25
IEC
Ion exchange
chromatography
TABLE IV
TOYOPEARL GigaCap resin base stability
RESIN
STORAGE
TEST CAPACITY STARTING WEEK 1 WEEK 2 WEEK 3
SOLUTION MOLECULE CAPACITY
TOYOPEARL GigaCap S-650M 1.0 mol/L NaOH h-IgG Dynamic
143 (g/L)
144
140
135
TOYOPEARL GigaCap CM-650M 0.5 mol/L NaOH h-IgG Dynamic 99 (g/L)
88
90
91
TOYOPEARL GigaCap Q-650M 0.5 mol/L NaOH BSA Static
166 (g/L)
NA 153*
136
*
12 days
Figure 12
TSKgel SuperQ-5PW (20) in 0.66 cm ID x 15 cm L
0
500
1000
1500
2000
2500
3000
3500
4000
0
10 20 30 40 50 60 70 80 90 100
Elution volume (mL)
mAU (254 nm)
purified oligo
>94% purity
n-1
n+1
C
TSKgel SuperQ-5PW (20) in 0.66 cm ID x 15 cm L
0
500
1000
1500
2000
2500
0
10 20 30 40 50 60 70 80 90 100
Elution volume (mL)
mAU (254 nm)
purified
FPLC peak recovery 60%
and 94% purity
oligo
>98% purity
n-1
n+1
A
Competitor Q (15 µm) in 0.66 cm ID x 15 cm L
0
200
400
600
800
1000
1200
1400
1600
1800
0
10 20 30 40 50 60 70 80 90 100
Elution volume (mL)
mAU (254 nm)
purified oligo
>98% purity
n-1
n+1
B
Competitor Q (15 µm) in 0.66 cm ID x 15 cm L
0
500
1000
1500
2000
2500
3000
3500
4000
0
10 20 30 40 50 60 70 80 90 100
Elution volume (mL)
mAU (254 nm)
purified
FPLC peak
recovery 45%
and 90% purity
oligo
>90% purity
n-1
n+1
D
Column:
0.66cm x 15cm (5.1mL) (resin as noted in figure)
Flow rate:
1.43mL/min (250 cm/hr)
Buffer A:
20mmol/L Tris-HCl + 10mmol/L EDTA (pH=9.0)
Buffer B:
20mmol/L Tris-HCl + 10mmol/L EDTA + 1.0mol/L NaCl (pH=9.0)
Sample loaded:
DNA based oligonucleotides were loaded as followed: 1mg/column panels A & B,
20mg/column panels C & D
Separation conditions:
Column was washed with 5CV 100% buffer A followed by 11mL injection. Column was then washed
with 3CV 100% buffer A followed by 6CV of linear gradient 35-53 buffer B.
Finally, column was washed with 5CV 100% buffer B.
Detection:
UV@254nm
Fractions:
0.5mL fractions were taken from peaks of interest and analyzed on a TSKgel DNA-NPR column
(1 mg load)
(20 mg load)
(1 mg load)
(20 mg load)
TSKgel SuperQ-5PW (20) maintains resolution at high olionucleotide load
Column: 0.66 cm x 15 cm L (5.1 mL) (resin as noted in figure); Flow rate: 1.43 mL/min (250 cm/h)
Buffer A: 20 mmol/L Tris-HCl + 10 mmol/L EDTA (pH= 9.0); Buffer B: 20 mol/L Tris-HCl + 10 mmol/L EDTA + 1.0 mol/L NaCl (pH= 9.0) Sample loaded: DNA based
oligonucleotides were loaded as followed: 1 g/column panels A & B, 20 mg/column panels C & D
Separation conditions: Column was washed with 5 CV 100 % b ffer A followed by 11 mL injection. Column was then wash d with 3 CV 100 % buffer A followed
by 6 CV of linear gradient 35-53 buffer B. Finally, olu n was wa hed with 5 CV 100 % buffer B.
Detection: UV @ 254 nm; Fractions: 0.5 mL fractio s were t ken from peaks of interest and analyzed on a TSKgel DNA-NPR column
TSKgel SuperQ-5PW (20) maintains resolution at high oli onucleotide load
