Rhone-poulenc Rorer Pharmaceuticals Inc.

The Immune Response Corporation

Michael E. Hrinda

David M. Weber

Pierre M. Bay

James J. Harter

Immunization Products Limited

Jonathan J. Mitschelen

Thomas W. Irish

Christopher P. Prior

Richard S. Gore

George C. Tarr

Jonathan J Mitschelen

Michael E Hrinda

Christopher P Prior

David M Weber

Richard S Gore

James J Harter

George C Tarr

Lida Y Akhnana

Thomas L Darling

Pierre M Bay

Thomas W Irish

This invention is directed to a continuous process for producing viral particles by providing in a perfused growth medium a population of viable virally infected non-lytic cells, and removing medium containing said cells at a rate to maintain the steady-state log-phase growth of cells remaining in the perfused growth medium. The invention is also directed to a process for purifying retroviral particles by passing a solution comprising the retroviral particles and contaminants through an anion exchange resin, and eluting the retroviral particles from the resin. The preferred exchange resin is a tentacle anion exchange resin.

Production and purification of retroviral particles using tentacle anion

Recovery of the 52/48 kDa tryptic fragment of vWF or peptide subfragments thereof produced in the form of inclusion bodies from recombinant host cells is carried out by providing a washed recombinant host cell suspension, adding a detergent and subjecting the cells to mechanical disruption. A second detergent is added, followed by another mechanical disruption and centrifugation to a pellet. The pellet is resuspended in a buffer and subjected to another mechanical disruption. Inclusion bodies are washed, resuspended and then recovered. In addition, endotoxins and DNA are removed from inclusion bodies containing the 52/48 kDa tryptic fragment of vWF or peptide subfragments thereof by mechanically disrupting the inclusion bodies in an aqueous buffer containing a detergent. A washed pellet is formed from these mechanically disrupted inclusion bodies and dissolved in a denaturant. Alkylation is then performed and the suspension is subjected to column chromatography, where the alkylated fragment or subfragment is recovered by elution.

Methods for purification of recombinantly produced proteins

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Perkiomenville, PA, United States of America

Jonathan J Mitschelen