The starting material for the present purification process is a supernatant, suspension, milk product, colostrum or crude plasma protein fraction containing MBL. The starting material is subject to few pre-processing steps in order to obtain an MBL containing solution. By this it is possible to perform an affinity chromatography as the first chromatographic process step employing a non-conjugated polysaccharide matrix, from which MBL of a high purity is eluted. Besides constituting the major purification step of the process, the affinity chromatography also serves as a virus-removal step. In the production process of a virus-safe product, a virus-inactivation step is also included. The product of the process of the present invention is ready for use as medicinal product.

The present invention relates to a novel purification process for production of mannan-binding lectin (MBL) (formerly designated mannan binding protein, MBP) preferably from donor plasma, to be used as an MBL medicinal product. The product is to be used for substitution or replacement therapy in patients with inherited or acquired MBL-deficiency associated with functional and/or clinical symptoms, i.e. where it is contemplated that said patients would benefit from the administration of MBL, e.g. for the treatment or prevention of infections.
Innate (also named natural or non-anticipatory) immune functions have recently received an increasing interest as important elements in defence mechanisms against potentially pathogenic microorganisms. Thus, attention has especially been given to a group of lectins, the collecting, which are believed to play an important role in the immediate defence against a wide range of microorganisms. The serum protein MBL is a collectin, i.e. it is built up as oligomeric structures, characterized by calcium-dependent, C-type carbohydrate-recognition domains (CRDs) attached to collagenous rods. The precise oligomerization of circulating MBL remains unclear; however, higher order oligomeric, bouquet-like structures such as hexameric MBL with multiple binding sites appear to be essential for the functional activity of MBL (for recent reviews, see references 1, 2) (a list of references is given at the end of this specification).
The cumulative knowledge about MBL indicates a future role for this protein in interventional therapy against serious infections. MBL is structurally similar to the complement component C1q, an essential component in the activation of the classical pathway of complement. MBL appears to activate the complement system by a mechanism analogous to that of C1q, i.e. via associated serine proteases, termed MASPs (MBL-associated serine proteases). This antibody-independent complement activation has been named the xe2x80x9cMB-lectin pathway of complement activationxe2x80x9d (3, 4).
MBL binds to carbohydrate structures on surfaces of bacteria, yeast, parasitic protozoa and viruses, and has been found to exhibit antibacterial activity through killing of the bacteria via the terminal, cytolytic pathway of the complement system, or through promotion of phagocytosis by opsonization. The level of MBL in plasma is genetically determined. Each individual has a constitutional MBL level reflected by the genomic structure in the controlling region as well as in the coding region. The concentration of MBL in plasma thus varies from about 10 xcexcg/ml to less than 10 ng/ml. Infants or adults with deficiency or very low levels of MBL are especially susceptible to infections. Recent information points to a role of MBL deficiency as a susceptibility factor in HIV infection, and also to MBL deficiency being associated with more rapid death following development of AIDS (1). MBL deficiency may also predispose to recurrent spontaneous abortions (5).
Mannan-binding lectin was first isolated from human serum in 1983 (6) by affinity chromatography on mannan-Sepharose (mannan coupled to a Sepharose matrix) in the presence of Ca-ions. Elution of MBL from the affinity column was performed by means of EDTA.
It appears from later publications that MBL has been purified essentially by the same procedure from serum and plasma. The purified MBL preparations recovered from this one-step procedure were heavily contaminated by antibodies with specificity for carbohydrates and serum amyloid p-component (SAP). To obtain MBL of higher purity, further chromatographic steps were included in the purification procedures, such as a Sepharose precolumn to the affinity column, and additional affinity steps using different carbohydrates either coupled to the matrix or added to the elution buffer; other chromatographic principles as ion exchange and gel filtration chromatography were employed as well (7, 8, 9, 10). In general, at least two affinity chromatographic steps have been employed in the procedures for obtaining highly purified MBL. Recently a procedure has been described, where a plasma protein fraction obtained by precipitation of human plasma with 7% PEG was used as the starting material for MBL purification (11). This procedure differed from those previously described in that the affinity chromatography was performed on non-conjugated Sepharose (Sepharose without immobilized carbohydrate-ligands): first the solubilized PEG-precipitate was subjected to batch adsorption on Sepharose, and after elution of MBL by EDTA, a subsequent affinity chromatography step on a Sepharose column was performed, with eluting of MBL by mannose. By this procedure employing two consecutive affinity steps, MBL was obtained at high purity. DNA encoding human mannose binding protein is disclosed in WO98/01519.
The present invention relates to a process for purifying mannan-binding lectin (MBL), preferably from a crude plasma protein fraction. The process of the invention will, among other elements, include at least two key elements: performing one affinity chromatography step on a non-conjugated polysaccharide matrix, and performing at least one validated virus-reduction step.
MBL can be purified from a wide range of starting materials containing MBL. In one embodiment, the starting material for the process of the invention is an MBL containing supernatant or a lysed cell suspension from a yeast or mammalian cell culture expressing MBL, said cell culture comprising cells coding for mammalian (e.g. human) MBL and optionally coding for the MBL Associated Serine Proteases (MASPs). The MBL expressing cell culture is grown in a medium providing the cell culture the nutrients needed with or without serum added to the culture medium. In another embodiment, MBL is purified from milk and/or colostrum from a mammal expressing a mammal (e.g. human) MBL gene. In one embodiment, the mammal is a transgenic non-human animal. In a preferred embodiment of the invention, the starting material for the process of the invention is a crude plasma protein fraction obtainable from industrial scale ethanol fractionation procedures, such as Cohn fraction I, II and III; Cohn fraction II and III; or Cohn fraction III. In a preferred embodiment, the plasma protein fraction is Cohn fraction II and III, where filter aid may or may not be present depending on the method employed for isolation of the Cohn fraction, i.e. by filtration or centrifugation. The use of Cohn fraction II and III as starting material has several advantages. These comprise, but are not limited to: no need of further ethanol fractionation, immunoglobulins can be recovered for an immunoglobulin product, and MBL is recovered from a fraction usually discarded.
Each of the starting materials will require a few pre-processing steps to obtain an MBL containing solution. The pre-processing steps will be discussed below.
The first key element of the present process, the affinity chromatography on a non-conjugated polysaccharide matrix, has several advantages. These comprise, but are not limited to: no need for prior protein precipitation, selectivity for functionally active MBL, a high degree of purification, removal of viruses, concentration by volume reduction.
The MBL containing solution is a complex protein mixture, where MBL may constitute less than 0.05% of the total proteins from the starting materials. Purification by means of chromatographic methods alternative to affinity chromatography would require further protein fractionation of the MBL containing solution e.g. by protein precipitation. The advantage of employing an affinity step is that no prior protein fractionation steps, such as precipitation and resuspension steps are needed, thus allowing the MBL containing solution to be applied directly to the column.
As a consequence of the pre-processing steps, e.g. the ethanol fractionation or the nature of the MBL expression system used, it is expected that the MBL containing solution contains MBL as native, oligomeric proteins as well as denatured and structurally impaired protein forms. Since the MBL product for use in medicine has to be constituted by functionally active MBL, it is of great importance to perform a purification step which selects for functionality. Affinity chromatography fulfils this requirement by selecting for functionally active, oligomeric ligand-binding MBL.
It is well known that affinity chromatography is the method of choice for purifying proteins from a complex protein mixture often resulting in several thousand fold of purification. By the affinity chromatography step of the present invention MBL is purified to a very high degree, i.e. more than 2500 fold. The affinity chromatography is the major purification step of the process, and contributes almost solely to the high purity obtained in the final MBL preparation of the process. Even purification to a minor degree is far beyond what is presently known in the art. Also, a purification which is 500 fold, i.e. 1000, 1500, 2000, or 2250 fold is acceptable. Purification to a minor degree is especially acceptable when less complex protein mixtures are used as the starting material. That is, if MBL constitutes more than about 0.05% of the total protein content.
Although the MBL containing solution applied to the column may have been concentrated, the concentration of MBL is still relatively low, and the affinity chromatography serves as a concentration step, by concentrating the MBL applied at least 3 fold, such as concentrating the MBL applied at least 4 fold.
The affinity chromatography step is performed on a non-conjugated polysaccharide based matrix. By a non-conjugated matrix is understood that no carbohydrate-ligands are coupled to the matrix. The advantages comprise, but are not limited to: The basic structure of the media used as matrix consists of bundles of polysaccharide chains, which act as ligands for MBL. There is no need for a special manufacturing of a matrix by chemically coupling of carbohydrate-ligands. Problems with an unstable matrix and/or uncontrolled leakage of ligands are avoided.
Furthermore, the matrix should preferably be cross-linked. The advantage of a cross-linked polysaccharide material is the rigidity and high physical stability, enabling the use of a large column with good flow properties in the process. The cross-linked matrix further has the advantage of a high chemical stability, enabling cleaning of the column with e.g. strong alkaline solutions. Preferred materials for the affinity chromatography step are gel materials containing agarose and/or dextran and/or cellulose such as Sepharose CL6B (Pharmacia), Ultrogel (Pharmacia), Bio-gel A materials, e.g. 0.5 m, 1.5 m, 15 m, and 50 m (all Bio-Rad), Sephadex gel materials, e.g. G-50, G-75, G-100, G-150, and G-200 (all Pharmacia), Sephacryl HR gel materials, e.g. S-300, S-400, S-500 (all Pharmacia), Superdex 200 prep grade (Pharmacia), Superose 6 prep grade (Pharmacia), and Cellulose gel material from Whatman, especially preferred materials for the affinity chromatography step is Sepharose CL4B (Pharmacia)
Preferably, the column is cleaned with 0.5 M NaOH in order to ensure aseptic production conditions and avoid batch-to-batch contamination. A person skilled in the art will appreciate the advantage of this cleaning procedure and the use of a matrix material for many cycles of chromatography.
After application of the MBL-containing solution to the affinity matrix, the column is washed. The buffers used for washing out protein contaminants from the affinity matrix are non-denaturing buffers having a composition, pH, and ionic strength resulting in elimination of the major proportion of protein contaminants without substantial elution of MBL. Initially, an equilibration buffer is used. This buffer could be a Tris buffer with a molarity within the range of 10-40 mM, preferably 10 mM, and a pH of 7.0-8.0, preferably 7.3, with a content of NaCl ranging from 100-250 mM, preferably 145 mM; and a content of CaCl2 of 3-15 mM, preferably 5 mM. Subsequently a buffer with a low content of CaCl2 is used. The low content of CaCl2 could be 0.2-2.0 mM, preferably 0.3-1.0 mM, such as 0.5 mM. Due to the employment of a matrix to which MBL binds with high affinity in a Ca2+-dependent manner, the concentration of CaCl2 can be lowered in the washing buffer after a stable adsorption of MBL to the matrix has been established without substantial elution of MBL. In this manner, contaminants binding Ca2+-dependently to the matrix with lower affinity, e.g. carbohydrate specific antibodies, are washed out.
The binding capacity of the column is defined as the total amount of MBL absorbed to and eluted from the matrix (calculated as volume of the eluate fraction times the concentration) divided by the volume of the gel-matrix. It is preferred that the binding capacity of the column is more than 20 xcexcg MBL/ml packed matrix, e.g. more than 25 xcexcg MBL/ml packed matrix, such as more than 30 xcexcg MBL/ml packed matrix, more than 35 xcexcg MBL/ml packed matrix, more than 40 xcexcg MBL/ml packed matrix, more than 42 xcexcg MBL/ml packed matrix, more than 44 xcexcg MBL/ml packed matrix, more than 46 xcexcg MBL/ml packed matrix, more than 48 xcexcg MBL/ml packed matrix or even more than 50 xcexcg MBL/ml packed matrix.
After washing the affinity chromatography column, the elution of MBL is performed with a selective desorbing agent in a neutral non-denaturing buffer capable of efficient elution of MBL. This buffer could be a Tris buffer with a molarity within the range of 10-40 mM, preferably 15 mM; and a pH of 7.0-8.0, preferably 7.3, with a content of NaCl ranging from 100-250 mM, preferably 100 mM. The desorbing agent could be a saccharide such as N-acetylglucosamine, mannose, N-acetylmannosamine or fucose and/or an agent chelating Ca-ions such as ethylene diamine tetra-acetic acid (EDTA). Optionally mannose is used, with a concentration within the range of 20-100 mM mannose, preferably 30 mM.
The second key element in the present process is performance of at least one validated virus-reduction step.
When discussing virus reduction steps, it is understood that a virus reduction step can be either a virus removal step and/or a virus inactivation step. More than one (e.g. two) virus removal steps and/or virus inactivation steps may be included in the present process.
The aim of validating a production step as a virus reduction step is to provide evidence that the production process will effectively inactivate/remove viruses which are either known to contaminate the starting materials, or which could conceivably do so. Validation studies involve the deliberate addition of a virus prior to the production steps to be validated and measuring the extent of its removal/inactivation after the production step or steps. GMP restraints prevent the deliberate introduction of any virus into the production facilities. Therefore, the validation should be conducted in a separate laboratory equipped for virological work on a scaled-down version of the production step and performed by staff with virological expertise in conjunction with the production engineers. The amount of virus added to the starting material for the production step which is to be validated should be as high as possible in order to determine the capacity of the production step to inactivate/remove viruses adequately. However, the virus spike should be added such that the composition of the production material is not significantly altered. Preferably, the volume of the virus spike will be equal to or less than 10%.
Quantitative infectivity assays should be performed according to the principles of GLP and may involve plaque formation, detection of other cytopathic effects such as syncytia or foci formation, end point titration (eg., TCID50 assays), detection of virus antigen synthesis or other methods. The method should have adequate sensitivity and reproducibility and should be performed with sufficient replicates and controls to ensure adequate statistical accuracy of the results.
Typically, a process step is challenged with 6 logs of virus, and if a reduction in the order of 4 logs or more is acquired, it is indicative of a clear effect with the particular test virus under investigation. Similarly, a reduction in the order of 4.5 logs, 5 logs, or even 5.5 logs, is indicative of a clear effect with the particular test virus under investigation, and the step can be classified as a validated virus reduction step
The virus validation studies should be performed with viruses resembling those which may contaminate the product as closely as possible and secondly to represent as wide a range of physico-chemical properties as possible in order to test the ability of the system to eliminate viruses in general.
Validation studies have shown that the present affinity chromatography step functions as a removal step for non-enveloped viruses and will be expected to remove enveloped viruses as well by a partition process. By this, the affinity chromatography constitutes a first virus reduction step in the present process (see example 4).
In a preferred embodiment, the validated virus reduction step is a virus inactivation step. Infectious enveloped viruses are preferably inactivated by addition of a virucidal amount of virus-inactivating agent to the MBL-containing eluate recovered from the affinity chromatography step. A xe2x80x9cvirucidal amountxe2x80x9d of virus-inactivating agent is intended to denote an amount giving rise to a solution in which the virus particles are rendered substantially non-infectious, and by this a virus-safe MBL-containing solution is obtained. Such xe2x80x9cvirucidal amountxe2x80x9d will depend on the virus-inactivating agent employed as well as the conditions such as incubation time, pH, temperature, content of lipids, and protein concentration.
The term xe2x80x9cvirus-inactivating agentxe2x80x9d is intended to denote such an agent or a method which can be used in order to inactivate enveloped viruses as well as non-enveloped viruses. The term xe2x80x9cvirus-inactivating agentxe2x80x9d is to be understood as encompassing both a combination of such agents and/or methods, whenever that is appropriate, and only one type of such agent or method.
Preferred virus-inactivating agents are detergents and/or solvents, most preferably detergent-solvent mixtures. It is to be understood that the virus-inactivating agent is optionally a mixture of one or more detergents with one or more solvents. Solvent/detergent (S/D) treatment is a widely used step for inactivating enveloped viruses (e.g. HIV1 and HIV2, hepatitis C and non A-B-C, HTLV1 and HTLV2, the herpes virus family, including CMV and Epstein Barr virus) in plasma derived products. A wide variety of detergents and solvents can be used for virus inactivation. The detergent may be selected from the group consisting of non-ionic and ionic detergents, and is selected to be substantially non-denaturing. Preferably, a non-ionic detergent is used as it facilitates the elimination of the detergent from the MBL preparation in the subsequent step. Suitable detergents are described, e.g. by Shanbrom et al., in U.S. Pat. No. 4,314,997, and U.S. Pat. No. 4,315,919. Preferred detergents are those sold under the trademarks Triton X-100 (t-octylphenoxypolyethoxyethanol) and Tween 80 (polysorbat 80) which may be used solely or in combination. Preferred solvents for use in virus-inactivating agents are di- or trialkylphosphates as described e.g. by Neurath and Horowitz in U.S. Pat. No. 4,764,369. A preferred solvent is tri(n-butyl)phosphate (TNBP). An especially preferred virus-inactivating agent for the practice of the present invention is a mixture of TNBP and Tween 80, but, alternatively, other combinations can be used. The preferred mixture is added in such a volume that a concentration of TNBP in the solution is within the range of 0.2-1.0% by weight, preferably at a concentration of about 0.3% by weight. The concentration of Tween 80 in the solution is within the range of 0.8-1.5% by weight, preferably at a concentration of about 1% by weight.
The virus-inactivation step is conducted under conditions inactivating enveloped viruses resulting in a substantially virus-safe MBL-containing solution. In general, such conditions include a temperature of 4-30xc2x0 C., such as 19-28xc2x0 C., 23-27xc2x0 C., preferably about 25xc2x0 C. incubation time found to be effective by validation studies. Generally, an incubation time of 1-24 hours is sufficient, preferably 4-12 hours, such as about 6 hours to ensure sufficient virus inactivation. However, the appropriate conditions (temperature and incubation times) depend on the virus-inactivating agent employed, pH, and the protein concentration and lipid content of the solution.
Validation studies of the present S/D treatment is presented in example 4.
It is contemplated that other methods for removal of or inactivating virus can also be employed to produce a virus-safe MBL product, such as the addition of methylene blue with subsequent inactivation by radiation with ultraviolet light.
In one aspect of the invention the affinity chromatography is a validated virus reduction step such that the two key elements are performed as one.
The preferred process for production of MBL from a crude MBL-containing plasma protein fraction contains the steps outlined below:
Step a) preparing an aqueous suspension of the crude MBL-containing protein fraction at acidic pH and substantially non-denaturing temperature,
Step b) eliminating the majority of immunoglobulins from the suspension of step a) and recovering an MBL-containing protein fraction,
Step c) solubilizing the MBL-containing fraction of step b., extracting MBL at neutral pH, and recovering an MBL-containing solution,
Step d) adding a mixture of a solvent and a detergent to the MBL-containing solution from step c),
Step e) applying the said MBL-containing solution of step d) to a non-conjugated polysaccharide based matrix under conditions promoting the binding of MBL to the matrix,
Step f) washing out protein contaminants from said polysaccharide based matrix of step e) with a non-denaturing buffer and/or buffers having a composition, pH, and ionic strength resulting in elimination of the major proportion of protein contaminants without substantial elution of MBL,
Step g) eluting MBL from the polysaccharide based matrix of step f) with a selective desorbing agent in a neutral non-denaturing buffer with efficient elution of MBL, which yields an MBL-containing eluate,
Step h) adding a virucidal amount of a non-denaturing virus-inactivating agent to the MBL-containing eluate of step g) resulting in a substantially virus-safe MBL-containing solution,
Step i) applying the MBL-containing solution of step h) onto an anion exchange matrix under conditions whereby MBL binds to the matrix,
Step j) washing the anion exchange matrix of step i) with a buffer having an ionic strength and pH sufficient to wash out the virus-inactivating agent from the matrix without causing substantial elution of MBL,
Step k) eluting MBL from the anion exchange matrix of step j) with a substantially non-denaturing buffer having an ionic strength and pH sufficient to cause efficient elution of MBL, which yields an MBL-containing eluate,
Step l) subjecting the MBL-containing eluate fraction of step k) to ultrafiltration, by this recovering an MBL-containing concentrate,
Step m) subjecting said MBL-containing concentrate of step l ) to gel filtration chromatography, by this recovering an MBL-containing solution of functionally active, oligomeric MBL proteins in a non-denaturing physiological buffer.
Steps a)-c) are the pre-processing steps of the process for purifying MBL from a crude plasma protein fraction.
Steps e)-g) and step h) are described above as the key elements of the process of the present invention.
Step a) Thus, the first step of the present process for production of MBL from plasma is the preparation of an aqueous suspension of the precipitated Cohn fraction, with a subsequent elimination of the majority of immunoglobulins from said suspension, thereby recovering a substantially immunoglobulin free MBL-containing protein fraction. It is preferred that the precipitated Cohn fraction is suspended in water and/or buffer at a sub-stantially non-denaturing temperature and pH. The term xe2x80x9csubstantially non-denaturingxe2x80x9d means that the condition to which the term refers does not cause substantial irreversible loss of functional activity of MBL nor of the immunoglobulins present. Advantageously, the plasma protein fraction is suspended in water acidified with at least one non-denaturing buffer system at volumes of from 6 to 9, preferably from 7 to 8, times that of the plasma protein fraction. The pH of the suspension is preferably maintained at a pH below 6, such as within the range of 4.0-6.0, preferably 5.1-5.7, most preferably about 5.4. Any suitable acidic buffer can be used, but the buffer system preferably contains at least one of the following buffers and acids: sodium phosphate, sodium acetate, acetic acid, HCI. Persons skilled in the art will appreciate that numerous other buffers can be used. The protein suspension is preferably maintained at a cold temperature, inter alia in order to prevent substantial protein denaturation and to minimize protease activity. The plasma protein suspension and water as well as the buffer system added preferably have the same temperature, within the range of 0-12xc2x0 C., preferably 0-8xc2x0 C., most preferably 1-4xc2x0 C.
Step b) The MBL-containing non-solubilized protein material, termed the xe2x80x9cresidual pastexe2x80x9d is isolated by means of depth filtration or by centrifugation. Preferably the suspension of the invention is filtered. The filtration is preferably performed through depth filters, e.g. C150 AF, AF 2000 or AF 1000 (Schenk), or similar filters. The majority of immunoglobulins in the suspension is eliminated by means of said filtration.
Step c) MBL is subsequently extracted from the residual paste under neutral conditions, preferably at a temperature from 1-8xc2x0 C., after addition of an essentially non-denaturing buffer. The buffer for extraction is preferably a Tris-buffered saline (TBS), with a concentration of Tris from 10-40 mM, preferably 10 mM, with a pH of 7.5-9.0, preferably 8.5, and a NaCl concentration of 100-200 mM, preferably 140 mM. Skilled artisans will appreciate that other non-denaturing buffers can be used to extract MBL. The by extraction obtained MBL containing solution is recovered by filtration through series of depth filters with decreasing pore sizes and a delipid filter, preferably as described in Example 1. This MBL containing solution can advantageously be concentrated by means of ultrafiltration before the affinity chromatography step.
Step d) Before the MBL containing solution is applied to the affinity column, a mixture of solvent and detergent such as 0.8-1.5% Tween 80 and/or Triton X-100 and 0.2-1.0% TNBP, is preferably added to the solution, most preferably 0.3% TNBP and 1.0% Tween 80, to reduce the content of lipoproteins in the MBL containing solution eluted in the subsequent affinity chromatography step. As a consequence of the high content of lipid and lipoproteins in the solution, this solvent/detergent treatment will not constitute a virus-inactivation step of the art. However, it will be expected that a high proportion of enveloped viruses will be inactivated by said treatment.
Step i) When performing the ion exchange chromatography step for the purification of MBL, it is preferred that the conditions, e.g. the pH and ionic strength, are chosen in such a way that substantially all of the MBL present in the solution applied to the anion exchange matrix binds to the matrix. Virus-inactivating agent or agents are removed in the subsequent washing of the anion exchange matrix.
As will be known by the person skilled in the art, ion exchangers may be based on various materials with respect to the matrix as well as to the attached charged groups. For example, the following matrices may be used, in which the materials mentioned may be more or less cross-linked: agarose based (such as Sepharose CL-6B(copyright), Sepharose Fast Flow(copyright) and Sepharose High Performance(copyright)), cellulose based (such as DEAE Sephacel(copyright)), dextran based (such as Sephadex(copyright)), silica based and synthetic polymer based. For the anion exchange matrix, the charged groups which are covalently attached to the matrix may e.g. be diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and/or quaternary ammonium (Q). Other anion exchangers can be used.
If, for instance, the chosen anion exchange matrix is Q Sepharose FF(copyright), then the column is advantageously equilibrated with a non-denaturing alkaline buffer having about the same pH and ionic strength as the MBL solution to be loaded. Any of a variety of buffers are suitable for the equilibration of the ion exchange columns, e.g. sodium phosphate, tris(hydroxymethyl)amino-methane. Persons skilled in the art will appreciate that numerous other buffers may be used for the equilibration as long as the pH and conductivity are about the same as for the applied MBL solution. A preferred buffer for the equilibration of the anion exchange column is a Tris buffer having a Tris concentration within the range of 10-40 mM, such as within the range of 20-30 mM, preferably about 15 mM. It is preferred that the pH of the Tris buffer used for equilibration is within the range of 7.0 to 9.0, such as within the range of 7.5-8.5, preferably about 8.0. The buffer used preferably contains a concentration of NaCl in the range of 10-40 mM such as 20-30 mM preferably 25 mM NaCl.
Step j) The initial washing is advantageously performed by using the equilibration buffer, even though other buffers, with a similar concentration and pH-value may be used for the washing. The washing is performed with a volume 10-20 times that of the column volume.
Step k) The elution of the MBL from the anion exchange matrix is preferably performed with a substantially non-denaturing buffer having a pH and ionic strength sufficient to cause efficient elution of the MBL, thereby recovering an MBL-containing eluate. In this context, efficient elution means that at least 80%, such as at least 90%, e.g. at least 95% of the MBL proteins are loaded onto the anion exchange matrix. The elution is advantageously carried out with a Tris buffer containing 5-25 mM, such as 10-30 mM, preferably 15 mM Tris and 0.1-1.0 M such as 0.3-0.7 M, preferably 0.5 M NaCl, with a pH in the range of 6.0-9.0, such as 7.0-8.0, preferably 7.4.
It is preferred that the salt concentration of the eluting buffer is sufficiently high in order to displace the MBL from the matrix. However, it is contemplated that a decrease in pH and a lower salt concentration can be used to elute the MBL from the matrix.
Step l) Subsequent to elution from the anion exchange column, the eluate is preferably concentrated. The membranes employed for the ultrafiltration advantageously have a nominal weight cutoff within the range of 10,000-100,000 Da. A preferred membrane type for the present process is a membrane with a nominal weight cutoff of 100,000 Da, obtained from Sartorius. Other ultrafiltration membranes of comparable porosity may be employed.
Step m) The last chromatography step of the process, the gel filtration step, can be regarded as a polishing step, whereby SAP, lgG, protein aggregates, and structurally impaired MBL, which might have formed during the steps subsequent to the affinity chromatography, are eliminated.
The preferred process for production of MBL from an MBL containing lysed cell suspension or supernatant comprises at least the pre-processing step of filtering the MBL containing lysed cell suspension or supernatant to clarify the solution and remove e.g. cell debris.
After this pre-processing step whereby an MBL containing solution is obtained, steps d)-m) are performed as described above. In another embodiment, the process for production of MBL from an MBL containing lysed cell suspension or supernatant comprises the pre-processing step and steps e)-m).
The preferred process for production of MBL from an MBL containing milk product or colostrum contains the following pre-processing steps:
Step 1) adding a water soluble, substantially non-denaturating protein precipitant to the MBL containing milk product or colostrum in an amount sufficient to cause precipitation of a high proportion of non-MBL components, without causing substantial precipitation of MBL, or precipitation of the majority of MBL without causing substantial precipitation of non-MBL components; thereby forming a mixture of a solid precipitate and a liquid supernatant.
Step 2) recovering a clarified MBL-containing supernatant from the said mixture of step 1) or a clarified resuspended MBL-containing precipitate from the said mixture of step 1).
Step 1) Substantially non-denaturating, water-soluble protein precipitants are well known in the field of protein purification. Such precipitants are used for protein fractionation, resulting in partial purification of proteins from suspensions. Suitable protein precipitants for use in the process of the present invention include various molecular weight forms of PEG, caprylic acid, and ammonium sulphate. Those skilled in the art will appreciate that several other non-denaturating water soluble precipitants may be used as alternative means for the precipitation. The term xe2x80x9cadding a protein precipitantxe2x80x9d and variants of that term implies the addition of one or more types of protein precipitation agents.
Step 2) After completion of the protein precipitation, an MBL containing supernatant or solution of resuspended precipitate is recovered. The first part of the recovery is performed by conventional techniques for separating liquid from solid phase, such as centrifugation and/or filtration. Preferably, a flow-through centrifuge with 1000-5000 g force is used. In another embodiment the first part of the recovery is performed by a depth filtration on a filter press. The MBL containing supernatant is hereby recovered.
The precipitate containing the majority of MBL obtained by the first part of the recovery is resuspended by addition of non-denaturing neutral buffer.
Optionally, the recovered MBL containing supernatant or resuspended precipitate is depth filtered to remove larger particles and aggregates. This is optionally followed by sterile filtration performed by use of a conventional sterilization filter (such as a 0.22 xcexcm filter from Millipore or Sartorius), which eliminates e.g. bacteria from the solution.
After the pre-processing steps 1) and 2) whereby an MBL containing solution is obtained, the preferred process for production of MBL from an MBL containing milk product or colostrum proceeds with steps d)-m) as described above.
The process of the invention is optimized to obtain a high yield of MBL with a high purity (see Example 3). The yield of the process of the present invention is calculated as the percent of the amount of MBL in the final product releative to the mean amount of MBL in the MBL containing solution applied to the affinity chromatography column. This yield is more than 40%, which is considered to be satisfactory, especially with the concomitant purity of the final MBL preparation prior to formulation, with MBL constituting about 60% of the total proteins. With other starting material also lower yields may be acceptable. Preferably the yield is at least 20%, such as 25%, 30%, 35%, 40% or even more than 40%.
The prior art procedures described in the introduction section have all been performed in small laboratory scale with the aim to obtain MBL for analytical research, i.e. employing up to about 1 litre of plasma as a starting material. The present invention aims at producing mannan-binding lectin in large production scale. By xe2x80x9clarge production scalexe2x80x9d is, when a crude plasma protein fraction is the starting material, understood that the starting material preferably is a plasma pool from more not fewer than 1,000 donors. Another, more general, conception of xe2x80x9clarge production scalexe2x80x9d is a binding capacity of the column in the first key step of more than 20 xcexcg MBL/ml packed matrix, e.g. more than 25 xcexcg MBL/ml packed matrix, such as more than 30 xcexcg MBL/ml packed matrix, more than 35 xcexcg MBL/ml packed matrix, more than 40 xcexcg MBL/ml packed matrix, more than 42 xcexcg MBL/ml packed matrix, more than 44 xcexcg MBL/ml packed matrix, more than 46 xcexcg MBL/ml packed matrix, more than 48 xcexcg MBL/ml packed matrix or even more than 50 xcexcg MBL/ml packed matrix.
The process of the present invention further aims at purifying MBL from a cell culture supernatant, from a lysed cell suspension, a milk product, or colostrum for subsequent use of the MBL product as a medicinal product in humans. Hence, the manufacturing process has to comply with requirements stated in Directives and guidelines from EEC to medicinal products such as biotechnological/biological products or products derived from human plasma, e.g. Note for guidance on plasma derived medicinal products, CPMP/BWP/269/95 or similar guidelines.
These requirements include, but are not limited to, the use of chemical agents in the purification process as well as in the final product. In the present process MBL is purified without the addition of protease inhibitors, such as PMSF, or bacteriostatic agents, such as azide and merthiolate. The product is thus totally free from added protease inhibitors and bacteriostatic agents.
The MBL product is manufactured according to GMP, under aseptic conditions in classified locations. To avoid proteolytic degradation of proteins during production, the process is mainly performed in cold rooms. The process of the invention is thus designed to produce an MBL product for use in medicine.
The MBL-product of the present purification process will, despite all efforts, contain other proteins than MBL. Using human plasma as the starting material plasma proteins such as IgM will be present.
It is of major importance for the clinical effect of the MBL product that the functional activity of MBL is maintained, i.e. the product is constituted by functionally active, oligomeric MBL. In this context a functionally active MBL is defined as an MBL capable of a) binding to carbohydrate on the surfaces of microorganisms (e.g. yeast mannan), b) by its ability to facilitate phagocytosis of MBL-bound microorganisms through interaction with collectin-receptors on phagocytic cells, and c) by its ability to activate complement as a consequence of binding to e.g. the surface of microorganisms. This activation of complement seems to occur via the MBL-associated serine proteases MASP 1 and 2, and elicits complement effector functions like inflammatory reactions, opsonisation, and cytolytic reactions.
MBL functional activity can be demonstrated in vitro by binding of MBL to mannan in mannan-coated ELISA-plates, a phagocytic assay where MBL-coated zymosan particles are ingested by phagocytic cells from peripheral blood, and by complement activation as visualised by deposition of complement factors (e.g. C3 or C4) after binding of MBL to carbohydrate in an ELISA-type assay.
In order to stabilize the MBL proteins during storage, the product is formulated by adding at least one protein stabilizing agent. Protein stabilizing agents are known to those skilled in the art, and include e.g. different sugar alcohols and saccharides (such as sorbitol, glucose, sucrose, trehalose, maltose), proteins (such as albumin), and amino acids (such as lysine, glycine). In the present invention albumin is preferred as a protein stabilizer, preferably at a concentration of 0.1-1% by weight, such as 0.5% by weight.
The MBL product is formulated as a liquid product for intravenous administration. An important aspect of the process of the invention is that the purified MBL becomes highly concentrated. It is thus possible to obtain a product with a concentration of at least 250 xcexcg of MBL per ml. The high concentration of MBL increases the stability of the liquid product. The MBL product can also be lyophilized in order to increase stability over time. The concentration of the lyophilized product is calculated by following the guidelines given by the manufacturer for reconstitution of the lyophilized product.
The primary indications for the MBL product is congenital and acquired MBL deficiency. In addition the MBL product has several indications:
Neurology: Chronic inflammatory demyelinating polyneuropathy (CIDP, Multifocal motoric neuropathy, Multiple sclerosis, Myasthenia Gravis, Eaton-Lambert""s syndrome, Opticus Neuritis, Epilepsy;
Gynaecology: Abortus habitualis, Primary antiphospholipid syndrome;
Rheumatology: Rheumatoid arthritis, Systemic lupus erythematosus, Systemic scleroderma, Vasculitis, Wegner""s granulomatosis, Sjxc3x8gren""s syndrome, Juvenile rheumatoid arthritis;
Haematology: Autoimmune neutropenia, Autoimmune haemolytic anaemia, Neutropenia;
Gastrointestinal: Crohn""s disease, Colitis ulcerous, Coeliac disease;
Others: Asthma, Septic shock syndrome, Chronic fatigue syndrome, Psoriasis, Toxic shock syndrome, Diabetes, Sinuitis, Dilated cardiomyopathy, Endocarditis, Atherosclerosis, Adults with AIDS and bacterial infections, Primary hypo/agammaglobulinaemia including common variable immunodeficiency, Wiskot-Aldrich syndrome and severe combined immunodeficiency (SCID), Secondary hypo/agammaglobulinaemia in patients with chronic lymphatic leukaemia (CLL) and multiple myeloma, Children with AIDS and bacterial infections, Acute and chronic idiopathic thrombocytopenic purpura (ITP), Allogenic bone marrow transplantation (BMT), Kawasaki""s disease, and Guillan-Barre""s syndrome.

Purification process for production of mannan-binding lectin and an MBL medicinal product


The invention relates to the use of ions of weakly basic oxides as linking ions for polyacids in cements, preferably polyelectrolyte cements. Suitable ions comprise elements of the scandium series, for example, Sc3+, Y3+, La3+, Ce4+ and all subsequent tri- and tetra-valent lanthanides and the ions Mg2+, Zn2+, Ga2+, In2+. The application of said ions permits a regulation of the cement reaction without surface treatment of the glass powder.

The invention relates to the use of unreactive glasses in dental materials, in particular dental cements, preferably polyelectrolyte cements, which can be used without pretreatment of the glass powder surface.
In the dental sector, glasses are used in particular for filling materials and for the fixing cements and composites for crowns, bridges and inlays.
Reactive glasses, i.e. glasses which participate in a chemical reaction, are used in what are known as polyelectrolyte cements, in particular glass-ionomer cements.
Polyelectrolyte cements of this type generally comprise three constituents, namely a polyacid, in particular a substance which contains carbonic acid, preferably in liquid form, a glass powder and water. If the three components are combined and mixed with one another, a reaction occurs, so as to form a solid body which hardens over the course of time (cement reaction).
Various raw materials are used for the production of glasses which are used in particular in glass-ionomer cements. These raw materials are firstly oxides, such as SiO2, Al2O3, CaO, fluorides, such as CaF2, SrF2, cryolite, hydroxides, such as Al(OH)3, phosphates, such as AlPO4, P2O5 or calcium phosphates. However, it is also possible to use silicates, such as mullite, or carbonates, such as Na2CO3, CaCO3 or other natural mineral raw materials. In principle, it is also possible for all the raw materials to be used in a form which contains water of crystallization.
In dental glasses, a considerable proportion of the oxygen is often replaced by fluorine. This is indicated by adding the element symbol F for fluorine to the description of the glass system.
Accordingly, glasses for glass-ionomer cements can usually be assigned to one of the following systems, in which P2O5 and Na2O in some cases are only present in small amounts or are not present at all:SiO2—Al2O3—CaO—(P2O5)—(Na2O)—FSiO2—Al2O3—SrO—(P2O5)—(Na2O)—FSiO2—Al2O3—SrO—La2O3—(P2O5)—(Na2O)—FSiO2—Al2O3—CaO—La2O3—(P2O5)—(Na2O)—F
The glasses which are used in dental cements are generally fluoroaluminosilicate glasses. The solubility of the glass in acid is a precondition for it to be used as a constituent of a polyelectrolyte cement. An acid-soluble glass structure is formed if silicon is partially replaced by aluminum. However, silicon can only be replaced by aluminum if basic oxides are present, in order to create charge equalization for the trivalent aluminum ion at positions of the tetravalent silicon ion.
When the polyacids and water are added, the glass structure is broken up, and in particular the ions with network-modifying properties are at least partially released as what are known as crosslinker ions.
The crosslinking manifests itself in hardening of the cement which increases over the course of time. All at least divalent basic ions, but also Al3+, are able to form polymeric structures of this type.
A distinction is usually drawn between the working time—the time during which the dentist is still able to work the still pasty cement material—and the hardening time—the time beyond which reworking is possible using rotating dental instruments.
It has been found that conventional glasses, which contain, for example, Ca2+ and Al3+ as crosslinker ions, in untreated form are too reactive and, on account of an excessive solubility, set too quickly with the polyacid, and consequently the dental cement which forms cannot reasonably be worked.
Although it is possible to slow the dissolution process by reducing the calcium content in the glass, it has been found that if the level of basic oxides, such as CaO or SrO, which can dissolve, is too low, the strength properties of the cement deteriorate as a result of insufficient availability of the ions. This means that the dentist has only a very short working time available to mix the filling material and apply it. At the same time, he has to accept the drawback of having to wait a very long time before he can start reworking the cement. This runs contrary to the demands which a dentist will impose on a dental cement.
The dentist usually requires a working time of from 1 to 4 min and a hardening time of from 5 to 8 min. The hardening time is usually determined according to ISO 9917 (First Edition) Part 7.3. The working time and the hardening time can be determined using a viscometer, as described in EP 0 023 013 A.
To achieve the desired working properties of the cement, i.e. to have sufficient working time and the shortest possible time to complete hardening, it is customary for the glass powders, after the milling process, to be subjected to surface treatment, as described, for example, in Clinical Materials 12, 113–115 (1993) or DE 29 29 121 A (EP 0 230 113 A). In this case, the glasses which react too quickly, on account of their composition, are adjusted to the desired level of reaction rate by reducing the levels of reactive ions at their surface.
EP 0 023 013 A describes the use of a calcium aluminum fluorosilicate glass powder for glass-ionomer cements to which further oxides may be added if they do not adversely affect the properties of the glass. According to the description, the surface of the glass has to be deactivated in order to obtain a glass which can be used for a dental cement. The deactivation represents a procedure whereby the reaction rate of a glass powder with an acid is delayed by a surface treatment, and in this way the desired working times of the cement are produced.
The deactivation of the surface can also be effected by means of other, relatively complex surface treatments, such as coating the surface, for example with a polymer.
In EP 0 023 013 A, this is achieved by means of a chemical treatment of the powder surface. The result is a cement with favorable working times combined, at the same time, with unchanged favorable mechanical characteristic data of the material.
However, this surface treatment of the glasses represents a complex process step.
Moreover, during the washing or conditioning processes, powder agglomeration may occur, having an adverse effect on the cement properties.
DE 38 06 448 A has disclosed a glass for a bone cement which comprises the elements Si, Al, Ca, Sr, F, Na and P and can be made visible to X-rays by the addition of La2O3. It is emphasized that the quantity of additives must not adversely affect the properties.
The glass powder described in DE 38 04 469 A is substantially free of alkali metal ions and alkaline-earth metal ions, with the exception of strontium which should be used in an amount from 15 to 40% by weight.
DE 20 65 824 B2 has described a fluoroaluminosilicate glass powder for self-hardening medical cements.
One object of the present invention is to provide a glass for a dental cement, in particular a reactive glass for a polyelectrolyte cement, which is simple to produce.
A further object can be considered to lie in that of directly using the glass immediately after the milling process without having to apply complex processes such as surface treatment, acid washing, coating and/or conditioning. The reactivity and therefore the working time and hardening time are then dependent only on the glass composition and the grain size distribution and are easy to produce reproducibly.
This object is achieved by a dental material containing a glass as described in the claims, and by the use of certain ions as crosslinker ions in a glass.
The invention also relates to hardenable materials, in particular cements, in particular polyelectrolyte cements, which contain these glasses.
In one embodiment, the invention relates to a process for producing a dental glass involving the steps a) providing oxidic substances, b) mixing the oxidic substances, c) melting the mixture from step b), d) quenching the molten material to form a solid, e) milling the solid from step d) to form a glass powder, the glass powder from step d) not being treated with acid before it is used in a dental cement.
In the context of the present invention, the term crosslinking is to be understood as meaning a reaction in which polyacids and at least divalent ions interact with one another in a chelate-forming reaction, preferably an acid-base-type reaction, leading to the formation of a polymeric network.
In the context of the present invention, the filling and fixing materials mentioned are to be understood as meaning substantially cements, and in particular polyelectrolyte cements. Accordingly, the glass described is preferably a reactive constituent rather than a conventional filler, unlike the glasses used in the composite sector, which are pure fillers and do not take part in a reaction.
Glasses for cements generally contain strongly basic ions, such as Li2+, Na2+, K+, Ca2+, Sr2+, Ba2+, Zn2+. It has now been found that by completely or partially replacing the strongly basic ions with weakly basic ions, such as Sc3+, Y3+, La3+, such as Ce3+/4+ or other divalent, trivalent or tetravalent ions from the lanthanide series, and/or Ga2+ or In2+, glasses which set significantly more slowly with polyacids are obtained.
Surprisingly, such glasses can be used to produce dental cements which, substantially without a conventional surface treatment of the glass powders, have setting characteristics which are desired by the dentist. Furthermore, it has been found that the setting times can be adjusted within a wide range by means of the glass composition.
In this context, the invention has the following advantages:
As a result of the strongly basic ions, such as Ca2+, Sr2+, being replaced by the weakly basic ions Sc3+, Y3+, La3+, Ce4+/3+ and other divalent, trivalent and tetravalent ions from the lanthanide series in glasses which are used in dental cements, it is possible to achieve a controlled setting reaction of the dental cement, in particular of a glass-ionomer cement, without the glass having to be surface-treated, for example by acid washing and/or conditioning, before being used in the cement. In addition to simplified production, the advantage also resides in the improved reproducibility of the working and hardening times. Although these times are not adjusted by a surface treatment, the desired setting profile, namely a relatively rapid transition from a state in which the cement can still be worked to a state in which the hardening begins and useful working is no longer possible, is surprisingly achieved.
Amazingly, it has been discovered that dental materials or cement in which the abovementioned glasses are used have mechanical properties which are identical or even slightly improved compared to cements in which glasses whose reactivity has been reduced by acid washing are used.
Furthermore, it has been found that cements according to the invention are hydrolytically stable with respect to water.
These properties were found in particular in glasses which, in addition to Al and Si, contain only Y and/or La or contain only relatively small amounts of relatively strongly basic-reacting ions, such as Ca2+ or Sr2+, Ba2+, Li+, Na+, K+.
Furthermore, some of the oxygen can be replaced by fluorine, which on the one hand improves the meltability of the glass and on the other hand improves the setting properties of the cement and makes it possible to release fluoride ions for secondary caries prophylaxis.
Therefore, the previously known cement systems are expanded by the addition of the following systems.SiO2—Al2O3—(SrO)—LnxOy—P2O5— (Na2O)—FSiO2—Al2O3—(CaO)—LnxOy—P2O5— (Na2O)—F
LnxOy stands for an oxide of the elements Sc, Y, La to Lu. x and y may adopt values of 1, 2 or 3 in this formula. The oxides which are inside parenthesis are used in only small amounts or are not used at all, since they would greatly accelerate the reaction. For example, DE 20 65 824 A describes a glass belonging to the systemSiO2—Al2O3—La2O3—P2O5—Na2O—Fhaving an Na2O content of approx. 12% by weight.
Tests have shown that with this glass powder the setting rate with polyacids can only be brought into a manageable range after conditioning for several hours at 400° C. (cf. Comparative Example 4). This is presumably attributable to the high level of a strongly basic oxide, in this case Na2O. A further drawback of a high Na2O content is the increased water solubility of the resulting cement.
Moreover, it has been found that the glasses described substantially do not have any phase-separation or crystallization effect within a wide range of compositions.
It is to be expected that the reproducibility of the setting rate of the cement containing the glasses will improve with clear glasses compared to segregated, i.e. opaque glasses, since their phase composition is not dependent on the cooling rate.
In addition to the standard components SiO2, Al2O3, P2O5, and Na2O, the glasses preferably mainly contain weakly basic and/or amphoteric-reacting ions, which act as crosslinker ions during the cement reaction.
Weakly basic trivalent and tetravalent ions are preferred, and the ions Sc3+, Y3+, La3+, Ce4+/3+ and all the following trivalent and tetravalent ions from the lanthanide series are particularly preferred.
Current teaching is that Al3+ also belongs to the weakly basic or amphoteric-reacting ions. However, this ion adopts a special position among the glasses. Aluminum is primarily responsible for the solubility of the glass structure in acid and has only a secondary function as a crosslinker ion. In the glasses which are suitable for dental cements, aluminum, unlike the abovementioned trivalent and tetravalent ions, which function as network modifiers, acts as a network former.
The glasses used generally have a BET surface area of from 1 to 15 m2/g, preferably 2 to 8 m2/g.
Furthermore, the glasses have a mean grain size (d50) of from 0.01 to 20 μm, preferably 1 to 5 μm.
It is preferable for 0 to 25% by weight of the oxygen in the glass used to be replaced by fluorine, particularly preferably from 8 to 18% by weight.
The pKB value is usually used to define the term basicity. A pKB of one can be taken as the limit between weakly and strongly basic. For example, the pKB of Mg(OH)2 is given as one in R. C. Weast: CRC Handbook of Chemistry and Physics, while Ca(OH)2 is classified as strongly basic, without being assigned a numerical value.
In the context of the present invention, oxides or hydroxides which only dissociate to a relatively minor extent in aqueous solutions are considered to be weakly basic.
In one embodiment, oxides with a pKB value of >1 are provided.
The following statements can be made in connection with the basicity:
The basicity increases from Sc through Y to La. La is to be classified as weakly basic compared to Sr, Ba or Na and K. At the same time, the basicity decreases again from La to Lu, and consequently the basicity of lutetium is approximately comparable to that of yttrium (lanthanide contraction).
Therefore, all oxides and hydroxides of the Sc series can be considered weakly basic in the context of present invention.
The elements from the 1st main group from Li through Cs and the elements from the second main group from Mg through Ba cannot be classified as weakly basic-reacting in the context of the present invention.
As has already been stated, it is assumed that, in addition to the base strength, the higher field strength of these ions also plays a certain role. This means that the ions described are anchored more strongly in the glass structure and are therefore dissolved out of it more slowly.
In the context of the present invention, the term polyacid is understood to mean a polyelectrolyte which includes a polymer with ionically dissociable groups, which may be substituents in the polymer chain and the number of which is so great that the polymers, at least in their (partially) dissociated form, are at least partially water-soluble. Substituents such as for example —COOH, —OH, —PO(OH)2, —OPO(OH)2, —SO2(OH) are particularly suitable for this purpose. Organic polyacids (DE 20 61 513 A), such as polymers and copolymers of acrylic acid, methacrylic acid (EP 0 024 056 A), itaconic acid, maleic acid, citraconic acid, vinylphosphonic acid (EP 0 340 016 A; GB 22 91 060 A) are particularly preferred. In addition, if a plurality of polyelectrolytes are present, water-insoluble polyelectrolytes may also be present in the polyelectrolyte cement. The only condition is that at least one of the polyelectroytes be at least partially water-soluble.
The polyelectrolytes should be able to react with the glass component as part of a chelate-forming reaction and/or an acid-base reaction/neutralization reaction.
The polyelectrolyte cement contains the at least partially water-soluble polyelectrolyte, which can be converted into the solid state, preferably in an amount from 0.5 to 30% by weight, particularly preferably 2 to 25% by weight and very particularly preferably 5 to 20% by weight.
In the case of polyelectrolyte cements, the addition of chelating agents in order to establish a suitable setting characteristic is particularly important (DE 23 19 715 A). There are numerous compounds which are suitable for this purpose, in particular those which contain hydroxyl or carboxyl groups, or both, which form the chelating agents. Particularly good results have been achieved with tartaric acid or citric acid, in particular in an amount of 5% by weight. Adding the substance in the form of a metal chelate also has the desired effect.
The polyelectrolyte cements contain from 0 to 10, preferably 0 to 5% by weight of a compound of this type, preferably tartaric acid.
Furthermore, the polyelectrolyte cement may include auxiliaries, such as dyes, pigments, X-ray contrasting agents, flow improvers, thixotropic agents, polymer thickeners or stabilizers.
Examples of standard fillers for dental materials are glass and quartz powder, plastic powder, pyrogenic highly dispersed silicas and mixtures of these components.
Other suitable fillers may include: aluminum oxide, mineral powders, feldspars and kaolin.
These other additives are usually present in the polyelectroyte cements according to the invention in amounts of from 0 to 60% by weight.
The abovementioned fillers may also be rendered hydrophobic by means of a surface treatment with organosilanes or organosiloxanes or by etherification of hydroxyl groups to form alkoxy groups.
In principle, the glass composition which has been described is also suitable for use in monomer-modified cements.
It has proven favorable for the glass to contain from 20 to 70% by weight, preferably from 30 to 60% by weight, of weakly basic oxides.
The cement according to the invention may if appropriate contain strongly basic oxides in an amount in the range from 0 to 25% by weight, preferably in the range from 0 to 10% by weight.
The cement according to the invention preferably has a flexural strength in the region of at least 25 MPa to 35 Mpa, particularly preferably of greater than 45 MPa, measured in accordance with ISO 4049.
The working time of the cement, which is determined using a viscometer, is 1 to 4 min, particularly preferably 2 to 3 min. The hardening time is 3 to 10 min, particularly preferably 4 to 8 min.
Preferred compositions of the glasses are given below.
In addition to the abovementioned weakly basic oxides from the scandium series, the glasses may also contain oxides from transition groups 4 and 5. Also, the aluminum oxide may be partially or completely replaced by boron oxide or gallium oxide. The melting conditions can be positively influenced by the addition of oxides from the first main group, phosphate, and/or basic oxides from the second main group or ZnO.
The oxides which are separated from one another by "+" in the table, may, according to the invention, also be present just individually. The crucial factor is the corresponding proportion by weight which the group forms in the glass.
TABLE 1OxideProportionY2O3 + La2O3 + other30 to 80% by weight, preferablylanthanide oxides35 to 60% by weightB2O3 + Al2O3 + Ga2O3 5 to 50% by weight, preferably10 to 40% by weight,particularly preferably 15 to 35%by weightSiO2 + GeO2 + SnO10 to 50% by weight, preferably15 to 50% by weightP2O5 0 to 15% by weight, preferably 0 to 10% by weight, particularlypreferably 0 to 2% by weightMgO + CaO + SrO + ZnO + BaO 0 to 10% by weight, preferably 0 to 8% by weight, particularlypreferably 0 to 5% by weightLi2O + Na2O + K2O + Rb2O + Cs2O 0 to 5% by weight, preferably 0 to 3% by weight, particularlypreferably 0 to 2% by weightTiO2 + ZrO2 + HfO2 0 to 10% by weight, preferably 0 to 4% by weight,V2O5 + Nb2O5 + Ta2O5 0 to 10% by weight, preferably 0 to 4% by weight,
Instead of the "Y2O3+La2O3+other lanthanide oxides", it is also possible for Sc2O3 to be present in an amount of from 20 to 50% by weight, preferably from 20 to 30% by weight. Glass compositions in which Sc2O3 is present in addition to the above constituent in a relatively small amount are also included.
In one embodiment, the invention relates to a dental cement comprising:                a) mineral solid in an amount of from 50 to 90% by weight;        b) water in an amount of from 5 to 50% by weight; and        c) polyacid in an amount of from 5 to 50% by weight.Further, the P2O5 of Table 1 may be provided in an amount of from 0 to 18% by weight.
The invention is explained in more detail below with reference to a number of examples.
None of the glasses described in the examples was treated with an inorganic acid leading to a reduction in the number of reactive ions at the surface of the glasses (acid wash) before being reacted with a polyacid.
Production of the Glass:
Glasses of the following oxidic compositions (in % by weight) were melted at temperatures in the range from 1300 to 1600° C. over a period of 30 min to 5 h. With the exception of Comparative Example 4, the fluorine content in the starting batch was 12 to 14% by weight.
In Comparative Example 4, a glass was melted in accordance with DE 20 65 824 A1 using the following composition:
9.5 g of SiO2, 10.0 g of Al2O3, 7.6 g of Na3AlF6, 9.4 g of LaF3, 7.3 g of AlPO4. The table gives the oxidic composition used in these examples.
TABLE 2123456789C1C2C3C4C5C6C7SiO220192017322713211920184623172629Sc2O3Y2O3365820415046494830La2O34863203422.523CaO71647316SrO17Al2O344262220283237282629343536263027P2O56105ZrO25Na2O11128.561Li2O13Milling
60 to 80 g of the glass granules obtained were dry-milled for 40 to 50 min a vibratory agate mill (produced by Siebtechnik, milling vessel 100 ml, 910 rpm). The glass powders obtained had a mean grain size in the range from 3 to 6 μm with a specific surface area of from 1.8 to 2.5 m2/g.


Apparatus and methods are described that enable concurrent transmission of multiple data signals including clock, synchronization, and power over a single-wire bus between a master device and one or more slave devices. A first transmission channel from the master device to the slave device may modulate the width of periodic pulses between a first voltage level and a second voltage level with respect to a reference potential. A second transmission channel may modulate the amplitude of at least one of the first and second voltage levels to at least one third voltage level. Concurrent communications between a master device and one or more slave devices over a single-wire bus can be achieved.

1. Field of the Invention
The present invention generally relates to data transmissions between two electronic circuits. The present invention more specifically relates to the simultaneous transmission, over the same wire, of a power supply, clock, and data signal between a master circuit and one or several slave circuits.
2. Discussion of the Related Art
Communication protocols between a master circuit and a slave circuit which transmit, over the same wire, a power supply, synchronization, and data signal, are generally called single-wire protocols. Most often, the master and slave circuits further have a common voltage reference (typically the ground).
U.S. Pat. No. 5,903,607 describes a system of transmission over a single-wire bus in bidirectional mode.
FIG. 1 is a simplified block diagram of such a transmission system.
FIGS. 2A, 2B, and 2C are timing diagrams illustrating the operation of the system of FIG. 1.
A master electronic circuit 1 (MD) is connected, by a single-wire bus B, to slave circuit 3 (SD). Circuits 1 and 3 further share the same ground or more generally the same reference potential (GND). Circuit 1 (for example a processing circuit of microprocessor type) is supplied with a voltage Vcc, which is, for example, positive with respect to the ground. Circuit 3 (for example, a memory or another processing circuit) is generally not autonomously powered and draws its power supply from the transmission over the communication bus. To make the presence of a common voltage reference clearer, bus B, although considered as a single-wire bus, has been shown in the drawings as including a ground wire 4 in addition to a wire 2 over which power supply, clock, and data signal S transits. The connection to ground 4 may be functional by the rest of the elements of the circuit or of the device.
Master circuit 1 comprises output and input interfaces illustrated in FIG. 1 respectively by output and input terminals OUT and IN connected to wire S. Slave circuit 3 also comprises input and output interfaces illustrated by input and output terminals IN and OUT.
FIGS. 2A to 2C are timing diagrams illustrating a data transmission in a bidirectional mode between master circuit 1 and slave circuit 3.
FIG. 2A illustrates an example of the shape of signal SM imposed by master circuit 1. FIG. 2B illustrates the shape of a signal SS imposed by slave circuit 3 to transmit data in return. FIG. 2C illustrates the shape of resulting signal S on the bus. Signal S corresponds to the signal present on terminals IN of circuits 1 and 3. For simplification, signals SM and SS have been schematized as respectively originating from the transmission circuits of master circuit 1 and of slave circuit 3 and it is considered that the reception circuits connected to terminals IN only see the level of signal S.
Master circuit 1 modulates signal SM in amplitude between two levels V1 and V0, for example, both positive, according to a predefined coding. In fact, circuit 1 modulates at the rate of a clock the pulse widths at level V1 according to the state of the bit to be transmitted. The slave circuit draws its power supply from a filtering at the level of signal S. The quiescent level of signal S for example is level V1. In the shown example, a transmission is initialized by a start bit START in which signal SM is positioned (time t1) at a level V0 while its quiescent level is level V1. This initializes slave circuit 3 and prepares it to receive data and to transmit data in return. Master circuit 1 modulates the level of signal SM at the rate of a clock signal which sets the transmission rate and which enables the slave circuit to extract a clock for sequencing its operation and for synchronizing the transmission with the master circuit. The transmission of a bit at state 0 occurs, for example, with a pulse of level V1 of a duration shorter than the half-period of the clock signal (for example, ¼ of the period) while a state 1 is coded with a pulse of level V1 of a duration longer than the half-period of the clock signal (for example, ¾ of the period). Slave circuit 3 detects the amplitude variation and the corresponding duration of the high and low pulses to determine the value of the transmitted bits. In the direction from slave circuit 3 to master circuit 1 FIG. 2B), the slave circuit modifies the load that it imposes on wire S according to the state of the bit that it wishes to transmit. In the example of FIG. 2B, the slave circuit does not modify the load to transmit a 1 (times t2 and t3). To transmit a 0 (times t4 and t5), it pulls (times t4 and t5) signal SS to the low level, which results in decreasing the duration of the pulse of signal S. This amounts, for the slave circuit, to modulating the width of the pulses of level V1 of signal S according to the state of the bit to be transmitted. The master circuit monitors the level of signal S and, if the level that it has transmitted has not been modified, considers a response by a state 1, while when it detects a modification in the duration of the pulse with respect to the one that it transmits, it considers the reception of a state 0.
A single-wire protocol such as discussed in relation with the above drawings has a rate limited by the period of the clock signal imposed by the master circuit. Further, the master circuit can only transmit a single signal to slave circuits.

Multiple-channel transmission over a single-wire bus


An optical touch sensing method includes sensing light corresponding to a touch region, comparing a first amount of the light sensed corresponding to the touch region with a first lower threshold, determining that a touch is made if the first amount is less than or equal to the first lower threshold, comparing the first amount of the light sensed corresponding to the touch region with an upper threshold, and determining that a touch is made if the first amount is greater than or equal to the upper threshold.

1. Field of the Invention
The present disclosure relates to a method for detecting a touch, and more particularly, to a method for detecting a touch and a flat panel using the same in which both an upper threshold level and a lower threshold level are set to make more sensitive detection in a structure which uses infrared cameras positioned at least at three corners for receiving and emitting light and retro-reflecting plates for reflecting light from the infrared camera to the infrared camera again.
2. Discussion of the Related Art
In general, a touch screen is one of interface types between a user and an information and communication device which uses various kinds of displays. A touch screen is an input unit which enables the user to interact with the device as the user personally touches a screen with a hand or a pen.
The touch screen is a device which allows for a conversational and intuitive handling of the device, since it may be used by anyone by touching a button displayed on a display device with a finger. Because of these features, the touch screen is applied to many fields, such as issuing machines in banks and public agencies, various medical apparatuses, tour and major facility guidance devices and traffic guidance devices.
In the touch screen, various types are available, such as, but not limited to, a resistive type touch screen, a micro capacitive touch glass, an ultrasonic wave touch glass, an infrared type touch screen, and so on depending on methods of detection used.
The resistive type touch screen typically has two transparent conductive layers where a lower layer thereof is formed of glass or plastic coated with a conductive material and an upper layer thereof is formed of a film coated with a conductive material. The two layers are spaced by micro printed spacers and are electrically insulated. The resistive type touch screen is a device which involves a change of resistance at each of an upper plate (X-axis) and a lower plate (Y-axis) when the upper plate is touched with a hand or a touch pen in a state where a fixed voltage is applied to the two layers with the conductive material coated thereon. In this instance, a controller calculates an X (the upper plate) position and a Y (the lower plate) position by detecting changes in resistances and displays the positions on a monitor or inputs as data.
The micro capacitive touch glass has a transparent glass sensor coated with a thin conductive material. Therefore, an electrode pattern is precisely printed along a periphery of a conductive layer and a transparent vitreous protective film is placed closely on the thin conductive coating for protecting and enclosing the sensor. In the micro capacitive touch glass, a voltage is applied to a screen, and an electrode pattern forms a low voltage field on a touch sensor surface through the conductive layer. When a finger touches the screen, a micro current flows at a touch point. A current from each corner is proportional to a distance from the corner to the finger, and a touch screen controller calculates ratios of current flows to find a position at which the touch is made.
The ultrasonic wave touch glass is not affected by surface damage or wear at all in comparison to other products, which are formed of 100% vitreous material and a lifetime of the expensive touch screens instantly come to an end even by small surface damage or wear. A touch screen controller forwards a 5 MHz electric signal to a transducer to generate an ultrasonic wave, and the ultrasonic wave generated passes a surface of the touch screen by reflected rays. In the ultrasonic wave touch glass, if a user presses a surface of the touch screen, a portion of the ultrasonic wave passing through a pressed point is absorbed by the user, a signal thus lost is identified instantly by a controller through a received signal and a digital map, and based on this, coordinates of a point having a change of the signal presently are calculated. Such a series of steps are performed independently on X- and Y-axes.
The infrared type touch screen utilizes an attribute of the infrared ray in which the infrared ray cannot travel if the infrared ray comes to an obstacle since an infrared ray travels in a straight line. A portion having a pressure applied thereto cuts off the infrared rays in a transverse direction and a longitudinal direction, and X and Y coordinates of a cut off portion are read for sensing. An infrared ray light type touch screen identifies a touched position by detecting a cut off of an infrared ray scan light at a front of the touch screen. The infrared type touch screen has an infrared ray emitted from one side and received at an opposite side for both x and y axes to form a lattice of the infrared ray.
Though above types have different advantages, recently, the infrared type touch screen is drawing attention due to a minimized pressure required to be applied to the touch screen, and convenience of arrangement.
A related art infrared type touch screen will be described with reference to the attached drawings.
FIG. 1 illustrates a plan view showing a touch detecting method of a related art infrared type touch screen.
Referring to FIG. 1, the related art infrared type touch screen is provided with infrared sensors 5 mounted to adjacent two corners of the panel 10 and reflective plates 7 mounted to three sides of the panel 10.
A touch to the infrared type touch screen is detected as follows. That is, lights from infrared sensors 5 at opposite ends of the panel 10 are reflected, lights cut off at the time of the touch are sensed, and angles thereof are calculated, to perceive the touch.
However, the infrared type touch screen has a dead zone with a range greater than a certain angle between the infrared sensors 5 in which the detection is not possible, making accuracy of the touch poor at a particular region to require adjustment. In order to adjust for the dead zone, the infrared sensors are positioned outside the corners of the liquid crystal display panel to form the dead zone at an outer side of the liquid crystal panel. In this case, a touch screen greater than the liquid crystal panel is required, resulting in increase of a non-effective area which does not contribute to picture display, thereby making efficiency of the display device poor.
In general, the liquid crystal panel is separate from the touch screen. If it is intended to enable touch capabilities, additional work is required to assemble the respective components and apply coordinates to the touch screen suitable to the liquid crystal panel, and to secure the touch screen to a liquid crystal module.
In the related art touch screen, selection of accurate coordinates is difficult, and only one touch point may be perceived at a time. In other words, if two points on the touch screen are touched at the same time, the touch screen fails to perceive the touches, or perceives one of the touch points touched first, causing an error.
FIG. 2 illustrates a graph showing a touch sensing method in a related art touch screen, and FIG. 3 illustrates a simulation diagram showing a light quantity, threshold level of each pixel and a touch perception according to the above on a panel by using the method of FIG. 2.
Referring to FIG. 2, in the related art touch screen, if there is a part having a light quantity reduced in sensing a light quantity of each region, it is determined that the part is touched.
That is, if the sensed light quantity is greater than a predetermined touch perception threshold level, the sensed light quantity is determined that no touch is made, and if the sensed light quantity is smaller than the predetermined touch perception threshold level, the sensed light quantity is determined that the touch is made.
FIG. 3 illustrates a graph for comparing an actual light quantity of each region sensed with an infrared camera to a touch perception level of the regions.
In actual infrared cameras, differences of quantities of lights reflected at the retro-reflecting plates and returned to the infrared cameras occurs due to differences of distances from the infrared cameras and differences of incident angles on the retro-reflecting plates, causing an intrinsic light quantity difference between regions which is not a change of the light quantity resulting from an actual touch. FIG. 3 illustrates that the touch perception threshold levels varies with regions, taking the above into account.
In this case, light quantities smaller than the touch perception threshold level are sensed at about 250 pixels and 300 pixels, and these parts are determined to be touched.
However, the related art touch sensing method has the following problems.
In the related art touch sensing method according to the above, if a touch means of a reflective material, particularly, having a highly reflective surface such as a finger nail, metal or a mirror makes a touch, a reflected light greater than the threshold level is detected, causing an incorrect determination that no touch is made.
Moreover, in the related art touch detection, a touch perception detection threshold level of each region is set to be about 80% of a quantity of the light of the case where light is emitted from the infrared camera with no touch being made at the region, reaches the retro-reflecting plate and returns to the infrared camera. However, if a particular part is touched with a thin pen or the like, a fine change light quantity caused by the touch cannot be detected if the touch is determined by the about 80% or below of the un-touched light quantity due to the fine change.
The intrinsic difference of the light quantity reflected at the retro-reflecting plate caused by arrangement of the infrared cameras and incident angles on the retro-reflecting plates regardless of the touch is also liable to cause failure in detection of a change of the fine touch at a part having a small light quantity.

Method for detecting touch and optical touch sensing system


A self curing activator for chemical polymerization of the interface of a dental bonding agent and a self curing or dual curing dental cement or restorative, includes an aryl borate compound, a polymerizable monomer, an optional acidic compound, an amine compound which exhibits a catalytic action, an organic peroxide containing material, and a metal compound wherein said metal compound promotes the decomposition of the organic peroxide.

Several catalysts for chemical polymerization of (meth)acrylate compounds have been identified. These systems primarily include and are not limited to the following materials:                1. A trialkylborane or a partial oxide of a trialkylborane such as tri-n-butyl borane.        2. A redox based self-curing initiator comprising a combination of an organic peroxide and a metal salt        3. A system utilizing a combination of an organic peroxide and a tertiary amine.        4. A combination of hydrogen peroxide and an Fe2+ compounds.        5. A barbituric acid, combined with a Cu2+ compound and an ionogenic halide species.        6. An aryl borate compound and an acidic compound        7. An aryl borate compound, an acidic compound and a transition metal compound
An unexamined Japanese Patent Publication (Kokai No. 169535/2000) discloses a self-curing resin composition that cures at ambient temperature obtained by blending a composition of a combination of a polymerizable unsaturated compound with a radical-generating catalyst, and a polymerization initiator. The initiator consists of an organoboron compound with an acid component or with an acidic, polymerizable unsaturated compound.
The trialkylboron or the partial oxide is an effective initiator for redox polymerization however, these materials are generally pyrophoric and are chemically very unstable. This catalyst requires special packaging and requires mixing with the monomer components immediately prior to use.
The organic peroxide and metal salt or tertiary amine along with the barbituric acid systems are primarily used in various dental materials due to availability and biocompatibility. The peroxide amine systems can affect the color/shading tinting the cured product due to oxidation of the amine compound and are generally unstable due to oxygen inhibition and thermal instability of the peroxide. The barbituric acid based catalysts have been determined to exhibit difficulties with controlling the curing time and are prone to oxidation reducing their activity.
In general the aryl borates are easy to handle, do not impart color to the cured product, and exhibit acceptable stability. According to Ibaragi et al (U.S. Pat. No. 6,660,784), the difficulty with the aryl borates is that these systems do not exhibit sufficient catalytic activity.
As discussed, Chemical polymerization of vinyl or acrylate based resins via a free radical polymerization mechanism at ambient temperature is traditionally achieved using a binary, redox curing system consisting of a peroxide and an aromatic tertiary amine. On the other hand, light-activated polymerization proceeds via the generation of free radicals from the activation of a photoinitiator, usually an α-diketone, to its excited triplet state. This is followed by the reduction of the activated photoinitiator by an amine accelerator to form an intermediate excited complex (exciplex), which releases the free radicals on dissociation. There was evidence to suggest that bond strengths of resin composites to dentin were influenced by the compatibility of the polymerization modes between adhesive systems and resin composites [Swift E J, May K N, Wilder A D. Journal of Prosthodontics 1998: 7: 256-60]. A recent report further revealed that common light-cured, self-etching adhesive systems were incompatible with chemical-cured composites [Miller M B et al. Realty 1999; 13:1-182-7], to the extent that no effective bonding was achieved for some systems. However, the systems that bonded poorly to the chemically cured composites exhibited high shear bond strengths with the use of light-cured resin composites. Generally, the incompatibility of self-etching adhesives with chemical-cured resins is attributed to the reaction of the amine accelerated by the acid components of the adhesive system. More specifically, these acid components of the bonding agent protonate the tertiary aromatic amine of the organic redox catalyst in the self-curing resin composite. Subsequently, the protonated amine (quaternary aromatic amine) does not react with the peroxide to form the complex, which decomposes into radicals capable of initiating polymerization under ambient conditions. Overall, the catalyst losses efficiency and the rate and degree of functional group conversion are significantly diminished compromising the performance of the dental adhesive. Based upon this amine protonation reaction, the dental restoratives to be used in combination are limited to those of the photo-curable type only.
Although light-cured resin composites have largely superseded the use of chemical-cured composites in esthetic dental applications, chemically activated composites still have important applications in contemporary restorative dentistry. The longer working time of chemically cured composites has been adopted in the 'directed shrinkage technique' for posterior resin composite restorations. In this technique, a slow setting, chemically cured composite was used either in bulk or as a basal layer to relieve the stress developed in a restoration by the flow of the partially polymerized material. Chemically cured resins are frequently used as restorative materials in areas that are not easily penetrable by light, and as auto- or dual-curing resin cements for luting of crown and bridges, inlays and onlays along with endodontic posts. In order to facilitate the use of light curing self-etching bonding agents with dual curing or chemically curing composites, a self-curing activator is required to overcome the incompatibility of the acid containing adhesive with the amine in the redox catalyst of the chemically cured system. In the Prime&Bond NT Dual-Cure bonding system, the regular light-cure bonding agent, Prime&Bond NT is mixed with Self-Cure Activator prior to use. The Prime&Bond NT Dual-Cure exhibits excellent bond strength when bonding a dual-cure cement, e.g. Calibra, in chemical-cure mode. Since the active ingredient in Self-Cure Activator is p-toluenesulfinate, which reacts with methacrylate resins slowly on storage, the Self-Cure Activator for Prime&Bond NT is a dilute solution of p-toluenesulfinate without the presence of any polymerizable resin. Upon adding the Prime&Bond NT with the Self-Cure Activator together, the adhesive is diluted with activator. Consequently, this allows excess (atmospheric) oxygen to permeate the adhesive inhibiting free radical polymerization. Other self-curing activators contain thermally unstable peroxides, which require refrigeration to inhibit decomposition. In order to minimize this dilution effect and provide a thermally stable system, a new self-curing activator system containing polymerizable resin is required.
Several articles indicate that the binary system of tetraphenylborate (TPB) salts and organic acids could effectively initiate free radical polymerization of vinylic compounds [T. Sato et al. Die Makromolekulare Chemie 162 (1972) 9-18]. Overall, the author concluded that the reaction between the TPB ion and the proton of the acid was important in the initiating radical production. Using dimethylbenzylanilinium TPB and trichloroacetic acid (TCA), the rate of polymerization of methyl methacrylate (MMA) was found to be proportional to the square roots of the concentrations of both TPB salt and TCA, confirming that the system induced radical polymerization. The copolymerization by this system with styrene resulted in a composition curve, which was in agreement with that obtained with ordinary radical copolymerization.
In 2001, Sato et al [T. Sato et al. Journal of Polymer Science, Part A: Polymer Chemistry (2001) 4206-4213] discuss the utilization of arylborates and an aryldiazonium compound in solvent. Several polymerizable monomer systems were investigated in conjunction with differing solvent types. This study presumes that phenyl radicals are generated and proposes the mechanism for initiation based upon kinetic and EPR studies.
In 1984 Mun et al [Y. Mun et al. Journal of Macromolecular Science, Chemistry (1984) A21 (5) 645-660] discusses the polymerization of methylmethacrylate and methylacrylate using a binary system of sodium tetraphenylborate with bis(ethylacetoacetato)copper (II). This study calculated the activation energy in acetone and suggests the participation of monomer in the initiation process.
In 1983, Mun et al [Y. Mun et al. Memoirs of the Faculty of Engineering, Osaka University (1983) 24 149-159] discuss the utilization of sodium tetraphenylborate with various metal salts. This study concluded that sodium tetraphenylborate in conjunction with Cobalt and Copper salts accelerated methylmethacrylate polymerization. Manganese, Titanium and Nickel salts exhibited little or no effects on polymerization. In addition, water, crown ethers and benzoquinone inhibited the polymerization process.
In 1970 Sato et al [T. Sato et al. Chemistry & Industry, (London, UK) (1970) 4 (125)] present a method for the preparation of the N-acyloxytrialkylammonium salt of tetraphenylborate. This paper determined that the tetraphenylborate salt produced thermally decomposed to initiate methacrylate polymerization at 60° C.
U.S. Pat. No. 6,660,784B2 [K. Ibaragi, H. Kazama and M. Oguri (Tokuyama Co., Japan), issued Dec. 9, 2003] disclosed a dental catalyst for chemical polymerization comprising an acidic compound, an organic peroxide such as cumene hydroperoxide, and aryl borate compound such as sodium tetraphenylborate, but without substantially containing an amine compound. This catalyst was chemically highly stable, was easy to handle, was highly active, was less likely to be impaired by polymerization, and did not cause the cured product to be tinted or discolored, and was very useful for the dental restorative.
What was claimed is a dental catalyst for chemical polymerization comprising: an aryl borate compound, an acidic compound, an organic peroxide, with the organic peroxide containing an amount from 0.1 to 10 mols per mole of the aryl borate, without substantially containing amine compound which exhibited a catalytic action and a metal compound wherein said metal compound promotes the decomposition of the organic peroxide.
U.S. Pat. No. 5,866,631 [H. Nakagawa and H. Ohno (Tokuyama Co., Japan), issued Feb. 2, 1999] disclosed a dental primer composition capable of obtaining high adhesive strength to both dentin and enamel, as a pretreatment material for a chemically polymerizable adhesive. This composition was a dental primer composition comprising a polymerizable monomer containing an acidic group, water, aryl borate and a transition metal compound.
JP Patent 09309811 [M. Oguri, H. Kazama and T. Sato Tokuyama Soda Co. LTD., Japan Dec. 2, 1997] discloses a dental adhesive containing acidic monomers and filler with good adhesion to dentin. The preferred composition contains acid group containing monomer arylborates and fillers along with other polymerizable monomers.
JP Patent Application WO2003027153 [M. Oguri, H. Kazama, M. Kimura, K. Ibaragi, K. Fuzinami and T. Sato Tokuyama Corp., Japan Apr. 3, 2003] discloses a polymerization catalyst comprising arylborates and vanadium compounds for use in dental adhesives. The preferred composition contains acid group containing monomer arylborates and vanadium compounds in which the vanadium is in the 4+ or 5+ oxidation state.