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Media	ITkc < li ml	Yiablc ccii density (.\IC^ cci U ml)
1. SI 90011 \V>/oVC$	4.49	11.8
2.ST90011 5%1‘CS	3.97	10.2
3.SI900II	3.46	7.5
4.45%SI90UI1 45% TC 100 H/MCS	3.11	12.0
5.47.5%.S1WJ1I 47.5 TCIOU II/*. I < S	2.55	9.5
622.5°. S 1*911011 • 67.5%TC100- 2.5%. FCS	2.47	5.9
7. 4«X.75% STWOII 48.75% TCIHO- 2.5% ICS	2.26	S.l
8. 75%MMS 20% SF900II 5%1CS	1.79	0.95
9. 75*.. MM8 yr .. TC 100- 5%. ICS	1.19	0.53
10. 203. MMK 75%. 1C 100 5% l*CS	1.14	l.l

2. Optimum conditions for dcngue envelope protein production

2.1 MOI and TOI tested in shake fiask Sf9 celis were infected at early exponential phase (I and 2x10⁶ cells/ml) with recombinant E baculovirus at \arious multiplicity of infection (0.1, I. 5 and 10). The nmimurn recombinant envelope proteins produced at various MOI were compared.

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Figurę I. Recombinant einelope protein production from yarious MOI at the initial celi concentration of (a) I x | O⁶ and (b) 2xl0⁶ cells/ml (I rai of celi culture was collected, centrifuged and celi pellet collected was dissoked in 100 ml lysis buffer) ((a) Sample No.l. MOI 0.1. TOI 7 days: Sample No.2. MOI 0.1. TOI 8 days: Sample No.3, MOI 1. TOI 7 days: Sample No.4. MOI 1. TOI 8 days: Sample No.5, MOI 5. TOI 3 days: Sample No.6. MOI 5. TOI 4 days: Sample No.7 MOI 10. TOI 3 days: Sample No.S.MOI 10. TOI 4 days (b) Sample No.l. MOI 0.1. TOI 7 days: Sample No.2. MOI I. TOI 5 days: Sample No.3. MOI 5. TOI 3 days: Sample No.4. MOI 5. TOI 4 days: Sample No.5. MOI 10. TOI 2 days: Sample No.6. MOI 10. TOI 3 days)

Figurę I showed that optimum conditions for protein production when infected at celi density of mo" and 2x10" cells ml were moi 5 and toi 3 days and moi io and toi 2 days, respecthely. Protein production at celi density of I > 10 cells/ml was highcr than that of celi density of 2x 10⁶ cells ml. Therefore. conditions obtained will then be used for all experiment in 2.5 I. stirred tank fermenter.

2.2 Recombinant einelope dengue protein production in 2.5 I. fermenter.

Preliminary results showed that stirring speed of 90

rpm was suitable. therefore, Sf9 cells were

cultivativated and infection at celi densit\ of |x|0⁶

*

cells ml MOI of 5 while dissoked oxygen was maintained in the rangę of 30-50 and 60-80% air saluration. From Figurę 2. the level of dissolved oxygen has a significant effect on protein production. The level of protein production at 30-50% dissoked oxygen is much lower than that

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Figurę 2. Level of recombinant einelope protein production in 2.5 I stirred tank reactor at stirring speed of 90 rpm. moi of 5 and dissoked oxygen of 30-50 (a) and 60-80% (b)

I his result is found to be in good agreement with that of Kloppinger et ul. (1990. 1991) who found that growth and level of protein production of Sf9 cells was affected by both agitation speed and level o f dissoked oxygen. At Ix |o⁶ cells/ml initial celi concentration. 90 rpm stirring speed and 60-80% dissolved oxygen. the highest concentrations of recombinant einelope protein produced are 13.0 mg I in shake fiask and 2.5 I fermenter, respectivelv.

Conclusion: Highest S1900 celi density could be achieved using medium containing 45% SF-900 supplemented with 45% TC100 and 10% FCS. For a 2.5 I. fermenter, oplimal conditions leading to the highest recombinant protein production of 13.0 mg I. was MOI of 5. 90 rpm. initial celi concentration of 1x10* cells/ml and 60-80 % dissoked oxygen while the optimum harvest time was 3 days post infection

Acknowledge:    T his work was partially

supported by National Center for Genetic Engineering and Biotechnology.

Refercnce

1.    Halstead. S.B. (1989) Antibody, macrophages, dengue virus infection. shock and haemorrhagic fever: A phatogenic Cascade. Rew Infect. Dis.. II: 830-839.

2.    Kloppinger. M.. Fertig. Ci.. Fraune. E. and Miltenburger. H.G. (1990) Multistage production oAutographa califoniica nuclear polyhydrosis virus in insect celi cultures, Cytotech.. 4: 271-278.

3.    Kloppinger. M., Fertig. Ci.. Fraune. E. and Miltenburger. H.G. (1991) High celi density perfusion culture of insect cells of production of bacuknirus and recombinant protein. In "Production of biologicals from ani mai celi culture” Spier. R.H.. Grilfiths. J.B. and Meignier. B. (eds)