Application Note USD3013. Monoclonal Antibody Production in Suspension CHO Culture Using Single-Use PadReactor Mini Bioreactor

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1 Application Note USD313 Monoclonal Antibody Production in Suspension CHO Culture Using Single-Use PadReactor Mini Bioreactor

2 Introduction Single use solutions are found in many applications as they offer straightforward alternatives to classical glass or stainless steel bioreactor systems. These ready-to-use systems require neither cleaning nor sterilization steps by the user, while facilitating the scale-up from bench scale to large scale productions. Within the range of its PadReactor bioreactors, Pall Life Sciences has introduced the PadReactor Mini bioreactor (Figure 1), as a scale-down model perfectly suited for feasibility studies, research, process development and small volume production of cells, proteins or viruses from both suspension and microcarriers cultures. The gentle mixing combined with efficient gas transfer allows the PadReactor Mini bioreactor to host suspension and anchorage-dependent cell cultures (e.g. on microcarriers). Critical cell culture parameters such as ph, DO, and temperature are monitored and controlled by the PadReactor Mini controller. This application note will present the transfer of a monoclonal antibody (mab) production process from a classical glass stirred-tank reactor (STR) to a single-use 1 L PadReactor Mini bioreactor. Material and Methods for Process Transfer in PadReactor Mini Bioreactor The mab production process is based on CHO cells (undisclosed cell strain). The process was applied in 3 systems for comparison: shake flask (ShF), Applikon glass STR with pitched-blade impeller and microsparger, and PadReactor Mini system (microsparger), a single-use bioreactor using patented mixing paddle technology. PadReactor Mini bioreactor was monitored and controlled using a dedicated controller (ez-control from Applikon adapted for Padreactor bioreactor). STR was controlled with Tryton controller (Pierre Guerin). Shake Flasks were incubated in INFORS shaker. Additional equipment used: Haemocytometer and trypan blue for cell counting and viability determination; Analyzer for Glucose (Roche) and Lactate (Scout) for metabolite follow-up; ELISA kit solutions (R&D Biotech) for mab titration; Figure 1 PadReactor Mini system Pre-cultures were done in shake flasks over 3 passages to amplify cells for simultaneous inoculation of the 3 systems. Cells were kept in culture for days with daily monitoring of cell density, cell viability, metabolites and mab titer.

3 On day 3, mab production was activated by sodium butyrate addition. In all systems temperature was maintained at 3 C. In the PadReactor Mini bioreactor and the STR, agitation, aeration and base addition were adapted to maintain a DO of % (air sat.) and a ph of.. All culture and operating conditions are summarized in Table 1. Table 1 Cell culture and operating conditions PadReactor Mini System Bioreactor Microsparger Glass Stirred-Tank reactor (STR) Shake Flask Cell type CHO CHO CHO Inoculation density.4x1 cells/ml.4x1 cells/ml.4x1 cells/ml Medium and reagents Power CHO- CD, w/o L-gln, PR or HT (Lonza) + 4mM Glu Sodium butyrate addition on day 3 (final concentration mm) Working volume 13 L 3 L 3 ml Agitation speed RPM RPM 13 RPM Temperature 3 C 3 C 3 C ph setpoint and regulation.. 9% Air - % CO Air/CO in overlay Air/CO in overlay Sodium hydroxide 1M Sodium hydroxide 1M Dissolved oxygen setpoint > % > % 9% Air - % CO and regulation O sparging in medium O sparging in medium Results and Discussion Cell growth Cell growth and viability as well as metabolites follow-up were monitored daily. The peak of cell density in the PadReactor Mini bioreactor was.9 x 1 viable cells/ml, reached after 4 days of culture. In the STR the maximum density achieved was similar,.1 x 1 viable cells/ml and is delayed compared to PadReactor Mini bioreactor culture. In both systems viability stayed near 1% for the first 3 days and decreased to 4% over the following days (Figure ). The decrease in cell density and viability corresponds to glucose depletion in the medium (Figure 3) which occurs after 4 days in the PadReactor Mini bioreactor and after days in the STR. These observations show that cell growth and metabolites evolution in the PadReactor Mini bioreactor exhibits a behavior equal to or better than the classical STR. Figure Viable cell density and viability monitoring in the PadReactor Mini bioreactor and STR. ) Cell concentration (million cell/ml) Cell Growth and Viability Monitoring in Padreactor Mini and STR Padreactor - Cell density STR - Cell density Viability (%) Figure 3 Glucose and Lactate concentrations follow-up in the PadReactor Mini bioreactor and STR. Glucose and Lactate concentration (mm) Cell Growth and Viability Monitoring in Padreactor Mini and STR Padreactor - Glucose STR - Glucose 3

4 Monoclonal Antibody production mab titer was measured from daily sampling by ELISA method. Follow-up shows a constant increase in titer up to day in both the PadReactor Mini bioreactor and the STR, reaching 18 and 188 mg/l respectively (Figure 4). The cell specific productivity, defined as mab titer divided by total cell concentration (viable and non-viable), was calculated for each system. By comparing the maximum reached in each system (Figure 4) we see that the PadReactor Mini bioreactor and the STR had very similar specific productivities. The shake flask performance, used only for final point reference is below the PadReactor Mini bioreactor and the STR. This is most probably due to the absence of culture condition control in this system. Figure 4 Volumetric production of CHO cells in PadReactor Mini bioreactor and STR (trends) and maximum mab cell specific productivity peak (bars). Error bars represent ELISA method error. MAb titer (mg/ml) Monoclonal Ab titers measured by ELISA Padreactor - mab titer STR - mab titer Monoclonal Ab spec prod Mab cell PAD Mab cell STR Mab cell SHF Mab specific productivity (mg/million cell) Conclusion In this study, a CHO-based monoclonal antibody production was transposed without any productivity loss from classical glass stirred tank reactor to PadReactor Mini bioreactor. The growth results suggest that culture conditions are more favorable thanks to the paddle-driven agitation. This demonstrates the suitability of the disposable PadReactor Mini system for suspension cell culture and bioproductions. Compared to classical STR, the PadReactor Mini bioreactor comes pre-sterilized and allows reduction of preparation time combined with ease of use. In addition, results generated in the small scale PadReactor Mini bioreactor are comparable to previous results in larger volume bioreactors, from to 1 L PadReactors systems 1,. This bioreactor offers a good platform for developing processes at benchtop scale before scale-up. References 1. Collignon F., CHO cells cultivation and antibodies production in a new L disposable bioreactor October.. Castillo J, Physical Characterization of a Cube-Shaped, Single-Use Bioreactor from ATMI and MAb Production at the 1L Scale, Poster ESACT 11. 4

5 Visit us on the Web at us at Corporate Headquarters Port Washington, NY, USA toll free (USA) phone European Headquarters Fribourg, Switzerland +41 () 3 3 phone Asia-Pacific Headquarters Singapore phone International Offices Pall Corporation has offices and plants throughout the world in locations such as: Argentina, Australia, Austria, Belgium, Brazil, Canada, China, France, Germany, India, Indonesia, Ireland, Italy, Japan, Korea, Malaysia, Mexico, the Netherlands, New Zealand, Norway, Poland, Puerto Rico, Russia, Singapore, South Africa, Spain, Sweden, Switzerland, Taiwan, Thailand, the United Kingdom, the United States, and Venezuela. Distributors in all major industrial areas of the world. To locate the Pall office or distributor nearest you, visit The information provided in this literature was reviewed for accuracy at the time of publication. Product data may be subject to change without notice. For current information consult your local Pall distributor or contact Pall directly. 1, Pall Corporation. Pall,, and PadReactor are trademarks of Pall Corporation. indicates a trademark registered in the USA and TM indicates a common law trademark. Filtration.Separation.Solution. is a service mark of Pall Corporation. Applikon is a trademark of Applikon Biotechnology BV and Infors is a trademark of Infors AG. /1, PDF, GN1.113 USD313

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