In-Depth Characterization of Cetuximab Charge Heterogeneity by Online Capillary Isoelectric Focusing-Mass Spectrometry (CAT#: STEM-ET-0351-ZJF)

Introduction

Cetuximab is a targeted anticancer drug. It is also known as Erbitux. It is a treatment for bowel cancer (advanced bowel cancer) and head and neck cancer that has spread. An online capillary isoelectric focusing/mass spectrometric (CIEF/MS) method for intact monoclonal antibody (mAb) charge variant analysis that uses an electrokinetically pumped sheath-flow nanospray ion source on a time-of-flight (TOF) MS with a pressure-assisted chemical mobilization was provided. The direct online CIEF/MS method exhibited excellent resolution of charge variants conforming to those of imaged capillary isoelectric focusing with ultraviolet detection (iCIEF/UV). However, for complex mAbs, CIEF/MS spectra of the intact charge variant peaks may be too convoluted to be effectively interpreted. In this sevice, a middle-up approach is used to enhance the capability of the CIEF/MS method for characterizing complex mAb charge variants by reducing sample complexity.

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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

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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