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3,060,865United States Patent OEce ,,,,,, . ,, ,,,,2

posed of 20 pounds of salt in 10 0 pounds of water and the3,6)60,165BRF,PARATICPNOF TOXIC RPCWHany L. Craig, Chcinnzti, and Otto H. Allderks, Wyo-ming, Ohio, and Alsoph H. Corwin and Sally H. Dieke,Baltimore, and Charlo$te L. Karel, Siher Spring, Md.,assignors to the United States of America as representedby the Secretary of the ArmyWiled July 3, 1952, Ser. No. 297,1422 Claims. (621. 260-123.5)

amount used was such that the sait content equalled 20%of the filtrate weight. This amount and concentration ofsalt solution was about optimum considering the factorsof cost and toxin recovery. Somewhat higher concentra-tions and larger amounts of solution can be used, however.The precipitation process is not limited to the use ofNa2S04, since a saturated solution of NaCl can be usedsuccessfully, but Na2S04 solution gives better nitrogen10 fractionation, more rapid precipitation, and can be oper-This invention relates to the method of preparing toxic ated under wider pH limits. It is desirable to raise thericin. pH to about 7-8 before precipitation as this gives betterRicin is a protoplasmic poison prepared from castor ecovery and greater non-toxic nitrogen removal. The pHbeans after the extraction of castor oil therefrom. It is was raised to this value by using NaOH o r Na2C03, themost effective as a poison when injected intravenously or 15 latter being preferred. The base used was quite diluteinhaled, the latter requiring extreme comminution and in order to prevent detoxification due to high local con-

small particle size to be effective. It is believed that the centrations in the solution. A 5% solution of NaOIl wastoxic action is catalytic rather than stoichiometric which used, whereas with Na2C03a 12% solution was preferred.probably accounts for the high toxicity of the agent. In general, this higher pH during precipitation gave aBecause of its relative instability, ricin must be handled 20 greater non-toxic nitrogen fractionation and at the samewith extreme care. In neutral aqueous solution it is stable time maintained the toxin loss at less than 2%.only up to 60"-75' C., and in solid for'm up to 100-l lOo After precipitation, the slurry was filtered using fromC., although for short exposures, temperatures up to 130' 1 to 4% filter aid, based on slurry weight, for satisfactorymay be tolerated. It is sensitive to acids, alkalis and filtration; the amount of filter aid needed being dependenthalogen and may also be inactivated by mechanical work- 25 on the type of press used. Washing the filter cake withing such as grinding or pulverizing. These factors are of Na2S04 solution removed additional non-toxic nitrogengreat importance in developing a satisfactory method for which is desirable. In this washing step a 16.7% soh-preparing the material. tion of Na2S04 was again used. This washing step re-Although ricin has been prepared in crystalline con&- moved an additional 15% of non-toxic nitrogen from thetion in the laboratory in small quantities, it becomes neces- 30 cake.sary, for purposes of toxicological warfare, to prepare After filtration the filter cake, which contains the ricinrelatively large quantities in a high state of purity. This in combination with the Na2S04,may be dried and slurriednecessitates that as much as possible of the non-toxic ma- with CCl, to separate the ricin by flotation. Separationterial present be removed in the process. of the ricin after a single precipitation and washing stepIn preparing the protein material, the castor beans are 35 is possible, but it is preferred to carry the process throughfirst ground and pressed to remove most of the oil. The an additional extraction and precipitation step. This ispressed cake still retains about 15% oil and this may be accomplished by slurrying the filter cake in three times itsremoved by means of solvents which will extract an addi- weight of water and the pH of the slurry is again broughttional 15 0 pounds of oil per ton of beans and reduce the to 3.8k .1 by means of 5% H2S04. The slurry is filteredoil retained in the cake to a little over 1% . In the event 40 and a second precipitation is brought about by addingthat the expressing step is supplemented by solvent extrac- Na2S04 olution. Although pH control here is not whollytion, it is important to prevent detoxification of the pro- essential it is advantageous to bring the pH to approximatetein during the solvent reinoval step. If residual solvent neutrality by adding 12% NazCO,. A precipitation timeis removed from the ground beans by blowing with steam, of 45 minutes was necessary to obtain complete removalconsiderable de to fi ca ti on results. Blowing with nitrogen 45 of the toxin. In filtering out the precipitate, no filter aideffectively prevents detoxification but is expensive when was used and the filter cake was washed with Na2S04solu-carried out on a large scale. tion on the filter whereby an additional amount of non-After the oi l has been removed, the pressed cake or toxic nitrogen was removed from the cake. This washingpomace is extracted by agitating with water at a pH of was effective only the first time and repeated washings had3.8k0.1 at 25" C. which removes substantially all of the little effect in removing further non-toxic nitrogen.toxic protein. The extraction process is operative within The ricin-NazS04precipitate was 'dried at about 50" toa pH range of about 3 to 4.5 although the preferred 60" C. on a hot air tray dryer. The dried product wasrange is about 3.5 to 4. The optimum operating point ground to pass a 40 mesh screen and agitated with 5 timesis a pH of 3.8e.1, as indicated above. A careful pH its weight of CC14, which served the separate the ricincontrol is essential in order that as much non-toxic protein Ii 5 from the Na2S04 by flotation. After settling, the ricinas possible may be eliminated and also that the filtration was skimmed off the top. This reduced the Na2S04con-rate may be held at a satisfactory value. Either HC1 or tent of the mixture from a previous 40 to 50% down toHzS04may be used to get the desired pH for the extraction 15 to 18%. About 1 to 2% of nitrogen remained in thewater, but HzS04 is preferred due to its lower corrosion Na2S04 salt which could then be used for subsequentrate and ease of handling in concentrated form. The acid precipitations.should be used in reasonably dilute form to prevent undue The final precipitation produced a particle size of 1-2local concentrations during its addition. A 5% concen- mu. On drying the wet cake, however, the ricin cementedtration is satisfactory. together forming larger particles. These could not beFollowing the extraction, the slurry is filtered using broken down to their original size by ordinary grindingeither a conveiltional recessed plate filter or a continuous 65 methods and since a very fine particle size was necessarystring discharge vacuum filter. With the latter about 7% in order that the product might be used as a toxic weapon,of filter aid, based on meal weight, was found necessary it was thought desirable to seek some method to preventfor satisfactory filtration. the agglomeration or cementing process that took placeThe filtrate from the water extraction step, which con- 70 on drying.tains the ricin, was treated with a 16.7% solution of To attempt t o affect this result, physical conditions pre-Na2S04 o precipitate the protein. This solution is com- vailing under the precipitation process were changed.

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3,080,1663 4This included changing the temperature of precipitation Numerous variations are possible in the several steps ofand the rate of agitation. Other changes included precipi- the process commencing with the water extraction and pre-tation with ony partial saturation of Na2S04 and the use cipitation which may be a single ,or multiple step. M-of wetting and seeding agents. None of these expedients though a single extraction step can be used, as indicatedproduced any significant improvement in particle size. 5 before, some process modifications are necessary forOrdinary dry ball and hammer milling of the dried ricin its successful operation on a plant scale. Double extrac-

produced considerable detoxifiiation perhaps due to the tion proved to be quite efficient but additional steps beyondgeneration of excess heat. Th e use of CC14 slurry plus the second extraction step were not found necessary.the use of low temperature and low moisture content of the The drawing is selfdescriptive and shows the variousricin reduced detoxification during ball milling. 10 steps of the process described.Spray drying proved to be an even better method of We claim:securing a reasonably small particle size. Best results 1. In a method of preparing toxic ricin from castorwere achieved by using a solution havinp about 20% solids, beans comprising slurrying an expressed castor bean cakean inlet temperature of 150" C. and an atomizing air with water to remove the water soluble ricin and precipi-pressure of 150 to 180 p.s.i. Theparticle size secured was 15 tating the r icin from the filtrate, the further steps which6 to 8 mu. include slurrying the precipitate with CC14 and separatingThe best means of securing a small particle size was by the ricin by flotation.air grinding. This was carried out in an apparatus having 2. A process in accordance with claim 1 in which thea chamber with conical top and bottom. The material precipitate is dried prior to slurrying.to be ground has been fed into this chamber and is with- 20drawn from the bottom and forced back into the center References Cited in the file of this patentof the chamber tangentially through a venturi. Com- Kabat et al.: J. Biol. Chem., vol. 168, 1947, pages 629-pressed air of about 100 p.s.i. was fed to the venturi to 39.provide the grinding force. The fines are drawn off the Kunitz et al.: J. Gen. Physiol, vol. 32 (1948), pagestop and the Iarge particles settle to the bottom to be re- 25 25-31.circulated and reground. This process produced particleshaving a mass median diameter of 2.5 to 3.5 mu.

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Oct. 23, 1962 H. L.CRAIG ETALPREPARATION OF TOXIC RICIN

Filed July 3, 1952I Castpr beans II Expressing 1

II Cake ]) Slurry with water: pH of 3.8 4 0.1 using 5%HSO 1

I Wash III Preci~itatewith NGSOA: pH of 7 using 12% NaCO III Wash fi lt er cake w ith 16.7% Na.SOA 1

Single extractionII Extract with water: pH df 3.8I 0.1 using 5% HaSO4 1 III Wash ]r Filtrate ]I Cake I II Precipitate with Na,S04 :pH of 7 using 12%No&I1 Filter I VOptional II) Wash fil ter cake w it h 16.7% NaSO 1

lI Settle and skim off Ricin 1Settled NaSO reuse& prindNvE;FJToRSHarry L. Craig0. . AlderksAlsoph H. CorwinSallv H: Dieke

By & C h p yATTORNEY