Abstract
Monoclonal antibody expression systems typically utilise a signal peptide to ensure secretion of the antibody into the cell culture medium. Although this reduces the complexity of purification and avoids the need for cell disruption, it does require the use of expensive and/or time-consuming techniques to remove cells from antibody-containing cell culture medium. In this study, we describe our tests of Sartoclear Dynamics® Lab V, a novel system for rapid clarification of cell culture media without the need for centrifugation or any other costly equipment.
Introduction
The Sartocear Dynamics® Lab V filtration system was tested and compared to the current standard method for removal of cells from antibody-containing cell culture medium. The standard method for clarification includes an initial centrifugation step followed by filtration of the cleared supernatant through a 0.45 μm vacuum driven bottle top filter (figure 1). The novel Sartoclear Dynamics® Lab V filtration system is designed for clarifying mammalian cell cultures. The addition of diatomaceous earth (DE) to cultures supports the formation of a porous filter cake to prevent blockage of the filter, allowing rapid removal of cells and cell debris from the sample (Figure 2). This avoids the need for a centrifugation step, circumventing issues around centrifuge capacity and availability as well as preventing filters from clogging.
Materials and Methods
Cell Culture
A two-liter culture of a Human IgG1 anti-EGFR antibody (Cetuximab; Absolute Antibody catalogue number Ab00279-10.0) was prepared in a two-litre HEK293 cell culture.
Conventional Clarification
Method 1-litre of cell culture was centrifuged at 3,500 g in a bench top centrifuge (2× 500 mL) for 45 minutes, followed by filtration of supernatant using, on average, three vacuum-driven PES 0.45 μm bottle-top filters. Several filtration units and pore sizes of 0.45 μm is used to avoid blocking of the filter.
Modified Clarification Method
In the modified downstream process, the conventional method is replaced with the use of Sartoclear Dynamics® Lab V. 20 g of DE filter aid was added to 1-litre of cell culture, mixed to a homogeneous suspension and then added directly to a 1,000 mL 0.22 μm PES Sartolab® RF vacuum-driven filter, which is included in the Sartoclear Dynamics® Lab kit.
Purification
Filtered supernatant was loaded onto a 5 mL Protein A column for antibody capture and elution in a low pH buffer. Antibody was neutralised before proceeding either to additional purification steps (e.g. cation exchange or size exclusion chromatography) or directly to quality control, depending on the requirements for the particular antibody batch.
Quality Control
Quality Control was performed by SDS-PAGE under non-reducing and reducing conditions, SEC-HPLC, endotoxin testing and, where required, an ELISA to measure binding activity (Figure 3).
Results
To compare the handling time and the quality of the antibody between the conventional and Sartoclear Dynamics® Lab V method, 1 liter of cell culture with a density of 2.4× 106 cells/mL and viability of 65% was clarified with both methods:
- Sartoclear Dynamics® Lab V reduced clarification time by 85% (Figure 4)
- Both methods provided the same quality and quantity of antibody (Figures 5, 6 and 7)
Conclusion
Sartoclear Dynamics® Lab V enables rapid clarification of mammalian cell cultures without the need for centrifugation. No meaningful differences in final product quantity or quality were detected, demonstrating that the diatomaceous earthbased system has no impact on the product. Importantly, the Sartoclear Dynamics® Lab V offered 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.
Additional resources:
Application note: Lab-Scale Clarification of Mammalian Suspension Cultures Using Sartoclear Dynamics® Lab V Kits
Application note: Centrifuge-Free Clarification of Antibodies from Cell Cultures Using Sartoclear Dynamics® Lab Decimates Working Time
Webinar: Body Feed Filtration – The Novel Method for Rapid Harvesting of Mammalian Cell Cultures
For more information on Sartoclear Dynamics® Lab V, click here.
References
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- Liu, H.F., Ma, J., Winter, C., and Bayer, R. (2010). Recovery and purification process development for monoclonal antibody production. MAbs 2, 480–499.
- 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.
