The present invention relates to a pharmaceutically acceptable dosage form of water insoluble porphyrins, particularly metal containing porphyrins such as tin porphyrin, which can be complexed with a lipid, reconstituted from a lyophilizate and administrated to a patient in the treatment of cancer and other diseases.

The present invention relates to a pharmaceutically acceptable dosage form of water insoluble porphyrins, particularly metal containing porphyrins such as tin porphyrin, which can be complexed with a lipid, reconstituted from a lyophilizate and administered to a patient in the treatment of cancer and other diseases.
Porphyrins are biologically active compounds, usually consisting of four pyrrole rings. Typically, they have a centrally located metal atom and are activated by light radiation of suitable wavelength. Porphyrins display antitumor activity in photodynamic therapy (PDT) where the porphyrins are administered to a patient and localize in neoplastic tissues. Typically, the neoplastic tissues are irradiated with light at a wavelength which corresponds to an absorption band of the porphyrin resulting in the activation of the porphyrin and preferential destruction of the neoplastic tissues. It has been suggested in U.S. Pat. No. 5,162,519 to Bonnett et al. that the mechanism involved is due to the production of the highly reactive singlet oxygen which is produced by transfer of energy from the light-excited porphyrin molecule to an oxygen molecule. Such photodynamic therapy is the subject of a series of articles making up a special issue of Photochemistry and Photobiology, Volume 46, number 5, November, 1987 (hereinafter xe2x80x9cPandP 46-5xe2x80x9d). According to these articles, photodynamic therapy has been used in the treatment of a wide variety of cancers including such solid tumors as cancers of the bronchial tubes, bladder, esophagus, lung, skin, head and neck, brain, and colon and intraocular and gynecologic cancers.
A major disadvantage to the use of porphyrins in the treatment of cancer is that when used in high concentrations to destroy the tumor cells, the porphyrins exhibit toxic side effects. The porphyrin is generally injected intravenously or intraperitoneally at a dose of about 2 mg per kg of body weight. At lower concentrations which might be more easily tolerated by the patient, the porphyrins tend to exhibit very little effect on the tumor cells.
It has been reported that porphyrins may be incorporated into a liposome and injected intraperitoneally. Spikes et al., xe2x80x9cPhotodynamic Behavior of Porphyrins in Model Cell, Tissue and Tumor Systemsxe2x80x9d; in Photodynamic Therapy of Tumors and Other Diseases (Edited by G. Jori and C. A. Perria); pages 45-53; Libreria Progetto, Padua (1985) and references cited therein.
There is a need for compositions containing porphyrins which are effective for the treatment of cancer and yet exhibit less toxic effects towards the patient.
It is an object of the present invention to provide a less toxic pharmaceutically acceptable dosage form of porphyrins for use in therapies such as chemotherapy and photodynamic therapy.
One manifestation of the present invention is complexes of a porphyrin and a phospholipid. Another manifestation of the invention is a lyophilizate of a complex of a porphyrin and a phospholipid, and a pharmaceutically acceptable excipient. Still another manifestation of the present invention is a method for preparing a lipid complex of a porphyrin.
The lipid complexes in accordance with this invention appear to be desirable for several reasons. It appears that the lipid complexes may sufficiently slow the release of the porphyrins that the porphyrins can be administered at higher dosage levels with less toxicity thereby improving treatment. The lipid complexes also appear to be more stable to ambient light and UV radiation than the porphyrin alone thus simplifying their handling. While it has not been confirmed, the lipid complexes may also preferentially accumulate in tumor tissues.
While the invention will hereafter be described with respect to the preparation of lipid complexes and lyophilizates of lipid complexes of tin porphyrin, those skilled in the art will appreciate that the methods taught herein are also applicable to the preparation of lipid complexes and lyophilizates of other porphyrins, particularly other metalloporphyrins.
In accordance with one embodiment of the invention, a lyophilizate of a phospholipid-porphyrin complex is prepared by a process comprising the steps of preparing a concentrated solution of a porphyrin and a phospholipid in an organic solvent, adding water to the solution to cause formation of a lipid complex of the porphyrin, removing the organic solvent to provide a dispersion of the lipid complex in water as an aqueous phase, dissolving a pharmaceutically acceptable excipient in the aqueous phase of the dispersion, and lyophilizing the dispersion of the lipid complex to form a lyophilizate.
In accordance with a preferred embodiment of the present invention, the lyophilizate is prepared by a method which comprises forming a concentrated solution of tin porphyrin and a mixture of dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG) in chloroform, adding an aqueous solution such as water for injection to form the lipid complex of tin porphyrin and provide a dispersion of the lipid complex in water as an aqueous phase, sparging the dispersion to remove the chloroform, reducing the particle size of the dispersion of the lipid complex, adding an aqueous solution of mannitol as a pharmaceutically acceptable lyophilization excipient to the dispersion, and lyophilizing the composition, wherein a lyophilizate is obtained which upon reconstituting with water provides a colloidal dispersion of a porphyrin-lipid complex. It is possible to prepare the solution of the prophyrin and the phospholipids by dissolving the porphyrin in one organic solvent, dissolving the phospholipids in another organic solvent, mixing the two solutions, and then removing the solvents after addition of water.
The term xe2x80x9cwater insoluble porphyrinxe2x80x9d as used herein means porphyrins having a solubility in water which is less than about 1.5 mg/ml at 23xc2x0 C. and, more typically, is less than 0.5 mg/ml at 23xc2x0 C. and still more typically less than 0.1 mg/ml at 23xc2x0 C.
The term xe2x80x9clipid complexxe2x80x9d is an art recognized term. Lipid complexes are characterized by a noncovalent bond between the lipid and the porphyrin which is observed by a phase change in differential scanning calorimetry. Liposomes are not lipid complexes within the meaning of the term.
The term xe2x80x9cpharmaceutically acceptable aqueous diluentxe2x80x9d as used herein refers to water for injection, saline, and other known aqueous vehicles.
The term xe2x80x9clyophilization excipientxe2x80x9d refers to a substance which is added to a solution prior to lyophilization to enhance characteristics such as the color, texture, strength, and volume of the cake. Examples of lyophilization excipients are provided below.
The term xe2x80x9cphotodynamic therapyxe2x80x9d (PDT) as used herein refers to a therapy treatment of a patient having a disease such as cancer wherein the patient is treated with a drug such as a porphyrin which has the tendency to preferentially accumulate in neoplastic tissues relative to normal tissues. The drug, when irradiated becomes toxic to the neoplastic tissue.
Porphyrins are biologically active nitrogen-containing compounds having a conjugated cyclic structure consisting of four pyrrole rings linked together through their 2- and 5-positions by methine bridges. The term xe2x80x9cporphyrinxe2x80x9d includes derivatives wherein a metal atom is chelated into the ring structure, presumably complexed or bonded to at least two of the nitrogens of the pyrrole ring. The metal atom inserted into the ring structure includes, e.g., tin, zinc, lanthanides, actinides, and those metals of the transition series of which chromium, manganese, iron, cobalt, nickel, and copper are exemplary. The preferred porphyrins for use in the present invention are metalloporphyrins, particularly tin porphyrins
Tin porphyrin and other porphyrins useful in this invention are described in the literature. It is known to irradiate tumors and cancerous tissues in the human body with intensive light following administration of a hematoprophyrin derivative in the wavelength range of 626 to 636 nanometers to reduce and, at times, destroy the cancerous cells (see PCT published specification WO83/00811). It is also known that porphyrins, especially the sodium salt of protoporphyrins, can maintain or promote the normal functions of cells and are useful for preventing the genesis, growth, metastasis, and relapse of malignant tumors. Japanese Published Patent Application No. 125737/76 describes the use of porphyrins as tumor inhibiting agents, exemplifying etioporphyrin, mesoporphyrin, protoporphyrin, deuteroporphyrin, hematoporphyrin, coproporphyrin, and uroporphyrin. U.S. Pat. Nos. 4,837,221 and 5,162,519 to Bonnett et al. teach the therapy of tumors susceptible to necrosis by a porphyrin when adminstered to locate in the tumor and illuminated with light of a wavelength absorbed by the porphyrin. U.S. Pat. No. 5,512,559 to Shalkos et al describes the use of porphyrins and metal complexes of prophyrin in the photodynamic treatment of cancer tumors. European Patent Publication 0 186 962 describes therapy of tumors susceptible to necrosis when pharmacological acceptable meso-porphyrins are administered followed by illumination with light of a wavelength absorbed by the porphyrin.
U.S. Pat. No. 5,407,808, discloses hematoporphyrin derivatives or a mixture of porphyrins derived therefrom (e.g. Phoytofrin II), to mammalian tumor cells and then subject such cells to light of appropriate wavelength to effect cell destruction. Any porphyrin can be used in the present invention provided that it complexes with a phospholipid and is pharmaceutically active. Theoretically, any of these therapies could be modified by a lipid complex of the water insoluble porphyrins and administering it to the patient instead of the uncomplexed porphyrin. In order to produce the lipid complex and administer the complex as a colloidal dispersion in water, the porphyrin must be essentially water insoluble as defined above.
In accordance with the invention the porphyrin, particularly a tin porphyrin, is provided as a concentrated solution of the porphyrin in an organic solvent. The most typical example of the solvent used to prepare this solution is chloroform. However, other organic solvents such as methylene chloride, carbon tetrachloride, ethylene dichloride, freons, DMSO (dimethyl sulfoxide), DMA (dimethyl acetamide), etc. can be used. Useful solvents must form stable solutions with the porphyrin, e.g., the solvent must not interact with, destabilize, or deactivate the drug. In addition, the solubility of the porphyrin in the solvent must also be high enough that the porphyrin can be dissolved in amounts sufficiently high to form commercially useful quantities of the lipid complex, and the solvent should be capable of being removed easily from an aqueous dispersion of the lipid complex as described hereafter. Preferably, a solution having a concentration of about 0.25 to 25 mg/ml, preferably about 2 to 25 mg/ml and most preferably about 15 mg/ml porphyrin is used. The concentration may vary depending upon the nature of the solvent and temperature, but it is advantageous to use a concentrated solution of the porphyrin in preparing the lipid-porphyrin complex. This minimizes the amount of solvent that must be removed later in the process, and it also assists in forcing the porphyrin out of solution and into lipid porphyrin complex formation with the addition of water.
Typically, the phospholipids are dissolved separately. The organic solvent used to prepare the solution of the phospholipids should meet similar requirements to those outlined for the porphyrin solvent. It must be compatible with the phospholipids and not destabilize them or the porphyrins. In addition, the lipids should be soluble enough in the solvent so as to be able to introduce enough of the lipid to form the complex yet minimize the amount of solvent that must be removed later. A solvent which can be readily removed from the dispersion of the lipid complex is most preferred. The solvent most typically used to prepare this solution is chloroform or methylene chloride. Typically the concentration of this phospholipid solution will range from about 10 to 250 mg/ml. Preferably, the same solvent is used to dissolve both the porphyrin and the phospholipid.
Phospholipids are amphophilic in nature, i.e., the molecules have a hydrophobic tail such as a long chain hydrocarbon, and a hydrophilic head. In an aqueous medium, such as water or saline, the tails of the molecules align with each other, away from the aqueous phase, while the heads of the molecules face outward into the aqueous phase. It is this nature of the phospholipids that makes them very useful for formulating soluble compositions from highly insoluble drugs like porphyrins.
The phospholipids used in the invention are preferably selected such that their phase transition temperature is equal to or below the body temperature of about 37xc2x0 C. and the complex releases the drug in the body. Representative examples of useful phospholipids include the synthetic phospholipids, dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidyl-glycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidycholine (DSPC), distearoylphosphatidylglycerol (DSPG), or a combination thereof. Other examples of phospholipids can be found in the CRC Handbook of Lipid Bilayers by Marsh, M. A., CRC Press (199). When DMPC and DMPG are used in a ratio of DMPC to DMPG of about 7:3 they mimic the cell membrane.
The porphyrin and the phospholipid are added to the solvent such that the weight ratio of the porphyrin to lipid is about 1/10 to 1/100 and preferably about 1/10 to 1/40.
In some applications, it has been found desirable to add cholesterol or its hemisuccinate derivatives or other salt forms to the lipid complex. The cholesterol is believed to slow down the release of the drug. This approach may be particularly desirable with subcutaneous formulations where severe necrosis can result if the drug is delivered too quickly. The cholesterol may be used in amounts up to about 50 parts per hundred parts phospholipid and preferably about 0.5 to 15 parts per 100 parts phospholipid.
Once the lipids and porphyrin have been added to the solvent and thoroughly mixed, water or an aqueous solution is added rapidly to the mixture with stirring for several minutes. Addition of the water is believed to cause the porphyrin and the lipid to come out of solution and complex with each other. The water is preferably added in an amount such that the porphyrin is present in an amount of about 10 mg to 100 mg per 100 ml water. It is desirable to limit the amount of water because higher amounts of water increase the amount of water that must be removed during the subsequent lyophilization process. It is believed that complexation may be complete in about 30 minutes. However, it is desirable to stir the dispersion for about one hour to insure that the complexation is complete.
The lipid complex dispersion described above, is treated to remove the solvents. Any of a variety of techniques can be used for this purpose. For example, it has been found that chloroform can be effectively removed if the dispersion is sparged with an inert gas such as nitrogen.
A pharmaceutically acceptable lyophilization excipient is dissolved in the aqueous phase of the dispersion. Mannitol it typically used as the excipient but other excipients which do not interact with the drug or the lipid complex may be used. Sodium or potassium phosphate, citric acid, tartaric acid, gelatin, and carbohydrates such as lactose, dextrose, dextran, hetastarch, etc. are common examples of excipients which are also believed to be useful herein. The excipients can be used alone or in combination to provide a cake of good quality which readily disperses in water upon reconstitution.
The excipients arc typically added to the dispersion as solutions in water. Again, it is desirable to use concentrated solutions to minimize the amount of water for removal by lyophilization. The amount of the excipient is adjusted in a manner that is well known in the art to provide a cake which does not crack or shrink and is porous so that it readily dissolves and has a good appearance. Mannitol has been found to be particularly useful. Mannitol is added to the dispersion as a solution having a concentration of about 0.01 to 0.15 g/ml. Mannitol is added in an amount of about 1 to 100 parts by weight per part tin porphyrin.
After removing the solvents and adding the excipient, the dispersion is passed through a homogenizer (e.g., a Tekmar rotor/stator homogenizer, Model T25, or a microfluidics submerged jet homogenizer, Model M110Y). As a general rule, the smaller the particle size of the dispersion, the faster the formulation can be dried during the lyophilization cycle. A dispersion having a particle size distribution ranging from about 10 to 4000 nm and averaging about 600 nm has been found to be satisfactory for lyophilization. The optimum particle size may vary depending on the mode of administration.
A typical lyophilization cycle useful in accordance with the present invention is provided below. The cycle may be varied depending upon the equipment and facilities available in a manner well known in the art.
The homogenized formulation can be poured into vials of a 5 to 50 mL nominal volume. The vials are placed into a lyophilization chamber at about 5xc2x0 C. The vial size will usually be selected such that each vial contains a single dosage of the porphyrin-lipid complex. The temperature of the chamber is reduced to xe2x88x9230xc2x0 C. over a period of one hour after which the temperature is maintained at xe2x88x9230xc2x0 C. for about four hours. The pressure in the lyophilization chamber is then reduced to 200-250 microns of pressure for the remainder of the cycle. After reducing the pressure in the chamber, the temperature is ramped up to +25 C. over a period of 15 hours and the product is held at +25xc2x0 C. for five hours. The temperature then is ramped up to +40xc2x0 C. over a period of 20 minutes and held at 40xc2x0 C. for two hours. The lyophilized product preferably has a final moisture content of less than about 5% and typically about 1 to 2%.
The lyophilizate prepared in accordance with the present invention can be reconstituted with water, saline, or another electrolyte or non-electrolyte diluent to give a colloidal dispersion for administraion in a conventional manner such as intravenous or intraperitoneal administration. The lyophilized product with the addition of water provides a colloidal dispersion of the lipid-porphyrin complex in an aqueous solution of the excipient. A colloidal dispersion consists of at least two discrete phases. The first is a dispersed or internal phase; the second is a continuous or external phase. Systems in the colloidal state contain one or more substances that have at least one dimension in the range of 10-100A to a few microns. See pp. 272-4 in Chapter 19, Disperse Systems, Remington""s Pharmaceutical Sciences, 18th Edition, 1990, Mack Publishing Company, Easton, Pa. 18042. In the colloidal dispersions of the present invention, the dispersed or internal phase comprises particles of the porphyrin/lipid complex having a particle size in the range of 10 nm to 5000 nm. In selecting the aqueous vehicle, it is recommended to use one having a specific gravity about equal to the lipid complex (est. 1.09 g/cc) to minimize the tendency for the dispersion to separate. The lyophilizate of the lipid complex can be reconstituted with water, saline, or another pharmaceutically acceptable aqueous diluent for intravenous administration. Upon reconstitution a dispersion is obtained which is suitable for injection. The lyophilizate can also be administered orally as an aqueous dispersion or as an encapsulated paste. While porphyrins are not generally administered subcutaneously because they cause necrosis, it has been observed that the lipid slows the release of the porphyrin into the tissue making it potentially feasible to administer the lipid-porphyrin complex subcutaneously. The preferred route of administration is intravenous or intraperitoneal.
For oral administration, the lyophilizate can be reconstituted to form an oral dispersion or formulated into a paste. Typically, the lyophilizate is filled into a soft gelatin capsule for oral administration.
Suitable dosages for lipid complexes of tin porphyrin range from about 10 to 200 mg/m2/hour. The drug is preferably administered as a continuous infusion over 0.5 to 5 hours using a programmable continuous infusion ambulatory pump or an IV infusion bag. After administration of the complex to the patient, the patient is exposed to radiation in photodynamic therapy in a known manner. This therapy is normally conducted by exposure to radiation in the range of 300 to 700 nm.
The invention will now be described in more detail with reference to the following non-limiting example.

Lyophilizate of lipid complex of water insoluble porphyrins


An elastomeric loop used as a suspension device, for example for suspending exhaust systems of motor vehicles, includes a closed loop of elastomeric material in which a link chain is embedded, whereby the link chain improves the suspension characteristics of the suspension device.

This invention relates to an annular, elastomeric loop for effecting suspension of various parts, for example, exhaust systems parts, of motor vehicles.
An annular elastomeric loop for effecting suspension in the sense of the invention is a closed loop or ring, closed within itself, which does not have to have an absolutely circular shape, that has in principle any cross section, and wherein the closed loop or ring, consisting substantially of an elastomer, serves to accept, dampen and buffer traction forces which may arise between two constructional parts or machine parts that are limited in their motions relative to each other.
Elastomeric loops of this kind find use in the field of machine construction for suspending various components that are exposed to vibrations or impact stresses, for instance for the suspending of tables, pipes or conduits. For example, exhaust pipes and mufflers and whole exhaust systems are suspended by such elastomeric loops onto supports which are solidly fastened to the chassis or the body of motor vehicles.
Such known elastomeric loops consisting exclusively of an elastomer, have too flat a spring characteristic curve and a short life expectancy. The too low grade of progression of the spring characteristic curve, particularly over large paths, causes at peak loads, particularly impact load peaks that the impacted constructional part describes, a relatively large lift relative to the bearing constructional part and is brought back towards the bearing construction part with relatively large acceleration when the load is removed. This happens when elastomeric loops for suspending are used consisting exclusively of an elastomer, the dimensions of the loops following economical precepts. Such aforementioned overly-large spring paths at limit loads lead to bumping and knocking of the parts against each other, thus to a considerable diminution of the service life of the elastomeric loop.
Therefore, it is known to reinforce such elastomeric loops with inserts. Textile fabrics (Federal Republic of Germany Pat. No. 15 05 498 C3) or spring steel (Federal Republic of Germany Pat. No. 26 58 358 C3) are used as reinforcing materials. The desired progression of the spring characteristic curve cannot be obtained by textile reinforcements, at least by far not as much as desired. Furthermore, the elastomeric loops, reinforced with textile inserts have an insufficient thermal stability of the characteristic spring curve. Such a thermal stability of the course of the characteristic spring curve is, though, important in many fields of use, for instance when suspending exhaust pipes in motor vehicle construction. When using a spring steel band as an insert for elastomeric loops of a suspension, practical experience has shown that particularly oscillatory loads burden the spring band insert frequently up to the limiting area of spring extension. That leads to comparatively fast fatigue of the material and quite often to breaking of the spring steel band.
Other experiments known from practice to reinforce the elastomeric loop by aid of inelastical or only slightly extendable reinforcing inserts have neither led to any success. The characteristic spring curves of such elastomeric loops show already in the region of proportionality and also following the proportionality region such a steep rise, that the desired dampening action of the elastomeric suspension is considerably weakened.
In view of this known state of the art, the invention has as an object to provide an annular elastomeric loop for a suspension, especially for suspension that dampens vibration and dampens sound, whose characteristic spring curve shows a proportionality region as broad and as flat as possible, and immediately adjacent to it a progression region changing into a steep rise of the characteristic spring curve.
In order to achieve this objective, the invention provides an elastomeric loop of the aforedescribed kind in which the elastomer is reinforced by an embedded link chain. A solid, adhering and positive connection exists between the link chain, or the individual links of the link chain, respectively and the elastomer which forms the ring cord of the elastomeric loop. The link chain or the link chain ring that is closed on itself and the elastomeric ring, respectively, thus form a homogeneous composite body. The link chain ring and the elastomeric ring are disposed at least substantially concentrical to each other or, respectively at least substantially coaxial with each other normal to the ring plane.
When the elastomeric ring is deformed, the angles between two adjacent links of the link chain change so that the links perform kinking motions relative to each other. Due to the fact that the links of the link chain are connected positively to the elastomer of the cord of the elastomeric loop, this kinking motion of the links of the link chain against each other concentrates the course of tension and the tension of deformation in the elastomer and amplifies it. This amplification and concentration results in an extension of the proportionality region of the spring characteristic curve, in other words the force-path-charcteristic line of the elastomeric loop, and this is followed by a steep rise of the spring characteristic curve after the aforementioned proportionality region. This steep rise is finally by itself limited by the tensile strength of the embedded link chain ring.
The substantial advantage of the elastomeric loop of the invention over comparable elastomeric loops with embedded textiles or spring steel bands lies in the practically completely long-lasting, fatigue-free continuous load capacity, particularly vibrational load capacity. Experiments have shown that the elastomeric loop lasts through millions of extension cycles up to the progression region of the spring characteristic curve completely undamaged, while at identical conditions, textile inserts have lost their effectiveness by stretching after less than 1 million extension cycles and likewise steel band inserts by breaking.
In principle, the link chain ring may be disposed peripherally or eccentrically relative to the core or the central or median line of the elastomeric ring cord. In most cases of use, the link chain ring is disposed preferably at least substantially coaxially to the central axis of the ring cord or is embedded therein, respectively, so that the elastomeric ring cord and the link chain ring, embedded therein, have substantially the same symmetry. The link chain ring is preferably completely surrounded by the elastomer even though this is not essential under all circumstances for attaining the objectives of the invention.
The elastomeric loop may have a toroidal configuration or the shape of an O-ring with a circular profile of the elastomeric cord particularly when used in the construction of motor vehicles and when used for the silencing mounting of a pipe.
The link chain embedded in elastomeric material may, in principle, consist of any material, as long as it is capable of forming a solid connection with the elastomer. The link chain may even consist of a synthetic resin when that is allowed by the tensile resistance expected from the elastomeric loop. Preferably, though, the link chain consists of steel. The connection between the steel links of the link chain or the link chain ring, respectively, may be improved by priming or by coating the link chain ring with an adhesion promoter as known in the art.
The surprising improvement of the service life of the elastomeric loop by embedding a link chain is caused by the fact that the link chain follows practically in any direction the deformations of the elastomer at the conditions of use due to its positive connection without showing by itself any fatigue or deterioration and moreover, particularly without losing its tensile strength in any way and thereby changing the limiting extensibility or tensile strength, respectively, of the whole elastomer. Therefore, the special construction of the link chain is in principle open to change as long as it does not reduce and impede the ductility of the elastomeric ring under conditions of use, but influences it only amplifyingly in the planned direction. The link chain embedded in the elastomeric cord according to the invention is preferably a bolt chain, particularly a stud chain or a bushing chain because these elastomeric loops are frequently stressed relative to their extension in the ring plane and are planned to accept such stresses.
The extensibility of the elastomeric ring in the composition of link chain ring and elastomeric ring of the elastomeric loop may be utilized better or more completely when the diameter of the ring of the link chain ring is larger than the diameter of the ring of the elastomeric cord ring in its relaxed state.
The link chain ring is reduced to the diameter of the elastomeric cord ring by the position of the individual chain links of the link chain ring that do not lie straight within the elastomer but are angularly disposed to each other at least in sections and that by forming preferably a sawtooth line that is at least substantially regular. The angle formed by two adjacent chain links of the sawtooth ring line formed by the order of the chain links lies preferably in the range of between approximately 20.degree. and 90.degree. and depends individually as well upon the parameters of the elastomer, particularly on the hardness of the elastomer as also on the lengths of the individual chain links. Optimal values for each special case are easily obtainable. The following standard is valid for such determinations: at a hardness of the elastomer in the region of about 50 to 60 Shore A and a length of the individual chain links of 4 mm and a diameter of the ring of the elastomeric loop in the range of about 5 cm, each angle formed by two adjacent chain links lies approximately in the range of between 40.degree. and 50.degree.. The angles become progressively smaller the softer the elastomer is compounded and reversively, with harder elastomeric compositions, the larger must be the angle formed by the individual chain links of the angularly disposed link chain. On the other hand, the longer the individual chain links, the larger the kinking angle of the sawtooth line.
Best results are obtained when the link chain is disposed in a stretched condition in those regions corresponding to the run of the elastomeric cord of the elastomeric ring where the mountings act upon the elastomeric loop, such mountings transferring the loads to the elastomeric loop. Simultaneously, the link chain remains at least essentially in an alternatingly regular sawtooth curve in the remaining sections of the elastomeric loop.
Other features which are considered characteristic of the invention are set forth in the appended claims.
Although the invention is illustrated and described in relationship to specific embodiments, it is nevertheless not intended to be limited to the details shown, since various modifications and structural changes may be made therein without departing from the spirit of the invention and within the scope and range of equivalents of the claims.
The construction and operation of the invention, however, together with additional objects and advantages thereof will be best understood from the following description of specific embodiments when read in connection with the accompanying drawings.


Organic-inorganic support matrices for immobilized enzymes comprise a solid, porous, inorganic, water insoluble support combined with a copolymeric material resulting from the reaction of aminopolystyrene and a bifunctional monomer. The matrix is prepared by depositing a salt of amino-polystyrene within the pores of the solid support from an aqueous solution at a pH less than 7 after which the resulting composite is then reacted with an excess of a bifunctional reactive monomer thus forming a copolymeric organic material in situ substantially entrapped in the pores of the support and containing functionalized pendent groups to which an enzyme may be coupled to form an immobilized enzyme conjugate.

It is known that enzymes, which are proteinaceous in nature and which are commonly water soluble, comprise biological catalysts which serve to regulate many and varied chemical reactions which occur in living organisms. The enzymes may also be isolated and used in analytical, medical and industrial applications. For example, they find use in industrial applications in the preparation of food products such as cheese or bread as well as being used in the preparation of alcoholic beverages. Some specific uses in industry may be found in the use of enzymes such as in the resolution of amino acids; in the modification of penicillin to form various substrates thereof; the use of various proteases in cheese making, meat tenderizing, detergent formulations, leather manufacture and as digestive aids; the use of carbohydrases in starch hydrolysis, sucrose inversion, glucose isomerization, etc.; the use of nucleases in flavor control; or the use of oxidases in oxidation prevention and in the color control of food products. These uses as well as many others have been well delineated in the literature.
As hereinbefore set forth, inasmuch as enzymes are commonly water soluble as well as being generally unstable and readily deactivated, they are also difficult either to remove from the solutions in which they are utilized for subsequent reuse or it is difficult to maintain their catalytic activity for a relatively extended period of time. The aforementioned difficulties will, of course, lead to an increase cost in the use of enzymes for commercial purposes due to the necessity for frequent replacement of the enzyme, this replacement being usually necessary with each application. To counteract the high cost of replacement, it has been suggested to immobilize or insolubilize the enzymes prior to the use thereof. By immobilizing the enzymes through various systems hereinafter set forth in greater detail, it is possible to stabilize the enzymes in a relative manner and, therefore, to permit the reuse of the enzyme which may otherwise undergo deactivation or be lost in the reaction medium. Such immobilized or insolubilized enzymes may be employed in various reactor systems such as in packed columns, stirred tank reactors, etc., depending upon the nature of the substrate which is utilized therein. In general, the immobilization of the enzymes provides a more favorable or broader environmental and structural stability, a minimum of effluent problems and materials handling as well as the possibility of upgrading the activity of the enzyme itself.
As hereinbefore set forth, several general methods, as well as many modifications thereof, have been described by which the immobilization of enzymes may be effected. One general method is to adsorb the enzyme at a solid surface as, for example, when an enzyme such as amino acid acylase is adsorbed on a cellulosic derivative such as DEAE-cellulose; papain or ribonuclease is adsorbed on porous glass; catalase is adsorbed on charcoal; trypsin is adsorbed on quartz glass or cellulose, chymotrypsin is adsorbed on kaolinite, etc. Another general method is to trap an enzyme in a gel lattice such as glucose oxidase, urease, papain, etc., being entrapped in a polyacrylamide gel; acetyl cholinesterase being entrapped in a starch gel or a silicone polymer; glutamic-pyruvic transaminase being entrapped in a polyamide or cellulose acetate gel, etc. A further general method is a cross-linking by means of bifunctional reagents and may be effected in combination with either of the aforementioned general methods of immobilization. When utilizing this method, bifunctional or polyfunctional reagents which may induce intermolecular cross-linking will covalently bind the enzymes to each other as well as on a solid support. This method may be exemplified by the use of glutaraldehyde or bisdiazobenzidine-2,2'-disulfonic acid to bind an enzyme such as papain on a solid support, etc. A still further method of immobilizing an enzyme comprises the method of a covalent binding in which enzymes such as glucoamylase, trypsin, papain, pronase, amylase, glucose oxidase, pepsin, rennin, fungal protease, lactase, etc., are immobilized by covalent attachment to a polymeric material which is attached by various means to an organic or inorganic solid porous support. This method may also be combined with the aforesaid immobilization procedures.
The above enumerated methods of immobilizing enzymes all possess some drawbacks which detract from their use in industrial processes. For example, when an enzyme is directly adsorbed on the surface of a support, the binding forces which result between the enzyme and the carrier support are often quite weak, although some prior art has indicated that relatively stable conjugates of this type have been obtained when the pore size of the support and the spin diameter of the enzyme are correlated. However, in such cases it is specified that the pore size of the support cannot exceed a diameter of about 1000 Angstroms. In view of this weak bond, the enzyme is often readily desorbed in the presence of solutions of the substrate being processed. In addition to this, the enzyme may be partially or extensively deactivated due to its lack of mobility or due to interaction between the support and the active site of the enzyme. Another process which may be employed is the entrapment of enzymes in gel lattices which can be effected by polymerizing an aqueous solution or emulsion containing the monomeric form of the polymer and the enzyme or by incorporating the enzyme into the preformed polymer by various techniques, often in the presence of a cross-linking agent. While this method of immobilizing enzymes has an advantage in that the reaction conditions utilized to effect the entrapment are usually mild so that often there is little alteration or deactivation of the enzyme, it also has disadvantages in that the conjugate has poor mechanical strength, which results in compacting when used in columns in continuous flow systems, with a concomitant plugging of the column. Such systems also have rather wide variations in pore size thus leading to some pore sizes which are large enough to permit the loss of enzyme. In addition, some pore sizes may be sufficiently small so that large diffusional barriers to the transport of the substrate and product will lead to reaction retardation, this being especially true when using a high molecular weight substrate. The disadvantages which are present when immobilizing an enzyme by intermolecular cross-linkage, as already noted, are due to the lack of mobility with resulting deactivation because of inability of the enzyme to assume the natural configuration necessary for maximum activity, particularly when the active site is involved in the binding process.
Covalent binding methods have found wide applications and may be used either as the sole immobilization technique or as an integral part of many of the methods already described in which cross-linking reactions are employed. This method is often used to bind the enzyme as well as the support through a bifunctional intermediary molecule in which the functional groups of the molecule, such as, for example, gamma-aminopropyltri-ethoxysilane, are capable of reacting with functional moieties present in both the enzyme and either an organic or inorganic porous support. A wide variety of reagents and supports has been employed in this manner and the method has the advantage of providing strong covalent bonds throughout the conjugate product as well as great activity in many cases. The covalent linkage of the enzyme to the carrier must be accomplished through functional groups on the enzyme which are non-essential for its catalytic activity such as free amino groups, carboxyl groups, hydroxyl groups, phenolic groups, sulfhydryl groups, etc. These functional groups will also react with a wide variety of other functional groups such as an aldehydo, isocyanato, acyl, diazo, azido, anhydro activated ester, etc., to produce covalent bonds. Nevertheless, this method also often has many disadvantages involving costly reactants and solvents, as well as specialized and costly porous supports and cumbersome multi-step procedures, which render the method of preparation uneconomical for commercial application.
The prior art is therefore replete with various methods for immobilizing enzymes which, however, in various ways fail to meet the requirements of economical industrial use. However, as will hereinafter be discussed in greater detail, none of the prior art compositions comprise the composition of matter of the present invention which constitutes an inorganic porous support containing a copolymer, formed in situ from a polyfunctional monomer, a low molecular weight polymer, a polymer hydrolysate, or a preformed polymer, of natural or synthetic origin by reaction with a bifunctional monomer, the copolymer formed being substantially entrapped within the pores of said support, and which contains terminally functionalized, pendent groups extending therefrom; the enzyme being covalently bound to the active moieties at the terminal reactive portions of the pendent groups, thus permitting the freedom of movement which will enable the enzyme to exercise maximum activity. A variable portion of the enzyme will also be adsorbed upon the matrix, but this will be recognized as an unavoidable consequence of almost all immobilization procedures involving porous inorganic supports and is not to be considered a crucial aspect of this invention. Furthermore, the bond between the inorganic support and the organic copolymer which has been prepared in situ in the pores of the support is not covalent but rather physico-chemical and mechanical in nature and the inorganic-organic matrix so produced presents high stability and resistance to disruption. As further examples of prior art, U.S. Pat. No. 3,556,945 relates to enzyme composites in which the enzyme is adsorbed directly to an inorganic carrier such as glass. U.S. Pat. No. 3,519,538 is concerned with enzyme composites in which the enzymes are chemically coupled by means of an intermediary silane coupling agent to an inorganic carrier. In similar fashion, U.S. Pat. No. 3,783,101 also utilizes an organosilane composite as a binding agent, the enzyme being covalently coupled to a glass carrier by means of an intermediate silane coupling agent, the silicon portion of the coupling agent being attached to the carrier while the organic portion of the coupling agent is coupled to the enzyme, the composition containing a metal oxide on the surface of the carrier disposed between the carrier and the silicon portion of the coupling agent. In U.S. Pat. No. 3,821,083 a water-insoluble polymer such as polyacrolein is deposited on an inorganic carrier and an enzyme is then covalently linked to the aldehyde groups of the polymer. However, according to most of the examples set forth in this patent, it is necessary to first hydrolyze the composite prior to the deposition of the enzyme on the polymer. Additionally the product which is obtained by the method of this patent suffers a number of disadvantages in that it first requires either the deposition, or initially the formation, of the desired polymer in an organic medium followed by its deposition on the inorganic carrier with a subsequent clean-up operation involving distillation to remove the organic medium. In addition to this, in another method set forth in this reference, an additional hydrolytic reaction is required in order to release the aldehyde groups from the initial acetal configuration in which they occurred in the polymer. Inasmuch as these aldehyde moieties are attached directly to the backbone of the polymer, the enzyme is also held adjacent to the surface of the polymer inasmuch as it is separated from the surface of the polymer by only one carbon atom of the reacting aldehyde group and, therefore, the enzyme is obviously subjected to the physico-chemical influences of the polymer as well as being relatively immobilized and inhibited from assuming its optimum configuration. Another prior art patent, namely, U.S. Pat. No. 3,705,084 discloses a macroporous enzyme reactor in which an enzyme is adsorbed on the polymeric surface of a macroporous reactor core and thereafter is cross-linked in place. By cross-linking the enzymes on the polymeric surface after adsorption thereof, the enzyme is further immobilized in part and cannot act freely as in its native state as a catalyst. The cross-linkage of enzymes in effect links them together, thereby preventing a free movement of the enzyme and decreases the mobility of the enzyme which is a necessary prerequisite for maximum activity.
U.S. Pat. No. 3,654,083 discloses a water-soluble enzyme conjugate which is prepared from an organic water-soluble support to which the enzyme is cross-linked and whose utility is limited only to cleaning compositions and pharmaceutical ointments. However, this enzyme composition also suffers from the disadvantages of the close proximity and interlocking of the enzyme and support, as well as the poor mechanical strength which is generally exhibited by enzyme conjugates based on organic polymeric supports.
U.S. Pat. No. 3,796,634 also discloses an immobilized biologically active enzyme which differs to a considerable degree from the immobilized enzyme conjugates of the present invention. The enzyme conjugate of this patent consists of an inorganic support comprising colloidal particles possessing a particle size of from 50 to 20,000 Angstroms with a polyethyleneimine, the latter being cross-linked with glutaraldehyde to staple the cross-linked polymer so formed as a monolayer on the surface of the colloidal particles, followed by adsorption of the enzyme directly onto this monolayer. Following this, the enzyme which is adsorbed as a monolayer on the surface of the colloidal particles is then cross-linked with additional glutaraldehyde to other adsorbed enzyme molecules to prevent them from being readily desorbed while in use. There is no indication of any covalent binding between enzyme and polymer matrix as is present in the present invention. By the enzyme molecules being cross-linked together on the surface of the support, this conjugate, therefore, is subjected to deactivation by both the cross-linking reaction and by the electronic and steric effects of the surface, said enzyme possessing limited mobility. Inasmuch as the product of this patent is colloidal in nature, it also possesses a very limited utility for scale-up to commercial operation, since it cannot be used in a continuous flow system such as a packed column because it would either be carried along and out of the system in the flowing liquid stream or, if a restraining membrane should be employed, the particles would soon become packed against the barrier to form an impervious layer. In addition, such a colloidal product could not readily be utilized in a fluidized bed apparatus, thereby limiting the chief utility to a batch type reactor such as a stirred tank type reactor from which it would have to be separated by centrifugation upon each use cycle. In contrast to this, the immobilized enzyme conjugates of the present invention may be employed in a wide variety of batch or continuous type reactors and therefore are much more versatile with regard to their modes of application.
In addition, another prior art reference U.S. Pat. No. 3,959,080 relates to a carrier matrix for immobilizing biochemical effective substances. However, the matrix which is produced according to this reference constitutes the product derived from the reaction of an organic polymer containing cross-linkable acid hydrazide or acid azide groups with a bifunctional cross-linking agent such as glutaraldehyde. However, this matrix also suffers from the relatively poor mechanical stability and other deficiencies which are characteristic of organic enzyme supports as well as the relatively complex organic reactions employed in preparing such polymeric hydrazides, etc.
As will hereinafter be shown in greater detail, the organic-inorganic matrix of the present invention will provide a support to which an enzyme may be covalently bound to provide a catalytic composite which will maintain its activity and stability over a relatively long period of use.
This invention relates to compositions of matter comprising support matrices for immobilized enzymes. More specifically, the invention is concerned with support matrices consisting of an organic-inorganic composite in which the inorganic support material is combined with an organic copolymer prepared in situ and substantially entrapped within the pores of the inorganic support. The copolymer is formed by the reaction of aminopolystyrene with a sufficient excess of a bifunctional monomer containing suitable reactive moieties to provide a copolymeric product containing terminally functionalized pendent groups capable of covalently binding enzymes at the terminal reactive portions thereof. In addition, the invention is also concerned with a process for preparing these matrices.
As hereinbefore set forth, the use of enzymes in analytical, medical or industrial applications may be greatly enhanced if said enzymes are in an immobilized condition, that is, said enzymes, by being in combination with other solids materials, are themselves in such a condition whereby they are not water soluble and therefore they may be subjected to repeated use while maintaining the catalytic activity of said enzyme. In order to be present in an immobilized state, the enzymes must be bound in some manner to a water insoluble carrier, thereby being commercially usable in an aqueous insoluble state.
It is therefore an object of this invention to provide novel compositions of matter in which enzymes may be covalently bound in an immobilized state.
A further object of this invention is to provide a process for preparing combined inorganic-organic support matrices which are utilized for covalently binding an enzyme to the functionalized pendent groups at the reactive terminal portions thereof.
In one aspect an embodiment of this invention resides in an organic-inorganic matrix comprising a porous, inorganic, water-insoluble solid support in combination with a copolymeric material resulting from the reaction of aminopolystyrene and a bifunctional monomer.
A further embodiment of this invention is found in a method for preparing an organic-inorganic matrix by depositing a salt of aminopoly-styrene on a solid support from an aqueous solution at a pH less than 7 and thereafter reacting the resultant aminopolystyrene-solid support composite with a bifunctional monomer to form the desired organic-inorganic matrix.
A specific embodiment of this invention is found in an organic-inorganic matrix which comprises gamma-alumina having combined therewith a copolymer resulting from the reaction of aminopolystyrene and an excess of glutaraldehyde.
Another specific embodiment of this invention is found in a process for preparing an organic-inorganic matrix which comprises depositing the hydrochloric acid salt of aminopolystyrene on gamma-alumina from an aqueous solution at a pH in the range of from about 1 to about 4, thereafter reacting the resultant aminopolystyrene-gamma-alumina composite with an excess of glutaraldehyde, and recovering the resultant organic-inorganic matrix.
Other objects and embodiments will be found in the following further detailed description of the present invention.
As hereinbefore set forth the present invention is concerned with support matrices which are used to immobilize enzymes, said matrices comprising an organic-inorganic composite consisting of an inorganic support material of the type hereinafter set forth in greater detail combined with a copolymeric organic material which, in the case of a porous support, is substantially entrapped in the pores of said porous support. The copolymeric composite will contain pendent groups extending therefrom, said pendent groups containing terminally positioned functional moieties which will enable an enzyme to be covalently bound to said group at the reactive terminal portions thereof. Furthermore, the invention is also concerned with a process for preparing these support matrices using relatively inexpensive reactants as well as utilizing more simple steps in the procedure for preparing said compositions. In addition, the mechanical strength and stability of enzyme conjugates which result from the covalent binding of enzymes to these support matrices will be greater than that which is possessed by the immobilized enzymes of the prior art. Therefore, it will be readily apparent that the compositions of matter of the present invention possesses economical advantages which are useful for industrial applications.
Examples of inorganic supports which may be utilized as one component of the support matrices of the present invention will consist of a wide variety of materials including porous supports such as alumina which possess pore diameters ranging from about 100 Angstroms up to about 55,000 Angstroms and which also possess an Apparent Bulk Density (ABD) in the range of from about 0.1 to about 0.6. The surface area of the particular inorganic porous support will also vary over a relatively wide range, said range being from about 1 to about 500 m.sup.2 /gm, the preferred range of surface area being from about 5 to about 400 m.sup.2 /gm. The configuration of the inorganic porous support material will vary, depending upon the particular type of support which is utilized. For example, the support material may be in spherical form, particulate form ranging from fine particles to macrospheres, as a ceramic monolith which may or may not be coated with a porous inorganic oxide, a membrane, ceramic fibers, alone or woven into a cloth, silica, mixtures of metallic oxides, sand particles, zeolites, mica, etc. The particle size may also vary over a wide range, again depending upon the particular type of support which is employed and also upon the substrate and the type of installation in which the enzyme conjugate is to be used. For example, if the support is in spherical form, the spheres may range in size from about 0.01" to about 0.25" in diameter, the preferred size ranging from about 1/32" to 1/8" in diameter. When the support is in particulate form, the particle size may also range between about the same limits. In terms of U.S. standard mesh sizes, such particles may range from about 2.5 to about 100 mesh, with about 10-40 mesh sizes preferred. Likewise, if the support is in the shape of ceramic fibers, the fibers may range from about 0.5 to about 20 microns in diameter or, if in the form of a membrane, the membrane may comprise a ceramic material which is cast into a thin sheet. It is to be understood that the aforementioned types of support configuration and size of the various supports are given merely for purposes of illustration, and it is not intended that the present invention be necessarily limited thereto.
It is also contemplated that the porous support materials may be coated with various oxides of the type hereinbefore set forth, or consist of mixtures thereof, or may have incorporated therein various other inorganic materials such as boron phosphate, etc., these inorganic materials imparting special properties to the support material. A particularly useful form of support will constitute a ceramic body which may have the type of porosity herein described for materials of the present invention or it may be honeycombed with connecting macro size channels throughout, such materials being commonly known as monoliths, and which may be coated with various types of porous alumina, zirconia, titanium oxide, etc. The use of such a type of support has the particular advantage of permitting the free flow of highly viscous substrates which are often encountered in commercial enzyme catalyzed reactions. One component of the organic portion of the support matrix comprises an aminopolystyrene which may be prepared by any manner known in the art and is deposited on the inorganic portion of the support material in a manner hereinafter set forth in greater detail. The composite is then treated with a bifunctional monomer to form the matrix upon which an enzyme may be immobilized. The bifunctional monomer reactant is present in sufficient excess as needed to produce pendent terminally functionalized groups, said bifunctional monomer being present in a range of from about 2 to about 50 moles or more relative to the reactive moieties of the support composite, the preferred range being from about 4 to about 25 moles of excess.
The functional groups which are present on the bifunctional monomer will comprise well-known reactive moieties capable of bonding readily with amino groups such as carbonyl, acyl, isocyanato, etc., moieties. As was also hereinbefore set forth, the reactive groups of the bifunctional compounds are preferably, but not necessarily, separated by chains containing from about 4 to about 10 carbon atoms. The reactive moieties of the bifunctional compounds are therefore capable of covalently bonding with both the aminopolystyrene component of the support matrix and subsequently, after washing out unreacted materials, also with the amino groups of the enzyme which is to be added in a subsequent step, said enzyme being then covalently bound to the reactive functional group at the terminal portion of the pendent chain. After addition of the enzyme to this composition, a relatively stable enzyme conjugate will be produced which possesses high activity and high stability. The unreacted enzyme can also be recovered for reuse. Due to the large excess of intermediate, or spacer bifunctional monomeric molecules which are used, the matrix will contain pendent groups comprising the spacer molecules, said molecules extending from the matrix and having reactive moieties available at the terminal portions thereof which are capable of reacting with and binding the enzyme to the aforesaid spacer molecules via covalent bonds. Therefore, it is readily apparent that a suitable organic-inorganic matrix which is applicable in binding enzymes will be formed, provided that a large enough excess of the bifunctional molecule is used to provide reactive pendent groups which are capable of subsequently reacting with the enzyme to be immobilized. By utilizing these functional pendent groups as a binding site for the enzymes, it will permit the enzymes to have a greater mobility and thus permit the catalytic activity of the enzyme to remain at a high level for a relatively longer period of time than will be attained when the enzyme has been immobilized by any of the other methods such as entrapment in a gel lattice, adsorption on a solid surface or cross-linkage of the enzyme with adjacent enzyme molecules by means of bifunctional reagents, etc. Not all formulations, however, will produce equivalent results in terms of stability or activity.
Examples of enzymes which may be immobilized by a covalent bonding reaction and which contain an amino group capable of reacting with an aldehydic, isocyanato, acyl, ester, etc., moiety of the pendent group which is attached to a polymeric material substantially entrapped in the pores of a porous support material will include trypsin, papain, hexokinase, betagalactosidase (lactase), ficin, bromelain, lactate dehydrogenase, glucoamylase, chymotrypsin, pronase, glucose, isomerase, acylase, invertase, amylase, glucose oxidase, pepsin, rennin, protease, xylanase, cellulase, etc. In general any enzyme whose active site is not involved in the covalent bonding can be used although not necessarily with equivalent results. While the aforementioned discussion was centered about pendent groups which contain as a functional moiety thereon an aldehydic or isocyanato group, it is also contemplated within the scope of this invention that the pendent group can contain functional moieties capable of reaction with carboxyl, sulfhydryl or other moieties usually present in enzymes. However, the covalent bonding of enzymes containing these other moieties with other pendent groups may not necessarily be effected with equivalent results and may also involve appreciably greater costs in preparing intermediates. It is to be understood that the aforementioned listing of porous solid supports, monomers, hydrolysates, polymers and enzymes are only representative of the various classes of compounds which may be used, and that the present invention is not necessarily limited thereto.
The preparation of the compositions of matter of the present invention is preferably effected in a batch type operation. In the preferred method of preparation, the inorganic support material will be treated with a solution, preferably aqueous in nature, of a salt of aminopolystyrene, the aqueous solution being maintained at a pH less than 7 and preferably in a range of from 1 to about 4. Examples of salts of aminopolystyrene which may be employed will include the hydrochloric acid salt, the sulfuric acid salt, the nitric acid salt, the phosphoric acid salt of aminopolystyrene, etc. The pH of the aqueous solution is maintained at the desired level by the addition of an acid such as those hereinbefore set forth, the amount of acid being added being sufficient, as hereinbefore set forth, to maintain the pH in a range of from 1 to about 4. Upon completion of the addition of the acid salt of the aminopolystyrene which in the preferred embodiment of the invention is effected at ambient temperature and atmospheric pressure, the mixture is placed under vacuum for a period of time which may range from about 0.5 up to about 4 hours or more in duration. Upon completion of the reaction time, the unadsorbed solution is removed and the treated support allowed to air dry until it is free flowing in nature. Thereafter the organic-inorganic composite is contacted with a sufficiently large excess of a bifunctional monomer of from about 3 to about 50 or more moles proportion relative to the amine content of the initial aminopolystyrene to provide pendent groups extending from the resultant copolymer, said pendent groups containing unreacted terminal functional moieties. The bifunctional monomer is also preferably added in an aqueous solution which, after reaction with the aminopolystyrene, is removed and the resultant matrix washed to separate any bifunctional monomer which may still be present.
As hereinbefore set forth, the use of an excess of the bifunctional monomer will result in pendent groups extending from the matrix which contain unreacted terminal functional moieties. The unreacted functional moieties are then available for covalent binding to the enzyme, which is added to the matrix, again usually in an aqueous solution. After removal of the unreacted materials by conventional means such as by treating, washing, etc., the enzyme which is covalently bound to the pendent functionalized groups remains attached at the terminal portions thereof. It is therefore readily apparent that the entire immobilized procedure can be conducted in a simple and inexpensive manner, for example, in a column packed with the inorganic supports, utilizing an aqueous or inexpensive solvent media. The procedure may be conducted over a temperature differential, if so desired, which may range from subambient (about 5.degree. C.) up to about elevated temperatures of about 60.degree. C., preferably at ambient (about 20.degree.-25.degree. C.) temperature, said procedure being effected by utilizing a minimal of operating steps and, in addition, permitting a ready recovery of the excess reactants, unbound enzyme and finished composition of matter, the excess reactants and unbound enzymes being available for reuse thereof.
It is also contemplated within the scope of this invention that the formation of the finished composition of matter may also be effected in a continuous manner of operation. When this type of operation is employed, a quantity of the solid support, either porous or non-porous in nature, is placed in an appropriate apparatus usually constituting a column. As in the case of the batch type operation, the solid support material may be in any form desired such as powder, pellets, monoliths, etc., and is charged to the column after which a preferably aqueous solution of a salt of aminopolystyrene is also charged and contacts the support until the latter is saturated with the solution. The aqueous solution is maintained at a pH less than 7 and preferably at a pH in a range of from about 1 to about 4 by the addition of an appropriate acid. After saturation of the support has been accomplished an excess is then drained and an intermediary spacer compound such as a reactive bifunctional monomer molecule, preferably in aqueous solution, is charged to the column, said bifunctional molecule being present in an excess in a range of from about 2 to about 50 moles or more relative to the amine content of the aminopolystyrene. While the formation of the matrix is effected during a period of time which may range from about 1 to about 10 hours or more in duration the formation is usually accomplished during a relatively short period of time. Following the completion of the desired residence time the excess spacer reactant such as glutaraldehyde is removed by draining followed by a thorough water washing to remove any unreacted materials.
To form an immobilized enzyme conjugate, an aqueous solution of an enzyme of the type hereinbefore set forth in greater detail is then passed through the column containing the thus formed support matrix thereby effecting a covalent bonding of the enzyme to the terminal reactive groups of the functionalized pendent moieties which extend from the matrix. This occurs until there is no further covalent binding of the enzyme to the pendent molecules. The excess enzyme is recovered in the effluent which is continuously withdrawn after draining and may be recycled to the column for further use. After washing the column, the column is then ready for use in chemical reactions in which the catalytic effect of the enzyme is to take place. These procedures are conducted within the time, temperature and concentration parameters hereinbefore set forth described in the batch type procedure and will result in comparable immobilized enzyme complexes. It is also contemplated within the scope of this invention that with suitable modifications of reaction parameters it will be obvious to those skilled in the art that the process may be applied to a wide variety of supports, bifunctional monomers and enzymes.

Support matrices for immobilized enzymes


Systems for controlling fluids in semiconductor processing systems are disclosed. The disclosed systems comprise a chemical blender, a reclaim tank, a dispense system, two chemical monitors, a controller, and a reclamation line in fluid communication with an outlet of the process station and coupled to the reclaim tank. The reclaim tank mixes solution from the chemical blender and the reclamation line. One of the two chemical monitors the mixed solution downstream from the dispense system. The controller is configured to flow the mixed solution to the process station upon determination by the first chemical monitor that at least one chemical compound in the mixed solution is at a predetermined concentration. The second chemical monitor monitors the reclaimed portion of the mixed solution to determine whether the at least one chemical compound is at a predetermined concentration before being reintroduced into the reclaim tank.

1. Field of the Invention
This disclosure pertains to methods and systems for the management of chemicals in processing environments, such as semiconductor fabrication environments.
2. Related Art
In various industries, chemical delivery systems are used to supply chemicals to processing tools. Illustrative industries include the semiconductor industry, pharmaceutical industry, biomedical industry, food processing industry, household product industry, personal care products industry, petroleum industry and others.
The chemicals being delivered by a given chemical delivery system depend, of course, on the particular processes being performed. Accordingly, the particular chemicals supplied to semiconductor processing tools depend on the processes being performed on wafers in the tools. Illustrative semiconductor processes include etching, cleaning, chemical mechanical polishing (CMP) and wet deposition (e.g., copper electroless, electroplating, etc.).
Commonly, two or more fluids are combined to form a desired solution for a particular process. The solution mixtures can be prepared off-site and then shipped to an end point location or a point-of-use for a given process. This approach is typically referred to as batch processing or batching. Alternatively, and more desirably, the cleaning solution mixtures are prepared at the point-of-use with a suitable mixer or blender system prior to delivery to the cleaning process. The latter approach is sometimes referred as continuous blending.
In either case, accurate mixing of reagents at desired ratios is particularly important because variations in concentration of the chemicals detrimentally affect process performance. For example, failure to maintain specified concentrations of chemicals for an etch process can introduce uncertainty in etch rates and, hence, is a source of process variation.
Therefore, there is a need for methods and systems for managing chemical conditioning and supply in processing environments.

Reclaim function for semiconductor processing system