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Figure 1. 150K Dalton protein before PEGylation. Notice theconstant molar mass across the entire peak.
Figure 2. The same protein that has been PEGylated. Notice thedramatic change in molar mass across the peak, depending on the
number of PEG attachments.
Protein PEGylation
he rise of biotechnology has spawned di�er-ent means of delivering drugs to the humanbody. Among the many problems encountered
is the rapid rejection or clearance of a protein before ithas accomplished its task.
A variety of methods exist for modifying pro-teins so that they are more e�ective in the bloodstream.Among them, glycosylation and PEGylation are per-haps the most popular. In the former, sugars are at-tached to the protein, in the latter, polyethylene gly-col strands are attached as “whiskers” to increase thehalf-life of the protein and make it less likely to behydrolized or cleared from the bloodstr eam.
In this application note, a monoclonal antibodywas measured before and after PEGylation. As illus-trated in Fig. 1, the antibody has a constant molar massof 150K Daltons across the entire peak.
Figure 2 shows what happens when the sameantibody has been conjugated with PEG molecules of5K Daltons each. Using a self-consistent, iterativemethod, the number of PEGs per protein was calcu-lated, and can be seen on the graph itself.
The chromatography set-up consisted of aminiDAWN multi-angle light scattering detector, Op-tilab DSP interferometric refractometer, HP 1050 HPLCsystem, and a Pharmacia Superose 12 30/10HRcolumn.
The results are, perhaps, most remarkable fortheir speci�city. Never before has it been so easy todetermine the number of PEG attachments to a pro-tein. Now, using the miniDAWN it has become astraightforward experimental procedure.
~52 PEG/protein
~38 PEG/protein
4-5 PEG/protein
0 PEG/protein
Optilab DSPsignal
Optilab DSPsignal
Mw = 150,000 g/mol
M.W. vs. Volume
Elution volume (mL)
Mw (
g/m
ol)
M.W. vs. Volume
Elution volume (mL)
Mw (
g/m
ol)
Protein SolutionsTM
