Jupiter® 300 C4 - PEGylation Reaction Time Course (Carbonic Anhydrase)

LC Conditions

Column

Jupiter 5 µm C4 300 Å, LC Column 150 x 4.6 mm, Ea

Brand

Jupiter

Part No

00F-4167-E0

Phase Name

C4

Elution Type

Gradient

Mobile Phase

A: 0.1% TFA in Water

B: 90% ACN 0.09% TFA in Water

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

80

20

2

25

45

55

Flow Rate

1.00 mL/min

Temperature

45°C

Detection

UV-Vis Abs.-Variable Wave.(UV) @ (22°C)

Detection Info

Sample Notes

Application Focus: To demonstrate the utility of using reversed phase chromatography with Jupiter 300 media for purifying PEGylated proteins. The addition of PEG groups to a protein complicates both the characterization and purification of such PEG/protein conjugates away from the "non-PEGylated" protein species. Protein PEGylation was performed using Methyl-PEO12-NHS Ester. A protein solution in PBS (pH 7.4) was reacted with an excess of PEGylation reagent dissolved in DMSO and incubated at 4C for up to two hours. The reaction was quenched with 50 mM Tris/1 % trifluoroacetic acid. In general, the PEGylation reaction concurrently occurs rapidly at several different protein sites in a fixed ratio. As the reaction continues, more heavily PEGylated (and later eluting) forms were observed. In every protein tested there was always more than one PEGylated protein peak observed by reversed phase HPLC; each seemingly ascribed to a different site of PEG modification. Unlike gel filtration which only separates PEGylated proteins by degree of modification, it appears that protein modification site influences the reversed phase separation of proteins much more than the degree of modification; such results show the utility of using reversed phase chromatography for purifying and analyzing PEGylated proteins.