Implementation of bioanalytical methods to quantify anti-VEGF-LAD (long acting delivery) Fabs in ocular matrices

Abstract

Purpose : Binding to hyaluronic acid is an attractive strategy to increase the residence time of anti-VEGF therapeutics in ocular compartments and reduce frequency of injections in AMD patients. Common bioanalytical methods failed to accurately determine the concentrations of certain hyaluronic acid (HA) bound anti-VEGF molecules (TSG6-Fab) in ocular matrices, making the determination of PK parameters challenging. Here, we compare generic Fab ELISA and quantitative mass spectrometry assays to determine which method is suitable for accurate measurements of HA bound anti-VEGF Fabs in vitreous humor compartment.

Methods : An anti-VEGF Fab was fused to one or two TSG6 modules (1X or 2X TSG6) and spiked into rabbit vitreous at known concentrations. Samples were analyzed using an optimized generic Fab ELISA or two newly implemented quantitative LC-MS/MS assays that use MRM method for quantification. The two LC-MS/MS assays differ from one another by the absence or presence of an affinity capture step and the use of different tryptic signature peptides as surrogates for quantification. After validation of these three assays in vitro, an in vivo study including 30 New Zealand White (NZW) rabbits receiving intravitreal injection of 0.3mg or 0.06mg/eye of control, 1X or 2X TSG6 anti-VEGF Fab was performed and pharmacokinetic profiles were determined.

Results : After extensive optimization, the generic Fab ELISA successfully measured both anti-VEGF LAD Fabs concentrations in rabbits vitreous in spiking experiments (100% recovery ± 20%). However, both ELISA and affinity capture LC/MS assay show under-recovery for the 2X TSG6 anti-VEGF Fab in vitreous collected from the NZW rabbits dosed in the vitreous chamber (10.1µg/ml and 13.7µg/ml, respectively; expected Cmax~42 µg/ml). The second LC-MS/MS assay without affinity capture step accurately measured 2X TSG6 Fab in vitreous (46µg/ml) and was used to assess pharmacokinetics.

Conclusions : The interaction of anti-VEGF LAD Fabs with ocular matrices is problematic for quantification as it prevents recognition by detection antibody. A comparative study revealed that a newly implemented LC-MS/MS assay omitting the affinity capture step accurately measured 2X anti-VEGF TSG6 Fab concentrations in vitreous and can be used for pharmacokinetic assessment. Other long-acting delivery platforms can benefit from this assay.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.