| Authors: Luisa Bortone, Dr. Benjamin Lehmann
Capillary Electrophoresis

A powerful technique for the characterisaiton and quality control of biopharmaceutical products
With the ever-increasing number of new biotechnologically produced drugs, the choice of an appropriate analytical technique for quality control is crucial. Earlier, we highlighted the many advantages of chip-based gel electrophoresis [link]. Notably, this method is an excellent alternative to classical SDS-PAGE, offering high reproducibility, resolution, sensitivity, and simplified handling.
For more complex analyses, capillary electrophoresis (CE) is applied. (1) This technique is based on classical electrophoresis and is known for its high resolution, efficiency and flexibility. CE-based approaches have been a part of the European and American Pharmacopoeias (Ph. Eur. 2.2.47 and USP <1053>) since 2001. These methods are recommended for the characterisation of size and charge heterogeneity, as well as for purity and stability testing of proteins, monoclonal antibodies, DNA and RNA. (2) For some products, such as EPO isomers, both pharmacopoeias even consider CE as the default method.

Figure 1: Electropherograms of a typical purity and identity test of (monoclonal) antibodies according to the USP. (3) Capillary gel electrophoresis (CGE) was used to analyse IgG under non-reducing (top) and reducing (bottom) conditions. The electropherograms enable us to recognise the intact antibody (IgG), individual fragments of the heavy chain (HC) and light chain (LC), as well as various aggregates and non-glycosylated antibody subunits.
The Versatility of Capillary Electrophoresis
In capillary electrophoresis (CE), two key mechanisms contribute to the effective separation of analytes:
- Electrophoretic mobility: Electrophoretic mobility is the driving force behind the movement of charged particles in a capillary. This enables the separation of molecules based on their charge-to-mass ratio.
- Electroosmotic flow (EOF): The EOF causes the migration of buffer ions in the electric field and often overlaps with electrophoretic migration, becoming a critical factor in separation. The EOF can be adjusted depending on the pH of the electrolyte and the charge on the capillary surface, resulting in a shallow flow profile in CE. This results in significantly less band broadening compared to HPLC, resulting in higher peak sharpness.
Depending on the research question, different detectors (UV-Vis, PDA, and laser-induced fluorescence) can be used for the quantification of analytes, similar to classical HPLC.
Capillary Zone Electrophoresis (CZE): CZE is the most commonly used method of capillary electrophoresis and can be applied to the analysis of small and large molecules (MW < 100,000 Da). Analytes move through the capillary at varying speeds in accordance with their charge, leading to the formation of distinct zones in the separation process. CZE is known for its efficient separation, allowing the precise analysis of molecules with even minor differences in their charge properties. Adding chiral selectors to the separation buffer makes CZE ideal for enantiomer analysis, e.g., the separation of D- and L-amino acids.(4)
Capillary Gel Electrophoresis (CGE): CGE is particularly suitable for the analysis of macromolecules such as oligonucleotides or proteins. In contrast to other separation techniques, CGE uses a capillary filled with a gel (e.g., polyacrylamide). The gel's molecular sieve effect allows additional separation based on molecule size, as smaller molecules move through the gel faster than larger ones. Therefore, CGE provides an effective means of characterising biopharmaceutical macromolecules.

Figure 2: Efficient separation of single-stranded DNA (40-60 base pairs) using CGE: With capillary gel electrophoresis, nucleic acids with minimal size differences (± 1 base pair) can be resolved in short analysis times with near baseline resolution (Rs ≥ 1.5).
Capillary Isoelectric Focusing (CIEF): CIEF allows the separation of analytes that differ not in their electrophoretic mobility but in their isoelectric point (pI).(5) The pI is the pH value at which the number of positive and negative charges of an amphoteric molecule (e.g., protein) are equal. At this stage, the analyte ceases to migrate in the electric field. To achieve this type of separation, an ampholyte-induced pH gradient is created within the buffer. CIEF is often employed for therapeutic monoclonal antibodies (mAbs) exhibiting natural heterogeneity. During production or storage, slight structural variations may arise, which can considerably influence the antibodies' efficacy. Therefore, analysing the charge variant ratios, for instance, via CIEF, is of great importance.
A Promising Future
Capillary electrophoresis is undoubtedly an indispensable tool for quality control and characterisation of biopharmaceuticals, particularly for investigating therapeutic proteins or oligonucleotides. It excels in high resolution, sensitivity, and flexibility, and is also a dependable method comparable to HPLC. Based on various separation techniques, this state-of-the-art approach offers a wide range of analytical possibilities for your biopharmaceutical products.
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(1) Engelhardt, H.; Beck, W.; Schmitt, T.: „Kapillarelektrophorese Methoden und Möglichkeiten“, 1994, Wiesbaden, Springer Verlag
(2) United States Pharmacopeia (2023), General Chapter, 〈1053〉 Capillary Electrophoresis.
(3) United States Pharmacopeia (2023). General Chapter, 〈129〉 Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies.
(4) X. Lu, Y. Chen, J. Chromatogr. A 2002, 955, 133–140.
(5) Sole, Marina: “Capillary Isoelectric Focusing (cIEF)- As a Platform Method for the Evaluation of Monoclonal Antibody Charge Variants”, Sartorius, https://www.sartorius.com/download/692314/cief-application-note-en-b-sartorius-pdf-data.pdf