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

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 bioreactorvirus 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

log

10HA

units

/ 10

0 µL

time (d)

HATCID50

TCID

50(v

irion

s/m

L)

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 bioreactorvirus 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

log

10HA

units

/ 10

0 µL

time (d)

HATCID50

+T +V

+V

+T

TCID

50(v

irion

s/m

L)

+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 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

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 2013Kröber 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 fee

d

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/k

HA

U

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

kHA

U/(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 fee

d

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 fee

d

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/k

HA

U

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

kHA

U/(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 fee

d

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 fee

d

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/k

HA

U

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 fee

d

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 fee

d

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

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

Max Planck Institute Magdeburg Continuous Production of Influenza Virus 28

Productivity Schemes

October 2013