Fast protein analysis of mucins using an AZURA® SEC System

Significance of the topic

Mucins are large glycoproteins that form the gel‐like matrix of mucus and play a critical protective role on epithelial surfaces. Their high molecular weight and complex glycosylation make them challenging targets for analytical characterization. Size exclusion chromatography (SEC) offers a gentle, non‐denaturing approach to determine molecular weight distributions, isolate purified fractions and preserve native conformations, which is essential for understanding mucin function and for biomarker development in biomedical applications.

Objectives and Study Overview

This study evaluates the performance of a KNAUER AZURA® SEC system coupled with an AppliChrom® VivoSep multistage hydrophilic SEC column. The primary goals are to demonstrate rapid, high‐resolution analysis of commercially available bovine submaxillary and porcine gastric mucins and to compare throughput and resolution with traditional polymer‐based SEC materials.

Methodology

Samples of bovine submaxillary mucin and porcine gastric mucin were prepared in aqueous buffer and injected directly into the SEC system under isocratic conditions. The column is based on a hydrophilic, polymer‐modified design enabling separation over 100 Da to 5 000 000 Da. Key chromatographic parameters include a 1 mL/min flow rate, ambient column temperature (25 °C), a 20 min run time, and detectors operating at 220 nm (DAD) and refractive index.

Used Instrumentation

• AZURA P 6.1L LPG Pump with 10 mL stainless‐steel head
• AZURA AS 6.1L high‐pressure autosampler
• AZURA DAD 2.1L diode‐array detector (190–700 nm)
• AZURA RID 2.1L refractive index detector
• AZURA CT 2.1 column thermostat
• AppliChrom® VivoSep SEC 350 column (300 × 8 mm, 10 µm particles, 2 500–1 000 000 Da range)
• ClarityChrom® 9.1.0 software with SEC/GPC extension

Main Results and Discussion

Using the VivoSep SEC column, both mucin types eluted within 15–20 minutes while maintaining clear, symmetrical peaks. This run time represents a dramatic improvement over conventional agarose or cross‐linked dextran SEC columns, which may require up to 300 minutes per analysis. The high pressure tolerance (50–200 bar) of the polymer‐based column and the use of 7–10 µm particles contribute to enhanced resolution and reproducibility.

Benefits and Practical Applications

• Time‐efficient profiling of mucin molecular weight distributions in under 20 minutes
• Preservation of native glycoprotein structure under mild, neutral pH conditions
• Potential integration into biomarker discovery workflows for cancer diagnosis and prognosis
• Scalability for high‐throughput quality control in pharmaceutical and bioprocessing environments

Future Trends and Potential Applications

Advances may include coupling SEC with multi‐angle light scattering or mass spectrometry for absolute molar mass determination and glycoform analysis. Miniaturized, high‐pressure systems and novel hydrophilic stationary phases could further reduce analysis times and sample requirements. Expanding applications span targeted mucin‐based biomaterials, drug delivery matrices, and real‐time process monitoring in biomanufacturing.

Conclusion

The combination of the KNAUER AZURA® SEC system and AppliChrom® VivoSep column delivers rapid, high‐resolution mucin analysis with significant time savings and preserved molecular integrity. This platform represents a powerful tool for both fundamental glycoprotein research and applied bioanalytical workflows.

Reference

1. Jumel K., Fiebrig I., Harding S.E., "Rapid size distribution and purity analysis of gastric mucus glycoproteins by size exclusion chromatography/multi angle laser light scattering", Int. J. Biol. Macromol., 18(1‐2), 133-139 (1996).
2. Schoemig V., Isik E., Martin L., Berensmeier S., "Solid liquid extraction of porcine gastric mucins from homogenized animal material", RSC Adv., 7(63), 39708-39717 (2017).
3. Johansson M.E.V., Larsson J.M.H., Hansson G.C., "The two mucus layers of colon are organized by the MUC2 mucin", Proc. Natl. Acad. Sci. USA, 108(suppl_1), 4659-4665 (2010).
4. Crouzier T. et al., "Modulating mucin hydration and lubrication by deglycosylation and PEG binding", Adv. Mater. Interfaces, 2(18) (2015).
5. Schömig V.J. et al., "Optimized purification process for porcine gastric mucin with preservation of native properties", RSC Adv., 6(50), 44932-44945 (2016).
6. Schuster-Little N. et al., "Immunoaffinity-free chromatographic purification of ovarian cancer biomarker CA125 (MUC16) from serum", Anal. Methods, 16(37), 6337-6348 (2024).
7. Felder M. et al., "MUC16 (CA125): tumor biomarker to cancer therapy, a work in progress", Mol. Cancer, 13(1) (2014).
8. Hall M., "Size exclusion chromatography (SEC)", Biopharm. Process., 421-432 (2018).
9. Hostettmann K., Marston A., Hostettmann M., "Separation of Macromolecules", Preparative Chromatogr. Tech., 202-216 (1998).
10. Sandberg T., Blom H., Caldwell K.D., "Potential use of mucins as biomaterial coatings: fractionation, characterization, and model adsorption", J. Biomed. Mater. Res. A, 91A(3), 762-772 (2008).