Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D; community with critical information on the tools, technologies, and trends that drive the biotech industry.
Issue link: http://gen.epubxp.com/i/695748
28 | JULY 2016 | GENengnews.com | Genetic Engineering & Biotechnology News Michael McGlothlen and Damon R. Asher, Ph.D. The future of infuenza vaccine production most likely lies with cell culture-based pro- cesses. Cell-based systems offer numerous advantages over traditional egg-based produc- tion, including speed of production, lack of dependence on egg supply, avoidance of egg allergens, higher initial purity, and ability to combine upstream and downstream into an automated process. Cell culture is well suited to rapid pan- demic response because time-consuming re- combination of the virus into an egg-adapted fu strain is not required. Cell-based process- es can therefore produce large quantities of vaccine in a relatively short period of time, provided they can scale up effciently. Growth of infuenza virus in single-use bioreactors and methods for scaling up pro- duction are the topics of this article. Infuenza virus is typically grown on ad- herent cells such as the Madin-Darby canine kidney (MDCK) cell line. These types of cells have traditionally been cultured using 2D tissue culture fasks or roller bottles that are ineffcient to scale to industrial volumes. The Mobius ® 3L single-use stirred-tank bioreac- tor is a benchtop vessel suitable for cell-based vaccine process development. This tutorial focuses on the optimization of an MDCK cell-based infuenza production process us- ing Cytodex ® 3 and Cytodex 1 microcarriers in a Mobius 3L single-use stir tank bioreac- tor and scale-up of this process to a Mobius 50L bioreactor (Figure 1). Effcient viral production in a stirred-tank bioreactor requires optimization of numerous parameters, including cell attachment to the microcarriers, suspension of the microcar- riers, cell growth on the microcarriers, virus infection of the cells, and viral output. Baffed shake fasks (125 mL with 40 mL work- ing volume) were found to be predictive of stirred-tank performance and were used for preliminary experiments. The parameters tested and the optimal conditions found are shown in Table 1. Cell Attachment Previous practices for cell attachment to microcarriers recommend using a reduced media volume to increase microcarrier density and intermittent agitation to produce cycles of microcarrier suspension and settling. However, this complex regimen requiring programmed agitation cycling and an additional media feed- ing was found to be unnecessary to achieve optimal microcarrier attachment. Effcient at- tachment was achieved by inoculating the Mo- bius 3 L bioreactor with 4 g/L of Cytodex 3 or Cytodex 1 microcarriers and MDCK cells at a density between 2 × 10 5 and 3 × 10 5 cells/mL in the full working volume of 2.0 L media. The bioreactor was simply agitated con- tinuously at 90 rpm for 24 hours to complete attachment. The media used for both attach- ment and cell growth was Dulbecco's Modi- fed Eagle's medium (DMEM) with glucose with 10% fetal bovine serum (FBS), 4mM L-glutamine, 1% nonessential amino acids (NEAA) and 1% sodium pyruvate. Cell Growth Agitation speed is a critical parameter for cell growth on microcarriers. As cells grow, the microcarriers will gain mass and tend to settle if undersuspended. However, overly vigorous agitation can cause deleterious sheer stress on the cells. Experimentation revealed that 90 rpm was the optimal agitation speed for cell growth. This speed equates to an impeller tip speed of 35.9 cm/s and a power input of 1.3 W/m 2 in the Mobius 3L bioreactor. Sparging strategy is another important consideration. Sparging needs to supply enough oxygen to support viable cell growth, but excessive sparging will impart shear stress on culture. The Mobius 3L bioreactor can be used with either a microsparger or open O 2 pipe. The latter was found to be op- timal, avoiding the foaming and sheer stress on the cells caused by the microsparger. Cells were grown for four days at 37°C to a density of 2 x 10 6 viable cells (vc)/mL. The culture was automatically controlled to main- tain dissolved O 2 (DO) at 50% ± 10% and pH at 7.0 ± 0.2 throughout the cell growth phase and the subsequent infection phase. Virus Infection Correctly timing the infection of the cul- ture is important for maximizing virus pro- duction. The optimal time for infection was found to be at ~4 days of culture growth, when the cells had reached a density of be- tween 2 × 10 6 and 3 × 10 6 vc/mL. At this time, agitation was turned off so that the Biomanufacturing on Adherent Cells Using a Single-Use Stirred-Tank Bioreactor Process Scalable Production of Infuenza Virus BIOPROCESSING Tutorial Table 1. Optimization of Cell-Based Flu Production at 3 L Scale Parameter Condition Tested Optimal Condition In Bafed Shake-Flask Format Inoculation density (cells/mL) 1x10 5 , 2x10 5 , 4x10 5 , 8x10 5 2x10 5 – 3x10 5 Microcarrier density (g/L) 2, 4, 6 4 Multiplicity of infection (pfu/cell) 1 x 10 -4 , 5 x 10 -4 , 1 x 10 -3 5 x 10 -4 Time of infection (days after cell inoculation) 2, 3, 4 4 (or cell density > 2 x 10 6 vc/mL) In Mobius® 3 L Bioreactor Agitation speed (rpm) 75, 90, 150 90 Starting volume (L) 1.0, 1.6, 2.0 2.0 Sparging O2 open pipe, O2 microsparger, Air/O2 microsparger O2 open pipe Figure 1. Mobius® 3 L and 50 L single-use bioreactor systems Figure 2. MDCK cell growth at 3 L and 50 L scales. VCD: viable cell density. Michael McGlothlen is manager, bioprocess solutions, upstream process innovation center, and Damon R. Asher, Ph.D., serves as associate director, Americas vaccine market, process solutions, at MilliporeSigma. Website: www.milliporesigma.com