PEGylated proteins: Optimizing conjugation reactions using triple detection SEC
In this application note, one protein, interferon α-5, was conjugated with two different PEG molecules in different reactions. The protein and PEG molecules were characterized individually using multi-detector SEC, followed by the PEGylated conjugates. Their composition, molecular weights and intrinsic viscosities were measured. This work was performed by Celares GMBH, Germany with samples provided by DIGNA BIOTECH, Spain
PEGylated proteins: Optimizing conjugation reactions using triple detection SEC
In this application note, a single protein, interferon α-5, was conjugated with two different PEG molecules in different reactions. The protein and PEG molecules were characterized individually using multi-detector SEC, followed by the PEGylated conjugates. Composition, molecular weights and intrinsic viscosities were measured. The work was performed by Celares GMBH, Germany, with samples provided by DIGNA BIOTECH, Spain.
Introduction
The last few years have seen a high level of growth in the biopharmaceutical market, using biological molecules as drugs. As this market has grown, so has the demand for higher levels of characterization of these molecules.
One possible modification of protein drugs, or biologics, is their conjugation with a polyethylene glycol (PEG) molecule. So called PEGylation is known to improve half-lives leading to greater efficacy and reduced dosing frequency.
Size-exclusion chromatography (SEC), also known as gel-permeation chromatography (GPC), is regularly used to characterize proteins in solution, for instance to study their molecular weight. In particular, light scattering is used to measure absolute molecular weight independent of column retention volume. This is particularly useful for conjugates that elute at radically different elution volumes than might be expected based simply on the protein's molecular weight. Furthermore, multi-detector SEC can also be used to characterize the composition of a conjugated molecule. This therefore allows the user to calculate the mass of each of the protein and PEG components. In this way, the PEGylated protein molecule can be completely characterized.
In this application note, one protein, interferon α-5, was conjugated with two different PEG molecules in different reactions. The protein and PEG molecules were characterized individually using multi-detector SEC, followed by the PEGylated conjugates. Their composition, molecular weights and intrinsic viscosities were measured.
