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Process:

1. Spin and centrifuge

2. Filtration

3. 24-hour stint in a capture column

4. another round of purification

5. dryers (drying insulin just above freezing for 24 hrs)

6. Packaging

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Powdered insulin is diluted with distilled water.

Packaged into glass vials.

To ensure vials are contamination free, they are washed dried and sterilized near 570 Degrees Fahrenheit

fill vials with insulin, computer-controlled precision and fitted with an aluminum cap.

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cell harvesting--> done by centrifugation or filtration

cell disruption-->lyse yeast cells to release intracellular contents

insulin extraction -->extract insulin from the lysed cells with solvent or detergent to break down membrane and release proteins

initial purification -->precipitation or filtration to remove large debris

chromatography

concentration-->concentrate the purifies insulin

analysis and quality control-->SDS-PAGE

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Yeast based expression systems from Saccharomyces cerevisiae, where inclusion bodies are directly secreted in the culture supernatant in its correctly folded conformation.

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Process using Tangential Flow Filtration

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Process using Tangential Flow Filtration

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Using TFF, we are able to recover IP from culture broth eliminating the laborious steps of centrifuging and secondary clarification of the supernatant.

Purifying the IP using a cation exchange resin at high loading capacity and performing IP elution in water/ethanol. The mixture was then ready for transpeptidation reaction.

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yeast is preferred host for insulin expression

faster, easier & less expensive production

high level of protein expression

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1. spin & centrifuge filtration

2. 24-hour stint inside a capture column -> from solution to a clay-like substance that contains 80-90% of insulin

3. insulin goes through a 2nd round of purification

4. the insulin will need to be dry, however, dryers that use heat will not be used as warm temperature causes the degradation of the insulin

instead, it will dry in temperature above freezing for 24 hrs

5. it will be packaged & transported to doctors and patients

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a vector will be used and electroplated into a P. pastoris strain. Transformants was then selected on plates with increasing concentration of antibiotic for selection of multiple clones. Which were then grown in a liquid culture.

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a preculture with a single colony made earlier was then made from the yeast extraact peptone dextrose plate. Dilution is then carried out and the pre-culture is transferred to the bioreactor with growth media containing glycerol. Once the amount of glycerol decreases and is in low amounts. methanol is then added to kick start IP production with the help of methanol and biotin, with an increased flow rate.

insulin precursor was then quantified with reversed phase- HPLC with a calibration curve being plotted. the culture broth was then passed through a 0.22 membrane to extract, filtrating it. Diafiltration was then carried out

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purification of IP was done using cation chromatography using resin. and eluted IP was then collected. Transpeptidation and purification of human insulin. Transpeptidation reaction by adding components to eventually form/elute insulin ester. eventually being crystallized and recovered by centrifugation and drying. It is converted to human insulin at the end of crystallization, and removal of butyl group in threonine by acidolysis.

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virus inactivation for insulin production

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virus inactivation for insulin production

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1) low pH inactivation method

2) chromatography

3) nanofiltration

4) Ultra violet-C radiation

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many viruses irreversibly denatured and destroyed at pH 5.0-5.5

some enveloped viruses are only destroyed at pH 3.5-4

however prolonged exposure to pH 3.5-4 can damage and inactivate insulin due to the prolonged exposure to pH stress

hence it is important to know the scope of reagent addition and time needed for the process in order to optimize the production

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using a column that can bind to the virus, like a charged membrane

solution is passed over it and virus is filtered out

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nanofiltration is a type of membrane filtration process that uses membranes with pore sizes in the range of 1-10 nanometers

it exludes viruses due to small size of the pores and may sometimes physically damage and inactivate certain viruses passing through

it can be selective and allow certain ions to pass through

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UVC (ultraviolet C) radiation is a form of ultraviolet light with wavelengths in the range of 200 to 280 nanometers.

damages the rna and dna of viruses and inactivates them

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however yeast is a prokaryotic cell and hence do not have eukaryote viruses which makes it safer as mammalian cells as hosts often come with a higher risk of being contaminated with viruses