Interferon Alpha intact 5% impurity on Jupiter 3u C18 and Jupiter 5u C4

LC Conditions

Column

Jupiter 3 µm C18 300 Å, LC Column 150 x 2 mm, Ea

Brand

Jupiter

Part No

00F-4263-B0

Phase Name

C18

Elution Type

Gradient

Mobile Phase

A: 0.1% TFA and 2% Acetonitrile in Water

B: 0.085% TFA, 90% Acetonitrile in Water

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

80

20

2

10

20

80

3

15

10

90

Flow Rate

0.30 mL/min

Temperature

25°C

Detection

UV-Vis Abs.-Diode Array (PDA) @ (25°C)

Detection Info

Sample Notes

Application Focus: To demonstrate Jupiter 300 utility for separating folded from unfolded biogeneric proteins Unlike other separation techniques, reversed phase can often visualize differences between intact and unfolded/ mis-folded protein states. Especially with E.Coli produced recombinant proteins, refolding analysis is often required as part of both manufacturing process analysis technology as well as quality control testing of a finished product. In this example Interferon alpha was exposed to chemical unfolding conditions at higher temperature in the presence of DTT (3 mM dithiothreitol (DTT) at 55°C for 30 min) and the chromatographic behavior of the unfolded protein was compared to the intact protein by analysis on the Jupiter 300 3u C18 and Jupiter 300 5u C4 columns. In App ID# 18073 5% of unfolded interferon was added to the intact protein. As one can see from the overlaid chromatograms, both columns could easily detect unfolded impurities lower than 5% with good, rapid resolution from the intact protein in fewer than 15 minutes. This application demonstrates the utility of Jupiter 300 media in doing refold analysis.