Purification of insulin from yeast by Pei Xin Yap

Process of obtaining insulin from yeast

kennice1112 — 2024-01-09 05:00:13 UTC

Process:

Spin and centrifuge
Filtration
24-hour stint in a capture column
another round of purification
dryers (drying insulin just above freezing for 24 hrs)
Packaging

Process of making insulin from powder

kennice1112 — 2024-01-09 05:02:51 UTC

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.

purification of insulin

2024-01-09 05:08:36 UTC

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

Expression of insulin in yeast

kennice1112 — 2024-01-09 05:09:59 UTC

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

Process using Tangential Flow Filtration

kennice1112 — 2024-01-09 05:18:02 UTC

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.

insulin from yeast capability

2024-01-09 05:18:22 UTC

yeast is preferred host for insulin expression
faster, easier & less expensive production
high level of protein expression

insulin purification

2024-01-09 05:20:33 UTC

spin & centrifuge filtration
24-hour stint inside a capture column -> from solution to a clay-like substance that contains 80-90% of insulin
insulin goes through a 2nd round of purification
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
it will be packaged & transported to doctors and patients

Strains and vectors

kennice1112 — 2024-01-09 05:30:02 UTC

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.

Growth

kennice1112 — 2024-01-09 05:30:23 UTC

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

IP purification and concentration

kennice1112 — 2024-01-09 05:54:32 UTC

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.

virus inactivation for insulin production

2024-01-09 12:21:01 UTC

1) low pH inactivation method

2) chromatography

3) nanofiltration

4) Ultra violet-C radiation

Low pH inactivation

2024-01-09 12:29:31 UTC

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

chromatography

2024-01-09 12:41:58 UTC

using a column that can bind to the virus, like a charged membrane
solution is passed over it and virus is filtered out

nano filtration

2024-01-09 12:44:09 UTC

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

UVC radiation

2024-01-09 12:45:13 UTC

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

Virus inactivation in yeast production

2024-01-09 12:49:12 UTC

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