The present invention relates to the field of carbohydrate crosslinked glycoprotein crystals. Advantageously, such crosslinked glycoprotein crystals display stability to harsh environmental conditions, while maintaining the structural and functional integrity of the glycoprotein backbone. According to one embodiment, this invention relates to methods for concentrating proteins that have been modified by carbohydrates and for releasing their activity at controlled rates. This invention also provides methods for producing carbohydrate crosslinked glycoprotein crystals and methods for using them in pharmaceutical formulations, vaccines, immunotherapeutics, personal care compositions, including cosmetics, veterinary pharmaceutical compositions and vaccines, foods, feeds, diagnostics, cleaning agents, including detergents and decontamination formulations. The physical and chemical characteristics of carbohydrate crosslinked glycoprotein crystals render them particularly useful as sorbents for separations, such as chiral chromatography, or affinity chromatographyxe2x80x94which are based on specific interactions between the active binding site of the glycoprotein component of the crystals and the substance or molecule of interest. Such characteristics also render carbohydrate crosslinked glycoprotein crystals useful as catalytic and binding components for the production of biosensing devices.

The present invention relates to the field of carbohydrate crosslinked glycoprotein crystals. Advantageously, such crosslinked glycoprotein crystals display stability to harsh environmental conditions, while maintaining the structural and functional integrity of the glycoprotein backbone. According to one embodiment, this invention relates to methods for concentrating proteins that have been modified by carbohydrates and for releasing their activity at controlled rates. This invention also provides methods for producing carbohydrate crosslinked glycoprotein crystals and methods for using them in pharmaceutical formulations, vaccines, immunotherapeutics, personal care compositions, including cosmetics, veterinary pharmaceutical compositions and vaccines, foods, feeds, diagnostics, cleaning agents, including detergents and decontamination formulations. The physical and chemical characteristics of carbohydrate crosslinked glycoprotein crystals render them particularly useful as sorbents for separations, such as chiral chromatography, or affinity chromatographyxe2x80x94which are based on specific interactions between the active binding site of the glycoprotein component of the crystals and the substance or molecule of interest. Such characteristics also render carbohydrate crosslinked glycoprotein crystals useful as catalytic and binding components for the production of biosensing devices.
Many proteins associated with the external surfaces of cell membranes or actively secreted from cells are commonly modified by the addition of one or more carbohydrate units to the side chains of particular amino acids [R. D. Marshall, Ann. Rev. Biochem., 41, pp. 673-702 (1972)]. Such proteins, known as glycoproteins, display the properties of proteins in general, as well as properties typical of the attached carbohydrate. The carbohydrate monomers typically attached to glycoproteins include galactose, mannose, glucose, N-acetylglucosamine, N-acetylgalactosamine, fucose, xylose, sialic acid and others. The carbohydrate units are usually attached through the hydroxyl groups of serine and threonine side chains, or the amide nitrogen atom of asparagine side chains. The carbohydrate side chains are arranged in a variety of chain lengths and branching patterns [P. V. Wagh and O. P. Bahl, Crit. Rev. Biochem., 10, pp. 307-77 (1981)].
Glycoproteins exhibit a range of protein functions, including catalysis of chemical transformations, proteolysis of proteins, binding of ligands and transport of ligands to and across membranes. Additionally, glycoproteins frequently perform functions associated with cellular communication, including protein-protein recognition, protein-carbohydrate recognition, protein-DNA recognition, pathogen recognition by antibodies, antigen presentation by CD4 and CD8 membrane glycoproteins, and targeting of proteins to specific locations.
Glucose oxidase exemplifies an enzyme glycoprotein that catalyzes the oxidation of xcex2-D-glucose to D-glucono-1,5-lactone. The reaction consumes one mole of oxygen and produces one mole of hydrogen peroxide per mole of glucose. The active glycoprotein glucose oxidase, or xcex2-D-glucose:oxygen 1-oxidoreductase [enzyme commission number 126.96.36.199] forms a dimer with a molecular weight of 150-180 kDa. Each monomer consists of 583 amino acids residues, one co-factor molecule of flavin adenine dinucleotide (FAD) and has a carbohydrate content of approximately 16% by weight. The three dimensional structure of the deglycosylated protein has been determined by X-ray crystallography [H. J. Hecht, H. M. Kalisz, J. Hendle, R. D. Schmid and D. Schomburg, J. Mol. Biol., 229, pp.153-72 (1993)]. Due to the importance of the quantitative determination of glucose in medicine and industry, glucose oxidase is considered a prime candidate for the development of biosensors [H. J. Hecht, D. Schomburg, H. M. Kalisz and R. D. Schmid, Biosensors and Bioelectronics, 8, pp. 197-203 (1993)]. Glucose oxidase may be advantageously used in the food, drug and cosmetics industries, because of its ability to interconvert oxygen and hydrogen peroxide. Commercially available glucose oxidase is usually isolated from Aspergillus niger.
The stereoselectivity and specific activity of enzymatic glycoproteins may be exploited for use in industrial syntheses. For example, glutaraldehyde crosslinked crystals of Lipase from Candida rugosa may be used to synthesize optically pure compounds [J. J. Lalonde, C. Govardhan, N. Khalaf, A. G. Martinez, K. Visuri and A. L. Margolin, J. Am. Chem. Soc., 117, (26) pp. 6845-52, (1995)].
In infectious diseases, glycoproteins are involved in the initiation and maintenance of infection, as well as host humoral and cellular immune responses against infection. The surface proteins of many viruses are glycoproteins. Examples of such glycoproteins include, for example, gp120 and gp41 of the Human Immunodeficiency Virus (HIV), which causes AIDS [H. Geyer, C. Holschbach, G. Hunsmann and J. Schneider, J. Biol. Chem., 263 (24), pp. 11760-67, (1988)] and the hemagglutinin (HA) and neuraminidase (NA) of Influenza Virus, which causes Flu.
Viral receptors, the cellular proteins recognized by invading viruses, are found on the surface of cells and therefore are frequently either glycoproteins or carbohydrates. The CD4 molecule, which is recognized by HIV-1 gp120, is a glycoprotein. Similarly, Influenza virus hemagglutinin binds to terminal N-acetylneuraminic acid residues of sialoglycoproteins and enters the cell through receptor mediated endocytosis [J. White, M. Kielian and A. Helenius, Ouart. Rev. of Biophys., 16, pp. 151-95, (1983)].
In spite of the tremendous medical, chemical, pharmaceutical and industrial potential of glycoproteins, their development has, in many instances, lagged far behind that of unglycosylated proteins.
As compared with unglycosylated proteins, the frequent association of glycoproteins with biological membranes and other membrane proteins, render glycoproteins significantly more difficult to purify and utilize for medical and industrial processes. The use of glycoproteins faces additional barriers because relatively little is known about their three-dimensional structure and the requirements for stabilization when faced with harsh environments. However, due to the specialized functions of glycoproteins, many benefits can be realized by overcoming the barriers to widespread large scale use of glycoproteins in industrial, chemical and medical applications.
One unique approach to overcoming barriers to the widespread use of proteins generally is crosslinked enzyme crystal (xe2x80x9cCLEC(trademark)xe2x80x9d) technology [N. L. St. Clair and M. A. Navia, J. Am. Chem. Soc., 114, pp. 4314-16 (1992)]. Crosslinked enzyme crystals retain their activity in environments that are normally incompatible with enzyme function. Such environments include prolonged exposure to proteases and other protein digestion agents, high temperature or extreme pH and organic solvents. In such environments, crosslinked enzyme crystals remain insoluble and stable.
One physical result of xe2x80x9cCLEC(trademark)xe2x80x9d technology is that the surface exposed amino acid side chains of the protein, in the crystal lattice, are covalently modified with the crosslinking agents. This modification stabilizes the crystal lattice, at the same time altering elements of the surface structure to gain the benefits of stabilization. For most applications involving protein crystals, any potential limitation resulting from minor surface modifications is overcome by the gains in stability achieved in the crystals. However, applications involving vaccines, and immunotherapeutics using glycoproteins, often require that the surface exposed protein structures, known as epitopes, precisely maintain the original structure. This may require stabilization without chemical modification of the amino acid side chains or perhaps with minor levels of chemical modification of the amino acid side chains.
The present invention specifically addresses the problems of stabilization of glycoproteins via crystallization and crosslinking for their use in industrial, chemical, and medical applications. Thus, the present invention relates to methods for crystallization of glycoproteins and for their subsequent stabilization through carbohydrate-to-carbohydrate crosslinking within the crystal lattice to produce carbohydrate crosslinked glycoprotein crystals.
Crosslinked glycoprotein crystals according to this invention may be produced by crosslinking glycoprotein crystals through one or more carbohydrate moieties on the glycoprotein or through both one or more carbohydrate moieties on the glycoprotein and one or more of the amino acid side chain functional groups in the glycoprotein.
The carbohydrate crosslinked glycoprotein crystals of this invention are useful in vaccines, pharmaceuticals and immunotherapeutic formulations for delivery of active, non-denatured, glycoproteins. They are also useful in biosensing devices and in methods, devices and systems for separating molecules of interest from mixtures or crude preparations thereof.
The present invention also includes methods for crystallizing glycoproteins in the absence of the potentially denaturing effects of chemical deglycosylation.
In order that the invention herein described may be more fully understood, the following detailed description is set forth. In the description, the following terms or phrases are employed:
Aaueous-organic solvent mixturexe2x80x94a mixture comprising n% organic solvent, where n is between 1 and 99 and m% aqueous, where m is 100-n.
Catalytically effective amountxe2x80x94an amount of carbohydrate crosslinked glycoprotein crystals of this invention which is effective to treat, protect, repair or detoxify the area to which they are applied over some period of time.
Chanae in chemical compositionxe2x80x94any change in the chemical components of the environment surrounding carbohydrate crosslinked glycoprotein crystals that affects the environment or the crosslinker, including addition of chemical reagents, solvent composition changes, chemical changes induced by application of energy in the form of light, microwave, or radiation to the environment, chemical events and parameters that affect the crosslinker and combinations thereof.
Change in shear force actina upon the crystalsxe2x80x94any change in factors of the environment surrounding carbohydrate crosslinked glycoprotein crystals under conditions of use, such as, changes in mechanical pressure, both positive and negative, revolution stirring, centrifugation, tumbling, mechanical agitation and filtration pumping.
Controlled dissolutionxe2x80x94dissolution of carbohydrate crosslinked glycoprotein crystals or release of the protein constituent of said crystals that is (1) triggered by a change in the environment surrounding said crystals, said change being selected from the group consisting of change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in shear force acting upon the crystals and combinations thereof and (2) controlled by a factor selected from the group consisting of the degree of crosslinking of said carbohydrate crosslinked glycoprotein crystals, the length of time of exposure of glycoprotein crystals to the crosslinking agent, the rate of addition of crosslinking agent to said glycoprotein crystals, the nature of the crosslinker, the chain length of the crosslinker, the surface area of said carbohydrate crosslinked glycoprotein crystals, the size of said carbohydrate crosslinked glycoprotein crystals, the shape of said carbohydrate crosslinked glycoprotein crystals and combinations thereof.
Formulations for decontaminationxe2x80x94formulations selected from the group consisting of: formulations for decontamination of chemical wastes, herbicides, insecticides, pesticides, environmental hazards.
Glycoproteinxe2x80x94a protein or peptide covalently linked to carbohydrate. The carbohydrate ay be monomeric or composed of oligosaccharides.
Glycoprotein activityxe2x80x94an activity selected from the group consisting of binding, catalysis, signalling, transport, or other activities which induce a functional response within the environment in which the glycoprotein is used, such as induction of immune response and immunogenicity, or combinations thereof.
Glycoprotein activity release ratexe2x80x94the quantity of glycoprotein dissolved per unit time.
Insoluble and stable form of a glycoprotxe2x80x94a form of a glycoprotein which is insoluble in aqueous solvents, organic solvents or aqueous-organic solvent mixtures and which displays greater stability than the soluble form of the glycoprotein. According to an alternate embodiment of this invention, the phrase xe2x80x9cinsoluble and stable form of a glycoproteinxe2x80x9d may be a protein which is insoluble in dry formulations but soluble in wet formulations. In any embodiment, carbohydrate crosslinked glycoprotein crystals may be active in insoluble form. And in one embodiment, carbohydrate crosslinked glycoprotein crystals according to this invention may be active in insoluble form, then dissolve or are removed or digested once their function is complete.
Immunotherapeuticxe2x80x94a protein or glycoprotein derived from a tumor cell with a protein activity of inducing protective immunity to said tumor. A protein or glycoprotein cytokine which stimulates the immune system to reduce or eliminate said tumor.
Oraanic solventsxe2x80x94any solvent of non-aqueous origin.
Pharmaceutically effective amountxe2x80x94an amount of carbohydrate crosslinked glycoprotein crystals which is effective to treat a condition in an individual to whom they are administered over some period of time.
Prophylactically effective amountxe2x80x94an amount of carbohydrate crosslinked glycoprotein crystals which is effective to prevent a condition in an individual to whom they are administered over some period of time.
Proteinxe2x80x94any peptide having a tertiary structure or any protein.
Separationxe2x80x94Separation of a substance from a mixture of two or more different substances or two or more forms of the same substance. According to another embodiment of this invention, xe2x80x9cseparationxe2x80x9d is defined as purification of a substance from a crude form thereof. Separation may be carried out by any means including, for example, chromatography, membrane separation, filtration and electrophoresis.
Therapeutic glycoproteinxe2x80x94A glycoprotein which is administered to a patient in a conventional pharmaceutical formulation and manner. Therapeutic glycoproteins include, for example, hormones, enzymes, antibodies, viral receptors, T-cell receptors, chemokines, chemokine receptors, MHC molecules, tumor antigens, mucins, inhibitors, growth factors, trophic factors, cytokines, lymphokines, toxoids, nerve growth hormones, blood clotting factors, adhesion molecules, multidrug resistance proteins, adenylate cyclases and bone morphogenic proteins.
Vaccine antiaenxe2x80x94a protein or glycoprotein derived from an infectious agent such as a virus, parasite, or tumor antigen. The protein activity of such vaccine antigens is to induce protective immunity against the infectious agent.
As a result of their crystalline nature, the carbohydrate crosslinked glycoprotein crystals of this invention achieve uniformity across the entire crosslinked crystal volume. This uniformity is maintained by the intermolecular contacts and chemical crosslinks between the carbohydrates attached to adjacent protein molecules constituting the crystal lattice, even when the crystals are exposed to buffers, organic or mixed aqueous-organic solvents and adjuvants. In such media, the glycoprotein molecules maintain a uniform distance from each other, forming well-defined stable pores within the carbohydrate crosslinked glycoprotein crystals that facilitate access of substrate or ligand to the glycoproteins, as well as removal of product.
The methods of this invention achieve stabilization of the crystal lattice by either exclusive crosslinking of attached carbohydrates of glycoproteins or a combination of carbohydrate and amino acid side chain crosslinking. In such carbohydrate crosslinked glycoprotein crystals, the lattice interactions, when fixed by chemical crosslinks, are particularly important in providing stability and preventing denaturation, especially in storage, under conditions including harsh environments created by components of compositions in which the crystals are used. The uniformity across crystal volume and enhanced stability of the constituent glycoproteins in carbohydrate crosslinked glycoprotein crystals creates novel opportunities for the use of glycoprotein vaccines, biosensors, and catalysis in harsh conditions, such as elevated temperature, and aqueous, organic or near-anhydrous solvents, as well as mixtures of these. Glycoprotein crystals may also be crosslinked in such a way that they dissolve or release their protein activity upon exposure to a trigger in their environment encountered under conditions of use. Thus, they may be substantially insoluble and stable in a composition under storage conditions and substantially soluble and active under conditions of use of said composition.
The methods of this invention advantageously accomplish the crystallization of glycoproteins, under large scale conditions, without the need for cumbersome and potentially denaturing effects of chemical deglycosylation. As a result, the carbohydrate moieties attached to amino acid side chains available for chemical crosslinking are maintained, while preserving those amino acid side chains not attached to carbohydrate in their unmodified form.
For those carbohydrate crosslinked glycoprotein crystals according to this invention which are enzymes, the entire crystal consists of active enzyme (and not inactive carrier). The specific activity per mg of immobilized protein product in carbohydrate crosslinked glycoprotein crystals is typically at least about 2 times higher than in conventionally immobilized proteins or catalysts, ranging from about 2 to 100 times higher. Such high glycoprotein densities are particularly useful in biosensor, analytical and other applications requiring large amounts of protein in small volumes.
Carbohydrate crosslinked glycoprotein crystals according to this invention offer several advantages over conventional protein/glycoprotein immobilization methods. For example, the crosslinked crystal matrix provides its own support. Expensive carrier beads, glasses, gels, or films are not required in order to tie down the enzyme catalyst, as they are in presently available immobilization methods. As a result, the concentration of glycoprotein is close to the theoretical packing limit that can be achieved for molecules of a given size, greatly exceeding densities achievable even in concentrated solutions.
In addition to their activity, carbohydrate crosslinked glycoprotein crystals according to this invention are particularly stable and insoluble under storage conditions, including the attendant storage temperature, storage pH, storage time, storage concentrate form, storage involving little or no shear force acting upon the crystals, or combinations thereof.
In addition to their stability under storage conditions, carbohydrate crosslinked glycoprotein crystals are particularly stable, at least about 2 times as stable, to thermal denaturation, digestion with proteases, for example pronase, and in mixed water:organic solvent mixtures. Specifically, the carbohydrate crosslinked glycoprotein crystals of this invention are between about 2 and 100 times as stable to thermal denaturation, between about 2 and 10,000 times more resistant to pronase digestion, and between about 2 and 1,000 times more stable to inactivation in 50% ethanol, than the soluble form of the enzyme.
The rate of dissolution of carbohydrate crosslinked glycoprotein crystals can be controlled by manipulating the conditions and extent of crosslinking. Controlled dissolution carbohydrate crosslinked glycoprotein crystals are slowly soluble and active under conditions of use, including conditions involving change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in shear force acting upon the crystals and combinations thereof. However such carbohydrate crosslinked glycoprotein crystals are insoluble and stable under storage conditions. Such properties make the carbohydrate crosslinked glycoprotein crystals of this invention particularly useful for delivery of pharmaceuticals, therapeutic glycoproteins, personal care agents or compositions, including cosmetics, vaccines, veterinary compositions, foods, feeds, diagnostics, cleaning agents, including detergents and formulations for decontamination.
According to one embodiment, the carbohydrate crosslinked glycoprotein crystals of this invention are characterized by a half-life of activity under storage conditions which is greater than at least about 2 times that of the soluble form of the glycoprotein that is crystallized to form the crystals that are crosslinked, as well as activity similar to that of the soluble form of the glycoprotein under conditions of use. Advantageously however, the carbohydrate crosslinked glycoprotein crystals of this invention display improved stability under storage conditions, as compared to their soluble or uncrosslinked crystallized counterpart glycoproteins.
Thus, carbohydrate crosslinked glycoprotein crystals according to this invention may be advantageously used instead of conventional soluble or immobilized proteins in pharmaceuticals, veterinary compounds, personal care compositions, including cosmetics, foods, feeds, vaccines, pulp, paper and textile processing, diagnostics, cleaning agents, including detergents and formulations for decontamination.
The carbohydrate crosslinked glycoprotein crystals of this invention are particularly advantageous because they are stable in harsh environments imposed by the formulations or compositions in which they are employed or conditions of their storage. At the same time, these carbohydrate crosslinked glycoprotein crystals are capable of (1) change to soluble and active form (an active form including, in one embodiment of this invention, a form which is active against macromolecular substrates) or (2) controlled dissolution or release of their activity when exposed to one or more triggers in their environment. Such triggers may be selected from the group consisting of change in temperature, change in pH, change in chemical composition, change from concentrate to dilute form, change in shear force acting upon the crystals and combinations thereof. Controlled dissolution or release of activity of carbohydrate crosslinked glycoprotein crystals according to this invention may also be triggered over a change in time.
Specific examples of such triggers include an increase or decrease in temperature, for example, an increase in temperature from a low temperature between about 0xc2x0 C. and about 20xc2x0 C. to a high temperature between about 25xc2x0 C. and about 70xc2x0 C. Other triggers include a change from acidic pH to basic pH and a change from basic pH to acidic pH. Examples of triggers of change from concentrate to dilute form include, for example, a change in solute concentration, a change in concentration of all solutes from about 2-fold to about 10,000-fold, a change in concentration of all solutes from about 2-fold to about 700-fold, an increase or decrease in salt concentration, an increase or decrease in water concentration, an increase or decrease in organic solvent concentration, a decrease in protein concentration and a decrease in detergent concentration.
Additional triggers involve changes in chemical composition of the environment surrounding the carbohydrate crosslinked glycoprotein crystals that affect the environment or the crosslinker itself. Such changes include, for example, addition of chemical reagents, increase or decrease in organic solvent concentration, chemical events that affect the crosslinker, chemical changes induced by application of energy, including light, microwave or radiation. As explained above, any of these triggers may act in combination or in sequence with one or more of the other triggers.
The glycoprotein constituent of the carbohydrate crosslinked glycoprotein crystals of this invention may be any glycoprotein, including for example, hormones, enzymes, antibodies, viral receptors, viral surface glycoproteins, parasite glycoproteins, parasite receptors, T-cell receptors. MHC molecules, immune modifiers, tumor antigens, mucins, inhibitors, growth factors, trophic factors, cytokines, lymphokines, toxoids, nerve growth hormones, blood clotting factors, adhesion molecules, multidrug resistance proteins, adenylate cyclases, bone morphogenic proteins and lectins.
Also included among the glycoproteins are the hormones and cytokines. Examples of hormones include follicle stimulating hormone, human chorionic gonadotropin, luteinizing hormone, thyrotrophin and ovine, bovine, porcine, murine and rat alleles of these hormones. Examples of cytokine glycoproteins include xcex1-interferon, lymphotoxin, and interleukin-2. Also included are glycoprotein tumor-associated antigens, for example, carcinoembryonic antigen (CEA), human mucins, her-2/neu, and prostate-specific antigen (PSA) [R. A. Henderson and O. J. Finn, Advances in Immunology, 62, pp. 217-56 (1996)].
Alternatively, the glycoprotein constituent may be selected from personal care glycoproteins, including cosmetic glycoproteins, veterinary glycoproteins, food glycoproteins, feed glycoproteins, diagnostic glycoproteins, glycoproteins used in chemical reactions, glycoproteins used in industrial methods, cleaning agent glycoproteins, including detergent glycoproteins, and decontamination glycoproteins. Included among such glycoproteins are enzymes, such as, for example, hydrolases, transferases, isomerases, lyases, ligases, transferases and oxidoreductases. Examples of hydrolases include lipase, cholinesterase, alkaline phosphatase, xcex2-amylase deoxyribonuclease, glucoamylase A and B, xcex1-galactosidase I and II, xcex2-fructofuranosidase, xcex2-glucouronidase, N-acetyl-xcex2-glucosaminidase, hyaluronidase, oxytocinase, kallikrein, bromelain, enterokinase, proteinase a, b, and c, pepsinogen and pepsin. Examples of oxidoreductases include glucose oxidase, peroxidase and chloroperoxidase. Examples of transferases include xcex3-glutamyltranspeptidase and ribonuclease.
In one embodiment of this invention, carbohydrate crosslinked glycoprotein crystals are produced by a method comprising at least one crosslinking reaction in which at least one carbohydrate moiety attached to the glycoprotein acts as or functions as the substrate for the crosslinking reaction. Multiple carbohydrate crosslinking reactions can be performed to modify further the characteristics of the carbohydrate crosslinked glycoprotein crystals.
The carbohydrate crosslinked glycoprotein crystals of this invention may be prepared using crosslinking reagents which are multifunctional or bifunctional agents. Such agents include the diamine group of compounds, such as, for example, hexamethylenediamine, diaminooctane, ethylenediamine, 4-(4-N-Maleimidophenyl)butyric acid hydrazide.HCl (MPBH), 4-(N-Maleimidomethyl)cyclohexane-1-carboxy-hydrazide.HCl (M2C2H), and 3-(2-Pyridyldithio)propionyl hydrazide (PDPH) and other amine alkenes.
In one embodiment of this invention, carbohydrate crosslinked glycoprotein crystals are produced by a method including at least one crosslinking reaction in which at least one carbohydrate moiety attached to the glycoprotein acts or functions as the substrate for the crosslinking reaction. One method of producing carbohydrate crosslinked glycoprotein crystals comprises an initial oxidation of the carbohydrate moieties, followed by crosslinking with at least a multifunctional reagent such as a diamine, followed by a reduction reaction using for example, NaBH4. Alternatively, additional carbohydrate crosslinking reactions can be carried out as described but using different crosslinking reagents.
In another embodiment of this invention, carbohydrate crosslinked glycoprotein crystals are produced by a method including an initial crosslinking reaction in which one or more amino acid side chain functional groups serve as substrate for a multifunctional crosslinking reagent, such as glutaraldehyde. The first crosslinking reaction is followed by additional crosslinking reactions in which at least one involves crosslinking through one or more carbohydrate moieties and using, for example, a diamine crosslinking reagent.
In another embodiment of this invention, at least one crosslinking reaction is performed, in addition to the crosslinking reaction in which at least one oxidized carbohydrate moiety attached to the glycoprotein acts as or functions as the substrate for the crosslinking reaction, in which the amino acid side chain functional groups act as or function as a substrate for the crosslinking reaction. This additional linking can be achieved using one or a combination of a wide variety of multifunctional crosslinking reagents, at the same time (in parallel) or in sequence. Such multifunctional reagents include bifunctional reagents. Examples of such crosslinking agents are glutaraldehyde, succinaldehyde, octanedialdehyde and glyoxal. Additional multifunctional crosslinking agents include halo-triazines, e.g., cyanuric chloride; halo-pyrimidines, e.g., 2,4,6-trichloro/bromo-pyrimidine; anhydrides or halides of aliphatic or aromatic mono- or di-carboxylic acids, e.g., maleic anhydride, (meth)acryloyl chloride, chloroacetyl chloride; N-methylol compounds, e.g., N-methylol-chloro acetamide; di-isocyanates or di-isothiocyanates, e.g., phenylene-1,4-di-isocyanate and aziridines. Other crosslinking agents include epoxides, such as, for example, di-epoxides, tri-epoxides and tetra-epoxides. For a representative listing of other available crosslinking reagents see, for example, the Pierce Catalog and Handbook, Pierce Chemical Company, Rockford, Ill. (1997) and also S. S. Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press, Boca Raton, Fla. (1991).
According to one embodiment of this invention, carbohydrate crosslinked glycoprotein crystals are produced by a method comprising at least one crosslinking reaction through one or more carbohydrate moieties in said glycoprotein, alone or in sequence with crosslinking via a multifuntional reagent such as glutaraldehyde or other crosslinkers that function by crosslinking one or more amino acid side chain functional groups in said glycoprotein.
According to an alternate embodiment of this invention, at least one non-carbohydrate crosslinking reaction may be carried out using reversible crosslinkers, in parallel or in sequence with the carbohydrate crosslinking reaction. The resulting carbohydrate crosslinked glycoprotein crystals are characterized by containing a reactive multifunctional linker, into which a trigger has been incorporated as a separate group. The reactive functionality is involved in linking together reactive amino acid side chains in a glycoprotein and the trigger consists of a bond that can be broken by altering one or more conditions in the surrounding environment (e.g., pH, temperature, or thermodynamic water activity).
Examples of reversible crosslinkers are described in T. W. Green, Protective Groups in Organic Synthesis, John Wiley and Sons (Eds.) (1981). Any variety of strategies used for reversible protecting groups can be incorporated into a crosslinker suitable for at least one crosslinking in producing carbohydrate crosslinked glycoprotein crystals capable of feversible, controlled solubilization. Various approaches are listed, in Waldmann""s review of this subject, in Anaewante Chemie Inl. Ed. Engl., 35, p. 2056 (1996).
Other types of reversible crosslinkers are isulfide bond-containing crosslinkers. The trigger reaking crosslinks formed by such crosslinkers is the ddition of reducing agent, such as cysteine, to the environment of the crosslinked protein crystals.
Disulfide crosslinkers are described in the Pierce Catalog and Handbook (1997). Examples of such crosslinkers include the symmetric homo-bifunctional, as for example DSS-dithiobis (succinimidyl-propionate), also know as Lomant""s Reagent and DTSSPxe2x80x943-3xe2x80x2-dithiobis (sulfo-succinimidylpropionate), a water soluble version of DSP and many more. Other examples include the heterobifunctional or asymmetric crosslinkers such as SPDPxe2x80x94N-succinimidyl-3-(2-pyridyldithio)propionate and LC-SPDPxe2x80x94succinimidyl-6-(3-[2-pyridyldithio] propionate)hexanoate and others.
In another embodiment of this invention, the dissolution, catalytic, antigenic and pharmaceutical properties of carbohydrate crosslinked glycoprotein crystals are modified by the specific combinations of crosslinking reactions performed.
Controlled dissolution of carbohydrate crosslinked glycoprotein crystals according to the present invention may also be effected by a change in time sufficient to permit a protein activity release rate between about 0.1% per day and about 100% per day, a change in time sufficient to permit a protein activity release rate between about 0.01% per hour and about 100% per hour or a change in time sufficient to permit a protein activity release rate between about 1% per minute and about 50% per minute.
Carbohydrate crosslinked glycoprotein crystals according to this invention, therefore, include those capable of releasing their protein activity at a controlled rate upon exposure to a change in their environment, said change being selected from the group consisting of change in pH, change in solute concentration, change in temperature, change in chemical composition, change in shear force acting upon the crystals and combinations thereof.
Factors contributing to the release rate of protein activity of carbohydrate crosslinked glycoprotein crystals according to this invention include the degree of crosslinking of the carbohydrate crosslinked glycoprotein crystals, whether the reduction step was performed after the crosslinking reaction, the pH used for the oxidation reaction, the pH used for the crosslinking reaction, the pH used for the reduction reaction, whether the multifunctional crosslinking agent was pretreated, the polymerization state of the crosslinking agent, the number of crosslinking reactions performed, the length of time of exposure of protein crystals to the crosslinking agent, the rate of addition of crosslinking agent or agents to the glycoprotein crystals, the length of time of exposure of glycoprotein crystals to the crosslinking agent, the nature of the crosslinker either carbohydrate specific or amino acid side-chain specific, the chain length of the crosslinker, the surface area of the carbohydrate crosslinked glycoprotein crystals, the size of the carbohydrate crosslinked glycoprotein crystals, the shape of the carbohydrate crosslinked glycoprotein crystals and combinations thereof.
According to this invention, any individual, including humans and other mammals, as well as birds and fish, for example, may be treated in a pharmaceutically acceptable manner with a pharmaceutically effective or a catalytically effective amount of carbohydrate crosslinked glycoprotein crystals for a period of time sufficient to treat a condition in the individual to whom they are administered over some period of time. Alternatively, individuals may receive a prophylactically effective or a catalytically effective amount of carbohydrate crosslinked glycoprotein crystals which is effective to prevent a condition in the individual to whom they are administered over some period of time.
Carbohydrate crosslinked glycoprotein crystals may be administered alone, as part of a pharmaceutical, personal care or veterinary preparation or as part of a prophylactic preparation, such as a vaccine, with or without adjuvant. They may be administered by parenteral or non-parenteral route. For example, they may be administered by oral, pulmonary, nasal, aural, anal, dermal, ocular, intravenous, intramuscular, intraarterial, intraperitoneal, mucosal, sublingual, subcutaneous, or intracranial route. In either pharmaceutical, personal care or veterinary applications, carbohydrate crosslinked glycoprotein crystals may be topically administered to any epithelial surface. Such epithelial surfaces include oral, ocular, aural, anal and nasal surfaces, to treat, protect, repair or detoxify the area to which they are applied.
Pharmaceutical combinations of the present invention may be formulated in a variety of conventional forms employed for parenteral administration. These include, for example, semi-solid and liquid dosage forms, such as liquid solutions or suspensions, suppositories, douches, enemas, gels, creams, emulsions, lotions, slurries, powders, and pastes. Standard formulation strategies for vaccines, immunotherapeutics and pharmaceuticals may be applied to carbohydrate crosslinked glycoprotein crystals in order to enhance the persistence and residence time of the active agent, and to improve the prophylactic efficacy achieved.
The present invention also includes controlled release formulations comprising carbohydrate crosslinked glycoprotein crystals. In such formulations, the carbohydrate crosslinked glycoprotein crystals are substantially insoluble under storage conditions and capable of releasing their protein activity in vivo at a controlled rate. For example, a pharmaceutical controlled release formulation according to this invention, administered by oral route, is characterized in that the component carbohydrate crosslinked glycoprotein crystals are substantially insoluble under gastric pH conditions and substantially soluble under small intestine pH conditions. Alternatively, for these and other uses according to this invention, the carbohydrate crosslinked glycoprotein crystals are biodegradable and may be active in the insoluble form and then dissolve and are removed or digested once their function is complete.
Pharmaceutical, personal care, veterinary or prophylactic compositions comprising carbohydrate crosslinked glycoprotein crystals according to this invention may also be selected from the group consisting of tablets, liposomes, granules, spheres, microparticles, microspheres and capsules.
For such uses, as well as other uses according to this invention, carbohydrate crosslinked glycoprotein crystals may be formulated into tablets. Such tablets constitute a liquid-free, dust-free form of carbohydrate crosslinked glycoprotein crystal storage, which are easily handled and retain acceptable levels of activity.
Alternatively, the carbohydrate crosslinked glycoprotein crystals may be in a variety of conventional depot forms employed for administration to provide reactive compositions. These include, for example, solid, semi-solid and liquid dosage forms, such as liquid solutions or suspensions, gels, creams, balms, emulsions, lotions, slurries, powders, sprays, foams, pastes, ointments, salves, balms and drops.
According to one embodiment of this invention, carbohydrate crosslinked glycoprotein crystals may be combined with any conventional materials used for controlled release administration of pharmaceutical glycoproteins. Such materials include, for example, coatings, shells and films, such as enteric coatings and polymer coatings and films.
The most effective mode of administration and dosage regimen of formulations or compositions comprising carbohydrate crosslinked glycoprotein crystals of this invention will depend on the effect desired, previous therapy, if any, the individual""s health status or status of the condition itself and response to the carbohydrate crosslinked glycoprotein crystals and the judgment of the treating physician or clinician. The carbohydrate crosslinked glycoprotein crystals may be administered in any dosage form acceptable for pharmaceuticals, personal care compositions or veterinary formulations, at one time or over a series of treatments.
The amount of the carbohydrate crosslinked glycoprotein crystals that may be combined with carrier materials to produce a single dosage form will vary depending upon the particular mode of administration, formulation, dose level or dose frequency. A typical preparation will contain between about 0.01% and about 99%, preferably between about 1% and about 50%, carbohydrate crosslinked glycoprotein crystals (w/w).
Upon improvement of the individual""s condition, a maintenance dose of carbohydrate crosslinked glycoprotein crystals may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced as a function of the symptoms, to a level at which the improved condition is retained. When the condition has been alleviated, treatment should cease. Individuals may, however, require intermittent treatment on a long-term basis upon any recurrence of the condition or symptoms thereof.
The properties of the carbohydrate crosslinked glycoprotein crystals of this invention are especially suited for applications involving vaccines, to protect humans and animals from infectious diseases.
The methods of this invention may be used to produce carbohydrate crosslinked glycoprotein crystals from virus, or parasite glycoproteins for use as antigens in a vaccine. Appropriately spaced and repeated parenteral inoculations of carbohydrate crosslinked glycoprotein crystals in combination with immune system modifiers such as adjuvants and/or cytokines are useful to induce patient antibody and T-cell immune responses to viral, or parasite glycoproteins. The patient immune responses can prevent or mitigate transmission or disease from of an enveloped virus infection, or parasite infections.
Such enveloped viruses include, for example, virus from the genera Retroviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Hepadnaviridae, Flaviviridae, Togaviridae, Rhabdoviridae, Poxviridae, Arenaviridae, Coronoviridae, Bunyaviridae and Filoviridae.
Retroviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by human immunodeficiency virus type 1 and the human immunodeficiency virus type 2 lentiviruses, foamy viruses and human T-cell leukemia viruses.
Herpesviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by herpes simplex type 1 and herpes simplex type 2 viruses, human herpes virus-6, Human herpes virus-8, varicella-zoster viruses, cytomegaloviruses, lymphoproliferative herpesviruses, Epstein-Barr virus and other herpes viruses.
Orthomyxoviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by influenza A, influenza B and influenza C viruses.
Paramyxoviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by respiratory syncytial virus, mumps virus, parainfluenza viruses and measles-like viruses.
Hepadnaviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by hepatitis B viruses.
Flaviviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by hepatitis C virus, yellow fever virus, dengue virus and tick-borne encephalitis viruses.
Togaviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by rubella virus.
Rhabdoviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by rabies virus and vesicular stomatitis virus.
Poxviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by vertebrate and avian poxviruses and vaccinia viruses.
Arenaviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by the arenaviruses.
Coronaviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by the coronaviruses.
Bunyaviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by the hantaviruses.
Filoviridae infections that may be prevented or treated using the methods of this invention include, for example, those caused by Marburg, Reston and Ebola viruses.
Preferably, the enveloped viral infection to be treated or prevented is one wherein the virus is from the genera Retroviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Hepadnaviridae, Flaviviridae, or Rhabdoviridae. More preferably, the virus is type 1 or type 2 human immunodeficiency virus, type 1 or type 2 herpes simplex virus, varicella zoster virus, Epstein-Barr virus, cytomegalovirus, influenza type A, B, or C virus, respiratory scincytial virus, mumps virus, hepatitis B virus, hepatitis C virus, encephalitis virus, rabies virus, or dengue fever-inducing virus. Most preferably, the virus is type 1 or type 2 human immunodeficiency virus or type 1 or type 2 herpes simplex virus.
Important examples of parasitic targets for which carbohydrate crosslinked glycoprotein crystal based vaccines include those in the kingdom Protozoa.
Protozoa infections that may be prevented or treated using the methods of this invention include those caused by representatives of the Sarcomastigophora (containing flagellates and amebas); Apicomplexa (containing the sporozoans) and Ciliophora (containing the ciliates).
Sarcomastigophora infections that may be prevented or treated using the methods of this invention include, for example, those caused by Trypanosoma cruzi, Toxoplasma gondii, Leishmania major.
Apicomplexa infections that may be prevented or treated using the methods of this invention include, for example, those caused by Plasmodium falciparum.
Ciliophora infections that may be prevented or treated using the methods of this invention include, for example, those caused by Balantidium coli.
Parasitic worm infections for which vaccines may be effective include, for example, those caused by the classes Cestoda (tapeworms) and Trematoda (flukes).
According to one embodiment of this invention, the use of dried carbohydrate crosslinked glycoprotein crystals permits routine handling and storage of these materials prior to use (dry storage at room temperature or above without refrigeration, for extended periods of time). The ability to transport the carbohydrate crosslinked glycoprotein crystals at ambient temperatures without denaturation of the glycoprotein advantageously overcomes the problem of inadequate refrigeration of vaccines often encountered during global distribution of vaccines.
Dried carbohydrate crosslinked glycoprotein crystals also allow for routine formulation by direct addition of adjuvants and immune modifying cytokines such as Type I interferon, immune interferon, tumor necrosis factor, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-8, interleukin-10, interleukin-12, colony stimulating factors and other immune modifiers.
Any conventional pharmaceutically acceptable carrier or adjuvant may be combined with the carbohydrate crosslinked glycoprotein crystals of this nvention. These carriers and adjuvants include, for example, Freund""s complete and incomplete, bacterial lipolysaccarides, cholera toxin, mono and di-phosphoryl lipid A, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium, trisilicate, polyvinyl pyrrolidone, cellulose-based substances and polyethylene glycol. Adjuvants for topical or gel base forms may be selected from the group consisting of sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxpropylene-block polymers, polyethylene glycol, natural and synthetic gum bases, and wood wax alcohols.
Formulations may include any excipient or carrier which may be added to carbohydrate crosslinked glycoprotein crystals or pharmaceutical salts thereof, without affecting their biological activity.
According to another embodiment, this invention provides a method for treating or preventing an enveloped virus infection in a patient comprising the step of administering to said patient a composition comprising: an amount of a carbohydrate crosslinked glycoprotein that is also an immune response modifier or antibody, sufficient to reduce or prevent viral replication in said patient via modification of patients immune response or direct interaction with said virus.
A carbohydrate crosslinked glycoprotein crystal or a combination of carbohydrate crosslinked glycoprotein crystals can be used as a component of a sensor, referred to as a biosensor, useful for detecting and/or measuring an analyte of interest in a fluid, such as body fluid (e.g., blood, urine), chemical and laboratory reaction media, organic media, water, culture medium and beverages. In some instances, the fluid in question can be a gas, as in an alcohol breath analyzer [E. Barzana, A. Klibanov and M. Karell, NASA Tech Briefs, 13, p. 104, (1989)]. In this application an appropriately-selected carbohydrate crosslinked glycoprotein crystal is brought into contact with a fluid to be analyzed for the analyte of interest. The analyte of interest can be measured directly (e.g., blood glucose) or indirectly (e.g., by detecting or measuring a substance which is a reactant (product or substrate) in a reaction in which the analyte of interest participates). In either case, the carbohydrate crosslinked glycoprotein crystal is able to act upon the analyte or the substance which is a reactant in a reaction in which the analyte also participates. The activity of the enzyme results in a detectable change (e.g., change in pH, production of light, heat, change in electrical potential) which is detected and/or quantified by an appropriate detecting means (e.g., pH electrode, light or heat sensing device, means for measuring electrical change) [J. Janette, et al., Anal. Chem., 62, pp. 33R-44R (1990)]. Any means useful for detecting the change resulting from the enzyme-catalyzed method can be used. A biosensor of the present invention includes a carbohydrate crosslinked glycoprotein crystal or a combination of carbohydrate crosslinked glycoprotein crystals and a retaining means for the carbohydrate rosslinked glycoprotein crystal which allows contact etween the carbohydrate crosslinked glycoprotein crystal(s) and the analyte of interest or the substance in the fluid which is a reactant in the reaction in which the analyte of interest participates.
The carbohydrate crosslinked glycoprotein crystals of this invention may be used in any of a number of chemical processes. Such processes include industrial and research-scale processes, such as organic synthesis of specialty chemicals and pharmaceuticals. Enzymatic conversion processes include oxidations, reductions, additions, including esterifications and transesterifications, hydrolyses, eliminations, rearrangements, and asymmetric conversions, including stereoselective, stereospecific and regioselective reactions.
Carbohydrate crosslinked glycoprotein crystals according to this invention may also be used in various environmental applications. They may be used in place of conventional soluble or immobilized proteins for environmental purposes, such wide area decontamination of environmental hazards.
Alternatively, the carbohydrate crosslinked glycoprotein crystals of this invention may be used in cleaning agents, selected from the group consisting of detergents, such as powdered detergents and liquid detergents, bleaches, household cleaners, hard surface cleaners, industrial cleaners and carpet and upholstery shampoos.
Cleaning agents containing carbohydrate crosslinked glycoprotein crystals according to the present invention may also comprise compounds conventionally included in such agents. See, for example, Soaps and Detergents, A Theoretical and Practical Review, Louis Spitz (Ed.), AOCS Press (Champlain, Ill.) (1996). Such compounds include anionic, non-ionic, cationic or zwitterionic surfactants, or mixtures thereof.
Anionic surfactants are exemplified by alkyl sulfates, alkyl ether sulfates, alkyl sulfonates, alkylaryl sulfonates, olefin sulfonates, alkyl ether phosphates, alkyl ether phosphates, fatty acid salts, soaps, isothionates and sulfonated unsaturated esters and acids.
Non-ionic surfactants are exemplified by products of condensation of an organic aliphatic or alkyl aromatic hydrophobic compound with an alkylene oxide, alkyl polyglucosides and sugar esters.
Cationic surfactants are exemplified by quarternary ammonium salts of tertiary alkyl amines, amino amides, amino esters or imidazolines containing al least one long chain (C8-C22) aliphatic group or an alkyl-aryl group, wherein alkyl comprises about 4 to 12 carbon atoms and aryl is preferably a phenylene group.
Zwitterionic surfactants are exemplified by derivatives of quarternary ammonium, quarternary phosphonium or tertiary sulfonium compounds, derivatives of secondary and tertiary amines and derivatives of heterocyclic secondary and tertiary amines.
Carbohydrate crosslinked glycoprotein crystals according to this invention may also be used as ingredients in personal care compositions, including cosmetics, such as creams, lotions, emulsions, foams, washes, compacts, gels, mousses, slurries, powders, sprays, pastes, ointments, salves, balms, drops, shampoos and sunscreens. In topical creams and lotions, for example, they may be used as humectants or for skin protection, softening, bleaching, cleaning, deproteinization, lipid removal, moisturizing, decoloration, coloration or detoxification. They may also be used as anti-oxidants in cosmetics.
An alternate embodiment of the present invention includes protein delivery systems comprising carbohydrate crosslinked glycoprotein crystals. Such a system may be used to deliver glycoproteins such as those included in personal care products, such as cosmetics, pharmaceuticals, veterinary compositions, vaccines, foods, feeds, diagnostics, cleaning agents, such as detergents, and formulations for decontamination. Glycoprotein delivery systems of this invention, which may be formulations or devices, such as implantable devices, may be microparticulate glycoprotein delivery systems.
In such systems, as well as in other embodiments of the present invention, carbohydrate crosslinked glycoprotein crystals have a longest dimension between about 0.01 xcexcm and about 500 xcexcm, alternatively between about 0.1 xcexcm and about 50 xcexcm. The crosslinked glycoprotein crystal components may have a shape selected from the group consisting of: spheres, needles, rods, plates, such as hexagons and squares, rhomboids, cubes, bipyramids and prisms. Advantageously, the crosslinked crystal form of the glycoproteins of this invention allow loading of up to between about 50% and about 90% protein per unit of weight.
According to one embodiment of this invention, carbohydrate crosslinked glycoprotein crystals are characterized by stability and integrity under elution conditions used in separations, particularly chromatography elution conditions, as compared with the soluble uncrosslinked form of the protein that is crystallized to form the glycoprotein crystals that are crosslinked. Alternatively, carbohydrate crosslinked glycoprotein crystals are characterized by stability and integrity in the presence of a solvent contained in the sample to be separated, as compared with the soluble uncrosslinked form of the glycoprotein that is crystallized to form the glycoprotein crystals that are crosslinked.
The carbohydrate crosslinked glycoprotein crystals may be used for high throughput screening in combinatorial chemistry, where large libraries may be screened for specific interaction with the glycoprotein component of the crystals.
An advantage of this invention is that chromatographic separations may be carried out in the presence of an aqueous solvent, an organic solvent, or an aqueous-organic solvent mixture.

Methods of preparing carbohydrate crosslinked glycoprotein crystals


A tube for use in a fixed angle centrifuge rotor having a rotor cavity inclined at a predetermined angle .THETA. with respect to the axis of rotation is characterized by one or more marking(s) thereon that indicates the level to which the tube may be filled with liquid without risk of spillage due to meniscus re-orientation. The markings may be graduated in terms of angles at which the rotor cavity may be inclined or in terms of the identity of each rotor into which the tube may be inserted.

1. Field of the Invention
The present invention relates to a tube for use in a fixed angle centrifuge rotor.
2. Description of the Prior Art
Tubes, whether capped or uncapped, are used to carry sample liquid in centrifugation protocols. Such tubes may have indicated thereon a rated nominal capacity that defines the full volume of the liquid contained within the tube. In addition, the tube may have graduations permanently formed thereon denoting the volume within the tube when the tube is filled to various heights. Such graduated centrifuge tubes are available from a variety of vendors, with the round or conical tubes as shown at page 41 of the Nalgene.TM. Labware Manual (1991) being representative.
When a tube is used in a fixed angle rotor, i.e., a rotor in which the axis of each rotor cavity is inclined at a predetermined angle .THETA. with respect to the axis of rotation, care must be taken to avoid filling the tube to a level that could, due to meniscus reorientation, cause spillage (in an uncapped tube) or cause challenge to the tube seal (in a capped tube, if challenge is a concern).
FIG. 1 permits a full understanding of the problem of meniscus reorientation encountered when a tube as shown is first inserted into a rotor and when the tube is thereafter spun. In FIG. 1 the rotor R is assumed to have a plurality of cavities C each of which is oriented at a predetermined rotor angle .THETA., shown as forty-five degrees (45.degree. ) with respect to the axis of rotation CL. When the axis A of the tube T is oriented vertically the tube T is filled with a volume of liquid reaching the level P.sub.f. When the tube T is first inserted into the rotor the liquid level re-orients to the static position indicated by the character P.sub.s. As the rotor R is spun centrifugal force causes the liquid to reorient to the dynamic level P.sub.d. Note that in a rotor having a forty-five degree angle and a tube sized and configured as shown in FIG. 1, the volume of liquid that would prevent spillage or seal contact in the static case (the static level P.sub.s) is equal to the liquid volume that would prevent spillage or seal challenge while spinning (the dynamic level P.sub.d). If the rotor angle is less than forty-five degrees the dynamic volume that prevents spillage or seal challenge is less than the corresponding static volume. Conversely, if the rotor angle is greater than forty-five degrees the dynamic volume that prevents spillage or seal challenge is greater than the corresponding static volume.
As noted in FORD and Graham, "An Introduction to Centrifugation", page 18, Bios Scientific Publishers, Ltd. (1991), uncapped tubes should be filled only to a level consonant with the degree of such meniscus reorientation. The appropriate level for a tube of given physical parameters and of a given configuration depends the magnitude of the angle .THETA. in the centrifuge rotor. The angle of inclination may be different for each fixed angle rotor able to be spun in a given centrifuge instrument, but is typically a value less than forty-five degrees.
The problem of overfilling of tubes may be avoiding by use of a filling gauge such as that shown at page 4-10, Beckman Instruments, "Rotors and Tubes for Preparative Ultracentrifuges--An Operator's Manual", LR--IM--12, March 1985. The filling gauge includes reference to rotor types. Prior to filling, each tube must be aligned with the filling gauge and suitably marked, as with a felt-tipped pen. Such a procedure may be viewed as laborious and time consuming. Moreover, since the marking on the tube may become obliterated when the tube is washed or sterilized the marking procedure using the filling gauge must be repeated each time the tube is re-used.
In view of the foregoing it is believed advantageous to provide a tube for use in a fixed angle centrifuge rotor that includes at least one, but preferably a series, of indicator marking(s) that are permanently provided thereon whereby the tube may be quickly and conveniently filled to a volume level appropriate for use in any one of a plurality of fixed angle rotors without risk of tube spillage or seal challenge due to meniscus re-orientation.

Tube for use in a fixed angle centrifuge rotor


The invention relates generally to processes for electrographic image development. An electrographic development apparatus is provided wherein a film is adjacent a cylindrical toning shell and a mixture of toner and carrier is particles disposed on the cylindrical toning shell in contact with the film. The cylindrical toning shell is closest to the film at a first location, the mixture of toner and carrier particles being movable through the first location with a flow direction. A magnetic core disposed within the cylindrical toning shell offset toward the cylindrical shell such that a magnetic field strength is greater at the second location than the first location.

The invention relates generally to processes for electrographic image development.
Processes for developing electrographic images using dry toner are well known in the art and are used in many electrographic printers and copiers. The term "electrographic printer," is intended to encompass electrophotographic printers and copiers that employ a photoconductor element, as well as ionographic printers and copiers that do not rely upon a photoconductor. Electrographic printers typically employ a developer having two or more components, consisting of resinous, pigmented toner particles, magnetic carrier particles and other components. The developer is moved into proximity with an electrostatic image carried on an electrographic imaging member, whereupon the toner component of the developer is transferred to the imaging member, prior to being transferred to a sheet of paper to create the final image. Developer is moved into proximity with the imaging member by an electrically-biased, conductive toning shell, often a roller that may be rotated co-currently with the imaging member, such that the opposing surfaces of the imaging member and toning shell travel in the same direction. Located adjacent the toning shell is a multipole magnetic core, having a plurality of magnets, that may be fixed relative to the toning shell or that may rotate, usually in the opposite direction of the toning shell.
The developer is deposited on the toning shell and moved into proximity with the imaging member, at a location where the imaging member and the toning shell are in closest proximity, referred to as the "toning nip." In the toning nip, the magnetic carrier component of the developer forms a "nap," similar in appearance to the nap of a fabric, on the toning shell, because the magnetic particles form chains of particles that rise from the surface of the toning shell in the direction of the magnetic field.

Electrographic development method and apparatus


A ferroelectric memory using ferroelectric capacitors, in which a plurality of electrodes are juxtaposed on each of the two sides of one ferroelectric substance, and the electric fields between the electrodes are controlled, so that more than two different amounts of charges are provided by the ferroelectric capacitors formed by the electrodes. Thereby, the quantity of storage per ferroelectric cell is increased without increase of the cell area.

1. Field of the Invention
This invention relates to a ferroelectric memory using ferroelectric capacitors.
2. Description of the Related Art
Recently, a ferroelectric capacitor has been employed as a memory to hold two valued charges. As for a memory of this type, a memory cell has been disclosed, for instance, by Unexamined Japanese Patent Application Hei-2-304796/(1991) FIG. 5 shows an electrical equivalent circuit of the memory cell, and FIG. 6 shows the structure of the same.
The memory cell, as shown in FIG. 5, comprises: a switching element, namely, a field-effect transistor 110, and a signal charge storing capacitor 120 using a ferroelectric substance. The field-effect transistor 110 has a gate electrode 111, a drain electrode 112, and a source electrode 113. The gate electrode 111 is connected to a word line WL, and the drain electrode 112 is connected to a bit line BL. The capacitor 120 comprises: a ferroelectric film 123; and two electrodes 121 and 122 formed on both sides of the ferroelectric film 123, respectively. The electrode 121 is connected to the source electrode 113 of the field-effect transistor 110, and the electrode 122 is connected to a ground line Vss or to a drive line DL. The ferroelectric film 123 is, in general, made of lead titanium zirconate (called "PZT").
The structure of the memory cell thus organized will be described with reference to FIG. 6 in brief.
A field oxide film 102 is formed by selective oxidation of the surface of a silicon substrate 101, thus defining a element forming region In the region, the field-effect transistor 110 is formed which consists of a gate electrode 111 covered by an oxide film 103, a drain region 112a, and a source region 113a. The lower electrode 121, the ferroelectric film 123, and the upper electrode 122 are formed on the source region 112a in the stated order, to form the capacitor 120. A metal conductor 104 is formed, as the bit line BL, on the drain region 112a, and a metal conductor 105 is formed, as the ground line Vss or the drive line DL, on the upper electrode 122.
The storage of charge of the ferroelectric capacitor in the above-described conventional non-volatile memory will be described with reference to FIGS. 7 and 8. FIG. 7 is an explanatory diagram showing the conventional capacitor formed on a semiconductor substrate. In FIG. 7, reference characters a and b designate the terminals of the capacitor. When voltage is applied across those terminals a and b of the capacitor, an amount of charge stored in the ferroelectric film 123 between the electrodes 121 and 122 is as shown in FIG. 8, in which the horizontal axis represents field strengths E and the vertical axis, amounts of polarization P. As the voltage between the terminals a and b changes, the amount of polarization of the ferroelectric film 123 changes as 0.fwdarw.A.fwdarw.B.fwdarw.C.fwdarw.D.fwdarw.E.fwdarw.F.fwdarw.G.fwdarw.B, thus showing a hysteresis characteristic.
When the field strength between the electrodes 121 and 122, after being raised to E.sub.sat much larger than E.sub.0, is returned to 0, then an amount of polarization P.sub.s (called "spontaneous polarization") remains in the ferroelectric film 123. Similarly, when the field strength between the electrodes 121 and 122, after being decreased to -E.sub.sat, is returned to 0, then an amount of polarization -P.sub.s remains in the ferroelectric film 123. With those positive and negative spontaneous polarizations corresponding to data "1" and "0" write states, the capacitor 120 provides a read signal charge Q represented by the following equation: EQU Q=2P.sub.s .multidot.S (Coulomb)
where S is the capacitor's area.
The spontaneous polarization P.sub.s is determined from the composition and thickness of the ferroelectric film 23.
In a conventional ferroelectric memory of this type, one ferroelectric capacitor holds binary charges. Hence, in order to provide multi-valued charges, it is necessary to provide as many ferroelectric capacitors as the number of charges required.

Ferroelectric memory with multiple-value storage states


A method of monitoring processor resources. To monitor a processor resource, first a set of needed resources is determined at the beginning of a block of code. A test is then performed to determine if the set of needed resources is available at the start of the block of code. An error is signaled if the needed resources are not available at the beginning of the block of code.

As new computer processors are designed, new computer architectural designs are created. The new computer architecture designs improve the processing performance of the new computer processors. However, the existing set of computer programs often can not directly execute on new computer architectures.
To run computer object code written for an older computer architecture on a newer computer processor, many computer systems use software that will "emulate" the older computer processor architecture. Specifically, software running on the newer computer processors simulates the actions of the older computer processor by interpreting each instruction. Software emulators work well for executing many legacy applications. However, software emulation wastes processor power due to the emulation software overhead.
Another method of running computer object code written for an older computer architecture on a new computer processor is to perform binary translation. A binary translator translates blocks of code written for an older source architecture into equivalent blocks of code in the newer target processor architecture. The translation can be done "on the fly" (while "executing" the older source architecture code). Translation could also be done before execution. By translating the old source architecture code into code for the newer processor architecture, the overhead of the emulation code is eliminated. Thus, binary translation often provides better performance than emulation.
When executing older code, both software emulation systems and binary translators must precisely duplicate the environment of the older processor architecture. Software emulators duplicate the behavior of the older processor architecture by providing an interpreter that interprets each microprocessor instruction as the original processor would have. Binary translators provide a duplicate environment by inserting extra instructions as necessary to ensure that various parameter limits of the older processor are not being exceeded. Since the extra instructions are not directly related to the goal of the original program, these extra instructions are "overhead" code that reduce performance. It would be desirable to implement binary translators in a manner that minimizes the amount of overhead code that needs to be added to duplicate the environment of the older processor architecture.

Method and apparatus for stack emulation during binary translation


Semiconductor devices having vertical field effect transistors with self-aligned source and drain contacts are provided, as well as methods for fabricating vertical field effect transistors with self-aligned source and drain contacts.

Traditional CMOS (complementary metal oxide semiconductor) techniques include process flows for constructing planar transistors. With planar transistors, transistor density can be increased by decreasing the pitch between transistor gate elements. However, with planar transistors, the ability to decrease gate pitch is limited by the required gate length and spacer thickness. In recent years, there has been significant research and development with regard to vertical transistor structures, which decouple the gate length from the gate pitch requirement and enable scaling of transistor density. In general, vertical transistors are designed to have gate structures that are disposed on multiple sides of a vertical semiconductor fin structure or vertical nanowire. With vertical transistors, device scaling is determined by how closely conductive via contacts to source and drain regions can be placed. Unlike planar transistors, however, where self-aligned contact processes (which determine the spacing between source/drain contacts and a gate electrode) can be used, there are no known techniques for forming self-aligned source/drain contacts for vertical transistors.

This patent is subject to a terminal disclaimer.

Self-aligned contacts for vertical field effect transistors

International Business Machines Corporation


A sound source playback unit plays back sound data from a sound source and outputs a playback signal. An amplification unit amplifies the playback signal and outputs the playback signal as an output signal converted to sound in a speaker. A fault detection unit including a first conversion circuit compares the playback signal to a predetermined first threshold, converts a waveform of the playback signal, and outputs the converted waveform as a converted playback signal. A second conversion circuit compares the output signal to a predetermined second threshold, converts a waveform of the output signal, and outputs the converted waveform as a converted output signal. A comparison circuit compares the converted playback signal to the converted output signal, and a determination circuit determines an output of the comparison circuit. Based on the determination, the fault detection unit detects a fault in the amplification unit.

Regarding fault detection, Japanese Patent Application Laid-Open Publication No. 2014-230016 discloses a fault detection device that detects an anomaly in an amplifier of an alarm device that includes an alarm sound emission means that emits an alarm sound, an alarm signal transmission means that transmits, to the alarm sound emission means, an alarm signal for emitting an alarm sound, and an amplifier that amplifies the alarm signal, the fault detection device having: a detection signal transmission means that transmits, to the amplifier, a fault detection signal having two pulse waves of differing polarities and phases; a first detection circuit for detecting a first detection signal attained by the amplifier amplifying the fault detection signal and outputting the amplified fault detection signal from a positive-side output terminal; a second detection circuit for detecting a second detection signal attained by the amplifier amplifying the fault detection signal and outputting the amplified fault detection signal from a negative-side output terminal; and a determination means that determines whether or not an anomaly has occurred in the amplifier on the basis of the detected first detection signal and second detection signal.
Also, Japanese Patent Application Laid-Open Publication No. 2005-202624 discloses an electronic device having: a sensor; a microcomputer that selects sound source data by controlling a sound source IC when it is determined according to a detection signal from the sensor that an anomaly has occurred; an amplification means that amplifies the sound source data outputted from the sound source IC; an alarm output means that is connected to the amplification means and that outputs an alarm sound based on the sound source data; and a fault test means that outputs a test signal to the alarm output means, the microcomputer including a fault diagnosis means that controls the fault test means so as to output the test signal, and determines that the alarm output means is in a normal state if the test signal is detected to be flowing to the alarm output means.
Meanwhile, Japanese Patent Application Laid-Open Publication No. H1-176952 discloses a fault diagnosis device for an audio device having: a voltage source that is connected to a prescribed circuit of an audio device when diagnosing a fault, and that supplies a prescribed voltage to the circuit; a means that is connected between the prescribed circuit of the audio device and the voltage source and forms a voltage-dividing circuit together with the circuit; a means for detecting the voltage of the means forming the voltage-dividing circuit and comparing the voltage to a reference value; and a means for performing notification according to comparison results from the means that performs comparison.

Semiconductor device and sound output device


Provided are methods, systems and computer program products for improving TCP stream transmissions by establishing a transmission schedule and making modifications to the timing and/or rate at which data packets are transmitted based on a timing of acknowledgment signals received in response to transmitted data packets.

This disclosure relates in general to content delivery over the Internet and, but not by way of limitation, to content delivery networks amongst other things.
Content delivery networks (CDNs) have a geographically distributed network of points of presence (POPs) such that one is likely to be close to an end user. A request for content is matched to a nearby POP using routing, domain name service (DNS) diversion, redirection, Anycast and/or other techniques. An edge server in the POP generally serves the content from a cache of the edge server, a host within the CDN, or an origin server depending on where the content is located. For content that is missing from the CDN, the request to the origin server can be costly in terms of quality of service (QoS). QoS can also be impacted by dropped packets and the associated delay, including due to the retransmission of packets.

Strategically scheduling TCP stream transmissions


An adapter for use with vehicle-mounted electronics includes an adapter body further including an input section having at least one main input terminal to which a video signal or audio signal is supplied and at least one secondary input terminal to which a video signal or audio signal is supplied, an output section having an output terminal from which a video signal or audio signal is delivered, a switching element switching an output signal from the input section between the main input terminal side and the secondary input terminal side, and a control section controlling the switching element. The control section includes a detector detecting a signal supplied to the secondary input terminal side. The control section controls the switching element based on signal detection by the detector so that the output signal from the input section is switched from the main input terminal side to the secondary input terminal side.

1. Field of the Invention
The present invention relates to an adapter suitable for use with vehicle-mounted electronics.
2. Description of the Related Art
Vehicles such as automobiles have conventionally been installed with genuine systems designed in compliance with vehicle specifications, such as genuine car audio systems or car navigation systems. On the other hand, portable audio/video devices are commercially available and comprise a recording medium such as a small-sized hard disc or flash memory with large capacity which is capable of storing a large amount of audio or video data. A frequency modulation (FM) transmitter is used in order that a portable audio/video device may be used via the aforesaid genuine system in a vehicle. In this case, for example, audio data is delivered from a car radio.
However, audio/video signals are delivered through a wireless circuit when the FM transmitter is used in the manner as described above. Accordingly, audio/video signals are susceptible to degradation or noise influences. Furthermore, since both portable audio/video device and car radio need to be powered up and then operated individually, the operation is troublesome. Still furthermore, digital versatile disks (DVDs) used with the aforesaid portable audio/video devices or car navigation systems have not unified to one standard including recording and reproducing systems and the like. However, users of the portable audio/video devices have strong demand for use of the portable audio/video devices or car navigation systems through the intermediary of the aforesaid genuine systems. However, since the genuine systems are designed in compliance with vehicle specifications, it is troublesome and expensive to install, into the genuine vehicle systems, vehicle-mounted electronics having different specifications of recording/reproducing system etc. from the genuine vehicle systems.

Adapter for use with vehicle-mounted electronics


The invention relates to an inertial sensor based on the magnetic levitation of an inertial mass comprising an active magnetic bearing unit arranged in such a way as to levitate the inertial mass and characterized by the fact that it furthermore comprises additional active magnetic bearings units arranged in such a way as to control the position of said inertial mass along three independent axis and to create, for any of said independent axis, restoring forces that can be oriented in any of the two directions of these independent axis.

Current inertial sensing systems such as seismometers, accelerometers, gravimeters and tiltmeters are based on the relative displacement between an inertial mass and the base of the instrument when the said base is subject to an external disturbance (vibration, modification of the "g" level, angle); and gyroscopes, which are another kind of inertial sensing systems, are made of an inertial mass which is rotated about one of its axis of inertia and the measurement principle relies on the relative movement between the said axis and the base of the instrument, or on the force generated by the said axis on the base of the instrument, when the said base is subject to an external movement.
All those inertial sensing systems will be limited by the friction between the inertial mass and the base of the instrument.
Indeed this friction will be responsible of imprecisions in the measurements, of wear between the mechanical parts in contact and it might also lead to failure due to mechanical fatigue.
In addition, inertial sensing systems, such as seismometers in seismology or inclinometers in civil engineering, are often placed to monitor structures or machines and the power consumption of such systems is sometimes a critical factor.
In seismology, it is relevant to study seismic waves within the following ranges:                Frequencies: From 1 mHz to 100 Hz        Accelerations: From 1 nano g to 5 g
Given this wide spread both of the relevant frequencies and the relevant accelerations that have to be recorded and analyzed, several classes of measuring instruments have been developed:                Short Period seismic sensors        Long Period seismic sensor        Broad band seismic sensors        Very broad band seismic sensor
All the corresponding products presently commercialized are designed around a damped mechanical mass-spring system made up of a mass detector linked both to a damping mechanism and, with a spring, to the frame of the instrument.
In the case of a seismic excitation, the frame of the seismometer follows the ground movement while the mass used as a detector, which we shall designate as the seismic mass, tends to remain in its initial position, thus moving relatively to the frame.
In the process the length of the spring changes and the displacement in relation to the frame can be measured as a function of time.
The response to a seismic excitation of instruments built according to this principle solely derives from the mechanical characteristics of such a damped spring mass system, i.e. the elastic constant k of the spring and the damping constant d.
Since, however, the spring characteristic k is not precisely constant upon the whole range of possible spring deformations, and is temperature dependant, some of the most recent seismometers are equipped with an electromagnetic counterforce system fed by a feed back loop, limiting spring deformations within a small range where k is assimilated to a constant value.
This design, however, does not eliminate distortions caused by spring inertia and friction and, for a given instrument, it is not possible to change its parameters k and d, a fact which limits its use to a chosen range of accelerations and frequencies.
In order to eliminate these shortcomings, some new designs have been recently patented: they use either magnetic or electrostatic forces or the force resulting from a special property of a super-conducting loop, called the Meismer effect, in order to levitate a seismic mass. The levitation is obtained through the action of one or several retroactive loops commanded by optical or capacitive sensors measuring the movements of the seismic mass in relation to the frame of the instrument.
Today's high quality seismometers and based on expensive multi-axis spring-damper elements with complex compensation systems. Electrostatic levitation of large spheres in high vacuum is the principle of some high precision gyroscopes.
A three axis active magnetic suspension seismometer, described in the U.S. Pat. No. 5,565,665 issued to Biglari et al., shows a limited sensitivity, caused by the sensing system, and a non-symmetric behavior of the vertical axis. In addition an upward acceleration can not be counter balanced since there are no electromagnets placed below the seismic mass.
In U.S. Pat. No. 4,947,067 issued to Habermann et al., a three axis magnetic suspension is presented but for the purpose of a vibrator/dampener and not for the purpose of an inertial sensor.
In U.S. Pat. No. 5,024,088 issued to Komatsu et al., the described accelerometer shows a complex inertial mass and a non optimal arrangement of the coils resulting in a hyperstatic system.
Magnetic suspension and rotation of small spheres was done by J. Beams, but with only one controlled axis and the purpose was to create high centrifugal fields.
Other Prior Art References:
Patent Documents
U.S. Pat. No. 5,955,800
U.S. Pat. No. 5,357,803
U.S. Pat. No. 5,224,380
U.S. Pat. No. 6,363,035
U.S. Pat. No. 5,983,699
Other Publications
    J. W. Beams, J. L. Young, J. W. Moore, the Production of High Centrifugal Fields, Journal of Applied Physics, Vol. 17, November, 1946.    J. W. Beams, Magnetic Suspension for Small Rotors, The Review of Scientific Instruments, vol. 21, No 2, 182-184, February, 1950.    E. F. Kinsey, J. W. Beams, M. J. Saunders, The Magnetically-Suspended Free Gyroscope, Naval Ordnance Research Laboratory, University of Virginia, December, 1951.    J. W. Beams, Magnetic Bearings, Automotive Engineering Congress, Detroit, Mich., January, 1964.    W. J. Bencze, Y. Xiao, D. N. Hipkins, B. W. Parkinson, G. F. Franklin, An Electrostatic Suspension and Orientation Control System for the Gravity Probe B Relativity Mission's Science Gyroscope, 3rd MOVIC, September 1996, Chiba, Japan.

Three axis active magnetic levitation for inertial sensing systems

Ecole Polytechnique Federale de Lausanne (EPFL)


A contextual keyboard allows users to bring in information not normally found on a keyboard, as though that information had been typed in afresh. It also allows users to take information entered on the keyboard, or simply available on the screen, or implicit in information on the screen, and perform actions on it, such as calling a phone number, entering contact or calendar information into a local database on the device, or using the information as search terms in an online query. The central novelty of this invention is moving the functionality into the keyboard layer, so as to be universally accessible on the device.

Conventional touchscreen devices typically require users to navigate between different applications using onscreen menus or by selecting icons representing applications or files from one or more pages of icons. The keyboard in any given application may only provide access to letters, numbers, and symbols, such as found on a typical physical keyboard. Users who wish to access or manipulate data, or perform functions available on the device, need to navigate through a series of onscreen menus and choices, which can be time-consuming and awkward.

Systems and methods for using entered text to access and process contextual information


A fluorine-containing copolymer obtained by copolymerizing tetrafluoroethylene, hexafluoropropylene and perfluoro vinyl ether as component monomers, wherein a weight ratio of tetrafluoroethylene, hexafluoropropylene and perfluoro vinyl ether units constituting the fluorine-containing copolymer is 70 to 95: 5 to 20: 0 to 10, respectively; the fluorine-containing copolymer having: a melt flow rate of 30 (g/10 minutes) or more; a volatile content index of 0.2% by weight or less; and a stress relaxation modulus G(t) (unit: dyn/cm2) which satisfies the following formula at t=0.1 second when measured at a temperature of 310° C.:G(0.1)>7×106×X−1.62−3000where X denotes the melt flow rate (unit: g/10 minutes). Also disclosed is an insulating material composed of the fluorine-containing copolymer and an insulated cable having a core conductor coated with the fluorine-containing copolymer.

Tetrafluoroethylene (TFE)/hexafluoropropylene (HFP) copolymer has superior heat resistance, chemical resistance, extrusion moldability and the like, and in addition, has superior electric insulating property and high-frequency property with a low dielectric tangent. Therefore, it is used for insulating cable such as a cable and a wire, and such insulated cable is suitably used as a communication cable. The communication cable includes a data transmission cable such as a LAN cable.
TFE/HFP copolymer also has low flammability and low smoking properties. Thus, insulated cable made from such a copolymer can be used as a plenum cable, which is laid, for example, on the back of a ceiling of a building (plenum area) and strictly regulated for preventing the spread of fire. The insulated cable comprises a core wire such as a cable and an insulating material formed from a resin such as a TFE/HFP copolymer coating the core wire. In general, the insulated cable is manufactured by extrusion coating in which molten resin is extruded in the shape of a tube, drawn down by inserting a core wire through the center portion of the resin tube in its axial direction, and the core wire coated with the resin is then taken up.
The term "draw-down" as used herein means extending a molten resin extruded from a die having an opening of relatively large sectional area to its final intended dimensions. The draw-down is characterized by a draw-down ratio (DDR), which is the ratio of the sectional area of the opening of the die to the sectional area of the insulated material of the final product. In general, the draw-down ratio is suitably from 50 to 150.
The term "insulated" cable as used herein means a cable or wire coated with an insulating material.
Preferably, the draw-down operation is carried out in so that the outer side and inner side of the molten resin, extruded from the die in the form of a tube, are evenly drawn down. This evenness is expressed as draw ratio balance (DRB).
Since in a conventional draw-down operation the resin is drawn as described above, a cone-break sometimes occurs during an extremely short period between the time the resin is extruded from an opening of the die until it contacts a core material. The cone-break is the largest cause of loss in productivity. This is because cone breaks, when they occur, requires restarting the extrusion process and machine utilization is decreased.
In recent years, an increase in molding rate has been desired to enhance productivity and to reduce cost, and there is a demand to increase the cable coating rate by increasing the winding rate of the insulated core wires. The trend of coating the cable at high speed generally increases the occurrence of cone-breaks even though the draw-down ratio is similar.
A technique for reducing the occurrence of cone-breaks is proposed in Publication No. WO 00/44797, in which a TFE/HFP copolymer having a melt flow rate (MFR) of 24 (g/10 minutes) is used and the coating rate of applying an insulating resin to the cable is lower than 2000 ft/minute. Indeed, this copolymer is effective at a coating rate of lower than 2000 ft/minute. However, this technique does not satisfactorily reduce the occurrence of cone-breaks at faster coating rates.
In the coating extrusion, the thickness of the insulating material of the insulated cable is determined by the desired electrical characteristic, and the size of the opening of the die is determined by selecting a draw-down ratio and draw ratio balance suitable for the structure of the insulated cable.
In the case of increasing the cable coating rate without changing the size of the opening of the die, the problem of developing a rough skin on the surface of the resin passing through the opening generally occurs when the velocity of the molten resin in the die exceeds a certain velocity limit. This rough skin is referred to as melt fracture, and it develops when the velocity of the resin exceeds the critical shear rate during melt fluidizing.
The trend of coating cable at high speed also involves the problem of exacerbating the extent of the melt fracture. Excessive melt fracture sometimes leads to a situation where the insulating material does not completely cover the core material, and causes an increase in molding faults such as spark-out and cone-breaks.
As one technique for delaying the occurrence of melt fracture and enabling high-speed coating of about 1.5 times faster than conventional rates, U.S. Pat. No. 5,703,185 discloses that vinyl ether for use as a third monomer component of a TFE/HFP copolymer is changed from perfluoro (propyl vinyl ether) to perfluoro (ethyl vinyl ether). However, this technique encounters difficulty in reducing molding faults such as cone-breaks and the like at high-speed coating rates.