Gel Electrophoresis in the Quality Control of Biopharmaceuticals

HWI pharma services GmbH offers a wide range of services in the field of biopharmaceutical analysis for human therapeutic applications. Given the significant impact that different production processes have on the properties of active substances, a variety of different instrumental methods are required for the quality control and characterisation of biopharmaceuticals.

Through our upcoming blog posts, we would like to introduce you to our various analytical techniques and the associated possibilities for analysing your biopharmaceutical products.

We will start with the established technique of electrophoresis, which is highly versatile in pharmaceutical quality control and is used for the examination of peptides, proteins, and nucleic acids. The separation principle of classical gel electrophoresis is based on the migration of dissolved and charged particles through a gel matrix in an electric field. The molecules migrate toward the oppositely charged electrode and are separated into discrete bands depending on their size, shape, and charge. Classical SDS-PAGE is widely used in numerous (research) laboratories worldwide.

Abbildung 1: Classic "gel image" generated by microchip-based gel electrophoresis. Positions 1-4 show an immunoglobulin G under non-reducing conditions, while positions 5-8 reveal the individual fragments of the reduced IgG, namely the Heavy Chain (HC) and Light Chain (LC).

Today, there are automated microfluidic-based gel electrophoresis systems available from various manufacturers, which offer numerous advantages over conventional gel electrophoresis. These advantages include drastically reduced sample (1 µL for nucleic acids, 4 µL for proteins) and reagent consumption, significantly shorter analysis time (30–40 minutes), simplified sample preparation, and faster data analysis. Furthermore, planar chip structures with rectangular channels and laser-induced fluorescence (LiF) enable low detection limits, improved resolution, and increased reproducibility. At HWI, we utilise the Agilent 2100 BioAnalyzer for this purpose, which is accompanied by a range of standardised analysis kits.

The available protein kits are suitable for content and purity testing of proteins in the size range of 5–230 kDa and offer a comparable sensitivity to the Coomassie Blue staining commonly used in SDS-PAGE. These assays are applicable, for example, for determining the degree of PEGylation (1) of biopharmaceutical agents or for the qualitative analysis of dairy products. Moreover, the system enables the identity and purity testing of antibodies with high sensitivity, as depicted in the figure illustrating the quality control of Immunoglobulin G.

Abbildung 2: Electropherogram of IgG (2 µg/µL), obtained using the Agilent Protein 230 Kit under reducing conditions. The individual fragments of the antibody are visible: the light chain (LC) at 26.1 kDa and the heavy chain (HC) at 58.1 kDa. Additional peaks may represent glycosylated sub-fragments or aggregates, which can be quantified as part of a purity assessment.

As an alternative to conventional SDS-PAGE with silver staining or immunological methods such as Western blotting, the High Sensitivity Protein 250 assay can be used.(2) This assay allows detailed examination of proteins in the molecular weight range of 10-250 kDa at concentrations as low as a few pg/µL.

In addition, the Agilent 2100 Bioanalyzer system and various DNA kits enable automated size determination and quantification of products generated through RT-PCR, gene expression, cloning, or restriction digestion. For instance, the High Sensitivity DNA kit allows the analysis of DNA fragments ranging from 50 to7000 bp with comparable sensitivity in the lower pg/μL range. Corresponding chips are also available for the examination of ribonucleic acids, which allow the assessment of typical quality control parameters such as RNA integrity, quality, and content.

By choosing microchip-based gel electrophoresis for the quality control of your biopharmaceuticals, you benefit from faster analysis times, precise results, and cost savings. Our state-of-the-art analytical methods and the expertise of our experienced team ensure that your products meet the highest quality standards. Take advantage of the benefits of microchip-based gel electrophoresis for the quality control of your biopharmaceuticals. Trust in our expertise and let us work together to ensure the safety and efficacy of your products. For further information and consultation regarding our analytical services, please feel free to contact us.

(1) “Analysis of PEGylated Proteins Using the Agilent 2100 Bioanalyzer System”, Agilent, https://www.agilent.com/cs/library/applications/5990-9593EN.pdf
(2) “Immunoprecipitation and the High Sensitivity Protein 250 Assay”, Agilent, https://www.agilent.com/cs/library/applications/5990-4097EN.pdf