High yield antibody production in a disposable WAVE Bioreactor™

Process intensification using perfusion culture

Christian Kaisermayer1, Jianjun Yang2 1 GE Healthcare Europe GmbH, Vienna, Austria (Christian.Kaisermayer@ge.com), 2 GE China Research and Development Center Co. Ltd. Shanghai First published in May 2011 at the 22nd ESACT meeting in Vienna, Austria

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Introduction

•  Perfusion processes provide high volumetric productivity by retaining a large cell number in a relatively small volume.

•  These characteristics can be used for process intensification to obtain the same amount of product from a considerably smaller bioreactor compared to a standard batch or fedbatch process.

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Introduction •  Drosophila Schneider 2 (S2) insect cells producing a

recombinant antibody were cultivated in disposable WAVE Bioreactor™.

•  Cell growth and protein production in a perfusion and a batch process were compared.

•  Additionally the implication of both cultivation modes on the cost of consumables in the upstream process was evaluated.

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Cell cultivation and reactor setups •  S2 cells were transfected to produce a monoclonal antibody

against hemagglutinin of influenza H5N1.

•  The cells were grown in commercial serum free medium. Inoculum was prepared in shake flasks.

•  Cultivation was done in disposable WAVE Bioreactor™ at working volumes of 0.85 L.

•  Oxygen was supplied by adjusting agitation and increasing O2 concentration in the headspace.

•  In perfusion, cell retention was achieved by a filter integrated into the bioreactor.

•  The perfusion rate was controlled via an integrated loadcell that allowed periodic addition and removal of equal amounts of feed and harvest.

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Results

Fig. 1: Cell concentration and viability. Batch culture in red, perfusion culture in blue. Circles: cell concentration, triangles: viability. Bold line indicates perfusion rate.

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Results •  Perfusion had a drastic effect on cell concentration and

viability.

•  As shown in Fig. 1, a tenfold higher cell concentration than in the batch culture was reached.

•  Additionally a viability higher than 95% was maintained throughout the process.

•  In combination this led to a 10 times larger viable cell integral for the perfusion culture.

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Results

Fig. 2: Cell specific productivity (bars), specific perfusion rate (line) and IgG concentration (dots) during perfusion culture

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Results •  Utilization of cell culture medium was optimized by adjusting

the perfusion rate to ensure maximum product concentration but at the same time avoid nutrient limitation of the cells.

•  The sudden decrease of the cell specific productivity on day 19 (Fig. 2) probably is the result of such a limit.

•  A residual glucose concentration of about 3 g/L had to be maintained to avoid depletion of other nutrients.

•  With the increased perfusion rate on day 20 and 21 cells recovered, reaching at least the same specific productivity as during the initial phase of the recombinant protein production.

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Results

Fig. 3: IgG concentration (circles) and volumetric productivity (bar graph). Protein production was induced on day 10. Batch culture in red, closed symbols, perfusion culture in blue, open symbols

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Results •  Volumetric productivity (STY) was considerably higher in

perfusion culture as shown in Fig. 3.

•  One of the reasons was the higher viable cell concentration, additionally also the cell specific productivity was improved more than twofold compared to the batch culture (Fig. 4).

•  The high and stable productivity in perfusion culture can most likely be attributed to the consistent metabolite concentrations.

•  As shown in Fig. 3 there were 6 days during the perfusion culture where a protein output of more than 1 g per liter working volume was achieved.

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Results

Fig. 4: Comparison of cell concentration, productivity and consumable cost in both culture modes

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Conclusions

•  Perfusion allowed a 20-fold increase of the volumetric productivity compared to batch culture

•  The upstream consumable cost per 100 mg IgG decreased by 85%

•  A viable cell concentration of about 1*108 c/ml was maintained for one week

•  Use of a disposable bioreactor allows short turnover times, improving equipment utilization

•  Despite the high cell concentration, no DO limits or clogging of the retention filter were observed during 3 weeks process time

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Acknowledgments The S 2 cell line used in this study, was generously provided by Lulan Wang and Paul Zhou, Institut Pasteur, Shanghai

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First published May 2011.