Résumé
Novel hydrophobic absorbents were synthesized by immobilizing butyl derivative onto the highly cross-linked agarose beads manufatured in China, which are used as matrix. The effect of the spacer arm length (3C, 8C and 10C) and ligand density (from 13 to 45 micromol/mL) on the hydrophobicity were investigated using purified Hepatitis B surface antigen (HBsAg) expressed by CHO cell lines. Also considering the effects of salt concentration and pH on HBsAg recovery and purification factor, orthogonal experiment design method was used to evaluated the absorbents. The results showed the butyl-S absorbent with the spacer arm length of C8, the ligand density of 22 micromol/mL gel showed the best performance for the separation of HBsAg. Approximately 100% HBsAg recovery and 60 as purification fold were achieved by this media under the operating condition of pH 7.0 and 9% of salt concentrateion.
Sujets)
Animaux , Cricetinae , Adsorption , Cellules CHO , Chromatographie sur agarose , Méthodes , Cricetulus , Antigènes de surface du virus de l'hépatite B , Interactions hydrophobes et hydrophiles , Protéines recombinantesRésumé
A new hydrophobic absorbent based on homemade highly cross-linked agarose beads was synthesized by immobilizing butyl derivative onto the matrix linkage. The density of ligand was controlled by adjusting the concentration of butanethiol and the synthesis route was optimized by evaluating the purification efficiency of recombinant Hepatitis B surface antigen (HBsAg) expressed by Chinese hamster ovary (CHO) cell line. A high performance absorbent was finally screened out with up to 80% of HBsAg recovery and purification-fold (PF) about 20. Furthermore, the column pressure was about 0.06 MPa under the flow rate of 500cm/h, and no leaked butyl were detected after exposing the gel in common buffers, chaotropic agents, high concentrations of denaturing agents such as guanidine hydrochloride, urea and polar organic solvents. These results demonstrated that the absorbent have high physico-chemical stability, so it was available for the downstream process. Finally, after scaled up to 2L wet gel/batch, the absorbent was applied to the integration of three-step chromatography and obtained the purified CHO-HBsAg with 95% purity by SDS-PAGE and HPLC, which meet the requirements of SFDA. The purification efficiency and the reproducible ability of the absorbents were also evaluated from batch-to-batch. The results demonstrated that the absorbent met the requirement of scalable, reproducible, economic effect as well. This absorbent is a promising alternative exported HIC gel for wildly being used in Chinese pharmaceutical industries.
Sujets)
Animaux , Cricetinae , Humains , Cellules CHO , Chromatographie sur agarose , Méthodes , Cricetulus , Antigènes de surface du virus de l'hépatite B , Génétique , Protéines recombinantes , GénétiqueRésumé
The dissociation of virus-like particles of Hepatitis B surface antigen (HBsAg) during the adsorption-desorption on the solid-phase of chromatography is a main challenge for its purification. Herein, poly (ethylene glycol) (PEG) was applied as an additive during the purification of HBsAg from recombinant Chinese hamster ovary (CHO) cell culture to improve the HBsAg recovery and protect its structural assembly. The presence of 1% of PEG10000 in the mobile phase of ion-exchange chromatography (IEC) of DEAE-Sepharose FF column could increase the recovery of HBsAg from about 55% to 80%, with a similar purification (-fold) (about 12) compared with the absence of PEG. Importantly, glycosylated protein forms of HBsAg were reserved well by PEG-accompanied chromatography. Furthermore, size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) analysis was performed on line to monitor the aggregates, particle size and molecular weight distribution of HBsAg. The results demonstrated that the HBsAg particle size and assembly are more homogenous after adding PEG in the mobile phase of IEC than no PEG added in the mobile phase.
Sujets)
Animaux , Cricetinae , Humains , Cellules CHO , Chromatographie d'échange d'ions , Méthodes , Cricetulus , Antigènes de surface du virus de l'hépatite B , Génétique , Polyéthylène glycols , Chimie , Protéines recombinantes , GénétiqueRésumé
In order to separate and purify the PEGylated recombinant human granulocyte stimulating factor (rhG-CSF) at large laboratory-scale level, a two-step ion-exchange chromatographic separation procedure was designed. Cation-exchange chromatography was applied first to separate PEGylated rhG-CSF from un-reacted rhG-CSF, followed by anion-exchange chromatography to dissolve individual PEG-rhG-CSF species (mono-, di- and tri-PEGylated rhG-CSF) and remove the free PEG. The molecular weight of individual PEGylated rhG-CSF was determined by MALDI-TOF and SDS-PAGE. MALDI-TOF mass spectrometry revealed that the molecular weights of mono-, di- and tri-PEGylated rhG-CSF are 23.8 kD, 28.6kD and 33.8kD, respectively. Cell proliferation activity was detected by MTT assay using NFS-60 cell. The in vitro residual bioactivity of mono-, di- and tri-PEGylated rhG-CSF were 90%, 75% and 43% respectively, comparing with the un-conjugated rhG-CSF. These results indicated that the un-conjugated rhG-CSF and excess free PEG can be removed completely and the three conjugate species can be purified into homogeneity by the two consecutive ion-exchange chromatographic steps. The purification procedure is easy to scale-up, high in performance and recovery.