BioHPLC Column Selection Guide Cover - Agilent Technologies

Separation of charge variants
of human IgG1 with pH gradient
Column: Agilent Bio MAb
2.1 x 150 mm, 5 µm
Mobile Phase: A: 10 mM Na2HPO4, pH 6.0
B: A + 0.5 M NaCl or just 0.5 M Na2HPO4, pH 6.0
Flow Rate: 2 mL/min
Gradient: 0.5 min hold with mobile phase A followed by a linear
gradient to 45% B in 15 min (elapsed time 15.5 min);
then 60% B at 15.6 min continued to 20 min.
Column flushed with 100% B for 15 min before
re-equilibration for the next run.
pH Gradient: A: 5 mM Na2HPO4, buffer pH 5.5 and
B: 40 mM NA2HPO4 (not buffered, pH 8.9). 2% B/min
at 1 mL/min for 15 min, followed by a column wash
with 90% B for 5 min.
Detector: UV at 220 nm
Sample: One mg each/mL in mobile phase A
Monoclonal antibodies (MAb) -human IgG1
(5 mg/mL stock solution) derived from CHO cells
Instrument: Agilent 1200 SL system with diode array detector
mAU
50
40
30
20
10
0
Biomolecule Separations
Before carboxypeptidase B digestion
After carboxypeptidase B digestion
10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 min
MAb c-terminal cleavage: Human IgG1 MAb, 1 mg/mL in 25 mM Na 2HPO 4 buffer, pH 7.5, was incubated with approximately 25 units of the
carboxypeptidase B for 18 hours and 10 µL samples were injected.
BioHPLC COLUMNS
7