Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Summary

Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

Abstract

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Introduction

Monoclonal antibodies (mAbs) are biotherapeutic proteins with increasing interest due to their capacity to act against several chronic and degenerative diseases¹. Like other biomolecules, mAbs are prone to undergo several physicochemical modifications at all stages of their life cycle (i.e., from biosynthesis to the final product). Such modifications include, but are not limited to: deamidation, glycosylation, oxidation, cyclization, isomerization, aggregation and proteolytic cleavage². Hence, analytical techniques capable to resolve intrinsic isoforms are needed to monitor mAbs heterogeneity and stability in order to establish quality s....

Protocol

1. Preparation of Solutions

1. Prepare the BGE solution.
   1. Prepare 100 ml of a solution composed of 0.05% (m/v) hydroxy propyl methyl cellulose (HPMC), 200 mM ε-amino n-caproic acid (EACA) and 30 mM lithium acetate.
      NOTE: As HPMC is a viscoelastic polymer, pour the powder into a glass beaker, add 80 mL of water and finally add the stirring bar. Continue adding the remaining reagents as normally. Wear safety glasses when handling lithium acetate as it can cause eye irritation.
   2. Adjust to pH value 4.8 ± 0.1 with 50% (v/v) acetic acid. Allow the solution to stabilize for 5 min and adjust as necessary.
   3. Add wat....

Results

Figure 2 shows the typical electrical current profile of a 200 mM EACA, 30 mM lithium acetate, pH 4.8 BGE with anti-TNFα mAb sample diluted with Tris buffer (50 mM, pH 8.0). As can be observed, the current is stable throughout the analysis and can oscillate between values of 30 to 35 µA. Figure 3 shows the CZE electropherogram of a blank sample where the detected peak corresponds to the histamine internal standard. It is expected for histamine to have a m.......

Discussion

In this tutorial, we highlight the importance of proper practices when conducting CZE analyses of mAbs in order to increase the probability to succeed. However, when CZE is used on a routine basis, issues arise inevitably¹².

For best results, it is important to follow the notes that were included throughout the protocol, as they will help the analyst to overcome and troubleshoot difficult steps. A major consideration to obtain optimum resolution at a given set of conditions is the c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glacial acetic acid	Tecsiquim	AT0035-7
ACS grade hydrochloric acid	J.T. Baker	9535-05
Histamine dihydrochloride	Fluka	53300
(Hydroxypropyl) methyl cellulose	Fluka	09963
Lithium acetate	Sigma-Aldrich	517992
6-Aminocaproic acid	Sigma-Aldrich	A2504
eCAP Tris Buffer, 50.0 mM,  pH 8	Beckman Coulter	477427
PA 800 Plus Pharmaceutical Analysis System	Beckman Coulter	A66528
eCAP Neutral capillary	Beckman Coulter	477441
Vial, Micro, 200 µl	Beckman Coulter	144709
Universal Vial Caps	Beckman Coulter	A62250
Universal Vials	Beckman Coulter	A62251
Cable, Optics, UV/Vis	Beckman Coulter	144093
UV/Vis Detector Module	Beckman Coulter	144733
Cartridge Assembly Kit, Blank	Beckman Coulter	144738