Interferon Alpha intact & oxidized 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: Using Jupiter 300 media for development of intact biogeneric protein assays for oxidation. Physical and chemical degradation of therapeutic proteins is a critical problem that can occur during production, purification, and storage. Such modifications can affect protein immunogenicity leading to serious consequences if the protein is being used as a therapeutic. Chromatograms comparing native to oxidized alpha interferon were overlaid to visualize chromatographic differences; oxidation of intact Interferon-alpha was performed by treating with 3% hydrogen peroxide and 50 mM sodium citrate pH 4.4 at 45°C for 120 min. Overlaid chromatographs of the intact versus oxidized alpha interferon clearly show good selectivity between the two samples; oxidized interferon elutes earlier than the intact protein and has a dramatically tailing peak. While both the C4 and C18 phases both had good resolution, the 3µm C18 clearly showed better performance than all other phases tested. These results indicate that the Jupiter media is an excellent solution with the required selectivity for quantitating very low levels of oxidized protein in less than 15 minutes.