Abstract
Objective
To develop a rapid process analytical technology (PAT) tool that can measure sialic acid content of an Fc-fusion protein from cell culture samples.
Results
A statistical significant correlation between the sialic acid content and size-exclusion chromatography (SEC)–HPLC retention time of an Fc-fusion protein was observed when analyzing the titer of the samples. Using linear fitting analysis, the data fit the model well with R 2 = 0.985. Based on the SDS-PAGE and oligosaccharide analysis, we speculate that the amounts of the glycans could expand the structure of the Fc-fusion protein. This was manifested by the SEC–HPLC method in which proteins were separated based on its molecular size. In order to development a robust PAT method, an internal standard was used to improve the precision of the method by reducing systematic errors. We found the change of SEC retention time (delta t) and sialic acid content were highly correlated (R 2 = 0.992). This method was further validated by a 1500 l production process.
Conclusion
SEC–HPLC is a promising PAT tool to monitor the sialic acid content of Fc-fusion protein during biomanufacturing or medium optimization processes.
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References
Butler M (2006) Optimisation of the cellular metabolism of glycosylation for recombinant proteins produced by mammalian cell systems. Cytotechnology 50:57–76
Glassey J, Gernaey KV, Clemens C, Schulz TW, Oliveira R, Striedner G, Mandenius CF (2011) Process analytical technology (PAT) for biopharmaceuticals. Biotechnol J 6:369–377
Hong P, Koza S, Bouvier ES (2012) A review size-exclusion chromatography for the analysis of protein biotherapeutics and their aggregates. J Liq Chromatogr Relat Tech R T 35:2923–2950
Hossler P, Khattak SF, Li ZJ (2009) Optimal and consistent protein glycosylation in mammalian cell culture. Glycobiology 19:936–949
Jedrzejewski PMJ, del Val IJ, Polizzi KM, Kontoravdi C (2013) Applying quality by design to glycoprotein therapeutics: experimental and computational efforts of process control. Pharm Bioprocess 1:51–69
Kuribayashi R, Hashii N, Harazono A, Kawasaki N (2012) Rapid evaluation for heterogeneities in monoclonal antibodies by liquid chromatography/mass spectrometry with a column-switching system. J Pharm Biomed 67:1–9
Markely LRA, Ong BT, Hoi KM, Teo G, Lu MY, Wang DI (2010) A high-throughput method for quantification of glycoprotein sialylation. Anal Biochem 407:128–133
Ngantung FA, Miller PG, Brushett FR, Tang GL, Wang DI (2006) RNA interference of sialidase improves glycoprotein sialic acid content consistency. Biotechnol Bioeng 95:106–119
Paris I et al (2006) Near infrared spectroscopy and process analytical technology to master the process of busulfan paediatric capsules in a university hospital. J Pharm Biomed 41:1171–1178
Park S et al (2010) Array-based analysis of secreted glycoproteins for rapid selection of a single cell producing a glycoprotein with desired glycosylation. Anal Chem 82:5830–5837
Petrescu A-J, Milac A-L, Petrescu SM, Dwek RA, Wormald MR (2004) Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding. Glycobiology 14:103–114
Rathore AS, Winkle H (2009) Quality by design for biopharmaceuticals. Nat Biotechnol 27:26–34
Rathore AS, Bhambure R, Ghare V (2010) Process analytical technology (PAT) for biopharmaceutical products. Anal Bioanal Chem 398:137–154
Royle L et al (2008) HPLC-based analysis of serum N-glycans on a 96-well plate platform with dedicated database software. Anal Biochem 376:1–12
Sethuraman N, Stadheim TA (2006) Challenges in therapeutic glycoprotein production. Curr Opin Biotech 17:341–346
Svennerholm L (1957) Quantitive estimation of sialic acids: iI. A colorimetric resorcinol-hydrochloric acid method. Biochim Biophys Acta 24:604–611
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 21106045, 21206040, 21406066), the National High Technology Research and Development Program of China (863 Program) (No. 2012AA02A303), the National Science and Technology Major Project (No. 2013ZX10004003-003-003), the Fundamental Research Funds for the Central Universities (WF1214035).
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Liu, J., Chen, X., Fan, L. et al. Monitoring sialylation levels of Fc-fusion protein using size-exclusion chromatography as a process analytical technology tool. Biotechnol Lett 37, 1371–1377 (2015). https://doi.org/10.1007/s10529-015-1815-3
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DOI: https://doi.org/10.1007/s10529-015-1815-3