Pharmaceutical antibodies are a class of therapeutics that plays an important role in controlling many types of diseases such as cancers, allergy, inflammation, infectious or autoimmune diseases. Monoclonal antibodies have become a major class of biopharmaceutical products. There is an increasing demand in the pharmaceutical industry for tools applicable to large scale production and for product characterization and control of the manufacturing process. To support analytical characterization, process development and quality control of therapeutic antibodies in the biopharmaceutical industry, capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) has been recognized as a significant alternative tool of SDS Page because of its ease of use and ability to automate.
This application note describes the analytical methodology for quality control analyses of IgG and their impurities using a 275nm LEDIF detector.
Method of Separation
Key features of the method | 275nm LED allows excitation of tryptophan and tyrosine contained in proteins with no labelling step. Electrokinetic injection -15kV for 15 seconds Separation: -15 kV for 35 min Temperature: 15°C Buffer: SDS-MW gel buffer (A10663, Sciex) |
Instruments | Capillary Electrophoresis: Agilent Technologies 7100 CE Detector: ZETALIF LED 275 Fused Silica Capillary: 50 µm ID Total Length = 33 cm Effective Length = 14 cm |
Limit of Detection (LOD)
Limit of Detection obtained with native fluorescence of IgG is 12 ng/mL. (LOD calculated for a ratio S/N=3)
Comparison of different detections in CE-SDS
CE-SDS | UV Absorbance | Native Fluorescence | Fluorescence Derivatization | |
Wavelength (nm) | 210-220 | 275 | 480 | 530 |
LOD | 500ng/mL | 12ng/mL | 150pg/mL | 50pg/mL |
Conclusion:
Native fluorescence detection with 275nm LED is a simple and sensitive solution for mAbs production like impurities assay thanks to the good resolution obtained.
Antibody degradation, molecular instability or manufacturing inconsistency can be easily followed with this method.