کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2021055 | 1069225 | 2011 | 9 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(Hc)] expressed in Pichia pastoris Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(Hc)] expressed in Pichia pastoris](/preview/png/2021055.png)
A purification process for the manufacture of a recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype C [rBoNTC(Hc)], a potential vaccine candidate, has been defined and successfully scaled-up. The rBoNTC(Hc) was produced intracellularly in Pichia pastoris X-33 using a three step fermentation process, i.e., glycerol batch phase, a glycerol fed-batch phase to achieve high cell densities, followed by a methanol induction phase. The rBoNTC(Hc) was captured from the soluble protein fraction of cell lysate using hydrophobic charge induction chromatography (HCIC; MEP HyperCel™), and then further purified using a CM 650M ion exchange chromatography step followed by a polishing step using HCIC once again. Method development at the bench scale was achieved using 5–100 mL columns and the process was performed at the pilot scale using 0.6–1.6 L columns in preparation for technology transfer to cGMP manufacturing. The process yielded approximately 2.5 g of rBoNTC(Hc)/kg wet cell weight (WCW) at the bench scale and 1.6 g rBoNTC(Hc)/kg WCW at the pilot scale. The purified rBoNTC(Hc) was stable for at least 3 months at 5 and −80 °C as determined by reverse phase-HPLC and SDS–PAGE and was stable for 24 months at −80 °C based on mouse potency bioassay. N-Terminal amino acid sequencing confirmed that the N-terminus of the purified rBoNTC(Hc) was intact.
Journal: Protein Expression and Purification - Volume 75, Issue 2, February 2011, Pages 177–185