Separation and Online Characterization of Intact Monoclonal Antibody Charge Variants by Capillary Isoelectric Focusing-Mass Spectrometry Method (CAT#: STEM-ET-0350-ZJF)

Introduction

This service provides an online capillary isoelectric focusing-mass spectrometry (CIEF-MS) method for monoclonal antibody (mAb) charge variant analysis using an electrokinetically pumped sheath-flow nanospray ion source and a time-of-flight MS with pressure-assisted chemical mobilization. Use of recombinant mAbs as therapeutics is rapidly growing throughout the worldwide biopharma industry. Unlike synthesized chemical drugs, biologics are cell-originated and are subject to post-translational modifications (PTMs) such as glycosylation, C-terminal lysine truncation, deamidation, disulfide-scrambling, glycation, and methionine or tryptophan oxidation. As these PTMs usually lead to changes in protein isoelectric point (pI), mAb drug substances are known to exhibit charge heterogeneity, represented by multiple charge variants with different pIs. Comprehensive characterization of mAb charge variants is crucial, as the variants may affect the in vitro and in vivo properties related to its activity, safety, developability, and quality. In addition, charge variant characterization is also important for developing biosimilar mAbs.

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Principle Applications Procedure Materials Notes Related Products

Principle

Isoelectric focusing (IEF) is a method of electrophoresis that separates proteins according to their isoelectric point. It performs in a particular pH gradient, in which proteins are separated by differences in their isoelectric points. The isoelectric point (pI) is the pH at which the net charge of the protein is zero. Protein is positively charged when its pI is above the solution's pH, so it migrates towards the cathode. Conversely, protein is negatively charged when its pI is below the solution's pH, so it migrates towards the anode. Protein has no net charge at its pl and stops migrating. Based on this principle, proteins can be separated into sharp bands with each protein positioned at a point in the pH gradient corresponding to its pI.

Applications

Alcohols, Biopolymers, Immunology, Peptides and proteins, Biopharmaceutics

Procedure

1. Preparation: Prepare the gel containing the sample. Mix the solution and add it to a glass tube to form a gel. Prepare the fixing solution, dyeing solution and decolorization solution.
2. Electrophoresis: Add the appropriate acidic solution in the positive electrophoresis tank, and the appropriate alkaline solution in the negative electrophoresis tank. Switch on the electrophoresis apparatus and set the voltage and current. Perform pre-electrophoresis for a period of time to form a pH gradient in the gel. Then set the electrophoresis apparatus to a higher voltage and perform electrophoresis for a period of time.
3. Determination: Different protein components focus on the corresponding pI. Fix, stain and decolorize the gel. Finally, dry it for storage.

Materials

• Isoelectric focusing apparatus
• Sample solution

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