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Arrive early and attend The Fourth Annual
Optimizing Cell Culture Development
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September 15-16, 2008
Boston, MA

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Drug Discovery & Development


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The Scientist

Baculovirus Technology - Day 1

Conference Proceeding CD Now Available
  • Speaker Presentations
  • Poster Abstracts
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7:30am Registration and Morning Coffee


8:20 Chairperson’s Remarks

Life, Viruses and the Molecular Revolution...Getting to Know Your Platform
undefined Loy Volkman, Ph.D., Professor Emerita, Plant & Microbial Biology, University of California, Berkeley and Board Member, Expression Systems
The molecular revolution has affected every aspect of biological science, but none as profound as the way we view life on planet earth.  Prior to the late nineties, based on morphological evidence, we thought life was organized into five kingdoms.  Now, based on molecular evidence, we think extant species are organized into three domains, all of them primarily microbial. Three previous “kingdoms”, plants, animals and fungi, are represented as small buds in the Eucarya domain.  Viruses, inert outside of their hosts, cannot be plotted on the tree of life; hence, viruses can be viewed as software in search of compatible hardware.  Baculoviruses appear to have been tracking their juvenile insect hosts since the radiation of the Lepidoptera (the most recent insect order to emerge) or for about 230 million years.  These hosts developed a formidable defense against pathogens by covering their exterior surfaces with chitinous cuticle, including their respiratory tubes, their foreguts and their hindguts.  Viruses need contact with living cells to insert their software (establish infection), and baculoviruses settled on accessing their hosts' columnar epithelial cells VIA THEIR MICROVILLI, an extraordinary biological feat.  Navigation through the dense, actin-based, cross-linking structures within microvilli is made even more challenging by columnar cells’ constant sloughing into the gut lumen and total replacement at each molt.  Baculovirus nucleocapsids, therefore, have been under constant heavy selection pressure to move rapidly within intracellular environments, even those devoid of microtubules, such as microvilli.  This actin-dependent, intracellular motoring activity is both unique among viruses and key to baculovirus infectivity.  Its understanding should lead to improved baculovirus vectors for specific commercial purposes.  

Improving High-Throughput Baculovirus Expression by Vector Design
Linda King, Ph.D., Professor, Dean and Head, School of Life Sciences, Oxford Expression Technologies, Oxford Brookes University
This presentation will review the success of current technologies that have been aimed at improving the through-put of recombinant protein expression using baculovirus vectors and will suggest how greater improvements could be achieved in future through re-design of current vectors based on detailed knowledge of the baculovirus genome. The presentation will also discuss how improvements to protein quality could be achieved in parallel with modifications to achieve high throughput

9:30 Towards Large-Scale Continuous Baculovirus Production in Insect Cell Culture: Overcoming Mutations in fp25k Baculovirus Gene
Lopamudra Giri, Ph.D., Research Assistant, Chemical and Biochemical Engineering, University of Iowa
Few polyhedra (FP) mutants and defective interfering particles (DIP) are commonly known to be responsible for the reduction in occluded virus yield and decreased infectivity of budded virus at late passages during continuous production in bioreactor. FP mutations are generally caused by insertion of transposons into the baculovirus fp25k gene and DIPs are caused by deletion of baculovirus genomic sequences. A novel recombinant baculovirus with removed transposon sites from the WT baculovirus fp25k gene (having a stabilized fp25k gene) reduced the formation of FP phenotype in late passages but could not eliminate the FP phenotype/DIPs in very late passages. Further genotypic and phenotypic analysis of late passaged virus showed that deletion of genomic sequences also contributed to the FP phenotype and reduced infectivity. Use of low MOI can greatly reduce the incidence of DIP mutant formation.

10:00 Networking Coffee Break with Poster and Exhibit Viewing


10:35 Chairperson’s Remarks

10:40 Generation of Antigen-Specific Antitumor Immunity Using Insect Cells Infected with Recombinant Baculoviruses
Jill Slansky, Ph.D., Assistant Professor, Immunology, Health Sciences Center, University of Colorado, Denver
With the ultimate goal of defining the characteristics of efficacious tumor vaccines, we have developed a method to immunize mice with insect cells infected with recombinant baculoviruses. These vaccines, including mainly baculoviruses encoding MHC molecules and antigenic peptides, result in both expansion of functional peptide-specific T cells that protect mice from tumor challenge and immunologic memory. We have also used this approach to elicit specific antibodies to other recombinant proteins. This method eliminates the need for protein purification and additional adjuvants. Peptide-specific T cell responses are similar in animals primed with soluble or membrane-bound antigen, and in animals primed in the presence or absence of MHC molecules. After vaccination, dendritic cells increase expression of maturation markers and stimulate proliferation of specific T cells ex vivo. Thus, dendritic cells likely “cross-present” antigens from this vaccine to T cells. This vaccine approach has proven to facilitate analyses of T cells elicited by different peptides, particularly those selected from peptide-MHC libraries encoded by baculoviruses. We selected peptides from the baculovirus-encoded library that stimulated tumor-specific T cells and immunized the mice. One complication that we encountered during selection of peptides from the libraries is that a retrotransposon, endogenous to insect cells, integrated into the baculoviruses along with a stimulatory peptide obscuring the identification and immune responses to some selected library peptides. Thus, specific cellular and humoral responses to this vaccine may provide an effective format for immunization as long as safety precautions are considered.

11:10 Preparation and Evaluation of Influenza Vaccines in Insect Cells
Sue Clarke, MIBiol, Head, Project Management, ImmBio
ImmBio have selected the Baculovirus expression system to facilitate the development of novel influenza vaccines. The BEVS offers a unique combination of a rapid manufacturing time line, high yield and secreted native product. ImmBio is developing a recombinant antigen-based product that is targeted to dendritic cells through the Fc receptor. This vaccine approach is versatile and has utility in the event of a pandemic. Data will be presented describing product characterization and in vivo efficacy. Process development and manufacturing scale-up will be discussed with an emphasis on the unique characteristics of the Baculovirus system that is essential for the preparation of this product.

11:40 A Novel Method for SARS Coronavirus-Like Particles Production from Insect Cells
Ye V. Liu, Ph. D., Scientist, Vaccine Development, Novavax Inc.
Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease caused by SARS coronavirus (SARS-CoV). A safe and effective vaccine is required to protect human population from SARS-CoV infection. We used baculovirus vectors to express SARS-CoV spike (S) glycoprotein in Sf9 insect cells. S protein was secreted into culture medium as a component of enveloped virus-like particles (VLPs). We developed a manufacturing friendly procedure to purify SARS-CoV VLPs as a vaccine candidate.

12:15pm Sponsored by  Blue Sky
Title To Be Announced
William Hermans, Manager Cell Culture, Blue Sky Biotech

12:40  Implementation of a Fast Titering Approach for the Quantification of Baculovirus Stocks 
Peter Jones, Andrew Fosberry, Research Investigators, Biological Reagents & Assay Development, GlaxoSmithKline Research and Development Ltd.
Baculovirus expression systems are an invaluable tool in protein production laboratories worldwide. Titration of baculovirus stocks is therefore a crucial procedure which routinely takes up valuable experimentation time. Traditionally, the titers of a recombinant baculovirus stock have been determined by a variety of methods that include, the plaquELISA assay, endpoint dilution, and immunoassays. However, all of these methods are cost and labour intensive; require a long processing time, and a certain degree of expertise in cell culture and virus handling. We describe efforts internally within BR&AD Harlow to reduce cycle times and provide timely titer data to support project/ program teams. In collaboration with a third party we demonstrate here a rapid and reliable indirect method for analysing baculovirus  stocks using capillary zonal electrophoresis with evaluation/comparison against the 'gold standard' plaquELISA assay. 



1:45 Chairperson’s Remarks

1:50 Featured Presentation:
Application of Baculovirus in GPCR and Ion Channel Protein Expression in Mammalian Cells
Simon Lee, Ph.D., Protein Sciences, Discovery Technologies, Roche Palo Alto LLC
Baculovirus expressing proteins under the control of a CMV IE promoter (BacMamTM) was adopted for protein production in mammalian cells. The mechanism responsible for uptake of Baculovirus into susceptible mammalian cells and subsequent transcription and translation events are not well understood. However, this protein expression protocol has provided an opportunity to produce sufficient amount of mammalian proteins for functional assays. We generated recombinant Baculovirus carrying 27 different single amino acid mutations of the 5HT6 receptor and transduced each one into 5x108 HEK 293 cells cultured in a shake flask. Expression of these mutated 5HT6 proteins was detected in cells one day post transduction by flow cytometry and western blotting. Specific activity of the receptors present in isolated membrane fractions was from 3 - 50 pmoles/mg protein in ligand binding assays. Mutation of several amino acid predicted by homology modeling to contribute to the binding pocket greatly reduced ligand binding. These data were consistent with previous literature observations obtained using membrane fractions derived from stable mutant cell lines. Compared to the conventional method of producing stable recombinant cell lines, this transduction protocol was rapid and less labor-intensive. Once the workflow was streamlined, 8 - 12 mutants could be processed each week. We extended this application to an ion channel protein and developed a transduction-based FLIPR assay for screening agonists and inhibitors from a compound library. Baculovirus transduction provides an alternative option in development of high throughput cell-based assays.

2:25 Engineering A Better CHO Cell Host for Baculovirus Transduction
Patrick Condreay, Ph.D., Research Investigator, GlaxoSmithKline Discovery Research

2:55 Technology Spotlight 1 Sponsored by emd logo 2

Dual-Purpose Insect Expression Vectors Facilitate Rapid Expression Screening as well as Baculovirus Generation
Katharine S. Kramer, Product Manager Cloning and Expression, EMD Chemicals, Novagen Brand
Dual-purpose pIEx/Bac™ vectors can be used to rapidly screen clones for expression by transient transfection in just 48 hours. For more robust protein expression, the same pIEx/Bac vectors can be used as transfer vectors for generating baculovirus.  Transfection-based expression results serve as a quick and reliable indicator of whether the same pIEx/Bac recombinant will be useful for protein production in the baculovirus system. 

 The following data will be shown:
·          Expression comparisons of pIEx/Bac vectors in transient mode vs. baculovirus mode.
·          Expression comparisons of pIEx/Bac vectors vs. traditional baculovirus transfer vectors.
·          Case studies using pIEx/Bac vectors for screening in transient mode and subsequent scale-up in baculovirus mode.

Technology Spotlight 2 (Sponsorship Available – Please contact Ilana Schwartz, ischwartz@healthtech.com)

3:25 Networking Refreshment Break with Poster and Exhibit Viewing


4:00 Isolation and Characterization of an Sf9 Cellgene Encoding a Key Enzyme Distinguishing Insect and Mammalian Protein N-Glycosylation Pathways
Donald Jarvis, Ph.D., Professor, Department of Molecular Biology, University of Wyoming
The major processed N-glycans produced by insect cells have paucimannose structures that are produced by an unusual N-glycan processing enzyme activity, which was first detected in Sf9 cells by Altmann and colleagues in 1995. This presentation will focus on our efforts to molecularly clone the cDNA encoding this Sf9 cell enzyme and to characterize the biochemical properties of the cDNA product. We also will report on our preliminary attempts to reduce theendogenous levels of this processing enzymeactivity, as part of our long-term effort to humanize the Sf9 cell protein N-glycosylation pathway.

4:30 A Recombinant Baculovirus-Expressed S Glycoprotein Vaccine Against SARS–CoV: Pre-clinical Development and Beyond
Zhimin Zhou, Ph.D., Project Manager, Process Development, Protein Sciences Corporation
The recombinant vaccine was formulated with or without adjuvant, aluminum hydroxide. Immune responses, including SARS-CoV neutralizing antibodies, were enhanced dramatically by the adjuvant. Safety and dose escalation was also demonstrated. 

5:00 Application of Baculovirus Technology for the Development of Sipuleucel-T, an Investigational Active Cellular Immunotherapy for the Treatment of Androgen-Independent Prostate Cancer
Samuel Li, Ph.D., Senior Scientist I, Product Development, Dendreon Corporation
Dendreon develops novel cellular immunotherapies for cancer using proprietary approaches to antigen discovery and engineering, and antigen presenting cell technologies. The goal of this approach is to stimulate an immune response against antigens expressed on cancer cells. Sipuleucel-T (Provenge®), an investigational active cellular immunotherapy for the treatment of androgen-independent prostate cancer, is in late stage Phase III clinical testing. It is comprised of autologous antigen presenting cells (APCs) stimulated ex vivo by the recombinant antigen PA2024, produced using BEVS technology. This presentation will describe the technology platform, the Antigen Delivery CassetteTM (ADC), PA2024 generation by the BEVS technology, Sipuleucel-T manufacturing, and product characterization.

5:30 Reception in the Exhibit HallSponsored by Delta Dot

6:30 End of Day One

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