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Clarification and sterile filtration of antibodies

Fast Cell Harvesting Without Using a Centrifuge

| Author / Editor: James Stephenson, Catherine Bladen & Ian Wilkinson* / Dr. Ilka Ottleben

Results and Discussion

To benchmark the Sartoclear Dynamics Lab V against the standard process, a two-litre culture of a human IgG1 anti-EGFR antibody (Cetuximab; Absolute Antibody catalogue number Ab00279-10.0) was prepared in HEK293 cells. Six days post-transfection the culture was split into two equal volumes. At the point of harvesting, the cell density was 2.4 × 106 cells/mL with a viability of 65%.

The first litre of cell culture was taken through the conventional process. This involved a 45-minute centrifugation step followed by filtration using three 0.45 µm PES 500 mL bottle-top filters. These filters typically block after about 400 mL of supernatant have been filtered, meaning on average three filters are required per litre of cell culture. No filters with a pore size of 0.22 µm were used in order to increase the supernatant volume that can be filtered prior to blockage. Filtering of 1 litre of cells took 9 minutes, 48 seconds, of hands-on time, which added up to an overall process time of approximately 55 minutes.

The second litre was taken through the Sartoclear Dynamics Lab V process. Two 10-gram pouches of DE were added to the cells, followed by vigorous mixing. The cells were then poured into a 1,000 mL 0.22 µm Sartolab RF filter and a vacuum was applied. The filtration ran to completion with no blockage and took a total of 8 minutes, 27 seconds, from applying the DE to completion of filtering. This represents approximately 15% of the time taken by the conventional approach, as illustrated in Figure 3.

To confirm that the Sartoclear Dynamics Lab V process had no effect on the quality of the antibody, the two samples proceeded through purification and quality control separately. The final purified antibodies showed no detectable differences in product quality as determined by SDS-PAGE (see Figure 5, online) or SEC-HPLC (see Figure 6, online). An endotoxin measurement was taken for each sample, with both giving a reading of < 0.05 EU/mg, which is the lower limit of detection of the testing kit routinely used. To confirm that the modified process had no effect on the function of the antibody, an indirect ELISA was performed to show binding to human EGFR-Fc (Absolute Antibody catalogue number Pr00117-10.9). As shown in Figure 4, the method of cell clarification had no impact on binding activity. Additionally, the final yields obtained by both processes were almost identical, showing that DE has no impact on both quality and quantity of antibody.

Conclusion

It was clearly demonstrated here that Sartoclear Dynamics Lab V enables rapid clarification of mammalian cell cultures without the need for centrifugation. This was done by taking two litres of cell culture transiently transfected with an antibody and comparing a standard centrifugation-based clarification process with the diatomaceous earth-based method.

Additional Information
 
Easy Filtration without Centrifugation

Any meaningful differences were unable to be detected in the final product quantity or quality as determined by a host of measurements (SDS-PAGE, SEC-HPLC, ELISA and endotoxin), demonstrating that the diatomaceous earth-based system has no impact on product quality. Importantly, the Sartoclear Dynamics Lab V process gave an impressive time saving of approximately 85%. This makes Sartoclear Dynamics Lab V an attractive option to increase productivity and throughput for the clarification step of secreted protein expression and purification systems.

References

[1] Kempken, R., Preissmann, A., and Berthold, W. (1995). Clarification of animal cell cultures on a large scale by continuous centrifugation. J. Ind. Microbiol. 14, 52–57.

[2] Liu, H.F., Ma, J., Winter, C., and Bayer, R. (2010). Recovery and purification process development for monoclonal antibody production. MAbs 2, 480–499.

[3] Riske, F., Schroeder, J., Belliveau, J., Kang, X., Kutzko, J., and Menon, M.K. (2007).

The use of chitosan as a flocculant in mammalian cell culture dramatically improves clarification throughput without adversely impacting monoclonal antibody recovery. J. Biotechnol. 128, 813–823.

* * J. Stephenson, C. Bladen, I. Wilkinson Absolute Antibody Ltd, Redcar, TS10 4RF, United Kingdom

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