Product Information
Phase Detail
bioZen WidePore C4
bioZen Widepore C4 HPLC columns can be used to characterize and profile impure biologic fragments
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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Application ID 24717

Columns:
Dimension:
150 x 4.6 mm
Mobile Phase:
50 mM Dipotassium Phosphate + 100 mM Sodium Sulfate, pH 5.0
Flow Rate:
0.3 mL/min
Temperature:
Ambient
Detection:
UV @ 280 nm
Sample:
1. γ-Globulin, 5 mg/mL
2. Ovalbumin, 1 mg/mL

Diverse mAb Comparison with Chromatographic Performance
Suitable for Intact MS Analysis 
  • 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

Columns:
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)
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

LC Conditions

Columns:
bioZen 2.6 µm WidePore C4
Dimension:
100 x 2.1 mm
Part No.:
00D-4786-AN
Mobile Phase:
A: 0.1 % TFA in Water
B: 0.1 % TFA in Acetonitrile
Gradient:
20-55 % B in 6 minutes (Intact)
25-45 % B in 6 minutes (Subunit)
Flow Rate:
0.5 mL/min
Temperature:
As Indicated
Detection:
UV @ 280 nm (Intact)
UV @ 214 nm (Subunit)
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

LC Conditions

Columns:
bioZen 2.6 µm WidePore C4
Dimension:
100 x 2.1 mm
Part No.:
00D-4786-AN
Mobile Phase:
A: 0.1 % TFA in Water
B: 0.1 % TFA in Acetonitrile
Gradient:
25-45 % B in 6 minutes
Flow Rate:
As indicated
Temperature:
90 °C
Detection:
UV @ 280 nm (Intact)
UV @ 214 nm (Subunit)
Injection Volume:
2 µL
Sample:
NIST mAb (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

LC Conditions

Columns:
bioZen 2.6 µm WidePore C4
Dimension:
100 x 2.1 mm
Part No.:
00D-4786-AN
Mobile Phase:
A: 0.1 % TFA in Water
B: 0.1 % TFA in Acetonitrile
Gradient:
As indicated
Flow Rate:
0.5 mL/min
Temperature:
90 °C
Detection:
UV @ 280 nm (Intact)
UV @ 214 nm (Subunit)
Injection Volume:
2 µL
Sample:
DTT-reduced NIST mAb (1 mg/mL)