Kansas State

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
(a) Polyclonal antibody production for
multiple sclerosis therapy

• 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.

(b) Vector production for gene therapy of
immunodeficiency

• An artificial plasmid contains the gene that
  balances a mutated human protein receptor gene.
• Virus is produced with a helper gene and is
  used to deliver the therapeutical gene.

Experimental system and approach
Cells and virus
Virus production
www.ncbi.nlm.nih.gov
www.insectscience.org
Autographa californica M nucleopolyhedrovirus
(40 x 300 nm)
SF-21 insect cells
Viral detection
Detection
Filtration
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
Membrane

• relative virus proportions vs. 100 and 300 kDa
  mem-branes
• b) 100 proteins (Hemoglobin, 68 kDa, 6 nm
  diameter)
• size exclusion (UF)
• statistic surface charge (IEX)
• shape exclusion
• competition
• adsorption
• back migration

Expected results
Effects on filtration are
and membrane-fluid
interactions.
Non-constant flow
Purity and concentration
Membrane resistance (serum-free medium)

Lower initial particle convection
(serum-containing medium)
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. All protein-images
www.en.wikipedia.org
membrane Johnson (2002) Diffusion
coefficients room temperature, diffusion in
water, 400 nm-virus, 6 nm-protein (Hemoglobin),
approximation (www.uni-ulm.de)