Max Planck Institute Magdeburg Continuous Production of Influenza Virus 1 October 2013 Max Planck Institute Magdeburg Options for continuous production of cell culture-derived viral vaccines MAX-PLANCK-INSTITUT DYNAMIK KOMPLEXER TECHNISCHER SYSTEME MAGDEBURG Frensing, T., Heldt, S., Jordan, I., Genzel, Y., Kröber, T., Hundt, B., Wolff, M., Seidel-Morgenstern, A., Reichl, U. Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg/Germany Chair for Bioprocess Engineering, Otto-von-Guericke University, Magdeburg/Germany Motivation: Influenza virus production Upstream processing o Host cells o Virus strains o Cultivations conditions Downstream processing o Batch versus SMB mode o Virus strains o Cultivations conditions o Summary Summary and outlook
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Max Planck Institute Magdeburg Continuous Production of Influenza Virus 1October 2013Max Planck Institute Magdeburg
Options for continuous production of cell culture-derived viral vaccines
MAX-PLANCK-INSTITUTDYNAMIK KOMPLEXER
TECHNISCHER SYSTEMEMAGDEBURG
Frensing, T., Heldt, S., Jordan, I., Genzel, Y., Kröber, T., Hundt, B., Wolff, M., Seidel-Morgenstern, A., Reichl, U.
Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg/GermanyChair for Bioprocess Engineering, Otto-von-Guericke University, Magdeburg/Germany
Motivation: Influenza virus production Upstream processing
o Host cellso Virus strainso Cultivations conditions
Downstream processing o Batch versus SMB modeo Virus strainso Cultivations conditionso Summary
Summary and outlook
Max Planck Institute Magdeburg Continuous Production of Influenza Virus October 2013
Max Planck Institute Magdeburg Continuous Production of Influenza Virus
Cell Culture-based Influenza Virus Production
3
cell line origin tissue
MDCK dog kidney
Vero monkey kidney
MDCK.sus dog kidney
Vero.SUS monkey kidney
HEK293.sus human embryonic kidney
CAP human amniocytes
AGE1.CRAGE1.CR.pIX
duck retinoblasts> 1 x 107 cells/mL
October 2013
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 4October 2013
Process Option: Continuous Cultivation?
Frensing & Heldt et al. (2013) Continuous Influenza Virus Production in Cell Culture Shows a Periodic Accumulation of Defective Interfering Particles PLOS ONE, 8(9), e72288
ureichl
DI particles probably in most animal virus systems, not found for poxviruses, parvoviruses, and coronaviruses(A.S., Huang (1973), Annu. Rev. Microbiol. 1973, 27, 101-118
Max Planck Institute Magdeburg 5Continuous Production of Influenza Virus October 2013
Propagation of Influenza Virus A/PR8/34 H1N1 in AGE1.CR (DUCK) Cells
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 6October 2013
viral proteins
Influenza virus genome segment
DI genome
Fluctuations Can Be Explained by Increase and Decrease of Defective Interfering Particles
Infectious virion
Defective interfering particles
Non-infectious virions
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 7October 2013FL = full-length; DI = defective interfering
Segment-specific PCR for the Detection of Full-length and Defective Interfering Genome Segments for 2nd cultivation
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 8October 2013
Mathematical model without DIPs
Mathematical model with DIPs
Model of Continuous Infection in the Presence and Absence of DIPs
Frensing & Heldt et al. (2013) Continuous Influenza Virus Production in Cell Culture Shows a Periodic Accumulation of Defective Interfering Particles PLOS ONE, 8(9), e72288
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 9
Summary / Outlook USP
• Continuous cultivations are possible but significant reduction of virus yield by formation of defective interfering particles (DI)
• Impact of DI particles depends on quality of virus seed, but DI particle formation cannot be completely prevented by conventional methods
• Formation of DI particles is reported for most animal viruses – except poxviruses (MVA), coronaviruses, and parvoviruses?
• Productivity of continuous cultivation superior if drop in HA titer is less than 0.6 log units, and
• we also need to consider genetic stability of product (virus, typically limited to max. 5 passages) and inactivation (14 d) if DSP is not on BSL 2/3
October 2013
Continuous cultivations useful• to investigate mechanisms of DI particle formation• to perform virus evolution studies, e.g.
- one strain over time (mutation rate, adaptation, etc.)- co-infections
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 10October 2013
Cell Culture(adh. MDCK cells,A/PR/8/34 (H1N1))
Filtration(5, 0.65, 0.45 µm)
Inactivation(ß-PL)
Concentration 10x(750 kDa, cross-flow)
Batch Chromatography
Batch limited in- Processing
volume- Throughput- ScalabilityKalbfuss, B. et al. (2007): Biotechnol. Bioeng. 96(5): 932-944;
Optiz et al. (2007) J. Biotechnol 131:309-317
50 kHAU/mL130 µg/mL protein9 µg/mL DNA
Downstream ProcessingGeneric Process
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 11October 2013
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 21
Productivity: Batch vs. SMB
October 2013
batch SMB• Max. flow rate mL/min
4.0 1.9
• HA yield % feed80 95
• Depletion % feed protein 60*47
DNA27* 5
• Protein contamination µg/kHAU 1.17 1.55
• Productivity kHAU/(mL*h)36
– max. flow rate 4551
– incl. CIP 8 22
* tailing,=> conservative cut-off
Extrapolation for 100 L concentrated cell culture broth:
Batch SMB
Max. flow ratecm/h
138 275
Column dimensions (di/L) m
0.5 / 0.3 0.4 / 0.1
Resin volumeL
593x13.4
Number of runs14
1
Process timeh
6.1* 5
*w/o CIP!
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 22
Summary DSP
• Comparison of batch and SMB– Similar separation performance
High HA yield Sufficient protein depletion (< 100 µg/strain)
– Productivity of SMB higher than batch, i.e. when CIP is considered– SMB needs lower total resin volume (i.e. validated backup columns)
• Combination of SEC and AEX removes 99% of DNA, however, contamination levels still exceed limits of regulatory guidelines for human influenza vaccines (<10 ng)
Outlook• Investigation of batch to batch variations and other viral strains• Benzonase® treatment to further reduce DNA contamination levels• Use of various other matrices in SMB (SEC, affinity, membranes)
October 2013
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 23October 2013
Special thanks toJun. Prof. Dr. T. FrensingS. HeldtPD. Dr. Y. Genzel
T. KröberL. FischerDr. M. Wolff
… of BPE GroupProf. A. Seidel-MorgensternMPI Magdeburg
and
IDT-Biologika GmbH, Dessau
ProBioGen AG, Berlin
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TMB (true moving bed)
October 2013
Seidel-Morgenstern 2008
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TMB Design: Triangle Theory
Mass balances:
Dimensionless flow rate ratio:
October 2013
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SMB Design: TMB Conversion Rules
• TMB conversion rules corresponding SMB
• BUT: SMB with only few columns doesn‘t meet the ideal TMB case
October 2013
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Calculation of productivity
for calculation regeneration with NaOH (2 CV) and reequilibration with buffer (3 CV) was considered:
batch:
SMB: Ncol = 7 (column 4 to 7: regeneration and reequilibration)
October 2013
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