Multi-arm Polyethylene glycol (PEG) molecules modified with functional groups and different molecular sizes are widely used in biomedical development. For instance, PEG-amine and PEG-maleimide are most useful as intermediates in PEGylation, bioconjugation and ADC drug development for pharmaceutical and biotech research and development. Developing a highly selective method to analyze purity and monitor degradation impurities of PEG intermediate is essential to ensure quality of these critical intermediates of the conjugation process. However, PEG polymers are challenging molecules for analysis due to their complexity. First, the heterogeneity of the PEG polymers causes a wide distribution profile of PEG species overlapped with different sized polymer distribution. Second, the mass difference between intermediate and degradant is almost negligible and therefore small modifications and degradants are hard to discriminate by common analytical methods. Third, the multiplicity of functional groups on these functionalized PEG chains leads to a complex mixture of degradants. Therefore, conventional one-dimensional liquid chromatography cannot separate all of these species with different properties. In this study, we developed a highly selective two-dimensional liquid chromatography (2D-LC) to overcome the method development challenges by using orthogonal methods in each dimension. A size exclusion chromatography (SEC) method using a UPLC protein BEH SEC column was developed to resolve the complex mixture of PEG polymer with different molecular weights. A reversed-phase LC was developed for the second dimension to resolve different functionalized PEG intermediates and related impurities. A reversed-phase HPLC method with stationary phase of charged surface hybrid (CSH) was developed for second-dimensional LC. CSH stationary phase has unique properties to separate various functional groups and degradants. Based on our results, 2D-LC of SEC coupled with reversed-phase LC, has proven to be effective and efficient in analyzing a high molecular weight PEG conjugation intermediate during its synthetic process.

Development of a two-dimensional liquid chromatography method for functionalized high molecular weight polyethylene polymer analysis

256th National Meeting (2018)

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