Comprehensive biophysical and functional study of ziv-aflibercept: characterization and forced degradation

Jesús Hermosilla,Raquel Pérez-Robles,Jonathan Bones,Salvador Casares,Natalia Navas,Jose Cabeza,Antonio Salmerón-García

doi:10.1038/s41598-020-59465-7

Abstract

Aflibercept (AFL) is an Fc fusion protein used in the treatment of colorectal cancers and different ophthalmological diseases. There are two medicines in which AFL is the active substance: Zaltrap and Eylea, referred as ziv-AFL and AFL respectively. No proper accelerated degradation studies were published on either AFL or ziv-AFL. These studies are essential during research, development and manufacturing stages. Here, we characterized ziv-AFL and submitted it to different stress conditions: light, 60 °C, freeze-thaw cycles, changes in pH, high hypertonic solution and strong denaturing conditions. We used an array of techniques to detect aggregation (SE-HPLC/DAD and DLS), changes in secondary structure (Far-UV circular dichroism), changes in conformation or tertiary structure (Intrinsic tryptophan fluorescence) and alterations in functionality (ELISA). Results indicate that aggregation is common degradation pathway. Two different types of aggregates were detected: dimers and high molecular weight aggregates attributed to β-amyloid-like structures. Secondary structure was maintained in most of the stress tests, while conformation was altered by almost all the tests except for the freeze-thaw cycles. Functionality, evaluated by its immunochemical reaction with VEGF, was found to be stable but with decrease when exposed to light and with likely partial inactivation of the drug when pH was altered.

Highlights

Aflibercept (AFL) is an Fc fusion protein used in the treatment of colorectal cancers and different ophthalmological diseases
A thermal stability study was performed by Circular Dichroism from 20 to 90 °C
The biological activity of the protein - measured by means of the ziv-AFL binding capacity to VEGFR- in solution was maintained at 60 °C, despite the appearance of HMW soluble aggregates, they are likely to impair the safety and the efficacy of the medicine

Summary

Introduction

Aflibercept (AFL) is an Fc fusion protein used in the treatment of colorectal cancers and different ophthalmological diseases. The top-selling biotherapeutics are monoclonal antibodies (mAbs), there is increasing use of therapeutic Fc-fusion proteins in medicine today[1] One of these is aflibercept (AFL), a recombinant human Fc-fusion protein that –as indicated in the assessment report of Zaltrap of the European Medicine Agency2– “acts as a high-affinity soluble decoy receptor that preferentially binds to vascular endothelial growth factor A (VEGF-A), VEGF-B and placenta growth factor (PlGF) and prevents these factors from activating their endogenous receptors. To the best of our knowledge, no forced degradation studies of ziv-AFL (Zaltrap) have been described to date These studies are essential in that they are an integral part of biotherapeutics research and development and serve a variety of objectives ranging from early stage manufacturability evaluation to supporting comparability assessments both prior to and after approval for sale[11]. These researchers are normally from academic institutions and biopharmaceutical companies

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