This application provides a fast and accurate way for biopharmaceutical chemists to monitor PEGylation reactions by using HPLC with complementary UV/Vis and charged aerosol detection.Separation on the MAbPac RP resolves PEGylation reagents, unreacted IgG, and PEGylated IgG products that elute as a broad hump.

The Thermo Scientific Dionex UltiMate 3000 system is applied for the determination of cyclophosphamide in formulation. The high resolution separation of cyclophosphamide from PEG was performed on a Thermo Scientific Syncronis C18 HPLC column, with a run time of 20 minutes.

In this work, polyethylene glycol mono-methyl ether has been labeled with CY5. The Thermo Scientific Acclaim SEC-300 column separates the labelled PEG from the reaction byproducts using size exclusion, but does not resolve labeled from unlabeled PEG. The CY5-labeled PEG is detected by both the FLD and UV; in contrast the CAD detects the labeled and unlabeled PEG equally. Both the UV and CAD detect some of the synthetic by-products. Using multiple detectors allows the reaction yield to be calculated.

The Thermo Scientific HPLC system is applied for the analysis of polyethylene glycols (PEGs) by size exclusion chromatography. The separation is performed on a Thermo Scientific Acclaim SEC-300 HPLC column, with RI detection. The separation achieved is compared to the separation obtained on a Thermo Scientific Acclaim SEC-1000 HPLC column. Both give excellent performance for measuring molecular weight and size distribution for this class of polymers.

The Thermo Scientific Acclaim Surfactant Plus HPLC column provides excellent resolution for the low molecular weight oligomers based on the degree of ethoxylation. The optimized pore size allows it to resolve polymers up to n = 30.