What is biotechnology?
A science driven industry sector that uses living organisms and their derivatives to produce products and processes
What were the principles of biotechnology restricted to before the 19th century?
to produce bread, beer, and wine using the natural process of fermentation
Name some key techniques used in biotechnology
- genetic engineering –> modify genetic makeup of organisms
- tissue culture
- therapeutics and diagnostics
What type of biologics are produced in bacteria and mammalian cells?
- enzymes
- rProteins: insulin, EGF, growth hormone, cytokines
- antibodies: mAb
- plasmid DNA vaccine
- ATP (advanced therapy products)
–> CAR-T
Briefly describe CAR-T
a new gene encoding a Chimeric Antigen Receptor (CAR) is incorporated into patient’s isolated T cells
Describe the steps involved in CAR-T therapy
- collection of T cells from patient
- genetic modification to express CAR
- expansion and activation: cell culturing
- infusion of CAR-T cells into patient’s bloodstream*
*chemotherapy may be given before CAR T cell therapy
What could be the potential side effects of CAR-T therapy?
cytokine release syndrome (CRS) and neurotoxicity
Briefly describe the E coli biologics production process
- transformation
- fermentation: large scale cultivation
- harvesting: collection of E coli biomass
- purification: isolation and purification of biologic
Briefly describe rProtein prodcution
- source DNA –> cloned into vector –> transfer to host cell –> screening & selection
- cell expansion –> cell production in bioreactors –> recovery thru filtration and centrifugation –> purification thru chromatography –> characterization and stability
What factors could be optimized in cell expansion and production in bioreactor phase?
- cell line
- growth media
- method of expansion
Name some elements of plasmid
- origin of replication
- restriction site
- promoter
- antibiotic resistance gene as selectable marker
Briefly explain the principle IPTG induction protein expression in prokaryote cells
w/o IPTG –> repressor protein bind to operator region of DNA –> inhibition of transcription
IPTG –> bind to repressor –> repressor undergo conformational change –> RNA polymerase bind –> transcription
What is IPTG?
Isopropyl β-D-1-thiogalactopyranoside
–> molecular mimic of lactose but not metabolized by cell
What are the main steps in plasmid manufacturing process?
- upstream processing
- fermentation
- downstream processing
What does ‘upstream processing’ involve in plasmid manufacturing process?
- vector construction
- strain selection: grow & replicate plasmid
- media optimization
What does ‘downstream processing’ involve in plasmid manufacturing process?
- cell lysis
- purification
- final product: supercoiled plasmid DNA
In what from does the plasmid DNA exist in by the end of plasmid manufacturing process?
supercoiled
What is the purpose of ‘clarification’ in plasmid manufacturing process?
performed after cell lysis and before purification
–> to remove insoluble particles, impurities, or debris from a liquid solution.
What are the steps involved in clarification and purification in processes like plasmid DNA production or protein purification?
- lysis
- flocculent treatment
- centrifugation
- filtration
What kind of centrifugation is used for industrial scale?
industrial centrifugation
–> denser or larger particles quickly move outward under centrifugal force
–> Lighter particles remain closer to the center and collected
Name 2 types of filtration in plasmid DNA production or protein purification
- Hollow fiber tangential flow filtration (TFF)
- Process scale cassette tangential flow filtration (TFF)
How is mRNA for mRNA vaccination produced?
- vaccine cloned into plasmid
- transformation, fermentation and plasmid isolation
- restriction enzyme linearize plasmid template
- in vitro transcription generates mRNA using RNA poymerase than stabilised by cap enzyme
- mRNA mixed with lipid nanoparticle
- mRNA vaccine produced
Who won the Nobel Prize in Physiology or Medicine awarded in 1986? Why?
Rita Levi-Montalcini and Stanley Cohen
for the discovery of nerve growth factor and epidermal growth factor (NGF and EGF)
For rProtein (such as EGF) production, what techniques are utilized to check for purity?
HPLC and SDS PAGE
