Preparation of Crystalline Insulin R. G . ROMANS, D. A. SCOTT, ANDA. M. FISHER University of Toronto, Toronto, Canada
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zinc-Insulin crystals became ANY p r o c e s s e s h a v e Insulin for the treatment of diabetes mellibeen published for preavailable in large quantities for tus can be made from crystalline or nonparing Insulin-soluchemical studies. In 1935 the crystalline preparations containing the Permanent Commission on Biotions containing the active diaactive antidiabetic principle. The protein logical Standardization, League betic principle of pancreas for use obtained by the hydrochloric acid-alcohol in the treatment of diabetes of Nations, replaced the old amorphous international standmellitus. Several papers have extraction of pancreas can be fractionally ard preparation of Insulin (8 also been concerned with laboraprecipitated by suitable concentrations of International units per mg.) by a tory methods for producing a acetone and zinc. From the physiologically preparation of zinc-Insulin cryscrystalline preparation of the active fraction, zinc-Insulin crystals can tals which, as a result of comactive principle. In 1926 Abel be readily prepared. The process described parisonwith the former standard, (I) published a method by was defined to contain 22 Interwhich he was able to prepare has had trial of about a year and a half and national units of Insulin per mg. such a c r y s t a l l i n e m a t e r i a l . has enabled the Connaught Laboratories ( 5 ) . The quantity of crystals The following year Abel and of the University of Toronto regularly to prepared a t that time amounted his associates (9) described an supply Insulin of crystalline, rather than to more than 100 g r a m . For i m p r o v e d m e t h o d . From a noncrystalline, origin for general clinical highly buffered aqueous solution several years, a t least a part of the Insulin supplied for clinical of an amorphous preparation use in the treatment of diabetes. use in various parts of the world these workers obtained sharply has been DreDared from zincdefined cubelike crystals which Insulin crystals. Use of Insulin of cr$stalline rather than have since been known as crystalline Insulin. noncrystalline origin will frequently eliminate hypersensitive Although the methods of Abel were not suitable for use on response occasioned in some diabetic patients by adminisa large scale, this early work was of great importance for it tration of a solution of the amorphous preparation. For the made available a means for the preparation of a crystalline treatment of patients who may exhibit allergic response, the product for chemical or clinical studies. Moreover, these supply of Insulin which has been made from a crystalline were the first experiments in which a hormone of protein preparation of the active principle is to be regarded as a nature had been prepared in crystalline form. In an endeavor desirable, and sometimes highly important, factor in the to add to the knowledge of the chemistry of Insulin and to treatment of diabetes mellitus. Moreover, from the standdevelop a method of preparation in which the yield of cryspoint of chemistry a crystalline preparation is usually to be tals would be greater than that of the early procedures, the preferred to an amorphous one. problem was taken up by various workers. In spite of conHeretofore in preparing zinc-Insulin crystals it was comtinued efforts, however, the yield of crystals remained small; mon practice first to prepare Insulin of a high degree of purity indeed, from some samples of Insulin it seemed to be impos(though still of an amorphous nature) by one of the wellsible to prepare any crystals whatever (3, 8). known methods. This material was then suitably diluted, In 1934 methods were developed in the Connaught Laborasterilized, etc., for supply as Insulin to be used in the treattories by which good yields of crystals could be obtained from ment of diabetes, or else further processed for the preparanoncrystalline Insulin prepared by well-known processes tion of zinc-Insulin crystals. I n the event of the second alter(6,?‘).The methods differed from previously published pronative, additional time was required and extra losses were cedures in that they required the addition of a small amount entailed before the Insulin was ready for clinical use. In the of zinc, or zinc salt, to the Insulin from which it was desired case of the method a t present in use for the production of to obtain crystals. The crystals produced by these methods zinc-Insulin crystals a t the Connaught Laboratories, it is not had the same microscopic appearance and the same chemical necessary to prepare Insulin of a high degree of purity as an and physiological properties as had the crystals obtained by independent step in the preparation of the crystals. A other processes. The crystals contained about 0.5 per cent crystalline preparation is now made in a shorter time and of zinc, a constituent that was also found in crystals prepared with smaller losses than were formerly encountered in the by any of the previously published methods. production of an amorphous preparation of sufficient purity In view of this, the term “crystalline Insulin” as applied for clinical use. to any crystalline preparation of antidiabetic principle yet described is somewhat inaccurate. The active principle has Preparation of Zinc-Insulin Crystals yet to be prepared in a crystalline form in which none of the The pancreatic glands are removed a t the abattoir as so011 metals zinc, nickel, cobalt, or cadmium is prescnt as an esas possible after slaughter and trimmed to remove excessive sential constituent. Crystals of the type known today are more correctly described by terms stlch as zinc-Insulin C ~ S - fat and connective tissue. The glands are stored a t refrigerator temperature for daily delivery to the laboratories or tals. Previous usage and convenience may, however, be placed in the freezer until several hundred pounds of glands factors in continued use of the expression “crystalline Inhave been collected. sulin”. The glands are minced and dropped into glass-lined extracWith the knowledge that a crystalline preparation of Intion tanks or glazed earthenware crocks containing acidified sulin can be obtained readily and in good yields from a sample alcohol. This extraction solution consists of 25 Imperial of Insulin to which a suitable amount of zinc has been added, 908
INDIJSTIIIAI, kND ENGINEERING CHEMISTRY
JULY. 19,tO
gallons (110 liters) of 83 per cent denatured alcohol (ethanol containing 10 per cent methanol) and 1000 cc. of concentrated hydrochloric acid for each 100 pounds (45.4 kg.) of glands. After being stirred slowly for at least 3 hours, the mixture is poiired into a nibher-lined basket centrifuge which separates the alcoholic extract from most of the insoluble material. The residue is re-extracted for about 2 hours with 22 gallons (IO0 liters) of 65-70 per cent alcohol for each 100 pounds of original matcrial. The mixture is centrifnged, and the first and second extracts are pooled and made ammoniacal (pII 8)
Fro-
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PtsoToniicHoosiAPIs Snowim A I k L D OF ZrS?-ISSul.Ih- CEvsTnLs
TYPICAL
The appearance of t h e crystals is independent of the specie8 from which the Insulin is obtained. with ammonium hydroxide. The heavy inactive precipitate which is formed is removed by filtration through a wooden filter press. The filtrate is acidified with sulfuric acid and concentrated in vacw at abont 20" C . to approximately one seventh of its original volume. The amount of sulfuric acid which is added should be such that the acidity of the concentrate is pFI 2.0-2.5. Immediately before removal from the still the concentrate is heated to 50" C. and then filtered through soft paper t.o remove fat and other insoluble material. The active principle is precipitated from the cooled filtrate by the addition of sodium chloride to a cancentration of 25 per cent. After standing for at least 2 hours, the active protein precipitate can he easily removed from the top of the brine. When the precipitates from 4800 pounds f2177 kg.) of pancreas have been collected, they are d d e d to 220 liters of water, and the mixtnre is acidified to pII 1.8-2.2 with hydrochloric acid. By stirring for abont 2 hours, the aetive principle is dissolved. Insoluble material is removed by filtrai tion through asbestos clarifying films. To the clear filtrate sodium chloride is added to a concentration of 15 per cent. After standing for at least 3 hours the precipitate is removed and allowed to drain on filter paper. The brine is discarded. The precipitate is then dissolved at pN 2.0-2.5 in 40 liters of mt,er acidified with hydrochloric acid. This solution is divided into two equal parts, and the process of purification continued. To 20 liters of the above solution, 25 cc. of a 90 per cent solntion of phenol arc n
