Monolithic Papain-Immobilized Enzyme Reactors for Automated Structural Characterization of Monoclonal Antibodies

Abstract

The characterization of monoclonal antibodies (mAbs) requires laborious and time-consuming sample preparation steps before the liquid chromatography–mass spectrometry (LC-MS) analysis. Middle-up approaches entailing the use of specific proteases (papain, IdeS, etc.) emerged as practical and informative methods for mAb characterization. This work reports the development of immobilized enzyme reactors (IMERs) based on papain able to support mAb analytical characterization. Two monolithic IMERs were prepared by the covalent immobilization of papain on different supports, both functionalized via epoxy groups: a Chromolith® WP 300 Epoxy silica column from Merck KGaA and a polymerized high internal phase emulsion (polyHIPE) material synthesized by our research group. The two bioreactors were included in an in-flow system and characterized in terms of immobilization yield, kinetics, activity, and stability using Nα-benzoyl-L-arginine ethyl ester (BAEE) as a standard substrate. Moreover, the two bioreactors were tested toward a standard mAb, namely, rituximab (RTX). An on-line platform for mAb sample preparation and analysis with minimal operator manipulation was developed with both IMERs, allowing to reduce enzyme consumption and to improve repeatability compared to in-batch reactions. The site-specificity of papain was maintained after its immobilization on silica and polyHIPE monolithic supports, and the two IMERs were successfully applied to RTX digestion for its structural characterization by LC-MS. The main pros and cons of the two supports for the present application were described.

Synthesis of Hydrophilic Polymeric Supports with Poly-HIPE Structures Based on Acrylates for Pharmaceutical Applications

“…In contrast to the lipophilic immobilized enzyme reactor (IMER) investigated by Rinaldi et al where a remarkable limitation of the IMER activity was assessed due to RTX absorption within the polymeric matrix, we herein demonstrated the quantitative recovery of the antibody.…”

“…This comparative experiment was aimed to prove that the polymeric composition can give rise to a monolithic material with enhanced hydrophilic properties with respect to that previously described . In fact, the previously described lipophilic poly-HIPEs failed in biocatalysis studies for the irreversible adsorption of the most hydrophobic drugs and Rituximab (data not shown).…”

“…In this context, previous investigations reported by our research group focused on the synthesis and characterization of lipophilic acrylate-based poly-HIPE supports for biomedical applications and enzymatic immobilization have been reported in literature …”

“…For this purpose, a papain-immobilized enzyme reactor based on a polyacrylate lipophilic poly-HIPE was prepared and used for Rituximab (RTX) digestion and online structural characterization by LC-MS. However, a significant adsorption of RTX on the hydrophobic poly-HIPE material was observed, suggesting the inclination of the hydrophobic porous material to irreversibly retain large proteins as antibodies in aqueous media …”

“…In this context, previous investigations reported by our research group focused on the synthesis and characterization of lipophilic acrylate-based poly-HIPE supports for biomedical applications and enzymatic immobilization have been reported in literature. 14 In detail, an online platform for therapeutic monoclonal antibody structural characterization was developed. For this purpose, a papain-immobilized enzyme reactor based on a polyacrylate lipophilic poly-HIPE was prepared and used for Rituximab (RTX) digestion and online structural characterization by LC-MS.…”