text:
|Publication number
|Publication type
|Application number
|Publication date
|Feb 8, 1994
|Filing date
|Jul 23, 1991
|Priority date
|Jul 25, 1990
|Fee status
|Also published as
|Publication number
|Otello Protasi, Donatella Mannucci
|Original Assignee
|Sclavo S.P.A.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (11), Non-Patent Citations (1), Classifications (14), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
The present invention relates to a process for the extraction and purification of human gamma-interferon from host cells transformed with recombinant DNA techniques and which produce the same in insoluble form. The present invention also relates to the use of the same for manufacturing pharmaceutical compositions suitable as antivirals, antitumorals and immunomodulators.
The interferons, briefly designated IFN, are glycoprotein substances which are produced by different animal or human cells (fibroblasts, leucocytes and lymphocytes) as a response to viral and nonviral agents during the first phases of an infection.
At present at least three types of interferon have been identified and characterized (alpha, beta, gamma), which differ from each other both for antigenic properties and biological activity.
In particular the human gamma-interferon (HuIFN-gamma) is an acid labile glycoprotein with molecular weight about 34,000 daltons and produced by leucocytes.
HuIFN-gamma exhibits, in addition to an antiviral and immunomodulator activity, an antitumor activity capable of inducing an in vivo regression of a tumor.
Therefore HuIFN-gamma is particularly suitable as an active agent for manufacturing pharmaceutical compositions useful as antivirals, antitumorals and immunomodulators. There is described in the prior art the production of human gamma-interferon in vitro by leucocytes in culture or by fermentation methods of host cells transformed with recombinant DNA techniques (see for example the EP-A-88 540).
Said methods, though enabling an improved yield in polypeptide production with regard to the in vitro techniques, are not free of important purification problems.
In fact the synthesis of redundant heterologous proteins or polypeptides, where heterologous indicates compound not naturally produced by the host cells, may affect the viability of the cells. Therefore sometimes the host organism provides a biological mechanism (proteolytic degradation or secretion) which allows the host cell to prevent interactions of the heterologous protein with the essential metabolic processes of the cell.
As a further option, for the product of heterologous gene expression may be produced in an altered conformation which limits its interference with the normal cellular pathways. In fact such heterologous products have been often observed in host organisms like E. coli or S. cerevisiae to be produced in non-native forms and to precipitate within the cells, forming protein aggregates known as "inclusion bodies" or "refractory bodies." Generally a process for isolating and purifying recombinant products (proteins or polypeptides) giving rise to insoluble aggregates within the host cells comprises disrupting said cells by means of conventional techniques to extract said aggregates, separating the cellular fragments from the supernatant containing said aggregates (which form a pellet) by filtration or centrifugation, solubilizing the pellet and purifying the solubilized recombinant product.
Methods for the purification of insoluble recombinant products, in particular of recombinant human gamma-interferon are known in the prior technique, which are essentially based on the use of denaturing agents like urea or guanidinium chloride for solubilizing the protein aggregates and on the use of substances intended to inhibit the endocellular proteases, activated during the breakage of the cell, to avoid the proteolytic degradation of the recombinant products.
By way of example of U.S. Pat. Nos. 4,511,502 and 4,620,948 disclose processes for the purification of insoluble heterologous proteins inter alia gamma-interferon, wherein the aggregate solubilization is carried out by means of a denaturing agent, preferably 9M guanidinium chloride or thiocyanate salts in presence of a detergent (0.01-0.02%) or of a reducing agent like β-mercaptoethanol.
Said methods however exhibit the following drawbacks:
production of soluble denatured products, thus products having a folding and a biological activity considerably different from those of the native products, which therefore need to be reactivated before the use;
incomplete reactivation of the denatured products by means of known techniques such as e.g. that disclosed in the U.S. Pat. No. 4,599,197;
activation of the endocellular proteases during the cell disruption phase;
unsatisfactory production yields in solubilized product (about 30-40%) due to a partial reaggregation during the chromatographic purification, the eluting being carried out in absence of denaturing agents.
An improvement in yield may be obtained by applying in the final elution a denaturing agent; however the resulting product would not be suitable for use as such because of the presence of said denaturing agent.
In order to prevent the enzymatic degradation, the U.S. Pat. No. 4,681,930 describes a method for the purification of recombinant gamma-interferon based on the use of cellular enzymatic protease inhibitors.
The use of such inhibitors, either alone or in combination with a solubilizing agent, does not allow the recombinant products with the desired features to be obtained.
In fact, though the endocellular protease catalyzed enzymatic degradation of the polypeptide is inhibited, an insoluble denatured product is nevertheless obtained.
For such reasons there are proposed in the prior art other purification methods for insoluble human gamma-interferon isolated from recombinant cells.
Said methods are based on the use of zinc or copper salts or polyethylenimine salts (WO 86/04067) or on the sequential removal of nucleic acids, of contaminating proteins and other contaminating substances of different molecular weights by means of different chromatographic techniques (U.S. Pat. No. 4,751,078). All of those cited methods are not yet fully satisfactory either because of the low production yields (WO 86/04067) or because of the number of necessary stages (U.S. Pat. No. 4,751,078).
In conclusion, despite of the great deal of performed works, no system turned out to be suitable for the preparation of a product satisfactory for a therapeutic application and, at the same time, convenient to allow an industrial scale up.
It has now been found that the shortcomings of the prior art may be avoided with a process based essentially on the use of nondenaturing agents for the solubilization of the pellet comprising the insoluble interferon-gamma.
Such a process enables the production of interferon-gamma, with unimpaired biological activity and unchanged chemical-physical properties, in high yield (65-75%) and purity, expressed as specific activity, of 1�108 International Units per mg protein (IU/mg). Therefore an object of the present invention is a process for the extraction and purification of human interferon-gamma from recombinant host cells producing the same in insoluble form. A further object of the present invention is an essentially pure polypeptide having biological activity and chemical-physical properties of the native human gamma-interferon obtained with the aforementioned process.
Still further objects of the present invention are pharmaceutical compositions suitable as antivirals, antitumorals and immunomodulators comprising a therapeutically effective amount of human gamma-interferon obtained with the process of the present invention.
Other objects of the present invention will be evident from the description and examples which follow.
In detail, the extraction and purification process of the present invention comprises:
a) suspending the recombinant host cells which produce human gamma-interferon in the form of insoluble aggregates in a buffer solution having pH comprised between 6.0 and 8.5, optionally complemented with an osmoprotector and disrupting the cells in conditions which do not activate the endocellular proteases;
b) separating the suspension of protein aggregates (pellet) which contain gamma-interferon from the cell debris produced in stage (a);
c) separating the pellet containing the insoluble recombinant human gamma-interferon from the supernatant;
d) solubilizing the recombinant human gamma-interferon by one or more extractions of the pellet with phosphate buffer having pH comprised between 6.5 and 8.5;
e) purifying gamma-interferon by means of one or more chromatographic techniques.
As for the gamma-interferon to be purified, it can be obtained from host cells, such as Escherichia coli or Saccharomyces cerevisiae, transformed with an expression vector containing the gene encoding the gamma-IFN as disclosed by e.g. E. Erhard and C. P. Hollenberg, (1983), Journal of Bacteriology, 156:625-635).
The cells recovered from the culture medium by means of centrifugation or filtration, are broken applying conventional techniques, e.g. osmotic shock, sonication, milling or chemical agents, in order to isolate the insoluble aggregates.
According to a preferred embodiment of the present invention, the cells are disrupted by homogenization in a bead-disrupter mill using commercial equipment.
In particular the cells are repeatedly washed with at least 30 volumes of buffer having a pH from 6.0 to 8.5. Thereafter they are suspended in the same washing buffer, optionally inpresence of an osmoprotector such as e.g. sorbitol at a concentration (weight/volume) comprised between 20% and 30%, preferably between 23% and 28%.
Buffers suitable for the purpose of the present invention are for instance Tris-HCl and phosphate buffer SORENSEN.
According to the present invention the homogenization is carried out in conditions suitable to obtain a high rate of cell breakage (98-99%), which is necessary to release the HuIFN-gamma-containing protein aggregates, without however collapsing the lysosomal vacuoles containing the enzymatic proteases. Disruption of the lysosomes would cause proteolytic degradation of the HuIFN-gamma. The homogenization is preferably carried out using lead-free glass beads having diameter from 0.25 to 0.5 mm with a rotation speed of the dishes from 2,000 to 4,000 revolutions per minute (rpm), preferably from 2,700 to 3,500, more preferably the speed is 3,000 rpm, i.e. 10 meters/second, for a period of time for 45 seconds to 2 minutes, preferably for one or about one minute. In fact, experimentation concerning the length of the homogenization time shows, see Table 1, that one minute homogenization is suitable to obtain a high percent rate of cell breakage, while avoiding the proteolysis problems which arise from prolonged homogenizations.
Finally the homogenate is recovered and added to the washes of the milling vessel made with the same buffer. The homogenate is then decanted and centrifuged first at a speed between 2,000 and 5,000 rpm, preferably between 3,500 and 4,500 rpm, with the purpose of separating the suspension of the gamma-interferon- comprising protein aggregates (pellet) from the cell debris and from the remaining glass beads and following at a speed about 100,000 rpm to sediment the aggregates which are thereafter removed from the supernatant. The so obtained pellet is repeatedly washed with the same homogenization buffer to completely eliminate the still present lipidic residues; said residues in fact could give rise to clarity problems in the following extractions, and cause obstruction of the adaptor membranes of the chromatographic column with resulting flow hindrance. The pellet is thereafter solubilized upon one or more extractions with phosphate buffer of pH comprised between 6.4 and 8.5, preferably 7.5 and 8.0, using a pellet/buffer ratio (weight/volume) from 2 to 20% preferably from 5 to 10%.
The extraction is accomplished at temperature comprised from 0� C. to 25� C., preferably from 4� C. for a period of 10 to 30 hours, preferably 15 to 20 hours. Buffers suitable for the purposes of the present invention are Tris-HCl and, preferably phosphate buffer SORENSEN prepared by mixing different amounts of the solutions A and B comprising respectively KH2 PO4, 9.08 g/l and Na2 HPO4 x2H2 O), 11.88 g/l. The purification of recombinant human gamma-interferon solubilized according to the process of the present invention may be achieved with one or more conventional chromatographic techniques, such as, e.g. ion exchange chromatography and/or hydrophobic chromatography. Preferably said purification is carried out with ion exchange chromatography. It was in fact observed that the isoelectric point (PI) of the recombinant human gamma interferon, solubilized according to the process of the present invention has a value lower than 8 unlike the product obtained with denaturing agents which has a PI>8. The combined mechanical disruption of the cells (homogenization) under the aforementioned conditions with the phosphate buffer, pH 6.5-8.5 extraction, enable a non-denatured, non-degraded product to be obtained in high final yield and purity, expressed as specific activity, of about 1�108 International Units/mg (IU/mg).
The experimental examples which follow are intended to illustrate, but not however to limit, the present invention.
S. cerevisiae cells capable of expressing recombinant human gamma-interferon in insoluble form and recovered from a fermentation run in a bioreacter, are washed twice, 5 minutes/wash, with 30 volumes phosphate buffer SORENSEN, pH 6.8 complemented with 1M sorbitol as an osmoprotector.
The cells (about 17 g wet biomass, corresponding to from 4.16�109 cells/ml) are suspended in 64 ml SORENSEN buffer, pH 6.8 complemented with sorbitol and transferred into a DYNO-MILL homogenizer type KLD equipped for discontinuous running as follows:
unleaded glass milling vessel, 0.15 liters;
VULCOLLAN stirring discs (64 mm diameter);
cooling by means of a refrigerator block (rectified glycol) for thermostated temperature within the milling vessel at 8� C.;
unleaded glass beads of 0.25-0.50 mm diameter in amount of 125 ml;
rotation speed of the disks 3,000 rpm, i.e. 10 meter/second.
The cell breakage rate and the corresponding enzymatic degradation are determined, at different homogenization times, by cell count per milliliter and by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis (SDS PAGE) of the proteins present in the supernatant after separation of the products of the cell lysis.
The results are reported in Table 1
TABLE 1______________________________________Homogen. yeast cell/mltime cell/ml after homog. % breakage______________________________________45" 7.02 � 109 1.05 � 109 85.01' 7.66 � 109 1.20 � 108 98.41' 30" 4.16 � 109 4.58 � 106 98.82' 5.07 � 109 1.12 � 107 99.7______________________________________
From such figures it can be appreciated that 1 minute homogenization is sufficient to obtain a high rate of cell breakage, while avoiding the proteolysis drawbacks raised by longer homogenization times.
S. cerevisiae cells are homogenized for one minute as described in example 1. The homogenate and the washes with SORENSEN buffer of the milling vessel are recovered and transferred into a glass vessel and decanted to separate the glass beads.
Thereafter the suspension of the protein aggregates which comprise the gamma-interferon is separated from the cell debris and from the still remaining glass beads by centrifugation at 4� C. for 10 minutes at 4,000 rpm with a Heraeus Mini Fuge T centrifuge.
The obtained suspension is further centrifuged at 4� C. for 60 minutes (Beckman L8-M centrifuge 100,000 rpm) to sediment the pellet of protein aggregates.
Thereafter the pellet is separated from the supernatant by decanting and washed twice with about 10 ml phosphate buffer SORENSEN, pH 6.8 to completely eliminate the lipidic residues present upon the pellet and the walls of the centrifuge tube. The aggregates containing the gamma-interferon are then solubilized by extraction of the pellet in phosphate buffer SORENSEN, at varying pH values. The extraction is actually carried out by suspending the pellet (about 1 g) having an activity of 3.9�106 IU/ml in 20 ml buffer and maintaining the resulting suspension at 4� C. for 20 hours with stirring.
The gamma-interferon present in the extract and in the remaining pellet is determined with an essay for antiviral activity after separation by 100,000 rpm centrifugation for 10 minutes. It can be appreciated from the results reported in Table 2 that the highest solubilization effectiveness is obtained at pH 8.0 and 7.6.
TABLE 2______________________________________Test IU/ml______________________________________Extract pH 8.0 108 000Extract pH 7.6 101 000Extract pH 7.0 8 000Extract pH 6.6 9 600Extract pH 6.0 14 000Extract pH 5.6 9 600Extract pH 5.0 9 000Pellet pH 8.0 480 000Pellet pH 7.6 672 000Pellet pH 7.0 720 000Pellet pH 6.6 720 000Pellet pH 6.0 768 000Pellet pH 5.6 864 000Pellet pH 5.0 1 008 000______________________________________
The process is carried out in the same manner as in preceding example 2, using SORENSEN buffer, pH 8.0 complemented with 1M sorbitol, in the homogenization phase. After separation of the supernatant (60 ml) with centrifugation at 100,000 rpm, the pellet (comprising 1,874,000 IU/ml gamma-interferon) is extracted three times with 90 ml buffer for a period of time of 24 hours. The results reported in Table 3 show that about 65% of gamma-interferon contained by the aggregates is solubilized upon extraction at pH 8.0.
TABLE 3______________________________________Test IU/ml mg/ml IU/mg tot. ml tot IU______________________________________a) 1.536 � 103 6.87 2.2 � 105 60 9.20 � 107b) 1.874 � 103 -- -- 56 1.05 � 108c) 247 700 0.356 6.9 � 105 90 2.20 � 107d) 365 400 0.47 7.7 � 105 90 3.30 � 107e) 130 000 0.167 7.7 � 105 90 1.20 � 107f) 380 000 -- -- 56 2.00 � 107g) 207 250 0.298 7.0 � 105 270 5.60 � 107______________________________________
a) is supernatant obtained after centrifugation at 100,000 rpm;
b) is the suspension of the pallet of aggregates in buffer SORENSEN pH 8.0;
c) is the first extract in buffer SORENSEN pH 8.0; d) is the second extract in buffer SORENSEN pH 8.0;
e) is the third extract in buffer SORENSEN pH 8.0;
f) is the pellet residue and
g) is the pool of extracts.
A chromatography column Pharmacia C-16 is used, which contains 1 ml of SP-Sephadex� resin (cation exchanger) equilibrated with 0.05M NaCl in SORENSEN buffer pH 8.0.
The column is loaded with 135 ml of the pool of the extracts obtained according to example 3 having a gamma-interferon titre of 2.8�107 total IU.
After loading, the column is eluted with 100 ml buffer having the aforementioned composition and the eluate is analyzed by assaying the antiviral activity to determine the presence, if any, of gamma-interferon. The results reported in Table 4 show that the eluate comprises the total amount of loaded interferon, proving that by such operative conditions the recombinant gamma-interferon is not bound to the resin.
TABLE 4______________________________________Test IU/ml mg/ml IU/mg tot. ml tot IU______________________________________a) 1.536 � 103 6.87 2.2 � 105 60 9.20 � 107b) 1.874 � 103 -- -- 56 1.05 � 108c) 207 250 0.298 7.0 � 105 135 2.80 � 107d) 238 620 0.286 8.3 � 105 135 3.20 � 107______________________________________
a) is the supernatant obtained after centrifugation at 100,000 rpm;
b) is the suspension of the pellet of aggregates in SORENSEN buffer pH 8.0;
c) is the pool of the loaded extracts and
d) the eluted pool.
The process is carried out in the same manner as in example 4, but using 12 ml of Sepharose-Q� resin (anion exchanger) equilibrated with 0.05M NaCl in SORENSEN buffer pH 8.0.
After loading, the column is eluted with 100 ml buffer having the aforementioned composition and the eluate [test (b)] is analyzed by assaying the antiviral activity.
The absence of gamma-interferon shows that this is bound to the anion exchanger. The gamma-interferon is thereafter eluted from the column with a NaCl gradient (from 0.05 to 0.5M); the different fractions are collected and submitted to gamma-interferon determination.
The results reported in table 5 show that the fractions 2 to 5 contain gamma-interferon having a specific activity, of 1.05�108 IU/mg and in yield of 75%.
Said results indicate that the isoelectric point (PI) of gamma-interferon solubilized with SORENSEN buffer pH 8.0, without using denaturing agents, is lower than 8.0 unlike the product obtained by urea extraction, which has PI higher than 8.0 on SP-Sephadex�.
______________________________________(NaCl 0.5M)______________________________________F122 1.40 � 105 0.013 3.07 � 106 10 1.40 � 106F123 1.18 � 105 0.023 7.80 � 105 10 1.18 � 106F124 9.90 � 104 0.022 4.30 � 104 10 9.90 � 105F125 5.76 � 104 0.024 2.60 � 104 10 5.76 � 105(NaCl 0.5M + Urea 6M)______________________________________
TABLE 5______________________________________Test IU/ml mg/ml IU/mg tot. ml tot IU______________________________________a) 207,250 0.298 7.00 � 105 135 2.80 � 107b) 18,600 0.051 3.60 � 105 135 2.50 � 106c) 8,500 0.007 1.20 � 106 5 4.20 � 104d) 1,050 � 103 0.01 1.05 � 108 20 2.10 � 107e) 8,300 0.038 2.20 � 105 100 8.30 � 105f) 136,600 0.027 5.00 � 106 15 2.00 � 106g) 71,200 0.029 2.45 � 106 15 1.06 � 106h) 47,000 0.061 7.70 � 105 10 4.70 � 105______________________________________
a) is the pool of loaded extracts;
b) pool of eluates;
c) fraction 1;
d) fractions 2 to 5 (NaCl 0.08M);
e) fractions 6 to 25;
f) fractions 26 to 28 (NaCl 0.23M);
g) fractions 29 to 31 (NaCl 0.32M);
h) fractions 32 to 33 (NaCl 0.50M).
The pellet of aggregates obtained according to example 1, is suspended in 80 ml 0.025M Tris-HCl buffer, 1 mM EDTA, 6M urea, pH 8.0 and the resulting suspension is extracted at 4� C. for one night with gentle stirring.
The suspension is then centrifuged at 100,000 rpm for 60 minutes at 4� C. to separate the remaining pellet from the supernatant containing the solubilized gamma-interferon. Said supernatant, which is not completely clear, is purified on cation exchange chromatography using a Pharmacia K 50/30 chromatography column packed with 25 ml SP-Sephadex� C50 equilibrated with 0.025M Tris-HCl buffer, 1 mM EDTA, pH 8.0 at 4� C. The column is fed with the extract at a flow rate of 100 ml/hour. After the loading, the column is washed with the same equilibration buffer (500 ml) fed at flow rate of 30 ml/hour until completely urea-free eluate is collected (monitored with enzymatic assay using the Kit Sclavo). Then a first elution from the column of human recombinant gamma-interferon is carried out by applying a gradient from 0.025M Tris-HCl buffer, 1 mM EDTA, 0.05M NaCl, pH 8.0 to 0.025M Tris-HCl buffer, 1 mM EDTA, 0.5M NaCl., pH 8.0 and at a flow rate of 100 ml/hour. Programmed time for the gradient: 1 hour.
Finally the column is eluted with 0.025M Tris-HCl buffer, 1 mM-EDTA, 0.5M NaCl and 6M Urea, pH 8.0. The eluted fractions are collected and screened to determined the antiviral activity. The results are reported in table 6.
TABLE 6______________________________________Test IU/ml mg/ml IU/mg tot. ml tot IU______________________________________Load 3.89 � 105 0.395 9.80 � 105 80 3.10 � 107Washes <9 000 0.17 -- 80 --F1-F16 <9 000 -- -- 150 --(NaCl 0.3M)F17 1.27 � 105 0.010 1.27 � 107 12 1.52 � 106F18 1.22 � 105 0.012 1.02 � 107 8 9.80 � 105F19 2.90 � 105 0.012 2.40 � 107 7 2.00 � 106F20 3.15 � 105 0.010 3.10 � 107 5 1.57 � 106F21 3.90 � 105 0.009 4.30 � 107 7 2.70 � 106F22 2.18 � 105 0.010 2.20 � 107 8 1.74 � 106F23 1.74 � 105 0.010 1.70 � 107 8 1.40 � 106F24 1.36 � 105 0.008 1.70 � 107 12 1.63 � 106F25-35 <9 000 -- -- 100 --(NaCl 0.5M + Urea 6M)F36 6.36 � 105 0.163 3.80 � 106 7 4.40 � 106F37 2.78 � 105 0.063 4.30 � 106 7 1.90 � 106F38 1.90 � 105 0.053 3.60 � 106 7 1.30 � 106F39 1.80 � 105 0.031 5.80 � 106 12 2.10 � 106______________________________________
It can be appreciated from the above figures that:
the elution with a salt gradient without urea at a NaCl concentration of 0.3M allows human recombinant gamma-interferon to be obtained in sufficient purity (specific activity 4.3�107 IU/mg) and with the yield of about 45%;
the subsequent treatment of the column with urea-containing buffer allow the elution of that part of gamma-interferon which has likely become segregated after washing the column with urea-free buffer and which has specific activity 4.3�106 IU/mg; and with the yield of about 45%;
the pellet remaining after such an extraction still exhibits a content of human recombinant gamma-interferon which can be estimated to be about 50% of to starting product.
The aggregate pellet obtained according to example 1, is suspended in 64 ml 0.025M Tris-HCl buffer, 1 mM EDTA, 6M urea, 0.05M NaCl, pH 8.0 and the resulting suspensions extracted at 4� C. for one night with gentle stirring. The suspensions then centrifuged at 100,000 rpm for 60 minutes at 4� C. to separate the remaining pellet from the supernatant containing the solubilized gamma-interferon.
The supernatant (57 ml) is loaded at a flow rate of 57 ml/hour (0.048 ml/minute�per cm2) onto a Pharmacia K 50/30 column packed with 100 ml SP-Sephadex� C50 equilibrated with the same buffer. After loading, the column is washed with about 500 ml of 0.025M Tris-HCl buffer, 1 mM EDTA, 6M Urea, 0.05M NaCl, pH 8.0 and the amount of interferon in the eluate is determined. Then the elution from the column of human recombinant gamma-interferon is carried out at a flow rate of 100 ml/hour.
(0.084 ml/minute�cm2) with 0.025M Tris-HCl, 1 mM EDTA, containing increasing molarity of NaCl (0.075M, 0.1M, 0.2 M, 0.3M, 0.4M and 0.5M). Finally, the column is eluted with 0.025M Tris-HCl buffer, 1 mM EDTA, 6M urea, 0.5M NaCl, pH 8.0. The eluted fractions are collected and analysed to detect the presence of gamma-interferon. The results are reported in table 7.
TABLE 7______________________________________Test IU/ml mg/ml IU/mg tot. ml tot IU______________________________________Load 5.10 � 105 1.417 3.6 � 105 57 2.9 � 107Eluate 6.400 0.403 -- 57 --F11-F15 <640 0.018 -- 50 --(NaCl 0.075M)F28-32 <640 0.013 -- 50 --(NaCl 0.1M)F48-52 <640 0.032 50(NaCl 0.2M)F69 2.97 � 105 0.009 3.30 � 107 10 2.97 � 106F70 3.82 � 105 0.007 5.40 � 107 10 3.82 � 106F71 2.45 � 105 0.010 2.40 � 107 10 2.45 � 106F72 3.17 � 105 0.008 3.90 � 107 10 3.17 � 106F73 1.96 � 105 0.006 3.30 � 107 10 1.96 � 106(NaCl 0.3M)F88 2.30 � 104 0.020 1.16 � 106 10 2.30 � 105F89 5.10 � 104 0.021 2.40 � 106 10 5.10 � 105F90 3.00 � 104 0.022 1.36 � 106 10 3.00 � 105F91 1.40 � 104 0.021 6.80 � 105 10 1.40 � 105F92 9.10 � 103 0.018 5.00 � 105 10 9.10 � 104(NaCl 0.4M)F103-106 <640 0.017 -- 40 --(NaCl 0.5M)F122 1.40 � 105 0.013 3.07 � 106 10 1.40 � 106F123 1.18 � 105 0.023 7.80 � 105 10 1.18 � 106F124 9.90 � 104 0.022 4.30 � 104 10 9.90 � 105F125 5.76 � 104 0.024 2.60 � 104 10 5.76 � 105(NaCl 0.5M + Urea 6M)______________________________________
From the above figures it can be derived that gamma-interferon of sufficient purity (specific activity of about 5.4�107 IU/mg) in yield of about 45% can be obtained upon elution with buffer comprising 0.3 NaCl. In this case too, the final elution with 6M Urea and 0.5M NaCl relates gamma interferon having specific activity of 3.07�106 IU/mg.
|Cited Patent
|Filing date
|Publication date
|Jun 1, 1984
|Apr 16, 1985
|Genentech, Inc.
|Purification and activity assurance of precipitated heterologous proteins
|Jun 1, 1984
|Apr 16, 1985
|Genentech, Inc.
|Purification and activity assurance of precipitated heterologous proteins
|Jun 1, 1984
|Jul 8, 1986
|Genentech, Inc.
|Purification and activity assurance of precipitated heterologous proteins
|Jun 1, 1984
|Nov 4, 1986
|Genentech, Inc.
|Purification and activity assurance of precipitated heterologous proteins
|Aug 10, 1984
|Jul 21, 1987
|Hoffmann-La Roche Inc.
|Immune interferon and a method for its extraction and purification
|Jul 3, 1985
|Jun 14, 1988
|Nagabhushan Tattanahalli L
|Process for the purification of gamma interferon
|Jun 4, 1985
|May 9, 1989
|Prior Chrisopher P
|Process for purifying a protein
|Dec 26, 1985
|May 9, 1989
|Naoki Higashi
|Method for purifying an interferon
|Dec 29, 1986
|Oct 17, 1989
|Suntory Limited
|Novel Host e. coli and use thereof
|Jan 9, 1987
|Mar 13, 1990
|New York University
|Novel polypeptide having gamma-interferon activity
|Nov 1, 1988
|May 18, 1989
|Genentech, Inc.
|Gamma interferon formulation
|Protein Purification Methods, Harris et al., Eds. IRL Press. 1989, pp. 88,92.
|U.S. Classification
|530/351, 530/427, 530/423, 424/85.5, 435/69.51
|International Classification
|Cooperative Classification
|European Classification
|Jul 23, 1991
Owner name: SCLAVO S.P.A.
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:PROTASI, OTELLO;MANNUCCI, DONATELLA;REEL/FRAME:005788/0600
Effective date: 19910712
|Sep 16, 1997
|Maintenance fee reminder mailed
|Feb 8, 1998
|Lapse for failure to pay maintenance fees
|Apr 21, 1998
|Expired due to failure to pay maintenance fee
Effective date: 19980211


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No one was more thankful last week than hip-hop fans.
Chris Brown Shares "Royalty" Album Tracklist
Chris Brown just dropped a 34-track mixtape this weekend, so it's hard to believe he still has a studio album coming in just a few weeks. Like clockwork, just after the mixtape impacted, we've now got a look at the tracklist for Brown's new album Royalty.Those looking for a large selection of features may be left a bit disappointed, as the only two guests on the album are Future (who shows up on a bonus track) and surprisingly, Atlanta up-an-comer Solo Lucci (the fact that -- like Chris -- he's an RCA signee, makes things a little more clear).
Future's Tour Rider Reveals He Makes $150K For 45-Minute Show
Undoubtedly one of the best parts of being a successful rapper is that you can request literally anything on your tour rider and the show promoters will do its best to deliver.
Lil Wayne's "No Ceilings 2" Tracklist Revealed
After hitting us with a trailer to increase the anticipation and excitement for No Ceilings 2 last night, Weezy F returns this afternoon with a tracklist, so we know it's real. The highly anticipated mixtape will serve as a follow-up to the much-beloved first No Ceilings, and thus it's got quite a reputation to live up too. The tracklist is stacked though, so let's hope Weezy didn't spare quality for quantity.
A HotNewHipHop Thanksgiving: 10 Things We're Thankful For
Thanksgiving is here, and while we gather with our families to reflect on what we are thankful for this year, we here at HotNewHipHop can't help but reminisce on the year thus far with gratitude. We've been spoiled with albums and mixtapes, as well as a healthy amount of "comebacks." As analysts of the genre, we've been overwhelmed with remarkable material from artists upcoming and legendary alike. As fans, our excitement has us feeling young again.
ASAP Ferg Talks "Huge Smash Song" With Future, Recording For A Week With 40
ASAP Ferg is getting ready to drop his sophomore album at the top of 2016, and from the sounds of his new interview with MTV, it's bound to be something special. "Early January you're gonna get a HUGE smash song called 'New Levels' with Future," said Ferg, promising the song will drop after we get to see the "Thought It Was A Drought" video he directed.
Big Sean Previews New Music With Future, Metro Boomin & Mike WiLL Made-It
Big Sean scored the biggest album of his career with 2015's Dark Sky Paradise, but from the sounds of his upcoming material, he's only just getting started. Sean was in L.A. last night for Red Bull Sound Select, and made sure to treat the audience to some samples of his new music. First up, he shared a preview of a track with Future and his go-to producer Metro Boomin, a recipe for success on paper, and judging by the video clip, also in practice.
Future On WATTBA: "It Never Happened"
Anyone who follows Future on Twitter knows that the man loves to remain cryptic, constantly tweeting vague phrases, any of which could probably be read as either a subtweet or a mixtape title under a magnifying glass. In a recent interview with Mass Appeal, Fewtch continued in that tradition of puzzling but highly hashtag-able phrases when asked about one of his projects, and it has the Future Hive in a frenzy.
Stream The Soundtrack For "Creed" Featuring Joey Bada$$, Future, Nas & More
The "Creed" movie, which stars Michael B. Jordan and Sylvester Stallone, hits movie theatres in five days. The official soundtrack for the film arrives a few days prior to the movie premiere, with a stream available via Spotify today. We've had a lot of album streams hit the web today already, so adding to that list, you'll find new (and old) music from the likes of Meek Mill, Future, Joey Bada$$, Nas, Childish Gambino, Jhene Aiko, Vince Staples, Krept & Konan and others.
Future Covers Mass Appeal, Talks Post-Breakup Comeback
It's hard to say anyone had a bigger 2015 than Future. His only competitor when it came to hitmaking was Drake, and by the end of the year, even he was knocking on Future's door. The Atlanta MVP is the star of the new Mass Appeal cover story, where he muses on his enviable mixtape run, rising out of a difficult breakup with Ciara, and throwing away his crossover dreams to return to his original gritty sound.
Charts Don't Lie: November 18
We just experienced our second week in a row with no notable rap and R&B debuts, but it's all leading up to the huge sales week that will include Jeezy, Logic, Ty Dolla $ign and Justin Bieber. In the meantime, the same major players have been keeping the charts populated, with The Weeknd, Fetty Wap, Drake, and Future making up a big chunk of sales, while Bryson Tiller continues to surprise with consistent numbers.Check out the full breakdown below.
Are Future & Mike WiLL Made-It Dropping "Ape Sh!t" On Thanksgiving?
Mike WiLL Made-It and Future have been prepping their joint mixtape Ape Sh!t since last December at least.
Top Tracks: November 9 - November 15
Last week saw a few highly anticipated releases, including new albums from Ty Dolla $ign, Jeezy, and Logic. Surprisingly, none of those guys made it into Top Tracks -- well, except Logic, who somehow ended up on a Juicy J track. Instead, Pusha T, the new prez of GOOD Music, came out of hiding and hit us with two top 10's. The other winner of the week has to be the astoundingly prolific Metro Boomin, who has three new productions in this week's list.
2 Chainz Speaks On Future's Current Hot Streak
One of the top stories this year has been Future's incredible string of releases. He's put out two stellar mixtapes and two No. 1 albums, including a joint effort with Drake. And he put out Monster, the tape that started it all in late October of 2014. Is his run historic? Well, some people have been saying that 2 Chainz had a period of similar productivity in 2012 and 2013.
Metro Boomin Confirms Young Thug Beef Is Squashed
Upon the release of the brand new Metro Thuggin collab, "Hercules," all is well between Young Thug and Metro Boomin. Of course, this wasn't the case a few hours before the release, but when Thug announced a Metro Thuggin collab on Instagram, it was assumed the whole thing had been a publicity stunt.
Did Young Thug & Metro Boomin Troll Us All? A Metro Thuggin Collab Is Coming Tonight
Well, damn. Young Thug may have just surpassed 50 Cent in the running for biggest troll of 2015. Fans really thought that Metro Thuggin was gone forever. But now, it seems, the whole beef might have been a means of promoting Thugger's new song, "Hercules," which drops tonight...produced by none other than Metro Boomin.
Metro Boomin's 20 Best Beats
Metro Boomin just turned 22 in September, and he's already racked up one of the most enviable discographies in modern hip hop. Everyone from Gucci Mane to Drake to Nicki Minaj has called upon the St. Louis-via-Atlanta beatsmith for some work, and it's no surprise that he's been able to find success with such a wide range of artists.
Charts Don't Lie: November 11
This was a bit of a dry week for rap and R&B releases. While we await projects from Ty Dolla $ign, Jeezy, and Logic next week, things are pretty quiet, with no big debuts this cycle. The good news is, there are still quite a few rap albums occupying the top ten, with The Weeknd, Fetty Wap, and Drake & Future's WATTBA continuing to do numbers. Meanwhile, Bryson Tiller continues his low-key but impressive presence within the top 30.Check out the full breakdown below.
Future Reportedly Has Six Albums And Three Mixtapes "Ready Right Now"
Future is white-hot at the moment, coming off a 12-month stretch that produced Monster, Beast Mode,


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Thomas Björn assumed outright possession of the 36-hole clubhouse lead of the 78th Masters at 3-under-par 73-68=141.
Francesco Laporta overcame an early wobble to shoot 2-under-par 35-35=70 and take a two shot lead into the weekend at the Trophée Hassan II.
Andrew Loupe settled for a 1-under-par 35-36=71 during the second round of the Wells Fargo Championship, staking an early claim of the clubhouse lead at 8-under 136.
Zac Blair self-reported a bent putter during the second round of the Wells Fargo Championship, disqualifying himself after 24 holes of play.
J.J. Henry withdrew from the Wells Fargo Championship prior to his second round tee time, he was 3-over through 18 holes. For now, no injury has been cited as the reason for the WD.
Steve Wheatcroft walked all over Quail Hollow Club on the way toward a 7-under-par 32-33=65 to grab a piece of the early lead at this week's Wells Fargo Championship.
Andrew Loupe torched Quail Hollow Club with a bogey-free, 7-under-par 31-34=65 to grab a share of the first-round lead at this week's Wells Fargo Championship.
Thomas Linard painted a 4-under-par 33-35=68 in round one of the Trophée Hassan II in Morocco, good enough for a share of the lead.
Rory Sabbatini withdrew from the Wells Fargo Championship prior to his round one tee time. Replacing him in the field is Martin Piller.
Anirban Lahiri put on a lag-putting clinic today in route to a bogey-free, 6-under-par 33-33=66 during the opening round of this week's Wells Fargo Championship.
Italian rookie Francesco Laporta took a share of the early clubhouse lead on day one of the Trophée Hassan II after hanging up a 4-under-par 35-33=68.
Jason Scrivener hit just five fairways on his morning lap of Royal Golf Dar Es Salam but posted the early lead at the Trophée Hassan II with a 4-under-par 33-35=68.


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Pixie Lott stars as Holly Golightly in BREAKFAST AT TIFFANY'S at the Theatre Royal, Haymarket
Pixie Lott stars in BREAKFAST AT TIFFANY'S at the Theatre Royal, Haymarket
Breakfast at Tiffany's, the classic tale of Holly Golightly written by Truman Capote and so memorably portrayed by Audrey Hepburn in the iconic 1961 film, is being given a new lease of life as a stage play with music in a sparkling, sophisticated production starring the multi award-winning Pixie Lott as Holly.
Adapted by Pulitzer Prize finalist and Tony and Olivier Award-winning playwright Richard Greenberg and directed by Nikolai Foster, this tale of vivacious, good-time girl Holly Golightly and her fantastical existence in 1940s New York City, is told through the eyes of a young writer, completely fascinated by this exquisite extrovert who every woman wants to be, and every man wants to be with.
With memorable songs from the era, this stylish production is set to capture the hearts of audiences and sparkle like a diamond in a Tiffany's window.
Based on the novel by Truman Capote
Adaptor Richard Greenberg
Director Nikolai Foster
Designer Matthew Wright
Music Grant Olding
Review: Phantom Of The Opera At Her Majesty's Theatre
My First Experience Of Phantom Of The Opera
The History Of Paris And Broadway Spectacular An American In Paris
AN AMERICAN IN PARIS, Broadway's Tony Award-winning New Musical To Open March 2017 At London's Dominion Theatre
Seating plan
Running time: TBC
Group rate 10+ Best available Stalls and Royal Circle (excluding premiums) reduced to £49.50. Valid Monday to Thursday subject to availability.
School group rate 10+ Upper Circle reduced from £42.50 to £25. Valid Monday to Thursday subject to availability. Maximum 20 tickets per booking.
© 1999-2016
London Theatre Direct Ltd. All rights reserved.
London Theatre Direct Limited |
207 Regent St,
London, UK
Tel. +44 845 505 8500 |


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Maruti Alto 800
Maruti Suzuki Alto 800 is the hatchback offering from the Indian car maker that has taken over from the Maruti 800. The Maruti Alto 800 is taller, has an improved interior space and a&better fit-and-finish.&The large headlamps upfront and the wide lower grille does looks pretty contemporary.&The tai ... read more
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J.C. Bamford Excavators Limited is globally known as JCB. This British multinational company has its headquarters based in Staffordshire. JCB manufact Read more detail
In what can be termed as a customer-centric move by Ford, the American auto major has commenced retail distribution of Ford Genuine parts in India Read more detail
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Audi Q3 in India : Launch, dates, price, first look
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Maruti Alto 800 launch
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Maruti Celerio Diesel Road Test Review
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|Killzone 2
#6405947, By saintedd What are you currently playing?
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Tom Clancy's The Division guide Our essential guide to Ubi's loot-stuffed shooter.
Dark Souls 3 guide UPDATED: Boss fights, bonfires and plenty more besides.
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text:
|Name: _________________________
|Period: ___________________
This quiz consists of 5 multiple choice and 5 short answer questions through Analysis.
Multiple Choice Questions
1. How many years have passed between the actual story and the narrator's telling of it?
(a) Twenty years.
(b) Thirty years.
(c) Five years.
(d) Sixteen years.
2. How does the narrator describe the farmers at Van Tassel's party?
(a) Good old boys.
(b) A group of well worn men.
(c) As a leather-faced race.
(d) Handsome hunks.
3. What does the narrator likens Brom's attempts at courting to?
(a) To the clawing of a wolverine.
(b) To the cawing of a crow.
(c) To the gentle caresses of a sword.
(d) To the gentle caresses of a bear.
4. What does Ichabod's physical features mimic?
(a) A crane.
(b) An elephant.
(c) A dolphin.
(d) A giraffe.
5. How old is Katrina Van Tassel?
(a) 21.
(b) 18.
(c) 30.
(d) 16.
Short Answer Questions
1. Where does Ichabod live for weeks at a time?
2. What is the name of the neighboring town described after the poem at the beginning of the story?
3. What does Ichabod Crane do for a living?
4. As Ichabod reaches the bridge, what item does the dark rider throw at him to knock him to the ground?
5. What success does the old farmer from New York state about Ichabod?
This section contains 240 words
(approx. 1 page at 300 words per page)


text:
Video: Samsung Checkout lets shoppers buy through their TVs
The new payment option from Samsung gives retailers another way to connect with customers.
Video: Samsung Checkout lets shoppers buy through their TVs
The new payment option from Samsung gives retailers another way to connect with customers.
Amazon China names a new president and links with a retailer
Electronics retailer Gome opens a storefront on Amazon China's e-commerce site.
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Eyewear retailer Visionworks, which operates 700 stores, began selling online for the first time this week.
Posted 04/21/2016Matt LindnerPost a comment
Nordstrom expects online sales to account for 30% of business by 2020.
Posted 03/10/2016Bloomberg NewsPost a comment
A shopping mall operator lets slip today that Amazon plans to open 300-400 bricks-and-mortar bookstores.
Posted 02/02/2016Don DavisPost a comment
The retail giant expects to double its online inventory in the second half.
Posted 08/15/2013Kevin WoodwardPost a comment
Related Content:Walmart Internet Sales
But the books and accessories retailer's store sales increase.
Posted 12/17/2012Kevin WoodwardPost a comment
The retailer will cut prices, reduce promotions and overhaul store design.
Posted 01/25/2012Allison Enright1 comments
Related Content:Sephora
E-commerce spending has increased 15% this year, comScore says.
Posted 12/20/2011Thad Rueter1 comments
The Boston Consulting Group projects spending to hit $315 billion within five years.
Posted 11/15/2011Thad Rueter1 comments
Best Buy lets some smartphone owners receive reward points when they visit stores.
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Related Content:Average Basket Size
Kantar predicts 13.5% growth for online holiday sales, a lower rate than for last year.
Posted 09/22/2011Allison Enright1 comments


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Howard Young on Coming Changes To The Healthcare Space
Latest Survey Highlights the Year's Top Trends in Class Actions
Real Estate E&O Policy Exclusions Arising from Bank-Owned Properties
Izzet Sinan on What Telecom Antitrust Regulation Means For EU Integration
Medical Apps and Devices: The Convergence of FDA, FTC, and State and Federal Regulations
Ben Indek on The SEC's Record-Setting Year
Turning IOT Data Into Opportunities
Can My Creditors Put me In Jail?
20 Things a Debt Collector Can't Do
5 Reasons Consumers Should File an FDCPA Lawsuit
Why You Should Enter into a Payment Plan with a Junk Debt Buyer
Beginners Guide to Debt Collection Lawsuits by Portfolio Recovery Associates
Quick Guide to Stopping Collection Calls.
3 Proven Strategies to Get the Lowest Debt Settlement Possible
3 Proven Strategies for Dealing with a Midland Funding Lawsuit
BakerHostetler Partner Alan Friel Talks Big Data and Data Collection
BakerHostetler's Tanya Forsheit Discusses Cloud Computing
The TCPA: Deep Dive: Details, Class Actions, Regulations, and Defense Strategies
The TCPA: Basics, Targeted Industries, and Trends
A Moment of Simple Justice - The ABA's Priorities
Out-of-state companies doing business in California would be well-advised to look closely at a new bill pending in the California State Legislature. If you are concerned about defending litigation cases in the most...more
New GAP Insurance Rules -
The FCA published new rules in June governing the sale of addon Guaranteed Asset Protection (GAP) insurance. The rules came into force on 1 September 2015 and apply to commercial customers as well...more
Some of the recent items on the Consumer Financial Protection Bureau's (CFPB) agenda have involved efforts to eliminate alleged discriminatory disparities amongst auto lenders and in connection with other private companies as...more
The Minnesota Legislature had a short week, with no official business on Friday due to their Easter recess. The legislature will return on Tuesday, March 29, at noon. This week, committees continued to hear budget details...more
Is the pick-off strategy to moot class actions still alive in the Southern District of New York? Possibly.
Last month we reported on Brady v. Basic Research, L.L.C. – the first decision to interpret the Supreme Court's...more
To read the full 2016 Class Action Survey report, please visit ClassActionSurvey.com.
The 2016 Carlton Fields Class Action Survey reveals surprising trends in class action litigation, based on insights provided by...more
Regulatory Whack-a-Mole: A Renewed Focus on Non-Hazardous Waste Recycling -
Why it matters -
In part due to a string of high-profile enforcements in multiple states, retailers by and large have evaluated the need to...more
The battle over the DOL's efforts to implement a new law that will impose a fiduciary standard on anyone giving retirement advice continues with some now questioning whether critics of the law really believe its effects will...more
We recently blogged about a new wave of class action litigation related to California's Transparency in Supply Chains Act. In December, Nestlé USA won the dismissal of a complaint against it alleging that the company was...more
An event data recorder (EDR), as defined in 49 CFR section 563.5, generally means a device or function in a vehicle that records the vehicle's dynamic time-series data during the time period just prior to or during a crash...more
The Health Insurance Portability and Accountability Act of 1996 (HIPAA), Public Law 104-191, contains extensive rules designed to limit access by non-health plan entities to certain individually identifiable health...more
Committee Decides Physician Acquisition Disclosure Bill Is Unnecessary -
The Senate Health and Welfare Committee has decided not to pursue consideration of S.245, a proposal to require disclosure of new affiliations...more
Similar to website browsers, many mobile applications collect a variety of information from the user, including, the user's identity, usage history, past log-ins, and location. This enables the application to provide various...more
Two new bills affecting credit reporting and the use of credit reports for employment purposes are working their way through Congress. If passed, the bills would amend the Fair Credit Reporting Act (FCRA). The first, The...more
Several companies now offer time and attendance solutions that incorporate the use of fingerprint identification technology and purport to eliminate "buddy punching" for hourly employees. In addition to time and attendance...more
While the sudden death of Supreme Court Justice Antonin Scalia creates an immediate vacancy on the bench, it also likely will leave the high court's docket in limbo on number of key consumer class actions awaiting the Court's...more
The EU Parliament Committee on Civil Liberties, Justice, and Home Affairs ("LIBE") finally released the text of the long anticipated new data protection law. While the law has not formally been enacted, its adoption at this...more
Fingerprint identification technology uses fingerprints to uniquely identify individuals. The technology has been used by law enforcement agencies for decades, and dozens of statutes regulate when government agencies may...more
Be Still, My Heart: New Suit Says Fitbits Fail to Track Heartbeats as Promised -
Why it matters -
Fitbit has been hit with another consumer class action asserting false advertising claims, this time alleging that...more
A 2009 Department of Justice ("DOJ") investigation of the employment and recruitment practices of a number of Silicon Valley technology companies resulted in DOJ lawsuits against seven companies, followed by consent decrees...more
The decision of the Court of Justice of the European Union (CJEU) to invalidate Safe Harbor in October 2015 sent shockwaves throughout the international business community. Safe Harbor was a certification mechanism that...more
In 2015, following numerous customer data security breaches at major U.S. companies, the IRS announced special tax relief to breach victims who were provided identity protection services as a result of the breach. In January...more
The Federal Trade Commission (FTC) has just released a report on Big Data: A Tool for Inclusion or Exclusion? Understanding the Issues (Report), January 6, 2016.
The FTC Chairwoman Edith Ramirez indicates in the FTC news...more
Two bills amending the Fair Credit Reporting Act (FCRA) intended to benefit consumers are making their way through Congress. H.R. 4172, "The Credit Access and Inclusion Act of 2015," has bipartisan support, although it is...more
The Regional Income Tax Agency of Ohio (RITA) announced on December 31, 2015, that it lost the personal data of approximately 50,000 individuals who filed tax forms with the agency in November. RITA provides tax collection...more
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Director of Catering, Bar Manager, and Restaurant Manager:. Minimum 5 years' experience as Chinese Restaurant Manager in a casual upscale Chinese restaurant.... - Indeed
Working knowledge of restaurant operations, seating. Keep floor in host area free of spills, food, trash. Performance of overall operations.... - Indeed
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Celebrating 30 years of providing fashionable comfort footwear and excellent service— Footwear etc. • Established 1986 —
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- Download games
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You're looking for some great games to play, but you have a device with a touch-screen and no mouse? No worries! Zylom is the best games website and offers incredible touch-screen games. Zylom has a wide variety of free online touch-screen g... More info »
You're looking for some great games to play, but you have a device with a touch-screen and no mouse? No worries! Zylom is the best games website and offers incredible touch-screen games. Zylom has a wide variety of free online touch-screen games for you. This page features the latest and most popular touch games. We have many interesting genres to choose from. Discover them now!Je bent op zoek naar geweldige spelletjes om te spelen, maar je hebt een apparaat met een touchscreen, zonder muis? Geen zorgen! Zylom is de beste spelletjeswebsite en heeft fantastische touchscreen games. Zylom heeft een uitgebreid aanbod van gratis online touch-screen games voor je. Op deze pagina vind je de nieuwste en populairste touchscreen spelletjes. We hebben veel interessante genres waar je uit kunt kiezen. Ontdek ze nu!
In Sharpe Investigations Death on the Seine Deluxe, you need to solve the mystery behind the curious death of a celebrity chef!
Join the fashion police and solve a criminal caper in Hostile Makeover - A Fashion Murder Deluxe, an exhilarating hidden object mystery!
Unravel a frightening conspiracy brought on by a ghostly riverboat in Midnight Mysteries - Devil on the Mississippi Deluxe.
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Play Super Collapse! 3 and experience the wonderful adventure in the exciting Quest mode or enjoy the classic game in Quick Play!
Buy and sell property with the fantastic Build-a-lot Super Pack! Enjoy these 7 real estate games for just one special price.
Feed the heroic soldiers of the Roman Empire as they go to war in Farm Frenzy - Ancient Rome Deluxe, a challenging new addition to the series.


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Eliezer Rodrigues
- BiographyDuring the course of his 14 year long pornographic career, Eliezer has filmed in 2 videos, having made his earliest appearance in Shore Thing in April 2002. The video content containing this model was requested 19,332 times in total, with well over 0.6 GB worth of exciting porno being downloaded during the last year. Some of the other actors co-starring together with Eliezer include Ali Parker, Igor dos Santos, Anna, Alexandre Senna, and Ali Moore. He was added to My Favorites folder 10 times by Gay Access members. On average, close to 92 percent of the Gay Access members have given Eliezer Rodrigues a plus rating. His most often performed activities are Guy On Guy, Blowjobs, Hardcore, Handjobs, and Anal Sex. This pornstar's newest video is featured in Brazilian Hard Hats.
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Auction Sat 7 May
- 2
- 1
- 1
Showing 1 - 3 of 3 properties for sale by Ray White - Port Phillip
Level 1,77 Acland StreetSt Kilda, Vic 3182
Ray White St Kilda has changed the way Melbourne real estate is bought and sold. Providing quality service and advice, combined with cutting edge technology and an international network, no-one is better placed to achieve a premium result in any market, like Ray White St Kilda. Led by Matt White who is a fourth generation member of the Ray White family who own and operate AustraliaâàƒÃ''€Â™s largest real estate agency business. The company has been in continuous family ownership for 110 years. Ray White will ensure that your move is a rewarding experience.
Auction Sat 7 May
Agent Matt White
Auction 11am Sat 28th of May
Auction Sat 28 May
St Kilda
...11am Sat 28th of May
In the Heart of the Action
Within a boutique block of just ten and situated on Acland Street's doorstep, this 2-bedroom apartment has an abundance of art deco period charm...
St Kilda
Potential aplenty on Chapel St!
The perfect opportunity to claim your own piece of the shopping, dining and entertainment mecca that is Chapel Street – this partially updated,...
$515,000 +
Great Location.. Exceptional Apartment!
This beautiful and spacious apartment is now available and surely wont last long. Boasting a bright, open plan living area, an extra large...


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Amazon AutoRip now supports vinyl
Amazon's AutoRip functionality which gives you a free digital copy of any CD you might buy, has now been extended to vinyl.
If you opt for an AutoRip-compatible vinyl record on Amazon, a digital file will instantly be added to your Amazon Cloud Player library. More impressive is that those who have purchased AutoRip compatible records since Amazon opened the gates to its music store in 1998 will find they now have a digital file waiting in their Cloud Player. All you need to do is look out for the AutoRip logo next to the record on Amazon's website.
"It's a fun experience to suddenly find CDs you purchased just today - or 15 years ago - added automatically and free of charge to your digital library. We're thrilled to extend this experience to vinyl records," said Steve Boom, Amazon's vice-president of digital music. "Many of our music customers are vinyl fans and it's traditionally been very difficult to make digital versions of vinyl records - now customers can enjoy the albums they buy wherever they are, not just when they have access to a record player."
AutoRip MP3s are stored instantly in Cloud Player libraries and have no impact on the storage limit of your Cloud Player. The music is uploaded in 256Kbps MP3 format files, which is a good high-quality format. Expect them to sound fairly different from the rich analogue sound of vinyl, however.
On top of AutoRip, Cloud Player users also benefit from a scan and match service. Importing tracks from iTunes or other music players to your Cloud Player will automatically grant you a 256 Kbps version from Amazon.
We are chasing Amazon on whether the AutoRip function applies to vinyl sales in the UK and not just the US. Right now, AutoRip for CDs is a US only affair, so don't hold your breath.
Here's what the Samsung Gear Fit 2 and Gear IconX cordless buds look like
Sonos now works with Amazon Prime Music properly
Harman Kardon Go + Play shows there's still interest in quality Bluetooth speakers
B&O Beoplay A1 Bluetooth stylish speaker lasts a whopping 24-hours
- Sonos now works with Amazon Prime Music properly
- Harman Kardon Go + Play shows there's still interest in quality Bluetooth speakers
- B&O Beoplay A1 Bluetooth stylish speaker lasts a whopping 24-hours
- The Denon Design Series: Real Hi-Fi for real lifestyles
- JBL Reflect Aware C will pipe noise-cancelled music from your USB Type-C port
- Vinyl fan? Audio-Technica AT-LP60BT Bluetooth turntable offers cool without the wires
- Naim Mu-so Qb review: An all-round success
- Cut the cord for quality and convenience with MMove Bluetooth Earbuds
- Astell & Kern AK380 is one bonkers but beautiful high-res portable player
- 5 Lightning headphones to buy when iPhone ditches the 3.5mm jack
- Samsung Galaxy S7 tips and tricks: Master your new Galaxy
- OnePlus 3 flagship might come with 'Dash Charge' speedy refueling mode
- 52 of the best tech life hacks
- What is HDR, what TVs support HDR, and what HDR content can I watch?
- Amazon Kindle Oasis vs Kindle Voyage vs Kindle Paperwhite: What's the difference?
- Which iPhone is best for you? iPhone SE, iPhone 6, iPhone 6S, iPhone 6 Plus or iPhone 6S Plus?
- OnePlus 3: What's the story so far?
- Apple iPad Pro 12.9 vs iPad Air 2: What's the difference?
- Best 4K TVs: Ultra HD televisions to buy in 2016
- WhatsApp tips and tricks: Amazing features you never knew about


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fix articles 63588, fled
US-Created Violence and Chaos in Yemen (tags)
South Central Activist Runs for City Council; Tells Radical Life Story (tags)
Ron Gochez: Life Story of a City Council Candidate from South Central, High School Teacher, and Community Organizer born to an undocumented mother who fled civil war in El Salvador. 33 Minutes Audio/Photography
Police Attack Peaceful May Day Rally (tags)
LOS ANGELES, May 2, 2007 – A massive police attack on a peaceful rally in MacArthur Park brought a violent end to yesterday's May Day celebrations.
Abandonment (tags)
On the encounter among the abandonments, truthful history.
U.S. Occupation forces Tortures Iraqi University Professors (tags)
12 Apr 2003 Iraqi scientists accused U.S. forces of torturing professors by poking their eyes out, and breaking limbs carried out by U.S. occupation forces also occupation forces are encouraging looting of universities to the extent of transporting looters to the universities and scientific institutions.
CIA has 2 sons of the 9/11 architect (tags)
In the finest traditions of third world despots the American Government has abducted 2 young boys and flown them to the U.S. for Interrogation. Aren't you so proud?
Why is Desmund Tutu silent on Egyptian aparthied. (tags)
Why is Desmund Tutu silent on Egyptian aparthied.
Maoist rebels kill 27 police and free political prisoners (tags)
Maoists killed 27 policeman at a Nepalese airport and in a separate incident took over a prison and freed all the prisoners, mostly Maoist comrades.


text:
Official Software Request Thread
Need Help? Ask Super Users & Experienced Site Members Here v7
how i met your mother s1 to 9
TV Show Request V2
ReLaXin !! V.2
Please request ebooks and audio books here. V14
KAT Member Remembrance Day
Q.121/ Kat Celebrities
Mafiatale - The Calm before The Storm
Active Strategies For Burning Winter Belly Flab
Tough times and vanishing titles. Be careful what you aspire to.
Q.120/Wild rose
Anon and me
the lost hero
Winter soldier
daughtry discography
bengali movie
Anal sweetness
remy lacroix
premonition 2007
sinbad legend of the seven seas swedish


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Great Place for lunch! The food is super good and very authentic. My Favorite is the brown stew chicken!
Sarasota Puerto Rican Food
1. Audrey's Jamaican Cuisine
Great Place for lunch! The food is super good and very authentic. My Favorite is the brown stew chicken!
2. A & G Caribbean RibsMenu
8471 Lockwood Ridge RdSarasota, FL 34243
3. Sunshine Caribbean Restaurant
6320 15th St E Ste B2Sarasota, FL 34243
4. Columbia RestaurantMenu
We do not live in Sarasota but visit often. We always go to Columbia each time we come to town and always will. Keep in mind, the restaurant is alwa…
5. Mi Tierra RestaurantMenu
1068 N Washington BlvdSarasota, FL 34236
6. Palmas De Cuba RestaurantMenu
1303 N Washington BlvdSarasota, FL 34236
7. Sofrito Mama's
935 N Beneva Rd. Unit#613Sarasota, FL 34232
8. Old Packinghouse CafeMenu
987 S Packinghouse RdSarasota, FL 34232
9. The Sandbar RestaurantMenu
I can't believe there is actually a place like this that exsists so close to my house. With white sandy beaches and emerald green translucent waters…
10. Jamaican & American Soul Food
624 Martin Luther King Jr Ave WBradenton, FL 34205
11. Caribbean Chicken
814 8th Ave WPalmetto, FL 34221
12. Banana Cabana Caribbean Seafood & GrillMenu
This place had a fun vibe but I really wasn't impressed by the food. My paella was OK, nothing spectacular. I found it overpriced ($24). Gratuity…
13. Caribbean Cafe
The Cubans and Cuban-style sandwiches are always pleasing. Just enough meat to make it an excellent sandwich. The black beans are always good, too…
14. Puerto Rican Cuisine
13220 Belcher Rd SLargo, FL 33773
15. Caribbean Grocery & Roti ShopMenu
Awesome hidden gem! Great food, fast and friendly service. Best curry in town. We had the chicken and goat curry. It was phenomenal.
16. Island Breeze
This little place boasts an amazing jamaican vegetarian dish full of cabbage, carrots, other veggies and good rice for 6 bucks. I've been 3 times i…
17. Mangos Restaurant & Tiki BarMenu
4 of us dined and all 4 meals were thorough enjoyed. Service good and beach atmosphere was pleasant. We ate out on the deck as opposed to the dini…
18. Marker 39
155 5th Ave NSafety Harbor, FL 34695
From Business: Marker 39 Floribbean fusion inspired cuisine is served in an old Florida setting with an innovative selection of food, wine & craft beers. Our food is Caribbean and…
19. D & G Caribbean RestaurantMenu
13672 Tamiami TrlNorth Port, FL 34287
20. Island Taste Jamaican Restaurant
3031 Placida RdGrove City, FL 34224
21. Golden Krust Caribbean Restaurant
1825 Tamiami TrlPort Charlotte, FL 33948
22. Caribbean Oriental Specialties
2745 Tamiami TrlPort Charlotte, FL 33952
23. Latin Quickie Restaurant
2200 Roy Hanna Dr SSaint Petersburg, FL 33712
24. Vibez Restaurant Caribbean
1836 49th St SGulfport, FL 33707
25. Yah MonMenu
301 W Platt St Unit CTampa, FL 33606
26. Bahama Breeze
3045 N Rocky Point DrTampa, FL 33607
27. Irie Bay Jamaican CuisineMenu
10041 E Adamo DrTampa, FL 33619
28. Ceci Cela Cuisine Antillaise
2140 Drew StClearwater, FL 33765
29. Caribbean Palms Restaurant
4220 N 34th StTampa, FL 33610
30. Sabor Latino
this place is wonderful WE went to Sarasota on vacation and we were looking for latin food and we ran into this amazing place ,food was very good …


text:
CHINA: Diageo's Don Julio to "take advantage" of new Tequila rules
Diageo has welcomed the lifting of China's ban on high-methanol Tequila and said its Don Julio brand is well placed to capitalise on the market's projected growth.
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text:
The Food and Drug Administration proposed the first regulations
for mobile health applications on July 19, focusing on products that act
like medical devices, The Washington Post reports.
The rapidly growing area, which includes apps such as heart
monitors and X-ray viewing boards, are causing some alarm because officials want to
make sure they are safe for patients.
The FDA won't enforce
the guidelines until it collects feedback from health care providers and
manufacturers during the next 90 days, the article said.
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Check out the digital edition of FCW magazine -- the federal IT community's premier publication.
In this issue: Profiles of every Fed 100 winner, the government and industry Eagle Award winners and more.
See the latest issue
Two key IT modernization bills could move in Congress, despite the compressed legislative calendar and the politically charged election year.
CyTech Services, the veteran-owned small business that may have played a significant role in discovering the OPM hack a year ago, says it's never been paid for that work.
Although most agencies are on track to meet a presidential directive to archive email records electronically, a few will need more time.
Limiting the impact of open source policy to a time- and scope-constrained pilot forgoes a unique and long-needed opportunity to modernize government, argues a leading open source advocate.
Agencies can put more money on mission by analyzing accounts payable data. The former leader of the U.K.'s Government Procurement Service explains what it takes.
This year's winners were honored in person at the April 7 Fed 100 Awards Gala. Click here to learn more about their accomplishments.
With a handful of questions to industry, the Department of Veterans Affairs signaled a big move to the cloud as part of its "buy first" initiative.
Managing a workforce that oversees 7,000 contracts and $3.5 billion in federal spending, Mary Davie sits in the catbird seat of the government's vast IT acquisition operation.
Dan Chenok, the IBM Center for the Business of Government's executive director, seems to have a hand in virtually every discussion devoted to making government run better.
By what stretch of the imagination is binary code running on a mobile platform FOOD or DRUG? You've got to be kidding me. Maybe we should get the EPA to regulate any website that advocates, uses, discusses, or mentions green technology, conservation, or the environment. Let's regulate the NRA website via the ATF. Let's regulate Ford's website through the DoT. I could go on, but I believe I've made my point.
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Pall of gloom over village after 12 charred to deathAurangabad: A pall of gloom has descended on Harinagar village under Daudnagar sub-division, 45km from the district HQ here, following the death of 12 persons in a fire on Friday. All the deceased belonged to one family of dalit community. Police ... Times of India, 1 week ago
Aurangabad police arrest three wanted Maoists
Aurangabad police on Thursday arrested three wanted Maoists from different localities under Daudnagar sub-division.Times of India, 2 weeks ago
Magadh fire: DMD for strict maintenance of Standard Operating Procedure
GAYA: Following major incidents of fire in Jahanabad and Aurangabad districts of Magadh division, the disaster management department has directed officials to strictly follow the standard operating procedure. Major loss of life and property has been ...Times of India, 1 week ago 13 killed in fire Bihar's Aurangabad Ani News, 1 week ago 13 killed in fire in Bihar`s Aurangabad Ani News, 1 week ago
More from: Sify, Yahoo! India...and 1 other sources
15 burnt alive, 3 injured in Aurangabad inferno
Aurangabad, Apr 22 (UNI) At least 15 people, including four women and 10 children, were burnt alive and three others sustained injuries, when a major fire broke out at Harinagar Tola in Tarar village under Daudnagar police station area in this ...United News of India, 1 week ago
Fire Kills 12 Guests Sleeping After Wedding In Bihar's Aurangabad
Twelve people including six children and two women were burnt alive as fire swept a row of houses, including one hosting a wedding party, in a village in Bihar's Aurangabad district, police said. Police officers, citing villagers, said the fire ...NDTV, 1 week ago 10 burnt alive in Bihar fire incident Prokerala, 1 week ago 10 charred to death in Bihar fire mishap Rediff.com, 1 week ago
Three children among 10 burnt alive in Bihar
At least 10 people, including five women and three children, on Friday were burnt alive at Harinagar Tola in Tarar village under Daudnagar police station of Aurangabad district when a fire engulfed hundreds of houses in the village. The victims ...The Hindu, 1 week ago Three children among 10 killed in Bihar fire The Hindu, 1 week ago
10 burnt alive in Bihar
Aurangabad, Apr 22: Altogether 10 people, including five women, were burnt alive at Harinagar Tola in Tarar village under Daudnagar police station when an accidental fire engulfed several villages in the district today. A dwelling unit constructed ...Newshence.com, 1 week ago 12 burnt alive in Bihar United News of India, 1 week ago
8 kids among 12 charred to death in Bihar's Aurangabad
AURANGABAD: At least 12 persons, eight of them children, were roasted alive in a fire that broke out at Harinagar village under the Daudnagar sub-division of the district on Friday evening. Strong wind was stated to be the reason behind the tragedy.Times of India, 1 week ago 8 kids among 12 roasted alive in Aurangabad village Times of India, 1 week ago
Three burned alive, 400 houses gutted in state
Motihari/Khagaria/Aurangabad/Darbhanga: Over 400 houses were reduced to ashes and at least three persons, including a woman and two boys, were charred to death in separate incident of fire, at different places in the state in the last 24 hours. ...Times of India, 2 weeks ago
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Severe drought reduces illegal marijuana and opium planting in Northern Mexico. The drought afflicting the area is described as the worst in 70 years.
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