Rinaldi Francesca, Tengattini Sara, Brusotti Gloria, Tripodo Giuseppe, Peters Benjamin, Temporini Caterina, Massolini Gabriella, Calleri Enrica
Department of Drug Sciences, University of Pavia, Pavia, Italy.
Instrumental Analytics R&D, Merck KGaA, Darmstadt, Germany.
Front Mol Biosci. 2021 Nov 9;8:765683. doi: 10.3389/fmolb.2021.765683. eCollection 2021.
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.
单克隆抗体（mAb）的表征在液相色谱 - 质谱（LC - MS）分析之前需要繁琐且耗时的样品制备步骤。采用特定蛋白酶（木瓜蛋白酶、IdeS等）的中观方法成为用于mAb表征的实用且信息丰富的方法。本工作报道了基于木瓜蛋白酶的固定化酶反应器（IMER）的开发，其能够支持mAb的分析表征。通过将木瓜蛋白酶共价固定在不同载体上制备了两种整体式IMER，这两种载体均通过环氧基团功能化：一种是默克集团的Chromolith® WP 300环氧硅胶柱，另一种是由我们研究小组合成的聚合高内相乳液（polyHIPE）材料。将这两种生物反应器纳入流动系统，并以Nα - 苯甲酰 - L - 精氨酸乙酯（BAEE）作为标准底物，对其固定化产率、动力学、活性和稳定性进行了表征。此外，还对这两种生物反应器针对一种标准mAb，即利妥昔单抗（RTX）进行了测试。利用这两种IMER开发了一个用于mAb样品制备和分析的在线平台，该平台只需最少的操作人员操作，与分批反应相比，可减少酶的消耗并提高重复性。木瓜蛋白酶固定在硅胶和polyHIPE整体载体上后仍保持位点特异性，并且这两种IMER已成功应用于RTX的酶解，以便通过LC - MS对其进行结构表征。描述了这两种载体在本应用中的主要优缺点。
