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Carolina Herrera doesn't understand why people think women in dresses "four sizes too small" are sexy.
The 73-year-old designer is known for wearing simple white shirts with well-fitting trousers or voluminous skirts.
Although she doesn't expect women around the world to follow her example, she does worry that a lot of them don't understand style.
"Everyone was very, very chic. You either went to Paris for your clothes or you had them made in Caracas. I would like women to feel they could be glamorous and elegant," she said of the '50s and '60s.
"Unfortunately there's a lot of confusion today over what is sexy and what is vulgar. It's horrifying. They say, 'Oh, that girl is so sexy', and she turns around and the dress is four sizes too small. Or she's wearing so much stuff, you wonder how long it took her to get ready... women don't want to be called elegant now because they connect it with old-fashioned. But elegance is not old‑fashioned."
Herrera is proud she is able to get ready for any event, whether a fashion show or a ball, in 10 minutes. She believes some women spend far too much time on their appearance, when there isn't really any need to do so.
The designer is also unimpressed by those who use celebrities to boost their appeal. She does have a solid relationship with Renee Zellweger, who is often seen in her fishtail gowns, but insists that is different as the dresses suit the actress so well.
"If you have to mention names for the collection to be successful, something is wrong," she told British newspaper The Telegraph.
"The right dress on the right actress is perfect. Otherwise, no. And Renee is the only star in Hollywood who doesn't have a stylist. She loves fashion and we work together on designs. She really knows what works and she moves around at the fittings to see the best pose for the photographers."
Herrera also slammed the public's penchant for trends, another reason she thinks modern fashion goes so wrong.
She implores women to dress for themselves rather than slavishly follow what they see in magazines.
"Too distracting. Women don't find their own style because they're too wrapped up in letting people know what they're wearing. It's like that handbag, the Hermes Birkin. Every woman has to have one now to express her status and style," she marvelled.
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|Publication number
|Publication type
|Application number
|Publication date
|Mar 15, 2005
|Filing date
|Jun 10, 2002
|Priority date
|Jul 6, 2001
|Fee status
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|Publication number
|Lucia Scapol, Giuseppe Claudio Viscomi
|Original Assignee
|Alfa Wassermann S.P.A.
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|Patent Citations (16), Non-Patent Citations (17), Referenced by (7), Classifications (28), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
A wide part of the biomedical sciences bases itself on the use of pharmacologically active proteins both of natural origin, obtained by means of extractive techniques, and of synthetic origin, obtained by means of techniques from recombinant DNA. The purity level of the interesting products is important in both cases, because the understanding of their activity is determined by the possibility of strictly binding the biological effect with the presence of a fixed amount of the protein.
In this context the purification processes of pharmacologically active proteins have gained an important part because the purity of the manufactured protein assumes remarkable significance when active principles or excipients contained in medicinal specialities are involved. The possibility of causing toxic effects and/or producing adverse effects during therapy have in fact pushed the authorities responsible for the registration and the authorization to the marketing of medicines based on proteins to introduce ever more stringent rules in order to determine the quality and the manufacturing consistency of active principles of proteic origin contained in marketed medicines.
From the above said the importance of the purification processes of the proteins clearly appears in which the main difficulty resides in the fact that the pharmacologically active proteins are always in composite mixtures together with many other proteins.
This fact is true both in the case natural sources are used for extracting the pharmacologically active protein, like for instance blood, extracts from animal or vegetal organs, and in the case recombinant DNA techniques are used because the proteins show chemical-physical properties similar between them independently from their origin.
Therefore from the above it comes that, in the case of purifications of pharmacologically active proteins, a protein mixed together with other proteins with similar properties and often abundantly exceeding the amount of the wanted protein has to be isolated with a high purity degree.
The task is exacting and several purification steps are normally used in order to gain the wanted purity levels. The purification processes become in this way very complex and the success of an industrial manufacturing of a protein is essentially bound to the efficiency of the purification process because this latter amply determines the manufacturing costs.
Many techniques are used for purifying proteins, like, for instance, selective precipitations in aqueous and organic solvents or with caotropic agents; separations by means of filtrations and/or dialysis; processes of immuno-precipitation with suitable antibodies; chromatographic processes.
These latter have gained in the recent years largely the greater importance mainly because they allow to get the requested purity degrees, as reported from Regnier F. E. on J. Chromatogr. 418, 115-143, (1987).
Many techniques are cited in the scientific literature and can be classified on the basis of the mechanism of action applied for separating the proteins like, for instance, separation on the basis of the molecular weight, absorption on polar matrices, also named normal stationary phases, absorption on non polar matrices, called reverse stationary phases, absorption by selective affinity with ligands bound to inert matrices, like heavy metals as copper, zinc, iron and platinum, chemical dyes like the brilliant blue, proteins like protein A and protein G, carbohydrates like the polysaccharides and the glucosaminoglycans, absorption by immuno-affinity with specific antibodies bound on inert matrices, absorption by ionic interaction with electrostatically charged ligands bound on inert matrices.
The selectivity, i.e. the capability to selectively recognize the wanted protein, the cost and the possibility to be used at industrial level are used as parameters to evaluate the performances of the different chromatographic techniques.
On the basis of these parameters, the immuno-affinity chromatography is considered that which warrants the greater selectivity, but it shows drawbacks like high costs of use, risks of denaturation of the antibody and risks connected to the end safety of the purified product because the antibody is of animal origin.
The chromatography which uses the ionic interaction, also called ionic exchange chromatography, is considered the less risky technique for keeping the pharmacological activity of the proteins and the easier to be carried out in industrial manner with low costs of management but it shows the drawback to be poorly selective.
Therefore it would be very advantageous to find conditions of execution of a ionic exchange chromatography that increase its selectivity so that to make it competitive with the other techniques from the point of view of the purity of the obtained product.
The ionic exchange chromatography is usually carried out by using columns of various sizes, filled with solid matrices containing chemical groups which, permanently or under particular conditions, are electrostatically charged.
A compound put into an ionic exchange column interacts by means of a coulomb attraction/repulsion with the charges bound to the matrix. Different compounds contained in a mixture will be able to bind themselves to the stationary phase in function of the amount of the possessed charge, and consequently they will be kept more or less, so defining their separation at the column exit.
The chromatography is named cationic exchange chromatography when the charges of the matrix are negative, because the cations are kept, while it is named anionic exchange chromatography when the charges of the matrix are positive.
The proteins are compounds having high molecular weight, higher than 10,000 Daltons, made by heterogeneous polymers of aminoacids; some aminoacids have in their side chain functional groups that can be ionized in function of the pH of the solution in negative manner, acidic aminoacids, or in positive manner, basic aminoacids, and therefore all the proteins possess a great number of negative and positive charges. The isoelectric point, pI, of a protein is the pH at which the protein is neutral because the contribution of the negative charges is equal to that of the positive charges, a protein put in an electronic field at pI is not attracted from any of the polarities of the electric field.
The number of the negative charges increases at pH higher than pI and the protein gains a net negative charge while the opposite happens at pH lower than pI and the protein gains a net positive charge. Every protein has its own characteristic pI which distinguishes it from the others and some proteins tend to become insoluble at the isoelectric point.
When a protein is in a solution at a pH lower than pI it has a net positive charge and therefore can interact with a negatively charged matrix and can be submitted to a cationic exchange chromatography while the protein can be submitted to an anionic exchange chromatography at pH higher than its pI, as reported from Regnier F. E., Science, 238, 319-323, (1987) and from Yamamoto S. et al., Chromatographic Science Series, 43, (1988), Marcel Dekker, Inc. Publisher, New York.
On the contrary we have unexpectedly found, and on this fact the object of the present invention bases itself, that it is possible to find a range of pH values higher than the corresponding pI of the protein at which pH the proteins still stay absorbed on matrices of cationic exchange chromatography so that it is still possible to carry out cationic exchange chromatographies. Such a situation is particularly important because a high selectivity between the proteins is gained under these conditions because also very small differences of pI between proteins become enough in order to get significant separations so affording a high efficiency of purification.
This latter aspect is particularly important in the purification processes of recombinant proteins wherein the wanted product is often accompanied from correlated impurities, i.e. made from very small structural changes of the product, like, for instance, different oxidation states, acetylations, loss of amidic functions and so on. This kind of impurities is very difficult to clear away also by means of immuno-affinity chromatographies because in most cases the antibodies are not able to distinguish them.
The mechanism which can explain the found phenomenon bases itself on the fact that the distribution of the charges along the external surface of the proteins is not uniform so that also when the pH is little higher than pI and the protein has a total net negative charge, there are still some positive charges located into the molecule than can interact with a negative stationary phase.
In order to make effective this mechanism it is important that the excess of negative charges is not too much accentuated otherwise the electric fields created from the negative charges would be so high as to prevent the interaction of the whole protein with the negatively charged chromatographic matrix.
Moreover its is necessary that the ionic strength of the solutions used as eluents is suitably controlled because a high ionic strength would have the effect of shielding the protein so preventing its interaction with the stationary phase.
Lampson G. P. et al., Anal. Biochem., 11, 374-377, (1965), in confirmation report the case of proteins like human gamma globulin, ribonuclease, hemoglobin, delta chymotrypsin, globin and lysozyme in which making small pH variations but keeping a too much high ionic strength, given by a 0.1 M phosphates solution, the elution in cationic exchange chromatographies happened at a pH of almost 0.4 unites lower than pI.
The above mentioned principle, on which the present invention bases itself, has never been used, to inventors' knowledge, in order to carry out efficient processes of purification of proteins.
The possibility of using differences of the isoelectric point of proteins in order to optimize the purification processes described by Kontturi A. K. et al., Acta Chem. Scand., 50 (2), 102-106, (1996) in fact refers to a conventional use of the ionic exchange chromatography wherein the cationic exchange chromatography is always carried out at a pH lower than the isoelectric point while the anionic exchange chromatography is carried out at a pH higher than the isoelectric point. The process described in the present patent application is such that the cationic exchange chromatographies are on the contrary carried out at a pH higher than the isoelectric point of the protein.
The process described in the present patent application can be considered of general nature as shown in the reported examples wherein it has been demonstrated how it is successfully applicable both to a protein of natural origin and to a protein from recombinant DNA. The difference between protein and protein is in the extent of the range of the field of the pH, higher than pI, useful for the purification of the interesting protein. In fact, for instance, as it will be shown in the following examples, such range is of about 0.2 pH units in the case of the interferon proteins while it is of about one pH unit in the case of albumin.
The application of the present invention to the purification of a recombinant alpha interferon (rIFNα) whose isoelectric point is 5.9, as reported from Thatcher D. and Panayotatos N., Methods Enzymol. 119, 166-177, (1986), will be reported among the examples and it will be shown how it is possible and advantageous to purify it in cationic exchange at a pH of 6.1. Moreover the example will be shown of the human seric albumin whose pI is 4.9, as reported by Rylatt D. B. et al., J. Chromatogr., 865, 145-153, (1999), and it will be shown how it is possible and advantageous to purify it in cationic exchange at a pH of 6.0.
The advantages of the process object of the present invention are very remarkable if compared with the results of the processes described in scientific publications and/or patents directed to the purification of α interferon and of human seric albumin, processes that usually require three or more subsequent treatments, fact that causes a high industrial cost and a decrease of the yields.
Thatcher D. and Panayotatos N. describe the purification of the human recombinant alpha interferon rIFN-α2, Methods Enzymol., 119, 166-177, (1986) through five subsequent treatments: a) cationic exchange chromatography; b) anionic exchange chromatography; c) affinity chromatography for heavy metals; d) treatment with a saturated solution of ammonium sulphate; e) molecular exclusion chromatography.
European Patent 0108585 describes for the purification of the interferon the subsequent use of three types of chromatography: a) immuno-affinity; b) cationic exchange; c) molecular exclusion.
U.S. Pat. No. 4,765,903 on the interferon purification describes the sequential use of four types of chromatography: a) immuno-affinity with a monoclonal antibody; b) inverted phase; c) cationic exchange; d) molecular exclusion.
European Patent 0679718 describes a process for the alpha interferon production that envisages the following four chromatographic steps: a) metal-chelating; b) cationic exchange; c) anionic exchange; d) gel filtration.
Other publications and patents describe three or more treatments necessary for the purification of the interferon proteins, for instance U.S. Pat. No. 4,732,683, International Patent Application WO 8604067 and the publication from Khan F. R. and Rai V. R., Bioprocess Technol., 7, 161-169, (1990).
The quoted examples cover the most relevant matter reported about the purification of interferon in general and of alpha interferon in particular. They show how the purification of this latter is particularly difficult and requires many purification steps. Moreover it has to be underlined how high purification levels are in particular obtained by means of immuno-affinity chromatographies by using monoclonal antibodies of murine origin. However the presence of such a chromatographic technique within processes of industrial production aimed at manufacturing active principles for pharmaceutic use in humans causes the risk of possible viral contaminations from viruses of murine origin because of the presence of possible immunogenic fragments coming from the murine immunoglobulins in the end product and because of the difficulties to validate the chromatographic matrices from the industrial point of view.
Moreover from the briefly illustrated examples the cationic exchange chromatography results to be widely used but never as unique separative technique because its performances are limited with regards to the increase of the purity levels.
The publications from Babu K. R. et al., Appl. Microbiol. Biotechnol., 53 (6), 655-660, (2000) and Bouyon R. et al., Biotecnologia Aplicada 14, 189-192, (1997), describe purification processes of alpha interferon in one step by means of ionic exchange chromatography in saline gradient. However in both cases to get a product sufficiently pure the authors have to isolate only some of the chromatographic fractions in which the alpha interferon is contained so obtaining very low yields, until a 7.5% minimum. Moreover the described purification processes of chromatography in saline gradient are not apt to be used at industrial level.
Many techniques of chromatographic purification are described also in the case of the human albumin starting from preparations of albumin obtained by fractionating human serum or by means of techniques of recombinant DNA, techniques complex and scarcely transferable at industrial level that confirm how the problem of an effective purification of human albumin, both of natural and of recombinant origin, is still existing.
U.S. Pat. Nos. 6,150,504 and 5,521,287 describe the purification of the albumin by means of ionic exchange chromatography and hydrophobic interaction. The purification scheme described in U.S. Pat. No. 6,034,221 envisages the albumin purification by means of two chromatographic steps, one ultrafiltration process and two further steps of chromatographic purification.
Less conventional methods of albumin purification in which anionic exchange chromatographies in fluid bed or affinity-chromagraphies interacting with commercially available matrices like those of Streamline®, or suitably prepared, like particles of modified zirconium or emulsions of perfluoro hydrocarbons, are used, are described in U.S. Pat. No. 5,962,649 and in the publications from Sumi A. et al., Bioseparation, 8 (1-5), 195-200, (1999), Mullick A. and Flickinger M. C., Biotechnol. Bioeng., 65 (3), 282-290, (1999) and Mc Creath G. E. et al., J. Chromatogr., 597 (1-2), 189-196. (1992).
Lastly techniques of purification of albumin on heavy metals have also been described from Yang L. et al., Sheng Wu Kung Cheng Hsueh Pao, 16 (1), 74-77, (2000) and techniques of affinity on matrices to which molecules of dyes like Cibacron Blue F3G are bound have been described from Compagnini A. et al., J. Chromatogr. A, 736 (1-2), 115, (1996).
All these techniques show in various manner problems of complexity of realization and of high costs so that the problem of individuating new purification processes of pharmacologically active proteins both of easy and efficient industrial feasibility and economically advantageous is not resolved.
The invention below described gives an answer to these important requirements by providing a process for the purification of pharmacologically active proteins of easy industrial exploitation and of low cost with remarkable economical advantages.
The present invention refers to a process for the purification of pharmacologically active proteins based on the use of the cationic exchange chromatography on a solid matrix under peculiar conditions which comprise, after the loading of the sample, conditioning the column with eluents of suitable pH and ionic strength so that in the column is uniformly present a more basic pH, i.e. higher, than the corresponding isoelectric point, pI, of the pharmacologically active proteins, at which pH however said proteins stay still absorbed on the solid matrix used for the cationic exchange chromatography. After this phase of conditioning the pharmacologically active proteins are eluted from the column by increasing the ionic strength and/or the pH of the eluents.
The effective performance of the present invention requires the individuation of the right combination among the chromatographic matrix to be used, the pH value higher then then pI and the ionic strength to be used in the chromatographic eluents because, once defined the chromatographic matrix, efficient purifications can be obtained by making limited variations of pH and/or ionic strength often of tenths of pH units and/or of variations of ionic strength of few hundreds of μS.
All the functionalized solid matrices commonly used as stationary phases for cationic exchange chromatographies can be used, in particular however the stationary phases named strong cationic exchange have to be preferred when the pI of the protein to be purified is lower than 6 while stationary phases with cationic exchange both strong and weak can be used without exception for proteins having pI higher than 6. Said stationary phases may have siliceous or polymeric matrix, functionalized by means of sulfonic or carboxylic groups both under proton or alkaline salts form. Stationary phases commercially available like, for instance, Source® S (Pharmacia Biotech), Sepharose® SP-Fast Flow, Sepharose® SP-High Performance, Sp Sepharose® XL (Pharmacia Biotech), Fractogel® S (Merck, Darmstadt), Mustang® S (Pall Corporate), CM Sepharose® FF (Pharmacia Biotech), Dowex®, Bio-Rad® AG (Bio- Rad), Poros® S (PerSeptive Biosystems), Shodex® -S, Toyopearl® SP (Tosohass) can be successfully used.
The range of the pH values at which the present invention can efficiently be carried out is very wide, depending upon the isoelectric points of the pharmacologically active proteins that have to be purified, and is comprised between 2 and 11, preferably between 4 and 8.5.
The extension of the range of the pH values higher than pI within which the process described in the present invention is applicable can vary from pH values corresponding to the pI of the pharmacologically active proteins to one pH unit over said pI, showing remarkable differences from protein to protein.
For instance, it has been found that in the case of the recombinant alpha 2b interferon (rIFNα-2b ) it is possible to obtain the absorption of the protein on a cationic exchange matrix till 0.2 pH units over its pI of 5.9 and consequently it is possible to carry out its purification by means of a cationic exchange chromatography while it has been found in the case of the human serum albumin that the protein stays absorbed till one pH unit over its pI.
The range of the saline concentrations of the aqueous solutions employed as efficiently usable eluents depends on the kind of pharmacologically active protein to be purified and it has been found comprised between values of 1 mM and 100 mM, preferably between 1 mM and 30 mM.
For instance, in the case of the purification of the recombinant alpha 2b interferon (rIFNα-2b) the concentration of the aqueous saline solutions is comprised between 1 mM and 30 mM, preferably between 5 and 15 mM.
The need to have fixed and stable pH values of the eluents used for the chromatographies object of the present invention makes very useful, even if not absolutely necessary, to employ aqueous solutions suitably buffered containing from 5 to 100 mM, preferably from 10 to 20 mM of buffered mixtures. Every chemical substance or mixture of chemical substances having a buffering power in the range of the pH between 2 and 11 can be advantageously employed because the pH values of the eluents that can be used are comprised between 2 and 11.
Many aqueous buffer solutions can be advantageously used in carrying out the present invention comprised those containing: glycine and sodium chloride, maleic acid and sodium hydroxide, malonic acid and sodium hydroxide, lactic acid and sodium hydroxide, formic acid and sodium or lithium hydroxide, succinic acid and sodium hydroxide, N-methylpiperazine and hydrochloric acid, piperazine and hydrochloric or acetic acid, L-hystidine and hydrochloric acid, 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid and sodium or lithium hydroxide, N-methyldiethanolamine and sulphuric acid, N-methyldiethanolamine and hydrochloric or acetic acid, pyridine and formic acid, dibasic sodium citrate and sodium hydroxide, monobasic potassium phthalate and hydrochloric acid, monobasic potassium phthalate and sodium hydroxide, monobasic potassium phosphate and dibasic sodium phosphate, bicine and sodium hydroxide, sodium barbital and hydrochloric acid, sodium borate and hydrochloric acid, sodium borate and sodium hydroxide, 1,3-diaminopropane and hydrochloric acid, citric acid and dibasic sodium phosphate, sodium acetate and acetic acid, imidazole and hydrochloric acid, triethanolamine and hydrochloric or acetic acid, tris(hydroxymethylaminomethane) and hydrochloric acid, sodium carbonate and sodium acid carbonate, ethanolamine and hydrochloric acid, piperidine and hydrochloric acid, trimethylamine and formic acid, pyridine and acetic acid, trimethylamine and acetic acid, trimethylamine and hydrochloric acid, ammonium hydroxide and formic acid, ammonium hydroxide and acetic acid, trimethylamine and sodium carbonate, ammonium carbonate and ammonium hydroxide.
In particular, in the case of the purification of the recombinant alpha 2b interferon (rIFNα-2b) all the buffer solutions that show a buffering power at the pH comprised between 5.9 and 6.1 can be used, preferably buffer solutions at pH between 5.9 and 6.1 containing monobasic potassium phosphate and dibasic sodium phosphate, monobasic potassium phthalate and sodium hydroxide, dibasic sodium citrate and sodium hydroxide, citric acid and dibasic sodium phosphate, imidazole and hydrochloric acid, while in the case of the purification of the human serum albumin buffer solutions can be used containing the same mixtures of chemical compounds showing a buffering power at the pH comprised between 4.9 and 6.0.
The aqueous solutions used as eluents can contain, in addition to the chemical substances used for buffering the pH, also chemical substances that have the task to modify the ionic strength of the solution. To this end both organic salts, such for instance carboxylates, alkylsulfonates, phthalates or inorganic salts, like for instance sulphates, chlorides, phosphates which can be salified with sodium, potassium, ammonium, primary, secondary, tertiary or aromatic amines, can be advantageously used.
These compounds can advantageously be used at a concentration comprised between values from 1 mM to 100 mM, preferably between 1 mM and 30 mM.
For instance, in the case of the purification of the recombinant alpha 2b interferon (rIFNα-2b) the concentration of these compounds can vary between 1 mM and 30 mM, preferably between 2 and 20 mM.
The efficiency of the purification can be increased, before the elution of the pharmacologically active proteins, by means of one or more washings carried out with eluents having suitable pH and ionic strength, so that the column is always at a pH higher than pI.
For instance, in the case of the human serum albumin whose pI is 4.9, washings can be carried out with buffer solutions at pH comprised between 5.5 and 5.8, while in the case of the recombinant alpha 2 b interferon (rIFNα-2b) whose pI is 5.9 washings can be carried out with buffer solutions at a pH comprised between 6.0 and 6.1.
The amount of eluent passed across during these washings is variable, normally comprised between 5 and 100 column volumes (CV).
For instance, in the case of the human serum albumin the washings executed are comprised between 20 and 40 CV while in the case of the recombinant alpha 2b interferon (rIFNα-2b) between 10 and 80 CV.
The amount of product to be purified that can be put in the column depends on the chromatographic matrices used, and can arrive until a maximum of 100 milligrams of total proteins for each milliliter of stationary phase even if usually lower amounts are used, comprised between 5 and 20 mg/ml.
The eluents can pass through the column at a linear speed compatible with the stationary phases until a maximum value equal to 10 cm/min.
The above illustrated purification process can be applied to all pharmacologically active proteins; the purification of the interferon proteins with particular regard to the interferons alpha, beta, gamma, delta, omega, tau, to the natural alpha interferon from leukocytes, to the recombinant alpha 2b and consensus interferons and the purification of the albumin with particular regard to the human albumin both of natural and recombinant origin are preferred in the execution of the present invention.
Scope of the above described purification process is to get in an industrial and economical manner pharmacologically active proteins at a purity degree such as to be directly used for the manufacturing of the medicinal specialities which contain them.
In particular, the medicinal specialities preferred within the scope of the present invention are those containing interferon, still more preferably recombinant alpha 2b interferon (rIFNα-2b), and albumin, still more preferably human albumin both of natural and recombinant origin.
Some illustrative examples of the process object of the present invention are reported hereinafter with the sole scope to make clearer the invention but they do not have to be considered in any way restrictive of the invention itself.
Production of the Recombinant Alpha 2b Interferon (rIFNα-2b)
A part of cells of the Escherichia coli BL21 DE3 strain has been transformed with 5 ng of the pET9a-IFNα-2b plasmid, obtained by cloning a synthetic gene reproducing the human gene sequence of IFNα-2b, suitably modified in order to apt the sequence to the codons more frequent in Escherichia coli, into the pET9a plasmid (Novagen).
The proteic sequence expressed from the Escherichia coli cells modified as above shown is equal to that reported in Methods in Enzymology, Interferons, part C, editor Pestka S., 119, 3-14, (1986), published from Academic Press Inc.
The Escherichia coli BL21 DE3 strain transformed by means of the pET9a-IFNα-2b plasmid has been put in culture in a flask in a suitable culture medium, for instance a solution containing 12 g/l of phytopeptone (Phyto peptoton, BBL), 24 g/l of yeast extracts (Yeast extract, DIFCO), 4 g/l of glycerol (BDH) and neomycin, at 37° C. for a time sufficient to arrive at a value of optical density at 600 nm equal to 0.6-0.8, usually 7-9 hours. The so obtained culture is then used at the dilution from 1 to 100 to inoculate a 5 l fermenter where a culture medium equal to that of the flask, previously described, was contained. The culture is kept 14 hours at 37° C. with a aeration equal to one air volume each minute in respect of the culture volume.
The bacterial cells are collected by centrifugation at 6000 rounds per minute (rpm) at the end of the culture, they are suspended in a suitable aqueous solution containing 1 mM of dithiothreitol (DTT) in amount not higher than 6 ml for each gram of wet weight of the bacterial centrifuged. The bacterial suspension is submitted to cell lysis by means of consolidated and described techniques, like for instance breaking by ultrasounds or by hydraulic pressure.
The resulting suspension is recovered by centrifugation and the solid part is suspended in a 50 ml saline solution containing 1 mM of DTT and again centrifuged.
The solid component, constituent the included bodies, is collected and suspended under vigorous stirring at room temperature into 450 ml of a solution containing 6M of guanidinium chloride, 50 mM of Tris-HCl at pH 8 and 0.1 mM of EDTA. The suspension is centrifuged and the supernatant is diluted in the ratio from 1 to 100 to 1 to 200 in a saline solution containing 50 mM of Tris-HCl at pH 8 and 0.1 mM of EDTA at pH 8 suitable for the renaturation of the protein. The solution for the renaturation can contain amino acids, like for instance glycine or arginine; mixtures of compounds containing sulfides in the oxidated and reduced form with the disulfide bridge formed, like for instance glutathione, ethanolamine, cysteine. The renaturation is carried out under vigorous stirring of the solution at 4° C. for almost 72 hours and then the solution is filtered and then concentrated by means of a process of dia-filtration versus a buffer made by 40 mM of Tris-HCl at pH 8 until an end concentration factor from 5 to 10 times. The end concentration of the solution is usually comprised between 0.4 and 1.0 mg/ml.
Purification of the Recombinant Alpha-2b Interferon (rIFNα-2b)
A 1M solution of sodium acetate is added until the 20 mM end concentration to the proteic mixture containing rIFNα-2b coming from example 1 and the mixture is brought to pH 5.5 with acetic acid. The so obtained solution is charged on a strong cationic exchange column containing the commercially available chromatographic matrix Mustang® S (Pall Corporate), a strong cation exchange medium consisting of a hydrophilic polymer with sulfonic acid groups crosslinked on a poly ether sulfone membrane. The cationic exchange column is conditioned, before the charge of the proteic solution, by means of a 20 mM sodium acetate solution at pH 5.5 in amount equal to 20 column volumes (CV).
The proteic solution is then charged at such amount that the 10 mg value of proteins charged for each milliliter of stationary phase is not exceeded, preferably in amounts comprised between 6 and 8 mg/ml.
After the charge, the products bound to the stationary phase are submitted to a first cycle of washing by means of a saline solution at pH 6.1 made by a mixture of monobasic potassium phosphate and dibasic sodium phosphate at an overall concentration comprised between 5 and 15 mM. The optimum concentration of the solution is anyway fixed by the fact that the conductivity has not to exceed about 1800 μS. A total amount of solution comprised between 5 and 60 CV, preferably between 25 and 35 CV, is used.
A second cycle of washing is then carried out by using the same solution of the first cycle of washing to which an amount of potassium chloride is added equal to an end concentration not exceeding 3 mM, preferably 2 mM; a total amount of solution comprised between 10 and 100 CV, preferably between 30 and 60 CV, is used.
After the washing cycles an elution phase is carried out by using a solution like that of the first cycle of washing with an end amount of potassium chloride at a concentration not lower than 10 mM, preferably at a concentration comprised between 15 and 25 mM. An overall amount of solution comprised between 15 and 40 CV, preferably between 20 and 30 CV, is used for the elution.
All the solutions and the sample charged pass through the column at a linear speed comprised between 0.1 and 1 cm/min, preferably between 0.4 and 0.7 cm/min.
Under these conditions rIFNα-2b is eluted from the column with a purity degree higher than 98%, while into the starting solution the purity degree was about 40%, with a yield of recovery of the wanted product higher than 80%.
The chromatographic profiles before and after the chromatographic purification are shown in
The chromatographic profiles have been carried out in HPLC by means of a liquid chromatograph HP 1090, by using a Vydac C18 column and a UV detector set at 214 nm. The elution has been carried out at a 1 ml/min flow by using a mixture made of two eluents, eluent A made of 700 ml of water, 298 ml of acetonitrile and 2 ml of trifluoroacetic acid and eluent B made of 198 ml of water, 800 ml of acetonitrile and 2 ml of trifluoroacetic acid. The two eluents have been mixed during the elution according to the following table:
Time (minutes)
The process is carried out according to the description of example 2 by using a buffer solution made of monobasic potassium phthalate and sodium hydroxide.
The process is carried out according to the description of example 2 by using a buffer solution made of dibasic sodium citrate and sodium hydroxide.
The process is carried out according to the description of example 2 by using a buffer solution made of citric acid and dibasic sodium phosphate.
The process is carried out according to the description of example 2 by using a buffer solution made of imidazole and hydrochloric acid.
Purification of Human Serum Albumin
The human serum albumin (HSA) has been purchased from Sigma (catalogue number A1653 of the 2000 year). The nominal title of this albumin preparation is stated 99.6%, but the RP-HPLC analysis shows a real title equal to 88% if the products albumin-like are considered as impurities.
A HSA solution has been prepared in a 20 mM citric acid solution at pH 3 at an end concentration equal to 1 mg/ml and has been charged on a strong cationic exchange column containing chromatographic matrices Mustangs® S (Pall Corporate) commercially available. The cationic exchange column is conditioned before the charge with a 20 mM citric acid solution at pH 3.0 in amounts equal to 20 column volumes (CV).
The amount of the charged solution is such that the value of 10 mg of charged proteins for each milliliter of stationary phase, preferably amounts comprised between 6 and 8 mg/ml, is not exceeded.
After the charge, the products bound to the stationary phase are submitted to the following cycles of washing:
1. washing cycle—40 CV with a 20 mM solution of sodium acetate at pH 5.5;
2. washing cycle—30 CV with a 20 mM solution of sodium acetate at pH 5.8.
The elution of the wanted product from the column is carried out by means of a saline solution at pH 6.0 made of a mixture of monobasic potassium phosphate and dibasic sodium phosphate at a concentration comprised between 5 and 100 mM depending on the composition of the mixture. However the conductivity of the solution has not to exceed 140 μS. A total amount of solution comprised between 25 and 35 CV is used.
All the solutions and the charged sample pass through the column at a linear speed comprised between 0.1 and 1 cm/min, preferably between 0.4 and 0.7 cm/min.
Under these conditions HSA is eluted from the column with a purity higher than 99% with a yield of recovery of the wanted product higher than 56%.
|Cited Patent
|Filing date
|Publication date
|Feb 26, 1979
|Oct 14, 1980
|Armour Pharmaceutical Company
|Purification of plasma albumin by ion exchange chromatography
|Dec 2, 1986
|Mar 22, 1988
|Biospectrum, Inc.
|Purification method for alpha interferon
|Oct 6, 1987
|Aug 23, 1988
|Interferon Sciences, Inc.
|Purification of monomeric interferon
|Sep 30, 1987
|Aug 6, 1991
|Cetus Corporation
|Pharmaceutical compositions of recombinant interleukin-2 and formulation processes
|Feb 27, 1992
|Oct 5, 1993
|Alpha Therapeutic Corporation
|Albumin purification
|Apr 12, 1993
|Nov 22, 1994
|Stolle Research & Development Corp.
|Large-scale purification of egg immunoglobulin
|Mar 26, 1993
|Jan 3, 1995
|Sclavo S.P.A.
|Process for the isolation of highly purified factors IX, X and II from prothrombin complex or human plasma
|Jul 21, 1994
|Jan 23, 1996
|Merck & Co., Inc.
|Purified herpes simplex virus protease and methods of purification
|Feb 25, 1994
|May 28, 1996
|The Green Cross Corporation
|Recombinant human serum albumin, process for producing the same and pharmaceutical preparation containing the same
|Jun 1, 1995
|Apr 1, 1997
|The Green Cross Corporation
|Process for producing albumin preparation
|Aug 31, 1995
|Oct 5, 1999
|Yoshitomo Pharmaceutical Industries, Ltd.
|Process for purifying recombinant human serum albumin
|Nov 14, 1997
|Mar 7, 2000
|Delta Biotechnology Limited
|High purity albumin
|Mar 6, 1997
|Nov 21, 2000
|Dsm Patents & Trademarks
|Process for the purification of serum albumin
|Oct 31, 1983
|May 31, 1989
|Genentech, Inc.
|Method for removing impurities from leukocyte interferon preparations, and purified leukocyte iterferon produced thereby
|Mar 29, 1995
|Sep 5, 2001
|F. Hoffmann-La Roche AG
|Process for producing alpha-interferon
|Title not available
|"A Simple Method for Estimating Isoelectric Points" by: Lampson, G.P. et al, pp. 374-377, undated.
|"Chromatography of Complex Protein Mixtures" by: Fred E Regnier; Journal of Chromatography, 418 (1987) 115-143 Biomedial Applications.
|"Electrophoretic transfer of proteins across polyacrylamide membranes" by: D.B. Rylatt, M. Napoli, D. Ogle, A. Gilbert, S. Lim, and C.H. Nair; Journal of Chromatography A, 865 (1999) 145-153.
|"Expanded Bed Adsorption of Human Serum Albumin from Very Dense Saccharomyces cerevesiae Suspensions on Fluoride-Modified Zirconia" by: Ashim Mullick, Michael C. Flickinger, Biotechnol. Bioeng, 65(3), pp 282-290 (1999).
|"Immobilized Ni<2+> -IDA Metal Chelating Affinity Membrane Chromatography for Purification of Commercial Human Serum Albumin" by: Yang Li, Jia Ling-yun, Zou Han-fa, Zhnag Yu-kui (National Chromatographic R & A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116011).Chinese Journal of Biotechnology, vol. 16 No. 1 Jan. 2000, pp 74-77.
|"Interferon from 1981 to 1986" by: Sidney Pestka; pp. 3-15 (inclusive), undated.
|"Ion Exchange Chromatography for the Purification of Recombinant Human Alpha-2B Interferon, Comparative Study of Medium and High Resolution Gels" by: Rebeca Bouyón, José R. Hernández, Tania Aleaga, Hector Santana, Miriela Gil, Raudel Sosa, Yai Cruz, Alberto Agraz, and Luis Herrera; Biotechnologia Aplicada 1997; 14:189-192.
|"Ion-Exchange Chromatography of Proteins" by: Shuichi Yamamoto, Kazuhiro Nakanishi, and Ryuichi Matsuno; Chromatographic Science Series, vol. 43 pp. 27-30 and 298-304, undated.
|"Isolation by gel-permeation chromatography of a non-covalent complex of Cibacron Blue F3G-A with human serum albumin" by: Anna Compagnini, Salvatore Fisichella, Salvatore Foti, Giuseppina Maccarrone, Rosaria Saletti; Journal of Chromatography A, 736 (1996) 115-123.
|"Large-Scale Production of Recombinant Proteins: Human Leukocyte Interferon" by: Fazal R. Khan and Vishva R. Rai; Hoffmann-La Roche, Inc., Nutley, NJ 161-169, inclusive, undated.
|"Novel affinity separations based on perfluorocarbon emulsions Use of a perfluorocarbon affinity emulsion for the purification of human serum albumin from blood plasma in a fluidised bed" by: Graham E. McCreath, Howard A. Chase Duncan R. Purvis and Christopher R. Lowe; Journal Chromatography, 597 (1992) 189-196.
|"Production of interferon-alpha in high cell density cultures of recombinant Escherichia coli and its single step purification from refolded inclusion body proteins" by K.R. Babu, S. Swaminathan, S. Marten, N. Khanna, and U. Rinas; Appl. Microbiol Biotechnol (2000) 53:655-660.
|"Purification of Recombinant Human IFN-alpha2" by: David R. Thatcher and Nikos Panayotatos; Purification of Interferons pp. 166-177, undated.
|"Purification of Recombinant Human Serum Albumin Efficient purification using STREAMLINE" by: Akinori Sumi, Kouzou Okuyama, Kaoru Kobayashi, Wataru Ohtani, Takao Ohmura, and Kazumasa Yokoyama; Bioseparation 8: 195-200, 1999.
|"Separation of Proteins of Nearly the Same Size but Having Different Isoelectric Points by Convective Electrophoresis" by: A.K. Kontturi, K. Kontturi, and M. Vuoristo; Acta Chemica Scandinaica, 1996: 50: 102-106.
|"The Role of Protein Structure in Chromatographic Behavior" Article by: Fred E. Regnier (pp. 319-323), 1987.
|Thatcher, Methods of Enzymol. 119, 166-177 (1986).*
|Citing Patent
|Filing date
|Publication date
|Apr 14, 2006
|Jul 8, 2008
|Agilent Technologies, Inc.
|Methods and system for protein separation
|Feb 8, 2005
|Nov 11, 2008
|Agilent Technologies, Inc.
|Methods and systems for protein separation
|May 17, 2011
|Agilent Technologies, Inc
|Methods and systems for on-column protein delipidation
|Feb 8, 2005
|Apr 6, 2006
|Martosella James D
|Methods and systems for protein separation
|Apr 14, 2006
|Aug 24, 2006
|Boyes Barry E
|Methods and system for protein separation
|Jun 22, 2006
|Oct 26, 2006
|Agilent Technologies, Inc.
|Methods and systems for on-column protein delipidation
|Dec 10, 2008
|Oct 14, 2010
|Yves Durocher
|Production of Recombinant Interferon Proteins
|U.S. Classification
|210/635, 424/85.5, 530/351, 530/416, 210/656, 435/811, 424/85.6, 530/413, 424/85.4, 424/85.7
|International Classification
|Cooperative Classification
|European Classification
|Jun 10, 2002
Owner name: ALFA WASSERMANN S.P.A., ITALY
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:SCAPOL, LUCIA;VISCOMI, GIUSEPPE CLAUDIO;REEL/FRAME:013006/0910
Effective date: 20020520
|Sep 2, 2008
|Fee payment
Year of fee payment: 4
|Oct 29, 2012
|Maintenance fee reminder mailed
|Mar 15, 2013
|Lapse for failure to pay maintenance fees
|May 7, 2013
|Expired due to failure to pay maintenance fee
Effective date: 20130315

subset: commoncrawl


text:
- Miss Florida 2017 stripped of crown days after win - US Weekly
- Lindsay Lohan steps out amid relationship drama - TMZ
- Was Taylor Swift snubbed by the VMAs? - Gossip Cop
- Rachel Platten sings "Fight Song" with lots of stars - Just Jared Jr
- Farrah Abraham sizzles in a revealing bikini - Radar
- New pics from the Clooneys' Italian summer - Lainey Gossip
- Fox News snubs major DNC moment - The Hollywood Reporter
26 November 2012 at 8:24 AM
Ryan Reynolds: 'R.I.P.D.' Reshoots with Mary Louise Parker!
About this photo set: Ryan Reynolds is dapper while filming reshoots for his upcoming movie R.I.P.D. with his paper bag carrying co-star Mary-Louise Parker on Sunday (November 25) in… Read More Here
Photo #: 2763812
Posted to Jeff Bridges, Mary Louise Parker, Ryan Reynolds
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(Mackinac Center Senior Legal Analyst Patrick J. Wright discussed the issue of eminent domain in an Oct. 4, 2005, interview on The Jack Lessenberry Show. Listen to a three-minute segment of the interview by clicking the audio download above or listen to the entire interview by visiting The Jack Lessenberry Show's Web site. This clip is posted with permission from the Jack Lessenberry Show and cannot be edited or reposted without the show's consent.)
Patrick J. Wright is senior legal analyst for the Mackinac Center for Public Policy, a research and educational institute headquartered in Midland, Mich.
Stay Engaged
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Hydroponic Systems
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This item has been shown times.
100% Customer Satisfaction Guarantee
To Be As Described Or Your Money Back!
Antique Vietnamese 15C. Hoi An Hoard Blue & White Porcelain Bottle Vase #1370
All of the items listed this week are from a single largeestate that we will be listing over time.
Please only offer if you are going to follow though with purchasing the item, non paying buyers will be reported to and blocked for future business... Items paid by PayPal are claimed for the full amount in customs.Items worth over $2500.00 will ship DHLfor full amount. All items must be shipped to the registered PayPal address, NO EXCEPTIONS! Also, we do combine shipping for multiple purchases.
Description : Antique 15C Vietnamese Ming Dynasty Hoi An Hoard blue & white porcelain shipwreck bottle vase.
Size : Measures 12 1/4" Tall by 5 3/4" Wide
Age : 15C.
Click here for Age/ Date Chart
Condition : Large chip on rim with small pieces reglued to rim, otherwise good condition
offer with confidence,our .99 cent salesare guaranteed as described!
Please read the very bottom of ad to see if any additional information was added during the course of the week prior to placing your final offer.We do combine shipping on multiple purchases.I will not take offers for Buy It Now, sale will run 10 day.I do not fraud customs forms.
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Draw bar, This was on 1957 Ford Tractor Work master, Pto Cap, repair manual and Spud bar,
Dodge 6 Lug 15" Wheel, this is a Alloy Rim for Durango and Dakota Trucks Pickup and SUV
Trades Considered? Mower, IPad, Air Compressor, Toolboxes, Workbench, 1911 Kahr S&W Trailers, Rims and?
Jun 20 -
Dayton :
Tipp City - Farm equipment
2001 Ford Excursion Limousine (White)
24 Passenger (240 inch stretch)
Current Mileage: 108,398 (still being used in businesses fleet)
Heated Mirrors
Privacy Divider
Electric Seats (driver/passenger up front)
Leather Seating
2 TV's - VCR&DVD
AM/FM Stereo with CD player
Jacks for MP3 players, ipods, iphones & ipads
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Ridgeline Gear, a direct dealer for the new line Goal Zero. Goal Zero manufactures compact batteries, laptop chargers, and even small silent generators. They provide various DC attachments for lighting allowing you to maximize your power without AC conversion. A full line of panels from compact Nomad 7 to mountable Boulder 30 and 90 watt panels are available to charge/power your power ...
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Nuix - Conshohocken, PA
Design and develop technical documentation using established standards; Improve documentation by evaluating internal process tools and interviewing technical...
Retention Sales Representative- Account Management $65-75K
Nutrisystem - Fort Washington, PA
Answer incoming calls or make out bound calls to customers who wish to cancel their program. This position receives program cancellation requests via inbound...
Auto Sales Representatives
Sussman Automotive - Jenkintown, PA
We hire sales people with or without automobile sales experience. Honda, Acura, Hyundai, Mazda, Kia 1940 Jenkintown RdJenkintown, PA 19046....
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BURDUMY MOTORS INC - Huntingdon Valley, PA
Required license or certification:. Valid Driver's license with no major violations within the past 3 years....
Inside Sales Representative
LRP Publications - Horsham, PA
We dissect the what, why and how of federal employment law to give our customers the guidance and tools they need but in the format they want....
Backroom Team Member
TARGET - Philadelphia, PA
Keep merchandise organized and accessible. Work as part of a fast, fun and friendly Backroom team. Maintain a clean, great-looking store. Quickly and...
Business Banking Relationship Manager
Santander Bank - Philadelphia, PA
Participates in joint calls with business partners to develop customer relationships and support specific product sales across business lines....
Assistant Fresh Beverage Development Manager
Wawa - Media, PA
Responsible for the Roasted Coffee Analysis, by systematically evaluating the aroma and taste of coffee beans for every production run, every sku....
Outside Sales Representative/Project Estimator
S&S Electrical Services, Inc - Warrington, PA
Required license or certification:. As we are an electrical contractor some electrical knowledge is a plus, though not mandatory....
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Cristo Rey Philadelphia High School - Philadelphia, PA
Process the financial aid application process with incoming Freshmen families. Process cash receipts. _" … to nurture and challenge young women and men to...


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Battle Dawn
Battle Dawn is a strategy game that plays differently than most browser games of the same genre. With a large map of either Earth, Mars, or a fantasy realm serving as the backdrop, players create colonies, set up outposts and vie for control of ancient relics. Alliances must be formed and resources extracted to muster enough troops to capture all 10 relics and declaring victory, causing a new round to begin. There are four key resources in each of the three settings and the map's geographically is generally limited to an aesthetic role; players receive no penalties for placing their colony in Antarctica nor any benefits for choosing a more fertile land.
- Status: Released
- Release Date: Jan 06, 2006
- Business Model: Free to Play
- Developer: Tacticsoft
- Publisher: Tacticsoft
- Category: Browser
- Genre(s): 2D, Strategy
- Platforms: PC, Mac, Linux
- +Three unique settings.
- +World map easily visible.
- +Optional, rewarding tutorial.
- +Customize avatar, flag, profile.
- -Servers reset, no permanent worlds.
- -Paying players receive large benefits.
- -Slow gameplay & progression.
- -Geographic location has limited effect on gameplay.
- Requires up to date Flash player.
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Plano Newswire
Comprehensive Real-Time News Feed for Plano, TX.
Results 1 - 20 of 2,194 in Plano, TX
With Shipt, Central Market reaches customers in Cedar Hill, McKinney and more w/Photo
... said Tuesday. In North Texas, Shipt said it will expand its current delivery area of Dallas, Carrollton, Garland and Plano to include Cedar Hill, DeSoto and McKinney. Shipt will also deliver groceries to Arlington, Grapevine, Southlake and Keller. ... Comment?
All Points: Overloaded police? w/Photo
Members of an honor guard carry the flag draped casket holding slain Dallas Police officer Michael Krol as Dallas Police Chief David Brown, left, looks on with Dallas Mayor Mike Rawlings, second from left, and Texas Lt. Gov. Dan Patrick after a funeral service at Prestonwood Baptist Church in Plano, Texas, Friday, July 15, 2016. Comment?
The STEM Education Challenge w/Photo
... new scholarship program that will help students earn STEM-related degrees. Masergy engineers have also volunteered in Plano, TX area schools. With a challenge this big, this stubborn, and this important, we all need to do our part. A former member ... Comment?
Plano poised to spend $12 million fast-tracking Cotton Belt rail... w/Photo
The Cotton Belt Railroad Line runs through the southern half of the city of Coppell as it connects Plano to DFW International. The Plano City Council last night reportedly agreed to spend $12 million in hopes of fast-tracking rail service on the Cotton Belt Line. Comment?
Torchlight Energy Meets NASDAQ Listing Requirements w/Photo
... consecutive trading day when the closing bid price of the Company's common stock traded over $1.00. : TRCH ), based in Plano, Texas, is a high growth oil and gas Exploration and Production (E&P) company with a primary focus on acquisition and ... Comment?
Collin-County 18 mins ago 12:23 a.m.New plans for Cotton Belt Corridor bought by DART w/Photo
... Worth International Airport, provided the cities it runs through can help pay for the line. It was an agenda item at Plano City Hall for city councilmembers on Monday. Plano's city manager Bruce Glasscock says the line is imperative to solving ... Comment?
Chuck JacksonChuck loved drag racing and his muscle cars. He was 69. Today at
... children Julie (Shelby) Kibler, of Grand Rapids, MI, Robert Jackson, of Wakeman, a brother James (Barbara) Jackson, of Plano, TX, two grandchildren, Alaina and Esau, two nieces and one nephew. Friends may call on Saturday July 30 from 2:00 pm until ... Comment?
Man arrested after leading police on chase from Plano to Seagoville w/Photo
Officers responded to a suspicious vehicle about 8:30 p.m. in the 900 block of Goodwin Drive. When they arrived, the van took off, police spokesman David Tilley said. Comment?
Past Pros Takes Show on the Road w/Photo
... sessions with former athletes, often where they live. Now, Greer is taking the players on the road. On August 13, the Plano Card Show will be hosting the first ever PastPros Road Show at the Comfort Inn and Suites Plano East (700 E Central Parkway, ... Comment?
Major Ken Paxton donor paying multi-million settlement after Medicaid fraud probe w/Photo
... felony fraud indictments. Webb has also given heavily to other North Texas Republicans, including Reps. Jeff Leach of Plano and Scott Turner of Frisco and House candidates John Keating and Thomas McNutt, both of whom lost primary races this year. ... Comment?
... easy guest access, and paired with existing hotel mobile offerings. OpenKey is a privately held company located in Plano, Texas. The app is available for both iOS and Android devices. For more information, please visit www.openkey.co . The Ashton ... Comment?
Pendrell Just Had A Great Quarter And Remains A Buy w/Photo
... came in at only 41% of what they were in the first half of last year. The termination of the company's office lease in Plano, TX at the end of June shows that these efforts are continuing. Pendrell has made clear in filings that its "primary focus ... Comment?
Funerals to begin for police officers shot dead in Dallas w/Photo
... Sergeant Michael Smith and Dallas Area Rapid Transit Officer Brent Thompson. Mr Ahrens' funeral will take place in Plano, Texas. Mr Smith's funeral at the Farmers Branch church he and his family attended will be followed on Thursday with a service ... Comment?
NTS Adds Reverberation Chamber Test Capabilities at Five US Laboratories
... best method to achieve their goals." The reverberation chambers have been added to NTS laboratories in Boxborough, MA; Plano, TX; Tempe, AZ; and Fullerton, CA. NTS Rockford, IL is adding a second reverberation chamber. These copper-lined chambers ... Comment?
Why Sustainable Manufacturing Facilities Are Green Building's Next Big Thing
... Just last month Toyota said it is seeking LEED Platinum certification for it's new North American headquarters in Plano, Texas. The $1 billion campus, slated to open in May 2017, will include a 7.7-megawatt solar array and will use 100 percent ... Comment?
MOOYAH Burgers, Fries & Shakes Launches Seriously Fun Redesigned Consumer Website w/Photo
... made-to-order burgers, French fries hand cut from Idaho potatoes, and real ice cream shakes. Founded in 2007, the Plano, Texas-based company serves fresh, lean American beef, all-natural Jennie-O turkey burgers, black bean veggie burgers, buns baked ... Comment?
Slim Chickens Continues Expansion in the Lone Star State, Brings Better Chicken to Plano w/Photo
Slim Chickens, a leader in the "better chicken" segment of fast-casual restaurants, will be bringing fresh chicken tenders and wings with unrivaled flavor to Plano, Texas. Restaurant development and operations veterans Courtney Lindley and Bob Gregg have assembled a team to develop 15 locations within the next eight years across North Texas. Comment?
... Palestine. Jerry is survived by his wife of nearly 60 years, Linnie (Davis); two sons, Jerry Jr. (Janeen) Foreman of Plano, Texas, and Michael (Shirley) Foreman of East Palestine; two brothers, Robert (Norma) Foreman of East Palestine and John ... Comment?
Jerry C. Foreman, of SR 154 Lisbon, passed away July 23, 2016 at Salem Regional Medical Center after a battle with cancer. He is survived by his wife Linnie ; two sons, Jerry Foreman Jr. of Plano Texas., Michael Foreman of East Palestine; two brothers, Robert Foreman of East Palestine, John Foreman of San Diego, Calif.; five grandchildren; four great grandchildren; many step grandchildren and great grandchildren. Comment?
Pacific Northwest golf: 50th Pacific Coast Amateur set for Seattle Golf Club w/Photo
... year ago. Not to be forgotten as an early favorite is two-time Trans-Miss and U.S. Junior champion Will Zalatoris of Plano, Tex. Playing in the Pacific Coast Amateur for the second consecutive year, the Wake Forest standout ranked 13th by Scratch ... Comment?
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Escape from Auschwitz: told for first time in EnglishBreaking News
Alfred Wetzler, a Slovak Jew, was one of the tiny number of people to escape from Auschwitz-Birkenau, Europe's heart of darkness during the Second World War, where an estimated 1.1 million Jews arrived of whom scarcely 7,000 survived the onslaught of the Nazis.
Wetzler and his companion Rudolf Vrba, also a Slovak Jew, were arrested by the Nazis in 1942 in Slovakia and sent to the death camp for slave labour. Wetzler was 24 and Vrba was just 18. Wetzler spent two years in the camp, witnessing some of the worst atrocities known to man. It was this experience that founded the basis for the memoirs he would later write: "It is incredible how tough human life can be, how quickly a person, even with a broken arm, a dislocated foot, a broken head and bitten by dogs, will do what is asked of him when over him hangs the cudgel waved by the goodwill of the Reich."
It was in the spring of 1944, with the assistance of other prisoners, that the pair managed to escape, initially by hiding under a huge woodpile for four days in the corner of the camp until the search for them was called off. The duo then escaped through a hole under a fence at nightfall. But it was what they did afterwards that was truly heroic. The two men had also smuggled out damning evidence - a ground plan of the camp, construction details of the gas chambers, crematoriums and, most convincingly, a label from a canister of Zyklon gas...
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Crazy Substitute Teachers
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Vacuum Salesman
This dude and his Megazord vacuum cleaner4.00 / 5.00 2,637 Views
Markiplier Animated | Mark Of The Survey Corps
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R | 162 min | Drama, Romance, War
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Writers: Michael Ondaatje, Anthony Minghella | Director: Anthony Minghella | Stars: Ralph Fiennes, Juliette Binoche, Willem Dafoe, Kristin Scott Thomas
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45. The Devil's Advocate (1997)
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48. Scary Movie 2 (2001)
R | 83 min | Comedy
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49. Some Like It Hot (1959)
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Writers: Billy Wilder, I.A.L. Diamond, Robert Thoeren, Michael Logan | Director: Billy Wilder | Stars: Marilyn Monroe, Tony Curtis, Jack Lemmon, George Raft
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50. True Lies (1994)
R | 141 min | Action, Comedy, Thriller
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Votes: 189,754 | Gross: $146.28M


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Rayman Legends (PlayStation Vita)
Rayman Legends is a great little 2D platform game that you can just dive in and play straight away
- Imaginative 2D art style and presentation
- Well balanced platforming gameplay.
- Some of the motion based mini-games are a bit iffy
- Inclusion of levels from Rayman Origins have limited appeal to seasoned fans
Bottom Line
Rayman Legends successfully recaptures the charm and wit of its predecessor. The traditional 2D platforming may not be for everyone, but fans will get a kick out of it.
Price$ 48.00 (AUD)
Best Deals (Selling at 1 store)
- Beyond the Bottom Line by Paula M. Rayman 55.13
Rayman Origins was one of the more imaginative titles to come out on the PlayStation Vita on launch, even though it was not an exclusive but a multiplatform title. That game received praise for its unique visuals and well-tuned 2D platforming gameplay, and the expectation is that the sequel, Rayman Legends, will do the same.
Originally announced as an exclusive for the Wii U, Ubisoft made the welcome decision to bring Rayman Legends to other platforms such as the PlayStation Vita, packing in the same gameplay and presentation to be found in the console version in a handheld package. This is the version we're looking at here.
Back to the Glade
Rayman Legends harks back to the old days of platforming where the focus was entirely on the gameplay. That means the story is more or less non-existent. All you need to know is that the game world, populated by limbless hero Rayman and his team of Globox pals, is called The Glade of Dreams. The core of the game consists of navigating the themed environments and defeating enemies, as well as collecting Lums and Teensies. The former are blue humanoid characters scattered throughout levels, and are used to unlock new levels in the game. Teensies are small pixels that are gathered to unlock medals, as well as scratch-it cards to unlock in-game bonuses.
Without any story exposition in between levels, you are able to dive in and play one level after the other. As you unlock more new characters, they can be selected and used in levels for a slightly different platforming experience. The look and feel of Rayman Legends is mostly unchanged from the earlier title, which is not such a bad thing considering how fun Rayman Origins was. What the game does have is a more varied and imaginative look to the game worlds, which are overflowing with activity. Bosses have also been upgraded to polygon models, which mesh surprisingly well with the game's 2D presentation.
Retooled modes
Some gameplay modes, such as flying levels on a mosquito, have been replaced with other types. One mode, with touch based gameplay reminiscent of Cut the Rope, requires you to remove obstacles as a Globox navigates the level. This mode is well implemented and makes good use of the Vita's touch functionality. When it comes to rescuing certain Lums, the player also has to use the Vita's motion controls to navigate the Globox through a maze-like level. The occasional imprecise nature of the handheld's motion sensor in these levels means that you will have to attempt it more than once.
As a bonus, Ubisoft has included the levels from Rayman Origins into the game, which aid in collecting additional Lums to unlock levels in the main game. Those who have already played Rayman Origins won't find much value in replaying these levels, but it's a nice bonus for those who didn't have a chance to play the first game.
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Slab Murphy has no chance of bail - judgeProminent republican Thomas 'Slab' Murphy would not "come close" to meeting the threshold required for bail pending an appeal, his lawyers have been told. Prominent republican Thomas 'Slab' Murphy would not "come close" to meeting the threshold ... Irish Independent, 22 hours ago
Slab Murphy not remotely close to bail threshold , judge says - Irish Times, 1 day ago
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Senate Elections: Patrick Murphy
Patrick Murphy Age: 33 Hometown: Miami Party affiliation: Democrat 1. Why are you running for U.S. Senate? Florida deserves a Senator who will show up to work, lead on the issues that matter, and will always put Floridians above their ...WJCT, 9 hours ago Obama Says Patrick Murphy Is A Strong Progressor He Can Count On (Video) Before It's News, 1 day ago Obama Tapes Television Ad for Murphy's Senate Campaign ABC News, 3 days ago Obama, Gabby Giffords Rally Democrats for Patrick Murphy Equilibrio Informativo, 2 days ago
The Packers are opening a concession stand dedicated to bacon
During the Green Bay Packers shareholders meeting on Thursday, team president Mark Murphy announced that the team would but more importantly: BACON. According to ESPN's Jason Wilde, the shareholders went nuts for marketing chairman Mike Weller's ...FOXNews.com, 1 day ago NFL to talk with 2 Packers over PED allegation ESPN.co.uk, 2 days ago Brett Favre Goes To Camp ESPN.com, 2 days ago Free agency could break up Packers' offensive line after this season ESPN.com, 1 day ago
Mount Sinai Researchers Identify Way to Predict and Prevent Damage in Donated Kidneys
A multicenter team of researchers led by Barbara Murphy, MD, of the Icahn School of Medicine at Mount Sinai has identified a panel of genes which can help predict whether a transplanted kidney will later develop fibrosis, an injury which can cause ...Infrosoft, 1 day ago Gene Studies May Forecast Success Of Kidney Transplant Surgical Products, 1 day ago Scientists discover group of genes that can help predict damage in transplanted kidney News-Medical.Net, 1 day ago
Spectrum music reviews: Roisin Murphy, Julie Ruin and Hans Hotter
The Julie Ruin HIT RESET (Hardly Art/Inertia) "I can play electric guitar while shaving my legs in a moving car," declares Kathleen Hanna in Hello Trust No One. It's a boast that's funny and righteous. With The Julie Ruin, her five-piece New ...Melbourne Age, 1 day ago
Good 401(K) Fiduciary Practices Are As Simple As 1-2-3
Last week Philip Murphy of Dow Indexes posted How Benchmark Selection Can Support A Retirement Plan's IPS , outlining the importance of having a formal Investment Policy Statement, and what that IPS should say. Most plan sponsors create an IPS for ...Seeking Alpha, 1 day ago
Greg Murphy hoping to abdicate as King of Pukekohe
Greg Murphy hugs a team member after winning at Pukekohe in 2002. New Zealand motor racing legendGreg Murphy hopes another Kiwi will soon take his crown as the King of Pukekohe. The launch of this year's Auckland Supercars Sprint took place ...The Press NZ, 5 days ago Watch: Daniel Murphy hits game-tying home run vs. Pirates SI.com, 6 days ago Murphy to the rescue for red-hot Nats ESPN.com, 6 days ago Daniel Murphy scratched from Nationals lineup vs. Pirates Washington Post, 1 week ago
Daniel Murphy remains sidelined Sunday
Updating a previous report, Washington Nationals 2B Daniel Murphy (hamstring) is not in the starting lineup against Pittsburgh Pirates SP Chad Kuhl Sunday, July 17. 1B Clint Robinson will bat fifth and play first base, while INF Stephen Drew will ...USA Today Fantasy Sports, 6 days ago Daniel Murphy likely for 1B Sunday USA Today Fantasy Sports, 6 days ago Daniel Murphy again out of Nationals lineup with sore hamstring Washington Post, 1 week ago Nationals' Daniel Murphy sat out Friday's game with left hamstring tightness Washington Post, 1 week ago
Starling Marte's home run trumps Daniel Murphy's in Nationals 2-1 loss
, The longest regular season game in Washington Nationals history grew out of one of the more uneventful ones they have played all season, a near-shutout loss transformed into a grueling 2-1, 18-inning loss Sunday to the Pittsburgh Pirates. The ...Washington Post, 6 days ago Daniel Murphy a late scratch from Washington Nationals lineup Washington Times, 1 week ago Daniel Murphy scratched USA Today Fantasy Sports, 1 week ago Nationals scratch Murphy with general leg soreness Equilibrio Informativo, 1 week ago
Del Taco Restaurants' (TACO) CEO Paul Murphy on Q2 2016 Results - Earnings Call Transcript
Del Taco Restaurants, Inc. (NASDAQ: TACO ) Q2 2016 Earnings Conference Call July 20, 2016, 05:00 PM ET Executives Raphael Gross - IR Paul Murphy - President and CEO John Cappasola - EVP and CBO Steven Brake - EVP and CFO ...Seeking Alpha, 2 days ago LivePerson to Announce Second Quarter 2016 Financial Results on July 27, 2016 TMC Net, 4 hours ago Gentex's (GNTX) on Q2 2016 Results - Earnings Call Transcript Seeking Alpha, 1 day ago
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