Print this out and save it. I woudln’t be able to do this, but I know people who would.
And from Frederick Banting’s Nobel Lecture in 1925:
Best and Scott who are responsible for the preparation of Insulin in the Insulin Division of the Connaught Laboratories have tested all the available methods and have appropriated certain details from many of these, several new procedures have been found advantageous have been introduced by them. The yield of Insulin obtained by Best and Scott at the Connaught Laboratories, by a preliminary extraction with dilute sulphuric acid followed by alcohol is 1,800 to 2,220 units per kg. of pancreas.
The present method of preparation is as follows. The beef or pork pancreas is finely minced in a larger grinder and the minced material is then treated with 5 c.c. of concentrated sulphuric acid, appropriately diluted, per pound of glands. The mixture is stirred for a period of three or four hours and 95% alcohol is added until the concentration of alcohol is 60% to 70%. Two extractions of the glands are made. The solid material is then partially removed by centrifuging the mixture and the solution is further clarified by filtering through paper. The filtrate is practically neutralized with NaOH. The clear filtrate is concentrated in vacuo to about 1/15 of its original volume. The concentrate is then heated to 50oC which results in the separation of lipoid and other materials, which are removed by filtration. Ammonium sulphate (37 grams. per 100 c.c.) is then added to the concentrate and a protein material containing all the Insulin floats to the top of the liquid. The precipitate is skimmed off and dissolved in hot acid alcohol. When the precipitate has completely dissolved, 10 volumes of warm alcohol are added. The solution is then neutralized with NaOH and cooled to room temperature, and kept in a refrigerator at 5oC for two days. At the end of this time the dark coloured supernatant alcohol is decanted off. The alcohol contains practically no potency. The precipitate is dried in vacuo to remove all trace of the alcohol. It is then dissolved in acid water, in which it is readily soluble. The solution is made alkaline with NaOH to PH 7.3 to 7.5. At this alkalinity a dark coloured precipitate settles out, and is immediately centrifuged off. This precipitate is washed once or twice with alkaline water of PH 9.0 and the washings are added to the main liquid. It is important that this process be carried out fairly quickly as Insulin is destroyed in alkaline solution. The acidity is adjusted to PH 5.0 and a white precipitate readily settles out. Tricresol is added to a concentration of 0.3% in order to assist in the isoelectric precipitation and to act as a preservative. After standing one week in the ice chest the supernatant liquid is decanted off and the resultant liquid is removed by centrifuging. The precipitate is then dissolved in a small quantity of acid water. A second isoelectric precipitation is carried out by adjusting the acidity to a PH of approximately 5.0. After standing over night the resultant precipitate is removed by centrifuging. The precipitate, which contains the active principle in a comparatively pure form, is dissolved in acid water and the hydrogen ion concentration adjusted to PH 2.5. The material is carefully tested to determine the potency and is then diluted to the desired strength of 10, 20, 40 or 80 units per c.c. Tricresol is added to secure a concentration of 0.1 percent. Sufficient sodium chloride is added to make the solution isotonic. The Insulin solution is passed through a Mandler filter. After passing through the filter the Insulin is retested carefully to determine its potency. There is practically no loss in berkefelding. The tested Insulin is poured into sterile glass vials with aseptic precautions and the sterility of the final product thoroughly tested by approved methods.
The method of estimating the potency of Insulin solutions is based on the effect that Insulin produces upon the blood sugar of normal animals. Rabbits serve as the test animal. They are starved for twenty four hours before the administration of Insulin. Their weight should be approximately 2 kg. Insulin is distributed in strengths of 10, 20, 40 and 80 units per c.c. The unit is one third of the amount of material required to lower the blood sugar of a 2 kg. rabbit which has fasted twenty four hours from the normal level (0.118 percent) to 0.045 percent over a period of five hours. In a moderately severe case of diabetes one unit causes about 2.5 grammes of carbohydrate to be utilized. In earlier and milder cases, as a rule, one unit has a greater effect, accounting for three to five grammes of carbohydrate.