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Lisa D. Orecchio, Michael Rivera, Edward Brissette, Cahil McGovern, Arne Nystuen, Rhett Schiffman, Konrad Kauper; Proteomic Mass Spectroscopy Profiling of Proteins Secreted from an Encapsulated Cell Technology (ECT) Implant Device. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):229.
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© ARVO (1962-2015); The Authors (2016-present)
Non-clinical and clinical studies have demonstrated the preliminary safety and efficacy of a novel implant device (NT-503-3) encapsulating a retinal pigment epithelial cell line (NTC-203-910) engineered to continuously produce a VEGF inhibitor (910-VEGFR). Analytical techniques, including proteomic mass spectroscopy, were used in the study to characterize both the 910-VEGFR protein in addition to naturally secreted NTC-203-910 proteins.
NT503-3 devices were pulsed in culture media for 24 hours. Device conditioned media (CM) was analyzed by ELISA, SDS PAGE and western blot. A buffer exchange and trypsin digest of CM, followed by nano-LS/MS/ mass spec was used to identify the most abundant proteins secreted from the encapsulated cell line. Relative quantification was performed by normalizing data for molecular weight and calculating relative percentage of total protein counts (NSAF). A coefficient of determination (R2) comparing the profile of secreted proteins was calculated for 15 manufacturing devices of NT-503-3 product.
VEGF direct binding ELISA determination of 910-VEGFR was within the specification range of manufacturing product stability. A prominent, visible band at 147 kDa was observed on non-reduced SDS PAGE gels. Western blots using anti-hVEGFR and anti-hIgG1(Fc) antibodies identified this band as intact 910-VEGFR. Proteomic mass spectroscopy identified over 300 proteins accounting for 96% of the total proteins detected. Preliminary analysis of the detected proteins secreted from 15 devices from 3 lots of NT-503-3 product appears to be consistent with normal retinal pigment epithelial cell line metabolic, development and organizational function. The coefficient of determination for the secreted proteins was always greater than 90 percent suggesting a consistency in the quality and quantity of the protein profile from NT503-3 device.
A single, intraocular ECT implant delivering 910-VEGFR was designed to provide comparable anti-VEGF therapy to standard-of-care treatments while eliminating the burden of frequent injections in patients with neovascular AMD. Proteome profiling of the most abundant proteins produced by NT-503-3, in addition to 910-VEGFR, identified low-level, stable secretion of naturally occurring proteins normal to human retinal pigment epithelial function.
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