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• 18 •
2008 vol. 2
Bioanalytical
PEGylation, the covalent attachment of polyethylene glycol (PEG) units to proteins and peptides, is an important tool in drug dis-
covery. PEGylation is used to enhance drug delivery, while maintaining the therapeutic function of the active compound. Viva Wide
Pore HPLC columns are ideal for the separation of large molecules, such as oxytocin
PEGylation reaction products, as the target analytes can enter the larger pores and access
more surface area, increasing retention and overall resolution. For analytes with molec-
ular weights larger than 3,000, pore diameters of 250-350Å offer the best combination
of retention and pressure stability, and Viva Wide Pore silica has the greatest available
surface area in 250-350Å pores. Here we demonstrate the suitability of Viva Wide Pore
HPLC columns for PEGylation reaction monitoring.
The PEGylation reaction mixture consisted of oxytocin with an excess of reducing agent tris(2-carboxyethyl)phosphine (TCEP) and
(methyl-PEO
12
)
3
-PEO
4
-maleimide. The oxytocin solution was mixed with ammonium bicarbonate buffer to pH 8. Excess TCEP was
added and the resulting solution incubated at 60°C for 1 hour. The test solution was cooled to room temperature and a molar excess
of (methyl-PEO
12
)
3
-PEO
4
-maleimide was added, followed by incubation in a water bath at 40°C for 1 hour. Approximately 6 nmoles
of oxytocin was injected in 20µL of deionized water with 0.1% formic acid. The extracted ion chromatograms in Figure 1 show excel-
lent resolution for the three compounds of interest. The added retention power of Viva columns allows separation of large, closely
related compounds, making it an ideal column for monitoring PEGylation reactions.
Easily Resolve Oxytocin PEGylation Reaction Products
Using Viva Wide Pore HPLC Columns
Julie Kowalski, Ph.D., Bioanalytical Innovations Chemist
• Ideal for PEGylation reaction monitoring.
• Easy isocratic method saves time, eliminating column equilibration time between injections.
• Largest available surface area in 250-350Å pores; engineered for proteins, peptides, and other large biomolecules.
Figure 1
Excellent resolution of oxytocin PEGylation reaction products on Viva Wide Pore HPLC columns.
Sample:
oxytocin PEGylation
reaction products
Inj.:
20µL
Conc.:
300pmoles/µL
Sample diluent: 0.1% formic acid in
water (v:v)
Column:
Viva Wide Pore C18
Cat.#:
9514561
Dimensions:
150mm x 1.0mm
Particle size: 5µm
Pore size:
300Å
LC_PH0467
Conditions:
Mobile phase: 0.1% formic acid in
water:0.1%formic acid in
acetonitrile, 60:40
Flow:
0.1mL/min.
Temp.:
ambient
Det.:
Micromass Quattro II
Interface:
ESI
Ion mode:
positive
Temp.:
200°C
Capillary:
2.25kV
Cone:
40V
Viva C18 Columns (USP L1)
5µm Column, 1.0mm
cat. #
price
150mm
9514561
ordering
note
For other dimensions and guard cartridges for these columns,
visit our website at
www.restek.com.
Viva columns reliably
separate large, closely
related compounds.