PEGylated alpha-Chymotrypsinogen A on Jupiter® 300 C18 3µm, reaction at different time-points

LC Conditions

Column

Jupiter 3 µm C18 300 Å, LC Column 150 x 4.6 mm, Ea

Brand

Jupiter

Part No

00F-4263-E0

Phase Name

C18

Elution Type

Gradient

Mobile Phase

A: 0.1% TFA, 2% ACN in Water

B: 90% ACN, 0.085% TFA in Water

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

80

20

2

25

35

65

Flow Rate

1.00 mL/min

Temperature

45°C

Detection

UV-Vis Abs.-Variable Wave.(UV) @ (25°C)

Detection Info

Sample Notes

Application Focus: Using larger (20KDa) PEGylation reagents and seeing better performance with Jupiter 300 3u C18. In other studies proteins were modified with relatively small polyethylene glycol moieties (PEG) which may not be applicable to all pharmaceutical applications. In this application chymotrypsinogen was PEGylated using two different 20 KDa PEGylation reagents that are commonly used in pharmaceutical drug development. Chymotrypsinogen was reacted with methoxy PEG suiccinimidyl carboxy methyl ester, 20 KDa (M-SCM-20K) or methoxy PEG propionaldehyde, 20KDa (M-ALD-20K). For the M-ALD-20K PEG, protein and PEG were dissolved in phosphate buffer pH 6.5 with 20 mM of sodium cyanoborohydrate. For the M-SCM-20K PEG, protein was dissolved in phosphate buffer pH 7.8 and M-SCM-20K was dissolved in DMSO then mixed and incubated at 4C. Reaction mixtures were quenched with 50 mM Tris/1 % TFA. Timepoints from each reaction were overlaid. Unlike GFC which can only separate PEGylated proteins based on degrees of polymerization, RP chromatography can also separate PEGylated species based on site of PEG attachment. Time course results for App ID 17514 shows multiple PEG/protein conjugates with unique retention by RP-HPLC dictated by both the site and degree of modification. Although other Jupiter phases have shown better results with small PEG modifiers, this study shows the advantages for separation of large PEGylated conjugates and un-reacted molecules using the Jupiter 300 3 um C18 media.