US3008877A

US3008877A - Purification of intrinsic factor

Info

Publication number: US3008877A
Application number: US693807A
Authority: US
Inventors: Kenneth C Robbins
Original assignee: Armour and Co
Current assignee: Armour and Co
Priority date: 1957-11-01
Filing date: 1957-11-01
Publication date: 1961-11-14
Anticipated expiration: 1978-11-14

Description

Unite. grates 3,008,877 PUREFICATIQN (BF TNTRINSIQ FACTGR Kenneth C. Robbins, Chicago, 111., assignor, by mesne assignments, to Armour and Company, Chicago, Ill., a corporation of Delaware No Drawing. Filed Nov. 1, 1957, Star. No. 693,807 7 Claims. (Cl. 167-74) This invention relates to a method of obtaining highly purified intrinsic factor preparations. It is particularly related to a method of purifying intrinsic factor utilizing diethylaminoethyl cellulose ion exchangers.

It is known that the duodenum and stomach tissues of animals contain a substance which is of value in the treatment of anemia in humans, and particularly in the treatatent Q process are dialyzed to remove salt and other crystalloid materials. Following the dialysis procedures, it has been found desirable to clarify the solution by centrifugation.

In the process of the invention intrinsic factor starting material is first put in solution. It is desired that this solution be one in which the conditions of pH and ionic strength are proper for later steps in the process hereinafter described. Following the placing of the intrinsic factor material in solution, the solution may be dialyzed and is thereafter clarified by centrifugation. The solution of intrinsic factor is then contacted with an anion exchange resin in the form of the diethylam-inoethyl ether of cellulose, i.e. DEAE-cellulose. DEAE-cellulose may be prepared according to procedures which are set forth in used to designate the component or components of animal duodenum or stomach tissue which is of value in the treatment of anemic conditions, particularly of the macrocytic type.

Heretofore various intrinsic factor preparations have 7 been utilized in the treatment of pernicious anemia, but all of them have suffered from the defects of requiring the patient to ingest objectionably large quantities of material. For example, only a short time ago the average daily requirement of whole hog duodenum by a pernicious anemia patient was A to /2 pound which is equal to at least 20 to 40 gms. when desiccated. Recently efforts have been made to improve this situation by preparing more concentrated intrinsic factor preparations. For example, in US. Patent No. 2,770,570 there is described a method for the preparation of intrinsic factor which is clinically active in daily oral doses on the order of as little as 120 mg. In the method of co-pending patent application Serial No. 497,401, filed March 28, 1955, now Patent No. 2,910,405, there is described a method for the preparation of intrinsic factor which is clinically active in daily oral doses of 35 mg. By daily oral dose is meant the minimum amount of intrinsic factor which, when administered orally together with 15 ,ug. of vitamin B to patients with Addisonian pernicious anemia in relapse, will produce a satisfactory clinical response. The procedures for this test are set out in the J. Am. Pharm. Assn. (PRACT), 1940, volume 1, p. 53.

It is therefore a general object of this invention to prepare intrinsic factor preparations of greatly enhanced potency. It is a particular object of the invention to provide a method for further purifying intrinsic factor preparations on DEAE-cellulose ion exhangers. Further objects and advantages will appear as the specification proceeds.

The preferred starting material for use in the process of this invention is an intrinsic factor preparation prepared according to conventional processes such as is illustrated below in Example I. Generally-- the potency of such preparations is approximately 35 mg. in a daily oral dose. The discussion following will be concerned primarily with the processing of such starting materials.

Intrinsic factor materials prepared according to the methods mentioned above contain considerable dialyzable inert materials. Therefore, such materials in the present the literature, for example, the reaction of alkaline cellulose with 2-chloro-N,N-diethylethylamine, JACS 76, p. 1711 (1954). Prior to contacting the solution with the ion exchanger it is desirable to equilibrate the ion exchanger to within the pH range of from about 5 to 8, and preferably within the pH range of from about 5.4 to 7.0. It is also desirable that the solution of intrinsic factor be at a pH within the range of from about 5 to 8 and preferably within the range of from 5 .4 to 7.0. I

The ionic strength of the solution of intrinsic factor during the adsorption step is determined by the pH of the solution. Where the pH of the solution is about 5 .4 the ionic strength of the solution must be below 0.3. Where the pH of the solution is increased (within the range of 5 to 8) the ionic strength may also be increased to a maximum of about 0.6. At higher ionic strength than about 0.6 the intrinsic factor is not adsorbed on DEAE-cellulose at any pH. The maximum ionic strength consonant with intrinsic factor adsorption is therefore seen to vary with the pH of the solution from about 0.3 at pH 5 to 5.4 to about 0.6 at pH 7 to 8. Preferably the ionic strength of the solution in the adsorption step should not be above 0.1.

The operable range of ionic strength of the elution solution is also relative to the pH of the solution. Where the pH of the eluant is about 5.4 the ionic strength must be in excess of 0.3. Where the pH of the eluant is increased the ionic strength must also be increased so that, for example, where the eluant is at a pH of 7.0 the ionic strength must be over 0.6 and preferably over 0.8. When eluting at a pH of 5 .4 it is preferred to employ an ionic strength of about 0.5. All the intrinsic factor is eluted from DEAE-cellulose by a solution having an ionic strength of 0.8 or greater where the pH is within the required range of from 5 to 8. It is preferred to carry out the elution procedure with successive solutions of increasing ionic strength and separate collection of the various fractions eluted at different ionic strengths.

The eluate from the adsorbate contains purified intrinsic factor along with various crystalloid materials. We have therefore found it desirable to dialyze the eluate to remove such crystalloid materials. Following the dialysis the eluate may be lyophilized to obtain an intrinsic factor preparation in a dry state which is active in daily oral doses of as little as about 10 mg.

The following detailed examples are set forth to more fully illustrate the invention.

Example I Intrinsic factor which was clinically active in daily oral doses of'about 35 mg. Was prepared by the addition of two 1. of ice-cold water to each kg. of fresh frozen ground centrifugation at 4000 rpm. at 2 C. for one hour. The extract of intrinsic factor can be concentrated by either lyophilization or in a Mojonnier pot still. The yield after lyophilization was 30 g. per kg. of pyloric tissue. The concentrate having a pH of 4 was adjusted to 7% total solids and to a pH of 5.2 with 2 N NaOH. A one molar sodium acetate-acetic acid buifer at a pH of about 5.2 was added to the concentrate to give an ionic strength of 0.15 molar sodium acetate at a solids concentration of 6% at 2 C. 95% ethanol at 20 C. was added to the extract to a final concentration of about 50% at 5 C. After standing overnight at 5 C., the precipitate was removed by centrifugation at 4000 rpm. at a temperature of 5 C. for a period of 30 minutes. It was suspended in 4 volumes (precipitate volume) of ice-cold distilled water and stirred overnight at 2 C. The solution of intrinsic factor prepared as indicated can be lyophilized and was clinically active in the daily oral dose of approximately 35 mg. The yield after lyophilization is about 5 to 6 g. per kg. of tissue. The clinical biological activity of this preparation is fully described in an article in Proc. Soc. Expe-r. Biol. and Med., vol. 95, page 781 (1957).

If the material prepared as above is dialyzed before lyophilization, the product will be active in a daily oral dose of approximately 20 mg. Dialysis of the intrinsic factor prior to lyophilization will result in the loss of approximately 60% of the solids.

Example I] Intrinsic factor which was effective in daily oral doses of about 35 mg. was fractionated on a DEAE-cellulose exchanger using gradient elution at pH of 7.0. g. of intrinsic factor prepared as illustrated in Example I were dissolved in 300 ml. of 0.005 M phosphate buffer at a pH of 7.0. The pH of the solution was readjusted to 7.0. The solution was then dialyzed against 8 liters of phosphate buffer (0.005 M) for 48 hours at 1 C. It was clarified by centrifugation to eliminate a slight precipitate which appeared during dialysis. This supernatant solution was then passed through a DEAE-cellulose ion exchange column (dimensions 41 x 2.5 c.) which had been equilibrated with 0.005 M phosphate buffer at a pH of 7.0 and which had 0.5 m. eq. per g. of basic groups. The column was washed with the buffer and the wash was collected in fractions 1 through 10. The intrinsic factor adsorbed on the column was eluted by continuous introduction into the column of 0.1 molar Na HPO plus 0.5 molar NaCl into a constant volume reservoir containing 100 ml. of 0.005 molar phosphate buffer at a pH of 7.0. The volume of each fraction was 6.0 ml. and the flow rate was 1.5 ml. per minute. The experiment was carried out at 25 C. The bulkof the intrinsic factor was removed in fractions 42 to 84 comprising fractions in pools A A and A The fraction pools, A comprising fractions 1 to 10, A comprising fractions 42 to 46, A comprising fractions 47 through 53 and A comprising fractions 54 through 84, were each dialyzed against distilled water at 1 C. for 48 hours and lyophilized. The Weights of the product from these fractions were as follows: A 1.50 g., A 0.36 g., A 0.56 g., A; 1.20 g. The total recovered intrinsic factor therefore amounted to 3.62 gm. or approximately 36% of the starting material. The material which was not adsorbed on the column was found to be inactive. original 10 g. material are lost on dialysis, the recovered fractions account for most of the non-dialyzable solids. The clinical activity of each of the adsorbed fractions was about 12.5 mg. per U.S.P. unit.

Example III Fraction A; which contained the bulk of the activity of the preparation of the previous example was rechromatographed on a carboxymethyl cellulose ion exchanger using gradient elution at a pH 5.4. Such a procedure Since 60% of the solids of the utilizing carboxymethyl cellulose is of no value in the purification of intrinsic factor since the activity appeared both in the unadsorbed and the adsorbed fractions with no purification of the starting material.

Example IV In another similar experiment intrinsic factor prepared as illustrated in Example I was fractionated on a DEAE- cellulose exchanger using gradient elution at a pH of about 5.4. The fraction was adsorbed at a pH of 5.4 and was collected in a single fraction C In the experimental procedures 10 g. of intrinsic factor prepared as indi* cated in Example I were dissolved in 250 ml. of 0.01 M acetate buffer at a pH of 5.4. The solution was dialyzed against 4 liters of the acetate buffer for 48 hours at 1 C.

It was clarified by centrifugation and the supernatant was thereafter passed through a DEAE-cellulose ion exchange column which was equilibrated with 0.01 M acetate buffer at a pH of 5 .4. The column was washed with the buffer and the wash was collected in fractions through 15 (each fraction containing 25 ml.). The gradient was begun at fraction 15 and produced with 0.01 molar acetate buifer at a pH of 5.4 containing 0.5 molar NaCl into a constant volume reservoir containing 200 ml. of 0.1 molar acetate buffer at a pH of 5 .4. The volume of each fraction after fraction 15 was 20 ml. and the flow rate was 1.5 ml. per minute. The experiment was carried out at 25 C.- Fractio-n pools 2 to 13, fraction C and fractions25 through 38, fraction C were dialyzed against distilled water at 1 C. for 48 hours and thereafter lyophilized. Fraction C weighed 2.1 g. and fraction C weighed 1.9 g. The results of the testing of these materials are sum= marized in Table I. The unadsorbed fraction C was not assayed since in a similar experiment carried on a smaller scale, the unadsorbed fraction was inactive. The calcu-- lated U.S.P. activity of C is about 9 mg. which indicates the purification of intrinsic factor of about 1.5 to times over the dialyzed preparation of Example I. There ap-' pears to be a slight loss in the units of activity in this procedure. The intrinsic factor (C obtained after lyophilization was found by urinary excretion tests to be clinically active in daily oral doses of approximately 9 mg, approximately /3 of the amount required to produce a satisfactory response of any of the previously known preparations of intrinsic factor. Fraction C was rechromatographed on DEAE-cellulose using gradient elution at pH 4.7, which is illustrated in the following Example V.

Example V 500 mg. of intrinsic factor preparation C obtained as indicated in Example IV was dissolved in 10 ml. 0.01 molar acetate at a pH of 4.7. The solution was dialyzed in 200 ml. of the acetate buffer for 48 hours at 1 C. It was then passed through a DEAE-cell-ulose column which had been equilibrated with 0.01 molar acetate buffer at a pH of 4.7. The column was washed with the same buffer solution and the gradient was begun at fraction 15 and was produced with a 0.01 molar acetate buifer containing 0.5 molar NaCl. The fraction volume was 6.0 ml. and the flow rate 0.75 ml. per minute. The experiment was carried out at 25 C. Fraction D contains fraction pools 3 through 6, fraction D contains fraction pools 7 through 11, fraction D contains fraction pools 22 through 24, and fraction D contains fraction pools 25 through 29. Each of these fractions were dialyzed against distilled water at 1 C. for 48 hours and lyophilized. Fraction D was found to weigh 138 mg, D weighs 54 mg, D weighs 50 mg, and D Weighs mg. The bulk of the activity was not adsorbed at this pH and the procedure resulted in no increase in activity in the most potent fraction collected.

All the materials resulting from the experiments described in Examples I to V were evaluated for clinical potency according to procedures described in an article entitled Preparation of Partially Purified Porcine Intrinsic Factor, K. C. Robbins and Jane Shields, Proc. Soc. Exper. Biol. and Med, vol. 95, pp. 781-785 (1957).

While in the foregoing specification and examples the invention has been described in specific detail, it will be understood that the invention is susceptible of many variations and modifications, all of which are within the spirit and scope of the invention and of the appended claims.

I claim:

1. A process for the purification of an intrinsic factor preparation comprising the steps of forming an aqueous solution of said intrinsic factor preparation at a pH of about 5.4 and having an ionic strength of from about 0.01 to 0.05; dialyzing said solution; clarifying said solution by centrifugation; contacting said solution with a diethylaminoethyl ether of cellulose ion exchanger equilibrated at a pH of about 5.4 whereby intrinsic factor is adsorbed from said solution on said ion exchanger; eluting said intrinsic factor from said ion exchanger with an aqueous eluant solution having an ionic strength of about 0.5; dialyzing the eluate which contains purified intrinsic factor; and lyophilizing said eluate to obtain intrinsic factor of enhanced potency.

2. A process for the purification of an intrinsic factor preparation comprising the steps of forming an aqueous solution of said intrinsic factor preparation at a pH of about 7.0 and having an ionic strength of from about 0.01 to 0.05; dialyzing said solution; clarifying said solution by centrifugation; contacting said solution with a diethylaminoethyl ether of cellulose ion exchanger equilibrated at a pH of about 7.0 whereby intrinsic factor is adsorbed from said solution on said ion exchanger; eluting said intrinsic factor from said ion exchanger with an aqueous eluant solution having an ionic strength of about 0.8; dialyzing the eluate which contains purified intrinsic factor; and lyophilizing said eluate to obtain intrinsic factor of enhanced potency.

3. In a process for preparing intrinsic factor of enhanced potency wherein the starting material is a clinically active intrinsic factor preparation, the steps of contacting a solution of intrinsic factor starting material with a diethylarninoethyl ether of cellulose ion exchanger equilibrated at a pH within the range of from about 5.4 to about 7.0 whereby intrinsic factor is adsorbed on said diethylaminoethyl ether of cellulose ion exchanger, said solution being at a pH within the range of from about 5.4 to about 7.0 and having an ionic strength of not more than about 0.1, and eluting said intrinsic factor from said diethylaminoethyl ether of cellulose ion exchanger with an aqueous solution having an ionic strength within the range of from about 0.3 to 1.0 whereby an aqueous solution of intrinsic factor of enhanced potency is obtained.

4. A process for preparing intrinsic factor of enhanced potency wherein the starting material is clinically active intrinsic factor comprising the steps of forming an aqueous solution of intrinsic factor starting material, adjusting the pH of said solution to approximately 5.4, adjusting the ionic strength of said aqueous solution to less than 0.1, dialyzing said solution against a sodium acetate buffer, clarifying said solution by centrifugation, contacting said solution with a diethytlaminoethyl ether of cellulose ion exchanger equilibrated at a pH of about 5.4 whereby the intrinsic factor from said aqueous solution is adsorbed on said on exchanger, eluting said purified intrinsic factor from said ion exchanger with an aqueous solution having an ionic strength of about 0.5, dialyzing said eluate and lyophilizing the resulting solution of purified intrinsic factor.

5. A process for the purification of intrinsic factor comprising the steps of forming an aqueous solution of intrinsic factor; dialyzing said solution; clarifying said solution by centrifugation; contacting said solution with a diethylaminoethyl ether of cellulose ion exchanger equilibrated at a pH within the range of from about 5 to 8 whereby the intrinsic factor from said solution is adsorbed on said ion exchanger, said solution being at a pH of from about 5 to 8 and having a maximum ionic strength ranging from 0.3 to 0.6 according to the pH of the solution, said diethylaminoethyl ether of cellulose ion exchanger being equilibrated at a pH and ionic strength approximately equivalent to the pH and ionic strength of said solution of intrinsic factor; and eluting said purified intrinsic factor from said ion exchanger with an aqueous solution of a salt at an ionic strength of more than 0.3, and dialyzing the resulting eluate.

6. In a process for the purification of intrinsic factor, the steps of contacting a solution of intrinsic factor with a diethylaminoethyl ether of cellulose anion exchanger equilibrated at a pH Within the range of from about 5 to about 8 whereby intrinsic factor is adsorbed on said anion exchanger, said solution being at a pH within the range of from about 5 to about 8 and having an ionic strength of not more than 0.3, and eluting said intrinsic factor from said anion exchanger with an aqueous eluant solution having an ionic strength in excess of 0.3 whereby an aqueous solution of intrinsic factor of enhanced potency is obtained.

7. In a process for the purification of intrinsic factor, the steps of contacting a solution of intrinsic factor with a diethylarninoethyl ether of cellulose anion exchanger equilibrated at a pH within the range of from about 5 to about 8 whereby intrinsic factor is adsorbed on said anion exchanger, said solution being at a pH within the range of from about 5 to about 8 and having an ionic strength within the range of from 0.01 to 0.05, and eluting said intrinsic factor from said anion exchanger with an aqueous eluant solution having an ionic strength in excess of 0.3 whereby an aqueous solution of intrinsic factor of enhanced potency is obtained.

References Cited in the file of this patent UNITED STATES PATENTS Robbins Nov. 13, 1956 OTHER REFERENCES

Claims

1. A PROCESS FOR THE PURIFICATION OF AN INTRINSIC FACTOR PREPARATION COMPRISING THE STEPS OF FORMING AN AQUEOUS SOLUTION OF SAID INTRINSIC FACTOR PREPARATION AT A PH OF ABOUT 5.4 AND HAVING AN IONIC STRENGTH OF FROM ABOUT 0.01 TO 0.05, DIALYZING SAID SOLUTION, CLARIFYING SAID SOLUTION BY CENTRIFUGATION, CONTACTING SAID SOLUTION WITH A DIETHYLAMINOETHYL ETHER OF CELLULOSE ION EXCHANGER EQUILIBRATED AT A PH OF ABOUT 5.4 WHEREBY INTRINSIC FACTOR IS ADSORBED FROM SAID SOLUTION ON SAID ION EXCHANGER, ELUTING SAID INTRINSIC FACTOR FROM SAID ION EXCHANGER, ELUTAQUEOUS ELUANT SOLUTION HAVING AN IONIC STRENGTH OF ABOUT 0.5, DIALYZING THE ELUATE WHICH CONTAINS PURIFIED INTRINSIC FACTOR, AND LYOPHILIZING SAID ELUATE TO OBTAIN INTRINSIC FACTOR OF ENCHANCED POTENCY.