Predictive In Vitro Vitreous and Serum Models and Methods to Assess Thiol-Related Quality Attributes in Protein Therapeutics

Abstract

In vitro models that mimic the in vivo environment can greatly facilitate and support criticality assessment of product quality attributes for therapeutic drugs to ensure product quality. An in vitro model is established to study and predict the impact of thiol-related attributes on safety or efficacy of intraocular antibody products. This model simulates the physiological redox environment of rabbit vitreous and maintains a steady-state redox potential using reduced and oxidized forms of glutathione. A similar in vitro model that mimics the thiol redox conditions of human blood has been previously established and has become a predictive tool to study intravenous (IV) therapeutic proteins. We utilized both vitreous and serum models to study the potential impact of antibody variants (trisulfides and free-thiols) on product qualities of different antibodies. The studies demonstrate that both models are effective tools to monitor changes of thiol-related attributes under physiological conditions, providing insights on these thiol-related attributes and allowing for more informed assessment of biological relevance and criticality of the attributes. Furthermore, we propose that the approach using an in vitro study for the product quality attribute assessment can be used to predict in vivo effects for future molecules during the development of biopharmaceuticals, reducing the need for live subject studies.

Expanding the Analytical Toolbox: Developing New Lys-C Peptide Mapping Methods with Minimized Assay-Induced Artifacts to Fully Characterize Antibodies

“…Between them, Dilution Scheme 1, with a 3-fold dilution (before digestion), performed well, while the digestion efficiency was decreased when a 9-fold dilution was used (Dilution Scheme 2). After the dilution, the Lys-C digestion was carried out at pH 7 to minimize artificial deamidation occurring during protein digestion [ 38 ]. Lys-C enzyme to protein ratios (enzyme: protein w / w ) of 1:20 and 1:10 and digestion times were evaluated.…”

Stability of monoclonal antibodies after simulated subcutaneous administration

“…Different in vitro models developed elsewhere, used static incubation 8,9,[35][36][37] or a dialysis setup. 7,10,11,13,27,38 Dialysis simulates a slow influx of the SC interstitial fluid 13,27 and allows to diffuse small molecules (excipients) across the membrane, whilst retaining the therapeutic protein locally. Such events have been argued to occur after SC administration.…”

“…Thus, a number of groups developed in vitro models simulating human in vivo conditions. For example using the desired biological fluid such as serum/plasma 7−9 (related to intravenous administration), vitreous humor 7,10,11 (related to intravitreal injections), artificial SC fluid, 12−15 or SC tissue homogenate 16 (related to SC administration). Simulating the SC tissue is particularly challenging as the SC interstitial fluid is not readily accessible.…”