bag-based rapid and safe seed-train expansion method for trichoplusia ni suspension cells nicole c....
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Bag-based rapid and safe seed-train expansion method for Trichoplusia ni suspension cells
Nicole C. Bögli1, Christoph Ries1, Irina Bauer1, Thorsten Adams2, Gerhard Greller2, Regine Eibl1, Dieter Eibl1
1 Zurich University of Applied Sciences, School of Life Sciences and Facility Management, Institute of Biotechnology, Biochemical Engineering and Cell Cultivation Technique,[email protected]; Grüental, CH-8820 Wädenswil, www.lsfm.zhaw.ch, www.bioverfahrenstechnik.ch and www.zellkulturtechnik.ch
2 Sartorius Stedim Biotech GmbH, August-Spindler-Str. 11, D-37079 Goettingen, www.sartorius-stedim.com
Conclusions
Cell line, medium and procedures
Results and discussion
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References:
[1] Eibl, R. and Eibl, D. (2010): Single-use technology in biopharmaceutical manufacture, Wiley VCH.
[2] Heidemann R., Mered M., Wang D. Q., Gardner B., Zhang C., Michaels J., Henzler H. J., Abbas N., Konstantinov K. (2002): A new seed-train expansion method for recombinant mammalian cell lines. Cytotechnology 38:99-108.
[3] Heidemann R., Lünse S., Tran D., Zhan C. (2010): Characterization of cell-banking parameters for the cryopreservation of mammalian cell lines in 100 mL cryobags. Biotechnology Progress 26:1154-1163.
[4] Beltrametti T., Bögli N. C., Ries C., Greller G., Eibl R., Eibl D. (2011): Zellkultivierung in einem wellendurchmischten, DO-regulierten Einwegbioreaktor. BIOForum 1: 22-23.
Contact:ZHAW Institute of Biotechnology, WädenswilNicole Bö[email protected]
Sartorius Stedim Biotech GmbH, GoettingenGerhard Greller [email protected]
In modern vaccine manufacture, savings in time and cost which result from a reduction in cleaning and sterilization steps are mainly due to implementation of single-use systems. These savings can be increased by new innovative working procedures. The one-step expansion procedure with large-scale cryopreservation in suitable bag systems is a promising approach. It is assumed that such procedures will become important for the development and production of personalized vaccines in cell-based pharmaceuticals, the biotherapeutics of the future.
Figure 1: BIOSTAT® CultiBag RM 20 perfusion.
www.sartorius-stedim.com
For the first time a cryobag working cell bank (WCB) was established and successfully used for rapid bag-based seed train expansion of Hi-5 suspension cells. Cell production in perfusion mode seemed to be the most effective method. Stepwise increase of the perfusion rate (0.24 - 2.64 vvd -1) produced on average maximum cell densities of 4 x 107 cells mL-1 (Fig. 5) and viabilities between 97 - 99 %. Problems with clogging and fouling of the perfusion membrane were not observed during cultivation. This also applied to foaming, which was negligible. One CultiBag RM 2 L delivered cells for at least 45 cryobags, with a 50 mL culture volume and a cell density of 1.2 x 107 cells mL-1. Moreover, the perfusion procedure mode was found to eliminate time-intensive and risky cell separation and concentration procedures. As shown in Fig. 6, Hi-5 cells were grown up to a cell density of between 7 and 8 x 10 6 cells mL-1 in the BIOSTAT® CultiBag RM 20 optical, independent of inoculum origin (cryobag or shake flask) [4]. In both cases the viabilities ranged from 95 to 99% and the growth rates from 0.039 to0.042 h-1. By applying the one-step expansion procedure (workflow depicted in Fig. 7) a minimum time saving of one week and a cost saving of between 20 and 30 % are achievable.
an internal 1.2 µm membrane was established. The BIOSTAT® CultiBag RM 20 perfusion was operated with pH- and DO probes (27 °C, 0.2 vvm, DO set point 50 %). The cascade regulation described by Beltrametti [4] proved to be suitable for this procedure. After harvesting and addition of 10 % DMSO, the cells were frozen under controlled conditions at a freezing rate of 1-5 °C min-1 (Fig. 3, Fig. 4). Long-term storage of prepared cryobags took place in nitrogen (vapour phase) at -196 °C. The frozen cells from the cryobags were either directly inoculated into the
CultiBag RM after thawing in a water bath or came from 500 mL shake flasks which had been inoculated with cells from vials. At 48 and 72 hours after inoculation, feeding took place. In order to validate the one-step cell expansion procedure, cell density and viability (NucleoCounter® NC100 from Chemometec) as well as substrate and metabolite concentrations (Bioprofile 100 plus from Nova Biomedical) of off-line samples were measured daily.
Figure 3: CBS Controlled Rate Freezing System from Custombio-genics.
www.custombiogenics.com
Figure 2: Cryobags (60 mL) from KryoSure®, American Fluoroseal Corporation.
http://www.toafc.com
Figure 7: Established seed-train expansion method suitable for Hi-5 suspension cells.
Figure 6: Typical growth of inoculated Hi-5 cells from cryobags (black line) and shake flasks (dotted line). Viable cell density (●) and viability (∆) are average values and includes the standard deviation.
Figure 5: Typical growth of Hi-5 suspension cells in perfusion mode. Standard deviation is indicated by error bars.
Trichoplusia ni suspension cells (High Five™, Hi-5) used in conjunction with the baculovirus vector expression system (BEVS) are regarded as a potential production system for new, recombinant virus-like particle (VLP) vaccines. In order to advance vaccine development and production, biomanufacturers use single-use technology when- and wherever possible. This particularly applies to upstream processing and seed train expansion, where wave-mixed bag bioreactors have become the new gold standard in the last five years [1].Our approach is based on Hi-5 suspension cells and the BIOSTAT® CultiBag RM 20 perfusion (Fig. 1) from Sartorius Stedim Biotech. Cells were grown in batch, feeding and perfusion mode and were subsequently transferred and frozen in cryobags (Fig. 2) before storage in nitrogen. We investigated the suitability of using a one-step cell expansion procedure [2, 3] for speeding up seed train production. Growth and metabolism of inoculated cells from shake flasks and cryobags were compared in the Biostat CultiBag RM.
Introduction
All experiments were performed with Express Five® SFM from Gibco Invitrogen. In a first experimental series Hi-5 cells (Gibco Invitrogen) from shake flasks were grown in batch and feeding mode in CultiBag RM optical 2 L in order to generate inoculum for 60 mL cryobags. In a second experimental series, a perfusion process using the CultiBag RM perfusion (1 L working volume) with
14 - 21 d
5 d
0.5 d
Figure 4: Freezing profile using the Controlled Rate Freezer (grey: sample -, black: chamber -, blue: target temperature).