Microvolume Analysis of Aflibercept in Aqueous Humor Using Mass Spectrometry

Purpose: To develop a microvolume analytical method for measurement of the aflibercept concentration in human intraocular fluid and plasma.

Methods: We analyzed trace amounts of aflibercept in human aqueous humor using Fab-selective proteolysis and nano-surface and molecular-orientation limited (nSMOL) proteolysis, coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Patients with age-related macular degeneration or diabetic macular edema were recruited. Just after an injection of 50 µL of aflibercept, regurgitate from needle holes was collected with a micropipette pressed to the side of the injection hole within 10 seconds. The median amount of regurgitate was 4 µL (range, 1-18 µL).

Results: In human plasma, the aflibercept concentration ranged between 0.195 and 50 µg/mL when using the quantitative signature peptide IIWDSR (aa. 56-61) present on the vascular endothelial growth factor receptor 1 domain of aflibercept. The method was validated by evaluating its linearity, carryover, selectivity, accuracy and precision, dilution effect, and sample/processing stability. As only a minimal amount of regurgitate through needle holes can be sampled, we performed and verified the aflibercept assay using patient samples after 1:10 dilution with control human plasma, a recognized diluent. The median concentration of aflibercept in the regurgitate was 240 µg/mL (range, 13-4300 µg/mL).

Conclusions: Our findings indicate that the aflibercept assay using human intraocular fluid can be reliably performed using nSMOL coupled with LC-MS/MS.

Translational Relevance: This technique for quantifying aflibercept in the regurgitate suggests that the amount of drug lost post-injection can be ignored, even in patients with a relatively large leak after vitreous injection. This new methodology suggests possible therapeutic responses and may be employed as a general analytical method for trapping many biologics, such as vascular endothelial growth factor, in various types of clinical samples, unaffected by proteinaceous or small organic pharmaceuticals.