A streamlined single-use solution for an intensified high-density cell culture processPeggy Lio1, Zhou Jiang1, Véronique Chotteau2, Marie-Francoise Clincke2, Carin Mölleryd2, Ye Zhang2, Puneeth Samani2, Eric Fäldt3, Kieron Walsh1, Eva Lindskog3, and Christian Kaisermayer3 1 GE Healthcare Life Sciences, Westborough, MA, USA; 2 The Royal Institute of Technology, KTH, Sweden; 3 GE Healthcare Life Sciences, Uppsala, Sweden

GE, and GE monogram are trademarks of General Electric Company. ActiCHO, Cellbag, ReadyToProcess, and WAVE Bioreactor are trademarks of General Electric Company or one of its subsidiaries. Express Five is a trademark of Thermo Fisher Scientific. CHO-CD XP is a trademark of Irvine Scientific. All other third party trademarks are the property of their respective owner. © 2014 General Electric Company – All rights reserved. First published Apr. 2014. All goods and services are sold subjects to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare, Bio-Sciences AB, Björkgatan 30, 751 84 Uppsala, Sweden

04/2014 29-1082-90 AA

IntroductionProcess intensification refers to the improvement of volumetric production capacity by the use of novel technologies. To maximize the biomanufacturing output, there is an emerging trend of implementing process intensification in upstream bioprocesses. Reported applications include intensified perfusion cultures, concentrated fed-batch cultures, and high-density seed trains. Compared with conventional batch and fed-batch cultures, significantly higher cell densities and/or product yields can be achieved with intensified processes. However, for successful implementation of intensified cell cultures, challenges such as increased hardware and operational complexity need to be overcome.

With advantages such as ease of implementation and hardware simplicity, single-use systems are often applied in batch and fed-batch cell culture processes. Given these benefits, we evaluated the feasibility of running an intensified perfusion cell culture using a streamlined single-use WAVE Bioreactor™ system. Either an internal floating filter or an external hollow fiber cartridge was used as a retention device. Evaluation studies were performed using multiple cell lines at various scales. The results demonstrate the simplicity and flexibility of this single-use solution for intensified cell culture, where high cell density (200 × 106 cells/mL) and volumetric productivity of up to 2 g/L/d were achieved.

Results

Materials and methodsCell culture studies were performed using a WAVE Bioreactor 20/50 system. Used Cellbag™ Bioreactor, cell line, culture volume, culture medium, temperature, and perfusion rate are listed in Table 1.

Cell retention was conducted using either an internal floating filter or an external hollow fiber cartridge (ReadyToProcess™ RTPCFP-2-E-4X2MS 850 cm2 with 0.2 μm pore size). Tangential flow filtration (TFF) was powered by a 620 pump (Watson-Marlow). Alternating tangential filtration (ATF) was driven by an ATF2 membrane pump (Refine Technology) controlled with an ATF C24 Control unit.

Conclusions• Implementation of

intensified cell culture processes can offer significant benefits in supporting extremely high cell densities and improvements in volumetric production capacities.

• Simple implementation of intensified cell culture was demonstrated on single-use WAVE bioreactor system, with an internal floating filter or an external hollow fiber filter.

• The system was tested with multiple cell lines in various commercial media and in different scales. Top viable cell density was 214 × 106 cells/mL and the IgG productivity reached 2 g/L/d.

Fig 1. Schematic setup of an intensified cell culture using a perfusion Cellbag bioreactor.

Fig 6. Schematic setup of intensified culture in WAVE bioreactor system with hollow fiber ATF retention.

Fig 7. Intensified CHO cell culture in WAVE bioreactor with hollow fiber ATF retention.

Fig 5. Filter performance under different cell densities. Fig 2. Growth curves of CHO cells (left) and Drosophila Schneider 2 (S2) cells (right) in perfusion Cellbag bioreactor.

Table1. Materials and culture conditions of four intensified cell culture experiments

Fig 4. Intensified CHO cell culture in WAVE bioreactor with hollow fiber TFF retention.

The perfusion Cellbag bioreactor is equipped with an internal floating filter that separates the cells from culture fluid by a retentive membrane. The rocking motion of WAVE Bioreactor system resulted in alternating tangential flow of liquid medium, which sweeps up the cells from the filter surface and prevents blockage. The filtrate is transported by a harvest pump to a harvest bag. Fresh medium from a feed bag is delivered to the bioreactor by a feed pump.

Intensified cultivations of CHO and S2 cells were performed in Cellbag bioreactors according to the setup shown in Figure 1. Peak viable cell densities were 53 × 106 cells/mL and 141 × 106 cells/mL for CHO and S2 cells, respectively (Fig 2). Monoclonal antibody (MAb) productivities were 0.8 g/L/d and 0.7 g/L/d for CHO and S2 cells, respectively.

The cell suspension on the retentate side of the membrane is constantly moving in one direction, tangential to the membrane. Only a relatively small portion of the fluid passes through the membrane and is withdrawn on the permeate side. The sweeping function of the tangential flow prolongs the filter lifetime. The TFF hollow fiber is disposable and can be replaced in the process.

Intensified cultivation of CHO cells was performed according to the setup shown in Figure 3. The viable cell density increased as the perfusion rate was increased (Fig 4A). Peak cell density was over 200 × 106 cells/mL at a 10 v/d perfusion rate. Cumulative IgG yield was 145 g (total harvest) over the 48 day culture, equivalent to 48.3 g/L volumetric productivity (Fig 4B).

Pressures at three locations were measured: Pin (pressure before filtration), Pper (pressure of permeate), and Pout (pressure of retentate). In Figure 5, measured pressures were plotted against viable cell densities. Dashed lines represent trendlines. A negative value of Pper is indicative of filter fouling. Filter fouling occurred when cell density was above 150 × 106 cells/mL.

Recirculation was performed using an ATF2 membrane pump controlled with an ATF C24 Control unit (Fig 6). In alternating tangential flow, cell suspension is drawn into the hollow fiber module and expelled in cycles. The change of the fluid direction increases clearance of the membrane and can improve the filter capacity compared to TFF mode.

Intensified cultivations of CHO cells were performed according to the setup shown in Figure 6. Comparable results were achieved in two consecutive runs. Peak cell density was over 120 million cells/mL in both runs.

Intensified cell culture

CHO cell perfusion culture

S2 cell perfusion culture

CHO cell with TFF retention

CHO cell with ATF retention

Cellbag bioreactor

2 L Cellbag bioreactor

2 L Cellbag bioreactor

10 L Cellbag bioreactor

10 L Cellbag bioreactor

Initial culture volume

0.7 L 0.85 L 3 L 3 L

Cell line CHO S2 CHO-K1 CHO-K1

Culture medium ActiCHO™ medium

ExpressFive™ SFM (Thermo Fisher)

CHO-CD XP™ (Irvine Scientific)

CHO-CD XP (Irvine Scientific)

Temperature 37˚C 27˚C 37˚C 37˚C

Perfusion rate 0.5–3.2 v/d 0.3–1.4 v/d 0.5–10 v/d 1–6 v/d

Harvest pump

Feed pump

Filtrate draw-off nozzleCell retentive membrane

with polyethylene top

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Fig 3. Schematic setup of an intensified culture in WAVE bioreactor with hollow fiber TFF retention.

Feed Pin

Retentate Pout

Retentate Pper

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(B)