Bio-Rad Bio-Scale™ Mini CHT™ 40 µm Cartridges User Manual
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hydroxyapatite media is available in larger amounts,
from 10 g bottles to bulk quantities, for scaling up
methods developed for using the cartridges. CHT
ceramic hydroxyapatite is fully supported by
Regulatory Support Files. In addition, Bio-Rad carries
an extensive line of empty chromatography columns
from laboratory scale to process scale.
Table 4. CHT stability in various buffers.*
C
CH
HT
T S
Su
uiittaab
biilliittyy
p
pH
H
B
Bu
uffffeerr
1
10
0 ccyycclleess
5
50
0 ccyycclleess
5.0
Acetate + 5 mM PO4
—
n/a
5.5
Acetate + 5 mM PO4
-
—
6.0
Acetate + 5 mM PO4
+
-
6.0
Succinate + 5 mM PO4
-
—
6.5
Succinate + 5 mM PO4
+/-
+/-
6.5
Acetate + 5 mM PO4
+
n/a
6.5
Phosphate (5 mM)
+
n/a
6.5
Acetate + 5 mM PO4
+
+
6.5
MES + 5 mM PO4
+
+
6.5
Imidazole + 5 mM PO4
+
+/-
6.5
Glycine + 5 mM PO4
+
+
6.5
Arginine + 5 mM PO4
+
+
6.5
Tris + 5 mM PO4
+
n/a
7.0
Phosphate (5 mM)
+
+
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calcium. Common buffers for hydroxyapatite
chromatography are listed in Table 4.
An appropriate starting point for purifying samples is
a linear gradient from 5–500 mM sodium phosphate
pH 6.8, spanning 10 to 20 column volumes at 2.0 ml/min
for the 5 ml cartridge. For separations of monoclonal
antibodies from aggregates, an alternative purification
protocol is eluting with a linear gradient from 0-2 M
sodium chloride in 5-20 mM sodium phosphate,
pH 6.5. The separation can be optimized by changing
the gradient profile. At the end of each run the
cartridge can be regenerated with 500 mM potassium
or sodium phosphate buffer, neutral pH; or 400 mM
trisodium phosphate, pH 11–12; followed by starting
buffer. Return to the desired flow rate and proceed
with the next separation.
4.3 Scaling Up the Separation
For quick scale-up, two or three cartridges of the same
type can be connected in series. Backpressure will
increase with cartridges in series, so care should be
taken to maintain pressures
£45 psi. CHT ceramic
16
+
No statically
significant mass
loss observed
+/-
Slight (0–1%)
loss
-
Small (1–2%) loss
—
Significant (>2%)
loss
n/a
Not applicable
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