Development of a fit-for-purpose semi-homogeneous competitive ELISA for total PEG quantitation in a PEGylated molecule.

AIM: The therapeutic effectiveness of a drug could be significantly enhanced by prolonging its retention time. Polyethylene glycosylation (PEGylation) technology is a method to increase the half-life of protein therapeutics. PEGylated anti-Factor D Fab (PEG-aFD) was developed as a potential therapeutic, targeting factor D to reduce the frequency of intravitreal injections in patients with geographic atrophy. To support a GLP Toxicology study, there was a need to develop a PEG assay to measure total PEG concentration.

RESULT: PEG-aFD consists of Fabs conjugated to a multi-arm PEG molecule, which posed challenges for PEG assay development. A sensitive fit-for-purpose, semi-homogeneous competitive ELISA (Enzyme-Linked Immunosorbent Assay) using a biotinylated version of the therapeutic as the competing molecule was successfully developed and validated with LLOQ (lower limit of quantification) at 200 ng/mL. The assay was used to measure total PEG concentrations in cynomolgus monkey serum samples from the study. The results revealed measurable PEG levels (ranging from 0.468 μg/mL to 188 µg/mL) in all samples across all dosage levels, but no PEG-related toxicity was observed in all the animals.

CONCLUSION: A competitive ELISA was successfully developed and validated to measure total PEG concentrations in cynomolgus monkey serum samples to support a GLP Toxicology study.