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/Germany
Chair 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
Influenza Virus / Cell Lines
http://www.virology.net/Big_Virology/BVRNAortho.html
Human Influenza- A/Puerto Rico/8/34/ (H1N1)- Seasonal strains (A/B)- Attenuated strains- del NS1 mutant
Equine / Porcine Influenza
Hemagglutinin
Neuraminidase
Lipid Envelope+ M2 protein
Matrix Protein M1
Ribonucleocapsid
Ø 70-120 nm
2
MDCK on Cytodex 1, LSM image
215 µm
- MDCK (Madin Darby Canine Kidney)- Vero (African Green Monkey Kidney)
- Epithelial cells, polarized- Adherent => Microcarrier culture! Process options: Suspension cells- MDCK.SUS2, HEK293.SUS- AGE1.CR, AGE1.HN (ProBioGen)- CAP (Cevec Pharmaceuticals
GmbH)
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
Max Planck Institute Magdeburg 5Continuous Production of Influenza Virus October 2013
Propagation of Influenza Virus A/PR8/34 H1N1 in AGE1.CR (DUCK) Cells
0.0E+00
1.0E+06
2.0E+06
3.0E+06
4.0E+06
5.0E+06
6.0E+06
0 2 4 6 8 10 12 14 16 18
viab
le c
ell c
onc.
(cel
ls/m
L)
time (d)
cell bioreactor
virus bioreactor
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
1.0E+08
1.0E+09
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 2 4 6 8 10 12 14 16 18
log10
HA u
nits /
100
µL
time (d)
HA
TCID50
TCID
50(v
irions
/mL)
0.0E+00
1.0E+06
2.0E+06
3.0E+06
4.0E+06
5.0E+06
6.0E+06
0 2 4 6 8 10 12 14 16 18
viab
le c
ell c
onc.
(cel
ls/m
L)
time (d)
cell bioreactor
virus bioreactor
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
1.0E+08
1.0E+09
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0 2 4 6 8 10 12 14 16 18
log10
HA u
nits /
100
µL
time (d)
HATCID50
+T +V
+V
+T
TCID
50(v
irions
/mL)
+V
1st continuous cultivation 2nd continuous cultivation
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 2013
FL = 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
Resin: Sepharose 4FF (GE)CV: 23 mL, Injection: 0.15 CV Kalbfuß et al. (2007) B&B 96(5):932
Batch Mode: Size Exclusion Chromatography
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 12October 2013
SMB System (Knauer), column switching valveColumns: Sepharose 4FF, 2 mL CV
1 2
3
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 13
SMB: Equipment
• SMB System (Knauer)column switching valve
• Columns: Sepharose 4FF, 2 mL CV
October 2013
Kröber et al. (2013) accepted
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 14
SMB: Design
October 2013
• Internal and external flow rates determine separation performance
• Dimensionless flow rate ratios (TMB):z=I, II, III; s=solid
• For linear adsorption the following Inequalities have to be fulfilled to achieve complete separation*
(mIV<Kv) Q I=QDQ II=Q I−QEQ III=Q II+QF=QR
flow rate balances:
"Triangle theory", Mazzotti et al. (1997) JChrA 769:3
Q I=QDQ II=Q I−QEQ III=Q II+QF=QR
flow rate balances:
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 15
SMB: Operating Points
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.2
0.4
0.6
0.8
1.0
(0.33; 0.47)
(0.45; 0.62)
(0.72; 0.93)
(0.20; 0.32)
mII
mIII
October 2013
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 16
Performance: 1 Column per Zone
October 2013
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80%
20%
40%
60%
80%
100%
HA yield (Raffinate)
mII
% o
f fe
ed
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80.0
1.0
2.0
3.0
protein contamination
mII
µg
/kH
AU
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.820
25
30
35
40
productivity w/o CIP
mII
kH
AU
/(m
L*h
)
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80%
20%
40%
60%
80%
100%
protein depletion
mII
% o
f fe
ed
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 17
SMB: Operating Points
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00.0
0.2
0.4
0.6
0.8
1.0
mII
mIII
October 2013
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 18
Performance: 1 and 2 Columns per Zone
October 2013
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80%
20%
40%
60%
80%
100%
HA yield (Raffinate)
mII
% o
f fe
ed
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80.0
1.0
2.0
3.0
protein contamination
mII
µg
/kH
AU
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80
10
20
30
40
productivity w/o CIP
mII
kH
AU
/(m
L*h
)
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80%
20%
40%
60%
80%
100%
protein depletion
mII
% o
f fe
ed
1 column per zone 2 columns per zone
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 19
SMB with Anion Exchange Column
• DNA co-elutes with virus in the raffinate
• Anion exchange (AEX) column to bind DNA(1 mL CaptoQ, GE)
• Virus flows through
October 2013
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 20
Performance: SMB and AEX
October 2013
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80%
20%40%60%80%
100%
HA yield (Raffinate)
mII
% o
f fe
ed
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80.000.501.001.502.002.503.00
protein contamination
mII
µg
/kH
AU
1 column per zone2 columns per zoneSMB and AEX
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80%
20%40%60%80%
100%
protein depletion
mII
% o
f fe
ed
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80%
20%
40%
60%
80%
100%
DNA depletion
mII
% o
f fe
ed
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 runs
141
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 to
Jun. Prof. Dr. T. FrensingS. HeldtPD. Dr. Y. Genzel
T. KröberL. FischerDr. M. Wolff
… of BPE Group
Prof. A. Seidel-MorgensternMPI Magdeburg
and
IDT-Biologika GmbH, Dessau
ProBioGen AG, Berlin
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 24
TMB (true moving bed)
October 2013
Seidel-Morgenstern 2008
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 25
TMB Design: Triangle Theory
Mass balances:
Dimensionless flow rate ratio:
October 2013
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 26
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
Max Planck Institute Magdeburg Continuous Production of Influenza Virus 27
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