10
IEC
IEC
TSKgel STAT SERIES
Antibody Analysis
The analysis profiles for five antibodies separated on a
TSKgel CM-STAT column were compared with the profiles
obtained on a competitive WCX column (Figure 9). Similar
or higher resolution profiles were obtained on TSKgel CM-
STAT in approximately half the time.
Column Selection for Antibody Analysis
Different antibodies were analyzed on a CM-STAT and SP-
STAT column at pH 7. Figure 10 shows that it is dependent
on the antibody which column provides the better resolu-
tion. The strong cation exchange column TSKgel SP-STAT
shows a better separation of charge variants of mAb A (up-
per chromatograms) while the weak cation exchange col-
umn TSKgel CM-STAT delivers a better separation of a ba-
sic variant from the main peak for mAb B.
0
10
20
30
40
0
10
20
30
40
B
C
E
D
A
Retention time (min)
mV
0
5
10
15
0
20
40
60
80
A
B
C
E
D
Retention time (min)
Columns:
A: TSKgel CM-STAT, 7µm, 4.6mm ID x 10cm
B: ProPac WCX-10, 10µm, 4mm ID x 25cm
Eluent:
A: 20mmol/L MES (pH6.0)
B: 20mmol/L MES + 0.5mol/L NaCl (pH6.0)
Gradient:
A: 10% B (0min), 30% B (15min), 100% B (15min),
100% B (17min), 10% B (17min), 10% B (21min)
B: 10% B (0min), 30% B (30min), 100% B (30min),
100% B (32min), 10% B (32min), 10% B (36min)
Flowrate:
A: 1.0mL/min
B: 2.0mL/min
Temp.:
Ambient
mV
B
A
figure 9
: A: TSKgel CM-STAT, 7 µm, 4.6 mm ID x 10 cm L; B:
Competitor WCX, 10 µm, 4.0 mm ID x 25 cm L;
t:A:20mmol/LMES(pH6.0);B:20mmol/LMES+0.5mol/LNaCl(pH6.0)
Gradient: A: 10% B (0 min), 30% B (15 min), 100% B (15 min), 100% B (17
min), 10% B (17 min), 10% B (21 min); B: 10% B (0 min), 30% B (30 min), 100%
B (30 min), 100% B (32 min), 10% B (32 min), 10% B (36 min)
Flow rate: A: 1.0 mL/min B: 2.0 mL/min; Temp.: Amb ent; Detection: UV @
280 nm; Inj. Vol.: 20 µL; Sample: monoclonal antibodies (mAb A through E)
Columns: TSKgel SP-STAT (7 µm, 4.6 mm ID x 10 cm);
TSKgel CM-STAT (7 µm, 4.6 mm ID x 10 cm)
Mobile phase A: 10 mmol/L sodium phosphate buffer pH 7.0
Mobile phase B: 100 mmol/L sodium phosphate pH 7.0 + 500 mmol/L NaCl
Gradient: 0-100 % B in 30 min; Flow rate: 1 mL/min; Detection: UV @ 280 nm
Injection vol.: 10 µL; Sample: mAb A (2 g/L); mAb B (2 g/L)
Comparison of TSKgel CM- and SP-STAT columns
figure 10
analysis of charge heterogeneity
