Next, the E Coli are lysed, the cell walls are dissolved to release the pre-proinsulin. This stage is filled with cellular debris, which is separated out through either centrifugation or other different forms of filtration. Next the signal peptide is cleaved from the preproinsulin to make proinsulin. This cleavage is done using cyanogen bromide in formic acid. Then the acid is removed from the solution and replaced with a different solvent.
Then the proinsulin is chemically modified to provide “handles” for purification. (Specifically, the proinsulin undergoes a sulfitolysis reaction. This puts strongly negative charges on certain parts of the molecule.) The purification step consists of several different types of columns. A column is a large tube, it can be over a meter in diameter, packed with small pebble-like particles. When the solution is passed through the column, most of it will pass by the pebble-like particles, but some specific parts proinsulin stick to the particles. After the complex solution containing proinsulin is passed through the column, a release buffer is passed through the column. The release buffer causes the proinsulin to release from the pebble-like particles, where the purified proinsulin is collected at the output of the column. In practice, there are multiple types of columns that are sequentially used to further purify the proinsulin.
The proinsulin is “folded” into the proper shape. It’s put into a particular solvent and
The proinsulin is transformed into insulin. An enzyme cleaves the proinsulin at two specific locations, cutting it into three pieces. Two of the pieces are bound together with the disulfide bonds, while the third piece (known as the C-peptide or connecting peptide) is separated from the insulin. Further purification is performed through columns to remove the c-peptide and any remaining contaminants.
A small amount of zinc is added to this pure form of human insulin. Six molecules of insulin arrange themselves around a single atom of zinc. These hexamers form a crystal in the solution. This final solution of crystalline insulin is spread out onto trays and a stack of trays, each separated by a few inches, is loaded into a special vacuum drying machine called a shelf dryer. Once the trays have been dried, a fine white powder consisting of insulin is left on the tray. The trays are moved to a packaging facility where insulin is packaged to be sold in different forms.
Although it is unlikely, perhaps even impossible, that these microbes would cause any problems if released, extensive safety measures are taken during the production of recombinant human insulin. The safety measures are a remnant of the controversy that played out in the late 1970’s about recombinant DNA. Exhaust air is treated to remove any water particles, which could contain microbes, then flows through an initial filter to remove any particulates, and finally goes through a sterilization procedure before being discharged into the atmosphere. All liquid that could contain organisms is heated to a high temperature before being discharged into the sewage system.