Large-scale purification of recombinant hepatitis B surface antigen from Pichia pastoris with non-affinity chromatographic methods as a substitute to immunoaffinity chromatography.
Prep Biochem Biotechnol
; 48(8): 683-692, 2018.
Article
in En
| MEDLINE
| ID: mdl-30265182
ABSTRACT
The costly media, inconsistent ligand density, ligand leakage, and possible destabilization of recombinant hepatitis B surface antigen (rHBsAg) particles are main drawbacks of using immunoaffinity chromatography (IAF) in the large-scale downstream processing. In this study, we aimed to use an efficient large-scale purification system as an alternative purification method for immunoaffinity chromatography. For this purpose, we suggested integrating non-affinity chromatographic methods of hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for cost-effective purification of rHBsAg expressed in P. pastoris. The optimization of such process is not trivial and straightforward since diverse molecular characteristics of expressed rHBsAg in each type of host cell cause different interactions in non-affinity chromatography processes. The working buffer composition and chromatography parameters are the most influential factors in hydrophobic interaction chromatography. The best result for lab-scale HIC was achieved by using ammonium sulfate buffer in 10% of saturation concentration in pH 7.0 with Butyl-S Sepharose 6 Fast Flow medium and with subsequent Tween-100 and urea elution. In this process, the recovery, purity, and total yield were about 84%, 82%, and 69%, respectively. By scaling-up the HIC and integrating it with Sephacryl S-400 SEC, we obtained highly pure, i.e., > 90%, rHBsAg virus-like particles (VLP).
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Pichia
/
Hepatitis B virus
/
Chromatography, Affinity
/
Hepatitis B Surface Antigens
Language:
En
Journal:
Prep Biochem Biotechnol
Journal subject:
BIOQUIMICA
/
BIOTECNOLOGIA
Year:
2018
Document type:
Article
Affiliation country:
Iran