Biomedical subjects

E M Dias

Publications and source records attributed to E M Dias.

3 recordsLinked to original sources

PubMed2002

Process development of a recombinant antibody/interleukin-2 fusion protein expressed in protein-free medium by BHK cells.

The production, purification and stability of quality (in terms of integrity and glycosylation) of an antibody/interleukin-2 fusion protein with potential application in tumour-targeted therapy expressed in BHK21 cells are described. Consistency of the product throughout time was determined by analysis of glycosylation of the fusion protein using MALDI-TOF mass spectroscopy and HPAEC-PAD combined with product integrity studies by SDS-PAGE and Western blotting. These investigations showed consistent expression in terms of integrity and of three major oligosaccharide structures of the fusion protein after 62 generations. The data obtained at this stage indicated the suitability of the cell line for production purposes. Different approaches for the production of this protein were subsequently carried out. The relative productivity of the recombinant fusion protein and general performance of the cells in two different protein-free medium (PFM) culture systems, continuous chemostat and continuous perfusion using a Centritech centrifuge as a cell retention device, were studied. The results indicate that the chemostat culture resulted in more stable and controllable nutrient environment, which could indicate better product consistency, in accordance with what has been observed under serum-containing conditions, in relation to the perfusion culture. Finally, product obtained from the chemostat culture was analysed and purified. The purification process was optimised with an increase in the overall yield from 38 to 70% being obtained, a significant improvement with important consequences for the implementation of an industrial-scale culture system. In conclusion, it was possible to produce and purify the recombinant antibody/interleukin-2 fusion protein assuring the quality and stability of the product in terms of integrity and glycosylation. Therefore, a candidate production process was established.

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PubMed2000

Metabolic shifts do not influence the glycosylation patterns of a recombinant fusion protein expressed in BHK cells.

BHK-21 cells expressing a human IgG-IL2 fusion protein, with potential application in tumor-targeted therapy, were grown under different nutrient conditions in a continuous system for a time period of 80 days. At very low-glucose (< 0.5 mM) or glutamine (< 0. 2 mM) concentrations, a shift toward an energetically more efficient metabolism was observed. Cell-specific productivity was maintained under metabolically shifted growth conditions and at the same time an almost identical intracellular ATP content, obtained by in vivo (31)P NMR experiments, was observed. No significant differences in the oligosaccharide structures were detected from the IgG-IL2 fusion protein preparations obtained by growing cells under the different metabolic states. By using oligosaccharide mapping and MALDI/TOF-MS, only neutral diantennary oligosaccharides with or without core alpha1-6-linked fucose were detected that carried no, one or two beta1-4-linked galactose. Although the O-linked oligosaccharide structures that are present in the IL2 moiety of the protein were studied with less detail, the data obtained from the hydrazinolysis procedure point to the presence of the classical NeuAcalpha2-3Galbeta1-3GalNAc structure. Here, it is shown that under different defined cellular metabolic states, the quality of a recombinant product in terms of O- and N-linked oligosaccharides is stable, even after a prolonged cultivation period. Moreover, unaffected intracellular ATP levels under the different metabolic states were observed.

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PubMed1997

Cell-dislodging methods under serum-free conditions.

In this work, a BHK21 clone producing a fusion protein consisting of a recombinant human IgG molecule with a cytokine tail, growing in a protein-free medium, was used to test several alternatives to avoid the use of serum for trypsin inactivation, currently used in cell dislodging. These included (1) trypsin inactivated with soybean trypsin inhibitor (STI); (2) cell dissociation solution instead of trypsin; (3) dispase instead of trypsin; (4) trypsin inactivated with fetal calf serum (positive control); (5) non-inactivated trypsin (negative control). Use of a centrifugation step was also tested for each alternative. Results indicate that the best method regarding cell growth, viability and adherent fraction is to use trypsin inactivated with STI followed by a centrifugation step. For all methods tested, the utilization of a centrifugation step always led to improved results. The optimal proportion for total trypsin inactivation is 1:1 trypsin (0.2% w/v) to STI (1 mg ml-1). equivalent to 2 mg trypsin to 1 mg STI. No toxic effect was observed for STI at the concentrations used. Long-term subculturing with this new, alternative dislodging method did not affect cell growth, viability and productivity.

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