1. PRACTICAL EXP. II BIBLIOGRAPHIC WORK STUDENT TIME SPENT IN THE REPORT Benito Rubio, Alberto8 h Fernández Fernández, Carolina8 h

2. Introduction Introduction Obtaining virus clearance is essential in the manufacture of protein-based biopharmaceuticals This is necessary approval a product for release to market

3. Efficacy Efficacy During the purification process, typically reduction is: ◦ - 103 to 105 monoclonal antibodies per mL ◦ - 1010 to 1015 retrovirus-like particles per mL Virus Removal Alternatives Virus Removal Alternatives Chemical methods (e.g., solvent detergents, low pH) physical methods (e.g., heat and radiation) FILTRATION SOMETIMES IT IS THE ONLY POSSIBLE!

4. MEMBRANE DESIGN Optimal virus filters must maximize: acceptable filtrate fluxes reject virus particles maximize protein passage Objective: - capacity - throughput - selectivity

5. MEMBRANE DESIGN membranes are designed to reject virus particles while allowing the product of interest to pass through the membrane pores - Very narrow pore-size distribution - Sturdiness depends on size exclusion - Some layers, need support low pH - Originally tangential flow mode - Today using disposable direct flow filters - Very narrow pore-size distribution compared to ultrafiltration membranes

6. OPERATION -Ultrafiltration membranes, on the other hand operated with the filtration surface (i.e. skin surface) facing the feed inlet -membranes are cleaned-in-place and reused -flux decline is dominated by osmotic pressure effects and gel layer formation -Asymmetric virus filtration membranes, virus particles often are trapped irreversibly within the more open support -Compaction and permeability effects

7. EXAMPLE For removal of: parvovirus ◦ Filters must reject virus particles as small as 20 nm Two ultrafiltration membranes have NMWCOs of 300 and 10 kDa. In addition, virus containing feed streams were spiked with BSA (1% (m/v) final concentration)

8. PATENTED MEMBRANES - Normal flow filtration experiments with: -Viresolve 180 -Large pore substructure acts as a depth pre-filter -Declined by nearly 50% -DV20 -10–20% reduction in permeability -DV50 -These particles could be removed using a small pore size filter placed directly in-line -Omega 300 -using different flow orientations -Less dramatic increases in flux

9. VIRUS REMOVAL BY FILTRATION: ANIMAL VIRUS : Minute canine parvovirus (CPV), risk of contamination is common to potable water supply GVS Speedflow® Positive equipped with a positively charged 0.2 μ m membrane. compared with : Mustang Q®, Speedflow® Positive and Mustang Q® Results employed virus models: Virus interact with negatively charged components of cell membranes such as GAGs and sialic acid, Able to attach to positively charged membranes providing new insights into their electrostatic properties.

10. PATENT -Methods for producing immunoglobulins and in particular anti-D immunoglobulin substantially free of virus and product resulting therefrom. Specifically provided are methods for nanofiltration of the anti-D immunoglobulin in high ionic strength buffer and with excipient such as polysorbate 80. Additional steps include diafiltration to concentrate the anti-D protein and reduce the concentration of excipient present.

11. PATENT 3449314June 1969Pollack 3916026October 1975Stephan 4021540May 1977Pollack et al. 4141887February 1979Seufert 4590002May 1986Zolton et al. 4880913November 1989Doleschel et al. 5115101May 1992Bloom et al. 5215681June 1993Truong et al. 5723123March 1998Karges et al. U.S. Patent Documents

12. BIBLIOGRAPHY 1 Understanding virus filtration membrane performanceUnderstanding virus filtration membrane performance Journal of Membrane Science, Volume 365, Issues 1-2, 1 December 2010, Pages 160-169 S. Ranil Wickramasinghe, Emily D. Stump, David L. Grzenia, Scott M. Husson, John Pellegrino http://www.sciencedirect.com/science?_ob=MiamiImageURL &_cid=271357&_user=857027&_pii=S037673881000699X& _check=y&_origin=search&_zone=rslt_list_item&_coverDat e=2010-12-01&wchp=dGLbVlk- zSkWz&md5=7f602c0374cdcbd7a5735d9ca727435e/1-s2.0- S037673881000699X-main.pdf http://www.sciencedirect.com/science?_ob=MiamiImageURL &_cid=271357&_user=857027&_pii=S037673881000699X& _check=y&_origin=search&_zone=rslt_list_item&_coverDat e=2010-12-01&wchp=dGLbVlk- zSkWz&md5=7f602c0374cdcbd7a5735d9ca727435e/1-s2.0- S037673881000699X-main.pdf Last visit: 17:30 (Spanish Hour) 28/09/2011

13. BIBLIOGRAPHY Indexed references [1] P.Y. Huang and J. Peterson, Scaleup and virus clearance studies on virus filtration in monoclonal antibody manufacture, W.K. Wang, Editor, Membrane Separations in Biotechnology, Marcel Dekker, New York (2001). [1] [2] A. Higuchi, M. Nemoto, H. Koyama, K. Hirano, B.-O. Yoon, M. Hara, M. Yokogi and S.-I. Manabe, Enhanced microfiltration of γ -globulin solution upon treatment of NaCl addition and/or DNase digestion. J. Membrane Sci., 210 (2001), pp. 369–378. [2] [3] T. Ireland, H. Lutz, M. Siwak and G. Bolton, Virus filtration of plasma-derived human IgG: a case study using Vireslove NFP. Biopharm International, 17 11 (2004), pp. 33–40. [3] …

14. BIBLIOGRAPHY Indexed references [1] P.Y. Huang and J. Peterson, Scaleup and virus clearance studies on virus filtration in monoclonal antibody manufacture, W.K. Wang, Editor, Membrane Separations in Biotechnology, Marcel Dekker, New York (2001). [1] [2] A. Higuchi, M. Nemoto, H. Koyama, K. Hirano, B.-O. Yoon, M. Hara, M. Yokogi and S.-I. Manabe, Enhanced microfiltration of γ -globulin solution upon treatment of NaCl addition and/or DNase digestion. J. Membrane Sci., 210 (2001), pp. 369–378. [2] [3] T. Ireland, H. Lutz, M. Siwak and G. Bolton, Virus filtration of plasma-derived human IgG: a case study using Vireslove NFP. Biopharm International, 17 11 (2004), pp. 33–40. [3] …

15. Thanks for your attention!