| Author: Elizaveta Guryeva
Comparison of different analysis methods

Comparison of different methods for analyzing monoclonal antibodies
For use as therapeutics, pharmaceutical products require suitable quality control after various production processes, e.g., production in transgenic organisms such as bacteria or cell cultures and subsequent isolation and purification. In connection with the testing of monoclonal antibodies, for example, checking the identity, purity and integrity of the products is of crucial importance. This is because possible impurities such as other proteins, cell components or degradation products can impair the activity, efficacy, safety and pharmacokinetic properties of antibodies.(1),(2)

Figure 1: The electropherogram of IgG (2 µg/µL), recorded with the Agilent Protein 230 Kit under non-reduced conditions. IgG intact at 160.2 kDa and the individual fragments of the antibody can be recognised: light chain (LC) at 27.1 kDa and heavy chain (HC) at 56.4 kDa. The signals numbered 3-6 indicate half-antibody fragments and dimers or other aggregates.
In combination with a specific sample preparation (e.g. reduced vs. non-reduced, deglycosylated vs. glycosylated), some analytical methods enable the investigation of a variety of properties and the quality of antibodies. Various techniques are available for quality attribute analysis of antibodies, e.g. capillary electrophoresis (CE), SDS-PAGE, UV-HPLC or LC-MS. In this blog post, analysis techniques such as microchip-based SDS-PAGE (2100 BioAnalyzer from Agilent) and RP-HPLC with UV (ACQUITY Premier System from Waters) are presented in more detail.
When analyzing intact monomers using microchip-based SDS-PAGE, additional polypeptide units such as heavy chain, light chain dimers, or glycosylated and non-glycosylated forms of the antibodies can be detected as impurities. The electropherograms have an excellent resolution between the signals and thus enable a quality check of the samples. A more precise determination of the smaller fragments is possible with the P230 kit and of larger fragments with the HSP250 kit. The signals indicate several different fragments and aggregates. However, for a more precise quality control of samples orthogonal analysis methods, such as RP-HPLC with UV or LC-MS, are required.
An ACQUITY Premier system, optimised for biomolecule analysis, can be utilized as an LC system for protein analysis. Polyetheretherketone (PEEK) is used for capillaries in this system instead of the usual stainless steel. This plastic is mechanically stable, chemically inert, and hydrophobic, which means that better peak shapes and higher recovery rates can be achieved. High separation performance, which enables precise quality control, can be obtained by using different stationary phases, optimized chromatographic parameters or different types of detection.

Figure 2: The chromatogram of the IgA sample under non-reducing conditions. Phenyl column (2.1 x 150 mm, 5 µm, 1000 Ǻ); A: water + 0.1 % TFA, B: ACN + 0.1 % TFA; 0.4 ml / min; 80 °C; 210 nm. The signals with the numbers 1-3 indicate antibodies with different glycan structures or different degrees of glycosylation.

Figure 3: The chromatogram of the IgA sample under reducing conditions. Phenyl column (2.1 x 150 mm, 5 µm, 1000 Ǻ); A: water + 0.1 % TFA, B: ACN + 0.1 % TFA; 0.4 ml / min; 80 °C; 280 nm. The signals indicate light chain (LC) and heavy chain (HC).
Antibodies are subject to post-translational modifications during production. The modifications and degree of modifications may vary due to minor changes in the production conditions such as the buffer composition, pH, or temperature. Particularly he glycans have a significant influence on the kinetics and stability of the antibody structure. Additionally, the pattern of different sugars is important for the recognition of the antibody by the immune system. Therapeutic antibodies with an incorrect glycosylation pattern can be recognized as foreign by the immune system. On the one hand, this can block the effect of the drug and, on the other hand, cause a severe reaction of the immune system. Methods for analyzing the glycan structure are therefore of great importance. Glycans cleaved and loaded by PNFase can be for example detected by UHPLC coupled with fluorescence detection and high-resolution mass spectrometry.
Other possible antibody modifications include oxidations or methylations of individual amino acids, conversions of the N-terminal glutamine and the absence of the C-terminal lysine due to the action of carboxypeptidases.2,3 The described modifications also contribute to the heterogeneity of therapeutic antibodies and can be characterized by high-resolution mass spectrometric analysis.
Microchip gel electrophoresis and RP-HPLC are two mutually orthogonal analytical methods that are well suited for analyzing proteins. If the identity and purity of the antibodies are to be determined, microchip gel electrophoresis offers significantly improved resolution and automated size determination compared to RP-HPLC. In addition, microchip technology offers significantly higher sensitivity, making it possible to detect fragments in the pg/µl range. By using a RP-HPLC method, only some of the fragments and aggregates could be detected in the µg/µl range. Another advantage of microchip-based gel electrophoresis over RP-HPLC is the speed of analysis. Here, individual samples can usually be analyzed with very short run times (< 1 min), whereas the established HPLC methods had run times of 3-15 min. Moreover, depending on the analytical question as well as a HPLC-system available, run times of 60 minutes or longer might be necessary. However, microchip gel electrophoresis has limitations as well. To perform a microchip-based gel electrophoresis analysis only prefabricated kits can be used. There are kits available for a specific protein molecular range (5-230 kDa or 10-250 kDa). Another disadvantage of microchip-based gel electrophoresis is that the sample preparation can be complex and requires derivatization. You can find further information regarding gel-electrophoresis and its applications in the blog "Gel electrophoresis in the quality control of biopharmaceuticals". In contrast to microchip-based gel electrophoresis, chromatographic techniques such as HPLC are very flexible. The separation efficiency can be influenced by many parameters, e.g., the composition of the mobile phase or various chemical properties of the stationary phase. In addition, HPLC enables the use of various detection methods such as PDA, UV, RI, CAD or coupled high-resolution MS.
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1. World Health Organisation. WHO-Guideline for Safe Production and Quality Control of Monoclonal Anibodies for Use in Humans; 2021.
2. Wang, W. H.; Cheung-Lau, J.; Chen, Y.; Lewis, M.; Tang, Q. M. Specific and High-Resolution Identification of Monoclonal Antibody Fragments Detected by Capillary Electrophoresis–Sodium Dodecyl Sulfate Using Reversed-Phase HPLC with Top-down Mass Spectrometry Analysis. MAbs 2019, 11 (7), 1233–1244. https://doi.org/10.1080/19420862.2019.1646554.
3. Huhn, C.; Selman, M. H. J.; Ruhaak, L. R.; Deelder, A. M.; Wuhrer, M. IgG Glycosylation Analysis. Proteomics. February 2009, pp 882–913. https://doi.org/10.1002/pmic.200800715