SEC
18
PEGYLATED PROTEINS
Chemical modification of therapeutic proteins is of increasing interest.
One of the most frequently used protein modification methods,
PEGylation, changes the biochemical and physicochemical properties
of the protein, which can result in several important benefits, among
them more effective target delivery, slower in vivo clearance, and
reduced toxicity and immunogenicity of therapeutic proteins. After
PEGylation reaction the mixture has to be purified in order to remove
non-reacted protein and undesired reaction products. A TSKgel
G3000SW
XL
column was used for the characterization of PEGylated
lysozyme, as shown in
FIGURE 13
. A random PEGylation of lysozyme
using methoxy PEG aldehyde of sizes 5 kDa, 10 kDa and 30 kDa was
performed. The retention volumes of PEGylated lysozymes were used
to assign the peaks based on a standard calibration curve. As a result
of PEGylation, a large increase in the size of lysozyme by size exclusion
chromatography was observed. The SEC elution position of lysozyme
modified with a 30 kDa PEG was equivalent to that of a 450 kDa globular
protein. There was a linear correlation between the theoretical molar
mass of PEGylated protein and the molar mass calculated from
SEC. This result illustrates the strong effect that PEG has on the
hydrodynamic radius of the resulting PEGylated protein.
figure 13
SEC analysis of PEGylation reaction mixtures
0
2
4
6
8
10
Retention time (minutes)
12
14
0
20
40
60
80
Detector response (mAU)
PEG5Lys
PEG10Lys
PEG30Lys
Column: TSKgel G3000SW
XL
, 5 μm, 7.8 mm ID × 30 cm
Mobile phase: 0.1 mol/L phosphate buffer, 0.1 mol/L Na
2
SO
4
, pH 6.7
Flow rate: 1.0 mL/min; Detection: UV @ 280 nm; Injection vol.: 20 μL
Sample: 5, 10, 30 kDa methoxy PEG aldehyde
