Process DEVELOPMENT
101
BULK
figure 2
Comparison chromatograms between ToyoScreen and semi-preparative columns
figure 3
Optimizing eluent pH in HIC
figure4
Optimizing salt conditions in HIC
Applications - ToyoScreen PROCESS DEVELOPMENT columns
0
20
40
60
80
100
0
10
20
30
40
50
(mV)
0
0.5
1
1.5
Ammonium Sulfate (mol/L)
0
50
100
150
200
250
300
350
0
10
20
30
40
50
(mV)
0
0.5
1
1.5
Ammonium Sulfate(mol/L)
Time(min)
Time(min)
Fr 1 Fr 2 Fr 3 Fr 4
1mL ToyoScreen column
10mL Semi-preparative column
Comparison chromatograms between ToyoScreen and Semi-preparative columns
Packing: Toyopearl Phenyl-650M
Eluent:
(A) 0.1mol/L phosphate buffer containing 1.8mol/L (NH
4
)
2
SO
4
, pH7.0 (B) 0.1mol/L phosphate buffer, pH7.0
Sample: Anti-TSH from cell culture supernatant (x4 diluted)
1mL ToyoScreen
10mL Semi-preparative
Column Dimensions:
6.4mm ID x 3cm
14.6mm ID x 6cm
Injection Volume:
500µL
5000µL
Flow Rate:
0.5mL/min; 0.5CV/min; 93cm/hr
2.5mL/min; 0.25CV/min; 90cm/hr
Gradient Profile:
25% B; 0-5min (isocratic)
25% B; 0-10min (isocratic)
50% B: 5min (step)
50% B: 10min (step)
50% to 100% B; 5-35min (linear)
50% to 100% B; 10-40min (linear)
Gradient Slope*:
0.06M/mL
0.012M/mL
* The gradient slope is the change in ionic strength per unit volume. Gradient volume is the product of flow rate and gradient time.
0
20
40
60
80
100
120
140
0
10
20
30
40
50
Time(min)
(mV)
Column: ToyoScreen Phenyl-650M (1mL)
Eluent A: 0.1mol/L phosphate buffer + 1.8mol/L ammonium sulfate (pH7.0)
Eluent B: 0.1mol/L phosphate buffer (pH7.0)
Flow Rate: 1mL/min Gradient: 30min linear (30CV)
Inj. Vol.: 200µL
Samples: Cell culture supernatant (x4 diluted) (antibody: Anti-TSH)
Optimizi g eluent pH in HIC
pH5
pH6
pH7
pH8
Optimizing salt conditions in HIC
0
20
40
60
80
100
120
140
0
10
20
30
40
50
Time(min)
(mV)
Column: ToyoScreen Phenyl-650M (1mL)
Eluent A: 0.1mol/L phosphate buffer containing 1.8mol/L each salt (pH7.0)
Eluent B: 0.1mol/L phosphate buffer (pH7.0)
Flow Rate: 1mL/min Gradient: 30min linear (30CV)
Inj. Vol.: 200µL
Samples: Cell culture supernatant (x4 diluted) (antibody: Anti-TSH)
Ammonium
Sulfate
NaCl
Packing: TOYOPEARL Phenyl-650M; Eluent: (A) 0.1 mol/L phosphate buffer containing 1.8 mol/L (NH
4
)
2
SO
4
, pH 7.0 (B) 0.1 mol/L phosphate buffer, pH7.0;
Sample: Anti-TSH from cell culture sup rnatant (x4 diluted)
1 mL ToyoScreen
10 mL Semi-preparative
Column Dimensions:
6.4 mm ID x 3 cm L
14.6 mm ID x 6 cm L
Injection Volume:
500 μ
5000 μL
Flow rate:
0.5 mL/min; 0.5 CV/min; 93 cm/h
2.5 mL/min; 0.25 CV/min; 90 cm/h
Gradient Profile:
25% B; 0-5 min (isocratic)
25% B; 0-10 min (isocratic)
50% B: 5 min (step)
50% B: 10 min (step)
50% to 100% B; 5-35 min (linear)
50% to 100% B; 10-40 min (linear)
Gradient Slope*:
0.06 M/mL
0.012 M/mL
* The gradient slope is the change in ionic strength per unit volume. Gradient volume is the product of flow rate and gradient time.
Column: ToyoScreen Phenyl-650M (1 mL); Eluent A: 0.1 mol/L phosphate
buffer + 1.8 mol/L ammonium sulfate (pH7.0); Eluent B: 0.1 mol/L phos-
phate buffer (pH 7.0); Flow rate: 1 mL/min; Gradient: 30 min linear (30 CV);
Inj. Vol.: 200 μL; Sample: Cell culture supernatant (x4 diluted) (antibody: Anti-
TSH)
Column: ToyoScreen Phenyl-650M (1 mL); Eluent A: 0.1 mol/L phosphate
buffer contai ing 1 8 mol/L each salt (pH7.0); Eluent B: 0.1 mol/L hos-
phate buffer (pH 7.0); Flow rate: 1 mL/min; Gradient: 30 min linear (30 CV);
Inj. Vol.: 200 μL; Sample: Cell culture supernatant (x 4 diluted) (antibody: Anti-
TSH)
0
20
40
60
80
100
0
10
20
30
40
50
(mV)
0
0.5
1
1.5
Ammonium Sulfate (mol/L)
0
50
100
150
200
250
300
350
0
10
20
30
40
50
(mV)
0
0.5
1
1.5
Ammonium Sulfate(mol/L)
Time(min)
Time(min)
Fr 1 Fr 2 Fr 3 Fr 4
1mL ToyoScreen column
10mL Semi-preparative column
Comparison chromatograms betwee ToyoScre n and Semi-preparative columns
Packing: Toyopearl Phenyl-650M
Eluent:
(A) 0.1mol/L phosphate buffer containing 1.8mol/L (NH
4
)
2
SO
4
, H7.0 (B) 0.1mol/L phosphate buffer, pH7.0
Sample: Anti-TSH from cell culture supernatant (x4 diluted)
1mL ToyoScreen
10mL Semi-preparative
Column Dimensions:
6.4mm ID x 3cm
14.6mm ID x 6cm
Injection Volume:
500µL
5000µL
Flow Rate:
0.5mL/ in; 0.5CV/min; 93cm/hr
2.5mL/min; 0.25CV/min; 90cm/hr
Gradient Profile:
25% B; 0-5min (isocratic)
25% B; 0-10min (isocratic)
50% B: 5min (step)
50% B: 10min (step)
50% to 100% B; 5-35min (linear)
50% to 100% B; 10-40min (linear)
Gradient Slope*:
0.06M/mL
0.012M/mL
* The gradi nt slope is the c ange in ionic strength per unit volume. Gradient volume is the product of flow rate and gradient time.
