Preparation and characterization of magnetic microspheres for the purification of interferon alpha-2b.
Magnetic agarose microspheres (MAMS), magnetic cellulose microspheres (MCMS), and magnetic poly(vinyl alcohol) microspheres (MPVAMS) were prepared by various different preparation methods. MCMS coupled with anti-IFN alpha-2b monoclonal antibodies (mAb) were selected for the purification of interferon alpha-2b (IFN alpha-2b) after performance characterization among microspheres. Parameters of immunomagnetic separation (IMS), including binding mAb, elution behavior, and sample pretreatment conditions, were optimized to improve the purification efficiency of the separation of IFN alpha-2b by MCMS. Size-exclusion HPLC (HPSEC) showed that the IFN alpha-2b was purified from crude cell lysate had an overall purity of 92.9%, while immunological and biological assays showed an activity recovery of 88.5% and specific antiviral activity of 2.7 x 10(8) IU/mg. Identity and molecular mass of purified IFN alpha-2b were confirmed by western blot and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis. This study illustrated the favorable separation media which combined desired properties for the development of magnetic separation of biological materials.
Cao
Yu
Y
Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, China.
Bai
Gang
G
Chen
Jiaqi
J
Tian
Wang
W
Wang
Shenqi
S
Yang
Wenbo
W
D015153:Blotting, Western; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000302:isolation & purification*; D008280:Magnetics; D008855:Microscopy, Electron, Scanning; D008863:Microspheres; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins