Date post: | 13-Apr-2017 |
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Synthetic Insulin – R&D and Industrial level Manufacturing process & requirement (overall idea)
Krishnasalini Gunanathan | Research Trainee|
Insulin
• Insulin –
• A hormone that regulates the amount of glucose in the blood
• Produced by cells in pancreas- islets of Langerhans
• types – Ultra short acting, short acting, intermediate acting, long acting
• History of synthetic insulin
• 1921 by Canadian sci Frederick G.Banting & Charles H.Best from Dog’s pancreas
• 1982 Eli lilly corp., produced genetically engineered human insulin
Structure - Insulin
• Insulin gene – a protein consist of two amino acid chains A above chain B held together with bonds
• Chain A - 21 amino acid , Chain B – 30 amino acids
• Formation of insulin –
• Preproinsulin proinsulin single chain without signaling sequence Active protein insulin without link between A & B
Types Insulin Production
• Type -1
• Two insulin chains are grown separately and inserted into plasmids and grown in E.coli
• Two chains are linked by oxidation-reduction reaction using lysosomes and cyanogen bromide
• Purified by chromatographic methods
• Type- 2
• Known proinsulin process
• Coded sequence are inserted to non-pathogenic E.coli bacteria & fermentation is carried out
• Zn2+ (additives) are added to delay the absorption in the body
• Type – 3
• Analog insulin is produced by changing its amino acid sequence & creating an analog, which
clumps less & disperses more readily into the blood. Eg: Glargine insulin
Materials Required (R&D level)
• Mother culture – Escherichia coli
• UV-Chamber
• Human protein with 20 amino acids which produce insulin
• Enzymes, antibiotics and primer
• PCR – cloning
• Fermentation flasks – bacterial culture growth
• Culture medium / nutrients – LB broth or any for the culture
• Centrifuge – screening process
• Culture shaker
• Chromatography (electrophoresis and paper chrom kit) – isolation and screening process
• X-ray crystallography – isolation and screening process
rDNA technology – Insulin gene production
rDNA technology – Insulin gene production
Isolation of human cells and grown in tissue
culture
Isolation of DNA from the human cells using restriction
enzyme
Meanwhile, Plasmid DNA is isolated
Recombinant plasmid is mixed with bacteria
Join the plasmid and human fragment
Use same restriction enzyme the plasmid DNA is cut to
create matching sticky ends
Allow the new bacteria recombinant plasmid into
the bacterial cell
Screen the bacteria to get the incorporated human gene for
insulin
Grow screened insulin producing bacteria (gene
cloning)
Insulin production – Industrial level
Involves two process
Upstream Process -
Cell Line preparation (Obtaining human insulin gene by complementary DNA from mRNA of the two chains
using enzyme reverse transcriptase or cloning of cDNA of both chains using PCR with amplification of cDNA seq) ,
insertion of cDNA in plasmids, transfection (electroporation – transferred cells )
Downstream Process –
Fermentation, Cell separation, Centrifugation, Analysis
Road map - Insulin productionProduct Idea (Type of insulin)
Selection of producing organism (E.coli or yeast)
Strain screening
Formulation medium requirement
Medium optimization
Small scale bioreactor cultures (batch fed, continuous)
Process control requirement
Scale – up (>100 liters)
Process kinetics (productivity etc)
Strain improvement (molecular tech)
Yield conversion, productivity test
Biomass- product separation
Product purification
Concentration, crystallization, drying
Fill finish
Storage properties, stability
Field trials
Approval, product license
Marketing & sales
Effluent recycle/disposal
USP DSP
Selections to be done
Choice of cell line – microbes
Bacterial cells – genetic ease(single molecule DNA
sequenced)
• High productivity, high µ
• Resistance to shear, osmotic pressure
• Negatives- poor secretors, glycosylation/post translational
modification
Yeast –
• High µ, cell concentration, productivity, secretion, post
translational modification, glycol engineered strains
available
• Non mammalian glycosylation, post translational
modifications, complexity of genetic manipulation
Types of Bioreactor Conditions
• Anchorage dependence / suspension adapted
• Mixing – homogeneous condition, absence of nutrients and
temperature gradients
• Mass transfer – OTR
• Cell density
• Shear resistance
Different bioreactors
• Shear tank reactor
• Membrane reactor
• Fixed bed reactor
• Disposable reactor
Selection of
• Substrate feeding
• Filtration, Purification, Polishing
Materials Required – Industrial production
Up Stream process Downstream process
Mother Culture (E.coli / Yeast) Bioreactor
cDNA(human insulin gene), mRNA sequence Fermenter
Primer, UV chamber CentrifugePCR Nitro homogenizer
Culture medium (LB broth) Cell separator
Antibiotic (Ampicillin, Lactose) Seed, harvest, receiver tank
Ligase, restriction enzyme, digestion enzyme Ion exchange chromatographyCulture shaker Electrophoresis kit
Cyanogen bromide (split the protein chain )Sodium dithionate, Sodium sulphite RP-HPLC
Manufacturing process – Industrial level
Upstream process
• Seed preparation – cell line selection,
genetic manipulation and characterization
• Equipment preparation – bioreactor
sterilization
• Media preparation – purification,
formulation, optimization & sterilization,
ingredients to sustain growth, raw
materials for the product
Bioreactor
• Inoculation / scaling up of seed culture
• Process monitoring and control
• Optimization of culture conditions
Downstream process
• Harvesting of cells (crude products)
• Isolation & centrifugation of
products
• Crystallization & purification of
product
• Analysis and packaging
The most successful manufactures - Insulin
Conclusion
Upon the fulfillment of the mentioned materials and equipment's manufacturing of synthesized insulin using rDNA technology is very much possible and will be successful .
Thank You