Webinar from the Biomanufacturing Education & Training Center at Worcester Polytechnic Institute

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

2. High Level Recombinant
Protein Production In
Insect Cell Culture
Kamal Rashid, Ph.D.
Director, Biomanufacturing Education & Training Center

3. 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

4. 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

5. Media
Optimization
Cell Culture Process Development
Expression
System
Bioreactor
Selection

6. 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

7. 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)

8. 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.

9. 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

10. 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.

11. 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).

12. 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

13. Baculoviruses-AcNPV
• Gene expression occurs at an early and late
phase 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

14. 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.

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

16. Baculovirus Replication Cycle
Source: Patel and Jones, 1995

17. Source: Rohrmann 2013

18. Rsource: Rohrmann 2013

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

20. 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

21. 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

22. 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

23. 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.

24. 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.

25. 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.

26. 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.

27. 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

28. 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

29. Protein Sciences Platform
Vaccine Production
Source: Manon Cox, Protein Sciences Corporation

30. 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
TRADITIONAL
METHOD
MODERN VACCINE
Source: Manon Cox, Protein Sciences Corporation

31. 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.

32. 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)

33. 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

34. Sf21 cells; an example of a
strongly attached cell line

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

36. Thank You
Questions?