A novel application of ion exchange chromatography in recombinant hepatitis B vaccine downstream processing: Improving recombinant HBsAg homogeneity by removing associated aggregates
| Autor: | Maryam Khatami, Zeinab Kimia, S. Siamak Ashraf Talesh, Amin Javidanbardan, Alireza Kavianpour, Seyed Nezamedin Hosseini |
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| Rok vydání: | 2019 |
| Předmět: |
HBsAg
Clinical Biochemistry Ion chromatography CHO Cells 030226 pharmacology & pharmacy 01 natural sciences Biochemistry Pichia Analytical Chemistry law.invention 03 medical and health sciences chemistry.chemical_compound Cricetulus 0302 clinical medicine law Cricetinae Escherichia coli Animals Hepatitis B Vaccines Vaccines Synthetic Hepatitis B Surface Antigens Downstream processing Chromatography Ion exchange Protein Stability Elution Chemistry 010401 analytical chemistry Virion Cell Biology General Medicine Chromatography Ion Exchange Recombinant Proteins 0104 chemical sciences DEAE-Sepharose Ionic strength Chromatography Gel Recombinant DNA |
| Zdroj: | Journal of Chromatography B. 1113:20-29 |
| ISSN: | 1570-0232 |
| DOI: | 10.1016/j.jchromb.2019.03.009 |
| Popis: | Production of recombinant HBsAg as a main component of the hepatitis B vaccine has already been established in commercial scale. So far, many studies have been performed to optimize the production process of this recombinant vaccine. However, still aggregation and dissociation of rHBsAg virus-like particles (VLPs) are major challenges in downstream processing of this biomedicine. The structural diversity of rHBsAg is dependent on many factors including cell types, molecular characteristics of the expressed recombinant rHBsAg, buffer composition as well as operation condition and specific characteristics of each downstream processing unit. Hence, it is not relatively easy to implement a single strategy to prevent aggregation formation in already established rHBsAg production processes. In this study, we examined the efficacy of weak anion exchange chromatography (IEC)- packed with DEAE Sepharose Fast Flow medium- on isolation of rHBsAg VLPs from aggregated structures. For this purpose, the influence of ionic strength of elution buffer as a key factor was investigated in isolation and recovery of rHBsAg VLPs. The elution buffer with electrical conductivity between 27 and 31 mS/cm showed the best results for removing aggregated rHBsAg based on SEC-HPLC analysis. The results showed that in the selected conductivity range, about 79% of rHBsAg was recovered with purity above 95%. The percentage of rHBsAg VLPs in the recovered sample was between 94% and 97.5% indicating that we could obtain highly homogeneous rHBsAg within the acceptable quality level. The TEM, SDS-PAGE and western blot analysis were also in agreement with our quantitative measurements. |
| Databáze: | OpenAIRE |
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