1st Workshop European Network of Viral Vaccines Processes, October 14-15, 2010, Frankfurt am MainStefan Fischer-Frühholz, Laura Chirica, Miyako Hirai, Rene Faber, Uwe GottschalkSartorius Stedim Biotech GmbH

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Virus Purification with Membrane Chromatography

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1. Outline single use Membranes

1. Outline single use Membranes

2. Virus removal2. Virus removal

Agenda

3. Virus purification3. Virus purification

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Diffusion limited gels versus convective limited membranes (high flow rate)

Average pore size 15 - 40 nm

Average pore size 3 - 5 μm

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Mainly ion exchangers for removal & purification of viruses

nano

1 ml

mini 5” 10” 20” 30” mega nano

3 ml

150 ml Jumbo 5 l

Q Q

S S

STIC

HIC

Contaminant removal: flowthrough mode to remove DNA, Host cell

proteins, endotoxins, viruses

Singe-use

Purification: bind & elute of viruses and virus like particles, large proteins

Single-use / intra-batch use

4 mm bed 8 mm bed

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Membrane Adsorbers - highest growth of single-use solutions

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15 reasons whysingle-use:

Reduction of complexity

No cleaning validation

Reduce time to get facility

running

Moreconvenient

Decreaseproduct

contamination

Assure sterilityIncreasefacilityoutput

Lowermaintanance

cost

Reduce space

Reduce waterand buffer

consumption

Product fasterto market

Moreflexible

Reducehazardouscleaningsolutions

Faster optimization

Simplifyoperations

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Further acceptance for single-use / intra batch reuse implies:

• Standardizaton of validation requirements

• Technology innovation & integration

• Scalable ranges & proof of scalability

• High volume, high flow systems from 2 to 2,000 L

• Product quality, robustness and integrity

• Delivery reliability & security of supply

• Cost improvements

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1. Outline singe use membranes

1. Outline singe use membranes

2. Virus removal2. Virus removal

Agenda

3. Virus purification3. Virus purification

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Virus removal study - polishing after CIEX column

Virus Enveloped LRV Run 1 LRV Run 2 Virus recovery (%)

≥ 6.03 ± 0.20 100

100

> 70

100

≥ 6.94 ± 0.24

≥ 5.52 ± 0.27

≥ 5.58 ± 0.22

≥ 6.03 ± 0.21

≥ 7.00 ± 0.31

≥ 5.35 ± 0.23

≥ 5.58 ± 0.28

MVM: Minute Virus of Mice no

Reo-3: Reovirus Type III no

yes

yes

MuLV*: Murine Leukemia Virus

PRV: Pseudorabies virus

pH: 7.2, conductivity: 4 mS/cm, flow rate: 450 cm/h, 1 % spikeprotein concentration: 4.3 g/l Mabload: 10.9 kg/l membrane (3 kg/m²) *LRV = 5.59 at 600 cm/hr

Zhou J and Tressel T: Basic Concepts in Q Membrane Chromatography for Large-Scale Antibody Production. Biotechnol Prog., 22 (2) 341–349, 2006

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Sartobind Q Sartobind STIC

Protein Binding [g/l]

BSA in 200 mM NaCl (20 mS/cm) 3.6 36

DNA Binding [g/l]

DNA in 50 mM NaCl (7 mS/cm) 7.3 22

LRV with Mouse Minute Virus (MMV)Fraction 1, 150 mM NaClFraction 2, 150 mM NaCl

2.101.81

3.82> 4.96

Salt tolerant anion exchanger Sartobind STIC (-NH2 ligand)

Sartobind STIC PA (primary amine)- provides higher binding capacity compared to Q anion exchanger at

higher conductivity- enables the polishing at broader operating conditions (high salt)

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Phosphate and other multivalent anions inhibit the binding of ΦX174 (virus surrogate)

Load 4*107 pfu/ml ΦX174, LP15, flow rate 20ml/min

00,5

11,5

22,5

33,5

44,5

5

25mMTRIS/HCl

25mMTRIS/HCl

25mMTRIS/HCl

25mMTRIS/HCl

20mMKPi

20mMKPi

20mMKPi

20mMKPi

7,5 7,5 8 8 7,5 7,5 8 8

0 150 0 150 0 150 0 150

LRV

Sartobind Q

Sartobind STIC

Buffer

pH

NaCl [mM]

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Phosphate inhibits phage binding but DNA contaminant is bound Sartobind STIC to be used at binding conditions of viruses

Ortho phosphate

mM

Phage in flowthrough

% of start material

DNA in flowthrough

% of start material

0 <0.00001 <1

2 <0.0001 <1

10 0.001 <1

30 75 - 83 <1

15 cm² (3 layers) Sartobind STIC PA were loaded with 150 ml ΦX174 1 x 107 PFU/ml, salmon sperm DNA 200 ng/ml at 10 ml/min was added.

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1. Outline single use membranes

1. Outline single use membranes

2. Virus removal2. Virus removal

Agenda

3. Virus purification3. Virus purification

Page 14

Virus capture / purificationSingle-use / intra batch reuse

• Influenza virus2,3

• Adenovirus4,5,6

• Lentivirus7

• Adenoassociated virus

• Baculovirus8

• Densonucleosis virus9

• Pseudorabies virus10

• Bovine herpesvirus1

• Foot and mouth desease virus11

• Rotavirus like particles12

• Bacteriophages13

• Norovirus (VLP)14

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Membrane Adsorbers vs. Density Gradient

Membrane Adsorbers Density gradient ultra centrifugation

2 hours 36 hours

Up to 1013 VP/ml 106 VP/ml

No carryover, disposable, no validation, simple

Carryover validation

No contaminants Toxic CsCl2, sucrose removal from finished product

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Purification of PRV Virions with S Cation Exchanger

- The virulent wild type NIA3 strain of Pseudorabies virus as well as its GFP expressing derivative were grown by infecting confluent PK15 cell monolayers

- After a 48 h growth period, the cell culture medium, containing progeny virions, was collected, chilled on ice and clarified by centrifugation at 4°C.

- Virions were purified by ion exchange chromatography on Sartobind S cation exchanger membranes as described previously [45] except that SingleSep minicapsules were used

Flori L et al., Transcriptomic analysis of the dialogue between Pseudorabies virus and porcine epithelial cells during infection. BMC Genomics 2008, 9:123INRA, DGA, UMR 314, Laboratoire de Radiobiologie et d'Etude du Génome, France

[45] Karger A, Schmidt J, Mettenleiter TC: Infectivity of a pseudorabies virus mutant lacking attachment glycoproteins C and D. J Virol 1998, 72:7341-7348

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Purification of Baculoviruses with Anion Exchanger

- Recombinant baculoviruses (rBVs) are widely used as vectors for the production of recombinant proteins in insect cells.

- A complete downstream process comprising three steps was successfully developed:- depth filtration - ultra/ diafiltration- membrane adsorption (Sartobind D)

- Global recovery yields reaching 40% (at purities over 98%) and, most importantly, relies on technologies easy to transfer to process scales under cGMP guidelines.

Vicente T et al, Purification of recombinant baculoviruses for gene therapy using membrane processes. Gene Therapy (2009), 1–10IBET/ITQB-UNL, Animal Cell Technology Laboratory, Portugal

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Purification of Influenza Viruses with Q Anion Exchanger

- Human and equine influenza A virus in cell culture supernatant (serum-free and serum-containing cultivation) was directly adsorbed to Sartobind Q and D 75 anion-exchangers.

- Elution of adsorbed virus from Sartobind Q by displacement with sodium chloride (up to 1.5 M, pH 7.0) resulted in average yields of 86% (based on HA activity).

- Due to their high productivity, ease of operation and acceptable yields Sartobind Q anion-exchangers can be considered promising candidates for the large-scale purification of cell culture derived influenza virus.

Kalbfuss B et al., Direct capture of influenza A virus from cell culture supernatant with Sartobind anion-exchange membrane adsorbers. Journal of Membrane Science 299 (2007) 251–260Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Germany

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Purification of PPV Virus Spike

PDA Technical Report No. 47: Preparation of virus Spikes used for Virus Clearance Studies 2010, p19-20

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Current applications for vaccines

Capture

Influenza vaccine

Rabies vaccine

Adenovirus-vectored vaccine

Polio vaccine

Conjugated vaccine

VLP

Polishing

DNA removal

HCP removal

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Summary

• Membrane chromatography is established in polishing applications

• Tool for virus removal in biopharmaceutical production as single-use solution

• Many applications for virus capture – first uses in the vaccine industry

• Driving force for using membrane chromatography: productivity and ease of use.

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References

1. Karger A, Schmidt J, Mettenleiter TC. Infectivity of a pseudorabies virus mutant lacking attachment glycoproteins C and D. J Virol 1998, 72, 7341-7348

2. Kalbfuss B, Wolff M, Geisler L, Tappe A, Wickramasinghe R, Thom V, Reichl U. Direct capture of influenza A virus from cell culture supernatant with Sartobind anion-exange membrane absorbers. J. Membrane Sci 2007, 299, 251-260

3. Opitz L, Lehmann S, Reichl U, Wolff MW. Sulfated membrane adsorbers for economic pseudo-affinity capture of influenza virus particles. Biotechnol Bioeng2009,103(6), 1144-1154

4. Zeidler R, Fischer-Frühholz S. Schnelle und einfache Reinigung von Adenoviren. F&D LP, 2004, Sep, 28-29

5. Delmdahl N. Fast, effective and safe adenovirus purification with Vivapure AdenoPACK kits. Nature Methods 2006, Aug

6. Peixoto C, Ferreira TB, Sousa MF, Carrondo MJ, Alves PM. Towards purification of adenoviral vectors based on membrane technology. Biotechnol Prog 2008, 24(6), 1290-6

7. Vivapure® Virus Purification and Concentration Kits. Brochure 85030-530-78, Sartorius Stedim GmbH

8. Vicente T, Peixoto C, Carrondo MJT, Alves PM. Purification of recombinant baculoviruses for gene therapy using membrane processes. Gene Therapy, 2009, 16, 766–775

9. Specht, R, Han, B, Wickramasinghe, SR, Carlson, JO, Czermak, P, Wolf, A, Reif, O-W. Densonucleosis virus purification by ion exchange membranes. Biotechnol Bioeng 2004, 88(4), 465-473

10. Karger A, Schmidt J, Mettenleiter TC. Infectivity of a pseudorabies virus mutant lacking attachment glycoproteins C and D. J Virol 1998, 72, 7341-7348

11. Oswald T. DSP in Vaccine manufacturing and development. Downstream Technology Forum, London, Nov. 2004

12. Vicente T, Sousa MFQ, Peixoto C, Mota JPB, Alves PM, Carrondo MJT. Anion-exchange membrane chromatography for purification of rotavirus-like particles. Journal of Membrane Science 311 2008 270–283

13. Sartorius Stedim Biotech Application Note Capture of Bacteriophage PR772. 85034-536-34, 2009

14. Taylor, R., Production and Downstream Processing of Norovirus Virus-Like Particles, Bio Process International conference, Providence, Rhode Island, 20-24 Sept. 2010

HCP removal: Ziegler T, Delvaille D. Contaminant removal in purification processes by membrane chromatography for recombinant proteins in early clinical development. American Pharmaceutical Review 2008 March/April

DNA removal: Chen B, Glynn J. Development of a Positive-Charged Membrane Chromatography Step. ACS Meeting, Boston, 19-23 Aug. 2007

Endotoxin removal: Clutterbuck A, Kenworthy J, Liddell J. Endotoxin reduction using disposable membrane adsorption technology in cGMP manufacturing. BioPharm Int. May 2007

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Thank you!

stefan.fischerf@sartorius-stedim.com

www.sartorius-stedim.com