CA2410654C - Method and device for concentrating and stabilizing conjugated estrogens from mares' urine - Google Patents
Method and device for concentrating and stabilizing conjugated estrogens from mares' urine Download PDFInfo
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- CA2410654C CA2410654C CA2410654A CA2410654A CA2410654C CA 2410654 C CA2410654 C CA 2410654C CA 2410654 A CA2410654 A CA 2410654A CA 2410654 A CA2410654 A CA 2410654A CA 2410654 C CA2410654 C CA 2410654C
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- C07—ORGANIC CHEMISTRY
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- C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
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- C07J—STEROIDS
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Abstract
The invention relates to a method and a device for concentrating and stabilising conjugated oestrogens from the urine of pregnant mares on solid supports in order to obtain a suitable starting material for producing pharmaceuticals that contain the natural mixture of these conjugated oestrogens as the active component. The inventive device is characterised by its suitability for practical, decentralised concentration and stabilisation of mixtures of conjugated oestrogens (CE) contained in the urine (PMU) of pregnant mares on a solid support (= adsorbent), especially in the proximity of the stable or field, in a column or a cartridge. The adsorber columns can be filled directly at the site of urine collection with the device. As a result, it is no longer necessary to transport large quantities of urine liquid daily to a central processing point. The filling process can take place continuously over a longer period of up to several weeks, until the adsorber column or cartridge is saturated. The leftover urine that runs off remains at the site, where suitable devices for disposal are already available. Only the filled column or cartridge is transported and the transport can take place at longer intervals and take longer, e.g., up to a few weeks. The excellent stability of the CE on the adsorber ensures that there is no risk of decomposition.
Description
Method and Device for Concentrating and Stabilizing Conjugated Estrogens from Mares' Urine Description The present invention relates to a method and to a device for concentrating and stabilizing conjugated estrogens from pregnant mares' urine onto solid supports in order to obtain a suitable starting material for producing pharmaceuticals that contain the natural mixture of these conjugated estrogens as the active component.
Estrogens are used in the medical field for hormone substitution therapy. In particular, estrogen mixtures are used for the treatment and prophylaxis of change-of-life conditions in women after natural or artificial menopause. In this regard, natural mixtures of conjugated estrogens present in pregnant mares' urine are particularly effective and well tolerated.
The dissolved solid content in pregnant mares' urine (hereinafter abbreviated as "PMU") can naturally fluctuate in a wide range, generally 40-90 g total solids per liter. In addition to urea and other typical components of urine, phenolic substituents in quantities of approximately 2-5 wt-% relative to total solids are contained in PMU solids. These phenolic substituents include cresols and dihydro-3,4-bis[3-hydroxyphenol)methyl]-2(3H)-furanone, known as HPMF. These substances may be present in the free or conjugated form. PMU contains a natural mixture of estrogens WO 112/09732 PCT/EPOi/08657 which are very prevalent in the conjugated form, as the sulfuric acid hemiester sodium salt, for example, (hereinafter abbreviated as bisulfate salt"). The content of conjugated estrogens (hereinafter abbreviated as ~~CE") can be calculated as estrogen sulfate salt, and ranges between 0.3 and 1 wt-~, relative to total solids.
Various methods have been described in the prior art for the direct preparation and recovery of conjugated estrogens contained in PMU. Extracts containing conjugated estrogens are usually obtained from PMU by extraction with a polar organic solvent immiscible or sparingly miscible in water, such as acetic acid ethyl ester, n-butanol, or cyclohexanol, for example. However, numerous problems arise in such liquid-liquid extractions, such as intense foam formation, sedimentation, emulsification, and strong phase separation. Several extraction steps are generally required, which results in losses and only partial recovery of the estrogen. To avoid these disadvantages, therefore, a number of solid phase extraction methods have been proposed in the prior art.
For the preparation of small quantities of urine and plasma liquids for the analytical determination of estrogens by gas chromatography, Heikkinnen et al. (Clin. Chem. 27/7, (1981), 1186-1189) and Shackleton et al. (Clinica Chimica Acta 107 (1980) 231-243) have described the solid phase extraction of estrogens using a cartridge comprising silanized silica gel containing octadecylsilane groups (Sep-Pak~ C1g cartridge, manufactured by Waters Ass. Inc., Milford, MA, USA). In this method the estrogens were eluted from the cartridge with methanol.
In 1968, H. L. Bradlow (see Steroids 11 (1968), 265-272) proposed the use of Amberlite XAD-2~, a neutral, nonpolar hydrophobic polystyrene resin from Rohm and Haas for the extraction of conjugated estrogens. The stated adsorption capacity is low. According to Bradlow, optionally diluted urine was led at a low throughput rate through a column containing the resin. The estrogens were eluted with methanol or ethanol.
Recent patent applications describe methods for obtaining an extract containing a natural mixture of conjugated estrogens from mares' urine by solid phase extraction of the mixture of conjugated estrogens from pregnant mares' urine on, for example, RP silica gel (International Patent WO 98/08525) or on nonionized, semipolar polymeric adsorbent resins (International Patent WO 98/08526).
In addition to the aforementioned optimization of the direct, complete preparation of pregnant mares' urine (PMU) for obtaining natural mixtures of CE, steps preceding the preparation, such as securing protective storage for the estrogens and handling the collected urine at the collection site, are of particular importance for the yield and quality of the estrogen-containing raw materials and of the natural mixture of conjugated estrogens isolated therefrom. In this regard, measures to optimize the transport of the collected estrogen-containing raw materials from the collection site to the site of actual preparation and isolation of conjugated estrogens are also desirable.
The complete processing of PMU, for example by solid phase extraction, requires qualified personnel trained in chemical and pharmacological methods in order to observe under well-controlled conditions the exacting requirements for purity and quality of a substance or substance mixture used as a pharmaceutical agent.
Collection of the urine (PMU), however, usually takes place in the normal environment of the pregnant mares, that is, at stud farms in rural and often remote locations. As a rule, only a simple infrastructure is present here, and the collection and handling of urine (PMU) is typically carried out by ordinary personnel following instructions, so that high standards for handling the collected urine cannot be expected.
However, conjugated estrogens (CE) in the composition excreted in pregnant mares' urine are a complex mixture containing in particular sodium estrone sulfate, sodium equilin sulfate, and other CE. It is very important in the isolation of CE that the PMU be processed as quickly as possible in fresh condition. Under extended storage urine rapidly begins to decompose, turning a dark color and giving off the odor of ammonia, with the CE content declining drastically. The rate of decomposition is a function of the storage conditions and the purity criteria. For this reason, the collected urine (PMU), which involves large volumes of liquid, hitherto has had to be transported daily from the collection site to a centralized processing point to assure the most rapid processing possible.
Hence, there is a pressing need for suitable methods and devices for concentrating and stabilizing conjugated estrogens (CE) contained in pregnant mares' urine (PMU) directly at the collection site, in a manner that is most protective of the product and allows the greatest ease in handling, and for the most efficient transport from the collection site to the site of processing and isolation.
The object of the present invention, therefore, is to develop a suitable technical method for the recovery of a natural mixture of conjugated estrogens and a suitable device for concentrating and stabilizing conjugated estrogens (CE) from the collected urine of pregnant mares (PMU), while avoiding the aforementioned disadvantages.
In particular, it is an object that the method and the device allow conjugated estrogens from PMU to be concentrated and stabilized at the site where the urine is collected, in a manner that protects the product and allows ease in handling, and allow the most efficient transport of the estrogen-containing raw material thus prepared, from the collection site to the site of actual preparation and isolation of the natural mixture of conjugated estrogens.
Surprisingly, it has been found that conjugated estrogens (CE) bound to solid supports (adsorbents) are very stable and durable in storage over long periods of time, with no signs of decomposition.
The present invention, therefore, relates to a method for concentrating and stabilizing mixtures of conjugated estrogens (CE) from pregnant mares' urine (PMU) in a manner that meets requirements in the field and is decentralized, that is, particularly close to the stable or pasture, characterized in that - the natural mixture of conjugated estrogens (CE) contained in pregnant mares' urine (PMU) is concentrated and stabilized on a solid support (adsorbent), and that - a predetermined maximum total amount of collected, optionally previously strained liquid urine is pumped from a supply vessel, continuously or in discrete portions, at a predetermined flow rate through an upright cartridge, and the liquid urine runoff is discarded, whereby - the cartridge is packed with a suitable adsorbent, which is (continuously) surrounded by liquid, for the adsorption of a predetermined quantity of the mixture of conjugated estrogens (CE) contained in the liquid urine, with - the maximum total amount of liquid urine pumped through the cartridge, which is predetermined as the end point check for the loading of the adsorbent, being matched to the maximum loading capacity of the adsorbent for the conjugated estrogens (CE) contained in the liquid urine.
The term "meets requirements in the field" as used here means that the method is adapted at the site to the requirements of the collection practice, and is therefore simple and safe to manage by trained personnel; that is, it is uncomplicated and capable of being carried out with the substantial exclusion of possible operator error.
The term "decentralized" as used here means that the method can be carried out at a particular site of the urine collection operation without specialized chemical or pharmaceutical apparatus, that is, particularly close to the stable and/or pasture. The term "cartridge" as used here means any type of closed column having a closeable inlet and outlet, in addition to connection elements such as quick couplings. Thus, within the scope of the present invention the term "cartridge" also encompasses columns as they are customarily used in laboratories, pilot plants, and the chemical industry, for example, and which are equipped to be used according to the invention as discussed above.
According to the inventive method, a raw or starting material is prepared which serves for the advantageous production of pharmaceuticals containing the natural mixture of conjugated estrogens from PMU as an active component, in which the natural estrogen mixture content of the PMU is essentially completely concentrated on the adsorbent (support) and can be stabilized over a rather long period under the normal environmental conditions of the particular collection site. After complete (maximum) loading, the cartridge can be easily exchanged and replaced with a cartridge containing an unloaded adsorbent. The cartridges with loaded adsorbents may be stored at the site for quite a long time at ambient temperature or optionally under refrigeration down to approximately 4°C, and, for specific requirements, also frozen down to approximately -20°C, for example, until a certain number of loaded cartridges is present for practical transport to the centralized processing point. The transport may take place at ambient temperatures or, for storage as described above, under cooling or refrigeration. The actual processing and isolation of the natural mixture of conjugated estrogens (CE) can then occur in a conventional manner in the centralized processing point for the particular adsorbent, for example, by washing and elution of CE from the adsorbent and further processing of the eluate by customary means. For example, the eluate may be further concentrated in a generally known manner to obtain a concentrate which is substantially or completely free of organic solvent and suitable for further pharmaceutical processing. If desired, a solids mixture that is free of elution agents may be prepared by other suitable drying methods such as spray drying, fluidized bed drying, freeze drying, or vacuum drying. The eluate containing.the estrogen mixture as well as a concentrate prepared therefrom or a spray-dried solid product may be incorporated by customary methods into solid or liquid pharmaceutical preparations such as tablets, dragees, capsules, or emulsions.
In addition to the inventive method, the invention also relates to a device that can be used in this method, said device taking into account in a useful manner the simple requirements at the location of use and assuring that the method is carried out in a manner that is safe and protective of the product. This device according to the invention is suitable for concentration and stabilization in a manner that meets requirements in the field and is decentralized, that is, particularly close to the stable or pasture. The following description contains a general discussion of several additional process parameters that are important in carrying out the method. With regard to specialized construction or apparative designs of suitable devices for carrying out the method according to the invention, reference is made to the detailed description of the inventive device which follows, which may be used to supplement the general description of the procedural method given below.
In the method according to the invention, the collected PMU
may be used as such, and to this end the daily collected quantity of PMU is first stored in a supply vessel. As an option, it recommended that coarse mechanical contaminants such as straw, hay, or the like be removed beforehand, such as by using a simple wide-mesh screen while filling the supply vessel with the freshly collected PMU. If desired, preservatives, germicides, bactericides, and/or anthelmintic agents may be added to the collected urine in the supply vessel to reduce the bacterial and viral counts.
The PMU is regularly pumped from the supply vessel to the adsorbent-containing cartridge once or twice a day, for example, depending on the amount of urine present, using a pump such as a hose pump. This cartridge typically has dimensions and weight which permit manual handling by a person of average strength. With regard to the method, it is recommended that the internal dimensions of the cartridge be designed for the receiving adsorption bed, such that the cartridge can accommodate approximately 30 to 50 L of adsorbent, and the PMU is preferably required to travel a path distance of preferably 80 to 120 cm from the inlet to the outlet. According to the invention, the conjugated estrogens are adsorbed onto the adsorbent by contact of PMU with the adsorbent, whereby the liquid urine is led, at either the head or base end, into an upright cartridge containing the adsorbent, and during throughput is held in contact with the adsorbent for a sufficient time for the estrogen to be adsorbed, until the leftover urine finally exits the opposite end of the cartridge. The PMU is led through the cartridge at the head or base end, for example, to assure that the adsorbent is continuously surrounded by liquid in order to eliminate to the extent possible undesired dry operation of the cartridge.
The contact time and the flow rate are matched to the particular adsorbent, and for adsorbents suitable for the method (loading velocity) should be in the range of 3 to 10 adsorber bed volumes/hr, preferably 4 to 6 adsorber bed volumes/hr. As an example, suitable loaded flow velocities are 4.5 to 5.5 adsorber bed volumes/hr.
Special contact times and flow rates for particularly preferred adsorbents are further described below.
The maximum total quantity of liquid urine that can be led through the cartridge is a function of the adsorption capacity of the particular adsorbent, and for adsorbents suitable for the method should be in the range of 20 to 60 adsorber bed volumes, preferably 30 to 40 adsorber bed volumes. The maximum total quantity of liquid urine that can be led through the cartridge for loading, depending on the adsorbent and the size of the cartridge, generally is in the range of 900 to 2,000 L. The particular end value of the throughput quantity is strictly specified for the operator at the collection site. Thus, a simple flow meter such as a water meter may be used to measure the quantity of liquid urine pumped through the cartridge.
After the maximum loading is reached, the operation is optionally switched to a parallel, second cartridge and the adsorption process is continued on this cartridge.
When the predetermined end value for the cartridge is reached, the cartridge with the adsorbed CE may be washed if desired WO x2/09732 PCT/EPO1/08657 with water, for example, and/or another suitable aqueous wash solution, before the liquid feed is interrupted. The liquid outlet and the liquid inlet are then closed and the connection, such as a simple quick coupling, at the liquid inlet to the cartridge is detached. The loaded cartridge can then be removed and stored at a suitable location until being transported to the centralized processing point, and a new, unloaded cartridge can be attached to the liquid inlet.
After loading with PMU, for example before replacing a cartridge which is completely loaded with PMU or before reaching the total loading capacity subsequent to any individual partial loading steps, the cartridge may be rinsed, if desired, with water and/or another suitable aqueous wash solution such as a basic wash solution, especially diluted aqueous sodium hydroxide, for example (such as aqueous 0.5-2N NaOH) to remove leftover liquid urine from the cartridge. The quantity of wash solution is not critical, and is preferably chosen to be sufficient to expel leftover urine from the cartridge without also washing out appreciable quantities of conjugated estrogens. It has proven useful to use, for example, 1 to 3, preferably approximately 2 bed volumes of wash liquid per bed volume of adsorbent. In this regard, it is useful to lead the wash water or wash liquid through the cartridge containing the adsorbent at a flow rate of 3 to 10, preferably 5 to 7 parts by volume wash water per 1 part by volume adsorbent per hour. The adsorbent loaded with CE in the cartridge may serve as the raw or starting material for the isolation of pure CE for the production of pharmaceuticals containing the natural mixture of conjugated estrogens.
In a variant of the invention, after washing with water and/or wash solution, to further stabilize the adsorbed CE the cartridge may undergo a final rinse with a preservative solution, such as a solution containing a preservative agent, a pH-adjusted aqueous solution, and/or an aqueous salt solution, before detaching the cartridge from the device and replacing it with a new cartridge. To prevent bacterial contamination of the column, particularly during storage or transport without refrigeration, customary preservatives and also germicides, bactericides, and/or anthelmintic agents, for example, may be used. A pH adjustment of the wash water depends on the particular adsorbent used. Aqueous solutions of inorganic salts in different concentrations may be used as salt solutions, for which examples are given in Table III.1. The use of salt solutions is particularly recommended when the cartridges loaded with CE are to be stored under cooling or in particular, under refrigeration.
Preferred salt solutions are sodium chloride solutions having salt concentrations of approximately 10 to 35 wt-~, preferably 25 to 33 wt-%.
In a preferred embodiment of the method according to the invention, the collected urine is preferably freed of viscous and fine-particle solids before being pumped from the supply vessel to the cartridge. It is therefore useful to first lead the liquid urine from the supply vessel through one or more prefilters before pumping the liquid urine through the cartridge. In this manner, the PMU can be led over at least one conventional separation device, for example, a filtration system having at least one deep-bed filter or precoated filter. As a separation device, a deep-bed filter having a sand bed, for example, or a commercially available filter cartridge may be used, or also commercially available precoated filters or plate filters, cartridge filters, filter bags, or filtration tubes.
The filters may be used singly or in any desired combination with one another; for example, connected consecutively in series or in parallel.
If desired, filtration aids may be added to the liquid urine (PMU) before filtration. Suitable filtration aids include those which bind calcium carbonate and/or mucin, thereby improving the urine filtration. The filter may also be optionally connected to a downstream particle separator-in the case of a sand bed, for example, connected to a downstream sand separator.
The principles and techniques of filtration are known to those skilled in the art. With regard to the object to be achieved by the invention, the conventional principles of clarification come into consideration. The object of clarification is to purify the liquid phase so that the purity of the filtrate is the significant parameter for achieving the process goal. The separation process is basically implemented by the interaction between the filter material and the suspension. This is important in the selection of the filter material for the process modeling as well as for the process control design of the filter apparatus. In most cases, the separated solid exerts a controlling influence on the course of the process. Thus, with a sufficient portion of solids and with the capability of said solids for bridge building (with particles supported over the filter material without plugging the filter), the filter cake which is forming and continuously growing assumes the function of the filter material. In this case for coated filtration and cake filtration, the structure of the filter cake determines the course of the process, whereas the actual filter material after an initial phase has only a supporting function. The site of the solids separation is a further significant feature for differentiating filtration processes. In surface filtration, the solids on the surface of the filter material are separated by a sieve effect, that is, as the result of the size ratio of the solid particles to the pores of the filter material. In contrast, in deep-bed filtration, the solid particles penetrate into the filter material and settle in the interior of same.
The adsorbents which are insertable in the cartridge can generally be any inorganic or organic adsorbent that has sufficient adsorption capacity for the natural mixture of conjugated estrogens contained in the PMU. Polymeric adsorber resins, silica gel, RP
silica gel, or, preferably, semipolar polymeric adsorber resins are thus suitable for use as adsorbents in the cartridge.
The hydrophobized silica gels which are insertable as adsorbents in the cartridge include, for example, generally known reverse phase silica gels (abbreviated as ~~RP silica gels"), which are chemically modified silica gels bearing hydrophobic functional groups or ligands. Silanized RP silica gels which contain n-octadecyldimethylsilyloxy, n-octyldimethylsilyloxy, or dimethylhydroxysilyloxy groups as hydrophobic functional groups, for example, are suitable. Silanized silica gels having average particle sizes of 15 to 500 ~.un, for example, are suitable. Silica gels containing dimethylhydroxysilyloxy groups having an average particle size ranging from 0.05 to 0.3 mm, such as silica gel 60/dimethylsilane derivative from Merck, have proven to be particularly useful. It is advantageous to lead the liquid urine through the silica gel-containing cartridge at a throughput rate such that the contact time is sufficient for adsorption of the estrogen. Throughput rates corresponding to a throughput of 5 to 20 parts by volume PMU per 1 part by volume silica gel/hour are suitable. The adsorption is preferably carried out at room temperature. It can be useful to control the throughput rate of liquid urine through the reactor by operating at a slight positive pressure (controlled by the power of the pump).
WO ~12I09732 PCT/EP01/08657 The quantity of hydrophobized silica gel to be used may vary, depending on the type of silica gel used and the solids content in the collected liquid urine. By using PMU which has been prefiltered, that is, freed of viscous and solid materials, 1 part by volume hydrophobized silica gel, for example, can be loaded with up to 80 parts by volume pretreated PMU without noticeable quantities of estrogen being detectable in the liquid urine runoff.
The semipolar polymeric adsorbent resins which may be used in the cartridge as adsorbents are preferably porous organic nonionic polymers which, in contrast to nonpolar hydrophobic polymeric adsorbent resins, have an intermediate polarity (for example, having a dipole moment of the active resin surface in a range of 1.0 to 3.0, particularly 1.5 to 2.0, Debye) and a slightly hydrophilic structure, for example, polycarboxylic acid ester resins. It is advantageous to use macroporous semipolar resins having a preferably macroreticular structure and average pore diameters ranging from 50 to 150, preferably 70 to 100 A, and a specific surface ranging from 300 to 900, preferably 400 to 500 mz/g. Macroporous crosslinked aliphatic polycarboxylic acid ester resins, especially crosslinked polyacrylic ester resins such as Amberlite XAD-7~ from Rohm and Haas, have proven to be particularly suitable. It is useful to lead the liquid urine through the cartridge containing the adsorbent resin with a throughput rate such that the contact time is sufficient for adsorption of the estrogen. Suitable throughput rates correspond, for example, to a throughput of 3 to 10, preferably 5 to 7 parts by volume PMU per 1 part by volume adsorber resin/hour. The adsorption is preferably carried out at room temperature. The throughput rate of liquid urine through the reactor may be advantageously controlled by operating at a slight positive pressure (controlled by the power of the pump). The quantity of semipolar adsorber resin to be used can vary depending on the type of adsorber resin employed and the solids content in the collected liquid urine.
By use of PMU, 1 part by volume adsorber resin, such as crosslinked aliphatic polycarboxylic acid ester adsorber resin, for example, may be loaded with up to 80 parts by volume pretreated PMU without noticeable quantities of estrogen being detectable in the liquid urine runoff.
In addition to the aforementioned preferred adsorbents, other types of adsorber resins or silica gels may also be used. In this regard, nonpolar, semipolar, and even polar adsorber resins are suitable adsorber resins. The quantity of resin that can be pumped through the adsorbent is determined beforehand, based on the particular adsorbent capacity. Examples of adsorber resins which may be used include commercially available types such as polymeric Amberlite adsorbents having a styrene divinylbenzene backbone chain (for example, XAD-1180, XAD-2, XAD-4, XAD-16), an acrylic ester backbone chain (for example, XAD-7), or highly polar backbone chains containing nitrogen and oxygen (for example, XAD-12). Other adsorber resins are Dowex resin (copolymers of styrene and divinylbenzene), such as Dowex 112, Dowex Optipore, Dowex Optipore V 493; Lewatits (crosslinked polystyrenes) such as Lewatit OC 1064, Lewatit OC 1066, or Lewatit OC 1163; and polyamine anion exchange resins such as Dowex resins. Adsorber resins XAD-7, XAD-16 (HP type), XAD 118, and Dowex Optipore, preferably Dowex Optipore V 493, and Lewatits OC 1064, OC 1066, and OC 1163 are particularly advantageous.
In addition to the aforementioned inventive method, the invention further relates to a device which may be used in said method, and which takes into account the simple conditions at the site of use in a useful manner and assures that the method may be carried out in a safe manner that protects the product.
The device according to the invention is characterized by its suitability for concentrating and stabilizing mixtures of conjugated estrogens (CE) contained in pregnant mares' urine (PMU) onto a solid support (adsorbent) in a manner that meets requirements in the field and is decentralized, that is, located near the stable or pasture, said device comprising:
- an upright cartridge packed with a suitable adsorbent, which is (continuously) surrounded by liquid, for the adsorption of a predetermined quantity of the mixture of conjugated estrogens (CE) contained in the liquid urine of pregnant mares, with the cartridge having either a) a liquid inlet situated at the base end and a liquid outlet situated at the head end or b) a liquid inlet situated at the head end and a liquid outlet situated at the base end, and - arranged in the stated sequence upstream of the cartridge and connected to one another by hose lines.
- a pump, a flow meter, and a throughput meter The device according to the invention is intended to carry out the aforementioned method for concentrating and stabilizing conjugated estrogens (CE) contained in PMU in a manner that meets requirements in the field and is decentralized. If desired, the device may also contain two cartridges connected in parallel which may be operated concurrently or alternately.
The cartridge used in the device according to the invention may be designed in a broad dimensional range with regard to its height and diameter. However, in order not to impair handling in the field, the size and weight of the cartridge should be designed so that it is easily handled and carried by hand, for example, during replacement of loaded cartridges with new, unloaded cartridges, by a person of average strength. For example, the cartridge should be designed so that the internal dimensions of the adsorption bed can accommodate 30 to 50 L
of adsorbent, and the internal height in particular is in the 80 to 120 cm range. The inner diameter of the cartridge generally ranges from 10 to 25 cm. Those skilled in the art are able to design precise dimensions of the cartridge to assure optimal flow conditions during operation.
Furthermore, the cartridge used in the device according to the invention preferably is made of robust, durable, and chemically resistant materials as customarily used in the production of devices for the chemical industry, such as impact-resistant laboratory glass, plastic, and/or metal such as sheet steel.
Any pump with a simple and robust construction may be used in the device according to the invention, such as a monopump, hose pump, or membrane pump. For example, a hose pump has proven to be a useful pump for the device according to the invention.
Any flow meter with a simple and robust construction may be used in the device according to the invention, such as a rotameter, vane air flow meter, or inductive flow meter. For example, a rotameter with a float has proven to be a useful flow meter for the device according to the invention.
Any throughput meter with a simple and robust construction may be used in the device according to the invention to measure the quantity of liquid urine pumped through the cartridge, such as a water meter or inductive flow meter. A water meter, for example, WO 02/09732 PCTlEP01/08657 has proven to be a useful flow meter for the device according to the invention.
In a useful variant of the device according to the invention, at least one or more prefilters, such as deep-bed filters or precoated filters, are connected in-line between the pump and the flow meter. The filter may be a deep-bed filter having a sand bed or a commercially available filter cartridge or a commercially available precoated filter, plate filter, cartridge filter, filter bag, or filtration tube. In addition, a particle separator may optionally be connected downstream of the prefilter, such as the example of a sand separator connected downstream of a sand bed being used as a prefilter. In variants of the invention, the filters may be used singly or in any desired combination with one another, such as connection in series and/or in parallel. An advantageous embodiment of these variants is characterized in that the prefilter is a module composed of two prefilters connected in parallel which are individually operated in alternation, and which may be replaced by new prefilters when the device is running. A further advantageous embodiment of this variant is characterized in that the filter system comprises a module composed of a prefiltration unit, connected in series, made of prefilters (filter bags, for example) in addition to one or two deep-bed filters and two absolute filters (membrane filters, for example) connected thereto in parallel, the absolute filters being individually operated in alternation, and which also may be replaced by new filters when the device is running. Furthermore, to check proper functioning of the prefilter it may be useful in the device according to the invention to connect an in-line manometer between the pump and the prefilter and an in-line manometer in the region downstream of the prefilter, preferably just behind the flow meter.
Figure 1, which shows a schematic device for stabilizing and concentrating CE from PMU, is used to further explain the device according to the invention in a preferred embodiment. Figure 2 provides a schematic representation of the method for a variant of filtering raw urine.
The method according to the invention and the device according to the invention have a number of advantages, of which the most important will be briefly stated below. Concentration of CE, that is, loading of the adsorption columns, may be carried out directly at the collection site, thereby eliminating the daily transport of large quantities of liquid urine to a centralized processing point.
Loading may be performed continuously over a rather long time, up to several weeks, until the adsorption column is saturated. For example, using adsorber resin XAD-7, the quantity of adsorbed urine is approximately 35 bed volumes. The leftover urine freed of CE
remains at the site, where suitable devices for disposal are already available. The loaded column, that is, only about 1/35 of the original weight of the liquid urine, is transported. Transport may take place at rather long time intervals and may take longer, for example, up to a few weeks. The excellent stability of the CE on the adsorption column ensures that there is no risk of decomposition.
There is no treatment of large quantities of urine in a separator and an ultrafiltration facility, since any viscous substances and sediment present are separated from the initially collected urine by using robust and maintenance-free precoated filters or deep-bed filters (for example, sand filters, filter cartridges, precoated filters, plate filters, cartridge filters, filter bags, or filtration tubes) which, if necessary, may be easily replaced.
The following figures and examples explain the invention in more detail without limiting the scope of same.
WO 02/09732 PCT/EPOl/08657 Figures Figure 1 Schematic, advantageous device for stabilizing and concentrating CE
from PMU. The elements of the device have the following meanings:
(1) inlet for the liquid urine from the supply vessel, (2) pump, such as a hose pump, (3) connecting lines, (4) manometers, (5) three-way line, (6) quick couplings, (7) closure valves, (8) filter, (9) flow meter with float (rotameter), (10) throughput meter, such as a water meter, (11) cartridge or column, (12) adsorber bed, (13) outlet.
Figure 2 Schematic representation of a method for filtering raw urine, with the filter system comprising a module composed of a prefiltration unit made of prefilters (filter bags, for example) in addition to one or two deep-bed filters and two absolute filters (membrane filters, for example) connected thereto in parallel.
Examples Example 1:
General operating specification for concentrating and stabilizing conjugated estrogens (CE) contained in pregnant mares' urine (PMU) directly at the PMU collection site by adsorption onto an adsorbent contained in a cartridge.
The collected PMU is filled into a supply vessel via a funnel fitted with a sieve. The sieve has a large mesh aperture and serves the sole purpose of separating coarse mechanical contaminants such as straw, hay, or the like.
The method is explained below by example, using an adsorption column (as a cartridge) containing a semipolar adsorber resin. If cartridges containing other adsorbents are used, the handling and especially the processing of the loaded cartridge may depend on the particular type of adsorbent. The loading process and final washing of the cartridge at the urine collection site were kept essentially as described under A) and B) or, optionally, minimally adapted to any special conditions at the site or to the particular adsorbent.
A) Adsorption of estrogen in PMU onto an adsorbent The adsorption of CE contained in PMU was carried out in an adsorption unit, as shown by example in the sketch in Figure 1.
A predetermined total quantity of PMU, relative to the capacity of the cartridge or the adsorption column, was pumped in whole or in part at ambient temperature and at a predetermined throughput rate through a column or cartridge packed with an adsorbent, after optionally straining and filtering beforehand.
As the adsorber, for example, a semipolar polyacrylic ester adsorber resin (Amberlite XAD-7 from Rohm and Haas, particle size 0.3 to 1.2 mm, dipole moment 1.8 Debye, average pore diameter 80 A, specific surface approximately 450 m2/g, dry) swollen in water, or other suitable adsorbents were used. After reaching the predetermined maximum loading quantity of PMU, the PMU feed to the column or cartridge was stopped and optionally pumped from the loaded column or cartridge to a free column or cartridge connected in parallel. The estrogen in the PMU was completely adsorbed on the adsorption column thus loaded.
B) Washing of the loaded adsorption column After the adsorption process had ended, the loaded adsorption column or cartridge was facultatively washed with water or another aqueous wash solution such as a basic wash solution, particularly diluted sodium hydroxide solution (0.5-2 N NaOH, for example). To this end, the wash water or wash solution was likewise pumped through the column or cartridge at a predetermined throughput rate of, for example, approximately 5.5 bed volumes per hour. The discharged wash water was discarded.
C) Storage and transport For transport or intermediate storage of the loaded adsorption column or cartridge, after conclusion of the adsorption process or after facultative washing (for example, with [water]or another aqueous, optionally basic wash solution, particularly sodium hydroxide solution, 0.5-2 N NaOH, for example), the column or cartridge [water] was closed on both ends, pressed out of the loading device, and stored in a suitable location.
Storage may occur at ambient temperatures, such as 15 to 30°C, in a standard storage room, or in a refrigerator at temperatures down to approximately 4°C. If the liquid in the adsorption column is protected from freezing, refrigeration can be carried out at lower temperatures, for example, down to approximately 20°C
[sic]. The loaded columns or cartridges were transported from the collection and storage site, in regular intervals of several days to a few weeks, to a centralized processing point for chemical processing and isolation of the conjugated estrogens by experienced personnel.
D) Desorption of conjugated estrogens from the adsorption column or cartridge The loaded columns or cartridges were processed in a customary manner for separation of accompanying substances and isolation of CE appropriate for the particular adsorbent. The processing can take place as described in International Patent Application WO 98/08526 by use of a semipolar adsorber resin of Amberlite such as XAD-7. To this end, the elution liquid was led at a suitable flow rate through the column or cartridge preheated to 45°C. The discharged eluate was collected in fractions. The first fraction was approximately 1 bed volume, while the remaining fractions were each approximately 0.75 bed volume. The individual fractions were analyzed by HPLC for estrone sulfate salt, cresol, and HPMF
content. The first fraction was collected as long as the eluate was colorless to pale yellow. This fraction consistently contained only traces of estrogen sulfate salt.
After the first bed volume of eluate had been discharged, the eluate turned an intense dark brown color. Approximately 80 to 90~ of the total amount of conjugated estrogens adsorbed on the column was consistently contained in the subsequent 2 to 4 fractions. The remaining fractions contained only slight amounts of estrogen sulfate salt. This was also clearly evident by the decrease in color intensity. After distilling off the solvent, the residual fractions may optionally be further processed and additional CE isolated.
The main fractions containing the conjugated estrogens each had a high TS content (total solids, determined by HPLC) of estrone sulfate salt, and were sufficiently freed of cresol and HPMF so that these fractions, as is, represented suitable extracts for further pharmaceutical processing.
E) Regeneration of the adsorber resin column The loaded columns or cartridges were regenerated in a customary manner appropriate for the particular adsorbent. The regeneration can take place as described in International Patent Application WO 98/08526 by use of a semipolar adsorber resin of Amberlite such as XAD-7. For the regeneration, the column or cartridge was first washed, for example with an ethanol/water mixture containing 50 wt-~ ethanol adjusted to pH 12, then with a 10 wt-% aqueous sodium citrate solution, once again with the ethanol/water mixture, and finally with distilled water. For the adsorber resin referenced as an example, the entire regeneration was carried out at a temperature of 45°C, whereby here as well the regeneration and wash solutions were led through the column or cartridge at either the base end or the head end. The column or cartridge can be loaded and regenerated multiple times, up to 40 times, for example.
Example 2:
Stability tests with loaded adsorption columns for the determination of desorbed CE
Stability tests were performed with adsorption columns loaded with initially collected urine to examine the column stability, that is, the stability of the adsorbed CE (conjugated estrogens). The adsorption columns were loaded in a customary manner, then the adsorber resin was taken from the columns and stored at two temperatures for different periods of time. At the end of the storage time, the adsorbate was eluted and analyzed for CE and accompanying substances. The results were compared to the elution results from control samples, that is, samples that had not been stored.
A) Test procedure The absorption [sic: adsorption] was carried out in a 3-L column packed in a known manner, using semipolar adsorber resin of type Amberlite XAD-7 (semipolar polyacrylic ester resin, that is, Amberlite XAD-7 from Rohm and Haas, with a particle size 0.3 to 1.2 mm, dipole moment 1.8 Debye, average pore diameter 80 A, and specific surface approximately 450 m2/9, dry). To this end, a quantity of 90 L (30 bed volumes) of previously ultrafiltered mares' urine (origin: Poland) was charged on the column at room temperature and a throughput rate of 5 bed volumes per hour. The estrogen in the PMU was completely adsorbed on the semipolar adsorber resin column loaded in this manner. After the column was loaded with the mares' urine, the column was subsequently washed with 3 L water, and the loaded resin was discharged from the column and divided into 10 samples of 300 mL each. These samples were transferred to smaller columns. One sample was discarded.
The following procedures were then carried out:
1. Immediate elution of the control sample, that is, without storage.
2. Storage of each of the remaining samples at 4°C or at 25°C.
3. Elution of samples 1 (4°C) and 2 (25°C) after one week of storage.
4. Elution of samples 3 (4°C) and 4 (25°C) after two weeks of storage.
Estrogens are used in the medical field for hormone substitution therapy. In particular, estrogen mixtures are used for the treatment and prophylaxis of change-of-life conditions in women after natural or artificial menopause. In this regard, natural mixtures of conjugated estrogens present in pregnant mares' urine are particularly effective and well tolerated.
The dissolved solid content in pregnant mares' urine (hereinafter abbreviated as "PMU") can naturally fluctuate in a wide range, generally 40-90 g total solids per liter. In addition to urea and other typical components of urine, phenolic substituents in quantities of approximately 2-5 wt-% relative to total solids are contained in PMU solids. These phenolic substituents include cresols and dihydro-3,4-bis[3-hydroxyphenol)methyl]-2(3H)-furanone, known as HPMF. These substances may be present in the free or conjugated form. PMU contains a natural mixture of estrogens WO 112/09732 PCT/EPOi/08657 which are very prevalent in the conjugated form, as the sulfuric acid hemiester sodium salt, for example, (hereinafter abbreviated as bisulfate salt"). The content of conjugated estrogens (hereinafter abbreviated as ~~CE") can be calculated as estrogen sulfate salt, and ranges between 0.3 and 1 wt-~, relative to total solids.
Various methods have been described in the prior art for the direct preparation and recovery of conjugated estrogens contained in PMU. Extracts containing conjugated estrogens are usually obtained from PMU by extraction with a polar organic solvent immiscible or sparingly miscible in water, such as acetic acid ethyl ester, n-butanol, or cyclohexanol, for example. However, numerous problems arise in such liquid-liquid extractions, such as intense foam formation, sedimentation, emulsification, and strong phase separation. Several extraction steps are generally required, which results in losses and only partial recovery of the estrogen. To avoid these disadvantages, therefore, a number of solid phase extraction methods have been proposed in the prior art.
For the preparation of small quantities of urine and plasma liquids for the analytical determination of estrogens by gas chromatography, Heikkinnen et al. (Clin. Chem. 27/7, (1981), 1186-1189) and Shackleton et al. (Clinica Chimica Acta 107 (1980) 231-243) have described the solid phase extraction of estrogens using a cartridge comprising silanized silica gel containing octadecylsilane groups (Sep-Pak~ C1g cartridge, manufactured by Waters Ass. Inc., Milford, MA, USA). In this method the estrogens were eluted from the cartridge with methanol.
In 1968, H. L. Bradlow (see Steroids 11 (1968), 265-272) proposed the use of Amberlite XAD-2~, a neutral, nonpolar hydrophobic polystyrene resin from Rohm and Haas for the extraction of conjugated estrogens. The stated adsorption capacity is low. According to Bradlow, optionally diluted urine was led at a low throughput rate through a column containing the resin. The estrogens were eluted with methanol or ethanol.
Recent patent applications describe methods for obtaining an extract containing a natural mixture of conjugated estrogens from mares' urine by solid phase extraction of the mixture of conjugated estrogens from pregnant mares' urine on, for example, RP silica gel (International Patent WO 98/08525) or on nonionized, semipolar polymeric adsorbent resins (International Patent WO 98/08526).
In addition to the aforementioned optimization of the direct, complete preparation of pregnant mares' urine (PMU) for obtaining natural mixtures of CE, steps preceding the preparation, such as securing protective storage for the estrogens and handling the collected urine at the collection site, are of particular importance for the yield and quality of the estrogen-containing raw materials and of the natural mixture of conjugated estrogens isolated therefrom. In this regard, measures to optimize the transport of the collected estrogen-containing raw materials from the collection site to the site of actual preparation and isolation of conjugated estrogens are also desirable.
The complete processing of PMU, for example by solid phase extraction, requires qualified personnel trained in chemical and pharmacological methods in order to observe under well-controlled conditions the exacting requirements for purity and quality of a substance or substance mixture used as a pharmaceutical agent.
Collection of the urine (PMU), however, usually takes place in the normal environment of the pregnant mares, that is, at stud farms in rural and often remote locations. As a rule, only a simple infrastructure is present here, and the collection and handling of urine (PMU) is typically carried out by ordinary personnel following instructions, so that high standards for handling the collected urine cannot be expected.
However, conjugated estrogens (CE) in the composition excreted in pregnant mares' urine are a complex mixture containing in particular sodium estrone sulfate, sodium equilin sulfate, and other CE. It is very important in the isolation of CE that the PMU be processed as quickly as possible in fresh condition. Under extended storage urine rapidly begins to decompose, turning a dark color and giving off the odor of ammonia, with the CE content declining drastically. The rate of decomposition is a function of the storage conditions and the purity criteria. For this reason, the collected urine (PMU), which involves large volumes of liquid, hitherto has had to be transported daily from the collection site to a centralized processing point to assure the most rapid processing possible.
Hence, there is a pressing need for suitable methods and devices for concentrating and stabilizing conjugated estrogens (CE) contained in pregnant mares' urine (PMU) directly at the collection site, in a manner that is most protective of the product and allows the greatest ease in handling, and for the most efficient transport from the collection site to the site of processing and isolation.
The object of the present invention, therefore, is to develop a suitable technical method for the recovery of a natural mixture of conjugated estrogens and a suitable device for concentrating and stabilizing conjugated estrogens (CE) from the collected urine of pregnant mares (PMU), while avoiding the aforementioned disadvantages.
In particular, it is an object that the method and the device allow conjugated estrogens from PMU to be concentrated and stabilized at the site where the urine is collected, in a manner that protects the product and allows ease in handling, and allow the most efficient transport of the estrogen-containing raw material thus prepared, from the collection site to the site of actual preparation and isolation of the natural mixture of conjugated estrogens.
Surprisingly, it has been found that conjugated estrogens (CE) bound to solid supports (adsorbents) are very stable and durable in storage over long periods of time, with no signs of decomposition.
The present invention, therefore, relates to a method for concentrating and stabilizing mixtures of conjugated estrogens (CE) from pregnant mares' urine (PMU) in a manner that meets requirements in the field and is decentralized, that is, particularly close to the stable or pasture, characterized in that - the natural mixture of conjugated estrogens (CE) contained in pregnant mares' urine (PMU) is concentrated and stabilized on a solid support (adsorbent), and that - a predetermined maximum total amount of collected, optionally previously strained liquid urine is pumped from a supply vessel, continuously or in discrete portions, at a predetermined flow rate through an upright cartridge, and the liquid urine runoff is discarded, whereby - the cartridge is packed with a suitable adsorbent, which is (continuously) surrounded by liquid, for the adsorption of a predetermined quantity of the mixture of conjugated estrogens (CE) contained in the liquid urine, with - the maximum total amount of liquid urine pumped through the cartridge, which is predetermined as the end point check for the loading of the adsorbent, being matched to the maximum loading capacity of the adsorbent for the conjugated estrogens (CE) contained in the liquid urine.
The term "meets requirements in the field" as used here means that the method is adapted at the site to the requirements of the collection practice, and is therefore simple and safe to manage by trained personnel; that is, it is uncomplicated and capable of being carried out with the substantial exclusion of possible operator error.
The term "decentralized" as used here means that the method can be carried out at a particular site of the urine collection operation without specialized chemical or pharmaceutical apparatus, that is, particularly close to the stable and/or pasture. The term "cartridge" as used here means any type of closed column having a closeable inlet and outlet, in addition to connection elements such as quick couplings. Thus, within the scope of the present invention the term "cartridge" also encompasses columns as they are customarily used in laboratories, pilot plants, and the chemical industry, for example, and which are equipped to be used according to the invention as discussed above.
According to the inventive method, a raw or starting material is prepared which serves for the advantageous production of pharmaceuticals containing the natural mixture of conjugated estrogens from PMU as an active component, in which the natural estrogen mixture content of the PMU is essentially completely concentrated on the adsorbent (support) and can be stabilized over a rather long period under the normal environmental conditions of the particular collection site. After complete (maximum) loading, the cartridge can be easily exchanged and replaced with a cartridge containing an unloaded adsorbent. The cartridges with loaded adsorbents may be stored at the site for quite a long time at ambient temperature or optionally under refrigeration down to approximately 4°C, and, for specific requirements, also frozen down to approximately -20°C, for example, until a certain number of loaded cartridges is present for practical transport to the centralized processing point. The transport may take place at ambient temperatures or, for storage as described above, under cooling or refrigeration. The actual processing and isolation of the natural mixture of conjugated estrogens (CE) can then occur in a conventional manner in the centralized processing point for the particular adsorbent, for example, by washing and elution of CE from the adsorbent and further processing of the eluate by customary means. For example, the eluate may be further concentrated in a generally known manner to obtain a concentrate which is substantially or completely free of organic solvent and suitable for further pharmaceutical processing. If desired, a solids mixture that is free of elution agents may be prepared by other suitable drying methods such as spray drying, fluidized bed drying, freeze drying, or vacuum drying. The eluate containing.the estrogen mixture as well as a concentrate prepared therefrom or a spray-dried solid product may be incorporated by customary methods into solid or liquid pharmaceutical preparations such as tablets, dragees, capsules, or emulsions.
In addition to the inventive method, the invention also relates to a device that can be used in this method, said device taking into account in a useful manner the simple requirements at the location of use and assuring that the method is carried out in a manner that is safe and protective of the product. This device according to the invention is suitable for concentration and stabilization in a manner that meets requirements in the field and is decentralized, that is, particularly close to the stable or pasture. The following description contains a general discussion of several additional process parameters that are important in carrying out the method. With regard to specialized construction or apparative designs of suitable devices for carrying out the method according to the invention, reference is made to the detailed description of the inventive device which follows, which may be used to supplement the general description of the procedural method given below.
In the method according to the invention, the collected PMU
may be used as such, and to this end the daily collected quantity of PMU is first stored in a supply vessel. As an option, it recommended that coarse mechanical contaminants such as straw, hay, or the like be removed beforehand, such as by using a simple wide-mesh screen while filling the supply vessel with the freshly collected PMU. If desired, preservatives, germicides, bactericides, and/or anthelmintic agents may be added to the collected urine in the supply vessel to reduce the bacterial and viral counts.
The PMU is regularly pumped from the supply vessel to the adsorbent-containing cartridge once or twice a day, for example, depending on the amount of urine present, using a pump such as a hose pump. This cartridge typically has dimensions and weight which permit manual handling by a person of average strength. With regard to the method, it is recommended that the internal dimensions of the cartridge be designed for the receiving adsorption bed, such that the cartridge can accommodate approximately 30 to 50 L of adsorbent, and the PMU is preferably required to travel a path distance of preferably 80 to 120 cm from the inlet to the outlet. According to the invention, the conjugated estrogens are adsorbed onto the adsorbent by contact of PMU with the adsorbent, whereby the liquid urine is led, at either the head or base end, into an upright cartridge containing the adsorbent, and during throughput is held in contact with the adsorbent for a sufficient time for the estrogen to be adsorbed, until the leftover urine finally exits the opposite end of the cartridge. The PMU is led through the cartridge at the head or base end, for example, to assure that the adsorbent is continuously surrounded by liquid in order to eliminate to the extent possible undesired dry operation of the cartridge.
The contact time and the flow rate are matched to the particular adsorbent, and for adsorbents suitable for the method (loading velocity) should be in the range of 3 to 10 adsorber bed volumes/hr, preferably 4 to 6 adsorber bed volumes/hr. As an example, suitable loaded flow velocities are 4.5 to 5.5 adsorber bed volumes/hr.
Special contact times and flow rates for particularly preferred adsorbents are further described below.
The maximum total quantity of liquid urine that can be led through the cartridge is a function of the adsorption capacity of the particular adsorbent, and for adsorbents suitable for the method should be in the range of 20 to 60 adsorber bed volumes, preferably 30 to 40 adsorber bed volumes. The maximum total quantity of liquid urine that can be led through the cartridge for loading, depending on the adsorbent and the size of the cartridge, generally is in the range of 900 to 2,000 L. The particular end value of the throughput quantity is strictly specified for the operator at the collection site. Thus, a simple flow meter such as a water meter may be used to measure the quantity of liquid urine pumped through the cartridge.
After the maximum loading is reached, the operation is optionally switched to a parallel, second cartridge and the adsorption process is continued on this cartridge.
When the predetermined end value for the cartridge is reached, the cartridge with the adsorbed CE may be washed if desired WO x2/09732 PCT/EPO1/08657 with water, for example, and/or another suitable aqueous wash solution, before the liquid feed is interrupted. The liquid outlet and the liquid inlet are then closed and the connection, such as a simple quick coupling, at the liquid inlet to the cartridge is detached. The loaded cartridge can then be removed and stored at a suitable location until being transported to the centralized processing point, and a new, unloaded cartridge can be attached to the liquid inlet.
After loading with PMU, for example before replacing a cartridge which is completely loaded with PMU or before reaching the total loading capacity subsequent to any individual partial loading steps, the cartridge may be rinsed, if desired, with water and/or another suitable aqueous wash solution such as a basic wash solution, especially diluted aqueous sodium hydroxide, for example (such as aqueous 0.5-2N NaOH) to remove leftover liquid urine from the cartridge. The quantity of wash solution is not critical, and is preferably chosen to be sufficient to expel leftover urine from the cartridge without also washing out appreciable quantities of conjugated estrogens. It has proven useful to use, for example, 1 to 3, preferably approximately 2 bed volumes of wash liquid per bed volume of adsorbent. In this regard, it is useful to lead the wash water or wash liquid through the cartridge containing the adsorbent at a flow rate of 3 to 10, preferably 5 to 7 parts by volume wash water per 1 part by volume adsorbent per hour. The adsorbent loaded with CE in the cartridge may serve as the raw or starting material for the isolation of pure CE for the production of pharmaceuticals containing the natural mixture of conjugated estrogens.
In a variant of the invention, after washing with water and/or wash solution, to further stabilize the adsorbed CE the cartridge may undergo a final rinse with a preservative solution, such as a solution containing a preservative agent, a pH-adjusted aqueous solution, and/or an aqueous salt solution, before detaching the cartridge from the device and replacing it with a new cartridge. To prevent bacterial contamination of the column, particularly during storage or transport without refrigeration, customary preservatives and also germicides, bactericides, and/or anthelmintic agents, for example, may be used. A pH adjustment of the wash water depends on the particular adsorbent used. Aqueous solutions of inorganic salts in different concentrations may be used as salt solutions, for which examples are given in Table III.1. The use of salt solutions is particularly recommended when the cartridges loaded with CE are to be stored under cooling or in particular, under refrigeration.
Preferred salt solutions are sodium chloride solutions having salt concentrations of approximately 10 to 35 wt-~, preferably 25 to 33 wt-%.
In a preferred embodiment of the method according to the invention, the collected urine is preferably freed of viscous and fine-particle solids before being pumped from the supply vessel to the cartridge. It is therefore useful to first lead the liquid urine from the supply vessel through one or more prefilters before pumping the liquid urine through the cartridge. In this manner, the PMU can be led over at least one conventional separation device, for example, a filtration system having at least one deep-bed filter or precoated filter. As a separation device, a deep-bed filter having a sand bed, for example, or a commercially available filter cartridge may be used, or also commercially available precoated filters or plate filters, cartridge filters, filter bags, or filtration tubes.
The filters may be used singly or in any desired combination with one another; for example, connected consecutively in series or in parallel.
If desired, filtration aids may be added to the liquid urine (PMU) before filtration. Suitable filtration aids include those which bind calcium carbonate and/or mucin, thereby improving the urine filtration. The filter may also be optionally connected to a downstream particle separator-in the case of a sand bed, for example, connected to a downstream sand separator.
The principles and techniques of filtration are known to those skilled in the art. With regard to the object to be achieved by the invention, the conventional principles of clarification come into consideration. The object of clarification is to purify the liquid phase so that the purity of the filtrate is the significant parameter for achieving the process goal. The separation process is basically implemented by the interaction between the filter material and the suspension. This is important in the selection of the filter material for the process modeling as well as for the process control design of the filter apparatus. In most cases, the separated solid exerts a controlling influence on the course of the process. Thus, with a sufficient portion of solids and with the capability of said solids for bridge building (with particles supported over the filter material without plugging the filter), the filter cake which is forming and continuously growing assumes the function of the filter material. In this case for coated filtration and cake filtration, the structure of the filter cake determines the course of the process, whereas the actual filter material after an initial phase has only a supporting function. The site of the solids separation is a further significant feature for differentiating filtration processes. In surface filtration, the solids on the surface of the filter material are separated by a sieve effect, that is, as the result of the size ratio of the solid particles to the pores of the filter material. In contrast, in deep-bed filtration, the solid particles penetrate into the filter material and settle in the interior of same.
The adsorbents which are insertable in the cartridge can generally be any inorganic or organic adsorbent that has sufficient adsorption capacity for the natural mixture of conjugated estrogens contained in the PMU. Polymeric adsorber resins, silica gel, RP
silica gel, or, preferably, semipolar polymeric adsorber resins are thus suitable for use as adsorbents in the cartridge.
The hydrophobized silica gels which are insertable as adsorbents in the cartridge include, for example, generally known reverse phase silica gels (abbreviated as ~~RP silica gels"), which are chemically modified silica gels bearing hydrophobic functional groups or ligands. Silanized RP silica gels which contain n-octadecyldimethylsilyloxy, n-octyldimethylsilyloxy, or dimethylhydroxysilyloxy groups as hydrophobic functional groups, for example, are suitable. Silanized silica gels having average particle sizes of 15 to 500 ~.un, for example, are suitable. Silica gels containing dimethylhydroxysilyloxy groups having an average particle size ranging from 0.05 to 0.3 mm, such as silica gel 60/dimethylsilane derivative from Merck, have proven to be particularly useful. It is advantageous to lead the liquid urine through the silica gel-containing cartridge at a throughput rate such that the contact time is sufficient for adsorption of the estrogen. Throughput rates corresponding to a throughput of 5 to 20 parts by volume PMU per 1 part by volume silica gel/hour are suitable. The adsorption is preferably carried out at room temperature. It can be useful to control the throughput rate of liquid urine through the reactor by operating at a slight positive pressure (controlled by the power of the pump).
WO ~12I09732 PCT/EP01/08657 The quantity of hydrophobized silica gel to be used may vary, depending on the type of silica gel used and the solids content in the collected liquid urine. By using PMU which has been prefiltered, that is, freed of viscous and solid materials, 1 part by volume hydrophobized silica gel, for example, can be loaded with up to 80 parts by volume pretreated PMU without noticeable quantities of estrogen being detectable in the liquid urine runoff.
The semipolar polymeric adsorbent resins which may be used in the cartridge as adsorbents are preferably porous organic nonionic polymers which, in contrast to nonpolar hydrophobic polymeric adsorbent resins, have an intermediate polarity (for example, having a dipole moment of the active resin surface in a range of 1.0 to 3.0, particularly 1.5 to 2.0, Debye) and a slightly hydrophilic structure, for example, polycarboxylic acid ester resins. It is advantageous to use macroporous semipolar resins having a preferably macroreticular structure and average pore diameters ranging from 50 to 150, preferably 70 to 100 A, and a specific surface ranging from 300 to 900, preferably 400 to 500 mz/g. Macroporous crosslinked aliphatic polycarboxylic acid ester resins, especially crosslinked polyacrylic ester resins such as Amberlite XAD-7~ from Rohm and Haas, have proven to be particularly suitable. It is useful to lead the liquid urine through the cartridge containing the adsorbent resin with a throughput rate such that the contact time is sufficient for adsorption of the estrogen. Suitable throughput rates correspond, for example, to a throughput of 3 to 10, preferably 5 to 7 parts by volume PMU per 1 part by volume adsorber resin/hour. The adsorption is preferably carried out at room temperature. The throughput rate of liquid urine through the reactor may be advantageously controlled by operating at a slight positive pressure (controlled by the power of the pump). The quantity of semipolar adsorber resin to be used can vary depending on the type of adsorber resin employed and the solids content in the collected liquid urine.
By use of PMU, 1 part by volume adsorber resin, such as crosslinked aliphatic polycarboxylic acid ester adsorber resin, for example, may be loaded with up to 80 parts by volume pretreated PMU without noticeable quantities of estrogen being detectable in the liquid urine runoff.
In addition to the aforementioned preferred adsorbents, other types of adsorber resins or silica gels may also be used. In this regard, nonpolar, semipolar, and even polar adsorber resins are suitable adsorber resins. The quantity of resin that can be pumped through the adsorbent is determined beforehand, based on the particular adsorbent capacity. Examples of adsorber resins which may be used include commercially available types such as polymeric Amberlite adsorbents having a styrene divinylbenzene backbone chain (for example, XAD-1180, XAD-2, XAD-4, XAD-16), an acrylic ester backbone chain (for example, XAD-7), or highly polar backbone chains containing nitrogen and oxygen (for example, XAD-12). Other adsorber resins are Dowex resin (copolymers of styrene and divinylbenzene), such as Dowex 112, Dowex Optipore, Dowex Optipore V 493; Lewatits (crosslinked polystyrenes) such as Lewatit OC 1064, Lewatit OC 1066, or Lewatit OC 1163; and polyamine anion exchange resins such as Dowex resins. Adsorber resins XAD-7, XAD-16 (HP type), XAD 118, and Dowex Optipore, preferably Dowex Optipore V 493, and Lewatits OC 1064, OC 1066, and OC 1163 are particularly advantageous.
In addition to the aforementioned inventive method, the invention further relates to a device which may be used in said method, and which takes into account the simple conditions at the site of use in a useful manner and assures that the method may be carried out in a safe manner that protects the product.
The device according to the invention is characterized by its suitability for concentrating and stabilizing mixtures of conjugated estrogens (CE) contained in pregnant mares' urine (PMU) onto a solid support (adsorbent) in a manner that meets requirements in the field and is decentralized, that is, located near the stable or pasture, said device comprising:
- an upright cartridge packed with a suitable adsorbent, which is (continuously) surrounded by liquid, for the adsorption of a predetermined quantity of the mixture of conjugated estrogens (CE) contained in the liquid urine of pregnant mares, with the cartridge having either a) a liquid inlet situated at the base end and a liquid outlet situated at the head end or b) a liquid inlet situated at the head end and a liquid outlet situated at the base end, and - arranged in the stated sequence upstream of the cartridge and connected to one another by hose lines.
- a pump, a flow meter, and a throughput meter The device according to the invention is intended to carry out the aforementioned method for concentrating and stabilizing conjugated estrogens (CE) contained in PMU in a manner that meets requirements in the field and is decentralized. If desired, the device may also contain two cartridges connected in parallel which may be operated concurrently or alternately.
The cartridge used in the device according to the invention may be designed in a broad dimensional range with regard to its height and diameter. However, in order not to impair handling in the field, the size and weight of the cartridge should be designed so that it is easily handled and carried by hand, for example, during replacement of loaded cartridges with new, unloaded cartridges, by a person of average strength. For example, the cartridge should be designed so that the internal dimensions of the adsorption bed can accommodate 30 to 50 L
of adsorbent, and the internal height in particular is in the 80 to 120 cm range. The inner diameter of the cartridge generally ranges from 10 to 25 cm. Those skilled in the art are able to design precise dimensions of the cartridge to assure optimal flow conditions during operation.
Furthermore, the cartridge used in the device according to the invention preferably is made of robust, durable, and chemically resistant materials as customarily used in the production of devices for the chemical industry, such as impact-resistant laboratory glass, plastic, and/or metal such as sheet steel.
Any pump with a simple and robust construction may be used in the device according to the invention, such as a monopump, hose pump, or membrane pump. For example, a hose pump has proven to be a useful pump for the device according to the invention.
Any flow meter with a simple and robust construction may be used in the device according to the invention, such as a rotameter, vane air flow meter, or inductive flow meter. For example, a rotameter with a float has proven to be a useful flow meter for the device according to the invention.
Any throughput meter with a simple and robust construction may be used in the device according to the invention to measure the quantity of liquid urine pumped through the cartridge, such as a water meter or inductive flow meter. A water meter, for example, WO 02/09732 PCTlEP01/08657 has proven to be a useful flow meter for the device according to the invention.
In a useful variant of the device according to the invention, at least one or more prefilters, such as deep-bed filters or precoated filters, are connected in-line between the pump and the flow meter. The filter may be a deep-bed filter having a sand bed or a commercially available filter cartridge or a commercially available precoated filter, plate filter, cartridge filter, filter bag, or filtration tube. In addition, a particle separator may optionally be connected downstream of the prefilter, such as the example of a sand separator connected downstream of a sand bed being used as a prefilter. In variants of the invention, the filters may be used singly or in any desired combination with one another, such as connection in series and/or in parallel. An advantageous embodiment of these variants is characterized in that the prefilter is a module composed of two prefilters connected in parallel which are individually operated in alternation, and which may be replaced by new prefilters when the device is running. A further advantageous embodiment of this variant is characterized in that the filter system comprises a module composed of a prefiltration unit, connected in series, made of prefilters (filter bags, for example) in addition to one or two deep-bed filters and two absolute filters (membrane filters, for example) connected thereto in parallel, the absolute filters being individually operated in alternation, and which also may be replaced by new filters when the device is running. Furthermore, to check proper functioning of the prefilter it may be useful in the device according to the invention to connect an in-line manometer between the pump and the prefilter and an in-line manometer in the region downstream of the prefilter, preferably just behind the flow meter.
Figure 1, which shows a schematic device for stabilizing and concentrating CE from PMU, is used to further explain the device according to the invention in a preferred embodiment. Figure 2 provides a schematic representation of the method for a variant of filtering raw urine.
The method according to the invention and the device according to the invention have a number of advantages, of which the most important will be briefly stated below. Concentration of CE, that is, loading of the adsorption columns, may be carried out directly at the collection site, thereby eliminating the daily transport of large quantities of liquid urine to a centralized processing point.
Loading may be performed continuously over a rather long time, up to several weeks, until the adsorption column is saturated. For example, using adsorber resin XAD-7, the quantity of adsorbed urine is approximately 35 bed volumes. The leftover urine freed of CE
remains at the site, where suitable devices for disposal are already available. The loaded column, that is, only about 1/35 of the original weight of the liquid urine, is transported. Transport may take place at rather long time intervals and may take longer, for example, up to a few weeks. The excellent stability of the CE on the adsorption column ensures that there is no risk of decomposition.
There is no treatment of large quantities of urine in a separator and an ultrafiltration facility, since any viscous substances and sediment present are separated from the initially collected urine by using robust and maintenance-free precoated filters or deep-bed filters (for example, sand filters, filter cartridges, precoated filters, plate filters, cartridge filters, filter bags, or filtration tubes) which, if necessary, may be easily replaced.
The following figures and examples explain the invention in more detail without limiting the scope of same.
WO 02/09732 PCT/EPOl/08657 Figures Figure 1 Schematic, advantageous device for stabilizing and concentrating CE
from PMU. The elements of the device have the following meanings:
(1) inlet for the liquid urine from the supply vessel, (2) pump, such as a hose pump, (3) connecting lines, (4) manometers, (5) three-way line, (6) quick couplings, (7) closure valves, (8) filter, (9) flow meter with float (rotameter), (10) throughput meter, such as a water meter, (11) cartridge or column, (12) adsorber bed, (13) outlet.
Figure 2 Schematic representation of a method for filtering raw urine, with the filter system comprising a module composed of a prefiltration unit made of prefilters (filter bags, for example) in addition to one or two deep-bed filters and two absolute filters (membrane filters, for example) connected thereto in parallel.
Examples Example 1:
General operating specification for concentrating and stabilizing conjugated estrogens (CE) contained in pregnant mares' urine (PMU) directly at the PMU collection site by adsorption onto an adsorbent contained in a cartridge.
The collected PMU is filled into a supply vessel via a funnel fitted with a sieve. The sieve has a large mesh aperture and serves the sole purpose of separating coarse mechanical contaminants such as straw, hay, or the like.
The method is explained below by example, using an adsorption column (as a cartridge) containing a semipolar adsorber resin. If cartridges containing other adsorbents are used, the handling and especially the processing of the loaded cartridge may depend on the particular type of adsorbent. The loading process and final washing of the cartridge at the urine collection site were kept essentially as described under A) and B) or, optionally, minimally adapted to any special conditions at the site or to the particular adsorbent.
A) Adsorption of estrogen in PMU onto an adsorbent The adsorption of CE contained in PMU was carried out in an adsorption unit, as shown by example in the sketch in Figure 1.
A predetermined total quantity of PMU, relative to the capacity of the cartridge or the adsorption column, was pumped in whole or in part at ambient temperature and at a predetermined throughput rate through a column or cartridge packed with an adsorbent, after optionally straining and filtering beforehand.
As the adsorber, for example, a semipolar polyacrylic ester adsorber resin (Amberlite XAD-7 from Rohm and Haas, particle size 0.3 to 1.2 mm, dipole moment 1.8 Debye, average pore diameter 80 A, specific surface approximately 450 m2/g, dry) swollen in water, or other suitable adsorbents were used. After reaching the predetermined maximum loading quantity of PMU, the PMU feed to the column or cartridge was stopped and optionally pumped from the loaded column or cartridge to a free column or cartridge connected in parallel. The estrogen in the PMU was completely adsorbed on the adsorption column thus loaded.
B) Washing of the loaded adsorption column After the adsorption process had ended, the loaded adsorption column or cartridge was facultatively washed with water or another aqueous wash solution such as a basic wash solution, particularly diluted sodium hydroxide solution (0.5-2 N NaOH, for example). To this end, the wash water or wash solution was likewise pumped through the column or cartridge at a predetermined throughput rate of, for example, approximately 5.5 bed volumes per hour. The discharged wash water was discarded.
C) Storage and transport For transport or intermediate storage of the loaded adsorption column or cartridge, after conclusion of the adsorption process or after facultative washing (for example, with [water]or another aqueous, optionally basic wash solution, particularly sodium hydroxide solution, 0.5-2 N NaOH, for example), the column or cartridge [water] was closed on both ends, pressed out of the loading device, and stored in a suitable location.
Storage may occur at ambient temperatures, such as 15 to 30°C, in a standard storage room, or in a refrigerator at temperatures down to approximately 4°C. If the liquid in the adsorption column is protected from freezing, refrigeration can be carried out at lower temperatures, for example, down to approximately 20°C
[sic]. The loaded columns or cartridges were transported from the collection and storage site, in regular intervals of several days to a few weeks, to a centralized processing point for chemical processing and isolation of the conjugated estrogens by experienced personnel.
D) Desorption of conjugated estrogens from the adsorption column or cartridge The loaded columns or cartridges were processed in a customary manner for separation of accompanying substances and isolation of CE appropriate for the particular adsorbent. The processing can take place as described in International Patent Application WO 98/08526 by use of a semipolar adsorber resin of Amberlite such as XAD-7. To this end, the elution liquid was led at a suitable flow rate through the column or cartridge preheated to 45°C. The discharged eluate was collected in fractions. The first fraction was approximately 1 bed volume, while the remaining fractions were each approximately 0.75 bed volume. The individual fractions were analyzed by HPLC for estrone sulfate salt, cresol, and HPMF
content. The first fraction was collected as long as the eluate was colorless to pale yellow. This fraction consistently contained only traces of estrogen sulfate salt.
After the first bed volume of eluate had been discharged, the eluate turned an intense dark brown color. Approximately 80 to 90~ of the total amount of conjugated estrogens adsorbed on the column was consistently contained in the subsequent 2 to 4 fractions. The remaining fractions contained only slight amounts of estrogen sulfate salt. This was also clearly evident by the decrease in color intensity. After distilling off the solvent, the residual fractions may optionally be further processed and additional CE isolated.
The main fractions containing the conjugated estrogens each had a high TS content (total solids, determined by HPLC) of estrone sulfate salt, and were sufficiently freed of cresol and HPMF so that these fractions, as is, represented suitable extracts for further pharmaceutical processing.
E) Regeneration of the adsorber resin column The loaded columns or cartridges were regenerated in a customary manner appropriate for the particular adsorbent. The regeneration can take place as described in International Patent Application WO 98/08526 by use of a semipolar adsorber resin of Amberlite such as XAD-7. For the regeneration, the column or cartridge was first washed, for example with an ethanol/water mixture containing 50 wt-~ ethanol adjusted to pH 12, then with a 10 wt-% aqueous sodium citrate solution, once again with the ethanol/water mixture, and finally with distilled water. For the adsorber resin referenced as an example, the entire regeneration was carried out at a temperature of 45°C, whereby here as well the regeneration and wash solutions were led through the column or cartridge at either the base end or the head end. The column or cartridge can be loaded and regenerated multiple times, up to 40 times, for example.
Example 2:
Stability tests with loaded adsorption columns for the determination of desorbed CE
Stability tests were performed with adsorption columns loaded with initially collected urine to examine the column stability, that is, the stability of the adsorbed CE (conjugated estrogens). The adsorption columns were loaded in a customary manner, then the adsorber resin was taken from the columns and stored at two temperatures for different periods of time. At the end of the storage time, the adsorbate was eluted and analyzed for CE and accompanying substances. The results were compared to the elution results from control samples, that is, samples that had not been stored.
A) Test procedure The absorption [sic: adsorption] was carried out in a 3-L column packed in a known manner, using semipolar adsorber resin of type Amberlite XAD-7 (semipolar polyacrylic ester resin, that is, Amberlite XAD-7 from Rohm and Haas, with a particle size 0.3 to 1.2 mm, dipole moment 1.8 Debye, average pore diameter 80 A, and specific surface approximately 450 m2/9, dry). To this end, a quantity of 90 L (30 bed volumes) of previously ultrafiltered mares' urine (origin: Poland) was charged on the column at room temperature and a throughput rate of 5 bed volumes per hour. The estrogen in the PMU was completely adsorbed on the semipolar adsorber resin column loaded in this manner. After the column was loaded with the mares' urine, the column was subsequently washed with 3 L water, and the loaded resin was discharged from the column and divided into 10 samples of 300 mL each. These samples were transferred to smaller columns. One sample was discarded.
The following procedures were then carried out:
1. Immediate elution of the control sample, that is, without storage.
2. Storage of each of the remaining samples at 4°C or at 25°C.
3. Elution of samples 1 (4°C) and 2 (25°C) after one week of storage.
4. Elution of samples 3 (4°C) and 4 (25°C) after two weeks of storage.
5. Elution of samples 5 (4°C) and 6 (25°C) after five weeks of storage.
6. Elution of samples 7 (4°C) and 8 (25°C) after eight weeks of storage.
The elution was carried out in a known manner by first washing the loaded adsorber resin with an aqueous sodium hydroxide solution. After each washing step, the respective wash liquid was analyzed for estrone (as the sulfate salt), equilin, cresol, and HPMF
content, using HPLC.
An ethanol/water mixture (30 wt-%, pH 12) made alkaline by the addition of sodium hydroxide was used as the elution liquid, and the resin was eluted at an elution temperature of 45°C. The eluate was collected in fractions, and the individual fractions were analyzed for estrone, cresol, and HPMF content, again using HPLC. The first fraction was collected as long as the eluate was colorless to pale yellow. This fraction contained only traces of estrone (estrogen sulfate salt). After the first bed volume of eluate had been discharged, the eluate turned an intense dark brown color. Approximately 80 to 90% of the total amount of conjugated estrogens adsorbed on the column was consistently contained in the subsequent 2 to 4 fractions. The remaining fractions for regenerating the resin (each at 45°C in the referenced sequence, with 50 wt-% ethanol at pH 12, 10 wt-%
sodium citrate, and water) contained only slight amounts of estrogen sulfate salt. This was also clearly evident by the decrease in color intensity.
B) Results The results of the column stability tests are presented for individual samples in Tables I.0 through I.9 and summarized in Table I.10, and are discussed in more detail below.
The loading of the column or cartridge with urine (see Table I.0) and elution of the control column (see Table I.1) showed a normal course of adsorption and desorption. However, elevated values of cresol and HPMF were found in the eluate, which could be attributed largely to incomplete laminar flow in the column or to the very high cresol levels (almost 1000 mg/L) in the initially collected urine.
Tables I.2 through I.9 give the results of elution after storage periods of one, two, five, and eight weeks. The basic objective was to determine that, as a rule, the adsorbed hormones (CE) were quantitatively desorbed. Occasionally, particularly after only one week's storage at 25°C, for example, quantitative desorption was determined only during regeneration with 50~
ethanol. However, this involved only desorption of CE residues, and the majority had already been isolated in the actual desorption. There appeared to be little or no dependency of desorption on the storage time and temperature, provided, for example, that possible non-optimal flow conditions did not play a greater role in the small columns. However, in all cases the eluate was essentially free of cresol and HPMF. To obtain optimal results, therefore, attention should be directed to optimal column throughput, regardless of the storage time. The columns or cartridges should therefore always be immersed in liquid; that is, dry operation should be avoided.
The results after five weeks' storage presented in Tables I.6 and I.7 show a satisfactory course of elution at a temperature of 4°C, whereas at T = 25°C, hormone still appears in the regenerate. The HPMF and cresol content in the eluate was very small in both cases.
Surprisingly, excellent results were obtained even after a very long storage time, as shown in Tables I.8 and I.9 for the eight-week storage. The elution peaks for estrone and equilin were very steep and sharp; that is, the volume of CE eluate was small. The cresol and HPMF values were practically nil.
Table I.10 gives an overview summary of all the tests. The sum of, for example, the estrone content of the eluate and regenerate was within approximately 1055 mg ~ 4% in all cases, in keeping with the expected mass balance.
In summary, it may be concluded that the one- to eight-week storage of the loaded XAD-7 resin at temperatures of 4°C and 25°C
shows differences in elution behavior and the height and width of the hormone peaks, but not in the sum of all hormones eluted, which taken together were in the range of 1055 mg ~ 4%, relative to the estrone content. It thus follows that the observed qualitative differences are based for the most part only on nonhomogeneous flow conditions in the relatively small columns used, and may be easily avoided by the use of columns or cartridges with dimensions meeting standards in the field.
Table I.O:
Column test for stability of column material Loading of column with native urine from Poland, ultrafiltered Adsorber: XAD-7, volume: 3 L
After charging on the column, resin was discharged and subsequently washed with 3 L water. The resin was divided into equal portions of 300 mL each and subjected to a column stability test. One resin parcel was eluted immediately, while the others were stored at room temperature and under refrigeration.
a) Samples Volume Estrone HPMF Cresol Equilin L mg/L mg/L mg/L mg/L
Starting solution (after ultrafiltration) Control solution Control solution Control solution - 30 BV - Average b) Vol. Time Flow Flow Estrone Equilin HPMF HPMF Cresol Cresol Samples L hr L/hr BV/hr mg/L mg/L mg/L mg mg/L mg Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run 7 Run 8 Run 9 [see source document for table values; commas in numerical values denote decimal points . ]
Tnl,l c T 1 .
Column test for stability of column material Elution without storage Adsorber: XAD-7, volume: 300 L
Starting solution: native urine from Poland, ultrafiltered '7 ~ e-(O W ~'0 r-~ M
~
N O tn t0CD~ 1n('~
O f~~ M r-et~
The elution was carried out in a known manner by first washing the loaded adsorber resin with an aqueous sodium hydroxide solution. After each washing step, the respective wash liquid was analyzed for estrone (as the sulfate salt), equilin, cresol, and HPMF
content, using HPLC.
An ethanol/water mixture (30 wt-%, pH 12) made alkaline by the addition of sodium hydroxide was used as the elution liquid, and the resin was eluted at an elution temperature of 45°C. The eluate was collected in fractions, and the individual fractions were analyzed for estrone, cresol, and HPMF content, again using HPLC. The first fraction was collected as long as the eluate was colorless to pale yellow. This fraction contained only traces of estrone (estrogen sulfate salt). After the first bed volume of eluate had been discharged, the eluate turned an intense dark brown color. Approximately 80 to 90% of the total amount of conjugated estrogens adsorbed on the column was consistently contained in the subsequent 2 to 4 fractions. The remaining fractions for regenerating the resin (each at 45°C in the referenced sequence, with 50 wt-% ethanol at pH 12, 10 wt-%
sodium citrate, and water) contained only slight amounts of estrogen sulfate salt. This was also clearly evident by the decrease in color intensity.
B) Results The results of the column stability tests are presented for individual samples in Tables I.0 through I.9 and summarized in Table I.10, and are discussed in more detail below.
The loading of the column or cartridge with urine (see Table I.0) and elution of the control column (see Table I.1) showed a normal course of adsorption and desorption. However, elevated values of cresol and HPMF were found in the eluate, which could be attributed largely to incomplete laminar flow in the column or to the very high cresol levels (almost 1000 mg/L) in the initially collected urine.
Tables I.2 through I.9 give the results of elution after storage periods of one, two, five, and eight weeks. The basic objective was to determine that, as a rule, the adsorbed hormones (CE) were quantitatively desorbed. Occasionally, particularly after only one week's storage at 25°C, for example, quantitative desorption was determined only during regeneration with 50~
ethanol. However, this involved only desorption of CE residues, and the majority had already been isolated in the actual desorption. There appeared to be little or no dependency of desorption on the storage time and temperature, provided, for example, that possible non-optimal flow conditions did not play a greater role in the small columns. However, in all cases the eluate was essentially free of cresol and HPMF. To obtain optimal results, therefore, attention should be directed to optimal column throughput, regardless of the storage time. The columns or cartridges should therefore always be immersed in liquid; that is, dry operation should be avoided.
The results after five weeks' storage presented in Tables I.6 and I.7 show a satisfactory course of elution at a temperature of 4°C, whereas at T = 25°C, hormone still appears in the regenerate. The HPMF and cresol content in the eluate was very small in both cases.
Surprisingly, excellent results were obtained even after a very long storage time, as shown in Tables I.8 and I.9 for the eight-week storage. The elution peaks for estrone and equilin were very steep and sharp; that is, the volume of CE eluate was small. The cresol and HPMF values were practically nil.
Table I.10 gives an overview summary of all the tests. The sum of, for example, the estrone content of the eluate and regenerate was within approximately 1055 mg ~ 4% in all cases, in keeping with the expected mass balance.
In summary, it may be concluded that the one- to eight-week storage of the loaded XAD-7 resin at temperatures of 4°C and 25°C
shows differences in elution behavior and the height and width of the hormone peaks, but not in the sum of all hormones eluted, which taken together were in the range of 1055 mg ~ 4%, relative to the estrone content. It thus follows that the observed qualitative differences are based for the most part only on nonhomogeneous flow conditions in the relatively small columns used, and may be easily avoided by the use of columns or cartridges with dimensions meeting standards in the field.
Table I.O:
Column test for stability of column material Loading of column with native urine from Poland, ultrafiltered Adsorber: XAD-7, volume: 3 L
After charging on the column, resin was discharged and subsequently washed with 3 L water. The resin was divided into equal portions of 300 mL each and subjected to a column stability test. One resin parcel was eluted immediately, while the others were stored at room temperature and under refrigeration.
a) Samples Volume Estrone HPMF Cresol Equilin L mg/L mg/L mg/L mg/L
Starting solution (after ultrafiltration) Control solution Control solution Control solution - 30 BV - Average b) Vol. Time Flow Flow Estrone Equilin HPMF HPMF Cresol Cresol Samples L hr L/hr BV/hr mg/L mg/L mg/L mg mg/L mg Run 1 Run 2 Run 3 Run 4 Run 5 Run 6 Run 7 Run 8 Run 9 [see source document for table values; commas in numerical values denote decimal points . ]
Tnl,l c T 1 .
Column test for stability of column material Elution without storage Adsorber: XAD-7, volume: 300 L
Starting solution: native urine from Poland, ultrafiltered '7 ~ e-(O W ~'0 r-~ M
~
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Cn (nfn ~ ~ ~ ~ ~ ~ _ _ _ _ _ _ Q' Q' U LL ILl1!lJJllJIll Table I.2:
Column test for stability of column material Elution after 7 days storage at 4°C
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered 'e, ~ n ~ ' o rio o n ~
c~iW v v v rsw n uio . .
r r r r r N N r r v in c ~ o 00o v 7 a0 N C'~ e-' r O r N O ch v ~ v v v ~
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Table I.3:
Column test for stability of column material Elution after 14 days storage at 25°C
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered 'n ~ co v O ~fo w n ccSriv d' CVe-~ N ~ COO ~Il t0 C M ~ M ~ N
J O d' ~ O O OO
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I.
Column est ity ofcolumn material t for stabil Elution after storageat days Adsorber : e:3 00mL
XAD-7, volum h h h ~ ' ~ h - M ~ o L ~ ~ ~ - ooco~ rio o v h O
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Column test for stability of column material Elution after 14 days storage at 25°C
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered 7 ~ O O N ~ QjN O tn O
E O O O ~ N N ~ M
LIJ ~ c' M (O ~f7 CO O
~
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E
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O ~
O tri O 000t~r N 00 00P
UE ~ ~ ~ ~ ~ 0 0 ~ N N 0 0 ~ N t f p n O
O
O. ~ ~ O f0O r O ~ N ~ O O
E
t0 ~ 00r O O O O O O O
M
~c 1nr M N O h h (VM O O
= ~ O r (p~ O c~M O r O O O
E Op W N
O
N O
J _ b E r O
, r N i17CVp 00 N P
E
LU ~ GrD ~ ~ N f~IN ~ ~ N ~ 4N ~ ~ O
w-O
N N
c~ rgcp W ~ O ~ O CD
O) O t0 E ~ t0N t0 O M ~ N
W p_r N c a0t~P a O
O
r O
~ _ U7t0CDM N
o C
O
c7r C O O
'S
O r r r r O ~Otn~ d' P O
M C~jCjM t! t~C~7C~N N N r 0 N ~ r r r r r ~-m O
~
lL FV O O O O O O O O O O O
m C
E
C ~ N
v ~ r J
M Q W O N O W O Z o W O
E f~ n O O Q N
N N N N N N N N N N N N V N N N
_ a U
.
E O ~ N N ~ N N ~ N N ~ N N N N N
Z r r r r r r r r ~
J \ O
a ~ O ~ O O O O O O O O O O O o O O O
J O O O
J I~ ~ O O O O O M O O O O O O t0 c0 (O
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O
~ ~ a ~
f9 N
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M
~ ~ ~ 'v'Ø...~.m ~ C C C C
a ~ ~ ~ ~ ~ ~ > 3 ~ ~ m ~ ~ ~
Column test for stability of column material Elution after 35 days storage at 4°C
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered ~7 ~ N ~0 f~~ ~ O O
O
E ~ tV '''-M ~ M 00.- N
e-tf~~ (Ve- V ~ M e- O
7 00 O ~ O O ODCD(Op M
Ol E ~ M M N O
W f0 ( r t0~ M eT M
D
f'~ ~ O f0 tDe-I~GO
rn N 41 p~
j v o o'~oc'oaic3 aov o uiN co E
~ W ~ e- N N ~ V N v-t0 GD
N ~-O ~ W t~ff~ O O ~ ~ O cDc0 M
V (O t0 01CCN f~~T~ N O
E
O t0 t0O ~t (O.-c0M N
O n Q N N ~ O O
E
. ~ ~ O O (OO O
(p N ~ ~ O O O O O O O
'~o o ~ o o M o 0 = ~ ~ '~' v rio uio o .-0 0 E
m O
J
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LJ ~ OrD N ~ N N Cf N n N cn0N
w O
N N N
b M O O M M O M N O
O
W e- ~ a0 N O>c0 c'~~ c0N c0 E
IJJ O . 00vt ~ 00h h N f~N (O
r O
O
~ _ (pO ~OM e-o C
N
c~N O O O
V
~S
Z C O ~ e-O O 1~M COl0 d y N cj cjfrjfh f'~j(V(VN N N
N ~ r r-v m O
j ~1- H O O O O O O O O O O O
(~
III~ Ilktnlf~ InIntn1A1f) C
C ~ N
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~ M y n ~n u o m m ~nu~ ui E
f9 N N N N N N N N N N N V N
_ o = ~
_N
v d O N V $ O
C_ E N ~ N N N N N ~ N N N N N ~ N U
O
r ~ ~7 IO
- -J Z ~ Z
$J 0070 .'x'0 0 0 0 0 0 0 0 0 0 0 o O o O O O O
> 1~ ~ O O O O O M O O p O
E 'C!~ ~ M M M M M ~ M M M M M ~ (D
' O C
p y, _ ~ N M Y N y w ::.O.N ~ N
~ f l9 7 7 O
~
O ~ fy0N f0f0 f00 ~ _7 _3_O_7_ _ ~S~ ~ ~ ~ l w W W W UJ
llJ fnfn l1.
Table I.7:
Column test for stability of column material Elution after 35 days storage at 25°C
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered ~ v w n ~r.'ty n r o E -~ M CDd; ~
,~
W !f7 ~ 4 ~ r r ~ N r h C~ M
C
7 00 ~ ~ N O ~ N ! M
O) "
n r t WE O ~ ~ r ~ - N N
( C'e V)<1 f 00~
p 7 - D
~ M ~ 00h M
E M 00 O !f) o U M ~ ~rM O ~ O ~ r N
c' V CG
~
~
M ~ C CCr d N N r ~
U ~ ~ o 'rn ~ o ~ ~ ~ c E ~' r (Or M r e-r h N
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c~~ o m o o N o w .~CD O~e-c r O f~O O (V O O
i f0 N
N
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LIJ ~ O N (VC3 ( r-p 0 O
N N
~O ~ ~ h V O O
b' Ih ~ O 00N h ~ r O
IWE ~ ~ n '~' r = ~ v a a m v cw d d o ce _ ~
I W n co~ M M
-'a rir o 0 0 d .s C O r r r O O f0V M M
4 v~ N c'~M c~M ciN tV(V N N
N ~ r r r r r r r r N
_O
j H o 0 0 0 0 0 0 0 0 0 o V
~iSri~iui~ci SriaiuiSri~i Sri m N
C ~
O
_O .~ a Z O
~ M - i ti E m ~n~ u ~w o u~ Sry N N
N N N N N N N N N N V N
r n v ~
_ E ~ o U 3 ' ~
4 r N r N ~ r r r r r ~ N
~p ~
Z r J \ N Z
~ ~ 4 J O O O O O O O O O O O W O W
t~ ~ O O O O O M O O O O O O
O p E P M M M M M M M M M M ~ (O
O
~ N $
j N
a .. ~, a '~~ '~ ~ '~~ '~~ = ::::?? a:m ~
m , ~ > > > 3 5 l v~ ~ac~ m m m ~om ca w W W W
vov~ ~ ~ ~ ~ ~ ~
w Table I.8:
Column test for stability of column material Elution after 56 days storage at 4°C
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered 'a v ~ coco E CON
~ chN ~ M ~ 0 M
t r ~ N ~ ~ a0c'~ c f> ~ 7 O7 a0 CD~ O a00EliN N O) E O ~ N r ~ r O N
( r l~~ M V
O ' a0 O
O N ~ O r' E ~ ~ ~ ~ QjN N O O O O O
1~ C~'-N e-h O O O O O
$ c~O
c~
M N
O In f~O CfO W I'~
E (p O O M c'~7 O O O O O
O N f~t0N sfO O O O O
Of 1~ ~ N N
E
M O O 1~O P O 00 ~ ~ ~ O
~ O O O
N
M O c'~a0O ~fO O
= 0 ~ M M
E
~ a V V O cMtriO CVO M
f0 N
N J
~d E ~ ~
v~
i..v a> u~o v E
lL ~ 0~0 N N N CVCV O f~ON t~DN N
O
N tn aoo r aooof ac'r E
W Q r~- ~ n ~ aDOD tI~N ~ N OMD
O
_O
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f~ OJO tn N e-N
M (VO O O
~S
Z C O ~ N r v-00V C7t~ N
~uf N C~f~c'~M c'~(Vc~If~N N
d ~ f m O
_ j ~m Fv 0 0 0 0 0 0 0 0 0 0 o V
c uiiriviSriui uiSrici ~ciSri Sri m c ~ f, _ ~
o ~
_ ~ Z o J a N N
M n C) f7A ~ O f7 f7 f0 N N N N N ~ N N N N N N
V
o Ri a v . o>
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E
f:.O Q ~ N ~ N N ~ N N N ~ N
~ r Z
J ~ N Z
~ 0 $J O ~ 0 0 0 0 0 r~ 0 0 0 0 0 \ O \
n 0 0 0 0 o 0 0 0 0 0 o > r~ r>r~c0c~c0. c0c~r~ r~r~,~ cn E o ~ ~ a c c m W
C N O m - ~
Q- ~ a L = ~ ~ N ~: .~.~ N N N
~ ~
N ~ N N fN4f N N 7 7 > > 7 7 N ~ N
~ 0 u v~ cn n ~ ~ ~ 3 3 ~ u W W a w <
Table I.9:
Column test for stability of column material Elution after 56 days storage at 4°C [sic: 25°C]
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered =v ~
E ~ ~.d~M N r M 000'NQCOc'~ t7 ,~-tf) r M C~r r 00M r-M r N
7 ap cp~.M,j O a00~1')CVN p C~
W (p0 r Is~ c~ N N ~ r ~ N
E
p N ~ O
O ajN N O O O O O
1~ M e-N r f~- r O O O O O
M O
cO
t!) ~ O CVM p !~
O O O O CO
V ~
E
~ r ~ ~ N v o 0 0 0 I' ~ N N
a E
M O O f~O P O OD
~ Ch~ O
d r O O O r N
a ~ ~n~ r o M o M aoo v o 0 E
= ~ ap0 ~V~ ~ ~!O ch~ O CVO ch N
N
N J
E '~ '~' n.v w o o v E
W M On0 N N CV(Y(V M ~ N (00N N
O
y b ~ o I c aoa0 r aooofao~ 't o i E
l1 a ~ o00n 0ia0op ~ N tODN OMO VN' O
r O
fn _ o COO ~ N r y M (VO O O
'S
'b Z C O r N r r 00etM M N
N W N c,~~,jch~j M CVCVfVCV N
d ~ r r r e- r r r N
O
j ~m Fv o 0 0 0 0 0 0 0 0 0 o V
SriSriSriSriui ~ciSriuiSriSri ~ci rr E m ~ o ' ' M _ I o a J Z m m m ~ n ti m n ~ iri E
t9 Q N N N N N ~ N N N N N N
V
R n v i c ca N N N N N N ~ N N N N N
r r r r ~ r r r r ~ N
Z N
J ~ N Z
~ ~ O
a O O O O O O O O O O O o ~ o J t~~ O O O O O M O O O O O
E p p I~ M C7M M M M M M M M
O
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m _ N
N W f0 f9 .. I v-N M ~ M
' ~' ~ ~ ~ '~~JSfTi'~,~T~ d m ~ m a?~ N N
~
~u ~ m m m m m i _> _>_>_>_>_>~ ~ y ~ '' m ~
~
M (n n ~ S ~ ~ ~ S W W W W W W
( U
Table I.10:
Summary of results of column stability tests a E
m m n E
co m a E
w a E
m v a~
a E
m M
m c.
E
m N
N
a E
m a~
a E
m m v m U
o 1 ~ ~ ~ Resin discharged, divided into portions J
t N
E T E E ~ E w , ~ ~ > i ~ ~ ~ ~ ~ ~ o m a c ~ ~ ~ ~ ~ c m ~ i w w ~i w o ~ w w w a W w n WO 02/09732 PCT/EPO1l08657 Example 3:
Stability tests with loaded adsorption columns for the determination of bacterial counts The microbiological quality and harmless effects of natural starting materials for the production of pharmaceutical preparations have become increasingly important in recent times. Therefore, analyses were performed to determine bacterial counts in samples after storage of columns for five or eight weeks, according to customary methods (pharmacopoeia).
The results of the bacterial count determinations are presented in Table II. The total bacterial counts at a storage temperature of 4°C
were characterized as small. At a storage temperature of 25°C, the column wash water had 1.2 to 4.6 * 106 organisms, and the eluate had 80 to 170 organisms. However, it is noted that the stored column adsorbents were not washed or treated with preservative beforehand, and thus were previously exposed to increased risk from bacterial growth. During storage without refrigeration, therefore, it is recommended that the loaded columns be thoroughly washed at least with water, and advantageously with additional stabilizing or preservative wash solutions such as sodium chloride solution.
m..y.i .. Tr _ Stability tests for bacterial count determination Storage Storage for for 5 weeks 8 weeks Storage time at C
Column wash water Basic wash water Ethanolic eluate *Data in CFU/mL (colony-torming um ts) [see source document for table values; commas in numerical values denote decimal points.]
Example 4:
Stability tests with refrigerated, loaded adsorption columns As the result of seasonal or regional conditions, temperatures may fall below the freezing point during the storage or transport of loaded columns or cartridges, with the undesired result that the aqueous contents of the columns or cartridges solidify.
Corresponding tests support the conclusion that the freezing process appears to have a negative influence on the elution of rethawed columns or cartridges. If the column cannot be tempered, or storage takes place at low temperatures, it is recommended that the last wash be performed with suitable freezing mixtures instead of water to prevent freezing.
A) Test procedure:
From the numerous possible freezing mixtures, of which several are given as illustrative examples in Table III.1, a sodium chloride solution, for example, was chosen for further tests.
The addition of 23 g NaCl per 100 g water resulted in a freezing point of -21°C.
The column loading proceeded in a conventional manner. After the urine was loaded, rinsing was performed with 3 bed volumes water, followed by 2 bed volumes of a 33 wt-% NaCl solution. The column was then stored for 9 days in a chest freezer at -19°C.
Table III. I:
Examples of liquid aqueous salt mixtures and freezing mixtures Achievable temperature g A/100 g in C ubstance A coolant ubstance B
water water ice water water water ice ice ice ice ice 66.1 HzS04 snow ice ice [see source document for table values; commas in numerical values denote decimal points.]
B) Results Even after 9 days of storage at -19°C, the contents of the column remained liquid, with no signs of partial solidification.
Processing of the column, that is, basic washing, elution, and regeneration, proceeded normally. The total hormone content per total solids was approximately 22 wt-~. The results are shown in Table IIT.2.
Negative effects of the high NaCl content on the CE content and the separation of cresol and HPMF were not detectable. In summary, it may be concluded that stabilization of urine-loaded columns or cartridges using a liquid freezing mixture such as an aqueous sodium chloride solution, and storage under refrigeration, for example, down to approximately -20°C, WO 02/09732 PCT/EPO1/0$657 have an advantageous effect, even for periods of 1.5 to 2 weeks.
The contents of the columns or cartridges remained liquid, and processing of the columns or cartridges to isolate CE by washing, elution, and regeneration proceeded normally. Negative findings were not detectable, even at high NaCl concentrations.
Table III.2:
Column test for stability of column material Elution after 9 days storage at -19°C
Adsorber: XAD-7, volume: 200 mL
N ~ pO O
O a0~a0.-O N O ON O
aE
z OO O t0N NN 0 O ~ M
1AO O~ N C
c 0 C C
m ~ M O O N ~ 1~n M
N
W
m ~ tD ~ r bp1 OO O OO 00 Oo 0~ 0 0 ~ p ~o . c C c p -N N
E
W
C
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m O~ ~ rN N(~~7 tD~ ~'~ ~ N a0 ~ N ~O N c O
~
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U
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~ ~
m U
~ w n _ c c ~ r VV ~~ w ~O N N N N h h NN
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C D 0G D tCtG
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a~
.
C ~ W ~ N ~'N N - N ~ ~ ~ V' E Nc0N (Om t0aD t0~ .- U ~ . ~ c9N N
tD fD MN
E ~ Z b N
O i1 V ~ t0 M Z y Z
C t0V ~ NN N N NO N N NN N o 0 j ' oc ~ cc o c ca c c o0 0 ,~o ~ c d E
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p . c .
C _~_ c N M V In~ N M~ inm~ ' N M
N M V~ f0h t ~ N N ~ CC c C
m d j ~~ ~ .~ ,~~l~,a I 7A 7 m I 3 ~ ~N N N !O~ O 7 N
U 33 N ~3 ~ ~ ~7 7 7 totoui w to WO ?2/09732 PCT/EPO1/08657 Example 5:
Concentration and stabilization of urine in a field test A) General The objective of a field test is to show that, with sufficient hormone content in the urine, satisfactory results can be obtained not only in the laboratory, but also under production conditions. A test was conducted under the same conditions that would occur during a sampling session at any given location.
B) Test procedure Fresh urine of sufficient quality was collected at a stud farm.
With three horses and 15 to 20 L urine/day, a total of approximately 150 to 200 L was obtained in 10 days. Therefore, a 5-L adsorption column was chosen for adsorption. The daily loading with urine, excluding weekends, was carried out according to the following program:
Start of urine collection on the weekend Monday: Charging of urinefrom Saturday, Sunday, and Monday Tuesday through Charging of urinefrom each day Friday:
Monday Charging of urinefrom Saturday, Sunday, and Monday Tuesday Elution The urine was charged directly on the column at a loading rate of about 4.5 bed volumes/hr, with only one cotton plug with a separation limit of 20 um serving as a prefilter. No water rinsing was performed between the individual charges in order to make the conditions more severe. In pretests it was possible to deliver up to 450 L urine through the cotton plug with no pressure loss. In this field test, filtration proved to be difficult on account of an unexpectedly high amount of calcium carbonate sediment and/or mucins.
After each 40 to 50 L, a blockage appeared, along with a pressure rise of approximately 4 bar, which required the filter to be replaced. Depending on the calcium carbonate and/or mucin content, the optional addition of filtration aids is recommended.
Although the plug allowed sediment > 20 um to pass through, [filtration aids) were not added to the adsorption column.
C) Results Hormone content The results of this field test are presented in Table IV.
The estrone and equilin content of 123 mg/L and 74 mg/L, respectively, in the raw urine were surprisingly high, corresponding to urine of average to good quality. However, the cresol values, at 531 mg/L, were unexpectedly high in fresh urine, which hitherto had not been observed. The issue of diet is a factor deserving consideration here. Although a basically higher cresol content, for example, can be overcome, in individual cases it may be problematic. Therefore, it may be necessary to prolong the alkaline washing process during processing to minimize the cresol values in the ethanolic eluate. Otherwise, the adsorption and desorption proceeded normally. The eluate showed a pronounced content of estrone and equilin. Eluates 2 and 3 contained more than 95°s estrone/equilin. The total hormone content relative to the total solids content was approximately 30 wt-~, thus meeting expectations for the test and achieving superior concentration to a hormone/total solids content greater than 15 wt-~ under severe test conditions.
Table IV.1:
Field test for stabilizing and concentrating CE
Test days: 1-10, column loading (on 7 working days) Adsorber: XAD-7, volume: 5 L
Starting solution: native urine, untreated The column was traversed from bottom to top.
c o0 oc co ccc o0o c c o0 oO cc cco co0 0 NN ~M ~~ aM0~m ~~m t00 O NN ~~ NW ~~m ~~m t~0 O OO OO OO OOO OOO O
n t0 ~V C~~fN~ ~O a0~< O
V V~fQ~ N~ VV~ ~f< ~
~ vv ~~ aoaoaoaon.ovv n v oo ~:o ~ cc oo ~
a cocnnlMM c~ d c~
a N_ NN NN N~ ~~N NNN N
t4 Z
O OO OO OO OOO OOO O
-r C NIh~N t0f~a0W~ ~r.M-.~-A CC CC CC CCG CCC C
C 7~ 77 77 777 777 7 7 d'd'~~ 2~ OCQ'~ ~d'~ ~
~
Table IV.2:
Field test for stabilizing and concentrating CE
Processing of column on 11th test day Adsorber: XAD-7, volume: 5 L
Starting solution: native urine, untreated The column was traversed from bottom to top.
w cv ~ ~ ~ 0 0 0 0 0 0 0 et N
C
O t!7O m ' ~n o, ~ o ~ c O
E , W c o V' r O o O O O O) N ~ 1~.O O O O
p1 N O N O ~ O O
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N
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m N
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N
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M f~D O C j f0 U
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c c ~ O O M M M , ~E
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v v v v v i: cciaiv d' o v o d o E
E
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d ~ ~ ~ y ( ~ O (Oet~ d O a.:O O 7 N ~ ~ ~
- M M M M M .( t ~ ~ ~ >
v p a v ~ y ' c E O tc)! ~ ~ inpp O N M M ~ ~ ~ ~ ~ C
c r ' E
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j ~ ~ ~C7In!l7~t7117~ ~ M tntnIn~ O ~ O O
J
r r N
~ r ~
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c r N M ~ III~ r N M ~ tO~ r ~ N M ~ U
c _ '~n ~ ~ ~ ~ m m m a;a;~ d ~
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f~ f9f0t0ID (0t0p _>_> 7 _7_77 ~
~SLU
UJ W LUW W ~ ~ ~ ~ ~ ~
Bacterial counts In addition to urine adsorption, possible growth of bacteria on the column was investigated. Under the following conditions, 1. Charging of contaminated raw urine (no bacteria were separated by cotton plug filtration), 2. No rinsing after daily charging, 3. Test carried out a room temperature, and 4. Test duration of 10 days, the bacterial density on the column and subsequent growth thereon were not precluded. Determination of the total bacterial count at different adsorption steps gave the following results:
Sample Total bacterial _ coun_t/mL
Raw urine Column wash water after 10 days Basic wash water Main eluate Isee source document for table values; commas in numerical values denote decimal points.]
D) Summary of results Fresh urine was charged on a 5-L adsorption column over a test period of 10 days. The entire volume of urine was 140 L, with an average estrone and equilin content of 123 mg/L and 74 mg/L, respectively.
Column rinsing with water after the daily urine adsorption was intentionally omitted. Both of the very pronounced main eluates contained approximately 93~ estrone and equilin. The total hormone content relative to total solids was approximately 30 wt-~. A further surprising result related to possible bacterial contamination of the column. In spite of charging highly contaminated raw urine, bacterial growth on the column itself was not detectable after 10 days. With l.5 organisms/mL, the eluate obtained was practically bacteria-free.
Hence, under severe conditions it was proven on a large scale which, with sufficient hormone in the raw urine, stabilization and concentration to a hormone/total solids content of greater than 15% and of good quality can be performed using an adsorption column.
Example 6:
Filtration tests using filter cartridges Filtration tests using filter cartridges were carried out in a further field test, the results of which are summarized below.
Related material: fresh urine (origin: a German stud farm) The urine of pregnant mares which had been collected in drums and occasionally agitated was pumped from the drums using a hose pump, through a 5u bag (prefilter), and into a container. Sediment of about 0.5 wt-% remained in the container. Figure 2 shows a schematic representation of the method for filtering raw urine.
~..~.. _ Bag 5 a Filter cartridges 5u, 3u, 1u connected in series Membrane filter 2 x 1.2u connected in parallel Column loading 95 L filtrate Sediment filtrate 0.000%
Bag 5 a Filter cartridges 5u, 3u, 1u connected in series Membrane filter 2 x 3u connected in parallel Column loading 142 L filtrate Sediment filtrate 0.001%
Bag 5 a Filter cartridges 3u, 1u connected in series Membrane filter 2 x 1u connected in parallel Column loading 160 L filtrate Sediment filtrate 0.000%
In each test, one sample was taken downstream of the membrane filter. After every 100 L a sample was taken from the outlet, downstream of the column.
Total column loading: 397 L filtrate The total solids (TS) content after processing was 5.9 wt-%.
m E c0 tha0O ~ O tUN (Ooff~ 00 (p tn~ M ~ IC) O ~
a0 O O
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p d N ~ ~ 0 0 p O O N ~ N
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C
n a N N N N pp ~DC~ N N N N N f07 7 7 7 7 0 ~ I
Cn (nfn ~ ~ ~ ~ ~ ~ _ _ _ _ _ _ Q' Q' U LL ILl1!lJJllJIll Table I.2:
Column test for stability of column material Elution after 7 days storage at 4°C
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered 'e, ~ n ~ ' o rio o n ~
c~iW v v v rsw n uio . .
r r r r r N N r r v in c ~ o 00o v 7 a0 N C'~ e-' r O r N O ch v ~ v v v ~
c~ou~
N O
E c'W - CVcMN IwN O O N
o N
aoc o aoui ado ofui cc f~ I~r etr-O M ~ N r ~
$ ~ O M
c~
th M O O N
1 ~
~ c'~o co~ ~ : ago N ~ o N o VE ~' O N ~ r lDr O ~ M N
O l~ r a ~ ~ o .
E
,~o , ~ 0 0 0 0 0 ~
(D N r ~ O r O O O O O
~
h to ~t E O M ODO O O O O
000 COOC'~'7cN7O ~f ~ O O C O O
N
N
N J
O E CO chr O
E O r M M
LJl~ a~0 N O CVC~CV l(j~ N r~COO
O
N N
c0 N N a0(DO tO
b f O n ~tcD h C'~r r E '~ O ~ M
l11p_ _ O ~ D1~ W 0 ' N
r O 0 Q
O
r I O
f~- d 1~ t0~ N
C~r C O O
.5 Z C r r r r r O tD
~N O M M ~''7c'~ c'Mc"~C~t~IC~ N
'1 ~ r r r- r r r r v. N
O
_ j 0 0 0 0 0 0 0 0 0 0 o V
C O O O 47O 10~ 1n10O O
$
O r ~' O
~ M Z
a J
LL
E a o o u m w w n ~mn o Sri (C N N N N N ~ N N N N N V N
d ~
' N Q
!9 E ~ U
O ~ N N ~ N ~ N N ~ N ~
.d.r Z r r r r r ~
J Z
m ~ 0 aJ 0 . 0 0 0 ~ 0 M 0 0 0 0 \ O \
!~. O O O c~O O O O O O p E f~ M Ihc'~C~M . M c'7l'~C~c'~p (D
O
' a ~ r.
N
j ~ C
N ~jJ N f9 C N M O
C C t ~II~ '~-'a0.m w N N N
~
N ~
..~~ c9 l9f9f9f9f9_7 _~_7 ~ _7_7C1 N N
fn fn ~ ~ ~ ~ ~ ~S~ ' (n U 11JL11llJLiJliJLU~ Q
Table I.3:
Column test for stability of column material Elution after 14 days storage at 25°C
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered 'n ~ co v O ~fo w n ccSriv d' CVe-~ N ~ COO ~Il t0 C M ~ M ~ N
J O d' ~ O O OO
y c aiSri ~ ~ ~ M ' .
y n M o N o~ o~o coao .- N
E ~ 'n 'r of ~ ~ cue,d.~ ~ ~ c v N
' c U r n y n ~no o u>co~ o 0 d tnO N O CV M CDODnj (D (V
~
n r M N M CO ~f M
O ~ V M O
O
p) I~ O ~ ~ N ~ ~ O O O O O
o ~ ~ ~ ofv rio 0 0 0 0 ' Q , ~
n M o o ~ cuo 0 0 0 0 E ~ N n r' ''0 0 0 0 0 = ~ , c, ~
~ O 00M h M O v-$ ~ I~ 00~ ~ (O O
p) ~ ~ V V
N J ~ N I ( ( C t~N N O O
E
Lll ~ a0 O
O
m M
O (O n ~ O M h O M O r M OD
0oadao aNOo v '- v c~
O O CO M
a o v n aom v H
o _ ri~ 0 0 0 c O O ~ e-M r-O CON ~ O
'S O C~cMCMM chM N (V N N
a C
m 3 ~ O O O O O O O O O O O
U7u7v!)~ ~ W f)~ N ~ 1A
m D
U
_ c c v N N N N N
N N N N N T N
U O
M a a ::
c0 N
o N N N N N ~ ~ N N N N
- - ~- v-~ N
y ~ e ~ d N
U ~ b o O v ~ 'c~
0 O Z ~ O O O O '~'O O O O O ~ O f6 E J n M CO~M CO~M o M ~ CO~CO~c'00O cOpZ
~ O o 0 M O
~O
of . w .
y _ ~ N M ~ O O N, . c _ ~ ~ ~ ~ '~~ '>m ''m w a~
m ~ m ~ m m m ~o> > > > > > ' ' 1 l ~ a ~ ~
~ ~~ ~ ~ ~ ~ ~ ~ w w ~ w ~ w Table 4:
I.
Column est ity ofcolumn material t for stabil Elution after storageat days Adsorber : e:3 00mL
XAD-7, volum h h h ~ ' ~ h - M ~ o L ~ ~ ~ - ooco~ rio o v h O
E ~ fV o r N h l1 ~Y
r C M
~ M t0 O
(p InCO r d'M h ~'j(pM r C~7 N M O
(p tn e-~ tri~ V r M 00N (VO
a0 O M
M M O
N M f0 O ~ ~ O M CD CO ~ O
E o n:
~ O ~ O
V f'~ r et r O r !DfV r O O
M
tN t~f>O ~ M t!7 N O ODO O O ~'O
V ~ r O ~ ~ ~ f'~~C l N GD ~ N O
E
C V
O
LL
h ~ v a ~ ~ r o 0 0 0 0 0 0 0 0 0 E
s co N r aio 0 0 0 0 0 0 0 0 r a ~ ' ~ '~'0 0 0 0 0 0 0 0 0 0 ' E
z r~ ~ ~ ~ o o uico0 0 0 0 0 0 ~, a~
O J
' E h a'g M M CO h N a c - v o ~ o ~ v cow o .i N
E h N
LJJ~ OhD N t0(VfV(V N n M r (VO
O
N rn O
~ N
M o ~ 0 0 t~ 00N O N P ~
E
(y r ~ N GOa0aD ~ W I r M N ~ O
r O
O
~ _ N OD~fr r o C
'fl C~r O O O
O
, a ~S
Z C N ODtDc0c0 tD sTV
~
Q yj M ~.jwjwj~j ~.jfvlN N N CV fVr O Qj r r r r r r r r r N
N
LL H O O O O O O O O O O O
m ~ tnInl~~f7 1nIl7lf~InIn C
E m C
Z O
O J
~
_ O ~f7M M ~ N U7M 47~ d tri'N M ~O
E N N
(p d N N N N N ~ N N N N N V N ~
' ~ ti a ' _ E O ~ N N N N N ~ N N N N N _ ~
r ~ r '-r r N N N
r r r r ~
Z
a ~ O ~ O O O O O O O ~ O O O o O o O O
J ' M o 0 0 0 0 0 0 0 J h ~ 0 0 0 0 o M M M M M 0 CO,_ (O(O
E h M M M M M O
O
~ ~ $
p C
f0 ~ ~ N M
N r N M r N M t X11 Y t ' ~ ~~ C ~ ~ ~ '~~ ~ c~~o~ c~~om A ~ d m ~
~ ' : ~ m co m m m m > > > 3 > > u~ d d ~ d r~ 1 l ~ ~~ ~ ~ ~ ~ ~ ~ w w ~ w ~ ~ ~ ~ ~ ~ ~
Column test for stability of column material Elution after 14 days storage at 25°C
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered 7 ~ O O N ~ QjN O tn O
E O O O ~ N N ~ M
LIJ ~ c' M (O ~f7 CO O
~
7 00 ~ ~ O ~''~~ p~sfc0 r h Ol M
to ~ ~ M i a ~ ~ v Tv m '~ o E
, c n ao 0 O f0N
d N ~ tD(V
M O pp I~ M N
E
V ~ ~ c~0N N O) O C O N CO ~ d~ O
O ~
O tri O 000t~r N 00 00P
UE ~ ~ ~ ~ ~ 0 0 ~ N N 0 0 ~ N t f p n O
O
O. ~ ~ O f0O r O ~ N ~ O O
E
t0 ~ 00r O O O O O O O
M
~c 1nr M N O h h (VM O O
= ~ O r (p~ O c~M O r O O O
E Op W N
O
N O
J _ b E r O
, r N i17CVp 00 N P
E
LU ~ GrD ~ ~ N f~IN ~ ~ N ~ 4N ~ ~ O
w-O
N N
c~ rgcp W ~ O ~ O CD
O) O t0 E ~ t0N t0 O M ~ N
W p_r N c a0t~P a O
O
r O
~ _ U7t0CDM N
o C
O
c7r C O O
'S
O r r r r O ~Otn~ d' P O
M C~jCjM t! t~C~7C~N N N r 0 N ~ r r r r r ~-m O
~
lL FV O O O O O O O O O O O
m C
E
C ~ N
v ~ r J
M Q W O N O W O Z o W O
E f~ n O O Q N
N N N N N N N N N N N N V N N N
_ a U
.
E O ~ N N ~ N N ~ N N ~ N N N N N
Z r r r r r r r r ~
J \ O
a ~ O ~ O O O O O O O O O O O o O O O
J O O O
J I~ ~ O O O O O M O O O O O O t0 c0 (O
E h M M M M M M M M M M
O
~ ~ a ~
f9 N
.. r N M etM ~ r N M
M
~ ~ ~ 'v'Ø...~.m ~ C C C C
a ~ ~ ~ ~ ~ ~ > 3 ~ ~ m ~ ~ ~
Column test for stability of column material Elution after 35 days storage at 4°C
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered ~7 ~ N ~0 f~~ ~ O O
O
E ~ tV '''-M ~ M 00.- N
e-tf~~ (Ve- V ~ M e- O
7 00 O ~ O O ODCD(Op M
Ol E ~ M M N O
W f0 ( r t0~ M eT M
D
f'~ ~ O f0 tDe-I~GO
rn N 41 p~
j v o o'~oc'oaic3 aov o uiN co E
~ W ~ e- N N ~ V N v-t0 GD
N ~-O ~ W t~ff~ O O ~ ~ O cDc0 M
V (O t0 01CCN f~~T~ N O
E
O t0 t0O ~t (O.-c0M N
O n Q N N ~ O O
E
. ~ ~ O O (OO O
(p N ~ ~ O O O O O O O
'~o o ~ o o M o 0 = ~ ~ '~' v rio uio o .-0 0 E
m O
J
E ,~ cuo o~n o 0 E
LJ ~ OrD N ~ N N Cf N n N cn0N
w O
N N N
b M O O M M O M N O
O
W e- ~ a0 N O>c0 c'~~ c0N c0 E
IJJ O . 00vt ~ 00h h N f~N (O
r O
O
~ _ (pO ~OM e-o C
N
c~N O O O
V
~S
Z C O ~ e-O O 1~M COl0 d y N cj cjfrjfh f'~j(V(VN N N
N ~ r r-v m O
j ~1- H O O O O O O O O O O O
(~
III~ Ilktnlf~ InIntn1A1f) C
C ~ N
U M ~ O
_O ~ r' ~
~ M y n ~n u o m m ~nu~ ui E
f9 N N N N N N N N N N N V N
_ o = ~
_N
v d O N V $ O
C_ E N ~ N N N N N ~ N N N N N ~ N U
O
r ~ ~7 IO
- -J Z ~ Z
$J 0070 .'x'0 0 0 0 0 0 0 0 0 0 0 o O o O O O O
> 1~ ~ O O O O O M O O p O
E 'C!~ ~ M M M M M ~ M M M M M ~ (D
' O C
p y, _ ~ N M Y N y w ::.O.N ~ N
~ f l9 7 7 O
~
O ~ fy0N f0f0 f00 ~ _7 _3_O_7_ _ ~S~ ~ ~ ~ l w W W W UJ
llJ fnfn l1.
Table I.7:
Column test for stability of column material Elution after 35 days storage at 25°C
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered ~ v w n ~r.'ty n r o E -~ M CDd; ~
,~
W !f7 ~ 4 ~ r r ~ N r h C~ M
C
7 00 ~ ~ N O ~ N ! M
O) "
n r t WE O ~ ~ r ~ - N N
( C'e V)<1 f 00~
p 7 - D
~ M ~ 00h M
E M 00 O !f) o U M ~ ~rM O ~ O ~ r N
c' V CG
~
~
M ~ C CCr d N N r ~
U ~ ~ o 'rn ~ o ~ ~ ~ c E ~' r (Or M r e-r h N
V.
g n v~
~ ~ ~ r ~ ~no M o o n o o~
E
(O N 1C7r c'~O N G O O O O
V.
c~~ o m o o N o w .~CD O~e-c r O f~O O (V O O
i f0 N
N
E ~ coo ~n ~ M m n N r M d ~ ~ r 0 N
LIJ ~ O N (VC3 ( r-p 0 O
N N
~O ~ ~ h V O O
b' Ih ~ O 00N h ~ r O
IWE ~ ~ n '~' r = ~ v a a m v cw d d o ce _ ~
I W n co~ M M
-'a rir o 0 0 d .s C O r r r O O f0V M M
4 v~ N c'~M c~M ciN tV(V N N
N ~ r r r r r r r r N
_O
j H o 0 0 0 0 0 0 0 0 0 o V
~iSri~iui~ci SriaiuiSri~i Sri m N
C ~
O
_O .~ a Z O
~ M - i ti E m ~n~ u ~w o u~ Sry N N
N N N N N N N N N N V N
r n v ~
_ E ~ o U 3 ' ~
4 r N r N ~ r r r r r ~ N
~p ~
Z r J \ N Z
~ ~ 4 J O O O O O O O O O O O W O W
t~ ~ O O O O O M O O O O O O
O p E P M M M M M M M M M M ~ (O
O
~ N $
j N
a .. ~, a '~~ '~ ~ '~~ '~~ = ::::?? a:m ~
m , ~ > > > 3 5 l v~ ~ac~ m m m ~om ca w W W W
vov~ ~ ~ ~ ~ ~ ~
w Table I.8:
Column test for stability of column material Elution after 56 days storage at 4°C
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered 'a v ~ coco E CON
~ chN ~ M ~ 0 M
t r ~ N ~ ~ a0c'~ c f> ~ 7 O7 a0 CD~ O a00EliN N O) E O ~ N r ~ r O N
( r l~~ M V
O ' a0 O
O N ~ O r' E ~ ~ ~ ~ QjN N O O O O O
1~ C~'-N e-h O O O O O
$ c~O
c~
M N
O In f~O CfO W I'~
E (p O O M c'~7 O O O O O
O N f~t0N sfO O O O O
Of 1~ ~ N N
E
M O O 1~O P O 00 ~ ~ ~ O
~ O O O
N
M O c'~a0O ~fO O
= 0 ~ M M
E
~ a V V O cMtriO CVO M
f0 N
N J
~d E ~ ~
v~
i..v a> u~o v E
lL ~ 0~0 N N N CVCV O f~ON t~DN N
O
N tn aoo r aooof ac'r E
W Q r~- ~ n ~ aDOD tI~N ~ N OMD
O
_O
~I W
f~ OJO tn N e-N
M (VO O O
~S
Z C O ~ N r v-00V C7t~ N
~uf N C~f~c'~M c'~(Vc~If~N N
d ~ f m O
_ j ~m Fv 0 0 0 0 0 0 0 0 0 0 o V
c uiiriviSriui uiSrici ~ciSri Sri m c ~ f, _ ~
o ~
_ ~ Z o J a N N
M n C) f7A ~ O f7 f7 f0 N N N N N ~ N N N N N N
V
o Ri a v . o>
' m E v I U ~ 1, W
E
f:.O Q ~ N ~ N N ~ N N N ~ N
~ r Z
J ~ N Z
~ 0 $J O ~ 0 0 0 0 0 r~ 0 0 0 0 0 \ O \
n 0 0 0 0 o 0 0 0 0 0 o > r~ r>r~c0c~c0. c0c~r~ r~r~,~ cn E o ~ ~ a c c m W
C N O m - ~
Q- ~ a L = ~ ~ N ~: .~.~ N N N
~ ~
N ~ N N fN4f N N 7 7 > > 7 7 N ~ N
~ 0 u v~ cn n ~ ~ ~ 3 3 ~ u W W a w <
Table I.9:
Column test for stability of column material Elution after 56 days storage at 4°C [sic: 25°C]
Adsorber: XAD-7, volume: 300 mL
Starting solution: native urine from Poland, ultrafiltered =v ~
E ~ ~.d~M N r M 000'NQCOc'~ t7 ,~-tf) r M C~r r 00M r-M r N
7 ap cp~.M,j O a00~1')CVN p C~
W (p0 r Is~ c~ N N ~ r ~ N
E
p N ~ O
O ajN N O O O O O
1~ M e-N r f~- r O O O O O
M O
cO
t!) ~ O CVM p !~
O O O O CO
V ~
E
~ r ~ ~ N v o 0 0 0 I' ~ N N
a E
M O O f~O P O OD
~ Ch~ O
d r O O O r N
a ~ ~n~ r o M o M aoo v o 0 E
= ~ ap0 ~V~ ~ ~!O ch~ O CVO ch N
N
N J
E '~ '~' n.v w o o v E
W M On0 N N CV(Y(V M ~ N (00N N
O
y b ~ o I c aoa0 r aooofao~ 't o i E
l1 a ~ o00n 0ia0op ~ N tODN OMO VN' O
r O
fn _ o COO ~ N r y M (VO O O
'S
'b Z C O r N r r 00etM M N
N W N c,~~,jch~j M CVCVfVCV N
d ~ r r r e- r r r N
O
j ~m Fv o 0 0 0 0 0 0 0 0 0 o V
SriSriSriSriui ~ciSriuiSriSri ~ci rr E m ~ o ' ' M _ I o a J Z m m m ~ n ti m n ~ iri E
t9 Q N N N N N ~ N N N N N N
V
R n v i c ca N N N N N N ~ N N N N N
r r r r ~ r r r r ~ N
Z N
J ~ N Z
~ ~ O
a O O O O O O O O O O O o ~ o J t~~ O O O O O M O O O O O
E p p I~ M C7M M M M M M M M
O
' $ M
m _ N
N W f0 f9 .. I v-N M ~ M
' ~' ~ ~ ~ '~~JSfTi'~,~T~ d m ~ m a?~ N N
~
~u ~ m m m m m i _> _>_>_>_>_>~ ~ y ~ '' m ~
~
M (n n ~ S ~ ~ ~ S W W W W W W
( U
Table I.10:
Summary of results of column stability tests a E
m m n E
co m a E
w a E
m v a~
a E
m M
m c.
E
m N
N
a E
m a~
a E
m m v m U
o 1 ~ ~ ~ Resin discharged, divided into portions J
t N
E T E E ~ E w , ~ ~ > i ~ ~ ~ ~ ~ ~ o m a c ~ ~ ~ ~ ~ c m ~ i w w ~i w o ~ w w w a W w n WO 02/09732 PCT/EPO1l08657 Example 3:
Stability tests with loaded adsorption columns for the determination of bacterial counts The microbiological quality and harmless effects of natural starting materials for the production of pharmaceutical preparations have become increasingly important in recent times. Therefore, analyses were performed to determine bacterial counts in samples after storage of columns for five or eight weeks, according to customary methods (pharmacopoeia).
The results of the bacterial count determinations are presented in Table II. The total bacterial counts at a storage temperature of 4°C
were characterized as small. At a storage temperature of 25°C, the column wash water had 1.2 to 4.6 * 106 organisms, and the eluate had 80 to 170 organisms. However, it is noted that the stored column adsorbents were not washed or treated with preservative beforehand, and thus were previously exposed to increased risk from bacterial growth. During storage without refrigeration, therefore, it is recommended that the loaded columns be thoroughly washed at least with water, and advantageously with additional stabilizing or preservative wash solutions such as sodium chloride solution.
m..y.i .. Tr _ Stability tests for bacterial count determination Storage Storage for for 5 weeks 8 weeks Storage time at C
Column wash water Basic wash water Ethanolic eluate *Data in CFU/mL (colony-torming um ts) [see source document for table values; commas in numerical values denote decimal points.]
Example 4:
Stability tests with refrigerated, loaded adsorption columns As the result of seasonal or regional conditions, temperatures may fall below the freezing point during the storage or transport of loaded columns or cartridges, with the undesired result that the aqueous contents of the columns or cartridges solidify.
Corresponding tests support the conclusion that the freezing process appears to have a negative influence on the elution of rethawed columns or cartridges. If the column cannot be tempered, or storage takes place at low temperatures, it is recommended that the last wash be performed with suitable freezing mixtures instead of water to prevent freezing.
A) Test procedure:
From the numerous possible freezing mixtures, of which several are given as illustrative examples in Table III.1, a sodium chloride solution, for example, was chosen for further tests.
The addition of 23 g NaCl per 100 g water resulted in a freezing point of -21°C.
The column loading proceeded in a conventional manner. After the urine was loaded, rinsing was performed with 3 bed volumes water, followed by 2 bed volumes of a 33 wt-% NaCl solution. The column was then stored for 9 days in a chest freezer at -19°C.
Table III. I:
Examples of liquid aqueous salt mixtures and freezing mixtures Achievable temperature g A/100 g in C ubstance A coolant ubstance B
water water ice water water water ice ice ice ice ice 66.1 HzS04 snow ice ice [see source document for table values; commas in numerical values denote decimal points.]
B) Results Even after 9 days of storage at -19°C, the contents of the column remained liquid, with no signs of partial solidification.
Processing of the column, that is, basic washing, elution, and regeneration, proceeded normally. The total hormone content per total solids was approximately 22 wt-~. The results are shown in Table IIT.2.
Negative effects of the high NaCl content on the CE content and the separation of cresol and HPMF were not detectable. In summary, it may be concluded that stabilization of urine-loaded columns or cartridges using a liquid freezing mixture such as an aqueous sodium chloride solution, and storage under refrigeration, for example, down to approximately -20°C, WO 02/09732 PCT/EPO1/0$657 have an advantageous effect, even for periods of 1.5 to 2 weeks.
The contents of the columns or cartridges remained liquid, and processing of the columns or cartridges to isolate CE by washing, elution, and regeneration proceeded normally. Negative findings were not detectable, even at high NaCl concentrations.
Table III.2:
Column test for stability of column material Elution after 9 days storage at -19°C
Adsorber: XAD-7, volume: 200 mL
N ~ pO O
O a0~a0.-O N O ON O
aE
z OO O t0N NN 0 O ~ M
1AO O~ N C
c 0 C C
m ~ M O O N ~ 1~n M
N
W
m ~ tD ~ r bp1 OO O OO 00 Oo 0~ 0 0 ~ p ~o . c C c p -N N
E
W
C
O N N M N ~t0N
y QE
w c m - " MN
"' 00 0 00 00 00 00 ~ > ~c c c c ~
m ~ ~ a0 t0~ NN ~
E t0W ~ ~N Nf~~ ~~ ~ ~
a _ e-~f7I'N ~~ o~ ~ f'O _ tDVN ~ O
.c ~ M
m O~ ~ rN N(~~7 tD~ ~'~ ~ N a0 ~ N ~O N c O
~
E
O O
~ V N GO O
o M m~ <f cYricvfc3c~I
U
m E W
~ Nf aDO~W ~~ OaD O~f7~ N ~ 0 0 0~70 O 0 0 if~ ~ ~~ V~ ~ 1riV Wl7~ N V7 ICJ1Al~1n~I7 tO 1n1~
~ ~
m U
~ w n _ c c ~ r VV ~~ w ~O N N N N h h NN
E - ' ~ ~ ~ ~
C D 0G D tCtG
~ mm (DfCtC~G~C tCtC ma0CCaDW t ( aa ~ Nn O
E c~'' O ~ M =m a . .-~ a n 3 ~ U
a~
.
C ~ W ~ N ~'N N - N ~ ~ ~ V' E Nc0N (Om t0aD t0~ .- U ~ . ~ c9N N
tD fD MN
E ~ Z b N
O i1 V ~ t0 M Z y Z
C t0V ~ NN N N NO N N NN N o 0 j ' oc ~ cc o c ca c c o0 0 ,~o ~ c d E
'~ ~
p . c .
C _~_ c N M V In~ N M~ inm~ ' N M
N M V~ f0h t ~ N N ~ CC c C
m d j ~~ ~ .~ ,~~l~,a I 7A 7 m I 3 ~ ~N N N !O~ O 7 N
U 33 N ~3 ~ ~ ~7 7 7 totoui w to WO ?2/09732 PCT/EPO1/08657 Example 5:
Concentration and stabilization of urine in a field test A) General The objective of a field test is to show that, with sufficient hormone content in the urine, satisfactory results can be obtained not only in the laboratory, but also under production conditions. A test was conducted under the same conditions that would occur during a sampling session at any given location.
B) Test procedure Fresh urine of sufficient quality was collected at a stud farm.
With three horses and 15 to 20 L urine/day, a total of approximately 150 to 200 L was obtained in 10 days. Therefore, a 5-L adsorption column was chosen for adsorption. The daily loading with urine, excluding weekends, was carried out according to the following program:
Start of urine collection on the weekend Monday: Charging of urinefrom Saturday, Sunday, and Monday Tuesday through Charging of urinefrom each day Friday:
Monday Charging of urinefrom Saturday, Sunday, and Monday Tuesday Elution The urine was charged directly on the column at a loading rate of about 4.5 bed volumes/hr, with only one cotton plug with a separation limit of 20 um serving as a prefilter. No water rinsing was performed between the individual charges in order to make the conditions more severe. In pretests it was possible to deliver up to 450 L urine through the cotton plug with no pressure loss. In this field test, filtration proved to be difficult on account of an unexpectedly high amount of calcium carbonate sediment and/or mucins.
After each 40 to 50 L, a blockage appeared, along with a pressure rise of approximately 4 bar, which required the filter to be replaced. Depending on the calcium carbonate and/or mucin content, the optional addition of filtration aids is recommended.
Although the plug allowed sediment > 20 um to pass through, [filtration aids) were not added to the adsorption column.
C) Results Hormone content The results of this field test are presented in Table IV.
The estrone and equilin content of 123 mg/L and 74 mg/L, respectively, in the raw urine were surprisingly high, corresponding to urine of average to good quality. However, the cresol values, at 531 mg/L, were unexpectedly high in fresh urine, which hitherto had not been observed. The issue of diet is a factor deserving consideration here. Although a basically higher cresol content, for example, can be overcome, in individual cases it may be problematic. Therefore, it may be necessary to prolong the alkaline washing process during processing to minimize the cresol values in the ethanolic eluate. Otherwise, the adsorption and desorption proceeded normally. The eluate showed a pronounced content of estrone and equilin. Eluates 2 and 3 contained more than 95°s estrone/equilin. The total hormone content relative to the total solids content was approximately 30 wt-~, thus meeting expectations for the test and achieving superior concentration to a hormone/total solids content greater than 15 wt-~ under severe test conditions.
Table IV.1:
Field test for stabilizing and concentrating CE
Test days: 1-10, column loading (on 7 working days) Adsorber: XAD-7, volume: 5 L
Starting solution: native urine, untreated The column was traversed from bottom to top.
c o0 oc co ccc o0o c c o0 oO cc cco co0 0 NN ~M ~~ aM0~m ~~m t00 O NN ~~ NW ~~m ~~m t~0 O OO OO OO OOO OOO O
n t0 ~V C~~fN~ ~O a0~< O
V V~fQ~ N~ VV~ ~f< ~
~ vv ~~ aoaoaoaon.ovv n v oo ~:o ~ cc oo ~
a cocnnlMM c~ d c~
a N_ NN NN N~ ~~N NNN N
t4 Z
O OO OO OO OOO OOO O
-r C NIh~N t0f~a0W~ ~r.M-.~-A CC CC CC CCG CCC C
C 7~ 77 77 777 777 7 7 d'd'~~ 2~ OCQ'~ ~d'~ ~
~
Table IV.2:
Field test for stabilizing and concentrating CE
Processing of column on 11th test day Adsorber: XAD-7, volume: 5 L
Starting solution: native urine, untreated The column was traversed from bottom to top.
w cv ~ ~ ~ 0 0 0 0 0 0 0 et N
C
O t!7O m ' ~n o, ~ o ~ c O
E , W c o V' r O o O O O O) N ~ 1~.O O O O
p1 N O N O ~ O O
E r N M (O O O O O
U N r a ~ O 47 '.1 O ~ O O tn O n N N O O O O O O
E o U
, ,n c O O O O O r M ~ ~ O p.
N
E
m N
O
O O O O p N
N
O CO O M O O
M f~D O C j f0 U
d O N
' E
c c ~ O O M M M , ~E
~ O O t0fC O O O
v v v v v i: cciaiv d' o v o d o E
E
N O U
C r M M r r r O O Iv(O(O= ~ ~ N
d ~ ~ ~ y ( ~ O (Oet~ d O a.:O O 7 N ~ ~ ~
- M M M M M .( t ~ ~ ~ >
v p a v ~ y ' c E O tc)! ~ ~ inpp O N M M ~ ~ ~ ~ ~ C
c r ' E
~. z v r ~ o ~ o o ~ ..-z ~ a M o o E
j ~ ~ ~C7In!l7~t7117~ ~ M tntnIn~ O ~ O O
J
r r N
~ r ~
~a N
c r N M ~ III~ r N M ~ tO~ r ~ N M ~ U
c _ '~n ~ ~ ~ ~ m m m a;a;~ d ~
' L, f E _ ~ m m m com o m m a~
f~ f9f0t0ID (0t0p _>_> 7 _7_77 ~
~SLU
UJ W LUW W ~ ~ ~ ~ ~ ~
Bacterial counts In addition to urine adsorption, possible growth of bacteria on the column was investigated. Under the following conditions, 1. Charging of contaminated raw urine (no bacteria were separated by cotton plug filtration), 2. No rinsing after daily charging, 3. Test carried out a room temperature, and 4. Test duration of 10 days, the bacterial density on the column and subsequent growth thereon were not precluded. Determination of the total bacterial count at different adsorption steps gave the following results:
Sample Total bacterial _ coun_t/mL
Raw urine Column wash water after 10 days Basic wash water Main eluate Isee source document for table values; commas in numerical values denote decimal points.]
D) Summary of results Fresh urine was charged on a 5-L adsorption column over a test period of 10 days. The entire volume of urine was 140 L, with an average estrone and equilin content of 123 mg/L and 74 mg/L, respectively.
Column rinsing with water after the daily urine adsorption was intentionally omitted. Both of the very pronounced main eluates contained approximately 93~ estrone and equilin. The total hormone content relative to total solids was approximately 30 wt-~. A further surprising result related to possible bacterial contamination of the column. In spite of charging highly contaminated raw urine, bacterial growth on the column itself was not detectable after 10 days. With l.5 organisms/mL, the eluate obtained was practically bacteria-free.
Hence, under severe conditions it was proven on a large scale which, with sufficient hormone in the raw urine, stabilization and concentration to a hormone/total solids content of greater than 15% and of good quality can be performed using an adsorption column.
Example 6:
Filtration tests using filter cartridges Filtration tests using filter cartridges were carried out in a further field test, the results of which are summarized below.
Related material: fresh urine (origin: a German stud farm) The urine of pregnant mares which had been collected in drums and occasionally agitated was pumped from the drums using a hose pump, through a 5u bag (prefilter), and into a container. Sediment of about 0.5 wt-% remained in the container. Figure 2 shows a schematic representation of the method for filtering raw urine.
~..~.. _ Bag 5 a Filter cartridges 5u, 3u, 1u connected in series Membrane filter 2 x 1.2u connected in parallel Column loading 95 L filtrate Sediment filtrate 0.000%
Bag 5 a Filter cartridges 5u, 3u, 1u connected in series Membrane filter 2 x 3u connected in parallel Column loading 142 L filtrate Sediment filtrate 0.001%
Bag 5 a Filter cartridges 3u, 1u connected in series Membrane filter 2 x 1u connected in parallel Column loading 160 L filtrate Sediment filtrate 0.000%
In each test, one sample was taken downstream of the membrane filter. After every 100 L a sample was taken from the outlet, downstream of the column.
Total column loading: 397 L filtrate The total solids (TS) content after processing was 5.9 wt-%.
Claims (33)
1. Method for concentrating and stabilizing mixtures of conjugated estrogens (CE) from pregnant mares' urine (PMU) at one or more decentralized locations for storage and transport to a centralized further processing location in a manner that meets requirements in the field, characterized in that - a natural mixture of conjugated estrogens (CE) contained in pregnant mares' urine (PMU) is concentrated and stabilized onto a solid adsorbent support, and that - a predetermined maximum total amount of collected PMU is pumped from a supply vessel, continuously or in discrete portions, at a predetermined flow rate through an upright cartridge, and the PMU runoff is discarded, - the upright cartridge being packed with a suitable adsorbent, which is surrounded by PMU, for the adsorption of a predetermined quantity of the mixture of conjugated estrogens (CE) contained in the PMU, and - the maximum total amount of PMU pumped through the upright cartridge, which is predetermined as the end point check for the loading of the adsorbent, being matched to the maximum loading capacity of the adsorbent for the conjugated estrogens (CE) contained in the PMU.
2. Method according to Claim 1, characterized in that the internal dimensions of the upright cartridge are designed for the adsorption bed such that the upright cartridge can accommodate 30 to 50 L of adsorbent, and the PMU is required to travel a path distance of 80 to 120 cm from the inlet to the outlet.
3. Method according to Claim 1, characterized in that the upright cartridge used in the method is made of impact-resistant laboratory glass, plastic, or metal.
4. Method according to Claim 1, characterized in that a monopump, hose pump, or membrane pump is used.
5. Method according to Claim 1, characterized in that a rotameter with a float, a vane air flow meter, or an inductive flow meter is used.
6. Method according to Claim 1, characterized in that a throughput meter is used to measure the quantity of liquid urine pumped through the upright cartridge.
7. Method according to Claim 1, characterized in that the flow velocity ranges from 3 to 10 adsorber bed volumes/hour.
8. Method according to Claim 1, characterized in that polymeric adsorber resins, silica gel, or reverse phase silica gel are used as the adsorbent.
9. Method according to Claim 1, characterized in that the maximum total quantity of PMU is 20 to 60 adsorber bed volumes.
10. Method according to Claim 1, characterized in that after loading of the adsorbent with a quantity of PMU that corresponds approximately to the total capacity of the adsorbent, or also before reaching the total loading capacity after any individual partial loading steps, the cartridge is rinsed with water or another suitable aqueous wash solution.
11. Method according to Claim 1, characterized in that after loading of the adsorbent, the upright cartridge is rinsed first with water or another suitable aqueous wash solution, and finally, for further stabilization, is rinsed with a preservative solution.
12. Method according to Claim 1, characterized in that the PMU from the supply vessel is first pumped through one or more prefilters before the PMU is pumped through the upright cartridge.
13. Method according to Claim 12, characterized in that the prefilter is a deep-bed filter having a sand bed, or a filter cartridge, a plate filter, a cartridge filter, a filter bag, or a filtration tube.
14. Method according to Claim 12 or 13, characterized in that a filtration aid is added to the PMU before filtration.
15. Device for concentrating and stabilizing mixtures of conjugated estrogens (CE) from pregnant mares' urine (PMU) onto a solid adsorbent support at one or more decentralized locations for storage and transport to a centralized further processing location, comprising:
- an upright cartridge packed with a suitable adsorbent, which is surrounded by PMU, for the adsorption of a predetermined quantity of a mixture of conjugated estrogens (CE) contained in the PMU, with the upright cartridge having either a) a liquid inlet situated at the base end and a liquid outlet situated at the head end or b) a liquid inlet situated at the head end and a liquid outlet situated at the base end, and - arranged in the stated sequence upstream of the upright cartridge and connected to one another by hose lines.
- a pump, a flow meter, and a throughput meter.
- an upright cartridge packed with a suitable adsorbent, which is surrounded by PMU, for the adsorption of a predetermined quantity of a mixture of conjugated estrogens (CE) contained in the PMU, with the upright cartridge having either a) a liquid inlet situated at the base end and a liquid outlet situated at the head end or b) a liquid inlet situated at the head end and a liquid outlet situated at the base end, and - arranged in the stated sequence upstream of the upright cartridge and connected to one another by hose lines.
- a pump, a flow meter, and a throughput meter.
16. Device according to Claim 15, characterized in that at least one prefilter is connected in-line between the pump and the flow meter.
17. Device according to Claim 16, characterized in that the prefilter is a module composed of two prefilters connected in parallel which are individually operated in alternation, and which may be replaced by new prefilters when the device is running.
18. Device according to Claim 16 or 17, characterized in that to check proper functioning of the prefilter a manometer is connected in-line between the pump and the prefilter, and a manometer is connected in-line in the region downstream of the prefilter.
19. Device according to Claim 15, characterized in that the upright cartridge has internal dimensions for the adsorption bed with an internal height ranging approximately from 80 to 120 cm and an inner diameter ranging approximately from 10 to 25 cm.
20. Device according to Claim 15, characterized in that the upright cartridge which is surrounded by the device is made of impact-resistant laboratory glass, plastic, or metal.
21. Device according to Claim 15, characterized in that the pump which is surrounded by the device is a monopump, hose pump, or membrane pump.
22. Device according to Claim 15, characterized in that the flow meter which is surrounded by the device is a rotameter with float, a vane air flow meter, or an inductive flow meter.
23. Device according to Claim 15, characterized in that the throughput meter which is surrounded by the device and which provides for the measurement of the quantity of PMU pumped through the cartridge is a water meter or an inductive throughput meter.
24. Method according to Claim 1, characterized in that the collected PMU is pre-purified by coarse mechanical means prior to be pumped through the upright cartridge.
25. Method according to Claim 6, characterized in that the throughput meter is a water meter or an inductive throughput meter.
26. Method according to Claim 7, characterized in that the flow velocity ranges from 4 to 6 adsorber bed volumes/hour.
27. Method according to Claim 8, characterized in that polymeric adsorber resins are used as the adsorbent.
28. Method according to Claim 27, characterized in that the polymeric adsorber resins are semipolar polymeric adsorber resins.
29. Method according to Claim 9, characterized in that the maximum total quantity of PMU is 30 to 40 adsorber bed volumes.
30. Method according to Claim 11, characterized in that the further stabilization is carried out by rinsing the upright cartridge with a solution containing a preservative agent, a pH adjusted aqueous solution or an aqueous salt solution.
31. Method according to Claim 12, characterized in that the one or more prefilters comprise at least one deep bed filter or a precoated filter.
32. Device according to Claim 16, characterized in that the at least one prefilter comprises a deep bed filter or a precoated filter.
33. Device according to Claim 18, characterized in that the manometer connected downstream of the prefilter is connected behind the flow meter.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10037389.5 | 2000-08-01 | ||
| DE10037389A DE10037389A1 (en) | 2000-08-01 | 2000-08-01 | Method and device for the enrichment and stabilization of conjugated estrogens from mare's urine |
| PCT/EP2001/008657 WO2002009732A2 (en) | 2000-08-01 | 2001-07-26 | Method and device for concentrating and stabilising conjugated oestrogens from the urine of mares |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2410654A1 CA2410654A1 (en) | 2002-11-26 |
| CA2410654C true CA2410654C (en) | 2011-09-20 |
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ID=7650908
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2410654A Expired - Fee Related CA2410654C (en) | 2000-08-01 | 2001-07-26 | Method and device for concentrating and stabilizing conjugated estrogens from mares' urine |
Country Status (25)
| Country | Link |
|---|---|
| US (3) | US7081451B2 (en) |
| EP (1) | EP1307210B1 (en) |
| CN (1) | CN100482239C (en) |
| AT (1) | ATE328600T1 (en) |
| AU (1) | AU785438B2 (en) |
| BG (1) | BG65554B1 (en) |
| BR (1) | BR0107062B1 (en) |
| CA (1) | CA2410654C (en) |
| CZ (1) | CZ303159B6 (en) |
| DE (2) | DE10037389A1 (en) |
| EE (1) | EE04541B1 (en) |
| ES (1) | ES2266221T3 (en) |
| HR (1) | HRP20020248B1 (en) |
| HU (1) | HU228396B1 (en) |
| IL (1) | IL148878A0 (en) |
| MX (1) | MXPA02002835A (en) |
| NZ (1) | NZ518645A (en) |
| PL (1) | PL202238B1 (en) |
| PT (1) | PT1307210E (en) |
| RS (1) | RS50238B (en) |
| RU (1) | RU2277241C2 (en) |
| SI (1) | SI1307210T1 (en) |
| SK (1) | SK287663B6 (en) |
| UA (1) | UA79067C2 (en) |
| WO (1) | WO2002009732A2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DE10159161A1 (en) * | 2001-12-01 | 2003-06-18 | Solvay Pharm Gmbh | Process for obtaining estrogens from mare's urine |
| US8349819B2 (en) * | 2002-10-09 | 2013-01-08 | Dr. Reddy's Laboratories New York, Inc. | Steroid extraction process from urine sources |
| ATE410435T1 (en) * | 2003-07-17 | 2008-10-15 | Solvay Pharm Gmbh | METHOD FOR OBTAINING A NATURAL MIXTURE OF CONJUGATED EQUINE ESRTROGENS |
| US20070161779A1 (en) * | 2003-10-24 | 2007-07-12 | Thorgard Pharmaceuticals (Aust) Pty Ltd | Estrogen Extraction From Pregnant Animal Urine |
| ITMI20052516A1 (en) | 2005-12-29 | 2007-06-30 | Evultis Sa | PROCESS FOR THE ISOLATION OF PHARMACOLOGICALLY ACTIVE PEGRINCIPES OF VEGETABLE AND ANIMAL ORIGIN |
| US20070212751A1 (en) * | 2006-01-18 | 2007-09-13 | Solvay Pharmaceuticals Gmbh | Microbial method for the 11beta hydroxylation of 9beta, 10alpha-steriods |
| CA2761844C (en) | 2009-05-14 | 2017-11-28 | University Health Network | Quantitative endoscopy |
| US10391419B1 (en) | 2015-02-12 | 2019-08-27 | Nostrum Pharmaceuticals. Llc. | Isolation and purification of conjugated estrogens |
| CA3023242A1 (en) * | 2016-05-05 | 2017-11-09 | Indiana University Research & Technology Corporation | Quantitative profiling of progesterone metabolites for the prediction of spontaneous preterm delivery |
| RU180071U1 (en) * | 2016-05-23 | 2018-05-31 | Федеральное государственное бюджетное учреждение науки "Федеральный исследовательский центр "Казанский научный центр Российской академии наук" | The line for obtaining the drug Na-, Fe-, Ca-polygalacturonate |
| US11014025B1 (en) * | 2020-03-06 | 2021-05-25 | Kevin-Steven Creagh Buford | Pressurized filtration system and device for rapid extraction and recycling of medication from body fluid |
| CN116920459B (en) * | 2023-09-15 | 2023-11-24 | 东营平明医药科技有限公司 | Purification device of 2-mercaptobenzothiazole crude product |
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|---|---|---|---|---|
| US2519743A (en) * | 1947-12-29 | 1950-08-22 | Ivy H Dameron | Adjustable protector cap |
| US2578729A (en) * | 1948-09-21 | 1951-12-18 | Upjohn Co | Extraction of hydrolyzed estrogens from urine |
| US2565115A (en) * | 1948-10-28 | 1951-08-21 | Squibb & Sons Inc | Method of obtaining a conjugated estrogen preparation |
| US2834712A (en) * | 1953-05-27 | 1958-05-13 | American Home Prod | Urinary estrogen compositions and methods for preparing them |
| GB821989A (en) | 1957-11-06 | 1959-10-14 | Ayerst Mckenna & Harrison | Urinary estrogen compositions and methods for preparing them |
| DE1245373B (en) * | 1964-02-06 | 1967-07-27 | Vni Chimiko Pharmazewtitschesk | Process for the isolation of steroids from culture fluids |
| NL7111863A (en) | 1970-10-13 | 1972-04-17 | ||
| US3769401A (en) * | 1970-11-19 | 1973-10-30 | Frosst & Co | Chemical process |
| US4379845A (en) * | 1981-07-23 | 1983-04-12 | Nabisco Brands, Inc. | Yeast process and product |
| GB2230856A (en) * | 1989-04-13 | 1990-10-31 | St Thomas Hosp Med School | Storing urine samples |
| HU9500322D0 (en) * | 1994-02-08 | 1995-03-28 | Solvay Deutschland | Process for separating estrogenes from the urine of mares with foal |
| HU9500323D0 (en) * | 1994-02-08 | 1995-03-28 | Solvay Deutschland | Process for separating estrogenes from urine of mares with foal |
| AU722084B2 (en) * | 1996-08-30 | 2000-07-20 | Abbott Products Gmbh | Process to obtain oestrogens from mare's urine |
| HU228763B1 (en) | 1996-08-30 | 2013-05-28 | Abbott Products Gmbh | Method to obtain oestrogens from mare's urine |
| CN1278432A (en) * | 1999-06-22 | 2001-01-03 | 中国科学院新疆化学研究所 | Method of extracting female hormone from pregnant woman's urine |
| DE10048524A1 (en) * | 2000-09-29 | 2002-06-06 | Solvay Pharm Gmbh | Cresol separation from mare urine |
| US6855704B2 (en) * | 2002-10-09 | 2005-02-15 | Apr Llc | Process for isolating conjugated estrogens |
-
2000
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