CA1043775A - Process for recovering glucagon - Google Patents

Abstract

ABSTRACT OF DISCLOSURE

The invention provides a process for the recovery of glucagon by A. isolating glucagon-containing protein from insulin process alkaline crystallization super-natant;
B. separating the glucagon from other proteins;
and C. purifying the glucagon obtained from step B.

Description

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The invention relates to a process for the recovery ; of glucagon from insulin process alkaline crystallization supernatant.
The invention provides a process for the recovery of glucagon by . isolating glucagon-containing protein from insulin process alkaline crystallization super-natant;
B. separating the glucagon from other proteins;
1~ and C. purifying the glucagon obtained from step B.
Shortly after the ~iscovery of insulin in 1921 by Banting and Best, several researchers [Murlin et al., J. Biol.
Chem., 56, 252 (1923) and Ximball and Murlin, J.
Biol. Chem., 58, 337 (1924)] noted that a hyperglycemic response was obtained with certain pancreatic extracts of insulin. The factor responsible for the hyperglycemic - ;
response was named glucagon.
At the present time, the most important use of glucagon is in the treatment of insulin-induced hyperglycemia.
As the world diabetic population grows, the demand for glucagon must increase. Additionally, the use of glucagon in cardiac disorders currently is being explored.
Consequently, these two applications are placing increasing demands on the production of glucagon, demands which are met best by improving the yield of glucagon from natural ~` sources.
The first preparation of crystalline glucagon was reported in 1953 [Staub, et al., Science, 117, 628 (19533;
see also, Staub, et al., J. Biol. Chem~, 214, 61g ~1955)].
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, 3~,3 The starting material was an amorphous fraction obtained during the commercial manufacture o~ insulin which involved an acid-alcohol extraction of pancreas tissue, concentration of the extract, precipitation with sodium chloride, isoelectric precipitation, several alcohol fractionations, decolorization, crystallization with ~inc from acetate buffer, washing to remove the amorphous fraction, and drying the zinc insulin thus obtained. The amorphous fraction contained about four weight percent glucagon and about seven weight percent insulin. Usually, the yield of amorphous material was in the range of from about five to about ten mg. per pound of pancreas.
The glucagon preparation in turn involved, optionally, fractionation at pH 6.7, acetone fractionation, fractional precipitation at pH 4.3, two fractional precipitations at pH 2.5, and two crystallizations from urea-glycine buffer at pH 8.6. The yield of crystalline glucagon was about 20 weight percent o~ the glucagon contained in dry amorphous material, which corresponded to a yield of from about 0.04 to about 0.08 mg. of glucagon per pound of pancreas.
The introduction of an intermediate crystallization of zinc insulin from citrate buffer, as taught by U.S.
Patent 2,626,228, resulted in an overall reduction in the total number of steps required in the insulin manufactur-ing process using beef pancreas. While approximately the same amount of amorphous fraction was obtained during the washing step as in the older process, glucagon content of the amorphous fraction had decreased to only about 0.2 weight percent. It was discovered that the glucagon was X-3572 ~3~

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retained in the citrate buffer during the intermediate crystallization- Recovery of glucagon from the citrate buffer required precipitation with excess zinc, followed by a salt precipitation and two precipitations at pH 5.1 and 5.6, respectively, to remove the zinc. The yield of crude glucagon from citrate buffer corresponded to about 0.8-0.9 mg. per pound of pancreas, from which source the yield of purified glucagon corresponded to about 0.1-0.3 mg. per pound of pancreas. The use of zinc as a precipitating agent made complete removal of zinc difficult and increased the amount of insulin carried over into the final puxified glucagon.
Studies on pancreas extractions have shown that the various acid-alcohol extracts usually employed in insulin manufacturing processes contain 4-6 mg. of glucagon per pound of pancreas. After concentrating and defatting the extracts, glucagon content decreases to 1.5-2.0 mg. per pound. However, as-discussed hereinabove, only about 25 to 50 weight percent of this amount is available for purifica-tion, depending upon the starting material.
The drawing is a flow dia~ram of a preferred em-bodiment of the present invention. The drawing also il-lustrates the relationship of the process of the present invention to the alkaline crystallization step of the ~ insulin process.
; The source of glucagon for the process is the supernatant from the insulin process alkaline crystallization step, disclosed in U.S. Paten~; 3,719,65S. This supernatant is an aqueous solution showing a pH from 7.2 - 10.0 and a cation concentration of 0.2 - 1~0 molar and has dissolved therein X-3572 _4 ~ ~ .

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protein which is about 1-10 percent insulin and about 0.2-1.5 percent glucagon, depending upon the source of pancreas employed in the insulin process. For example, with beef pancreas this alkaline crystallization supernatant protein usually contains about 5 - 10 percent insulin ~-~
and 1.0 - 1.5 percent glucagon; with pork pancreas, the supernatant protein usually contains about 1-2 percent insulin and about 0.2 - 0.3 percent glucagon. It should be noted, however, that the actual yields of glucagon on a batch-to-batch basis can vary over a wide range because of the susceptibility of glucagon to enzymatic degradation.
Separation of glucagon-containing protein from the alkaline crystallization supernatant comprises the first step of the process. In general, such a separation can be accomplished by any known method. For example, the pH
of the supernatant can be adjusted to about 3.0 and 20 percent, weight per volume, of sodium chloride added to precipitate glucagon and other proteins. Alternatively, precipitation can be accomplished by the addition of excess zinc chloride at a pH o~ about 6Ø A third procedure involves the use of isoelectric precipitation. Yet another procedure involves the use of ion-exchange chromatography. ;~
The second step of the process comprises separating glucagon from other proteins. This separation in general can `
be accomplished by any known method, such as gel filtration, ion-exchange chromatography, isoelectric focusing, and glu-cagon fibril formation, of which methods glucagon fibril for-mation is preferred.
The third and final step of the process comprises purifying the glucagon obtained from step two. In general, 3~r~

such purification can be accomplished by any of the methods known to those skilled in the art, such as crystallization, ion-exchange chromatography, and the like. Crystallization is the preferred method.
The use of isoelectric precipitation or ion-exchange chromatography, or some combination thereof, is preferred for separating glucagon-containing protein from alkaline crystallization supernatant. In general, isoelectric precipitation requires that the supernatant pH be in the range of from about 4.2 to about 6.6. The preferred pH
range is from about 4.6 to about 5.2, while the most preferred range is from about 4.7 to about 5Ø
, Because the alkaline crystallization supernatant is at a pH of from about 7.2 to about 10, it is necessary to acidify the supernatant to the desired pH. Such acidifica-tion normally can be accomplished simply by adding a dilute solution of an inorganic or organic acid which will not degrade or interact with glucagon. Examples of suit-able acids include hydrochloric acid, phosphoric acid, ~' ~ 20 formic acid, acetic acid, propionic acid, and the like.
Hydrochloric acid is preferred.
Optionally, and preferably, up to about 20 volume pexcent of an alcohol can be added to reduce the solubility , - of the glucagon~containing protein in the alkaline ?
crystallization supernatant. By the term "alcohol" is meant a saturated aliphatic monoalcohol having fewer than about six carbon atoms. Preferably, the alcohol will have fewer than about four carbon atoms, such as methyl alcohol, ethyl alcohol, propyl alcohol, and isopropyl alcohol. The most preferred alcohol is ethyl alcohol.
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When employed, the alcohol normally is added to the super-natant before acidification.
Upon acidifying the supernatant to the desired pH, precipitation of the desired glucagon-containing protein ; begins quickly, usually within minutes. To ensure complete precipitation, the solution is allowed to stand, usually at a temperature no higher than ambient temperature.
Preferably, the solution is chilled to a temperature of from about 3C. to about lO~C. Although precipitation generally is complete after about 24 hours, the mixture can be allowed to stand indefinitely without deleterious effects.
When precipitation is complete, the precipitate, referred to hereinafter as isoelectric precipitate, is isolated by any convenient known method, such as centrifugation or filtration; filtration usually is preferred. The isoelectric precipitate thus obtained need not be-washed or dried, although such procedures can be employed if desired.
Another procedure well-suited to the separation of glucagon-containing protein from alkaline crystallization supernatant is ion-exchange chromatography. Of the known ion-exchange procedures, the process of U.S. Patent 3,715,345 is ; especially effective and is preferred. Briefly, the process of ion-exchange chromatography consists of passing the glu-cagon-containing solution over an alkali metal form of a sul-fonated macroreticular styrene-divinylbenzene copolymer resin at pH 7-8. The glucagon is adsorbed onto the resin. The ; ;
glucagon is then eluted with a dilute base such as 0.1 N
ammonium hydroxide~ The glucagon containing eluant is -~
adjusted to pH 2.5 and then the glucagon is precipitated by a standard "salt precipitation" method. The resulting glucagon- ;~
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r~5 containing precipitate has been substantially separated from insulin proteins but the precipitate still contains other non-glucagon materials.
Subjecting alkaline crystallization supernatant to the process of U.S. Patent 3,715,345 results in an insulin-containing eluant and a glucagon-containing salt cake. The insulin-containing eluant, if desired, can be returned to the insulin process. The glucagon-containing salt cake is re-dissolved for further processing; for convenience, the solution ~-pH and protein concentration in general are made approximatelyequivalent to that of alkaline crystallization supernatant.
It frequently may be advantageous in carrying out the first step of the process i.e., separation of glucagon-containing protein from alkaline crystallization supernatant, to employ isoelectric precipitation and ion-exhange chroma-tography in sequence. For example~ a]kaline crystalline super- -natant can be subjected to the process of ion-exchange chroma-tography. The~glucagon-containing salt cake thus obtained is .
re-dissolved and subjected to isoelectric precipitation as described hereinbefore. Alternatively, the alkaline crystal-lization supernatant is subjected to isoelectric precipitation;
the precipitate obtained therefrom can be dissolved in a slightly alkaline aqueous medium and subjected to the process of ion-exchange chromatography. In each case, the insulin-containing eluant from the ion-exchange chromatography can be returned, if desired, to the insulin process. -As stated hereinbeore, the preferred method for the separation of glucagon from other protein is glucagon fibril formation. In general, the formation of glucagon fibrils is carried out in acidic, aqueous solution. Normally, X-3572 -~_ ~ . .

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the glucagon-containing protein is dissolved in an acidic, aqueous medium having a pH below about 2.7. While the pH
generally can range from about 1.5 to about 2.7, the preferred pH range is from about 2.0 to about 2.5. While any of the acids suitable for use in acidifying alkaline crystallization supernatant prior to an isoelectric precipitation can be employed, hydrochloric acid again is preferred. With hydrochloric acid, however, a preservative, such as phenol, should be employed, usually at a concentra-tion of about 0.2 percent, weight per volume. Typically,the glucagon-containing protein is dissolved in 0.01 N hydro-chloric acid containing 0.2 percent phenol, weight per volume.
The concentration of glucagon-containing protein in solution in general can range from about 2.5 to about 30 ~`
mg./ml. The preferred range is from about 5 to about 20 mg./ml., and the most preferred range is from about 5 to about 10 mg./ml~
Optionally, a water-soluble inorganic salt can be added to the acidic glucagon-containing protein solution to initiate fibril formation. The term "water-soluble" is meant to include inorganic salts which are soluble in water at the concentrations employed as described hereinafter.
Because the inorganic salt is believed primarily to have a salting-out effect relative to the initiation of fibril formation, the choice of inorganic salt is not critical.
Practically, however, the use of radioactive, toxic, or colored salts, or salts which are oxidizing agents, reducing agents, or strongly acidic or basic, is not desired for obvious reasons. Thus, the suitable inorganic salts ~-3572 _9_ ;

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generally include the water-soluble ammonium salts, the water-soluble salts of alkali metals up to and including period 6 of the periodic table of the elements (Robert C.
Weast, Ed.-in-Chief, "Handbook of Chemistry and Physi~s,"
53rd Edition, The Chemical Rubber Co., Cleveland, Ohio, 1972, p. B3), and the water-soluble salts of the alkaline earth metals up to and including period 6 of the periodic table of the elements. Examples of such salts include, among others, ammonium bromide, ammonium chloride, ammonium fluoride, ammonium iodide, ammonium magnesium sulfate, ammonium manganese sulfate, ammonium nitrate, ammonium sulfate, lithium bromide, lithium chloride, lithium fluosilicate, lithium fluosulfonate, lithium iodide, lithium molybdate, lithium nitrate, lithium potassium sulfate, sodium ammonium phosphate, sodium ammonium sulfate, sodium bromide, sodium chloride, sodium iodide, sodium hexafluorophosphate, sodium fluosulfonate, sodium magnesium sulfate, sodium nitrate, sodium hex~-metaphosphate, sodium dihydrogen orthophosphate, s~dium monohydrogen orthophosphate, sodium sulfate, sodium hydrogen ~:
sulfate, potassium bromide, potassium calcium chloride, potassium chloride, potassium fluoride, potassium iodide, potassium magnesium chloride sulfate, potassium ma~nesium sulfate, potassium magnesium chloride, potassium molybdate, ..
potassium orthophosphate, potassium sodium sulfate, rubidium bromide, rubidium chloride, rubidium fluoride, ;
rubidium iodide, rubidium nitrate, cesium bromide, cesium chloride, cesium fluoride, cesium iodide, cesium nitrate, cesium sulfate, beryllium bromide, beryllium chloride, beryl- -lium fluoride, beryllium nitrate, beryllium orthophosphate, X-3572 -10~

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magnesium bromide, magneslum chloride, magnesium iodide, magnesium nitrate, magnesium silicofluoride, calcium bromide, calcium chloride, calcium iodide, calcium nitrate, strontium bromide, strontium chloride, strontium iodide, barium bromide, barium iodide, water-soluble hydrates thereof, and the like. Ammonium salts are preferred, with ammonium sulfate being most preferred. The suitable inorganic salts in general can be employed in concentrations up to about 0.5 M. Preferably, such salts will be present 10in concentrations of from about 0.01 to about 0.05 M.
The temperature range in which glucagon fibril formation occurs normally is from about 20 to about 30C.
The preferred temperature range is from about 24 to about 26 C. The most preferred temperature is 25C.
Fibril formation, which is aided by agitation, usually begins within about 3 to 4 hours from the time preparation of the acidic, aqueous glucagon solution has been completed, and is complete in about 48 hours. Formation times in excess of about 48 hours usually are not necessary. ~ -- When fibril formation is complete, the glucagon - ; -fibril~ oan be collected by any known method, such as filtratio~ or centrifugation, the later method being preferred.
If desired, the glucagon fibrils can be washed by ~uspending ; ~-the fibrils in aqueous acid medium which may or may not .
contain inorganic salt. The temperature of the wash medium can be in the range of from about 20 to about 30C., with ~ 25C. being preferred. The fibrils then are collected g as be~e and kept cold, usually at a temperature below $
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If desired, a chelating agent such as ethylene-diaminetetraacetic acid can be included in the fibril-forming medium. The concentration of chelating agent normally will be less than about 0.01 M., the preferred concentration being 0.004 M. However, the use of a chelating agent is not preferred unless zinc or other divalent metal ions are known to be present.
As stated hereinbefore, crystallization is the preferred method for purifying the glucagon obtained in the second step. In general, crystallization of glucagon is carried out by dissolving the glucagon in an alkaline ~ -aqueous medium and acidifying to a slightly alkaline or acidic pH to initiate crystallization; i.e., to a pH in the range of from about 4.5 to about 8.5.
Dissolution of the glucagon can be accomplished --by either of two ways. First, the glucagon can be dissolved directly in an alkaline aqueous medium. Or, the glucagon can be suspended in distilled water and the pH adjusted by the addition of aqueous base. Suitable bases in ~ 20 general are the alkali metal and ammonium hydroxides, of ; which potassium hydroxide is preferred.
In general, the resulting glucagon solution can have a pH in the range of from about 9.0 to about 11.5, with the preferred range being from about 9.5 to about 10.5.
;~ The concentration of glucagon in the alkaline solu-tion should be within the range of from about 2 to about 10 mg./ml. The preferred concentration range is from about 4 to about 8 mg./ml., with the most preferred concentrat~on boeing about 5 mg./ml.
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Because acidification frequently results in the immediate precipitation of a small amount of non-glucagon protein, the following acidification procedure, while not essential, is preferred.
The alkaline glucon solution is heated to a temperature of from about 55 to about 65C. The solution then is acidified to a pH of from about 4 to about 6 with 10 percent phosphoric acid.
While any of the acids suitable for use in previous steps can be employed, the use of phosphoric acid is preferred. The resulting solution then is filtered while still at the initial elevated temperature. In general, the filtration step is optional and frequently can be omitted when the amount of precipitate resulting from the acidification is minimal. Furthermore, procedures other than filtration, such as centrifugation, can be employed if desired.
If the glucagon solution is discolored, decolorizing carbon can be added either before or after acidification, preferably before.
After acidification (and filtrat:ion, if employed), the glucagon solution is allowed to stand at a temperature within the range of from about 2 to about 10C. and for a crystallization time of from about 24 hours to about 120 hours. me preferred temperature is 4C. Preferably, the crystallization time will be in the range of from about 72 to about 120 hours, with the most preferred crystallization time being 72 hours.
Most of the supernatant is decanted from the resulting glucagon crystals, usually through a filter. The , :
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remaining supernata~-t is removed from the glucagon crystals, usually by centrifugation. The purified glucagon then is washed successively with dilute saline (usually 0.001 percent) and water. The glucagon then is lyophilized and stored in the cold.
Depending upon the purity of the glucagon obtained from the second step and the purity desired in the final purified glucagon, one or more additional crystallizations may be employed. It has been found, however, that a total of two crystallizations usually is sufficient to yield glucagon having a purity of at least 80 percent, based on biological assay in cats.
; When two crystallizations are employed, the fol-lowing procedure is preferred: The first crystallization is carried out as described hereinabove and the crystals are dis-solved in an alkaline solution. The second crystallization employs acidification to a pH of from about 7.0 to about 8.5, pre~erably from about 7.3 to about 7.5, and most preferably ; about 7.4. Dissolution and the filtration procedure, if em-ployed, preferably are carried out at a temperature of from about 35 to about 45~C., most preferably at a temperature of about 40C. The glucagon concentration preferably is about . ,. ~ ..
lO mg~/ml~ -If desired, the supernatants from any one or all of the crystallization procedures can be recycled. For example, ~ll dissolved protein contained in any supernatant can be precipitated by adjusting the pH with dilute hydrochloric acid to 2.5 - 3.0 and adding 20 percent of sodium chloride, ~` weight per volume. The precipitate thus obtained can besubjected to the first step of the process of the present ~, .... :~ , - , .

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invention either separately or dissolved in alkaline crystallization supernatant.
As discussed hereinbefore, the alkaline crystal-lization supernatant normally contains dissolved therein substantially more insulin than glucagon. This insulin is a component of the glucagon-containing protein obtained by isoelectric precipitation. While the second step in the process i.e., separation of glucagon from other proteins, effectively separates glucagon from insulin, the separa-tion procedure can be detrimental to the insulin component of the protein mixture. Consequently, it often is desirable to carry out the first step of the process by means of a pro-cedure, such as ion-exhange chromatography, which also will -result in the separation of insulin from glucagon.
It should be pointed out, however, that ion-exchange chromatography is not the only means of separating insulin from glucagon prior to separating glucagon from other proteins.
For example, the precipitate from an isoelectric precipitation can be subjected to what is referred to in the art as a hyper-glycemic factor fractionation. Briefly, such a procedure in-volves salting out glucagon from a slightly alkaline, phenolic ~ `
aqueous medium. Hyperglycemic factor fractionation has been described by Staub, et al, supra. The insulin-containlng supernatant is recycled in the insulin process, while the pre-cipitated crude glucagon is employed in step two of the present process.
While not essential to the process of the present invention, it is preferred that the supernatant from the fibril-formation step be recycled in the insulin process, usually at the beginning of the alkaline crystallization ;

:. . : , - -,: -step disclosed in said U.S. Patent 3,719,655. Of course, such recycling is not necessary if insulin has been separated from glucagon, as described hereinbefore, prior to fibril formation. In such an instance, however, the insulin thus separated would be recycled in the insulin process.
The present process and its relationship to the insulin process perhaps is better understood by referring to the drawing which illustrates as a flow diagram one em-bodiment of the present invention. For the sake of simplicity,the supernatants obtained after the first and third steps of -the present process are shown as being discarded, it being ~-understood that such supernatants can be recycled as described hereinbefore.
The alkaline crystallization procedure of U.S. Patent 3,719,655 is shown at the top of the drawing. The alkaline crystallization procedure gives alkali metal or ammonium insulin and a supernatant, as shown. Because the alkaline -crystallization procedure and the al~ali metal or ammonium insulin obtained therefrom are a part of the insulin process, the blocks representing both the procedure and the insulin are enclosed by a broken line and labeled "Insulin Process." Evexything without said broXen line, therefore~ is a part of the present process and is labeled "Glucagon Process."
The process begins, as shown, with the alkaline crystallization supernatant. The preferred steps comprising the present process then are carried out, giving the iso-electric precipitate, glucagon fibrils, and crystallized glu-cagon, respectively, as shown. The drawing also indicates ' '` ' . .
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'7~75 :' return of the supernatant from the fibril formation step to the insulin process.
Unless otherwlse stated, all temperatures are in degrees centigrade.
Example 1 Alkaline crystallization supernatant, 14.75 liters, from the processing of 13,000 lbs. of beef/pork pancreas, -having a solids content of 40.7 mg./ml., was diluted with 1.45 liters of absolute ethanol. The pH of the resulting ~
solution was adjusted to 5.2 with 3 N hydrochloric acid. ~ -~ . .
The solution was chilled at 5 overnight. The precipitate which had formed was collected by filtration, dissolved in 11.28 liters of 0.01 N hydrochloric acid containing 0.2 percent phenol, weight per volume, (referred to hereinafter as acid-phenol water) and the resulting solution assayed:
Total solids: 512.6 g. (45.4 mg./ml.) Insulin: 87.43 Units/ml. (1.93 Units/mg. solids) Glucagon: 463.7 mcg~/ml. (L.02 percent of total ~`~
., .
solids) An 870-ml. portion o~ the solution was removed for testing, `
leaving 10.4 li~ers containing about 473 g. of solids.
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~ To carry out a hyperglycemic factor fractionation~
i the above solution was diluted with 111.8 liters of acid~
~` phenol water, giving a total volume of 122.2 liters with -', a solids content o~ 0.387 percent. To the diluted solution ;~
were added 245.8 ml. of liqui~ied phenol, 941 g. of sodium . chloride, and sufficient 40 percent aqueous sodium hydroxide to adjust the pH to g.~ in order to aid dissolution of all solids. The pH then was adjusted to 7.5 with 3 N hydro- -chloric acid. The resulting solution was chilled ~t 5 for : :

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1-2 days, during which time a precipitate formed. The supernatant was decanted and filtered under suction. The precipitate was collected by centrifugation. The solids obtained by filtration and centrifugation were combined and dissolved in 10 liters of acid-phenol water; the resulting solution assayed as follows:
Total solids: 75.0 g. (7.5 mg./ml.) Insulin: 6.33 Units/ml.
Glucagon: 2-75 mcg./ml. (5.01 percent of total solids) The solution was diluted to a solids concentration of 5.0 mg./ml. by adding an additional 5 liters of acid-phenol water. A 5.0-liter portion of the resulting solution was removed for testing, leaving 10.0 liters of solution contain-ing about 50 g. of solids.
To the remaining solution were added, with agitation, 8.0 ml. of 0.5 M aqueous ethylenediaminetetraacetic acid (as the tetrasodium salt) and 60 ml. of 50 percent aqueous ammonium sulfate. Agitation was continued for 16 hours at ambient temperature. The glucagon flbrils which had formed were collected by centrifugation and washed twice with acid-phenol water which contained ethylenediaminetetraacetic acid and ammonium sulfate as before.
The glucagon fibrils were suspended in 10 liters of water containing 0.2 percent phenol, weight per volume. The -~
mixture was heated to 40 and the pH adjusted to 10.5 by ~ -addiny 150 ml. of 10 percent aqueous potassium hydroxide. ~ -The solution then was heated to 60 while the pH was adjusted to 7.8 by the addition of 68 ml. of 10 percent phosphoric acid.
After the solution temperature- reached 60, the pH was further adjusted to 5.0 by adding an additional 68 ml. of 10 ., .. . .
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percent phosphoric acid. The solution then was gravity filtered while ho-t and the filtrate was chilled, with agitation, ~-at 5 for 72 hour~. The glucagon which had precipitated `
was isolated by filtration. The solid thus obtained was dissolved as described above for the glucagon fibrils; how-ever, upon adjusting the pH to 10.5 the total volume was 870 . . .
ml. The solution then was heated to 60, the pH was adjusted to 5.0 with 10 percent phosphoric acid, and the resulting solution was gravity filtered while hot. The filtrate was cooled and agitated as before. The precipitated glucagon was collect~d by centrifugation and then lyophilized, giving .:
1.84 g. of purified glucagon. This corresponds to 74 -percen~ of the glucagon available prior to fibril formation, and to 39 percent of the glucagon available after precipitation of protein from the alkaline crystalli~ation supernatant tallowance being made for sample removals) Example 2 .
~ The alkaline crystallization mother liquors from ~ ~
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- three crystallizations of insulin derived from 65,257 lbs., ~20 82,006 lbs., and 108,397 lbs. of a 2:1 mixture of beef/pork ~pancreas were separated from the insulin crystals by centxifugation. The respective mother liquors measured -~ ~60 liters, 515 liters, and 680 liters, to which were added .
; Sl liters, 57.2 liters, and 75.5 liters of absolute alcohol, ~;~
respectively, and each was adjusted to pH 5.0 with 3N

HCl, agitated 15 minutes, and chilled for at least 24 hours.
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3 Assays of the mother liquors before precipitation and the r~ 501uti~ns Qf the precipitates were tl) 1150 mcg. glucagon . , . : - :
and 228 U~its insulin/lb. original pancreas (O.P.) in moth~r liquor; 839 mcg. glucagon and 291 Units/lb. O.P, 59.7 mg.

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solids/lb. O.P. in solution of the precipitate, 158 liters;

(2) 736 mcg. glucagon and 139 U insulin/lb. O.P. in mother liquor; 345 mcg. glucagon and 123 U insulin/lb. O.P. and 27.8 mg. solids/lb. O.P. in solution of the precipitate, 150 liters.

(3) 1214 mcg. glucagon and 218 U. insulin/lb. O.P. in mother liquor; 946 mcg. and 191 U insulin/lb. O.P. and 59.0 mg.
solids/lb. O.P. in solution of precipitate, 156 liters. The precipitates were collected by filtration and dissolved in acid-phenol water (prepared as described in Example 1).
The solutions of the pH 5.0 precipitates were combined to give 464 liters (12,570 gm. solids), and diluted to 630 liters with acid-phenol water to make the solution 2.0 percent solids, and adjusted to pH 2.1 with additional 3N HCl. The solution was treated with 0.3 percent ammonium sulfate, which was added as a 50 percent solution ~6 ml/l, 3,780 ml), and stirred slowly 24 hours at 25C. during fibril formation, then allowed to stand 48 hours at 15C.
The glucagon fibrils were separated from the remaining solution by centrifugation. The supernatant solution was assayed (contained 222 mcg. glucagon and 114 U insulin/lb.
O.P.) and returned for insulin processing. The glucagon fibrils were suspended in 450 liters of cold water containing 0.2 p~rcent phenol, 2,700 ml. of 50 percent ammonium sulfate solution was added, and the mixture was stirred slowly for 30 minutes to wash the fibrils; the fibrils were again --collected by centrifugation and the wash discarded. The glucagon fibrils were suspended in 275 liters of water contain- ;
ing 0.2 per~ent phenol and adjusted to pH 3.85 using 10 percent phosphorlc acid, and assayed: Solids, 5.48 mg./lb.
O.P. (0.52 percent; 1,404 g. from 255,660 lbs. O.P.); ~ -. - :- . . -: . . . . .

glucagon, 212 mcg./l~. O.P. (54.2 g.).
The glucagon fibril suspension, 275 liters, was divided into two portions of (l) 135 liters and (2) 140 liters, respectively~ for the first crystallization. The first portion was diluted to 140 liters with 0.2 percent phenol-water to make a 0.5 percent solids concentration;
the other portion was left at 0.52 percent solids. Each fibril suspension was warmed to 60~C. and adjusted to pH 9.0-ll.0 l(l) 9.9, (2) 9.7] with lO percent potassium hydroxide to obtain a clear solution; 281 g. of "Norite Al'* (0.4 g/g solids) was added, and after 10 minutes agitation the solu-tion was readjusted to pH 5.0 using lO percent phosphoric acid, and filtered while hot on funnels with ED No. 613 filter paper. The filtrate tcrystallization solution) was further agitated slowly for 16-20 hours while chilling at 4C. to promote uniform glucagon crystallization. The precipitate separated by filtration at pH 5.0, 60C., was reprocessed for additional glucagon crystals by suspending in 120 liters of 0.2 percent phenol-water at 0.5 percent solids concentration (solids assay, 8.0 g.), warming to 60~C., adjusting to pH
lO.0 to dissolve the solids, agitating lO minutes, and readjusting the pH to S.0 with lO percent phosphoric acid and filtering while hot using gravity filtration. The filtrate was agitated slowly for 16-20 hours while chilling to 4C. Crystallization usually is complete after 72-120 hours. The second pH 5.0 precipitate was discarded. The ~; bulk of the crystallization mother liquors from the 1st and 2nd crystallization mixtures was decanted and filtered by ` .
suction. The 1st mother liquor was treated with 20 percent (w/v3 sodium chloride in each case to precipitate any glucagon X 3572 *Trademark for purified activated charcoal of vegetable origin, used as a decolorizing agent.

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~3Lq ~r,J~, that failed to crystallize. These precipitates were pooled with other lots and reprocessed after reprecipikating at pH 4.6 through another crystallization to provide an additional yield of glucagon crystals. The glucagon crystals from the two portions providing the first crop and the crystals obtained from reworking the combined pH 5.0 precipitates from the first crystallizations that gave the second crop, ~ -were collected by centrifugation for 3 minutes, separated from the mother liquors, transferred to lyophilization con-tainers using cold water as the suspending medium, and freeze-dried. The glucagon crystals from the intermediate crystallization were weighed and samples assayed. Glucagon intermediate crystals: yields, (lst crop) (1) 41.0 g. (91.7 percent pure) and (2) 44.0 g. (86.8 percent pure) for 85.0 g.
(89.1 percent pure), 75.8 g. glucagon, 292 mcg./lb. O.P. (pure~
or 332 mcg./lb~ (as is); first rework crystals (2nd crop), yield, l9.0 g. (100.0 percent pure), 75 mcg./lb. O.P. (pure and as is); second reqork crystals (lst mother liquors), yield, 22.0 g. (46.0 percent pure) or 9.9 g., 39 mcg./lb. O.P.
(pure) or 85 mcg./lb. (as is). Total yield: 126.0 g.
solids (476 mcg./lb. O.P.) of 83.1 percent purity, or 104.7 g. glucagon (409 mcg./lb. O.P.).
Recrystallization was performed in conjunction with ~ ;
other intermediate crystals accumulated. The final glucagon crystals represented 85 percent of the glucagon weight present in the intermediate crystals. Recrystallization was done at pH 7.5 after dissolving the intermediate crystals at } p~ 8.0 - 10.0 with lO percent potassium hydroxide, 40C~
at l.0 percent solids, and readjusting to pH 7.5 with lO
percent phosphoric acid, filtering, and chilling. The final " :
, . . . . . .
".
.,: ,...... . '' -' : .

-~ :

crystals were collected by centrifugation after decanting the bulk of the mother liquor (which was reprocessed for~
some additional yield), washed with 0.001 percent sodium chloride solution twice, cold distilled water once, and freeze-dried. The calculated yield was 89.0 g. or 348 mcg./lb. O.P. and the recovery from the aIkaline crystal~
lization mother liquor 33.2 percent.

~ .

X-3572 -23- ~
~ ' " ~

Claims (22)

The embodiment of the invention for which an ex-clusive property or privilege is claimed are defined as follows:

1. A process for the recovery of glucagon, which comprises:
A. isolating glucagon-containing protein from insulin process alkaline crystallization supernatant;
B. separating the glucagon from other proteins; and C. purifying the glucagon obtained from step B.

2. The process of claim 1, wherein step A is accomplished by means of isoelectric precipitation at a pH
from about 4.2 to about 6.6.

3. The process of claim 2, wherein said isoelectric precipitation is carried out in the presence of up to about 20 volume percent of a saturated aliphatic monoalcohol having fewer than about six carbon atoms.

4. The process of claim 3, wherein said monoalcohol is ethyl alcohol.

5. The process of claim 1, wherein step A is accomplished by means of ion-exchange chromatography.

6. The process of claim 1, wherein step A is accomplished by means of isoelectric precipitation and ion-exchange chromatography in any order.

7. The process of claim 1, wherein step A is accomplished by means of isoelectric precipitation, followed by a hyperglycemic factor fractionation.

8. The process of claim 1, wherein step B is accomplished by means of glucagon fibril formation.

9. The process of claim 8, wherein said fibril formation is carried out at a pH in the range of from about 1.5 to about 2.7.

10. The process of claim 8, wherein said fibril formation is carried out in the presence of an inorganic salt.

11. The process of claim 10, wherein said salt is ammonium sulfate.

12. The process of claim 8, wherein ammonium sulfate is present at a concentration of from about 0.01 to about 0.05 M.

13. The process of claim 1, wherein step C is accomplished by means of crystallization.

14. The process of claim 13, wherein said crystallization is carried out at a pH in the range of from about 4.5 to about 8.5.

15. The process of claim 13, wherein said crystal-lization is carried out with a glucagon concentration of from about 2 to about 10 mg. glucagon per ml. of solution.

16. The process of claim 14, wherein two successive crystallizations are carried out.

17. The process of claim 14, wherein a first crystallization is carried out at a pH in the range of from about 4.5 to about 5.5, and a second crystallization is carried out at a pH in the range of from about 7.0 to about 8.5.

18. The process of claim 1, wherein step B is accomplished by means of glucagon fibril formation and step C is accomplished by means of crystallization.

19. The process of claim 18, wherein step A is accomplished by means of isoelectric precipitation.

20. The process of claim 18, wherein step A is accomplished by means of ion-exchange chromatography.

21. The process of claim 18, wherein step A is accomplished by means of isoelectric precipitation and ion-exchange chromatography in any order.

22. A process for the recovery of glucagon which comprises:
A. isolating glucagon-containing protein from insulin process alkaline crystallization super-natant by isoelectric precipitation at pH 5.2 followed by hyperglycemic factor fractionation at pH 7.5;
B. separating the glucagon from other proteins by forming glucagon fibrils in hydrochloric acid-phenol water in the presence of ethylene-diamine-tetraacetic acid and ammonium sulfate;
and C. purifying the glucagon obtained from step B by crystallization at pH 5Ø