Transcript Slide 1
Purification of a Prototype Baculovirus by Membrane Filtration R. Michalsky1, 2, 3, P. Czermak1, 2, P. H. Pfromm2, A. L. Passarelli3 1University of Applied Sciences Giessen, Germany, 2Department of Chemical Engineering, 3Division of Biology, Kansas State University, Manhattan, Kansas, USA Kansas State University Purpose: to obtain virus-free solutions Requirements for filtration membrane virus Affinity Chromatography filtrate (proteins, salts) miscellaneous proteins www.aml-benzene.com antibody virus fragments UF antigen www.oceancolor.gsfc.nasa.gov www.yorku.ca (a) Polyclonal antibody production for multiple sclerosis therapy www.actden.com www.waterforlife.ca Membrane filtration reusable & sterile membranes non denaturing methods Proteins (antigens) from multiple sclerosis patient are used to obtain antibodies from a rabbit. Membrane filtration will securely retain contaminant virus and virus fragments from purified antibodies and safely used for human therapy. www.kotaku.com Gel Filtration Chromatography supernatant (b) Vector production for gene therapy of immunodeficiency shaker, 3d freeze and thaw cycles www.sbs.utexas.edu An artificial plasmid contains the gene that balances a mutated human protein receptor gene. virus genes with therapy gene + helper genes IEX www.biochemsoctrans.org therapeutical vector www.biochem.wisc.edu www.oceancolor.gsfc.nasa.gov miscellaneous proteins www.oceancolor.gsfc.nasa.gov www.waterforlife.ca permissive cells cell debris Membrane filtration elution buffer ~1ml/min Virus is produced with a helper gene and is used to deliver the therapeutical gene. Experimental system and approach Cells and virus Virus production Scaleup virus to 50 m l Sf-21 cells in serum-free media virus cvir, out ~ 107 108 pfu / m l t 4d www.biochemj.org V 200 ml + www.chemicalgenomics.iu.edu www.biochemj.org www.insectscience.org www.ncbi.nlm.nih.gov SF-21 insect cells Autographa californica M nucleopolyhedrovirus (40 x 300 nm) Viral detection Vout ~ 180 m l / spinner + www.ncbi.nlm.nih.gov www.biochem.wisc.edu 106 cells / m l MOI Vend ~ 2m l (35 m m plate) 27C 0.1 pfu / cell www.chemicalgenomics.iu.edu t 10 15m in a 1000g www.faculty.clintoncc.suny.edu www.faculty.clintoncc.suny.edu cell debris cell debris Detection Filtration eGFP flow SDS-PAGE Passarelli research group, Kansas State University Passarelli research group, Kansas State University Cells infected with a virus expressing the enhanced green fluorescent protein (eGFP) Cells infected with a virus carrying a capsid protein-eGFP EtBr www.evolutionary-research.net Membrane TEM TMP ~ 0.5 bar (Wickramasinghe (2005)) a b a) relative virus proportions vs. 100 and 300 kDa membranes b) 100 proteins (Hemoglobin, 68 kDa, 6 nm diameter) www.pharmaceuticalonline.com Expected results a flow – – – – – – size exclusion (UF) statistic surface charge (IEX) shape exclusion competition adsorption back migration Effects on filtration are V , p, T , t, , [ prot], dmemb , Amemb and membrane-fluid interactions. Non-constant flow: Purity and concentration: Membrane resistance (serum-free medium) 300 nm d pore 300nm ( MWCO 30kDa) d vir, real 40 / 300nm Lower initial particle convection (serumcontaining medium) d prot 6nm d macromol 1 10nm D prot 7 1011 m 2 / s , Dvir 1 1011 m 2 / s Three graphs: Grzenia, Carlson, Czermak, Han, Specht, Wickramasinghe; Purification of Densonucleosis Virus by Tangential Flow Ultrafiltration. Biotechnology Progress (2006), 22(5), 1346-1353. Acknowledgements: The authors thank Chris Lehiy for his assistance in laboratory work. membrane: Johnson (2002); Diffusion coefficients: room temperature, diffusion in water, 400 nm-virus, 6 nmprotein (Hemoglobin), approximation (www.uni-ulm.de) All protein-images: www.en.wikipedia.org