Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 17, Num. 1, 2000, pp. 51

R Valdés, B Reyes, N Ibarra, R Hernández, M González, S Padilla, A Tamayo, J Montero, C García, J García, L Dorta, T Álvarez, J García, EG Fernández, D Fernández, A Figueroa, O Herrera, R Alemán, J Zubiaurrez, A Marrero, M Alonso, H Gómez, G Calás, A Agraz, L Herrera

Monoclonal Antibodies Division. Center for Genetic Engineering and Biotechnology, PO. Box 6162, Havana 10 600, Cuba. Telephone: (53-7) 21 8466, 21 8008; Fax: (53-7) 21 8008, 33 6008; E-mail: rodolfo.valdes@cigb.edu.cu

Introduction

Several reasons compel to the replacement of in vivo production technology by in vitro technology, for instance: using animals is not easy to scale-up, potential pathogen contamination will always be present, hard validation work should be done and in vivo production has relatively high costs compared to the invitro [1] procedure. However, sometimes it turns out difficult to obtain high levels of antibody secretion in cell culture technologies. This article describes an in vivo large-scale production of the CB.Hep-1 monoclonal antibody, a pharmaceutical preparation, specific for the rHBsAg.

Materials and Methods

Ascitic fluid production

106 cells, previously growing in spinner flasks, were inoculated in mice.

IgG purification

Ascitic fluid was filtrated and two saline precipitation steps with (NH4)2SO4 at 50% of saturation conditions were respectively carried out. The ascitis fluid was desalted by Gel Filtration Chromatography and purified by Protein A affinity chromatography.

Immunosorbent

Sepharose CL-4B was activated by the CNBR method and CB.Hep-1 MAb was covalently immobilized on the support. In order to calculate the total virus clearance factor, the validation of the purification system using five different model viruses was done.

Results and Discussion

All batches were released under WHO regulations, thus guaranteeing safety [2]. Since we introduced the large scale-up process, we have kept a steady production, purity levels are higher than 90% mean, while the yield is higher than 1mg/mL. Assays performed on the process showed the appropriate quality and purity. The immunosorbents were prepared at large scale with high yield. Consistent values of coupling efficiency greater than 95% were observed in all batches, the recovered rHBsAg from the immunosorbents, showed consistence in its purity above 85% ( Figure), which demonstrated the high purification power of the designed immunoafinity chromatography system; while the ligand leakage was below 0.5ng MAb/pg rHBsAg.

The process validation study using viral models is showed in the Table. This result provides a high level of assurance that the final product will be free of contaminants.

Table. Reduction factors (RF) when viral validation was carried out.

In conclusion, the large-scale production process permits to obtain a great amount of the CB.Hep-1 monoclonal antibody, guaranteeing the biosafety of the product.

Copyright 2000 Elfos Scientiae

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