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llMlliI United States Patent Office 1 2,952,585 IMMUNIZING AGAINST AND TREATMENT OF DISEASES John H. Heller, Wilton, Conn., assignor to New England last|tufa for Medical Research, Rldgefield, Conn., a corporation of Connecticut No Drawing. Filed June 19, 1957, Ser. No. 666,/76 13 Claims. (CI. 167--78) This invention relates to improvements in method and means for immunizing mammals and birds against various infections to which they are susceptible and for treating mammals and birds for alleviating diseases, It has long been known that immunity, for instance, may be developed in nlammals and birds against various toxins by introducing a corresponding toxoid into the body. Numerous efl'cctive toxoids arc known and are being successfully used. Various antigens and allergens, hormones and therapeutic agents are also being success- fully used in this way for the prevention and treatment of various infections, diseases or dysfunctions. Though various specifics arc known and in general use for the treating of various conditions and for immuniz- ing against them, frequently the effectiveness of the spe- cific is of short duration, so that repeated treatment is necessary, or else the agent must be administered in doses of such size as to cause undesirable side-effects. It is a purpose of my present invention to increase or prolong the effectiveness of toxoids, antigens, antiallergic agents, hormones, therapeutic agents and the like, in gen- eral, in preventing or alleviating infections and diseases, and to accomplish this with a materially reduced dosage of the agent. This purpose is accomplished by my present invention in accordance with a new principle of depot medication whe, chy there is induced significantly greater and longer lasting immunity, and in accordance ~'ith which I ad- minister the toxoid, or other selected agent, in a state of adsorption in or on carbon black. It has beeen gcnerally recogni?ed that a primary de- fense of mammals and birds against infection is the retie- uloendothelial system, commonly referred, to as the RES, and so designated herein for brevity. The RES is composed essentially of phagocytic cells, located primarily in the liver, spleen and bone marrow, but to a laser (x- tent scattered throughout most of the body as fixed :or "wandering" cells. One of the functions of these cells is .to clear the blood stream of bacteria, and other par- ticdlate material, which may be present in the blood stream or in the lymph system, by phagocytosis. Where infectious foreign matter, bacteria for instance, enters the body of a host, these phagocytic cells of the RES in some way envelop and destroy the bacteria, thus rendering the bacteria impotent. The development of antibodies against the invading bacteria appears to be closely related to, and a sequel of, this phagocytosis, in some way not fully understood. In the extensive study of the RES, it has been estab- lished that where an appropriate aqueous suspension of colloidal carbon black is in cctcd intravenously into a host, this foreign particulate matter is quickly taken-up from the blood stream by the phagocytic action of the RE cells, i.e. the rcticulocndothelial cells of the RES. The particles are phagocytized preferentially by RE cells in the liver and by the spleen cells, as well as other loci, according to particle size and electro-charge. I utilize this selective phagocytic activity of the RE 2,952,585 Patented Sept. 13, 1960 i i it i i Jill i ill Jll Ill "-- 2 cells toward carbon black to direct selectively an anti- gun, toxoid, hormone therapeutic agent or the like to that portion of the body where it is most effective by adsorb- ing the antigen or the like in or on carbon black par- ticles prior to their injection, so that the agent is con- veyed by the carbon black particles directly to the RE cells and is more quickly and more effectively taken up by the ceils, together with the carbon black particles. A major limiting factor in the development of any new therapeutic agent is that the entire body must be "drenched" with the substance so that the cells or organ which one specifically desires to reach will have an ade- quate amount. The side-effects, toxic or otherwise, of "drenching" the entire body are a major limiting and interdicting obstacle to the use of innumerable otherwise useful agents. Critical aspects of this invention are the particle size, or range of particle sizes, the charge, adsorptivity, state of aggregation of, and suspending agent for, the colloidal carbon black. "[he adsorbed agent must not be too light- ly adsorbed and hence lost before it reaches the RE cells,- neither must it be too tightly adsorbed and hence un- available for its function. Colloidal carbon black hav- ing a particle diameter of from 1 to 100 millimicrons (Paint, Oil and Chemical Review, vol. 115, No. g, page 22, April 10, 1952) is admirably suited as far as its range of particle sizes is concerned. In the previous investigations of the RES involving the injection of colloidal carbon, difficulties have been ex- perienced due to the accumulation of the carbon in the lungs. Early experimenters used India ink as their source of carbon black. It was not known until 1953 that the shellac and other gums present in the ink were not toler- ated by the body. By careful control of particle size, by choice of original material and by ~ltracentrifuglng and avoiding excessive agglomeration of the colloid in the environment of plasma or other body fluids, I can avoid any significant deposition of carbon black in the lungs and selectively bring about its deposition largely in the liver, spleen and bone marrow. In the presently preferred aspect of the invention, the carbon blafk having the toxoid, antigen or the like l~- &~,~ the r~n is injected parenterally in suspension in a physioio'g]~:al ~aque~ous~sus~nsion which must, of course, be free from pyrogens which might cause shock, chills or high fever. The colloidal suspension must be so pre- pared that the particles retain an electro-negative charge in the body fluids, and do not agglomerate significantly, in the plasma, lymph or other body fluids. It is also es- sential that the toxoid, antigen or the like be adsorbed in or on the carbon black particles in such a way that it is retained thereby until phagocytosis of the particle has occurred. Various procedures may be employed in the preparation of the colloidal suspension so long as de- naturation of the antigen or the like is avoided by the selected procedure. It is essential that the adsorption force be sufficiently great to prevent desorption en mute to the RE cells. On the other hand, it must not be so great as to prevent 60 desorption once the particle is in the RE cell or, in/he case of macmmolecules such as a protein, the adsorptive force must not be so great as to uncoil or otherwise de- nature the protein. The size of the carbon black particles thereof may be 65 varied, depending primarily upon the location in the body to which it is desired selectively to direct the agent. They may be as small as 5 millimlcrons, or even 2 millimicrons. Usually the size of the largest particle should not exceed about 80 millimicrons. I have found 70 that, where the particle size of the carbon black colloid does not exceed this maximum, and excessive agglomera. tion after injection is avoided, the carbon black Im no 10 15 go ~5 30 $5 4o 45 50 55

g,osg,585 3 noticeable toxic effect and may be administered in sur- prisingly large amounts without ill effect on the host, providing, of course, that the suspension used for intra- venous injection is sterile, isotonic, and free from pyrogens and other contaminants, as indicated above. 5 The colloidal carbon used in accordance with the pres- ent invention, herein designated "carbon black" is not to be confused with the various chars produced by de- structive distilling of animal or vegetable matter. It is produced by the pyrolysis of liquid or gaseous hydro- 10 carbons and is substantially free from mineral matter. The proportion of the toxoid, antigen or the like ad- sorbed on the carbon black may vary over a considerable range and will usually be determined by the adsorptive capacity of the particular carbon black for the particular 15 agent adsorbed. The amount of carbon black admin- istered will, in large measure, depend upon the amount of the agent adsorbed thereon. In general, the carbon black having the agent adsorbed thereon or therein to be administered by injection will be prepared in colloidal 20 suspension in a physiological isotonic solution. The sus- pension may be administered in appropriate amounts, usually proportionate to the body weight, by injection intravenously, subcutaneously or intraperitoneally. It may also be administered intramuscularly, or as a trans- 25 fusion, or intravenous feeding or by inhalation in a dry powder form or aerosol. The invention has been applied effectively, without deleterious side-effects, to lower animals and birds and is believed to be effective in the treatment of human be- 30 ings, though conclusive clinical evidence concerning the latter is not presently available. I,arge doses of carbon black have been injected intra- venously into human beings with no toxic slde-effects. But in my controlled testing of immunity, in which I 35 have used botulinum toxin, the most virulent protein toxin known to man, I have carried out the tests on lower animals, as is customary in this field of research, before proceeding with tests on human beings. As will be described below, protection of such lower animals by 40 the above-described methods has been obtain.ed. For example, I have immunized mice against this botulim, m infection, which is also a serious problem with respect to duels, pheasants and other fowl, both domestic and wild. 45 As previously noted, botulinum is recognized to be one of the most toxic, deadly poisons known. Effective botulinum toxioid has been prepared by treating the botulinum toxin with formaldehyde. This toxoid has been used effectively in accordance with the present in- 50 vention in developing in mice an immunity for this deadly toxin. More particularly, a suspension of carbon black, of a particle size within the range of 18-25 millimicrons, and having the botulinum toxoid adsorbed thereon, was pre- 5b pared in a physiological solution and, at intervals one week apart, 20-gram albino mice were twice injected in- travenously with ~i0 cc. of the suspension containing 0.7 milligram of the carbon black having deposited thereon 70 micrograms of the protein toxoid. No ill effects 60 were apparent. One week following the last injection, the mice were given ten 100% lethal doses of the botulinum toxin without ill effect, while the controls died within 30 minutes, showing that by this method there was developed within the mice an effective ira- 65 munity for this deadly poison. The size and potency of the injection should be varied depending upon the weight of the mammal or bird treated. Where a 20-gram mouse is immunized by ad- ministering the toxoid protein in the customary manner, 70 i.e. not adsorbed on carbon black, 8.5 milligrams of the toxoid would normally be required to establish compara- ble immunity. In the treating of wild fowl, e.g. ducks and pheasants, care must be exercised to avoid domesticating them. 7~ venously. 4 For this reason, handling and confining of the fowl must be reduced to a minimum. Preferably the immunizing should be effected by a single handling. By my present invention, this may be accomplished by a single injectieya of the toxoid. In the immunizing of pheasants, for instance, against Clostridium botulinum, type C, the toxoid is with ad- vantage adsorbed on carbon black and the fowl Injected with an aqueous suspension thereof. To develop in a 4-pound fowl, for instance, a hotulinum immunity by administering the toxoid in the conventional manner, re- quires 275 milligrams of the toxo|d. In accordance with my present invention, comparable immunity can be af- fected by intravenously injecting the fowl with a suspen- sion of carbon black, in the particle size range of 18-25 millimicrons, in physiological, isotonic solution, on which there is adsorbed 1Ao0 that amount of the toxold. The invention will be further illustrated by the fol- lowing specific example of immunizing against botulinum infection, it being understood that the invention is not restricted to the particular aspects thereof illustrated. Clostridium botulinum type C toxin is harvested from a ten-day culture of CI. botulinum, type C, grown within a cellophane dialyzing sack, the medium used being cleared cornsteep liquor enriched with 6.5% glycerin and 0.1% CaCI2. The crude toxin is freed from the organisms by centrifugatlon and subsequent filtration. The toxin is then concentrated to ~Ao of its original value by lyophilization. On dialysis of the concentrate, the toxin is precipitated. The precipitate is dissolved in a sterile sodium chloride aqueous solution (0.9%) and ad- justed to pH 4.5 at which point the toxin flocculates. This toxin so prepared contains a 2,000,000 MLD per 0.1 cc., or, for a 20-gram mouse, and is shown by chemi- cal analysis to contain 0.1 rag. of protein nitrogen per ml. To produce the toxoid, formaldehyde is added to the toxin until the concentration of the formalin is 0.5%. The toxin is then incubated at 37' C. for 10 days. After that period, the resultant toxoid is tested in mice for residual toxicity. If the mice survive injections of I mi. of the undiluted toxoid daily for 4 days, the toxold is ready for use. A colloidal suspension of carbon black of a mean particle size of 25 millimicrons and containing 90 mg. of solid material per ml. is diluted with water to contain 20 mg. of solid per ml. An aliquot of the purified toxoid is added to 5 times the volume of the carbon black sus- pended at pH 6.8, at which pH the carbon black re- mains in colloidal suspension. The mixture is then left at 4° C. for 12 to 16 hours. The toxoid-carbon black mixture is then adjusted to pH 2, causing the carbon black agglomerates to drop out of the suspension. The mixture is then centrifuged at 12,000 r.p.m, for 30 minutes (24,500 g.), and the resultant supernate is do- canted and saved. The sediment is resuspended in sodium phosphate buffer solution at a pH of 6.8. The suspension becomes colloidal again at this pH and does not sediment. The above procedure of precipitating, separating and resuspending is repeated three times. After each wash- ing, the supernate is tested to determine the amount of toxoid remaining in the supernate. Ordinarily the second and third washings contain no toxoid. The final suspen- sion of the carbon black adsorbed toxoid in the sodium phosphate buffer at pH 6.8 is filtered through a clarifying type sintered glass filter and analyzed for the amount of toxoid present by measuring the amount of protein present on the carbon black. The suspension is now ready for injection. Mode el administration o! the toxold Two routes of administration are possible. The first is conventional~intramuscularly. The second, since the amount of carbon to be injected is innocuous, h intra. At present, the recommended procedure with

9,952,585 5 botulinum toxoid is at least 6 injections over a period of two to three weeks. But, since, in accordance with my invention, the carbon black retains the adsorbed toxoid and releases it gradually in the RES, two injec- tions of 1 ml. each, spread one week apart will usually be found sufficient. By the intravenous route, the carbon black toxoid is very rapidly taken up by the reticuloendothelial cells of the liver and speen, thus forming a depot source of antigenic material without the need for replenishing by additional inoculation. Te,st /or immunity in the immnnized mammals or birds Two types of challenge may be used. One is by the injection of graded lethal doses of the toxin into the immuni.-,ed mammal or bird to determine the highest dose they can tolerate. The second is by feeding the mammals and birds graded amounts of toxin added to their drinking water. The latter is believed to be a better index of immunity since it involves the natural route of botulinum intoxication. I claim: I. As a composition of matter, colloidal carbon black having adsorbed thereon an agent selected from the group consisting of toxoids, antigens, anti-allergic agents, hormones, natural and synthetic, and therapeutics. 2. As a composition of matter, colloidal carbon black of a mean particle size within the range of 2-25 milli- microns and having adsorbed thereon an agent selected from the group consisting of toxoids, antigens, anti- allergic agents, hormones, natural and synthetic, and therapeutic,~. 3. A stable suspension, in physiological, isotonic solu- tion, sterile and pyrogen free for intravenous injection, of colloidal carbon black of a particle size within the range of 2-25 millimicrons and having adsorbed thereon an agent selected from the group consisting of toxoids, antigens, anti-allergic agents, hormones, natural and syn- thetic, and therapeutics. 4. ]'he st,spension of claim 3 in which the particle size of the carbon black is within the range of 18-25 miUi- ' microns. 5. A stable suspension, in physiological, istonic solu- tion, sterile and pyrogen free for intravenous injection, of colloidal carbon black of a particle size within the range of 18-25 millimicrons and having adsorbed there- on an agent selected from the group consisting of toxoids, antigens, anti-allergic agents, hormones, natural and syn- thetic, and therapeutics, the liquid phase of the suspen- sion being sttbstantially free from said agent. 6. A stable suspension, in physiological, isotonic solu- tion, sterile and pyrogen free for intravenous injection, of colloidal carbon black of a particle size within the range of 18-25 millimicrons and having adsorbed thereon an agent selected from the group consisting of toxoids, antigens, anti-allergic agents, hormones, natural and svn- 6 thetic, and therapeutics, the liquid phase of the suspen. sion being substantially free from said agent and the carbon black particles with the agent adsorbed thereon having electro-negatively charged surfaces. 5 7. A stable suspension, in physiological, isotonic solu- tion, sterile and pyrogen free for intravenous injection, of colloidal carbon black of a particle size within the range of 18-25 miUimicrons and having botulinum toxoid adsorbed thereon. l0 g. A stable suspension, in physiological, isotonic solu- tion, sterile and pyrogen free for intravenous injection, of colloidal carbon black of a particle size within the range of 18-25 millimicrons and having botulinum toxoid adsorbed thereon, the liquid phase of the suspension being 15 substantially free from the toxoid. 9. In the method of administering toxoids, antigens, anti-allergic agents, hormones, therapeutic and like agents to mammals and birds, the steps of adsorbing the agent on non-toxic colloidal carbon black particles, and intro- 20 ducing the agent into the body in the state of adsorption on the carbon black. 10. The method of claim 9 in which the agent is ad- sorbed on carbon black particles of a particle size within the range of 18-25 millimicrons, 25 11. Method of conveying toxoids, antigens, anti- allergic agents, hormones, therapeutic and like agents, selectively, to the RE cells of mammals and birds which comprises adsorbing the agent on non-toxoid colloidal carbon black particles, suspending the particles in physio- 30 logical, isotonic solution and parenterally injecting the suspension into the body. 12. The method of claim 11 in which the agent is ad- sorbed on carbon black of a particle size within the range of 2-25 millimicrons. 85 13. The method of immunizing mammals and birds against bacterial toxin which comprises de-toxifying the toxin, adsorbing the de-toxified toxin on carbon black particles of a particle size within the range of 2-25 milli- microns, suspending the carbon black particles having 40 the de-toxified toxin adsorbed thereon in the physiological isotonic solution and parenterally injecting the resulting colloidal suspension into the body. References Cited in the file of this patent 45 UNITED STATES PATENTS 2,143,088 Rockwell .............. Jan. 10, 1939 2,897,115 Dullaghan .............. July 28, 1959 OTHER REFERENCES 50 Kato et al.: Internfl. J. Leprosy, vol. 23, No. 4, pp. 406-417, 1955 (as cited in April 1947, Biol. Abstr. 11462 and 11463). Rossman: Ind. and Eng. Chem., vol. 35, No. 9, Sep- 55 temher 1943, pp. 972-976. Wiegand: India Rubber World, December 1941, p. 270. Development of Carbon Black: 1945, p. 90.

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United States Patent O/t ce 2,787,579 Patented Apr. 2, 1957 I 2,787,S79 $HAFED ACTIVATED CARBON MEDICINAL COATED WITH CARBOXYALKYL ETHER Thonis Johannes van der Weel, Zandvoort, Netherlands, assignor to N. V. Algemeene Norit MaatschappiJ, Amsterdam, Netherlands, a corporation of the Neth- erlands No Drawing. Application June 15, 1953, ~rlal No. 361,869 Claims priori/y, application Netherlands June 17, 1952 4 Claims. (CI. 167---82) ' It is a well-known fact that gastric and intestinal dis- turbances, nutritional poisonings and the like can be successfully combated by means of activated carbon, which to this end is often taken in the form of tablets, such as the well-known medicinal Norit tablets. Practical experience has shown, however, that these tablets present some drawbacks. In the first place they tend to give off powder, so that they blacken hands and mouth. Furthermore the swallowing of these tablets may be found to be difficult, especially for children. This is, apparently, due to the adsorptive properties of the activated carbon. In many cases the gullet veristalsis is not capable of immediately conveying the tablet to the stomach, so that the patient has the disagreeable feeling that it "sticks halfway in his throat." With other tablets, which are not composed of activated carbon, these draw- backs are not encountered at all, or only to a very slight degree. The drawbacks in question have been met by dredging the activated carbon, but it is, of course, undesirable to add to a substance, whose activity is based on its surface- active properties, extra large amounts of sugar. According to the invention it is now possible to pro- duce tablets, granules and similar moulded articles (which for convenience sake will be called tablets hereinafter) from activated carbon, which do not at all show the above-mentioned drawbacks, by coating the tablets with a thin film of carboxymethyl cellulose or of a similar carboxyalkyl ether of cellulose. It appears that such a film substantially entirely prevents the carbon particles from giving off powder when ,one touches the tablets, while as a result of the hydrophilic properties said film will rapidly absorb water when the tablets are taken, so that these will get a slippery surface and can easily pass the throat and the gull©t. The carboxyalkyl cellulose film may be very thin, and in many cases its thickness is less than 0.01 ram. Very good results can be obtained, for instance, by applying on a carbon tablet of a normal size and of a weight of e. g. 500 milligrams an amount of carboxymethyl cellu- lose of e. g. 1-2 milligrams, which corre,ponds to a thickness of a coating of about 0.003-0.006 nun. This is a very favorable drcmnstance, as it is undesirable to 2 add considerable mounts of colloidal substances to the activated carbon. It is very surprising that it is thus possible to coat a surface consisting of more or less porous, strongly ad- sorptive carbon particles with an extremely thin film and to obtain in this manner a "closed" surface, while unlike the case of dredging, only very small amounts of foreign substances are used and the adsorptive power does not decrease in any respect. 10 The film can be applied by treating the finished tablets with an aqueous solution of the carboxyalkyl cellulose, preferably by spraying this solution on the tablets and drying the latter, if necessary. The carboxyalkylcellulose preferably used is carboxy- 15 methylcellulose. C.arboxyalkylcelluloses of a similar character which may also be used according to the in- vention are those which are soluble in water to a viscous solution, e. g. carboxy ethyl cellulose and carboxy propyl cellulose. 9.0 I claim: 1. A medicinal preparation comprising a shaped prod- uct consisting essentially of activated carbon, said shaped product being coated with a thin hydrophilic film to substantially eliminate its tendency to give off carbon 25 particles upon contact with other surfaces, said film con- sisting of water-soluble carboxyalkyl ether of cellulose and being of a thickness of less than 0.01 ram., the amount of said carboxyalkyl ether of cellulose being such that the adsorptive properties of said carbon are 30 not materially decreased. 2. A medicinal preparation as defined in claim I, wherein said carboxyalkyl ether of cellulose is carboxy. methyl-cellulose. 3. A medicinal preparation as defined in claim 1, 35 wherein said film thickness is from about 0.003 to 0.006 mm. 4. A medicinal preparation comprising a shaped prod- uct consisting essentially of activated carbon, said shaped product being coated with a thin hydrophilic flint to sub- 40 stantially eliminate its tendency to give of/carbon par- ticles upon contact with other surfaces, said film con- sisting of carboxymethylcellulose and being of a thick- ness of from about 0.003 to 0.006 ram., the amount of said carboxymethylcellulose being such that the ad- 45 sorptive properties of said carbon are not materially decreased. 50 t;5 References Cited in the file of this patent UNITED STATES PATENTS 1,542,006 Sauer ................. June 16, 1925 1,552,549 Eicher ................ Sept. 8, 1925 2,196,768 Hlatt .................. Apr. 9, 1940 2,502,809 Vogelsang .............. Apr. 4, 1950 OTHER REFERENCES Hollabaugh et al.: Ind. and Eng. Chem., October 1945, p. 945.

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/ Patented Jan. 28, 1941 / 2,229,876 UNITED STATES PATENT OFFICE 2,229,876 MANUFACTURE OF A COMPLEX ADSORBATE Ilenry A. Smith, Berkeley, Calif., assignor, by mcsne assignments, to National Oil Products Co., Harrison, N. J., a corporation of New Jersey No Drawing. Application August 13, 1937,' Serial No. 158,953 6 Claims. (CI. 167--$1) • • This invention relates to the manufacture of a precipitates and floats to the surface of the liquid, vitamin adsorbate containing at least two vita- from whence it is readily removed, dried and rains which adsorbate is of a high plural vitamin ground. potency per unit volume. The residual liquor, now free from casein and 6. In manufacturing vitamin adsorbates contain- albumen, Is concentrated for milk sugar recovery. ing two or more vitamins, the usual practice is to Either before or after the concentration step a adsorb a vitamin on one adsorbent and then corn- preliminary vitamin G or lactoflavln adsorbate blne this with another adsorbent carrying an- can be secured.. For ,example, 40 pounds of other vitamin. While the result is satisfactory fuller's earth is added to 1000 gallons of liquid t0. in that a plural vitamin content is contained in prior to concentration. This mixture is agitated the mixture, the mass is of a low vitamin potency for about 9.0 minutes and then filtered, the filtrate per unit volume, as can be readily appreciated, being returned to the plant, where it goes Into the because of the use of separate masses of adsor- next step, vacuum' evaporation, for milk sugar .. bent for each vitamin. I have dlscovered that production. The adsorbate removed from th~ 151 adsorbent materials selectively adsorb vitamins filter press is washed, dried and ground in a ball and that even though an adsorbent has practical- mill, this concentrate containing approximately ly exhausted its adsorbent capacity for a given 60 gamma of riboflavin per gram. vitamin, this adsorbent still has the capacity to Instead of the foregoing, the liquid, free from adsorb an appreciable quantity of another vita- casein and albumen, can be concentrated in 20 rain. For example, I am able to successfully in- vacuum evaporators. Prior to transportation of £0 corporate vitamins G and B~ on the same ad- the material to the milk sugar crystallizer, the sorbcnto thus reducing materially the unit volume material is contacted with fuller's earth to secure of the adsorbate, a first vitamin G adsorbate. This process hos the This selective adsorption enables a compound advantage that the color of the subsequent sugar U vitamin adsorbate to be manufsctured In which precipitated from the liquid is improved, a whiter £5 the individual vitamins are present in selected sugar being obtained on the first precipitation values and at a'high unit volume potency. For when the fuller's earth treatment follows the example 1500 gamma of riboflavin (also termed concentration step. lactoflavin and vitamin G) per gram can be It is also possible to" recover the vitamin G by~ |0 placed on an adsorbate per gram thereof while a adsorption on fuller's earth from the mother~30 .comparable high Bz value per gram can also be liquor obtained from the lactose crystallizersl placed on the adsorbate. The milk sugar when delivered from the evapora- As suitable adsorbent materials, I can use tors is in the form of a heavy syrup. This ma- fuller's earth, bentonite, and other adsorbent 'terial is transferred to crystallizers in which the $~l earths, _~ctivated carbon and the like. These sugar is slowly crystallized. These crude crystals: U materials operate-suecessfuliy in the liquid phase are separated from the mother liquor in the cen- and in the presence of water, trifuge. This mother liquor is reconcentrated Hereinafter I have disclosed the manufacture and again crystallized to give as high a recovery of an adsorbate containing lactoflavin and vlta- of sugar as is possible. The mother liquor ob- 40 rain B~. Of course, any other vitamin which is tained by centrifuging the raw crystals is of a 40 adsorbed on these materials can be substituted dark brown color containing some sugar and the .in place of these, these vitamins being mentioned minerals originally in the milk and any addi- as examples, tlonal salts added in the proce~ of manufacture A suitable source of lactoflavin is skim mtlk together with the vitamin G contained in the 45 and its products. The lactoflavin manufacturing milk. It is possible to treat this mother liquor 45 operation Of this invention includes precipitation with fuller's earth and secure an adsorbate con- of the casein from the skim milk by the addition raining up to 9.00--300 gamma of riboflavin'per " of an acidifying medium as hydrochloric, sulfuric . grani, i or lactic acid. This causes casein coagulation, After the adsorbate has been prepared by any 60 the casein being separated from the remaining one of the foregoing processes, i~ should be 50 liquid, washed, dried, and ground for market, thoroughly washed to free it from all occluded Albumen is removed by adjtmting the pH of the milks, salts, or other form contaminates picked up remaining liquid to approximately 6.8, calcium in the process of manufacture. The adsorbate hydroxide being used for this, and the material may then be used wet as is, or dried, ground and 65 thereafter Is brought to a boil. The albumen stored for future use. /~6 ~li, • .. " ~ !'~i

2 This adsorbate0 containing vitamin G, can be treated with a liquid containing vitamin B~, or any other water soluble vitamin which has the property of being adsorbed on fuller's earth. The 5 resultant prodt.~ct is an adsorbate containing two or more water soluble vitamins of the B complex. To make an adsorbate containing vitamin G in a higher concentration than is po~ible by ad- sorption of riboflavin from crude substances by I0 clay, the following method may be employed. This enables a low potency adsorbate (50 to 250 gamma of riboflavin per gram) to be utilized in Producing an adsorbate containing up to aP- proximately 3000 gamma per gram. This rea- l6 terial can be used as such or for making a com- bination adsorbate by an additional adsorbate with vitamin B, Be, etc. The adsorbate is elutriated with a suitable solvent such as 70% (60% to 90%) acetone, or gO with pyridine; 250 pounds of the adsorbate con- taining approximately 60 gamma of riboflavin, for example, are mixed with 500 gallons of 70% acetone in a ribbon type mixer. This mix is preferably made by adding approximately 100 g~ gallons of acetone to the mixer; 250 pounds of clay are then added, and the mix is agitated until a uniform pasty consistency results. The balance of the acetone is then pumped into the mixer, after which it is agitated for two hours and then 30 filtered. The filtrate is pumped back to a feed tank, and the cake is returned to the mixer. The second extraction is made by adding 100 gallons of acetone to the mixer. Filter cake is added slowly so that it is broken up in the mixer, .~$; When the mix has a uniform pasty consistency, the balance of the acetone (about 150 gallons) is added and the mixture is agitated for two hours. A filter aid as Filter-cell can be added about 5 minutes before the material is pumPed 40 into the filter, to insure quick fltratlon. The second nitrate is pumped to the feed tank and the cake is discarded. The acetone filtrate is then concentrated, in the absence of light. The still is oPerated to first 45 drive off the acetone and then the water. When the clutriate from the treatment of about 1000 pounds of the clay has been passed into the still, it is concentrated down to as small a volume as possible and drained into a receiving tank. The O0 still is then washed down with water and the wash is drained into the receiving tank. The ma~ terial In the receiving tank is now concentrated under a vacuum to a volume of approximately 24 liters. The 24 liters is then filtered and the 05 filtrate adsorbed on fuller's earth, the 24 liters requiring about 18Y~ pounds. The resulting earth will contain approximately 1500 gamma of ella- rain O per gram. A second adsorption made from this liquid using 6 pounds of fuller's earth will" 00 give a potency of about, 100 gamma of vitamin O per gram. Th~ clay is fl.nally separated from the liquid by filtration after which it is air dried and ground in a ball mill. Thereafter, the high po- tency adsorbate may be used as is or may be 0~ treated with a solution of vitamin B~ or Be or both to make a combination adsorbate, For example: i00 pounds of rice bran, or an equivalent amount of other cereal products con- talning vitamin B~ (thiamine), is mixed with 500 ~$ pounds of water. When thorough mixing has been secured and sufficient time has.been allowed for the thorough penetration of the material by the water, the solid material is then separated by passing the material through a suitable filte~ device. It is desirable to maintain the tempera- ture at approximately 125° F. during the mixing period. The filtrate obtained from the mixture of bran and water carries a suspended solid content of a 5 colloidal nature which is only difficultly sepa- rated. In fact centrifuging and pH adjustment are not effective and the only satisfactory way in which the solids can be ©aimed to coalesce is by heating. The solids are preferably removed be- 10 fore treatment with the adsorbent. The filtrate is usually heated to a temperature between 60 and 100° C. Under these conditions the protein and comparable material in suspension is caused to coagulate and rise to the top of the liquid. 1~ The clear liquid can then be withdrawn from the bottom of the tank, leaving a cake of coagulated material. This clear liquid may now be tlsed as a source of vitamins B~, Be, and the filtrate factors. The cake of coagulated material may 20 either be washed to secure a better yield of vita- min B complex or may be discarded. The dried and ground adsorbate containing |' laotofiavin is now contacted with the described liquid containing vitamin B~. Be, and an absorp- tion of B~ and Be occurs upon the adsorbate which contains lactofiavin. Thereafter, this clay now containing both vitamins adsorbed thereon Is dried and ground. By suitably regulating the amounts of vitamin B Per pound of adsorbate ~0 used, the amount of vitamin B~ adsorbed upon the clay containing the lactoflavin can be regu- lated to provide wherever varlation is desired. I claim: 1. A method for producing a high potency vlta° $45 rain adsorbate of low bulk comprising adsorbing '" on an adsorbent material a vitamin from a solu- tion thereof, separating the adsorbent from the solution, drying the adsorbent, adsorbing a second vitamin on said adsorbent from a solution there- 40 of, seParating the adsorbent from the second solution and drying the same toprovide a vitamin adsorbate having at least two vitamins In an amount greater than the vitamin content repre- senting the saturation value of said adsorbent 45 with respect to' either one of the adsorbed vita- • rains. 3. A method for producing a vitamin adsorbate of high plural vitamin potency per unit volume comprising adsorbing on an adsorbent material 50 vitamin G from a solution thereof, separating the adsorbent from the solution and adsorbing vita- min B thereon from a solution thereof to provide an adsorbent material bearing vitamins B and G in an amount greater than the vitamin content representing the saturation value of said ad- • sorbent with respect to either vitamins B or O. • 3. A method for producing a vitamin adsorbate of high plural vitamin potency per unit volume comprising adsorbing on an adsorbent material 60 vitamin B from a solution thereof, separating the - adsorbent from the solution and adsorbing vita- min O thereon from a solution thereof to provide an adsorbent .materl~l bearing vitamins B and G in an amount greater than the vitamin content e~ representing the saturation value of said ad- ' sorbent with respect to either vitamins. B or O. 4. The process as setforth In claim 2 wherein fuiler's earth is used as the adsorbent. 5. The process as set forth in claim 9. wherein ~O bentonite is used as the adsorbent. 6. The process as set forth In claim 3 wherein ., bentonite is used as the adsorbent, 'HENRY A, SMITH.

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Patented Sept. 29, 1936 Z,OSS,47S UNITED STATES PATENT OFFICE M~4~S Tm~TMimT OF FLUID oxidizing szent in activated ee~tmn from aqueous solutio~ I hsve tow~ that by such method s high eoncentmtJon of oxidisi~ agent may be obtained in the m~, such as to render it hiZhly effective, and that the material is quail- J fled to retain its effecttvonm for extended pe- invention relates to the treatment of fluids for modification or removal of sub~anees carried in them, and to a new material and the method of preparing same for such and other 5 tmeL A general object of the invention is the pro- vision of a new material having eharacteristies qualifying it for industrial use in the oxidation, sterilization or modification of fluids or of sub- 10 stances carried in them in suspension or solution. Another object is the provision of a method for treatment of fluids for oxidation, sterilization or modification of them or their components. 8till another object is the prov~on of a rea- l5 terial and method for the separation and re° covery of solutes by oxidation. Another object is the provision of a method for the separation of oxldizl~ agents from fluidL ~0 ~ another object is the provision of a new oxidising material I~ceptlble of regeneration. Other and further objects will be pointed out or indicated hereinafter or will be apparent to one skilled in the art upon an undemtsnd~ 95 of the invention. It is in many ~ces desirable to separate or convert various oxidizable substances which are carried in solution or in suspension in different fluids. This may be desirable for the purpose 80 of freeing the fluid from such substances or for the recovery of the mzlmtances themselves. For -example, it may be desirable to free water from iron or manganese carried in solution, or to sub- Ject It to sterilizing trcatment for the Imrpose 85 of killing or incapacitating bacteria or other micro-organisms which it may carry. Proce- dures heretofore employed for such p~0 in the treatment of water on a commercial or in. dustrlal scale for example, involve the dosing of 40 the water, as with coagulants, precipltants and bactericidal agents, as practiced for example in the aluminum sulphate, lime and soda, and chlorination metho~ In these various dosing procedures, the success of the treatment and the 45 proper condition of the water are dependent upon accuracy in the dosing and treatment, which ac- curacy it is difficult to maintain. By utiliza- tion of the present invention, all uncertalntiem in and necessity for accurate dosage control are 50 obviated. Described generally, the present invention coro- t prises a new treating material consisting of acti- rated carbon having an adsorbed oxidizing agent. The invention also comprises a method of form- 55 lng such treating material by adsorption of an rlods even when in Contact with liquids. Ttm, material may be of granular, puiveruiemt or other form ~ptinz it for suitable in~ of con- tact with the fluids or other substances which 10 are to be treated. T'nk material, suitably pared with an oxldistnz agent selected with ref- erenco to the ecnteml~ted use or mal0mtanec to be trested, will reset effectively with the con- tempiated oxidisable substances canted in solu- 141 tion or in suspension in a fuid subjected to con- ta~'t with the trestieG mtm'l~ Depending ms the nature of the oxidim~e substance in the fluid, it may unite with the trea~inZ material. and thus be removed from the ~ or it may reme~ in the fluid in Its oxidized ¢o~ditlo~ De- pendinz upon the nsture of the o~ldizinZ ue~t used in the prelmmtlon of the treatl~ material, the latter may have sterfisin~ or b~zrlcidal powers effective to kill or inc~actt~te micro- ormmim~ in the treated fluid, or other powers effective to modify substane~ contained In the trested flu~L • A more defied un~ of the inven- tion may be had from the followinS specific ex- SO smplec, which are presented merely by w~ of liltmtmtlon, as they do not exhatmt the mnse of particular applications of the invention. The activated carbon used for preparation of the treating material may be of vea~o~ kinds 85 now known. I have found an activated carbon or char made from Texas l~zites, and marketed under the trade-name 'q)areo", to prove gener- ally effective and satl~s~zry. I have found that in its fresh and activated Condition, and in 40 a~nular or pulvertdent form, this material will I adsorb quantities of a number of oxldisl~ a~ente [ from aqueous solution, and with its adsorbed '[ content or loading or charge of the oxidiMng | agent so acquired, will react subsequently with [1~ oxidizable or other substances carried in fluids, -- either in solution or in suspension and will re- tain this power over an extended period of use in treatment of liquids as well as pses. For example, for preparation of the tnmting ma- 50 terial, a tenth-normal solution of potuslmn per- manganate may be percolated thro~h a bed of the activated carbon in sueh quantity that the • carbon will acquire by adsorption an effective charge or losdin~ of the ~ ~ Xn 1