Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 1, 2000, pp. 50

Alejandro Beccaria, Marina Etcheverrigaray, Ricardo Kratje

Instituto de Tecnología Biológica (INTEBIO) - Facultad de Bioquímica y Ciencias Biológicas. Universidad Nacional del Litoral - CC 242-(3000) Santa Fe - Pcia. Santa Fe. Argentina. Tel./Fax: + 54 342 4575214 E-mall: rkratie@fbcb.url.edu.ar

Introduction

Erythropoietin (EPO) is a sialoglycoprotein hormone that regulates red blood cell production. This hormone is mainly produced by the kidney in human adults and anemia associated with renal failure often results from decreased level of EPO. This observation has led to the recombinant expression and subsequent therapeutic use of EPO for patients with chronic renal failure. Since the sugar chains of EPO play an important role in the expression of its biological activity, animal cells are selected as host cells for the production of recombinant human EPO (rhEPO). For its commercial production several methods have been described. In the present study, recombinant BHK cells producing human EPO were adapted to suspension growth and culture conditions were optimized in order to carry out the scaling up without carriers in a perfused stirred tank bioreactor.

Materials and Methods

Cell line. BHK 21 cells were manipulated by genetic methods to produce human EPO constitutively under the control of SV 40 promoter [1].

Media. Culture medium consisted of a 1:1 mixture of DMEM and Ham's F 12 (Gibco BRL, USA) supplemented as described for Medium 1 [2]. Other media used consisted of Medium 1 supplemented with different concentration of fetal calf serum (FCS) (Bioser, Argentina) and/or 100 ppm Antifoam (Dow Corning, USA).

Culture systems and conditions. Adherent culture was achieved in T flasks (Nunc, USA) in Medium 1 containing 1% FCS, in order to estimate the rhEPO specific productivity (qrhEPO) and the monolayer performance during long term culture with medium changes every 2 days. Cells were adapted to suspension growth in spinner flasks (Techne, UK) by successive subcultures of the free growing cell-enriched fractions determined by measurement of the pellet's diameter. Once adapted to suspension growth, qrhEPO was evaluated in spinner flask under different conditions: antifoam supplementation, volume/area ratio, FCS concentration and culture pH. Dense cell cultivation was carried out in a perfused 25 1 stirred tank bioreactor employing a spin sieve system (MBR, Switzerland). Oxygen supply was performed by a sparger provided with sintered stainless steel diffusers. The set point for dissolved oxygen was adjusted to 40% air saturation. This value corresponds to an oxygen concentration of 2.7 mg/L. The stirred speed was adjusted to 70 rpm.

Analysis of samples. Glucose and lactate content were determined with YSI 2700 glucose and lactate analysers (Yellow Springs Instruments, OH, USA). The total cell number was determined by nuclei staining method. The proportion of dead cells was estimated by trypan blue exclusion. The rhEPO concentration was determined by DOT BLOT with own monoclonal antibodies [3].

Results and Discussion

The qrhEPO in adherent cultures was 0.2 pg/cell/d and remained constant over the whole 48 days culture period assayed. During the adaptation to suspension growth a gradual decrease in the pellet number and size was observed. Finally, after 29 subcultures (5 months) a suspension of majority single cells was obtained, with qrhEPO of 0.5 pg/cell/d. This value represents a 2.5-fold enhancement in comparison to the adherent culture. Similar effects were informed for other recombinant protein [2]. qrhEPO increased linearily with the pH of the culture, reaching a double value when the pH ranged from 6.4 to 7.4. Supplementation with antifoam up to 100 ppm had no effects on cell proliferation and on rhEPO productivity. A 2.7 times increase in the volume/area ratio showed no effects either on the specific growth rate (m  = 0.36 d-1) or on the qrhEPO value. The results also indicate that the dependence of m with FCS concentration follows a simple hyperbolic kinetic. As reported by other authors [4] qrhEPO values increased parallely with the decrease of m .

The figure shows the results obtained during 50 days of continuous fermentation with different operation modes: repeated batch, perfusion and continuous culture with cell removal (c.c.). In all cases, cell density varied in parallel to the oxygen uptake rate (OUR). Therefore, the culture state should be followed only by on-line oxygen concentration monitoring. Medium perfusion was adjusted according to on-line redox potential and off-line glucose and lactate measures. High cell densities up to 107cells/mL were achieved. Besides, qrhEPO resulted directly proportional to the perfusion rate. As previously reported [5], maximal rhEPO production was 80 mg/d with a perfusion rate of 1 volume reactor/d.

In conclusion, despite the long time spent by the cell adaptation in the suspension growth mode, this procedure is convenient to diminish the costs and steps of the production process.

References

1. Beccaria J, Etcheverrigaray M, Kratje R. Terceras Jornadas de Investigación de la Asociación de Universidades del Grupo Montevideo. Concordia, Argentina y Salto Uruguay 1995.

2. Kratje R, Wagner R. Biolech Bioeng 1992;39:233-42.

3. Didier C, Pereira D, Kratje R, Etcheverrigaray M. ll Encuentro de Jóvenes Investigadores de la Universidad Nacional del Litoral. Santa Fe, Argentina 1998.

4. Lao M, Toth D, Danell G, Schalla C. Cytotechnology 1996;22:43-52.

5. Beccaria J, Etcheverrigaray M, Kratje R. Vlll Pan-American Association for Biochemistry and Molecular Biology (PAABMB) (Congress. Pucón, Chile 1996.

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