1.

Antiviral vaccines:
 Animal cell culture tech since 1950s.
 Cell culture replaces live animals, advancing bioprocess.
 DNA tech enables recombinant vaccines (e.g., HBV).
 Many vaccines, incl. HBV, in final clinical trials.
2. Viral particle production by cell culture:
 Differs from protein/enzyme production.
 Product formation via secondary metabolic pathways.
 Virus production post-infection directs cell machinery.
 Two stages:
1. Cell culture system for substrate conversion.
2. Virus production with unique nutritional/metabolic needs.
 Immortalized cell lines used, listed in Table 14.5
3. Virus-like particles (VLPs) as a new type of vaccine:
 Traditional vaccines may carry risks like incomplete inactivation or
reversion of attenuated strains.
 Viruses with segmented genomes can undergo genetic exchange,
leading to the production of new variants.
 Some live virus vaccines can have harmful effects, like teratogenicity.
 Recombinant DNA (rDNA) technology enables the development of a
new vaccine type without typical side effects.
4. Effectiveness and advantages of VLPs:
 VLPs mimic virus structure but lack infectious genetic material.
 Capsid proteins can form core-like particles without nucleic acids,
stimulating B-cell-mediated immune responses.
 VLPs elicit CD4-proliferative and cytotoxic T-lymphocyte responses.
 VLPs are nonpathogenic, replication-deficient, and do not require viral
inactivation, reducing epitope modifications.
 Their structural similarity to viruses ensures presentation of
conformational epitopes to the immune system, enhancing immune
response.
 VLPs induce strong B-cell responses and can be adapted for broader
protection.
5. Vaccines based on virus-like particles (VLP):
 The FDA has approved VLP-based vaccines for HBV and HPV.
 HBV vaccine was approved in 1986, and the HPV vaccine in 2006.
 To generate immunogenic VLPs, the S gene is cloned and expressed in
a eukaryotic expression host such as yeast or mammalian cells (e.g.,
CHO cell line).
 Mammalian cell culture allows easy recovery because cells can secrete
the HBsAg antigen.
 Two major CHO-based vaccines are Gene Hevac B and Sci-B-Vac.
6. Human papilloma virus vaccine (HPV):
  HPV can cause lesions, warts, and cervical cancer.
 FDA approved vaccines include Gardasil and Ceravarix.
 Gardasil is manufactured by Merck and Co., Inc., while Ceravarix is by
Glaxo Smithkline.
7. Other VLP-based vaccines:
 Several VLP-based vaccines are in clinical trials, including anti-influenza
A, anti-malarial, and anti-AngIIQβ vaccines.
 VLPs are produced in mammalian cell lines and Baculo cell lines
infecting humans and other animals.
8. Proteins in Biochemical Processes:
 Proteins facilitate biochemical reactions, molecule transport, receptor
formation, and structural support.
 Post-translational modifications like glycosylation, phosphorylation,
and others significantly increase protein functional diversity.
 Structural abnormalities or mutations in proteins can lead to diseases.
 Protein therapeutics offer promising solutions for disease management.
9. Main Therapeutic Protein Groups:
 Seven main groups include cytokines, growth factors, hormones, blood
products, enzymes, and antibodies.
 These proteins often undergo post-translational modifications to
ensure full biological activity.
 Glycosylation is a prominent modification, crucial for proper protein
function.
 Eukaryotic cells, especially mammalian cells like CHO cells, are preferred
for protein production due to their capability for complex
modifications.
10. Cytokines:
 Cytokines are immune system proteins vital for immune response.
 Produced in response to immune stimuli by various white blood cells.
 Interferons (IFNs) were the first cytokines discovered and utilized as
biopharmaceuticals.
11. Applications of Interferons:
 IFNα is used to treat hepatitis, leukemia, and certain cancers.
 IFNβ is utilized for multiple sclerosis under names like Avonex, Belaseron,
and Rebif.
 IFNγ treats chronic granulomatous disease.
 Interleukin regulates cell growth, differentiation, and motility, with
recombinant IL-2 used for renal cell carcinoma.
12. Growth Factors:
 These proteins bind to cell surface receptors to promote cell proliferation or
differentiation.
 Types include TGF, insulin-like growth factor, and EGF.
 Examples like Osigraft/Eptotermin alfa and InductOS/Dibotermin are used for
bone fractures, commercially grown in CHO cells.
13. Hormones:
 Insulin, glucagon, gonadotropins, and growth hormones are common
therapeutic hormones.
 Insulin and recombinant human growth hormones were the first approved
biopharmaceuticals.
 Gonadotropin hormones like Gonal-F and Luveris are produced in CHO cells
and used for treating female infertility.
14. Therapeutic Enzymes:
 Recombinant tissue plasminogen activator (tPA) is expressed in
mammalian cells and used as a thrombolytic agent for dissolving blood
clots.
 Commercially known as Alteplase and Tenectplase, they are used to
treat acute myocardial infarction.
 Fabrazyme, approved in 2001, treats Fabry disease by providing
recombinant α-galactosidase A, produced in genetically modified CHO
cells.
15. Blood Coagulation Factors:
 Hemophilia A, B, and C result from deficiencies in clotting factors VIII,
IX, and XI, respectively.
 Recombinant factor VII products like Recombinate and Kogenate are
expressed in CHO and BKH cells.
 Recombinant factor IX, sold as BeneFIX, is produced in recombinant
CHO cells.
16. Antibodies:
 Therapeutic antibodies treat cancer, cardiovascular disease, infections,
and autoimmune diseases.
 Avasin (Bevacizumab), approved in 2004, inhibits vascular endothelial
growth factor for metastatic colorectal cancer treatment.
 Zenapax, produced in NSO cells, is used to prevent organ transplant
rejection and was approved for human use in 1997.
17. Importance of Cell Culture in Gene Therapy:
 Gene therapy involves manipulating genes to cure diseases or slow
their progression.
 Animal cell culture technology is crucial for gene therapy
advancements.
 Monogenic diseases caused by single gene defects are primary targets.
 Steps include identifying faulty genes, isolating genes, generating
expression constructs, integrating genes, and delivering genetic
material in vivo or ex vivo.
18. Clinical Correlation:
 Numerous clinical studies and trials for gene therapy are approved
worldwide, with a significant focus on cancer treatment.
  Gendicine, the first gene therapy product, treats head and neck
carcinoma by expressing the tumor suppressing gene p53 using
recombinant adenovirus.
 Gendicine is produced using the SBN-cel cell line, derived from human
embryonic kidney (HEK) cells.
19. Biopesticides:
 Biopesticides have gained importance due to environmental and food
safety concerns.
 They provide effective control of insects and plant diseases while being
environmentally safe.
 Baculoviruses are promising biocontrols against caterpillars but face
challenges in large-scale production.
 Developing cost-effective in vitro production processes for
baculoviruses can provide safe and efficient insect control.
20. Factors for Successful Baculovirus Production:
 Large-scale production at competitive costs is crucial.
 Economic production involves low-cost media and culture running
expenses.
 Effective cell lines with high virus productivity are essential.
 Minimizing virulence loss and mutant formation during virus passage is
necessary.
 Quality of polyhedra produced in cell culture should match those from
caterpillars.
21. Advantages of Insect Baculovirus Cell System:
 Produces functional and immunologically active recombinant proteins
with post-translational modifications.
 Utilizes a potent promoter, polyhedrin.
22. Cell Lines for Biopesticide Production:
 Commonly used cell lines include Sf21 and Sf9, derived from ovarian
tissues of the fall armyworm.
 Sf9 cells exhibit faster growth rates and higher cell densities compared
to Sf21, making them preferred.
 High Five cell lines (BTI-Tn-5BI-4) from Trichoplusia ni embryonic tissue
are also utilized.
23. Viral Mutant Formation in Cell Culture:
 Continuous culturing for virus production leads to instability and the
"passage effect", causing decreased virulence, polyhedral production,
and mutations.
 Common mutations include defective infective particles (DIPs) and few
polyhedra (Fp) mutations.
 Fp mutations result in reduced polyhedra, increased BV production, and
reduced pest infectivity.
  DIPs occur due to prolonged passaging, resulting in reduced filtering of
infectious virus.
24. Monoclonal Antibodies (mABs):
 Majority of mABs are produced in animal cell cultures due to their
glycosylation and structural conformation capabilities.
 Hybridoma technology is widely used for mAB production, but they
have limited therapeutic applications due to adverse immune
responses.
 Various cell lines including CHO, NSO, Sp 2/0, HEK-93, and BHK are
used for recombinant antibody production.
 Factors influencing mAB production include cell line productivity,
protein productivity, and scalability.
25. Stem Cells:
 Stem cells have the potential to differentiate into various cell types and
tissues.
 Their ability for self-renewal and differentiation into specialized cells
makes them promising for organ replacement therapy.
 Human embryonic stem cells (hESCs) are traditionally cultured on
mouse embryonic fibroblast feeder layers but animal-free culture
systems have been developed.
 Recent methods allow subculturing of embryonic cells without feeder
layers, using substances like Martigel for effective attachment and
differentiation.
26. Introduction to 3D Cell Culture and Microfluidics:
 Traditional 2D cell cultures lack in vivo environment representation and
physiological relevance.
 Recent advancements in 3D cell culture and imaging technologies allow
better analysis and observation of individual cells.
 3D cell culture systems provide a biomimetic environment, improving
cell differentiation and function.
 Microfluidics technology, manipulating fluids at the micron-scale, has
integrated with 3D cell culture, offering precise control and promoting
biologically relevant functions.
27. Advantages of Microfluidics in 3D Cell Culture:
 Microfluidic devices recreate the microenvironment of the vasculature,
allowing precise spatial control over gradients and medium exchange.
 They promote biologically relevant functions not seen in 2D cultures
and offer high-throughput capabilities.
 Microfluidic platforms can provide continuous nutrient supply,
investigate dynamic cell culture characteristics, and utilize various
substrates for fabrication.
28. Applications of Microfluidics Technology:
  Microfluidics is utilized in tissue engineering for regenerative medicine,
organ-on-a-chip technology, and toxicological studies.
 It serves as a robust platform for developing in vitro models to mimic
physiological conditions and has shown promise in clinical applications.
29. Introduction to Organ-on-a-Chip Technology:
 Current in vitro and in vivo models have limitations in simulating
intricate cell interactions.
 Organ-on-a-chip devices, microfluidic cell culture platforms, mimic 3D
tissue microenvironments and physiology.
 They offer biological relevance and potential for high-throughput
applications, impacting drug discovery and toxicity testing.
30. Tissue Models on a Chip:
 Various tissue models have been developed, including liver, kidney,
lungs, intestines, muscles, fat, blood vessels, and tumors.
 Liver-on-a-chip devices are crucial for studying drug-induced liver
toxicity and metabolic activity.
 Tumor-on-a-chip platforms facilitate the study of cancer cell behavior,
drug transport, and resistance to chemotherapy.
31. Liver-on-a-Chip:
 Microfluidic devices mimic physiological and mechanical environments
of hepatocytes, supporting growth and functional integrity.
 Multiwell micropatterned co-culture systems maintain phenotypic
functions of liver cells for extended periods.
32. Tumor-on-a-Chip:
 Microfluidic platforms detect circulating tumor cells for early cancer
diagnosis.
 Various designs of microfluidic devices culture solid and liquid tumors,
study drug resistance, and optimize treatment parameters.
 Microfluidic systems facilitate photodynamic therapy-based
measurements for cancer treatment optimization.