Performance Comparison of CHO-S Cell Cultivation and mAb Production in the Allegro™ STR 200 Single-Use Bioreactor and in Conventional Single-Use Bioreactor

Pall: Odette Becheau, Emmanuelle Cameau , John H. Welsh , Kyle Jones, Joe Capone and Marie-Laure Collignon

ZHAW: Sören Werner , Cedric Schirmer, Nina Steffen, Jan Müller, Regine Eibl and Dieter Eibl

INTRODUCTION

Today, the benefits of single-use (SU) technologies for upstream processing are much more prevalent and heavily adopted within biopharmaceutical industry. Pall has developed the Allegro™ STR stirred single-use bioreactor family which is currently available in 4 sizes: STR 50, STR 200, STR 1000, and STR 2000 (Figure 1). The direct-bottom mechanically driven impeller allows large range of power inputs from 0.002 W/kg up to 0.3 W/kg, while the macro sparger seated below the three 45-degree elephant ear blades results in high oxygen transfer rates (kLa up to 40 h^-1) and short mixing time (minimum t_M~10 s).

Previous studies highlighted that the critical parameters to be kept constant from one scale to another are the volumetric power input (P/V) for agitation and the superficial gas velocity (vs) for aeration. This will successfully scale up a process at constant kLa, keeping equivalent mixing time and shear stress environment in the bioreactor.

MATERIALS AND METHODS

Cell Line, Medium and Supplements

• Chinese hamster ovary (CHO)-S clone 38 cell line, producing a human IgG antibody
• ActiPro♦ medium (GE Healthcare) supplemented with:
  • 4 mM UltraGlutamine♦ 1 (Lonza)
  • 1x Gibco♦ HT Supplement mixture (Thermo Fisher Scientific)
  • Puromycin (1.25 g/L, 10 mg/mL)
• Feed with HyClone♦ Cell Boost 7a and Cell Boost 7b (GE Healthcare) start in day 3
• Glucose solution (45%, 450 g/L) for feed if glucose was below 4 g/L
• Antifoam C emulsion (Sigma Aldrich)

Bioreactors

• 125 and 500 mL Erlenmeyer shake flasks (Corning) for inoculum production
• Flexsafe♦ RM wave bioreactor bag (Sartorius) for inoculum production
• Allegro STR 200 single-use bioreactor system with heat mat (Pall)
• Conventional single-use bioreactor

Operating parameters for the cultivation for Pall’s Allegro STR 200 single-use bioreactor and for the conventional single-use bioreactor are shown in Table 1

Analytics

• Polarographic oxygen sensor InPro♦ 6800 (Mettler-Toledo) for Allegro STR 200 single-use bioreactor
• pH sensor (EasyFerm Hamilton) for Allegro STR 200 single-use bioreactor
• Single-use pH and oxygen sensor incorporated in conventional single-use bioreactor
• BioProfile♦ 100 Plus ID 2 (Nova Biomedical)
• Cedex HiRes and Cedex Bio Analyzer (Roche)
• Prominence HPLC System (Shimadzu)
• Xevo♦ G2 LC-MS and Acquity H-Class Bio UPLC systems (Waters)

RESULTS

• Simplified handling of a novel bioreactor system
• Inoculum production with shake flask and wave-mixed bag is an appropriate method
• Average peak cell density in the Allegro STR 200 single-use bioreactor at the end of the growth phase was 2.15 · 10⁷ cells·mL^-1 (Figure 2a)
• Cell metabolism in terms of glucose uptake and lactate formation is very comparable (Figure 2b)
• Average monoclonal antibody (mAb) titer of 2.18 g·L^-1 at the culture harvest day (day 10) (Figure 2c)
• The main glycan composition shows a similar finger print for both bioreactors flor species G0F, G1F and G2F (Figure 2d)
• Aggregates in both systems and amount of molecular weight species (LMW) (Figure 2e)

CONCLUSION

• The Pall Allegro STR 200 stirred single-use bioreactor demonstrated excellent performance to successfully cultivate this CHO culture
• The large elephant ear impeller exhibits efficient mixing and effective oxygen transfer. No limitations could be detected during the cultivations
• The profiling of mAb quality and the mAb quantity show that the single impeller's shear profile does not influence the antibody
• Further, using one single impeller only, feeding processes can be applied with high degree of possible working volume

References

1. Schirmer, Müller J., Steffen N., Werner S., Eibl R. & Eibl D., “How to produce mAbs in a cube-shaped stirred single-use bioreactor at 200 L scale”. R. Pörtner, editor. Animal Cell Biotechnology. Methods in Molecular Biology (Methods and Protocols), Fourth Edition. New York, Springer Science+Business Media, LLC (Publication forthcoming)
2. “Characterization and engineering performance of the Allegro STR 1000 stirred single-use tank bioreactor” Pall Application Note USD 3136
3. “Cultivation of chinese hamster ovary (CHO) cells in Allegro STR 1000 stirred single-use tank bioreactor system” Pall Application Note USD 3135