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High Level Recombinant Protein Production In Insect Cell Culture [Webinar Series]

High Level Recombinant Protein Production In Insect Cell Culture Kamal Rashid, Ph.D.

Director, Biomanufacturing Education & Training Center

About Your Presenter

• Kamal has over thirty years of academic experience in both research and biotechnology educational program development plus an additional focus on training at universities including WPI, Utah State University, and Penn State University.

• Prior to WPI, he was the Associate Director and Research Professor of Toxicology at Utah State University’s Biotechnology Center where he developed and equipped the bioprocess facility at the Center with the most advanced bench top bioreactors and fermenters that are utilized in both research and training programs. His grant work included a multi-year, multimillion dollar grant from the US Department of Health and Human Services, Biomedical Advanced Research and Development Authority (BARDA) to train employees of vaccine manufacturing facilities from fourteen countries in the latest advances in cell-based vaccine production with emphasis of Influenza vaccines.

• Dr. Rashid was a faculty member in the Department of Biochemistry and Molecular Biology at Pennsylvania State University where he conducted research on the impact of environmental pollutants on human health. He developed and taught biotechnology undergraduate courses, biotechnology training programs, directed the Summer Symposium in Molecular Biology and was the key faculty in the development of the biotechnology undergraduate degree and the course curriculum.

• He has delivered numerous lectures and training programs in several countries, including Canada, China, Dominican Republic, Egypt, Indonesia, Iraq, Korea, Malaysia, Philippines, Puerto Rico, Vietnam, Thailand, Taiwan, Turkey, Singapore and US. He received a national Faculty Service Award in 1997 from US University Continuing Education Association for meritorious service to Penn State University. Additionally, he was honored in 2012 as the international professor of the year in College of Agriculture at Utah State University. His present research emphasis is on bioprocessing utilizing mammalian cell systems

Disclaimer

• Content is the sole responsibility of the presenter

• Products and vendors listed are provided for reference and illustration and are not meant to constitute a complete list or endorsement

Media

Optimization

Cell Culture Process Development

Expression

System

Bioreactor

Selection

Expression Systems

• A vector based system that transfer genes into cells for production of recombinant proteins:

• Types of expression systems:

– Bacterial Expression Systems

– Yeast Expression Systems

– Mammalian Expression Systems

– Baculovirus-Mediated Insect Cell Expression Systems

– Plant Expression Systems

Baculovirus Expression System• BEVS has been used extensively for expression of a wide

variety of mammalian proteins including human.

– High level of expression

– Provide an Eukaryotic environment – post trans. Mod.

– It can also infect mammalian cells in culture

– Could be used for gene therapy- xeno-vectors

• BEVS is a powerful and versatile expression system

becoming widely known in recent years.• Used in heterologous gene expression.

• Insect cells grow without CO2

• Insect cells grow at room temp. (25-280 c)

Historical Background

• Insect cell culture began in 1915 with Goldschmidt’s observation of in vitro spermatogenesis in the silk moth.

• Baculoviruses especially NPV (Nuclear Polyhedrosis Viruses) are heavily used in biological control of Gypsy Moth.

Baculovirus Expression System

• The first published report of baculovirus expression system appeared in literature in 1983 by Gale Smith and Max Summers in the Journal of Molecular and Cell Biology.

• They described production of human beta interferon in insect cells coupled with baculovirus expression system.

• Since then interest in the system has been growing steadily.

• The baculoviruses are host specific and cause no harm to humans

Baculoviruses

• One of the largest groups of viruses.

• Capable of infecting more than 500 species of insects.

• Seven families of insect viruses are known, with the most common insect viruses belonging to the family Baculoviridae

• Members of Baculoviridae are characterized by having double stranded circular DNA of 80-200 kb within a rod shaped envelop virion.

• Non-toxic to cells at even high level of MOI.

Baculoviruses• Within the family there are three morphologically

distinct subgroups:

– Nuclear ployhedrosis viruses (NPV)

– Granulosis viruses

– Non-occluded viruses

• NPV is of the greatest interest to researchers and

the most extensively studied virus is Autographa californica isolated from alfalfa looper (AcNPV).

Baculoviruses-AcNPV

• It can replicate in the cell nuclei of over 30 lepidopteran species.

• AcNPV is bi-phasic and differ from other DNA animal virus groups in having a second phase of replication.

– Yields two distinct forms of progeny:• Extracellular (budded) virus particles

• Polyinclusion bodies - Polyhedra

Baculoviruses-AcNPV

• Gene expression occurs at an early and latephase of replication:

– Extracellular (budded) virus (ECV) particles produced in the early phase. Spread infection from cell to cell within the insect host.

– Occluded virus particles also known as ployinclusion bodies (PIB). They are embedded polyhedra.

– The polyhedron matrix is comprised primarily of polyhedrin protein, a viral structural protein of 29 kd that protect the viruses from environmental factors that would otherwise inactivate ECV. Spread infection from insect to insect

Polyhedrin– Polyhedrin has evolved two highly specialized functions.

– forms a protective crystal around the virus

– it resists solubilization except under strongly alkaline conditions similar to those found in the insect midgut.

– Both of these properties allow the virus to remain viable for many years outside the insect host.

– Strong transcriptional promoter.

– Virus-encoded transcriptional complex.

• None-essential for BV replication in vitro.

• The crystalline part of baculovirus polyhedra consists of ∼29 kDa polyhedrin.

Source: Rohrmann 2013:Baculovirus Molecular Biology 3rd Ed.

Baculovirus Replication Cycle

Source: Patel and Jones, 1995

Source: Rohrmann 2013

Rsource: Rohrmann 2013

• Two adjacent dissolved polyhedra showing rod-shaped virions trapped by the collapsed polyhedron envelope.

Photo by K. Hughes

BEVS Life Cycle in vivo vs in vitro• in vivo :

– two distinct populations are formed in the insect cell

• Occluded and budded virions

• Occluded virions are protected in the environment and infect new larva.

• in vitro :– Foreign genes are cloned into a transfer vector

– Vector contain flanking sequences

– These sequences are homologous to the BEV genome

– Vector is co-transfected into insect cells

– Recombination will take place within the insect cell

– The recombinant virus produces r-proteins

Most Common Cell Lines

• Sf 9 & Sf21 From Spodoptera frugiperda– Frozen in serum containing medium

– Can be thawed directly to serum free medium (Ex-Cell 401)

– From the Moth Army Worm Spodoptera frugipedra ovarian tissue.

– Reaches 6x10.6/ml in suspension after 6 days with 98% viability

– Sf9- Faster growth and higher densities than Sf21

• For virus expansion these two cell lines are preferred over High Five

• Common name: Fall Armyworm

Most Common Cell Lines

• BTI- Tn-5B1-4 ( High Five) from Trichoplusia ni– Reported to have 5-25 fold greater expression

of secreted proteins compared to other cell lines

– Strongly anchorage dependent but can be adopted to suspension easily

– Cells sub-cultured from anchorage dependent to suspension exhibit a tendency to clump especially in early passage.

– Reaches 8x106 cells/ml in 6 days with 98% viability

• Common name: Cabbage Looper

Factors affecting insect cell scale-up and infection with baculovirus

• Medium nutrients

• pH

• Oxygen tension

• Cell Density

• Stage of Growth

• The multiplicity of infection or MOI

– is the ratio of infectious agents (e.g. phage or virus) to infection targets (e.g. cell). For example, when referring to a group of cells inoculated with infectious virus particles, the multiplicity of infection or MOI is the ratio defined by the number of infectious virus particles deposited in a well divided by the number of target cells present in that well.

Insect cell scale-up and infection with baculovirus

• Stirred tank reactors in batch mode are suitable for scale-up. Single use bioreactors will be economical

• Protein expression is limited by nutrient depletion.

• Cultures are infected with an MOI of 0.5-10 PFU per cell at 2-3x106 cells /ml.

• Usually cell growth stops shortly after virus infection.

Advantages of BEVS

• Insect cells and Baculoviruses are easily handled in vitro.

• The late expressed polyhedrin is under control of a strong promoter, is nonessential for viral replication.

• Thus, it is suitable for replacement by a foreign gene.

• The rod-shaped capsid can expand to accommodate large DNA inserts and the dsDNA is easily modified through recombinant DNA technology.

Advantages - Continued

• Plaque purified recombinant virus can be obtained within 4-6 week leading to rapid scale-up.

• BEVS are host specific. They propagate only in Lepidopteran species and are not known to infect vertebrates or plant cells.

• Natural biological containment results because without polyhedra, viruses can’t survive in the environment.

• Co- and post-transnational modifications.

• Recombinant viruses replicate like wild type viruses in established cell lines resulting in high level expression of protein products.

Insect Cell/ BEVS & Vaccines

• According to Manon Cox, President and CEO of Protein Sciences Corporation:

– BEVS has matured into a commercial manufacturing technology

– Vaccines can be produced in a “plug and play” process

– Economically feasible system

– Sustainability of vaccine supply

BEVS as a Vaccine Manufacturing Platform

• Human vaccines:

– Cervical Cancer- CERVARIX GSK

– Prostate Cancer- PROVENGE Dendreon

– Influenza - Flublok Protein Sciences

• Vetrinary vaccines:

– PCV2 - Porcilis PCV Merck

– PCV2 - CircoFLEXB. Ingelheim

– Classical Swine Fever Procilis Pesti Merck

Source: Manon Cox, Protein Sciences Corporation

Protein Sciences PlatformVaccine Production

Source: Manon Cox, Protein Sciences Corporation

A New Approach to Vaccine Manufacture

Traditional BEVS Grow pathogen (in eggs)

Inactivate pathogen

Purify antigen (i.e., the active ingredient)

Formulate vaccine

Produce antigen (in cell culture)

Purify antigen

Formulate vaccine

Fast Pure Egg-free

TRADITIONALMETHOD

MODERN VACCINE

Source: Manon Cox, Protein Sciences Corporation

Bac-to-Bac Baculovirus Expression System

• From Thermo Fisher

• High titer ~ 108 pfu suitable for large scale recombinant protein production.

• Polyhedrin promoter- High yield

• Specific transcription in E. coli– Faster and easier than homologous recombination in insect

cells

– DH10Bac E.coli competent cells

– Possibly eliminate the need for plaque assay

Source: Luckow VA, Lee SC, Barry GF, Olins PO J1993.

FlashBAC Expression Vectors

• From Oxford Expression Technologies.

• Several genes deleted from baculovirus genome to suit expression of several proteins.

• These genes include chitinase and cathepsin

– Suitable for genes expressed in the cytoplasm or nucleus

– Suitable for secreted proteins (falshBACGOLD) or membrane bound proteins.

– Suitable for release and subsequent purification of intracellular VLPs and protein complexes (flasBACPRIME)

BHK

HEK 293

Mammalian cells transduced With BacMam virus Expressing GFP

According to Kost, et.al,. (2005)Transduction frequency in these cell lines is higher than 90% based on the number of fluorescent green cells

BEVS can serve as Mammalian cell gene-delivery vectors

Source: Kost, Condreay and Jarvis. Nature Biotechnology. 2005

Sf21 cells; an example of a strongly attached cell line

TN-368 cells; an example of a loosely attached cell line

Thank You

Questions?