Background: Polyethylene glycolylation (PEGylation) technology is a long-acting delivery platform used to increase the half-life of protein therapeutics. Quantitation of PEGylated anti-Factor D Fab (PEG-aFD) poses bioanalytical challenges. Results: An ELISA was developed to determine total Fab concentration in cynomolgus monkey serum following intravitreal administration of PEG-aFD. However, assay characterization showed a low recovery of about 25% for free unconjugated Fab whereas recovery for PEG-conjugated Fab was within 80–120%. To overcome this challenge, an immunoaffinity liquid chromatography tandem mass spectrometry (IA LC–MS/MS) assay was developed, achieving recovery within 80–120% for both free and conjugated Fab. Conclusion: Immunoaffinity LC–MS/MS is more suitable than ELISA to accurately quantify the total protein concentration of PEG-aFD in cynomolgus monkey serum.
Keywords:
- ELISA
- IA LC–MS/MS
- immunoaffinity LC–MS/MS
- intravitreal
- ITV
- LAD
- long acting delivery
- PEGylation