PROCESS DEVELOPMENT FOR THE LARGE SCALE PRODUCTION OF CLINICAL GRADE HUMAN MONOCLONAL ANTIBODY USING A CYTOPILOT FLUIDIZED BED REACTOR
G. Klima, G. Kreismayr, D. Müller, C. Schmatz, S. Wiederkum, W. Steinfellner, A. Assadian, G. Lhota, G. Blüml, O. Doblhoff-Dier, H. Katinger
Institute of Applied Microbiology, University of Agriculture, Vienna/AUSTRIA
URL: htt://www.boku.ac.at/iam/iam.html E.mail:iam@mail.boku.ac.at
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Introduction
Monoclonal antibodies for passive immunotherapy demand for cheap large scale manufacturing technologies. Previous studies showed that fluidized bed reactors (FBR) are a promising production system considering scale up potential and volumetric productivity.
r-CHO cells were cultivated on 200-400 ml settled volume of macroporous microcarriers (Cytoline 1, Pharmacia Biotech) in a Cytopilot-Mini FBR to optimize relevant parameters for large scale production of clinical grade material in a 120 l FBR (20-30 l of packed bed).
Main focus was put on i) balancing medium composition to enable reasonable perfusion rates, ii) optimized carrier preparation, iii) high efficiency of cell attachment during inoculation iv) fast change from serum supplemented propagation medium to protein free production medium.
Materials & Methods
r-CHO cell line
Human anti-HIV-1 monoclonal antibody IAM CHO2F5
Cytopilot fluidized bed reactor (Pharmacia Biotech)
Cytoline 1 macroporous microcarriers (Pharmacia Biotech)
DMEM :HAMS-F12 1:1
4-l stirred tank reactor (STR) for inoculum preparation (Chemap AG)
HPLC analysis of glucose and amino acid concentration
Carrier Preparation
The microcarriers were prepared and sterilised outside the FBR (e. g. in a modified 50 l vessel) and subsequently transferred into the bioreactor. This simplified the handling of large quantities of microcarriers (20-30 l) as well as the sterilisation of the pilot scale fermentation system (e. g. homogenous temperature distribution in the fermenter, separate sterility testing of fermenter and microcarriers).
Inoculation
To achieve the large number of cells needed for the inoculation of a FBR (~2*1010 cells for a 20 l carrier bed) a stirred tank reactor - operated as fed batch - was used for inocula preparation. The cells were growing as aggregates and were transferred into the FBR through the bottom valve.
To achieve a shorter lag phase the STR was fed batched again and the FBR was inoculated a second time. After reaching a minimum cell density of >1*107 cells/ml packed bed protein free perfusion was started (Fig. 1, Tab. 2).
Balancing Medium Composition
Preoptimization was carried out in T-flask batch cultures.
Due to different amino acid consumption rates in different culture systems the final formulation (Tab. 1) was determined during continuous culture in a stirred tank reactor. No amino acid limitations occurred in the FBR down to residual glucose concentrations of ~500 mg/l.
Fig. 2.a & b: Fluidized bed fermentation of CHO2F5 with 400 ml of packed Cytoline 1
Fig.2a: Cell number and perfusion rate versus time
Fig.2b: Productivity and hIgG titer versus time
Summary
Several fermentations lasting between 700-1200 hours were performed in Cytopilot fluidized bed reactors, achieving cell densities around 4-7*107 cells/ml of settled carrier volume and a productivity of approximately 100-120 mg/l carrier*d.
Perfusion of serum supplemented medium can be reduced to 50 - 100 hours depending on the cell density of the starter culture.
Acknowledgments
Part of this work was supported by Pharmacia Biotech.& Polymun Scientific Immunbiologische Forschung GmbH
5.95/96 C by IAM
