US3434927A - Highly purified intrinsic factor - Google Patents

Description

United States Patent US. Cl. 1952 5 Claims ABSTRACT OF THE DISCLOSURE A process of purification is described in which intrinsic factor or the complex of intrinsic factor and a physiologically active cobamide is subjected to hydrolysis with a proteolytic enzyme such as trypsin, chymotrypsin and papain and then subjected to the successive steps of exclusion chromatography and ion exchange chromatography.

Generally stated, the subject matter of the present ininvention relates to a process of obtaining intrinsic factor and the complex of intrinsic factor and a physiologically active cobamide, in a substantially pure state. The complex of intrinsic factor and a physiologically active cobamide shall hereinafter be referred to as intrinsic factor complex. More particularly, the invention relates to a process of purification in which intrinsic factor or intrinsic factor complex, which has been partially purified, is subjected to hydrolysis by a proteolytic enzyme and subsequently to the successive steps of exclusion and ion exchange chromatography. Furthermore, the invention relates to the substantially pure intrinsic factor and intrinsic factor complex obtained by the process of the present invention.

Intrinsic factor is a heat labile component of normal human gastric juices which was first discovered by Castle and his associates, (American Journal of Medical Science, vol. 178, page 748, 1929). Evidence presently indicates that intrinsic factor is involved in the utilization of vitamin B in the classic condition in which a deficiency of intrinsic factor is found, namely pernicious anemia, small oral doses of vitamin B are ineffective unless a source of intrinsic factor is administered simultaneously.

The study of gastrointestinal absorption, with radioactive vitamin B in pernicious anemia patients and healthy individuals has proven that intrinsic factor is essential for this absorption (Ellenbogen, Williams, Rabiner, and Lichtman, Proceedings of the Society for Experimental Biology and Medicine 89, 357, 1955), and although hematopoietic responses in pernicious anemia may follow the oral administration of massive doses of vitamin B given without the intrinsic factor, the absorption of amounts comparable to those found in an average diet requires the participation of intrinsic factor.

Heretofore, various intrinsic factor preparations have been utilized in the treatment of pernicious anemia, however, most preparations require the administration of objectionably large quantities of an unpleasant material, for example, the average daily requirement of whole hog duodenum by a pernicious anemia patient is from about onefourth to one-half pound, and in its desiccated form from at least 20 to 40 grams are required. Various efforts have been made to improve this situation by attempting to prepare a more concentrated intrinsic factor preparation, and this has resulted in preparations which are therapeutically adequate in daily quantities of as low as 20 mg. as, for example, in United States Patent No. 2,848,367, to Williams et al., there is described a process for preparing intrinsic factor concentrate having such potency that 30 mg. provides the minimum daily requirement. In

3,434,927 Patented Mar. 25, 1969 addition, the product demonstrated an augmentative effect which had not hitherto been obtained, that is, the intrinsic factor concentrates which had been previously available inhibited the absorption of vitamin B by healthy individuals, although same were useful in treating pernicious anemia. Unexpectedly the augmentative intrinsic factor concentrate of Williams et 211., does not have this undesirable effect, but increases the absorption of vitamin B in healthy individuals, as well as in those suffering from pernicious anemia.

With regard to the intrinsic factor complex, same may be defined as that intrinsic factor which has been complexed with any physiologically active cobamide.

The present invention represents the culmination of a long series of investigations conducted largely by the inventors and their associates, and directed to the achievement of a substantially pure intrinsic factor, or intrinsic factor complex. The inventive concept resides in the discovery that substantially pure intrinsic factor, or intrinsic factor complex can be obtained by subjecting a partially purified intrinsic factor, or intrinsic factor complex to hydrolysis by proteolytic digestion and the successive steps of exclusion, and ion exchange chromatography.

Therefore, the invention relates to a process of purifying partially purified intrinsic factor and partially purified intrinsic factor complexed with a physiologically active cobamide, which comprises hydrolyzing the partially purified material with a proteolytic enzyme, which will not adversely effect intrinsic factory activity, at a temperature of from about 25 to 40 C., for a period of time sufficient to digest the inactive protein present in the partially purified material, that is, non-intrinsic factor protein, or nonintrinsic factor complex protein, subjecting the hydrolysate to exclusion chromatography, utilizing a medium which is capable of separating that matter having a molecular weight of approximately 10,000 and less from the active material; the material having a molecular weight of greater than 10,000 is then subjected to ion exchange chromatography using a gradient of increasing ionic strength and a dextran gel substituted with diethylaminoethyl groups as an ion exchange adsorbent, the active material is separated from the bulk of impurities, and subjected to the successive steps of exclusion and ion exchange chromatography as hereinabove set forth, the active material is again separated from the bulk of impurities, and lastly subjected to the above exclusion chromatography step.

Broadly stated, the manner of carrying out the present invention is as follows: the partially purified material is dissolved in an aqueous solution, the pH of same being dependent on the proteolytic enzyme to be utilized in the hydrolysis step, for example, when utilizing either trypsin or chymotrypsin either alone or in combination, the aqueous solution is one that has a slightly alkaline pH, approximately 7.8 such as a phosphate buffer, barbital buffer, or the like, and the concentration of partially purified material may, for ease of handling, be anywhere from 2 to 10 percent. The solution is then hydrolyzed by proteolytic digestion, utilizing proteolytic enzymes which will not adversely affect intrinsic factor activity such as trypsin and chymotrypsin. The proteolytic digestion is preferably carried out at a temperature of approximately 37 0., however, temperatures ranging from 25 to 40 C. may be employed. The reaction is allowed to proceed for a period of time sufficient to digest the inactive protein,

The hydrolyzed, partially purified material is then subjected to exclusion chromatography, employing materials such as a dextran gel or a copolymer of acrylamide and methylenebisacrylamide, in which the active material is separated from that matter having a molecular weight of 10,000 or less. The active, high molecular weight material, that is, having a molecular weight greater than 10,000 is concentrated as, for example, by lyophylization, and redissolved in an essentially neutral buffer of low ionic strength such as a phosphate buffer of 0.02 M having a pH of 7. This material is then subjected to ion exchange chromatography using a gradient of increasing ionic strength, and employing as the ion exchange adsorbent a dextran gel substituted with diethylaminoethyl groups. The active material, which is determined by the Schilling urinary excretion test (as modified per Ellenbogen et al., Blood 13, 582 (1958)), is separated from the bulk of impurities and successively subjected to the above steps ofexclusion and ion exchange chromatography and lastly exclusion chromatography.

The advantages which inure to the art as a consequence to the advent of the present invention should be readily apparent from the following: namely, the minimum daily requirements of intrinsic factor can be provided by administering approximately 25 to 100 mcg. of the substantially pure intrinsic factor, or intrinsic factor complex obtained by the novel process of the present invention. This, in effect, constitutes a ten-fold increase in potency over the partially purified material, which requires the administration 0.2 to 2 mg. to provide the minimum daily requirement. Furthermore, the novel process of the present invention provides, for the first time, a substantially pure intrinsic factor and intrinsic factor complex.

As presently used in the instant specification and claims, the term dextran gel shall be generally defined to mean those gel materials which comprise uncharged, insoluble organic substances consisting of a three-dimensional macroscopic network of molecules bonded together by covalent linkages in the form of ether bridges of the general type --O-XO, wherein X represents an aliphatic radical containing from 3 to carbon atoms, and having a content of hydroxyl groups corresponding to at least 12 percent, preferably 15 percent or more, of the weight of the dry gel.

These gel substances are obtained by polymerization of uncharged organic hydroxyl group containing substances, with bifunctional organic substances containing halogen atoms and/or epoxy groups. With regard to dextran gel, the suitable hydroxyl group containing substance is a polysaccharide, that is, dextran. The bifunctional compound is epichlorohydrin.

The copolymerization of the organic hydroxyl group containing substance with the bifunctional substance readily takes place by reacting same in an aqueous solution in the presence of an alkaline reacting substance as a catalyst such as an alkali metal hydroxide. The concentration of the organic hydroxyl group containing substance in said aqueous soltuion may suitably be within the range of from about 10 to 70 percent by weight. The molecular proportion of the organic hydroxyl group containing substance to the bifunctional substance should be 1 to at least 10. The reaction time and temperature may be varied within rather wide limits, although a temperature range of about 15 to 90 0, preferably a range of to 70 C., has been found to be most satisfactory. Depending on the precise reaction temperature, as well as the reactants themselves, the formation of a gel usually occurs in less than 4 hours, Subsequent to gel formation, the gel copolymerisate is subjected to a curing step, preferably carried out at an elevated temperature within the range of about 30 to 90 C., and for approximately 5 to 48 hours.

In particular, the dextran gel may be obtained by reacting dextran having an average molecular weight within the range of from 5,000 to 100,000 with epichlorohydrin while maintaining the reaction conditions indicated above. The reaction results in a copolymerizate consisting of a three-dimensional macroscopic network built up of chains of mainly alpha-16-glycosidically bonded glucose residues bound together by ether bridges of the type said gel having a content of hydroxyl groups of at least 15 percent of the weight of the dry gel and a water regain within the range of from about 1 to 50 g./ g. of the dry gel product.

With regard to the dextran gel substituted with the diethylaminoethyl groups same may be defined as the diethylaminoethyl ether of dextran gel which is obtained by the introduction of diethylaminoethyl groups into dextran gel. The reaction is carried out in the presence of alkali and, as a consequence to hydrolysis occurring as a side reaction, the amounts of water have to be kept low in order to obtain a high degree of substitution. It has been found to be advantageous to perform the sub stitution with swollen gel particles dispersed in an inert solvent such as a hydrocarbon. Effective temperature control is thereby obtainable in this fashion, and the ultimate product becomes more evenly substituted.

The partially purified materials, that is, either the partially purified intrinsic factor, or the partially purified intrinsic factor complex employed as the starting material in the novel process of the present invention may be defined as that intrinsic factor, or intrinsic factor complex, which is capable of providing the minimum daily requirement by the administration of 0.2 mg. to 2 mg.

Intrinsic factor is partially inactivated by the process of the present invention, and said inactivation can be obviated by .saturating the binding capacity of the intrinsic factor with any physiologically active cobamide prior to initiating the process of the present invention. Furthermore, when intrinsic factor complex is to be subjected to the process of the present invention it is deemed advisable to saturate the binding capacity of same to obviate the inactivation of any portion thereof. It appears that the complex formed by the interaction of intrinsic factor and a physiologically active cobarnide possesses greater stability than intrinsic factor alone.

The binding capacity of either intrinsic factor, or intrinsic factor complex can be ascertained by employing the method of Rosenblum et a1. (Procedures of the Second Radio Isotope Conference, J. E. Johnston, Ed., page 287, Butterworths Scientific Publication, London, 1954). Once the binding capacity of the intrinsic factor, or the intrinsic factor complex is ascertained, same is saturated by the addition of a sufiicient amount of a physiologically active cobamide.

With regard to the proteolytic enzymes employed in the hydrolysis step of the present invention, same may be defined as those enzymes which will not adversely effect intrinsic factor activity such as trypsin, chymotrypsin and papain among others. Furthermore, in addition to the foregoing criteria of compatability of the enzyme and the intrinsic factor an additional criteria is utilized, that is, the substrate specificity of the enzyme or enzymes employed. In particular the ability of the enzyme or enzymes to hydrolyze the inactive protein present in the partially purified material. Therefore, an enzyme may be used alone or in combination with other enzymes, as demonstrated in the following examples, wherein both trypsin and chymotrypsin are employed, and as such constitute a preferred embodiment of the instant invention. However, each of the enzymes may be used alone to achieve the desired result of hydrolysis.

The following examples are provided for illustrative purposes and may include particular features of the invention. However, the examples should not be construed as limiting the invention many variations of which are ppssible without departing from the spirit or scope there- 0 EXAMPLE I stantially pure intrinsic factor complex from a partially purified material.

A 3 gm. sample of partially purified intrinsic factor complex was dissolved in 50 cc. of a slightly alkaline, pH 7.8, 0.007 M phosphate buffer solution. The binding capacity of the complex, deter-mined by the Rosenblum et a1. method, was saturated by the addition of a sufficient amount of cyanocobalamine.

The complex was then subjected to hydrolysis by proteolytic digestion for 3 hours at 37 C., employing a mixture of trypsin and chymotrypsin, 1.5 mg. of each enzyme per 100 mg. of complex. The hydrolysate is then subjected to exclusion chromatography on a dextran gel column, the volume of same being five times the total volume of the aqueous solution. All matter having a molecular weight of 10,000 or less was thereby separated from the active material. The high molecular weight material, that is, having a molecular weight greater than 10,000 was concentrated by lyophilization.

The concentrated material, 1.375 gm., was dissolved in 50 cc. of a 0.025 M phosphate buffer, pH 7.0, and subjected to ion exchange chromatography on a column of dextran gel substituted with diethylaminoethyl groups, the volume of same being five times the total volume of the aqueous solution, using a linear gradient of increasing ionic strength. The gradient was composed of two solutions, first, a 0.025 M phosphate buffer solution, pH 7.0, and second, a 0.15 M phosphate buffer solution, pH 7.0. The active intrinsic factor complex, determined by the Schilling urinary excretion test (as modified by Ellenbogen et al., Blood 13, 582 (1958)), was recovered from the effluent, and again subjected to exclusion chromatography as hereinabove set forth and concentrated by lyophilization.

The concentrated material was dissolved in 50 cc. of a 0.025 M phosphate buffer, pH 7.0, and again subjected to both ion exchange chromatography and exclusion chromatography, as hereinabove set forth. The elfiuent was concentrated by lyophilization, and approximately 80 mg. of substantially pure intrinsic factor complex was obtained, which was capable of providing the minimum daily requirement by the administration of about 50 mcg.

EXAMPLE II Preparation of substantially pure intrinsic factor This example demonstrates the preparation of substantially pure intrinsic factor from a partially purified material.

A 3.5 gm. sample of a partially purified intrinsic factor was purified following the procedures set forth in Exampic I; and approximately 90 mg. of substantially pure intrinsic factor was obtained, which was capable of providing the minimum daily requirement by the. administration of about 75 mcg.

EXAMPLE III Utilization of other adsorbents, as well as enzymes in the preparation of substantially pure intrinsic factor and intrinsic factor complex Substantially pure intrinsic factor and intrinsic factor complex were obtained by substituting for the dextran gel of the exclusion chromatography step, a copolymer of acrylamide and methylenebisacrylamide. In addition, the same results were realized by substituting the enzyme papain for the enzymes trypsin and chymotrypsin in the hydrolysis step.

Although the invention has been described and illustrated by reference to particular embodiments thereof, it will be understood that in its broadest aspects the invention is not limited to such embodiments, and that variations and substitution of equivalents may be resorted to within the scope of the appended claims.

We claim:

1. A process of purifying partially purified intrinsic factor and the partially purified complex of intrinsic factor and a physiologically active cobamide, which comprises (a) hydrolyzing the partially purified material with a proteolytic enzyme, which will not adversely effect intrinsic factor activity, at a temperature of from about 25 to 40 C., for a period of time sufficient to digest the inactive protein present in the partially purified material,

(b) subjecting the hydrolysate to exclusion chromatography, utilizing a medium which is capable of separating that matter having a molecular weight of approximately 10,000 and less from the active material,

(c) the material having a molecular Weight of greater than 10,000 is chromatographed on a dextran gel, substituted with diethylaminoethylene groups, using a gradient of increasing ionic strength,

(d) separating the active material from the bulk of impurities,

(e) subjecting the active material to the successive steps of exclusion chromatography and ion exchange chromatography as set forth in steps b and c,

(f) separating the active material from the bulk of impurities, and

(g) subjecting said active material to exclusion chromatography as set forth in step b.

2. A process according to claim 1, in which the partially purified material is hydrolyzed at a temperature of 37 C.

3. A process according to claim 1, in which the proteolytic enzyme is an admixture of equal parts of trypsin and chymotrypsin.

4. A process according to claim 1, in which the proteolytic enzyme is papain.

5. A process according to claim 1, in which the medium employed in the exclusion chromatography is dextran gel.

References Cited UNITED STATES PATENTS ALVIN E. TANENHOLTZ, Primary Examiner.

US. Cl. X.R. l28; 424-96