Cristina Peixoto

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.

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.

Product quality of a recombinant fusion protein expressed in immobilised baby hamster kidney cells grown in protein-free medium

Baby hamster kidney cells producing a recombinant IgG-IL2 fusion protein were grown as spinner batch cultures in a protein-free medium, with cells immobilised in porous and non-porous supports at different support concentrations and agitation rates. Product quality, i.e., integrity, decreases up to 60% with the increase in agitation rate or support concentration and from porous to non-porous supports. LDH activity is a good indicator for the monitoring of product degradation, and thus product quality. Optimal conditions regarding titre of intact product were achieved at 60 rpm with 2 g porous support l−1.

Product Quality of a recIgG Under Different Culture Systems of BHK Cells

In the present work, BHK cells producing a recombinant protein with potential application in tumor target therapy were used in order to study the effect of operating conditions upon growth, production and product quality. Cells were grown in a protein-free medium, SMIF6, as spinner batch cultures with cells immobilized in porous and nonporous supports. Two different support concentrations and two distinct agitation rates were used. Results obtained indicate that increasing support concentration led to slightly higher titers, particularly for non porous supports; increasing agitation rates always led to lower final titers. Product quality decreased with increasing support concentration and agitation rates, this being more evident for non porous support cultures. No significant difference in specific growth rates for the different supports was observed, although higher cell concentrations were obtained with porous supports. Specific productivities were higher for non porous supports, but final titers as well as product quality were superior with porous supports.