In this study, it was aimed to investigate the effects of different co-carbon sources on
therapeutically important glycoprotein, recombinant human erythropoietin (rHuEPO) production by
Pichia pastoris by designing feeding strategies which were applied in the production phase of the
bioprocess. During the experiments, the cell growth, sorbitol, mannitol, and methanol consumptions,
recombinant human EPO production, alcohol oxidase activity, total protease concentrations and the
by-products organic acid concentrations were analyzed.
In this context, firstly, laboratory scale air filtered shake bioreactor experiments were
performed by P. pastoris Mut+ strain to investigate the effects of mannitol and sorbitol. 50 gL-1 initial
concentration of co-substrates was found more affordable and appropriate for cell concentration and
recombinant protein production. Thereafter, six pilot scale bioreactor operations were designed and
performed. In the first designed strategy (named as SSM strategy), batch-wise 50 g L-1 sorbitol was
fed at t=0 h of the production phase and then sorbitol concentration was kept constant at 50 g L-1 by
fed-batch feeding with a pre-determined specific growth rate of &mu / Srb0=0.025 h-1 within t=0-15 h of the
production phase together with fed-batch methanol feeding with a pre-determined specific growth rate
of &mu / M0=0.03 h-1. In the following bioreactor experiments co-substrate mannitol was fed to the system
with different feeding strategies together with fed-batch methanol feeding with a pre-determined
specific growth rate of &mu / M0=0.03 h-1. In the second strategy (MM), only 40 g L-1 mannitol was added
to the system at t=0 h of the production phase. In the third strategy (MMM), after adding 50 g L-1
mannitol at t=0 h, mannitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a
pre-determined specific growth rate of &mu / Man0=0.11 h-1 within t=0-9 h of the production phase when
the same cell concentration was attained in SSM strategy. In the fourth one (MLM), limiting amount
of mannitol, 3 g L-1, was added at t=0 h and then mannitol concentration was kept constant at 3 g L-1
by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.005 h-1 within t=0-10 h of
the production phase. After these strategies, several pulses, batch-wise, mannitol feeding strategies
were performed. In the fifth strategy (MPM), besides 50 g L-1 initial mannitol feeding at t=0 h, adding
second batch-wise mannitol at t=6 h, and third one at t=12 h were applied. In the last strategy
(MPMG), four 50 g L-1 pulse feeding of mannitol were performed at t=0 h, 7 h, 14 h, and 24 h,
containing glycerol, with an initial concentration in the fermentation medium being 8 g L-1. The
highest extracellular rHuEPO production was achieved in the fifth strategy MPM as CrHuEPO=645 mg
L-1 at t=9 h while the highest cell concentration was achieved in the first strategy SSM as Cx=109 gL-1
at t=48 h. The overall cell and product yields on total substrate were calculated as YX/St=0.22 g g-1 and
YP/St=2.23 mg g-1 in the highest rHuEPO production case.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/12615356/index.pdf |
| Date | 01 January 2013 |
| Creators | Eskitoros, Sukran Melda |
| Contributors | Calik, Pinar |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | Access forbidden for 1 year |
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