Virus Purification with Membrane Chromatography

Transcription

1 Virus Purification with Membrane Chromatography 1 st 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

2 Agenda 1. Outline single use Membranes 2. Virus removal 3. Virus purification Page 2

3 Diffusion limited gels versus convective limited membranes (high flow rate) Average pore size nm Average pore size 3-5 μm Page 3

4 Mainly ion exchangers for removal & purification of viruses 4 mm bed 8 mm bed Q S STIC Q S HIC nano 1 ml mini mega nano 3 ml 150 ml Jumbo 5 l 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 Page 4

5 Membrane Adsorbers - highest growth of single-use solutions Page 5

6 15 reasons why single-use: Product faster to market Reduction of complexity Reduce space No cleaning validation Decrease product contamination More flexible Simplify operations Reduce hazardous cleaning solutions More convenient Increase facility output Assure sterility Reduce water and buffer consumption Lower maintanance cost Reduce time to get facility running Faster optimization Page 6

7 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

8 Agenda 1. Outline singe use membranes 2. Virus removal 3. Virus purification Page 8

9 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 ± ± Reo-3: Reovirus Type III no 7.00 ± ± MuLV*: Murine Leukemia Virus yes 5.35 ± ± 0.27 > 70 PRV: Pseudorabies virus yes 5.58 ± ± 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) , 2006 Page 9

10 Salt tolerant anion exchanger Sartobind STIC (-NH 2 ligand) Protein Binding [g/l] Sartobind Q BSA in 200 mm NaCl (20 ms/cm) DNA Binding [g/l] DNA in 50 mm NaCl (7 ms/cm) LRV with Mouse Minute Virus (MMV) Fraction 1, 150 mm NaCl Fraction 2, 150 mm NaCl Sartobind STIC 3.82 > 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

11 Phosphate and other multivalent anions inhibit the binding of ΦX174 (virus surrogate) Load 4*10 7 pfu/ml ΦX174, LP15, flow rate 20ml/min LRV 5 4,5 4 3,5 3 2,5 2 1,5 Sartobind Q Sartobind STIC 1 0,5 0 Buffer 25mM TRIS/HCl 25mM TRIS/HCl 25mM TRIS/HCl 25mM TRIS/HCl 20mM KPi 20mM KPi 20mM KPi 20mM KPi ph NaCl [mm] 7,5 7, ,5 7, Page 11

12 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 0 < <1 2 < < < <1 DNA in flowthrough % of start material 15 cm² (3 layers) Sartobind STIC PA were loaded with 150 ml ΦX174 1 x 10 7 PFU/ml, salmon sperm DNA 200 ng/ml at 10 ml/min was added. Page 12

13 Agenda 1. Outline single use membranes 2. Virus removal 3. Virus purification Page 13

14 Virus capture / purification Single-use / intra batch reuse Influenza virus 2,3 Adenovirus 4,5,6 Lentivirus 7 Adenoassociated virus Baculovirus 8 Densonucleosis virus 9 Pseudorabies virus 10 Bovine herpesvirus 1 Foot and mouth desease virus 11 Rotavirus like particles 12 Bacteriophages 13 Norovirus (VLP) 14 Page 14

15 Membrane Adsorbers vs. Density Gradient Membrane Adsorbers Density gradient ultra centrifugation 2 hours 36 hours Up to VP/ml No carryover, disposable, no validation, simple No contaminants 10 6 VP/ml Carryover validation Toxic CsCl 2, sucrose removal from finished product Page 15

16 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: Page 16

17 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

18 Purification of Influenza Viruses with Q Anion Exchanger - Human and equine influenza A virus in cell culture supernatant (serum-free and serumcontaining 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) Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Germany Page 18

19 Purification of PPV Virus Spike PDA Technical Report No. 47: Preparation of virus Spikes used for Virus Clearance Studies 2010, p19-20 Page 19

20 Current applications for vaccines Capture Influenza vaccine Rabies vaccine Adenovirus-vectored vaccine Polio vaccine Conjugated vaccine VLP Polishing DNA removal HCP removal Page 20

21 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

22 References 1. Karger A, Schmidt J, Mettenleiter TC. Infectivity of a pseudorabies virus mutant lacking attachment glycoproteins C and D. J Virol 1998, 72, 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, 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), Zeidler R, Fischer-Frühholz S. Schnelle und einfache Reinigung von Adenoviren. F&D LP, 2004, Sep, 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), Vivapure Virus Purification and Concentration Kits. Brochure , 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, 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), Karger A, Schmidt J, Mettenleiter TC. Infectivity of a pseudorabies virus mutant lacking attachment glycoproteins C and D. J Virol 1998, 72, Oswald T. DSP in Vaccine manufacturing and development. Downstream Technology Forum, London, Nov 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 Sartorius Stedim Biotech Application Note Capture of Bacteriophage PR , Taylor, R., Production and Downstream Processing of Norovirus Virus-Like Particles, Bio Process International conference, Providence, Rhode Island, Sept 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, Aug Endotoxin removal: Clutterbuck A, Kenworthy J, Liddell J. Endotoxin reduction using disposable membrane adsorption technology in cgmp manufacturing. BioPharm Int. May 2007 Page 22

23 Thank you! Page 23