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APPENDIX
APPENDIX
Column Storage
When the column will be used the next day, allow it to run overnight at
a low flow rate in a buffer that does not contain a halide salt. When the
column will not be used for more than a day, clean it first, then flush salt
from the column and store in 0.05 % sodium azide or 20 % ethanol. Seal
tightly to prevent the column from drying out.
Scaling Up
for Size Exclusion Chromatography
Tosoh Bioscience offers semi-preparative (21.5 mm ID), preparative
(55 mm ID), and larger ID stainless steel columns packed with TSKgel
SW-type or PW-type resin for seamless scale-up to commercial
production of therapeutic proteins and other biopharmaceuticals.
These packing materials have a larger particle size that is appropriate
for use in process scale equipment. The packing materials, however,
have the same pore size and provide the same selectivity as the
corresponding TSKgel analytical column. The column volume (CV) of
the preparative column that is needed to produce the required amount
of product (per injection) is given by the relationship:
(CV)pc / (CV)ac = (mg product)pc / (mg product)ac
in which pc and ac refer to the preparative and analytical column
respectively. The volume of a column is equal to 1 /4
π
(ID)
2
L, in which
ID is the internal diameter and L the length of the column. In scaling up,
column length (L) is usually kept constant. If so, to achieve a 100-fold
increase in product per run, the ID of the prep column should be 10
times larger than that of the analytical column. As noted, the particle
size in the preparative column is usually larger, and one should select
a larger ID column than predicted by the above equation. As a rule of
thumb, a 2-fold increase in particle size reduces resolution and thus
output by the square root of 2.
Since scale-up from analytical columns is relatively straightforward,
preparative TSKgel SW columns may be an economical route for the
rapid production of biomolecules for clinical testing. See the SEC
section of this catalog for more information and request a copy of the
process media catalog. For more detailed analysis of your scale-up
requirements, please contact Tosoh Bioscience’s Technical Service
Specialists .
for Hydrophobic Interaction and Ion Exchange
Chromatography
Tosoh Bioscience provides various ID preparative columns for
hydrophobic interaction (HIC) and ion exchange (IEC) chromatography.
As shown above, to calculate the sample capacity of a larger column,
multiply the capacity obtained on a 7.5 mm ID column by the ratio of the
column volumes. The table below lists the column volumes for TSKgel
HIC and IEC columns and their ratios relative to the 7.5 mm ID x 7.5 cm L
column.
Dimensions
(mm ID x cm L)
Volume (mL)
Volume ratio*
5 x 5
1.0
0.3
7.5 x 7.5
3.3
1.0
8.0 x 7.5
3.8
1.2
20 x 15
47.1
14.3
21.5 x 15
54.4
16.4
55 x 20
474.9
143.6
108 x 20
1831.2
554.8
* Relative to 7.5 mm ID x 7.5 cm L column
Based on a 1 mg capacity for a 7.5 mm ID x 7.5 cm L column, the capacity
for a 55 mm ID x 20 cm L column is expected to be about 150 mg. Much
larger amounts of crude sample can be injected as long as impurities do
not co-elute from the column with the compound of interest.
