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Virus Purification with Membrane Chromatography

Presentation · October 2010

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Fruehholz Consultancy Sartorius Stedim Biotech, Germany, Goettingen
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 Virus Purification with Membrane Chromatography
1st Workshop European Network of Viral Vaccines Processes, October 14-15, 2010, Frankfurt am Main
Stefan Fischer-Frühholz, Laura Chirica, Miyako Hirai, Rene Faber, Uwe Gottschalk
Sartorius Stedim Biotech GmbH
_
Agenda

1.
1. Outline
Outline single
single use
use
Membranes
Membranes
2.
2. Virus
Virus removal
removal

3.
3. Virus
Virus purification
purification

Page 2
Diffusion limited gels versus convective limited membranes (high flow
rate)

Average pore size Average pore size

15 - 40 nm 3 - 5 μm
Page 3
Mainly ion exchangers for removal & purification of viruses

4 mm bed 8 mm bed

Q Q
S S
STIC
HIC
nano mini 5” 10” 20” 30” mega nano 150 ml Jumbo 5 l
1 ml 3 ml

Contaminant removal: flowthrough Purification: bind & elute of viruses and

mode to remove DNA, Host cell virus like particles, large proteins
proteins, endotoxins, viruses
Singe-use Single-use / intra-batch use

Page 4
Membrane Adsorbers - highest growth of single-use solutions

Page 5
15 reasons why
Decrease
single-use: Reduce space
product Simplify
contamination operations
Product faster
to market
Reduce
No cleaning validation More hazardous
flexible cleaning
Reduction of solutions
complexity
Reduce water
Increase Assure sterility and buffer
More
facility consumption
convenient
output

Lower Reduce time to

maintanance get facility Faster optimization
cost running
Page 6
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

Page 7
Agenda

1.
1. Outline
Outline singe
singe use
use
membranes
membranes
2.
2. Virus
Virus removal
removal

3.
3. Virus
Virus purification
purification

Page 8
Virus removal study - polishing after CIEX column

Virus Enveloped LRV Run 1 LRV Run 2 Virus

recovery
(%)
MVM: Minute Virus of Mice no ≥ 6.03 ± 0.21 ≥ 6.03 ± 0.20 100

Reo-3: Reovirus Type III no ≥ 7.00 ± 0.31 ≥ 6.94 ± 0.24 100

MuLV*: Murine Leukemia Virus yes ≥ 5.35 ± 0.23 ≥ 5.52 ± 0.27 > 70
PRV: Pseudorabies virus yes ≥ 5.58 ± 0.28 ≥ 5.58 ± 0.22 100

pH: 7.2, conductivity: 4 mS/cm, flow rate: 450 cm/h, 1 % spike

protein concentration: 4.3 g/l Mab
load: 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
Page 9
Salt tolerant anion exchanger Sartobind STIC (-NH2 ligand)

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 NaCl 2.10 3.82
Fraction 2, 150 mM NaCl 1.81 > 4.96

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)

Page 10
Phosphate and other multivalent anions inhibit the binding of ΦX174
(virus surrogate)

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

5
4,5 Sartobind Q
4 Sartobind STIC
3,5
3
LRV

2,5
2
1,5
1
0,5
0
25mM 25mM 25mM 25mM 20mM 20mM 20mM 20mM
Buffer TRIS/HCl TRIS/HCl TRIS/HCl TRIS/HCl KPi KPi KPi KPi

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

pH
NaCl [mM] 0 150 0 150 0 150 0 150

Page 11
Phosphate inhibits phage binding but DNA contaminant is bound
Sartobind STIC to be used at binding conditions of viruses

Ortho phosphate Phage in flowthrough DNA in flowthrough

mM % of start material % 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, buffer 20 mM Tris/HCl pH 7.5, 150 mM NaCl
plus phoshate.

Page 12
Agenda

1.
1. Outline
Outline single
single use
use
membranes
membranes
2.
2. Virus
Virus removal
removal

3.
3. Virus
Virus purification
purification

Page 13
Virus capture / purification • Influenza virus2,3
Single-use / intra batch reuse
• 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

Page 14
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 Carryover validation
validation, simple
No contaminants Toxic CsCl2, sucrose removal from
finished product
Page 15
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, 9:123
INRA, 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
Page 16
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), 1–10
IBET/ITQB-UNL, Animal Cell Technology Laboratory, Portugal
Page 17
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)
251–260
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems,
Germany
Page 18
Purification of PPV Virus Spike

PDA Technical Report No. 47: Preparation of virus Spikes used for Virus Clearance
Studies 2010, p19-20
Page 19
Current applications for vaccines

Capture
Influenza vaccine
Rabies vaccine
Adenovirus-vectored vaccine
Polio vaccine
Conjugated vaccine
VLP

Polishing
DNA removal
HCP removal

Page 20
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.

Page 21
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 Bioeng
2009,103(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

Page 22
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Thank you!

stefan.fischerf@sartorius-stedim.com
www.sartorius-stedim.com
Page 23