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