IL30448A - Protein metal chelate composition stabilized with saccharide,and preparation thereof - Google Patents

Description

PROTEIN METAL CHELATE COMPOSITION STABILIZED WITH SACCHARIDE AND PREPARATION THEREOF This invention relates to mixtures of a protein and a stabilized saccharide, more particularly an isolated, soluble, globulin-type, alpha-helical, non-antigenic metal chelated protein and sucrose or other stabilized saccharide as defined herein.

The invention relates to a method for stabilizing a protein metal chelate as defined herein by forming an admixture thereof with (a) sucrose; (b) pentose, hexose or a heptose monosaccharide having a hydroxy group on the carbon atom adjacent to a free keto or aldehyde carbonyl group and whose spatial configuration is opposite that of two hydroxy groups on the next two adjacent carbon atoms; (c) alky! acetals of pentose, hexose and heptose monosaccharides; (d) glucose; or (e) mannose. In more specific aspects, the invention relates to the production of aqueous solutions and lyophilized solid mixtures thereof storable at room temperature, particularly sterile injectable solutions thereof and lyophilized sterile mixtures thereof hermetically sealed in a container suitable for storage of the solution prior to use and for reconstitution of the lyophilized mixture into an injectable form thereof, respectively. In a process aspect, the invention relates to a process for drying a solution of the protein in stab lizing admixture with such a saccharide. In another process aspect, the invention relates to purifying such a protein metal chelate while in the presence of such a saccharide.

It is an object of this invention to provide a lyophilized protein as defined herein as a mixture with sucrose or other stabilizing saccharide as define herein. Another object is to provide such a mixture in solid form, particularly in sterile, solid form. Another object is to provide such a mixture as a sterile injectable solution. Still another object is the provision of an article of manufacture comprising such a solid, sterile mixture hermetically sealed in a container suitable for reconstituting the mixture into a form suitable for injection. Still another object is the provision of an article of manufacture comprising a sterile injectable solution thereof sealed in a container suitable for storage of the solution prior to use. naturation. Other objects will be apparent to those skilled In the art to which this invention pertains.

The protein metal chelate component of this invention is unstable in its pure state. For example, partial denaturization of the purified protein occurs during lyophilization, irrespective of how it was carried out. This is not unusual for pure proteins and has been observed and described for numerous other proteins once they pass a certain degree of purification, particularly proteins which are non-rigid, i.e., not held together by intramolecular covalent bonds, such as S-S bridges.

It is well-known that highly purified proteins are best stabilized with small amounts of other proteins, such as albumin. Other high-molecular weight soluble compounds of biological origin often work equally well, e.g. marine colloids (carrageenin), starch, dextrane and other polysaccharides, and phospolipids. However, because the protein component of this invention is administered by injection, all" these products are unsuitable because of allergenicity, non-isotonicity and in some cases potential toxicity.

• In addition, and often more important, other proteins and/or lipoproteins lower the biological efficacy of the protein when given in admixture with it.

Similar limitations apply to many other chemicals that have been used successfully as protein stabilizers, such as nucleotides, glutathione and other thiols and sulfites. Still others include the common buffers, terpenes, polycations and polyanions. Polycations include protamine-sulfate, polylysine, polyvinyl mide, and 1 ,10-diaminodecane. Polyanions include poly- styrene-sulfonates , polyvinyl sulfates, sodium-dodecylsulfate, dextran fsulfate and others. P. Burnfeld et al., Arch.Biochem. Bioph.,111 , 31(1965) is a representative example of the use of polycations and polyanions in the stabilization of various highly-purified proteins. Again, while several of these compounds might work well as stabilizers, they are useless for the novel protein because of toxicity, allergenicity and isotonicity considerations.

Another example is the publication of Wakid and Mansur, J.MoT. Pharmacology, 1_, 53 (1965) which explores the protective effect on phos-phofructokinase of hexose-phosphates, necleotides, glutathione and sulfate or phosphate ions, either alone or preferentially in combination.

Examination of the results shows that the Wakid-Mansur approach does not solve the problems for the novel protein either. The nucleotides, to be even partially effective, must be used at least at 5 times excess on a weight per weight basis, eliminating them for isotonicity and allergenicity reasons, ignoring costs. Glutathione cannot be used because of its inter-action with the novel protein and the consequent loss of solubility and efficacy. Of the hexose-phosphates, fructose-1 ,6-diphosphate works best and in principle could have been a satisfactory, albeit very costly choice for the novel protein. Its protective effects depend, however, on the presence of glutathione, mercaptoethanol , and inorganic ions, all of which make the combination useless for the novel protein. The statement of the authors that the free monosaccharides had no protective effect, -2 even at 10 M or 250 times excess of stabil zer over protein, in fact contra-indicates sugars as potential stabilizers for the novel protein.

Various saccharides have previously been used in combination with proteinaceous materials. For example, Glycerol together with tricresol has been used as a bacteriostatic preservative for a sarcoma antigen. Vitamin A and other autooxidizable compounds of low molecular weights have been protected from oxidation by formation of inclusion-compounds with high molecular weight polysaccharides such as dextrins and starch.Sensitive materials of biological origin have been protected during freeze-drying with dextran, a very high molecular weight complex polysaccharide. A protein hydrolysate for intraveneous use has been stabil zed by a gelatin hydrolysate. A trypsin has been solubilized and stabilized by mixtures of polyethylene glycol, vegetable gums, sugar alcohols, soluble starches, soluble cellulose derivatives, dextrose, levulose, inositol, arabinose and beta-lactose. Injectable low molecular drugs have been given "depot-action" after injection thus causing the depot effect and using glycerol to prevent coagulation prior to injection. Measles vaccine has been stabilized with lactose-glutamate mixtures containing very large amounts of lactose. Baccilli Calmette-Guerin vaccine has been stabilized with dextran. Polio vaccine is conventionally absorbed into a sugar cube for oral administration. Vaccinia virus (pox) vaccine has been stabilized by the addition of a mixture of lactose and calcium lactobionate. A measles vaccine has been stabilized by sorbitol. Preparations of chlordiazepoxide for parenteral injection have been dispersed in a medium containing maleic acid, polyoxyethylene and an OH containing aliphatic Cg-C^ compound such as glycerine, propylene glycol , sorbitol , glucose.

It is apparent a wide variety of compounds of highly diverse structure have been used with varying degrees of success as stabilizers for various proteins. However, no pattern of operability exists. Each protein requires a different type of stabilizer. A stabilizer which is highly effective with one protein often is completely ineffective with others.

In Israeli patent Application no.26551 , filed 1966 Sept. 21 , there is disclosed a novel isolated injectable protein metal chelate having unique pharmacological activity which is produced by a process which optionally includes its Tyophilization to a dry powder. This pure protein is highly susceptible to denaturation during lyophilization. Moreover, aqueous solutions of and lyophilized forms of protein are not sufficiently stable to be storable at room temperature, except for brief periods of time. Both of these problems are serious disadvantages from a commercial point of view. The isolated protein is very expensive to produce since final yields of only about 0.01 percent, calculated on the dry weight of the natural source of the protein, are common. Thus, the 25 percent or more loss of the purified protein on lyophilization is a serious economic loss. Further, the denatured protein produced during lyophilization or on storage at room temperature is Insoluble and requires filtration of the reconstituted solution before injection another serious problem associated with its use commercially.

The lyophilized mixtures of this invention comprising the above-described protein metal chelate and sucrose or other saccharide as defined herein are substantially and ordinarily completely free of insoluble protein. They readily reconstitute to a clear solution. Equally important, both dry mixtures and solutions of the protein and stabilizing saccharide are storable at room temperature and higher at 37° C. for much longer periods of time than the protein alone and mixtures of the protein and other types of saccharides. The compositions of this invention thus represent an improvement over the lyophilized pure protein of Israeli application No. 26 551 of great technical and commercial importance.

It was found that certain but only certain water soluble polyhydroxy compounds are surprisingly effective as stabilizers against denaturation of the protein metal chelate described herein. Not only do they protect the protein against denaturation during its isolation from other proteins with which its precursor form is associated in natural sources thereof, it also increases the resistance of the pure protein to denaturation on storage at temperatures above 4°C, either as a solution or a dried solid. This latter ability is particularly important because it permits the isolated protein, particularly in its lyophilized form, to be shipped and stored at room temperature something not possible with the pure protein alone.

The saccharide stabilizers used in the compositions of this invention are, with a few exceptions, those which produce a granular lyophilizate when a solution thereof and the protein metal chelate are lyophilized. These saccharides include (a) sucrose, (b) the aldose and ketose pentose, hexose and heptose monosaccharides in which the carbonyl group is in the form of an alky! acetal, e.g., methyl, ethyl or other lower alkyl acetal , such as methyl glucoside, methyl galactoside, (c) aldose and ketose pentose, hexose and heptose in which the hydroxy group adjacent the free carbonyl group has a spacial configuration opposite that of the next two adjacent hydroxy groups, e.g., galactose, fructose, fuccose, arabinose, aldose galactoheptulose, sedoheptulose etc., (d) glucose, and (e) mannose. For a listing of saccharides which include Interscience Pub. , 0954) Vol .13, pages 228-236. It was first bel ieved a non-reducing sugar would be superior to reducing sugars in view of the wel l -known Mail lard (."Browning") reaction. This reaction frequently has been observed to occur during lyophi l ization of basic proteins in the presence of reducing sugars , resulting in brown insoluble products by virtue of interaction of £ -amino groups , (lysine) with the aldehyde or keto groups of the reducing sugars . Since the novel protein is rich in lysine , has an isotonic point of pH 7.9 and needs an al kal ine pH for retention of conformation , it was assumed a "frowning reaction" would occur and , therefore, make impossibl e the use of reducing sugars as stabil izers .

Surprisingly, not only do two reducing sugars (galactose , fructose) function as excel lent stabilizers , but two non-reducing sugar alcohol s (sorbitol , manitol ) and the non-reducing inositol were less effective. This phenomena appears to be related to the steric configuration of the stabil izers Both fructose and galactose haye two adjacent hydroxyl s sterical ly located on the same side of the molecule . In addition, these two adjacent hydroxyl s are non-adjacent to the functional group, i .e. , the aldehyde or the keto group. This steric arrangement may permit the sugar molecule to al ign itself with the protein molecule in such a way that the £ -amino groups of lysine would be spatial ly unable to react with the aldehyde or the keto group of the reducing sugar.

I a disaccharide the steric arrangement of the sugar molecule makes such an al ignment as described for the two hexoses l ess readily possibl e, which may explain the less effectiveness of mal tose and lactose , which also are reducing sugars , Sucrose is non-reducing in character and there is no free carbonyl group for interaction; hence steric configuration is not crucial Consistent with this hypothesis , fuccose and arabinose are very effective. Their steric arrangement is analogous to the very effective galactose.

Absorbance values at 280 m μ are of interest. The higher Δ A^ values , the higher the eyentual degree of denaturation of the protein . This a arentl means the de ree of denaturation to be ex ected in the resence the native protein molecule which are signalled by the absolute values for the respective A ^280' n some °^ ^e dissaccharides and in two sugar alcohols the trend is the same, although the absolute values are not quite as valid because the turbidity of the solution interferes with the reliable determination of A . In the samples of the protein chelate without stabili- 280 zer, reliable determination of A ■ is not possible because of the presence 28Q of insoluble flakes in the turbid solution.

The physical appearance of the lyophilized protein stabilized with the preferred galactose, fructose or sucrose, is granular rather than fluffy. It dissolves exceptionally readily both in water and common buffers resulting in a sparklingly clear solution. The rate of solubility in fact is considerably better than that of the protein alone.

The addition of saccharide stabilizer as defined herein, especially sucrose, galactose or fructose, also increases the shelf life of solutions of the protein. The stability at 4° C. of a solution in isotonic saline containing two parts of sucrose per part of protein was compared with a solution of protein in isotonic saline alone. Both solutions were periodically checked by A absorption and by visual inspection for turbidity. After one week the 280 protein solution without sucrose was slightly turbid and the A as well as the dry-weight determination indicated a protein loss of 15-20 percent. The solution with sucrose was perfectly clear even after three weeks and the dry weight and the A_ were the same at beginning and end within the limit of 280 experimental error. 2:1 maltose and dulcitol solutions showed similar stabilities for a week or more.

Amounts of 1 part by weight or less of sugar per part of protein appear to be insufficient for optimum protection. Two parts of sucrose per part of protein work very well. Larger amounts of the less efficient stabilizers are sometimes required to obtain optimum stabilization.

Practical upper limits are defined in terms of isotonicity in . addition to the effectiveness of the selected saccharide as a stabilizer.

The following tabulation illustrates this. Calculations are based on milligrams of sugar per milliliter of solution. The sugar weights in turn are calculated on an average protein dose of 1 mg/ml .

TABLE I At Isotonicity Isotonic! ty Increase Due to mg/ml Stabilizer mg/mg of Protein 2.0 4.92 5.05 9.25 NaCl 9.0 — Fructose 50.5 3.96% 10% Galactose 49.2 4.07% 10% — Sucrose 92.5 2.16% 10% Two parts of sugar per part of protein chelate represent an increase in isotonicity of 3.96%, 4.07% and 2.16% for fructose, galactose and sucrose, respectively. Pharmacologically, a 10% increase above isotonicity is acceptable in intramuscular injections particularly for such readily diluted and absorbed products as sodium chloride and sugars. At 10% hypertonic! ty the practical upper limits of stabilizer are Fructose 5.05 parts; Galactose 4.92 parts; and, Sucrose 9.25 parts per part of protein.

In terms of isotonicity increase, sucrose is the preferred of the above three stabilizers because the relative degree of hypertonic ity is the smallest on a weight-per-weight basis.

Isotonic solutions containing larger amounts of sugars can be readily produced by using saline at less than 0.9 percent concentration.

However, in practice, this would be cumbersome since it would call for dilutio of sterile isotonic saline with sterile water in appropriate ratios, thus requiring calculation and extra operations.

If for stability or other purposes, even larger amounts of saccharide were desirable then saline could be replaced altogether by isotonic solutions made up solely from the respective sugars or mixtures thereof. In this instance the maximum amounts would be fructose, 50.5 mg/ml; galactose, 49.2 mg/ml; and sucrose 92.5 mg/ml.

The protein component of the compositions of this invention are metal chelates substantially free from other proteins associated with the precursor of the proteins as they exist in the sources thereof and are the subject matter of Israeli application no.26 551, filed 1966 September 21. They are white powders soluble in water, saline, and buffer solutions and injectable without manifesting the antigenic reactions typical of foreign-body proteins. Their elemental, infrared, ultraviolet, spectrographs, optical rotary dispersion and other analyses are consistent with their metal lo-protein chelate structure. They possess the inherent use characteristics of ameliorating and mitigating in mammals the adverse effects of a stress condition, and exerting anti- inflammatory and anti-viral activity in mammals and other animals as evidenced by pharmacological and clinical evaluation.

Pharmacological and clinical data has established the compositions of this invention are useful in the treatment of a variety of ailments and diseases in mammals, particularly those which result in a stress condition manifesting itself in the afflicted mammal, i.e., in alleviating or ameliorating the stress condition the protein chelate facilitates the termination of the ailment or diseased condition or partially or completely relieving the symptoms thereof. This utility has shown no specificity as to any particular species of mammal.

More specifically, the compositions are efficacious in ameliorating acute inflammatory conditions and mitigating the effects thereof, especially those involving the urinary tract and joints, in various mammals with about 0.2 mg. intramuscular doses. They also possess wide-spectrum anti-viral activity. Surprisingly, a very low dose, e.g., 0.004 mg/kg calculated on the protein, has a period of effectiveness of several weeks in chronic conditions, after the patient has initially been brought to a plateau. The compositions are also useful in treating allergic states, e.g. ,penicill in reaction, multipl wheals, indurations, erythemas, endemas and itching. The compositions are highly efficacious in treating chronic and intractable bacterial infections, e.g., mixed staph, strep, col and enterococci infections of the genito The compositions of this invention can be prepared in pharmaceutical forms suited for injection by lyophilizing a solution of the protein and stabilizing saccharide in a conventional manner. Solutions containing the protein and the saccharide only or in combination with bacterio-stats, bacteriocidal agents, systemically active steroids, e.g., the progestational, estrogenic, androgenic and anti- inflammatory steroids, thickening agents, preservatives and pharmaceutically-acceptable coloring agents can be used. Salts, e.g., sodium chloride, in an amount which will provide an isotonic solution when the mixture is reconstituted with sterile distilled water can also be present.

The protein-saccharide mixture can also be formed into sterile solutions, suspensions, etc., suitable for injection without further modification or reconstitution.

The dry solid mixtures of this invention are stable for many weeks at room temperature without detectable denaturation of the protein in marked contrast to the protein alone, which badly deteriorates in a matter of several days at room temperature. The improved stability of aqueous solutions of the mixtures of this invention at room temperature is equally striking, the protein alone deteriorating in a matter of hours whereas the mixture is stable for several days or weeks. For example, a solution of the protein and sucrose is stable without measurable denaturation at both room temperature and 37°C. for over a month. Even dulcitol and maltose, which are less effective than sucrose in protecting the protein against denaturation during lyophilization, protect solutions of the protein for at least a week at room temperature before significant deterioration begins.

The improved stability of the protein at room temperature when in admixture with sucrose or other saccharide defined herein renders topical and oral administration of the protein feasible. Ordinarily, such routes of administration are not commercially feasible when a pharmaceutical is not storable at room temperature because there are not suitable commercial means for storing such pharmaceutical forms prior to its sale and subsequent administration. The protein is remarkably resistant to proteolytic enzymatic degradation, which makes oral administration feasible.

Thus, the protein and saccharide mixture can be formulated in a preparation suitable for oral or topical administration in conventional manner with the aid of one or more carriers or excipients. Examples of such types of topical preparations include ointments, lotions, creams, sprays, powders, drops (e.g., ear drops and eye drops), suppositories or retention enemas (e.g., for the treatment of rectal or colonic inflammations), and aerosols. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Such bases may thus, for example, include water and/or an oil such as liquid paraffin or a vegetable oil. Thickening agents which may be used according to the nature of the base include soft paraffin, aluminum stearate, cetosterayl alcohol, polyethylene glycols, woolfat, hydrogenated lanolin, beeswax, etc.

Lotions may be formulated with an aqueous or oily base and generally also include one or more of stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes and the like.

Powders may be formed with the aid of any suitable powder base, e.g. talc, lactose, starch, etc. Drops may be formulated with an aqueous base or non-aqueous base also comprising one or more dispersing agents, suspending agents, sol ubili zing agents, etc.

These pharmaceutical compositions may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxy benzoate, propyl hydroxy benzoate, chlorocresol , benzalkonium chlorides, etc. These may also contain other active ingredients such as antimicrobial agents, particularly antibiotics and/or steroidal anti- inflammatory compounds.

The proportion of the protein in the compositions according to the invention depends on the precise type of formulations to' be prepared but will generally be within the range of from 0.001 percent to 5 percent by weight. tion used will be within the range of from 0.001 to 0.5 percent and preferably 0.01 to 0.25 percent.

Examples of such types of oral preparations include the conventional liquid preparations, e.g., solutions, suspensions, and the solid form, e.g., tablets, pills, capsules, encapsulated micro-spheres .Enteric coated tablets which disintegrate in the intestine, particularly the illium, are preferred. In the preparation of such tablets, the conventional enteric coatings and fillers, e.g., cornstarch, lactose, talc, gums, etc., can be used.

Preparation: Protein Metal Chelate.

All operations, unless otherwise indicated, are carried out in a cold room (2-5°C).

Fresh beef liver is ground into a suitable plastic container. Cold distilled water (two liters per kg. of liver) is added with stirring and the mixture is adjusted with 0.1 N sodium hydroxide to pH 7.5 to 7.6. Sufficient 2M manganese sulfate is added to bring the molarity of the mixture to 0.05. The pH is adjusted to 7.6 and fresh cold water is added to bring the water to three liters per kg. of liver. Thereafter, 50 ml. of toluene per kg of liver are added and the mixture is stirred in the cold room overnight.

The next morning the suspension is passed, through plastic gauze and to the filtrate an equal volume of cold acetone (-10°C) is added with gentle stirring. The acetone is added rapidly through a glass tube extending well below the surface of the mixture. The ensuing precipitate is immediately collected by centrifuging and then right away suspended with about 25 percent ++ (V/V) of 0.05 M maleate-Mn buffer, calcuiated upon the volume of the filtrate before addition of the acetone. The mixture is stirred in the cold room for several hours, passed through plastic gauze and clarified by centrifuging.

The supernatant is heated rapidly to 60°C. with stirring in a stainless steel kettle and maintained at 60°C. until 20 minutes have elapsed from the start of the heating. Thereafter, the mixture is cooled to 5°C. as rapidly as possible and the bulky precipitate is separated by centrifugation or centrifugate is brought to 2-5°C. and 0.9 volume of denatured ethanol (-10°C.) are added rapidly from a funnel through a glass tube extending well below the surface of the mixture. Effective stirring is essential and the temperature must remain at 2°C. or lower.

After the addition of the alcohol has been completed, the mixture is kept in the cold room just long enough to permit the precipitate to compact and to settle. The precipitate is recovered by centrifugation or filtration ++ at low vacuum and immediately dissolved in cold 0.001 M maleate- n buffer, pH 7.0. The amount of buffer is approximately 4 v/wt. The solution is clarified by centrifuging,the supernatant decanted, the precipitate re-extracted using small amounts of cold buffer, the supernatants combined and lyophilized. Prior dialysis to remove buffer ions is not necessary at this point. The resultant powder is stable for several months at room temperature but preferentially is kept in the cold room. It represents a mixture of the desired protein, arginase and other enzymes, albumin and other non-essential proteins.

This powder is dissolved in about twelve times the volume of cold 0.2 M Tris-0.001 M Mg^buffer.pH 7.8. This solution is treated with cold saturated ammonium sulfate solution, 0.001 M in Mg++. Five increments of 375 ml. each are added per 1,000 ml. of buffer solution. The respective states of saturation achieved by this technique are 15 percent, 30 percent, 45 per-cent, 60 percent and 75 percent. In each instance the addition of the ammonium sulfate solution is carried out drop by drop at 0-5°C. with stirring.

Stirring is continued for another 30 minutes and the resulting precipitate s collected by centrffuging at 4500 rpms for thirty minutes at 0° C.

Of the five precipitates obtained, the first one (A) is discarded. It represents high-molecular weight-protein impurities. The second and third precipitates (B and C) are combined. They represent arginase and other enzymes which can be processed separately for the isolation of these products. The fourth and fifth (D and E) are also combined. They contain the desired protein, in a still crude state, contaminated with albumin and various other proteins both of lower and higher molecular weight. The final supernatant is discarded.

Precipitates D and E are dissolved in 0.03 M Tris-0.001 M Mg buffer, pH 7.8 at a concentration as close to 10 percent (w/v) as possible and dialyzed against cold buffer until negative to sulfate ion. The dialyzed solution is clarified by centrifuging and the supernatant is passed through a Millipore filter. The filtrate is applied directly to the head of chromatography columns (3 x 18 inches) filled with Sephadex G-100 (epichlorohydrin cross-linked dextran resin; Pharmacia, Sweden). The Sephadex has been swelled, defined and washed by standard techniques described in literature of the ++ manufacturer. The packed columns are equilibrated with 0.03 Tris-O.OOl g buffer, pH 7.8 and adjusted to a flow rate of about 20 ml. per hour.

After application to the column, the sample is permitted to equilibrate within the first 3 cm of the resin bed for approximately 30-45 minutes when fractionation is started. Individual fractions of 10 ml. are collected. The emergence of peaks is determined by measuring the protein concentration by the absorbance at 280 millimicron.

Two peaks emerge from the column prior to the emergence of the desired protein. They represent albumin and other undesirable protein impurities of similar or larger molecular weight. Fractions representing these peaks are discarded. The desired protein generally emerges in the range of 130-170 ml. of total eluate. These fractions from the columns are combined for further processing. Residual, lower molecular weight protein impurities emerge from the column on further elution, particularly on increasing the ionic strength of the buffer. They are removed to clear the column for a subsequent run.

The combined fractions containing the desired protein are dialyzed ++ -6 against deionized hyD-O.OOl M Mg until they contain less than 10 M Tris buffer. Thereafter, dialysis is continued against deionized water containing 1-5 x 10 M ortho-phenanthrol ine or ethylenediamine tetraacetic acid salts until the concentration of Mg++ has been reduced to less than 10"7 M. If a protein chelate is desired whose predominant metal is other than magnesium, e.g zinc, calcium, copper or iron, continue dialysis at 4°C.for several days and choice of a molarity which maintains the protein in solution, and then remove excess metal ion in the manner described above. The resultant solution is clarified by centrifuging. 75 kg. of fresh beef liver (22.5 kg dry weight) yields about 200 grams (1 percent) of the Mn protein chelate intermediate from which 12.5-17.5 grams (0.06-0.08 percent) of the desired protein is obtained from the combined D and E ammonium sulfate fractions. On Sephadex chromatography, these amounts of D and E fractions yield 2.4 to 2.9 grams of the desired protein, equivalent to an over-all yield of 0.011 - 0.014 percent calculated on the dry weight of the liver.

Examples.

A. Lyophil ization Stabil ization Fifteen parts of the isolated protein described in the preparation and thirty parts of the selected sugar or sugar derivative are weighed and mixed. The mixture is dissolved in 30 parts of demineral ized water that has been adjusted to pH 9.4 by gaseous ammonia. The solution is then filtered with slight vacuum through an 0.45 μ pre-wetted Millipore filter. The volume of filtrate is measured and the weight of protein therein calculated as follows 2 ml of the filtrate is mixed with 3 ml Biuret Reagent and the mixture in-cubated for 15 minutes at 37° C. Absorbance at 555 μ of the mixture is measured against a water (buffer) blank. Concentration in mg/ml is determined by multiplying absorbance at 555 μ by 9.1. This conversion factor was obtained by plotting the following data obtained from samples of known concentration on a graph: Protein Cone. Absorbance mg/ml* A555 1.8 0.9 0.45 0.22 The sample is then shell -frozen, and thereafter lyophil ized. The lyophilized material is reconstituted to approximately the initial concentration by adding the appropriate volume of demlneralized water brought to pH 9.4 with ammonia. The solution is checked whether it is clear or not, then filtered through a pre-wetted 0-45 μ Millipore filter and its absorbance at 280 ji measured. The ^80^' 1s determined. Tne weight of protein lost is expressed by the difference between the protein concentration calculated from the weight of protein and volume of solution before lyophilization and the weight of protein and volume of solution after lyophilization. Percent loss is expressed as percent of the calculated protein present before lyophilization. The same procedure is repeated for successive lyophilizations.

Tables II and III list the results of stabilization studies with various polyhydroxy stabilizers following the above procedure. Protein loss after one and four lyophilizations were determined. It can be seen the saccharides listed in Table II are excellent stabilizers, reducing protein loss after four lyophilizations to about 15 percent or less, compared with 61-80 percent for the unstabilized protein and, except for o -methyl -D-glucosid producing a crystal clear solution when the protein was redissolved. The saccha rides listed in Table III improve the stability of the protein to a lesser extent and usually produce a turbid solution when the mixture is reconstituted with water or isotonic solution. The latter result renders the saccharides listed in Table III less desirable from a commercial point of view, even though they also substantially reduce protein loss.

TABLE Π EFFECT OF LYOPHILIZATION Cone, of Appearance of re¬ Appearance of Starting Initial 1 4 constituted solution Percent Stabilizer *■ Lyophilizate Protein •^200 lyop ili- lyophili- 1 4 .- ' 1 Solution zation zations lyophili- lyophili- lyoph (mg/nil) zation zations zati I. 'Monosaccharides a. ) arabinose II 0.55 0.527 0 0.011 II t» 0 e. D-fucose II . 0.53 0.528 0/004 0.051 . II II o . f. D-glucose II 0.51 0.606 0.053 0.211 II II . 5.9 g. O{-r') mannose II . 0.48 0.568 . 0.028 0.222 II 2.0 Π. Disaccharides a. sucrose 0.49 0.604. . 0 0.058 tl II 0 ΙΠ. Alk l glycosides a. cC-met yl-D- II 0.54 0.681 0 0.072 tl V.S1. 0 glucoside turbid b. 2-methyl-D- ' fluffy Ό.42 0.681 0.014 0.054 tl Clear 0 galactoside IV. Control a. no stabilizer II 0.48 0.662 turbid with turbid 12.5 b. no stabiliz r 11 0.46 0.6Q3 — . insoluble with in•10.9 1 flakes soluble flakes EFFECT OF LYOPHILIZATION B. Solid Storage Stability mg. of freshly prepared protein metal chelate and 50 mg. of sucrose, fructose or galactose were dissolved in 50 ml. of demineral ized water, brought to an alkaline pH (around 9) with ammonia water, filtered and lyophilized. The lyophil izates were kept under air at room temperature along with the sample of the starting protein metal chelate. The storage stability of the mixtures at room temperature for various periods of time is shown in Table IV, as determined by the .A of solutions of the samples prepared after the sample had been stored for the indicated time.

TABLE IV.

ROOM TEMPERATURE STABILITY OF SOLID MIXTURES.

Stabilizer A280/mg. A555(Corr.

Sucrose initial 0.637 Ί week 0.595* 1 month 0.633 2 months 0.637 4 months 0.602 Fructose initial 0.631 1 week 0.589* 1 month 0.768 2 months 0.666 4 months 0.758 Galactose initial 0.630 1 week 0.615 1 month 0.649 2 months 0.689 4 months 0.673 * Temporarily slightly lower readings after about 1-2 weeks storage are consistently observed. All samples reconstituted after storage into clear precipitate-free solutions.

These data indicate no significant protein degradation after four months storage of the lyophilized protein-saccha ide mixture. The protein To determine stability at 37° C, 50 mg. of protein metal chelate was dissolved in 100 ml. of a 1 mg/ml solution of sucrose in water, the pH adjusted to 9.4 with ammonia water and filtered through a 0.45 Millipore filter. An aliquot was withdrawn to determine A280 and protein concentration. The remainder was lyophilized for 4 days and stored at 37° for various periods of time. The results are shown in Table V.

TABLE V.

STORAGE STABILITY AT 37°C.

Appearance of Reconstitute Solution "555 Stabil zer A /mg . (Corr. ) Sucrose initial 0.5194 0.038 Clear 1 week 0.4846 0.041 2 weeks 0.5216 0.039 1 month 0.5081 0.039 2 months 0.5324 0.039 These data show no significant protein degradation has occurred after two months storage of the lyophilizate at 37° C. The protein alone shows a 50 percent loss in 11 days.

C. Solution Storage Stability mg. of protein metal chelate and 50 mg.of sucrose, maltose or dulcitol were dissolved in 50 ml. of demineral ized water in the manner described above. The solutions were maintained at room temperature for various periods of time and protein loss determined. The results are shown in Table VI.

TABLE VI ROOM TEMPERATURE STABILITY OF SOLUTIONS Stabilizer A280/mg A555 Appearance of (Corr. ) • Solution 1. Sucrose initial 0.562 0.055 Clear 1 week 0.640 0.052 II 3 weeks 0.626 0.045 II weeks 0.702 0.051 V.S1.turbid* 2. Dulcitol initial 0.618 0.052 Clear 1 week 0.637 0.054 II 3 weeks 0.696 0.049 II weeks 0.660 0.055 II 3. altose initial 0.603 0.065 Clear 1 week. 0.725 0.061 II 3 weeks 0.759 0.063 II * possibly due to bacterial contamination These data show no significant protein degradation after three weeks or more storage at room temperature of an aqueous solution of the protein-sucrose and protein-dulcitol mixtures. The protein-maltose mixture shows signs of protein degradation. A solution of the protein alone shows a protein loss of 70 percent within 36 hours.

Claims (15)

WHAT IS CLAIMED IS:

1. The isolated water-soluble globulin-type non-antigenic protein of appl cation No. 26551 as a mixture with a stabilizing amount of a saccharide selected from the group consisting of (a) sucrose, (b) pentose, hexose or heptose having a hydroxy group on the carbon atom adjacent a free keto or aldehyde carbonyl group and whose spacial configuration is opposite that of two hydroxy groups on the next two adjacent carbon .atoms, (c) a lower-alkyl acetal of a pentose, hexose or heptose, (d) glucose, and (e) mannose.

2. A mixture according to Claim 1 wherein the stabilizing saccharide is sucrose.

3. A mixture according to Claim 1 wherein the stabilizing saccharide is a pentose, hexose or heptose having a hydroxy group on the carbon atom adjacent the carbonyl group whose spacial configuration is opposite that of two hydroxy groups on the next two adjacent carbon atoms.

4. A mixture according to Claim 1 wherein the weight ratio of saccharide to protein is at least 2:1.

5. A mixture according to Claim 1 as a dry solid, substantially free from denatured protein.

6. A mixture according to Claim 5 wherein the stabilizing saccharide is sucrose.

7. A mixture according to Claim 5 wherein the stabilizing saccharide is a pentose, hexose or heptose having a hydroxy group on the carbon atom adjacent the carbonyl group whose spacial configuration is opposite that of two hydroxy groups on the next two adjacent carbon atoms.

8. A mixture according to Claim 5 wherein the weight ratio of saccharide to protein is at least 2:1.

9. A method of producing a composition according to Claim 5 which comprises the step of freeze-drying an aqueous solution thereof which is substan

10. A mixture according to Claim 9 wherein the stabilizing sacclw^ ride is sucrose.

11. A mixture according to Claim 9 wherein the stabilizing saccharide is a pentose, hexose or heptose having a hydroxy group on the carbon atom adjacent the carbonyl group whose spacial configuration is opposite that of two hydroxy groups on the next two adjacent carbon atoms.

12. A mixture according to Claim 9 wherein the weight ratio of saccharide to protein is at least 2:1.

13. A mixture according to Claim 1 wherein the mixture is in the form of a biological preparation, suitable for injection, substantially free from denatured protein, and hermetically sealed in a sterile condition, in a vial or ampule.

14. A mixture according to Claim 13 wherein the biological preparation is in the form of a dry solid.

15. A biological preparation according to Claim 14 wherein the saccha ride is sucrose, present in a proportion to the protein of at least 2:1. ' :'r^:..:::. .