E Coli containing the insulin gene grow in a fermenter

A recombinant insulin plant is fully self contained, unlike the old insulin plants that first had to take delivery of tons of animal pancreases to produce grams of insulin. Eli Lilly froze samples of the initial cloned proinsulin gene in E Coli back in the 1980’s.  Each new batch of insulin that they produce originates with a frozen sample.  It is important that an industrial process be repeatable with consistent properties over decades[Handbook of Industrial Cell Culture: Mammalian, Microbial, and Plant Cells]

The large scale production of insulin progresses in stages.  First, a frozen sample of the original culture is thawed and some drops are place onto an agar plate.  These drops are smeared onto the surface, and the plate is left in a warm (body temperature) moist oven overnight.  Cultures appear on the plates as distinct opaque areas.  Several of these cultures are picked off the plate and put into several shake flasks. Shake flasks contain a liquid broth optimized for E Coli growth.  They are placed on integrated shaker/incubator where both the temperature and the rate at which the flask is shaken is controlled.  After a period of growth in the shaker/incubator, the broth is transferred into a seed tank.  The seed tank is a small version of the fermenter.  When the E Coli have outgrown the seed tank, they are transferred into the main 40,000 gallon fermenter.

E coli will reproduce about every 20 minutes, under ideal conditions.  Exponential growth under these ideal conditions cannot last forever.  Soon the density of E Coli will reach saturation.  Saturation occurs when the waste that the bacteria secrete reaches such high levels that it inhibits further growth.  Insulin production will be non-ideal in under saturation conditions.  To optimize the amount of insulin that the E Coli produce, the reaction must continue as long as possible, but terminate before the E Coli reach saturation.

The density of E Coli in a solution can be measured by shining a light through the solution.  At very low density, most of the light will travel across the solution, but as the density of bacteria increase, light will be scattered by E coli and not travel across the solution.  Using measurements like light scattering, the process controllers can identify the ideal density at which to stop the reaction.

Each bacteria produces insulin, but retains it inside the cell within inclusion bodies.  The next step is to harvest the insulin from the bacteria. Harvesting begins by concentrating the E Coli, removing it from the fermentation broth.  This concentration is performed by machines called disk stack centrifuges.  A disk stack centrifuge creates an artificially high gravitational field (about 13,000 times earth’s gravity) by rotating.  This high gravity-like field causes the heavier particles (E Coli) to move the edge of the machine, while the lighter material (the broth) moves away from the edge.  The disk stack centrifuge allows this process to be continuous, a hose is connected from the top of the disk stack centrifuge to the large fermenter.  All 40,000 gallons of the fermenter are passed through a disk stack centrifuge.  The heavier particles, the bacteria containing the insulin, are periodically discharged out the side of the centrifuge.