sec
SEC
15
figure 7
Analysis of heat induced forced denatured, large hydrophobic
metalloprotein, apoferritin
0
5
10
15
20
25
30
5
6
7
8
9
10
Detector response (mAU)
Retention time (minutes)
RT
5min
20min
30min
45min
60min
0
5
10
15
Retention time (minutes)
Monomer
8.354 min
Trimer
6.589min
Dimer
7.190min
Tetramer
6.245min
Aggregate(s)
apoferritin
Rs: 1.736
Rs: 0.824
Protein
Molecular weight (kDa)
Monomer Dimer Trimer Tetramer
ferritin and apoferritin
450
900 1350
1800
Column: TSKgel UltraSW Aggregate, 3 μm, 7.8 mm ID × 30 cm
Mobile phase: 50 mmol/L potassium phosphate (monobasic), 50 mmol/L
sodium phosphate (dibasic), 100 mmol/L sodium sulfate, 0.05% NaN
3
, pH 6.7
Flow rate: 1.0 mL/min; Detection: UV @ 280 nm
Temperature: 30 °C; Injection vol.: 10 μL
Samples: ferritin – Sigma, 4.7 g/L, in saline (0.9% NaCl in water) solution,
stored at 2-8 °C apoferritin – Sigma, 5.0 g/L, in 50% glycerol and 0.075 mol/L
sodium chloride, stored at -20 °C
3
5
7
9
11
13
15
0
10
20
30
Retention time (minutes)
Detector response (mAU)
Monoclonal
antibody-2
Dimer
Monomer
Trimer
Aggregate
Column:
TSKgel UltraSW Aggregate, 3 µm, 7.8 mm ID
×
30 cm
Mobile phase: 0.2 mol/L phosphate buffer, pH 6.7 + 0.05% NaN
3
Flow rate:
0.8 mL/min
Detection:
UV @ 280 nm
Temperature:
25 ˚C
Sample:
monoclonal antibody-2
(mouse-human chimeric IgG, Erbitux), 10 µL
Rs (3mer/2mer) = 1.40
Rs (2mer/monomer) = 2.89
Column: TSKgel UltraSW Aggregate, 3 µm, 7.8 mm ID × 30 cm
Mobile phase: 0.2 mol/L phosphate buffer, pH 6.7 + 0.05% NaN
3
Flow rate: 0.8 mL/min; Detection: UV @ 280 nm
Temperature: 25 ˚C; Sample: monoclonal antibody-2
(mouse-human chimeric IgG, Erbitux), 10 µL
figure 6
Separation of mAb trimer and dimer
Applications of TSKgel SW-type Gel Filtration columns
SEPARATION OF HIGHER AGGREGATES
TSKgel UltraSWAggregate has a smaller particle size than the SuperSW
material, and offers high resolution separation of mAb multimers.
FIGURE 6
shows the analysis of a mouse-human chimeric IgG using
the TSKgel UltraSW Aggregate column. Superior resolution of the mAb
trimer and dimer is obtained. The smaller particle size (3 μm) and higher
molecular weight exclusion limit (2,500 kDa, globular proteins) of the
TSKgel UltraSW Aggregate column, compared to the TSKgel SuperSW
mAb HR and HTP columns, allows for high resolution separation of mAb
multimers and aggregates.
SEPARATION OF LARGE PROTEINS
TSKgel UltraSW Aggregate provides a larger pore size than TSKgel
SuperSW3000. It is therefore not only suited for the analysis of
mAb aggregates but can also be used for the analysis of other large
proteins and their aggregates. The analysis of a heat denatured, large
hydrophobic metalloprotein, apoferritin, is shown in
FIGURE 7
. A set of
six, 0.3 mL HPLC vials each containing 100 μL stock solution of apoferritin
was used for protein thermal denaturation. Thermal denaturation was
carried out at 60°C using an electric heating block. Individual sample
vials were tightly capped and exposed to the heat for 5, 20, 30, 45, and
60 minutes. Samples were analyzed using a TSKgel UltraSW Aggregate
column at the end of each incubation time period. The TSKgel Ultra SW
Aggregate column yielded high resolution between the monomer and
dimer. The trimer, tetramer and higher order aggregates of apoferritin
were well separated.
