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.
Highlights
Biopharmaceutical market is rapidly growing, with monoclonal antibodies representing the most widespread products
Since the aim of the work is the application of papain-immobilized enzyme reactors (IMERs) to antibody digestions, reaction conditions were studied on a commercial monoclonal antibodies (mAbs), namely, RTX
The presence of ethylenediaminetetraacetic acid (EDTA) and cysteine in the buffer was found to be essential for papain activity since their removal from the reaction buffer resulted in a complete suppression of the digestion
Summary
Biopharmaceutical market is rapidly growing, with monoclonal antibodies representing the most widespread products. Their therapeutic indications include a large variety of diseases, such as cancer, inflammation, diabetes, cardiovascular and genetic disorders, autoimmune diseases, and infections. From small molecules, mAb drugs present heterogeneous and complex structures, and their production and characterization require the development of challenging and long processes (Sandra et al, 2014). The complexity of these macromolecules implies the investigation of several critical quality attributes (CQAs) with the consequent application of appropriate methods for their. Papain-IMERs for mAb Characterization analytical control at intact, subunit, peptide, amino acid, and glycan levels (Fekete et al, 2013; Sandra et al, 2014)
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