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Vol. 17. No. 3
The Preparation of Insulin' By D. A. Scott and C. H. Best CONNAUGHT LABORATORIES, UNIVZRSITY OF TORONTO, TORONTO, CANADA
HE discovery by Banting and Best's* of the pancre- hour 25 gallons of 95 per cent alcohol are added for each 100 atic hormone, insulin, has added another biological pounds of glands. (The alcohol is ethyl denatured with 10 product to the already long list. The preparation per cent methyl alcohol.) Slow agitation of the mixture is and purification of this hormone, however, differs greatly continued in order to facilitate the extraction. At the end from that of the other biological products. of 2 hours the acid-alcohol mixture is poured into a large I n a previous communication* on the preparation of in- rotary centrifuge which separates the alcoholic extract from sulin the writers referred to many investigators who did the solid materials. The solid materials remaining in the pioneer work in attempting to isolate the internal secretion centrifuge are re-extracted with a volume of 60 per cent albf the pancreas. They also coho1 equal to that of the described, in some detail, liquid obtained during the The method used for the preparation of insulin in this some of the earlier expericentrifuging. After this laboratory has been reported in detail. This method has ments and the methods of mixture has stood overbeen in practice for some time and has worked very satispreparation of the earlier night, it is centrifuged as in factorily. The essential features of the process are, first, e x t r a c t s used b y t h e the first extraction. The the extraction of the pancreas by acid alcohol; second, original investigators in extracts from the first and the precipitation of the concentrate with ammonium Toronto. Since that coms e c o n d e x t r a c t i o n s are sulfate; and third, the precipitation in hot alcohol. This munication they have modimixed and filtered through step in the process is very essential, since it removes the fied, in certain details, their large glass funnels which fat and lipoid substances as well as much coloring mateprocess of manufacture. have been fitted with fluted rial. These substances interfere with the isoelectric preThis has resulted in a purer filter papers. The filtrate cipitation. The fourth step is the fractional precipitation is partially neutralized with product and higher yields at different acidities. of insulin. There have also a solution of sodium hydroxApproximately 80 per cent of the total amount of insulin been numerous contribu(240 cc. of 50 per cent ide extracted by the alcohol from the pancreas is purified. tions on the preparations of sodium hydroxide solution Practically the only loss which occurs in the process is insulin by other investifor each 50 gallons of filthe incomplete precipitation of the insulin at the isogators. Dudley and Startrate). The alcoholic exelectric point. The average nitrogen of the different lots ling3 have obtained very tract is then concentrated to of insulin prepared during the past year is 0.010 mg. per satisfactory yields of insulin about one-fifteenth of its unit of insulin. Much lower nitrogen values have been by alkali-alcohol extraction original volume in an effiobtained by modifying the purification, but this has of the pancreas. Dodds cient vacuum still. During always resulted in a much decreased yield. Moreover, it and Dickens4 report large the distillation the temperais doubtful whether there is any particular advantage in yields of insulin by a picrateture of the distillate is not a further purification for clinical use. allowed to rise above 30" C . acetone method of extracAfter the completion of tion. Doisy, Somogyi, and Shaffer6 have reported further details of the purification of the distillation the concentrate is quickly heated &I 50" C. At this temperature lipoid and other materials readily rise insulin by fractional precipitation a t different acidities. The method of preparing insulin in the writers' laboratories to the surface and are removed by filtration. The concenhas many details in common with the Doisy, Somogyi, and trate has an acidity of p H 2 to 2.5. It is important that Shaffer6 process. However some new features have been the acidity be within this range in order to insure rapid filtraintroduced which are believed to be very important. The tion and to avoid loss of potency. After the concentrate has separation of fats and lipoid material from the concentrate, cooled, ammonium sulfate is added to half saturation (37 (1) by filtering a t 50" C. and (2) by treatment with 90 per grams per 100 cc.). This mixture is stirred, and almost cent alcohol, seems most advantageous for obtaining a solu- immediately protein material separates out and readily rises to the top of the liquid. After standing for 0.5 hour the tion from which to secure an isoelectric precipitate. protein precipitate is skimmed off and as much liquid as The Present Process possible is pressed out. The weight of the moist precipitate Fresh pancreatic glands from the ox are obtained from from 300 pounds of glands is approximately 200 grams. This precipitate is dissolved in hot acid alcohol. Usually abattoirs. As much fat and connective tissue as possible are separated from the glands, and they are collected hourly and 1 liter of 50 per cent alcohol containing 5 cc. of 5 N hydroplaced in a refrigerator a t -4' C. The glands representing chloric acid is sufficient. The temperature is kept between the morning's kill are collected and taken t o the laboratory 35" and 45" C. When the precipitate has completely dissolved, 10 volumes of warm alcohol are added. The solution at 12 o'clock noon. The glands are weighed. They are then run through a is then neutralized by adding 5 cc. of 5 N sodium hydroxide. power meat chopper, in wljch they are finely minced. The After the solution has cooled to room temperature it is minced material drops into 50-gallon earthenware crocks placed in a refrigerator a t 5" C. for 2 days. At the end of which contain acid water. (For every 100 pounds of glands this time the dark-colored supernatant alcohol is decanted there are 4 gallons of water containing 500 cc. of concentrated off. The alcohol contains practically no potency. The sulfuric acid.) The glands are extracted for 1 hour, during precipitate is dried in vucuo to remove all traces of alcohol. which time they are slowly agitated. At the end of the It is then dissolved in acid water, in which it is readily soluble. The solution is made alkali with sodium hydroxide to p H 1 Received February 9, 1925. 7.3 to 7.5. At this alkalinity a dark-colored precipitate * Numbers in text refer to bibliography at end of article.
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I .YD USTRI A L A S D ENGISEERISG CHE. IfIS T R Y
settles 0ut,5 and is immediately centrifuged off. This precipitate is washed once or twice with alkali 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 alkali solution. The acidity is adjusted to pH 5.0 and a white precipitate readily separates out. Tricresol is added to a concentration of 0.3 per cent in order to assist in the isoelectric precipitation and to act as a preservative. After standing 1 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 preeipitation is carried out by adjusting the acidity to a p H of approximately 5.0. After standing overnight the resultant precipitate is removed by centrifuging. The precipitate, which contains the active principle in a comparatively pure form, is dissolved in acid ~ a t e and r the hydrogen-ion concentration adjusted to p H 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 cc. Tricresol is added to secure a concentration of 0.1 per cent. Sufficient sodium chloride is added to make the solution isotonic. The insulin solution is then 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.
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fairly satisfactor; index of purity. Obviously it is not an absolute index. It the present time the writers believe it is highly advisable that all estimations of potency should be made by the rabbit test as used by the Insulin Committee. It is quite possillle that better methods will be elaborated. However, as it is impossible to correlate the results from the variety of proce lures of testing which have been used by different groups )f investigators, a useful purpose would be served by the ge ieral adoption of one method. It is hardly necessary to emriliasize the fact that large numbers of rabbits should be used. Variations of 100 per cent or more may be found in the resiilts of assays in which a limited number of animals were U S I d. It should be remembered that a large dose of insulin may produce no greater effect upon the test animal than a mi ch smaller amount. The situation is further complicated whe 1 the investigator fails to specify whether the material under t ,st is in the crude form or in a more or less purified form. Equipment t
Because of thl colloidal nature of insulin in aqueous solution it is extrer iely difficult to remove metals completely from an insulin olution. This fact is very important in the manufacture of insulin, because here one is generally dealing with insulin in ai id solutions; hence great care must be taken during the proccwing to avoid as far as possible direct contact of the insul n solutions with metallic substances. The glands a e minced through a power meat chopper. This chopper h:s a capacity of approximately 300 pounds Standardization per hour. The I ierforations in the chopper plate are '/s inch. The method of estimating the potency of insulin solutions Finer mincing h \ S not appreciably increased the yield. The is based on the effect that insulin produces upon the blood minced glands d op into 50-gallon glazed earthenware crocks sugar of normal animals. Rabbits serve as the test objects. which are suppc rted on small portable trucks. During exThey are starved 24 hours before the administration of traction the gla ids are agitated by wooden paddles, which insulin. Their weight should he approximately 2 kg. The are driven from a power shaft. After extraction the acid details of this method have recently been published by Mac- alcohol is rem0 -ed by means of a large centrifuge. This leod and Orr,6 from the laboratories of the Insulin Committee, centrifuge is rubt)er-lined and has a finely perforated basket. University of Toronto. Insulin is distributed in strengths However, to in-tire a clearer filtrate a coarse linen bag is of 10, 20, 40, and 80 units per cc. The unit is one-third of placed inside tht: centrifuge basket. The alcoholic extract the amount of material required to lower the blood sugar of is somewhat tur )id. Attempts to clarify it by a high-speed a 2-kg. rabbit which has fasted 24 hours from the normal rotary centrifugc or by a filter press have only been partially level (0.118 per cent) to 0.045 per cent over a period of 5 successful. Bes results have been obtained by filtering hours. through fluted filter papers fitted in large glass funnels. Yields of Insulin These funnels :ire arranged in rows on large tables. The Various investigators have reported very different yields filtrate from thc funnels runs into rubber-lined troughs, the of insulin from the pancreas. Murlin' by hot acid water ends of which art' connected by another trough which runs the obtained a yield of 5500 units of insulin per kg. of pancreas. clear filtrate into glazed earthenware crocks. a picrate acetone method of exDodds and D i ~ k e n susing ,~ The filtrate is concentrated in a glass, enamel-lined still, of traction, report a yield of 5500 units of crude insulin per kg. 60 gallons capar ty. This still is heated from the bottom by Moloney and Findlays extracted 4500 units by means of a steam jacket. It is very important that the heating surface benzoic acid alcohol. Dudley and Starling,3 by extracting be a t the base the still, as this avoids superheating and with sodium bicarbonate alcohol, obtained as the finished charring of the rganic matter when the volume of concenhydrochloride 1500 units per kg. Doisy, Somogyi, and Shaf- trate becomes >mall. During distillation from 60 to 75 fer,6 using sulfuric acid alcohol, extracted from 1500 to 2200 pounds of stean are used. The still is equipped with peep units per kg. At the Connaught Laboratories, by a pre- grasses, a recording thermometer, an intake, and a drain pipe. liminary extraction with dilute sulfuric acid followed by The alcohol filtiate is sucked into the still, which is under alcohol, 1800 to 2200 units per kg. are obtained. vacuum by me ns of a rubber hose connecting the intake pipe and the ea thenware crock. Distillation is carried out Note-In the writers' early experiments on the extraction of insulin from the pancreas, hydrochloric acid alcohol was found to give very satisfactory a t 25" C. Thi> is made possible by means of a large rotary results, in so far as purity of product and yields of activity were concerned vacuum pump ai id a brine-cooled tubular condenser. During Because o f the di5culties encountered in the filtration of the pancreasdistillation the b i n e circulates in the condenser a t 0' C. The alcohol mixture, hydrochloric acid was not used. In recent experiments the distillate is collc %ed in a brine-cooled receptacle a t the base difficulty in filtration has been overcome by the alkalization of the extract before filtration through paper. It is expected that this modification will of the condenser At intervals this is emptied and the alcohol shortly be introduced into the routine procedure, since the yields of insulin is transferred b;\ means of suction to a storage tank. Later are much greater than those which the writers report with sulfuric acid. this alcohol is rtbctified in a still. Any alcohol remaining in Undoubtedly, there are numerous factors to be considered the extracted glmds is recovered by heating them in a boiler in the interpretation of the significance of the yields of insulin t The writers ar : greatly indebted to the engineering staff of Eli Lilly as repopted by different groups of inve&tigators. The nitro- and Company for d signing much of the equipment n e d s a r y for the comgen content of the solution per unitfof activity has been a mercial preparation insulin. 1,f
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with live steam and condensing the alcohol that is driven off. The refrigerating system, besides supplying cold brine to the condensers, chills a cold storage room and manufactures ice for numerous small refrigerators throughout the laboratory. Precipitations of the final product with alcohol are carried out in 12-liter balloon flasks. Such precipitates can be readily dried under vacuum without being removed from the flasks.
Vol. 17, No. 3
Bibliography 1-Banting and Best, J . Lab. Clin. M e d . , 1, 251 (1921-22). 2-Best and Scott, J . Bid. Chem., 67, 709 (1923). 3-DudIey and Starling, Biochcm. J . , 18, 147 (1924). 4-Dodds and Dickens, Brit. J . E z p f l . Path., 6, 115 (1924). 5-Doisy, Somogyi, and Shaffer, Am. J . Biol. Ckem., 60, 31 (1924). B-Macleod and Orr, Lab, Clin. M e d , , 9, (1g24), 7-Clough, Allen, and Murlin, A m . J . Physrd., 68, 213 (1924). 8-Moloney and Findlay, A m . J . Pkys. Chcm., 18, 402 (1924).
J.
Application of Enzymes to Beet Sugar Factory Control’ By H. S. Paine and R. T. Balch CARBOHYDRATE
LABORATORY, BUREAUOF
CHEMISTRY, WASHINGTON, D. C .
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VAST amount of energy has been expended by a large sucrose and raffinose. The determination of sucrose is of even number of chemists in investigations designed to re- greater importance in beet sugar factory control than the duce the errors of the Clerget procedure for analytical determination of raffinose. determination of sucrose by polarization before and after If top yeast extract is added to a portion of the sample and inversion by acid. The literature of the subject is quite bottom yeast extract to a duplicate portion, sucrose is hyvoluminous. In suite of drolvzed in the first uortion to invert sugar and raffinose these efforts, the me’thod in A n analytical procedure involving the use of the enzymes to fructose and melibiose, its most approved form is invertase and melibiase has been adapted to practical use whereas in the second porgreatly lacking in precision for precise determination of the sugars sucrose and raffition sucrose is hydrolyzed to for many purposes, and nose in the chemical control of beet sugar factories. By invert sugar and raffinose to this is particularly true in comparing the data obtained with those resulting from dif r u c t o s e , d e x t r o s e , and the case of d e t e r m i n a rect polarization and acid hydrolysis, information is gained galactose; the difference betion of the sugars sucrose relative to the polarization and distribution of optically tween the corrected polariand raffinose in mixture by active nonsugar compounds at successive stages of the zations of the two portions the procedure of Creydt.* factory process. The comparison explains in part the after complete hydrolysis is The advantages of enzymes frequent decrease in factory operating efficiency toward a measure directly of the as hydrolytic reagents for the end of the campaign, and brings to light certain errors melibiose content and incertain analytical deterin sucrose accounting as now practiced. The data also directly of the raffinose conminations have been dishave a distinct bearing on the matter of “unaccountable” tent. The two stages of cussed by a number of insucrose losses and the extent to which these are only apthe hydrolysis may be exvestigators, but such proparent and are due to errors of polariscopic analysis. pressed as follows: cedures have not come into general use, owing largely to (invertase present) + invert sugar I/Sucrose the difficulty of securing sufficiently active enzyme prep\Raffinose (invertase present) -+ fructose melibiose arations. I1 Melibiose (melibiasepresent) -+ galactose dextrose I n connection with a beet sugar manufacturing problem unThe use of enzymes such as invertase and melibiase as hyder investigation in this laboratory, it was necessary to determine percentages of sucrose and raffinose with greater precision drolytic reagents in the determination of sucrose and raffinose than is possible by means of analytical methods depending has been rendered quite practicable by the ultrafiltration upon acid hydrolysis of these sugars. Recourse was there- method devised by F. W. Reynolds5 of this laboratory for fore had to the use of the enzymes invertase and melibiase. concentration of enzyme solutions. Invertase (top yeast Bau3has described a method in which top yeast, containing extract5) and invertase-melibiase (bottom yeast extract5) invertase, and bottom yeast, containing invertase and meli- solutions of high activity were prepared by this procedure biase, are used for the analytical determination of raffinose; from ordinary bakers’ yeast and from brewers’ yeast, rehis procedure, however, is time-consuming, since complete spectively, and constituted the hydrolysts employed in the fermentation of the sugars attacked by top and bottom investigation here described. The top yeast extract was yeasts is required. Hudson and Harding‘ developed this tested in order to make certain that it contained no melibiase. idea further by using purified extracts of top and bottom The analytical technic presented no difficulties and was simyeasts which they applied only to determination of raffinose in pler in some respects than that of methods involving acid mixture with certain other sugars in pure aqueous solutions. hydrolysis; the degree of precision obtained was much Their procedure was not elaborated to the point where it greater, and it is possible, if desired, to accomplish the enzymic could be used in a practical manner for determination of raffi- hydrolysis in as short a period as in the rapid form of the acid nose in beet sugar factory ,products. Furthermore, their inversion procedure. I n the work of this laboratory acid inversion methods for method does not provide for simultaneous determination of estimation of sucrose and of sucrose and raffinose in mixture 1 Presented by Mr. Balch under the title “Determination of Sucrose and have been abandoned and enzyme methods have been used Ra5nose in Beet Products by the Enzyme Method” before the Division of exclusively for over two years. By using a highly active Sugar Chemistry at the 67th Meeting of the American Chemical Society, invertase solution concentrated by the ultrafiltration method Washington, D. C., April 21 t o 26,1924. 2 2. Vn.dcut. Zuckcrind., 81, 153 (1887); Ibid.. 40, 194 (1890). the writers have been able to operate the invertase inversion
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Chcm. Zfg., 18, 1794 (1894). THISJOURNAL, 7,2193 (1915).
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THISJOURNAL,
16.169 (1924).
