• Buffer exchanges
• Removal of alcohol or other organic solvents
• Removal of aromatic compounds (e.g., phenol) in purification
of nucleic acids
• Removal of detergents used to solubilize proteins (e.g.
Triton® X-100, SDS)
• Permits use with all commonly used solvents and buffers
without shrinkage or swelling
• Removal of chaotropic agents, (e.g. urea, guanidine) |
High Speed Desalting
Although the rigidity of Cellufine GH-25 makes it ideally suited
to large scale column use, its ability to operate at very high
flow rates enables the use of smaller columns running multiple
cycles giving similar throughput to lower flow rates on larger
columns. Unlike conventional chromatography, the performance (as
measured by sample load, salt removal, and dilution) of desalting
chromatography can actually improve as the flow rate increases,
due to decreased sample dilution at high volumetric loads.
Protein Desalting
| Packing : |
Cellufine GH-25 |
| Column : |
105 x 587 mm Vt = 5086 ml |
| Mobile Phase : |
0.1M NaCl |
| Flow : |
1250 ml/min, 870 ml/h/cm2 |
| Sample : |
5 % (w/v BSA) in 1.5M NaCl |
| Sample Volume : |
1272 ml (25 % of column volume) |
| Protein Recovery : |
99.5 % |
| Salt Exchange : |
99 % |
| Dilution : |
1.13x |
|
Alcohol Removal
Fractionation of human blood and the subsequent removal of alcohol
from the albumin are two important elements of albumin production.
Table 1 compares alcohol removal from albumin with GH-25 gel at
two different flow rates. Increasing the flow rate does not affect
de-alcoholization efficiency, albumin recovery or sample dilution.
Concentration of the remaining alcohol was below 0.01 % for either
flow rate. Cycle time was reduced to one quarter by increasing
flow from 29 cm/h/cm² to 100 cm/h/cm² .
| Run
Number |
1 |
2 |
| Gel type |
GH-25 |
GH-25 |
| Column diameter (mm) |
50 |
50 |
| Column diameter length
(mm) |
680 |
670 |
| Gel volume (ml) |
1335 |
1320 |
| Flow rate (ml/hr) |
570 |
2010 |
| Linear velocity (ml/h/cm²) |
29 |
102 |
| Time needed for a cycle
(hr) |
2.3 |
0.6 |
| Product
Applied |
| Process volume (ml) |
310 |
310 |
| Process volume (% Vt) |
23 |
23 |
| Albumin concentration (%) |
12 |
12 |
| Ethanol concentration (%) |
4.8 |
4.8 |
| Product
Collected |
| Recovered volume of product
(ml) |
546 |
525 |
| Dilution factor |
1.8x |
1.7x |
| Recovered albumin concentration
(%) |
6.6 |
6.9 |
| Remaining alcohol concentration
(%) |
0.002 |
0.01 |
| Mass recovery of albumin
(%) |
97 |
98 |
Table 1. Alcohol removal from human albumin fractionation
procedure |
Industrial Desalting
The advantages of desalting biological molecules chromatographically
are clearly realized in large-scale applications. From research
to pilot and production facility, the scale-up of Cellufine GH-25
has proven to be direct and trouble-free. Mechanical stability
of GH-25 allows the use of high flow rates in large columns.
A typical large scale desalting application requiring the processing
of 225 liters per day can be accomplished by 5 liters of Cellufine
GH-25 in a 105 x 587 mm column (see Table 2). This rigid gel withstands
flow rates to 1250 ml/min in that column geometry (870 ml/cm/cm²).
Cycle time between injections is 8 minutes. The high volume load
(25 % of Vt), is accommodated by the packing
without sacrificing resolution or purity.
| Packing |
Cellufine
GH-25 |
| Column (mm) |
105 x 587 |
| Vt |
5.1 liters |
Flow rate (ml/min)
(liters/hr) |
1250
(75 liters/hr) |
| Linear velocity |
870 ml/h/cm² |
| Product |
5 % w/v
protein in 1.5M NaCL |
| Product volume |
1.27 liters
(25 % of Vt) |
| Product mass/cycle |
63.6g |
Cycles/day
|
180 |
| Product mass/day |
11.4kg |
| Protein mass recovery |
99.5 % |
| Salt exchange |
99 % (0.015M
final conc.) |
| Sample dilution |
1.13 |
| Total product volume recovered |
257 liters |
| Total product volume processed per day |
225 liters |
Table 2. Throughput analysis for high speed desalting |
|
|
| Description |
Quantity |
Catalogue
No. |
| Cellufine
GH-25 |
100
ml
0.5 L
5 L
10 L |
6700
003 27
19711
19712
6700 003 35 |
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