METHOD FOR RECOVERING BIOTIN

Abstract: The present invention is directed toward recovering biotin, particularly toward recovering biotin produced by biotin-producing microorganisms. The recovery method includes adsorbing biotin from a biotin-containing medium onto an anion exchange resin, eluting biotin from the resin, precipitating biotin eluted from the resin, dissolving the precipitated biotin, and crystallizing biotin from the solution.

Description: METHOD FOR RECOVERING BIOTIN Field of the Invention This invention provides an improved method for recovering biotin, and more particularly to a method for recovering essentially pure biotin from a medium containing biotin-producing microorganisms. Background of the Invention Biotin, also known as vitamin H, is an essential, water-soluble, sulfur-containing vitamin that is widely distributed in nature. Biotin serves as a prosthetic group (co-factor) in a number of carboxylation and transcarboxylation reactions that play essential roles in both eukaryotic and prokaryotic carbohydrate metabolism fatty acid synthesis, amino-acid diminution, purine synthesis and nucleic acid metabolism. Biotin deficiency in both animals and humans causes skin and hair abnormalities as well as locomotor and reproductive problems. Since animals are unable to synthesize biotin, the vitamin is an important supplement to the diet. However, the price of biotin is currently too high to make vitamin supplementation practical, particularly in animal feeds. Biotin has commercial applications in the biotechnology field as a component in biotin-avidin-based labelling systems to identify specific nucleotide sequences, proteins, and other compounds. Currently, biotin is produced synthetically at relatively high costs and low yields. Since only one optical isomer of biotin is active, chemical synthetic methods must separate active and inactive isomers or prepare intermediates that yield only the active isomer. The ability to produce biotin by microbial fermentation is potentially attractive because microbes only produce the active isomer of biotin and biosynthetic biotin meets with the expanding preference for products produced by fermentation as opposed to those produced by chemical synthesis. Although several microorganisms are known to produce some biotin and/or biotin vitamers, the inventors are not aware of any method in the art to obtain substantially pure biotin from microbial fermentations. Investigators have described methods to analyze biotin produced by microorganisms. For example, Ogata ("Microbial Synthesis of Dethiobiotin and Biotin", Methods in Enzymology, Vol. 17a, pp. 390-394, 1970) discloses a method which includes the use of activated carbon and anion exchange chro atographies to obtain partially- purified solutions ofmicrobially-producedbiotin suitable for assay. The method is not suitable for a large-scale commercial purification process nor does it provide substantially pure biotin. Ogata also describes a small-scale method to purify the biotin vitamer dethiobiotin from a fermentation medium in which a Bacillus sphaericus strain is used to produce small amounts of dethiobiotin (0.1 g dethiobiotin per liter medium) . In Ogata's multi-step process, the medium is submitted to lead acetate precipitation, sulfuric acid precipitation, and activated carbon chromatography prior to anion exchange chromatography. Following anion exchange chromatography, dethiobiotin is precipitated from the eluent and crystallized three times. Using this method, Ogata was able to recover only 10% of the dethiobiotin present in the fermentation medium. Methods developed until now are therefore ill suited for use in large fermentation processes. A need exists for a simple, inexpensive method to obtain high recoveries of essentially pure biotin crystals f-rom a medium containing biotin-producing microorganisms. Summary of the Invention The present invention is directed toward recovering biotin from a biotin-containing medium by adsorbing biotin from the medium onto an anion exchange resin, eluting biotin from the resin, precipitating biotin eluted from the resin, dissolving the precipitated biotin, and crystallizing biotin from the solution. Precipitation and crystallization steps may be performed more than once. In accordance with an embodiment of the present invention, biotin is recovered from a medium containing biotin-producing microorganisms by (a) separating the medium from the biotin-producing microorganisms to obtain a substantially cell-free, biotin-containing medium, (b) adsorbing biotin from said substantially cell-free medium onto an anion exchange resin, (c) eluting biotin from the resin to obtain a biotin-containing eluent, (d) concen¬ trating the eluent to obtain a first biotin-containing solution, (e) precipitating biotin by contacting the first solution with an acid to obtain a first biotin precipitate, (f) dissolving the first biotin precipitate to obtain a second biotin-containing solution, (g) precipitating biotin by contacting the second solution with an acid to obtain a second biotin precipitate, (h) dissolving the second biotin precipitate to obtain a third biotin-containing solution, and (i) crystallizing biotin from the third solution. Recovery of biotin according to the present invention typically yields biotin crystals having a purity of at least about 90%, and preferably at least about 97%. In one embodiment of the present invention, the biotin-producing microorganisms are recombinant biotin- producing prokaryotes or eukaryotes. According to one embodiment, biotin can be eluted from the anion exchange resin using an effective concentration of a volatile acid or salt thereof. Preferably, the step of concentrating the eluent includes solubilizing any biotin that has precipitated in • the eluent, drying the solubilized eluent to obtain a biotin composition, a,nd dissolving the biotin composition in an alkaline aqueous solution to obtain a first biotin- containing solution. Biotin solubilization can be conducted by elevating the pH of the eluent to a pH of from about pH 9 to about pH 12 or by raising the te perature of the eluent to a temperature of from about 30°C to about the boiling point of the eluent. Preferably, the acid precipitating steps include adjusting the pH of the biotin-containing solutions to a pH of from about pH 1 to about pH 4, incubating the pH- adjusted solutions at about 4°C for at least about one hour to maximize biotin precipitation, and collecting the biotin precipitate. In one embodiment of the present invention, the medium containing biotin-producing microorganisms is heated to an effective temperature for an effective amount of time to kill essentially all the microorganisms prior to separating the medium from the microorganisms. Preferably the medium is heated to a temperature of about 80°C for at least about 1 hour. In another embodiment of the present invention, the biotin crystals are further purified by dissolving the crystals to obtain a fourth biotin-containing solution, and crystallizing biotin from the fourth solution • to recover biotin crystals typically having a purity of at least about 96%, and preferably at least about 99%. In accordance with a preferred embodiment of the present invention, biotin is recovered from a medium by (a) adsorbing biotin from the medium onto an anion exchange column, (b) eluting biotin from the column witli formic acid to obtain a biotin-containing eluent, (c) drying the pH-adjusted eluent under reduced pressure to obtain a biotin composition, (d) dissolving the composition in an alkaline aqueous solution to obtain a firstbiotin-containing solution, (e) precipitating biotin by contacting the first solution with an acid to obtain a first biotin precipitate, (f) dissolving the first biotin precipitate in an alkaline aqueous solution to obtain a second biotin-containing solution, (g) precipitating biotin by contacting the second solution with an acid to obtain a second biotin precipitate, (h) dissolving the second biotin precipitate in water to obtain a third biotin-containing solution, and (i) crystallizing biotin from the th-rd solution to recover biotin crystals typically having a purity of least about 90%. In one embodiment, biotin crystals obtained by this method are dissolved to obtain a fourth biotin-containing solution and crystallized to recover biotin crystals typically having a purity of at least about 96%. Detailed Description of the Invention The present invention relates to a simple method for the recovery and purification of biotin and is particularly useful in the recovery of essentially pure biotin from a medium in which microorganisms produce biotin. The present invention is capable of recovering at least about 73% of the biotin in the biotin-containing medium wherein the recovered biotin has a purity of at least about 90%. Addition of a secondary recovery step results in an overall biotin yield of at least about 61%, the biotin having a purity of at least about 96%. In accordance with the present invention, a "biotin- containing medium" is a medium containing biotin. Preferably, the biotin-containing medium is a fermentation medium in which microorganisms have produced biotin. Biotin-producing microorganisms can be prokaryotic or eukaryotic and can be natural isolates, mutants, variants, or recombinant microorganisms. In a preferred embodiment of the invention, the concentration of biotin in the biotin-containing medium is at least about 0.3 grams of biotin per liter of medium. More preferably, the medium contains at least about l gram of biotin per liter of medium. When biotin is present in a medium at a concentration of less than 0.3 grams biotin per liter, additional precipitation and crystallization steps may be required to obtain biotin that is at least about 90% pure. In accordance with the present invention, standard techniques are utilized to culture microorganisms in an effective fermentation medium to produce biotin. The effective fermentation medium can be any medium that promotes production of biotin. Surprisingly, the biotin recovery method of the present invention allows for the recovery of essentially pure biotin from a fermentation medium containing complex nutrients, such as bactotryptone or yeast extract. After culturing, the biotin-containing fermentation medium may be separated from the microorganisms using a variety of techniques, such as centrifugation, filtration, icrofiltration, or ultrafiltration. As used herein, a "substantially cell-free biotin-containing medium" is a biotin-containing medium from which sufficient micro¬ organisms have been removed so as not to substantially interfere with the recovery of biotin. If too many microorganisms remain in the medium, they may interfere with one or-more recovery steps. For example, too many microorganisms in a medium may reduce the ability of biotin to bind to an anion exchange resin. Preferably, at least about 95% of the microorganisms are removed during the separation step. To comply with various governmental regulations, microorganisms (particularly recombinant microorganisms) often must be killed prior to their disposal. In order to prepare the biotin-containing medium for the present invention, microorganisms are preferably heat-killed prior to their removal from the medium. Microorganisms are killed by heating the fermentation medium to an effective temperature for an effective amount of time to kill essentially all the microorganisms. The use of heat may also advantageous to solubilize any biotin that has precipitated from the medium. However, it has been found, surprisingly, that biotin has a solubility of about 3 g/1 in a fermentation medium at room temperature. (The reported solubility of biotin in water is only about 0.2 g/1.) Without intending to be bound by theory, it is believed that biotin exists as a salt in the fermentation medium. The present invention provides a method for recovering high yields of essentially pure biotin. Essentially pure biotin is typically at least about 90% pure, preferably at least about 96% pure, and more preferably at least about 99% pure. Although biotin vitamers exhibit some biotin-like activity, one aim of the present invention is to produce biotin of a known consistency, activity, and purity. Until now, it has been difficult to recover biotin essentially free of its vitamers, such as dethiobiotin. In one embodiment of the present invention, biotin is produced by recombinant microorganisms that have been genetically engineered to produce biotin. Although recombinant microorganisms are likely to produce biotin to the exclusion of biotin precursor vitamers, it is possible that under nutrient-limiting conditions, biotin vitamers and other biotin precursors may accumulate. For example, a low sulfur concentration during a fermentation may lead to the accumulation of dethiobiotin, which will not be converted to biotin if there is not enough sulfur present. Surprisingly, the recovery method taught by the present invention provides essentially pure biotin even if the starting biotin-containing medium contains biotin and dethiobiotin at a biotin:dethiobiotin weight ratio of 10:4. At higher ratios of biotin to dethiobiotin, recovery of biotin typically becomes easier to accomplish. In accordance with the present invention, biotin is recovered from a biotin-containing solution by adsorbing biotin from the biotin-containing medium onto an anion exchange resin under conditions that permit binding of the biotin to the resin, eluting biotin from the resin, precipitating biotin from the eluent at least once, dissolving the precipitated biotin, and crystallizing the biotin at least once. The present invention obviates the need to pretreat the fermentation medium prior to loading it onto an anion exchange column, thereby lowering overall production costs. Thus, the present invention is a teaching away from Ogata (id) , who teaches the need to pretreat a biotin- or dethiobiotin-containing medium using a lead acetate precipitation, sulfuric acid precipitation, and activated carbon chromatography. Such techniques are cumbersome and add to the cost of the recovery process. In one embodiment of the present invention, anion exchange chromatography is used to separate biotin from biotin vitamers and other contaminants in the medium. In accordance with the present invention, anion exchange chromatography is carried out by contacting the biotin- containing medium with an anion exchange resin to allow biotin to adsorb to the resin. As used herein, adsorption refers to the attachment, or binding, of a substance to a surface, such as the surface of an ion exchange resin. The biotin-bound resin is washed to remove unbound material, and biotin is subsequently eluted from the resin substantially free of biotin vitamers to obtain a biotin- containing eluent. Eluent fractions are collected and aliquots thereof are assayed to identify those that contain biotin. The preferred anion exchange resin for use in the present invention is Dowex-1-formate packed in a column. Effective eluents include acids and salts thereof present in concentrations effective to elute biotin substantially free from biotin vitamers. Such eluents include, but are not limited to, hydrochloric acid, formic acid, acetic acid, ammonium acetate, and ammonium formate. When acid concentrations are too low, biotin elutes from the column in a large volume. At high acid concentrations, biotin is not separated from biotin vitamers in the eluent. Therefore, preferred eluent concentrations are selected to obtain optimum recovery yields while at the same time effectively separating biotin from biotin vitamers. Dilute concentrations of acids are most preferred. Preferred acids for elution of biotin include volatile acids such as formic acid and hydrochloric acid because it is easy to evaporate from the biotin. Sulfuric acid is not preferred because it may be difficult to remove the sulfuric acid from the biotin. Nitric acid is not preferred because nitric acid may oxidize the sulfur in biotin. In a preferred embodiment, an eluent comprising about 12 mM formic acid is used to elute biotin from the anion exchange resin. Methods to assay biotin include, but are not limited to, microbiological, chromatographic, chemical and enzymatic assays. In a preferred embodiment, eluent fractions which contain biotin are identified by reverse phase high performance liquid chromatography (HPLC) of fraction aliquots. According to the present invention, eluent fractions containing biotin are pooled and concentrated prior to acid-precipitation. When the concentration of biotin in the eluent fractions is high, some of the biotin may precipitate and must be solubilized prior to pooling and concentration. Biotin may be solubilized by raising the pH of the biotin-containing eluent. Preferred pH values for biotin solubilization range from about pH 9 to about pH 12. A number of bases including, but not limited to, sodium hydroxide, potassium hydroxide, and ammonium hydroxide can be used to adjust the pH. Sodium hydroxide is preferred in the present invention. In another embodiment, biotin may be solubilized by raising the temperature of the biotin-containing eluent. Preferred solubilization temperatures range from about 30°C to about the boiling point of the eluent. If the biotin-containing eluent is heated to the boiling point, it is preferable that the pH of the eluent be less than about pH 9, as biotin may degrade if both high pH and high temperature conditions are used. In a preferred embodiment, the pH of biotin- containing eluent fractions is adjusted to a pH of from about pH 9 to about pH 12 to ensure biotin solubilization. The solubilized, pH-adjusted eluent is subsequently concentrated to near dryness to obtain a biotin composition. As used herein, the term drying refers to removing at least about 90% of the moisture of the eluent. Preferred methods of drying include heating the biotin- containing eluent to a temperature of from about 40°C to about 50°C under reduced pressure in a rotary evaporator, lyophilizing the eluent, and spray drying. After drying, the biotin composition is dissolved in an alkaline aqueous solution to form a biotin-containing solution. As used herein, an alkaline aqueous solution is a solution that is sufficiently basic to solubilize biotin. Preferably, the pH of the alkaline aqueous solution is from about pH 9 to about pH 12. In a preferred embodiment, water is added to the biotin composition, most preferably at a ratio of about 5 ml water per 100 mg biotin. A concentrated base, preferably 5 N sodium hydroxide, is then slowly added dropwise until the biotin dissolves. The resultant pH of the biotin-containing alkaline aqueous solution is typically from about pH 9 to about pH 12. In accordance with the present invention, biotin is precipitated from a biotin-containing solution by contacting the solution with an acid. As used herein, contacting a biotin-containing solution with an acid refers to adjusting the pH of the solution with an acid to a pH of from about pH 1 to about pH 4, and preferably to a pH of from about pH 2 to about pH 3. The preferred acid for use in the present invention is hydrochloric acid. To maximize biotin precipitation, the acidified biotin- containing solution is incubated at a temperature of from about 0°C to about 10°C for a time effective to precipitate essentially all of the biotin contained in the solution. In a preferred embodiment, the acidified biotin-containing solution is incubated at about 4°C for at least one hour. The biotin precipitate can be collected by any of a variety of effective methods, including, but not limited to, filtration and centrifugation. An advantage of centrifugation is that it collects fine particles that might be trapped in a filter, and thus lost. An. advantage of filtration is ease of washing the precipitate and of removing the aqueous phase. In a preferred embodiment of the present invention, the collected biotin precipitate is submitted to a second precipitation step. Briefly, the precipitate is dissolved in an alkaline aqueous solution, precipitated with acid, and collected, as described above. In accordance with the present invention, the precipitated biotin is prepared for crystallization by dissolving the biotin in a solution. The precipitate can be dissolved in a variety of solvents, including, but not limited to, water, methanol, and ethanol, of which water is preferred. In order to crystallize the biotin, the biotin-containing solution is typically (a) heated to a temperature sufficiently high to dissolve biotin; (b) cooled slowly until crystals form; and (c) incubated at a temperature of from about 0°C to about 10°C for a time effective to maximize crystal formation. Biotin crystals can be recovered by a variety of techniques, including centrifugation and filtration. Biotin crystals are typically from about 90% to about 98% pure at this stage. Preferably, the crystals are about 97% pure biotin. Typically, from about 73% to about 86% of the biotin in the biotin-containing medium is recovered after the first biotin crystallization step. A second biotin crystallization step can be performed in which the biotin crystals are dissolved, preferably in water, and crystallized as described above. Biotin crystals recovered after this step are typically from about 96% to about 100% pure. Preferably, the crystals are about 99% pure biotin. From about 82% and about 89% of the biotin recovered in the first crystallization step is recovered after a second biotin crystallization step. The following experimental results are provided for purposes of illustration and are not intended to limit the scope of the invention. Example 1 Escherichia coli strain MM294 was grown to stationary phase in a complex growth medium (10 g Bacto-tryptone, 5 g yeast extract, and 10 g sodium chloride per liter of water) and the resulting cell suspension, at near neutral pH, was divided to conduct experiments (A) through (D) . In experiment (A) , 100 mg of biotin were added to 100 ml of the cell suspension, and the mixture was incubated at room temperature for 1 hour. In experiment (B) , 100 mg of biotin plus 40 mg of dethiobiotin were added to 100 ml of the cell suspension, and the mixture was incubated at room temperature for 1 hour. In experiment (C) , 100 mg of biotin were added to 100 ml of the cell suspension, and the mixture was incubated at 80°C for 1 hour. In experiment (D) , 100 mg of biotin plus 40 mg of dethiobiotin were added to 100 ml of the cell suspension, and the mixture was incubated at 80°C for 1 hour. After incubation, each of the four cell suspensions (A) through (D) were treated identically. For each experiment, the cell suspension was centrifuged at 4000 x g for 15 minutes at 20"C. The supernatant was collected, and the cell pellet was washed with 100 ml of water. The washed cell pellet was centrifuged and the supernatant was collected and pooled with the first supernatant. The pooled biotin-containing supernatant was loaded onto a Dowex-1-formate column (1.2 cm x 45 cm), after which the column was washed with 4 to 5 bed volumes of water to remove unbound material. Biotin was then eluted from the column with 12 mM formic acid. About 20 ml fractions were collected. Biotin-containing fractions were identified by assaying an aliquot of each fraction by C18 reverse phase high performance liquid chromatography (HPLC) . Biotin-containing fractions were then pooled. If necessary, any biotin that had precipitated from solution in the fractions was solubilized prior to pooling by adjusting the pH of the fraction(s) to a pH of from about pH 9 to about pH 12 by slowly adding 5 N sodium hydroxide dropwise until the biotin dissolved. The pooled biotin-containing fractions were concentrated to near dryness under reduced pressure in a rotary evaporator at about 40°C to 50°C to form a biotin- containing composition. Five ml of water was added to the dried biotin-containing composition, and 5 N sodium hydroxide was slowly added dropwise until the biotin dissolved. The pH of the resultant biotin-containing solution was then adjusted to a pH of from about pH 2 to about pH 3 with 12 M hydrochloric acid to precipitate the biotin. A white precipitate appeared immediately. Precipitation was maximized by incubating the acidified biotin- containing solution at 4°C overnight. The biotin precipitate was collected by centrifugation. The biotin precipitate was then redissolved as described above, the pH adjusted to a pH of from about pH 2 to about pH 3, and the acidified biotin-containing solution incubated as described above to effect a second acid precipitation. The resultant biotin-precipitate was dissolved in 10-15-ml of water and crystallized using standard procedures. Biotin formed white/clear needle crystals under this condition. The collected biotin crystals were redissolved in water and recrystallized as described above. The yield and purity of biotin crystals after both the first and second crystallization steps are presented for each experiment in Table I. Table I Recovery of Biotin from Biotin- and Biotin-plus-Dethiobiotin-Containing Media (A) Cell plus 1 g/1 biotin (B) Cell plus 1 g/1 biotin and 0.4 g/1 dethiobiotin (C) Cell plus 1 g/1 biotin and heated at 80"C for an hour (D) Cell plus 1 g/1 biotin and 0.4 g/1 dethiobiotin and heated at 80"C for an hour Commercial biotin with a purity of 100% has a melting point (m.p.) of 228-229°C using our instrument ** Yield based on recovery on the second crystallization step only *** From HPLC analysis Example 2 Table II presents the results of a second study carried out in exactly the same manner as described in Example 1, except E_. coli strain W3110 was used in place of E. coli strain MM294. Table II Recovery of Biotin from Biotin- and Biotin-plus-Dethiobiotin-Containing Media (A) Cell plus 1 g/1 biotin (B) Cell plus 1 g/1 biotin and 0.4 g/1 dethiobiotin (C) Cell plus 1 g/1 biotin and heated at 80°C for an hour (D) Cell plus 1 g/1 biotin and 0.4 g/1 dethiobiotin and heated at 80°C for an hour Commercial biotin with a purity of 100% has a melting point (m.p.) of 228-229°C using our instrument ** Yield based on recovery on the second crystallization step only *** From HPLC analysis While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in. the following claims:

Claims: What is claimed is: 1. A method, for recovering biotin, comprising: (a) adsorbing biotin from a biotin-containmg medium onto an anion exchange resin; (b) eluting biotin from said resin to obtain a biotin-containing eluent; (c) precipitating biotin by contacting said biotin-containing eluent with an acid to obtain a biotin precipitate; (d) dissolving said biotin precipitate to form a solution; and (e) crystallizing biotin from said solution. 2. A method for recovering biotin from a medium containing biotin-producing microorganisms, comprising: (a) separating said medium from said biotin- producing microorganisms to obtain a substantially cell-free, biotin-containing medium; (b) adsorbing biotin from said substantially cell-free, biotin-containing medium onto an anion exchange resin; (c) eluting biotin from said resin to obtain a biotin-containing eluent; (d) concentrating said biotin-containing eluent to obtain a first biotin-containing solution; (e) precipitating biotin by contacting said first solution with an acid to obtain a first biotin precipitate; (f) dissolving said first biotin precipitate to obtain a second biotin-containing solution; (g) precipitating biotin by contacting said second solution with an acid to obtain a second biotin precipitate; (h) dissolving said second biotin precipitate to obtain a third biotin-containing solution; and (i) crystallizing biotin from said third solution to recover biotin crystals. 3. A method according to Claim 2, wherein said microorganisms are selected from the group comprising recombinant biotin-producing prokaryotes and eukaryotes. 4. A method according to Claim 2, wherein said step of eluting is performed using an effective concentration of a volatile acid or salt thereof. 5. A method according to Claim 2, wherein said step of concentrating comprises: (a) solubilizing any biotin precipitate in said biotin-containing eluent; (b) drying said solubilized eluent to obtain a biotin composition; and (c) dissolving said composition in an alkaline aqueous solution to obtain said first biotin- containing solution. 6. A method according to Claim 5, wherein said step of solubilizing comprises elevating the pH of said eluent to a pH of from about pH 9 to about pH 12 or raising the temperature of said eluent to a temperature of from about 30°C to about the boiling point of said eluent. 7. A method according to Claim 2, wherein said steps of precipitating comprise: (a) adjusting the pH of said biotin-containing solutions to a pH of from about pH 1 to about pH 4; (b) incubating said pH adjusted solutions at about 4°C for at least about one hour to maximize biotin precipitation; and (c) collecting the biotin precipitate. 8. A method according to Claim 2, wherein recovered biotin crystals are at least about 90% pure. 9. A method according to Claim 2, wherein recovered biotin crystals are at least about 97% pure. 10. A method according to Claim 2 further comprising heating said medium containing biotin-producing microorganisms to an effective temperature for an effective amount of time to kill essentially all of said microorganisms prior to said step of separating. 11. A method according to Claim 2 further comprising: (a) dissolving said biotin crystals to obtain a fourth biotin-containing solution; and (b) crystallizing biotin from said fourth solution to recover biotin crystals having a purity of at least about 96%. 12. A method according to Claim 11, wherein said recovered biotin crystals are at least about 99% pure. 13. A method for recovering biotin rom a medium, comprising: (a) adsorbing biotin from said medium onto an anion exchange column; (b) eluting biotin from said column with an acid or salt thereof to obtain a biotin-containing eluent; (c) drying said pH-adjusted eluent under reduced pressure to obtain a biotin composition; (d) dissolving said composition in an alkaline aqueous solution to obtain a first biotin-containing solution; (e) precipitating biotin by contacting said first solution with an acid to obtain a first biotin precipitate; i- , (f) dissolving said firs'ϊ biotin precipitate in an alkaline aqueous solution to obtain a second biotin-containing solution; (g) precipitating biotin by contacting said second solution with acid to obtain a second biotin precipitate; (h) dissolving said second biotin precipitate in an aqueous solution to obtain a third biotin- containing solution; and (i) crystallizing biotin from said third solution to recover biotin crystals having a purity of least about 90%. 14. A method according to Claim 13 further comprising: (a) dissolving said biotin crystals to obtain a fourth biotin-containing solution; and (b) crystallizing biotin from said fourth solution to recover biotin crystals having a purity of at least about 96%. 15. Biotin crystals having a purity of at least about 90% wherein said crystals are recovered from a biotin-containing medium by a method comprising: (a) adsorbing biotin from said medium onto an anion exchange column; (b) eluting biotin from said resin to obtain a biotin-containing eluent; (c) concentrating said biotin-containing eluent to obtain a first biotin-containing solution; (d) precipitating biotin by contacting said first solution with an acid to obtain a first biotin precipitate; (e) ' dissolving said first biotin precipitate to obtain a second biotin-containing solution; (f) precipitating biotin by contacting said second solution with an acid to obtain a second biotin precipitate; (g) dissolving said second biotin precipitate to obtain a third biotin-containing solution; and (h) crystallizing . biotin from said third solution to recover biotin crystals having a purity of at least about 90%. 16. Biotin crystals having a purity of at least about 96%, wherein said crystals are recovered from a biotin-containing medium by a method comprising: (a) adsorbing biotin from said medium onto an anion exchange column; (b) eluting biotin from said resin to obtain a biotin-containing eluent; (c) concentrating said biotin-containing eluent to obtain a first biotin-containing solution; (d) precipitating biotin by contacting said first solution with an acid to obtain a first biotin precipitate; (e) dissolving said first biotin precipitate to obtain a second biotin-containing solution; (f) precipitating biotin by contacting said second solution with an acid to obtain a second biotin precipitate; (g) dissolving said second biotin precipitate to obtain a third biotin-containing solution; (h) crystallizing biotin from said third solution to obtain first biotin crystals; (i) dissolving said first biotin crystals to obtain a fourth biotin-containing solution; and (j) crystallizing biotin from said fourth solution to recover biotin crystals having a purity of at least about 96%.

Inventors: CHEUNG ALEX, CHEUNG, ALEX
Applicants: ZEAGEN INC, ZEAGEN, INC.
Application year: 1993
Main classification: C07D495
IPC codes: C07D495, C12P17