HPLC
22
875
biozenlc-hplc-column
biozen-widepore-c4
Biozen WidePore C4
Impurity profiling and characterization of intact biologic fragments is a challenging undertaking because of the need to identify very small differences between variants. Both Biozen Intact columns contain skillfully manufactured large pore core shell particles that provide narrower, taller peaks in conjunction with higher resolution between the target HC/LC, Fc/Fab, or isoforms
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Column:
bioZen 1.8 μm SEC-3
Dimension
150 x 4.6 mm
Mobile Phase:
50 mM Dipotassium Phosphate + 100 mM Sodium Sulfate, pH 5.0
Gradient:
20-50 % in 5 min
Flow Rate:
0.3 mL/min
Temperature:
Ambient
Detection
UV @ 280 nm
Sample:
- γ-Globulin, 5 mg/mL
- Ovalbumin, 1 mg/mL
Diverse mAb Comparison with Chromatographic Performance
- High throughput and single method for multiple mAb analysis
- High temperature leads improved peak shape for all mAbs
- Formic acid mobile phase ensures sensitivity for intact mass by high resolution MS
LC Conditions
Column:
bioZen 2.6 µm WidePore C4
Dimension
100 x 2.1 mm
Part No.:
00D-4786-AN
Mobile Phase:
A: 0.1 % Formic acic in Water, B: 0.1 % Formic acic in Acetonitrile
Gradient:
10-90 % B in 4 minutes
Flow Rate:
0.3 mL/min
Temperature:
80 °C
Detection
UV @ 280 nm
Sample:
mAbs, Various (1 mg/mL)
Improved peak shape for intact mass applications by high resolution MS.
Sample
Retention Time
(min)
(min)
Width @ 50%
Rituximab
3.580
0.0233
Infliximab Biosimilar
3.606
0.0272
Cetuximab
3.696
0.0270
Infliximab
3.615
0.0222
Temperature Effects on Intact and Subunit Analysis
- Displays improved temperatures for Intact Influximab and Reduced NIST mAb at 70, 80, and 90 ºC
- 80 ºC exhibits narrowest and most ideal peak shape for both the intact and subunit analysis
Column:
bioZen 2.6 µm WidePore C4
Dimension
100 x 2.1 mm
Part No.:
00D-4786-AN
Mobile Phase:
A: 0.1 % Formic acic in Water, B: 0.1 % Formic acic in Acetonitrile
Gradient:
10-90 % B in 4 minutes
Flow Rate:
0.3 mL/min
Temperature:
80 °C
Detection
UV @ 280 nm
Sample:
Infliximab (1 mg/mL)
DTT-reduced NIST mAb (1 mg/mL)
Flow Rate Effects on Intact and Subunit Analysis
- Comparing 0.5, 0.6, 0.7, 0.8 mL/min flow rates for peak capacity of NIST mAb
- Faster flow rates help overcome slow diffusion rates and provides sharp, narrowing peaks
- Higher flow rates lead to higher throughput
Column:
bioZen 2.6 µm WidePore C4
Dimension
100 x 2.1 mm
Part No.:
00D-4786-AN
Mobile Phase:
A: 0.1 % Formic acic in Water, B: 0.1 % Formic acic in Acetonitrile
Gradient:
10-90 % B in 4 minutes
Flow Rate:
0.3 mL/min
Temperature:
80 °C
Detection
UV @ 280 nm
Sample:
Infliximab (1 mg/mL)
DTT-reduced NIST mAb (1 mg/mL)
Gradient Effects on NIST mAb Subunit Analysis
- Comparing gradient effects of changing 20-55% B to 25-45% B
- Shallowing of gradient slope provides improved resolution and enable characterization of closely eluting impurities
Column:
bioZen 2.6 µm WidePore C4
Dimension
100 x 2.1 mm
Part No.:
00D-4786-AN
Mobile Phase:
A: 0.1 % Formic acic in Water, B: 0.1 % Formic acic in Acetonitrile
Gradient:
10-90 % B in 4 minutes
Flow Rate:
0.3 mL/min
Temperature:
80 °C
Detection
UV @ 280 nm
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