text:
|Publication number
|Publication type
|Application number
|Publication date
|Jul 30, 2002
|Filing date
|May 24, 1999
|Priority date
|Jun 26, 1991
|Fee status
|Also published as
|Publication number
|Felix Franks, Ross Henry Hatley, Sheila Frances Mathias
|Original Assignee
|Inhale Therapeutic Systems, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (76), Non-Patent Citations (60), Referenced by (34), Classifications (39), Legal Events (7)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation of U.S. application Ser. No. 08/241,457, filed on May 11, 1994, now U.S. Pat. No. 5,928,469, and claims the foreign priority benefits under U.S.C. �119 of Great Britain Patent Application No. 9113798.4, filed Jun. 1991 and of Great Britain Patent Application No. 9207839.3, filed Apr. 9, 1992.
This invention relates to the stabilisation and storage of materials. The principal envisaged field of application is materials employed in the biochemical field and some pharmaceuticals.
A few biologically active materials (e.g. some proteins) are sufficiently stable that they can be isolated, purified and then stored in solution at room temperature. For most materials however this is not possible and some more elaborate form of stabilisation/storage procedure must be used.
As discussed in our co-pending European application published as EP-A-383569 a number of storage techniques are known but are not universally applicable to materials which give rise to a storage problem.
That pending application discloses the storage of materials by incorporating them into a water-soluble or water-swellable substance which is in an amorphous, glassy or (much less preferably) rubbery state.
That application discloses the preparation of storable compositions by preparing a solution of the substance(s) to be stored and a water-soluble or swellable substance, then evaporating water from the solution at room temperature or with some heating. Temperatures of 37� C. and 60� C. are exemplified. The solutions were simply held in a stationary container during drying.
It is of course considered prudent to minimise the application of heat when drying a material which is not particularly stable. Freeze drying is a prime example of this.
Spray drying is a known process for drying a solution or suspension to a solid, particulate form. The process entails delivering the solution or suspension into a flow of preheated gas, usually air, whereupon water rapidly evaporates from the droplets. It is widely used in the manufacture of detergent powders and in that field it is well known that certain materials are not stable to spray-drying conditions.
Spray drying has been used to kill microbial cells, for instance in dairy products as disclosed by A. Chopin et al, Can J. Microbiol 23, 716 (1977). EP-A-366303 discloses the use of spray drying to dry a cell composition with the intention that the cells will be killed but cell components such as enzymes will be recoverable from the dried composition. Spray drying has been used in attempts to dry microbial cells to a state of suspended animation from which viable cells can be recovered, but even in a relatively favourable case losses of 97% were recorded after 30 days storage at room temperature as disclosed by I. A. Abd el Gawad et al Egyptian Journal of Dairy Science, 17 273 (1989).
Surprisingly, we have now found that spray drying can be used to make storable compositions by drying mixtures of the material(s) to be stored and aqueous solutions of a water-soluble or water-swellable substance which forms a glassy (or possibly rubbery) state on drying.
According to this invention, therefore, we provide a process of rendering a material suitable for storage comprising spraying into a hot gas stream, an aqueous mixture of the material and a carrier substance which is water-soluble or water-swellable, thereby drying the mixture to particles in which the said carrier substance is in a glassy or rubbery state, and separating the particles from the gas stream.
This process is of course also a process for preparing a storable composition.
The aqueous mixture of the material for storage and the carrier substance will generally be formed by mixing the material with the carrier substance in the presence of water. However, it is possible that the material to be stored will be provided as a solution which already contains a substance which is able to form a glass and so is suitable as a carrier substance, so that deliberate addition of a carrier substance is unnecessary.
As will be explained in more detail below it is preferred that the composition produced by the drying procedure displays a glass transition temperature of at least 20� C., preferably at least 30� C. and possibly well above this e.g. at least 50� C.
The invention may be utilised for stable storage of a single material, or for a mixture of materials which have little or no effect on each other.
However, a further possibility is that the invention is used to produce a composition which contains a plurality of materials which (when in contact with water) form part or all of a reacting system. These materials may be fairly simple chemicals.
Yet another possibility is that the material comprises viable biological cells.
Material Stored (i) inanimate materials
The material(s) stabilised for storage may potentially be any of a wide range of materials which are ordinarily liable to undergo chemical reaction, and so are not stable during storage at ambient temperature of 20� C.
One category of materials to which the invention is applicable is proteins and peptides, including derivatives thereof such as glycoproteins. Such proteins and peptides may be any of: enzymes, transport proteins, e.g. haemoglobin, immunoglobulins, hormones, blood clotting factors, other blood plasma components and pharmacologically active proteins or peptides.
Another category of materials to which the invention is applicable comprises nucleosides, nucleotides, dinucleotides, oligonucleotides (say containing up to four nucleotides) and also enzyme cofactors, whether or not these are nucleotides. Enzyme substrates in general are materials to which the invention may be applied.
The material for stabilisation and storage may be isolated from a natural source, animal, plant, fungal or bacterial, may be produced by and isolated from cells grown by fermentation in artificial culture, or may be produced by chemical synthesis. Such cells may or may not be genetically transformed cells.
The material will need to be soluble in aqueous solution, at least to the extent of forming a dilute solution which can be used for incorporation into the carrier substance.
As mentioned above, a possibility is to store more than one component of a reacting system in a glass. This can be useful for materials which will be required to be used together in, for example, an assay or a diagnostic kit.
Storing the materials as a single glassy preparation provides them in a convenient form for eventual use. For instance, if an assay requires a combination of one or more substrates, and/or a cofactor and an enzyme, two or all three could be stored in a glass in the required concentration ratio and be ready for use in the assay.
If multiple materials are stored, they may be mixed together in an aqueous solution and then incorporated together into a glass. Alternatively they may be incorporated individually into separate glasses which are then mixed together.
When multiple materials are stored as a single composition (which may be two glasses mixed together) one or more of the materials may be a protein, peptide, nucleoside, nucleotide or enzyme cofactor. It is also possible that the materials may be simpler species. For instance a standard assay procedure may require pyruvate and NADH to be present together. Both can be stored alone with acceptable stability. However, when brought together in aqueous solution they begin to react. If put together in required proportions in the glassy state they do not react and the glass can be stored.
Material to be Stored (ii) cells
In a significant development of this invention we have found that the material which is stored may comprise viable biological cells. The composition obtained by spray drying can then contain the cells in a state of suspended animation, and viable cells can be recovered from storage. Cells which may be placed in a storable condition by the method of the invention will preferably be existing as single cells, being either a single cell organism or being cells which are in culture as individual, undifferentiated cells. In particular the cells may be a bacterial culture, which may be isolated from nature or may be a laboratory or industrial bacterial strain including genetically transformed bacteria. The cells may be eukaryotic cells, notably including yeasts but also other fungal cultures. Again the cell culture may be a natural isolate or a laboratory or industrial culture produced by fermentation including genetically transformed strains.
The Carrier Substance
A glass is defined as an undercooled liquid with a very high viscosity, that is to say at least 1013 Pa.s, probably 1014 Pa.s or more.
Normally a glass presents the appearance of a homogeneous, transparent, brittle solid which can be ground or milled to a powder. In a glass, diffusive processes take place at extremely low rates, such as microns per year. Chemical or biochemical changes involving more than one reacting moiety are practically inhibited.
Above a temperature known as the glass transition temperature Tg, the viscosity drops rapidly and the glass turns into a rubber, then into a deformable plastic which at even higher temperatures turns into a fluid.
The carrier substance employed in this invention must be hydrophilic—either water-soluble or water-swellable—so that water will act as a plasticiser. Many hydrophilic materials, both of a monomeric and a polymeric nature either exist in an amorphous state or can be converted into such an amorphous state which exhibit the glass/rubber transitions characteristic of amorphous macromolecules. They have well defined glass transition temperatures Tg which depend on the molecular weight and on molecular complexity of the substance concerned. Tg is depressed by the addition of diluents. Water is the universal plasticiser for all such hydrophilic materials. Therefore, the glass/rubber transition temperature is adjustable by the addition of water or an aqueous solution.
It will generally be preferred to employ a carrier substance which, on its own, forms a glass rather than a rubber at ambient temperature. Hence it will be preferable that the carrier substance, on its own, is able to exist in a glassy amorphous state with a glass transition temperature Tg above 20� C.
Mixtures of substances may be used as carrier substance if the components are miscible as a solid solution. If so, material(s) of lower Tg serve as plasticiser(s) for material(s) of higher Tg.
A composition prepared by a process of this invention will generally have a glass transition temperature Tg below that of the pure carrier substance.
If the dried composition is stored in the glassy state (below Tg) the deterioration of the active material is retarded to the extent that, on practical time-scales, even substances which in solution are extremely labile are found to possess long shelf-lives.
Full biochemical activity is maintained, but locked in, throughout this period at temperatures below Tg and can be rapidly released by resolubilization of the glass in an aqueous medium.
If the material to be stored is inanimate, then in order to provide a long storage life, it will generally be desirable that the dried composition has a glass transition temperature of at least 20� C. For achieving this it is desirable that the glass-forming carrier substance, when anhydrous or nearly so, displays a glass transition temperature Tg of at least 40� C., better at least 50� C. There is no theoretical upper limit on Tg. In practice suitable materials have values of Tg below 250� C., usually below 200� C. A desirable range is therefore 50� to 200� C., preferably 60� or 80� C. to 150� or 180� C.
If the material to be stored comprises biological cells, the composition containing the cells may well be in an amorphous rubbery state. Thus a composition obtained by the method of the invention and comprising biological cells may have a glass transition temperature Tg below 20� C. for instance in the range between 0� C. and 20� C. even though its carrier substance has a glass transition temperature above 20� C. Such compositions could readily be stored with refrigeration to approximately 0� C. so as to be stored in the glassy rather than rubbery state.
If Tg of the final composition made according to the invention is sufficiently high, storage can be at room temperature. However, if Tg of the composition is close to or below room temperature it may be necessary or desirable to refrigerate the composition if storage is for a prolonged period. This is less convenient but still is more economical than freeze-drying.
If a composition is heated above its Tg during storage, it will change to its rubbery state. Even in this condition stored materials are stable for a considerable period of time. Consequently, it may well do no harm if the temperature of a stored composition is allowed to rise above Tg for a limited time, such as during transportation.
If a composition is maintained slightly above its Tg (and therefore in a rubbery condition) the storage life will be limited but still considerable and the benefit of the invention will be obtained to a reduced extent.
Conversely, if Tg of the composition is well above room temperature, the composition is better able to withstand storage at an elevated temperature, e.g. in a hot climate.
The carrier substance should be sufficiently chemically inert towards an inanimate material which is to be incorporated in it. An absolute absence of chemical reactivity may not be essential, as long as it is possible to incorporate the material, store the glass, and recover the material without serious degradation through chemical reaction.
If the material to be stored comprises biological cells, the carrier substance should not be toxic to these cells. Indeed the carrier substance may be a nutrient for the cells and able to support cell division so long as the drying operation is carried out at sufficient rapidity that the carrier substance is not consumed by the cells.
As mentioned above, the carrier substance, which very preferably forms a glass, may be deliberately added to form the mixture which is spray dried. For instance it may be added to a solution of the material to be stored.
Many organic substances and mixtures of substances will form a glassy state on cooling from a melt.
In this context carbohydrates are an important group of glass forming substances: thus candy is a glassy form of sugar (glucose or sucrose). The Tg for glucose, maltose and maltotriose are respectively 31, 43 and 76� C. (L. Slade and H. Levine, Non-equilibrium behaviour of small carbohydrate-water systems, Pure Appl. Chem. 60 1841 (1988)). Water depresses Tg and for these carbohydrates the depression of Tg by small amounts of moisture is approximately 6� C. for each percent of moisture added. We have determined the Tg value for sucrose as 65� C.
In addition to straightforward carbohydrates, other polyhydroxy compounds can be used, such as carbohydrate derivatives and chemically modified carbohydrates (i.e. carbohydrates which have undergone chemical reaction to alter substituents on the carbon backbone of the molecule but without alteration of that backbone).
Another important class of glass forming substances are water-soluble or water-swellable synthetic polymers, such as polyacrylamide.
Yet another class of substances which are suitable are proteins and protein hydrolysates. Thus albumin can be used, and so can hydrolysis products of gelatin.
A group of glass forming substances which may in particular be employed are sugar copolymers described in U.S. Pat. No. 3,300,474 and sold by Pharmacia under the Registered Trade Mark "Ficoll". This U.S. patent describes the materials as having molecular weight 5,000 to 1,000,000 and containing sucrose residues linked through ether bridges to bifunctional groups. Such groups may be alkylene of 2, 3 or more carbon atoms but not normally more than 10 carbon atoms. The bifunctional groups serve to connect sugar residues together. These polymers may for example be made by reaction of the sugar with a halohydrin or a bis-epoxy compound.
The suitability of an intended carrier substance, and the amount of material which can be incorporated into it can both be checked by preparing a glassy or rubbery composition with the material incorporated, and then recovering the material without any substantial period of storage.
Tg values can be determined with a differential scanning calorimeter and can be detected as a point at which a plot of heat input against temperature passes through an inflection point—giving a maximum of the first temperature derivative.
As was also mentioned above, a further possibility is that the material which is to be stored may occur in a form which incorporates a suitable carrier substance. It is envisaged in particular that this situation may arise with products derived from blood plasma where the material to be stored is a relatively minor component of the blood plasma and other components which naturally occur in the blood plasma, notably albumin, are able to form a glass on drying. In such a situation there would be no need for separate addition of a glass-forming carrier substance although the possibility is not ruled out.
The first stage is to provide an aqueous mixture of the material to be stored and the water-soluble or water-swellable carrier substance. This may be done by mixing the carrier substance, as a powder or as an aqueous solution, with a solution or suspension of the active material to be stored. Alternatively a suitable solution may be available from some other process, without requiring deliberate addition of glass-forming carrier, as mentioned above.
When the invention is applied to the storage of cells, a possibility which has been found suitable for some cells is to suspend the cells in a dilute aqueous solution containing the carrier substance and then subject this to the drying step. To arrive at the suspension, solid carrier material may be dissolved in a suspension of the cells in a dilute aqueous buffer solution. This can lead to a composition with a glass transition temperature above ambient and temperature having very good storage stability.
For some cells, it has been found that survival during drying is better if the cells are dried from a mixture which is rather closer to their normal growth medium. This may for example be carried out by adding carrier substance to an aqueous culture of the cells in their growth medium and drying the resulting mixture. Many bacteriological growth media have a relatively high electrolyte content and this electrolyte or other components is effective to lower the glass transition temperature of the dried product. If this procedure is followed the composition is likely to have a glass transition temperature below ambient temperature making it desirable to store the composition under refrigeration. In this situation the need for refrigerated storage is accepted for the sake of greater survival of cells during the drying operation.
In order to determine whether any cell species can be dried from a simple aqueous suspension or whether it should be dried from something akin to its growth medium, a test can be made by drying some cells by each procedure, then recovering the cells without storing for any substantial period of time and determining the quantity of cells which have survived.
After arriving at a mixture containing the material to be stored and a carrier substance the next step is a spray drying operation in which the above aqueous mixture is sprayed into a hot gas stream. The gas will generally be air but could be some other gas such as nitrogen.
Apparatus to carry out spray drying on a fairly small scale is available from various manufacturers. One is Drytec Ltd, Tonbridge, Kent who manufacture a pilot plant scale dryer. Another manufacturer is Lab-Plant Ltd of Longwood, Huddersfield, England who manufacture a laboratory scale dryer.
Process plant to carry out spray drying on a larger scale is also well known.
The sole drawing is a diagrammatic illustration of laboratory scale spray-drying apparatus.
In this apparatus air from the atmosphere is drawn in by a blower 10 and passes over an electric heater 12 after which the air passes down a main chamber 16. The aqueous mixture to be sprayed is drawn up from a supply vessel 18 by means of a peristaltic metering pump 20 and delivered to a spray nozzle 22 which discharges the aqueous mixture as a fine spray into the stream of hot air coming from the heater 12.
The droplets of spray are dried to solid powder form as they pass down within the main chamber 16. The powder is entrained in the air which has passed down the main chamber 16. This leaves by an exit tube 26 at one side delivering to a cyclone separator 28 which serves to remove entrained solid particles from the air stream. The solid particles which are separated from the air stream in this way are collected as the product in a detachable vessel 30 while the air passes out to atmosphere through an exhaust tube 32. Solids which stick to the wall of the main chamber fall into waste container 24.
A significant parameter in the operation of any spray drying apparatus is the temperature of the gas stream which is admitted to the main chamber and into which the spray is delivered. For the present invention this inlet temperature of the gas stream will generally exceed 80� C., will usually exceed 90� C. and may well lie in a range from 100 to 250/300� C.
The aqueous mixture which is delivered into the gas stream may typically contain from 10 up to 50 or even 250 grams per liter of the carrier substance. The content of material to be stored can vary widely but will often lie within a broad range from 10−3% to 10% by weight of the carrier substance. Much lower levels are possible. A substance whose desired activity is present when very dilute might be stored at a concentration as low as 10−5% by weight of the dried composition. By contrast storage at a concentration of stored material of up to 50% by weight or even more can also be envisaged.
In many instances the carrier substance will form at least 20%, better at least 25% or 30% by weight of the particulate composition formed by drying and usually at least 50% by weight of that particulate composition.
The particulate solid compositions produced by discharging an aqueous mixture into a heated gas stream in accordance with the invention will frequently be sufficiently dry for storage without further processing. However it is within the scope of the invention to subject these compositions to further drying such as by subjecting them to sub-atmospheric pressure possibly accompanied by moderate heating so as to reduce any residual moisture content.
When the material to be stored comprises biological cells the moisture content of a composition produced by a method of the invention would typically be in the range 3 to 9% by weight. A low moisture content enhances stability.
The suitability of conditions for preparing a storable composition can be checked by carrying out the preparation but recovering the material without any substantial period of storage and determining the proportion which has survived. As mentioned above storage stability can if desired be tested by storage at a temperature above ambient, although not above the Tg value of the glass.
Recovery from storage
Recovery of stored material from a composition produced by drying in accordance with this invention can be effected by simply adding water or aqueous solution to a quantity of the composition with the active material therein. If the carrier substance is water-soluble the result is a solution of the material and the carrier substance.
Separation by chromatography to isolate a stored, active, inanimate material from the carrier substance is possible. However, in general it will be neither desirable nor necessary. Instead the carrier substance is chosen so that it will not interfere with the use (e.g. an assay) of the stored, active material.
In the case of a water-swellable carrier substance, it will remain out of solution, perhaps as a gel, and the solution of the material can be separated by centrifugation if required.
A further aspect of this invention is use of a composition prepared according to the invention to provide a solution of the stored material, by addition of water or aqueous solution to the composition. The application to which the solution of recovered material is put may or may not be a therapeutic application.
The active material to be placed in a storable form was lactate dehydrogenase (LDH) type XI (ex rabbit muscle) from Sigma Chemical Co. The glass forming carrier substance employed was Ficoll 400 DL (Pharmacia, Reg. Trade Mark) which is a copolymer of sucrose and epichlorohydrin. It is water-soluble and has a Tg of 97� C.
8 g of Ficoll was added to 200 ml of 0.01 M phosphate buffer pH 7 and stirred at ambient temperature until a clear solution was obtained. This solution was then cooled to, and stored at, 4� C. until use. All solutions were used within 72 hr. To 200 ml of the phosphate buffer/Ficoll solution (at 4� C.) 10 mg of LDH was added. The resulting solution was then passed through a spray-drier (Drytec, pilot scale drier) to give a dry powder containing 1.25 mg LDH/g powder. An air inlet temperature of 210� C. (at the air inlet to the drying chamber) was employed; this produced an air temperature of 70� C. at the entry to the cyclone separator.
The dried material was a particulate solid. This was divided into 2 g portions and placed into vials. The vials were sealed under a normal atmosphere and stored at ambient temperature (fluctuating between 10 and 35� C.). Portions of powder were periodically removed and the vials resealed. Assays were performed following drying, and periodically thereafter. An assay of the solution prior to drying was used as the control.
The actual enzyme activity was determined by the following procedure (Hatley, Franks and Mathias, Process Biochemistry, December 1987 page 170) and based on a minimum of nine replicates. The powder was dissolved in phosphate buffer to give a test solution calculated to contain 1 μg protein per ml (for the control sample a portion of the solution to be dried was taken and diluted to 1 μg per ml).
Activity of the test solution was then measured: 2.7 ml of 0.01 phosphate buffer pH 7, 0.1 ml of 2 mg/ml NADH and 0.1 ml of 10 mM pyruvate were placed into a cuvette of light path 10 mm. The cuvette was capped and shaken. 0.1 ml of the test solution was added and the cuvette again capped and shaken. The absorbance at 340 nm was recorded at 30 s intervals for a total of three minutes. The temperature of the solution was also noted. The absorbance change per minute, δA, was calculated. The enzyme activity was then calculated as follows:
δA=the absorbance change per minute
6.25=a correction factor for the molar absorbance of NADH.
TCF=a temperature correction factor applied to all assays performed at temperatures other than 25� C.
C=the concentration of protein in mg/ml
The control (unprocessed) solution had an activity of 322 U/mg protein. This was taken as 100% and all subsequent assays quoted relative to this value. Enzyme activities were:
Storage period after drying (days)
Product Tg was determined throughout the storage period. The value decreased from 79� C. to 64� C. as moisture entered the product as it was repeatedly opened and resealed. However, throughout the experiment the product remained in the form of a glass at the storage temperature.
The results show that enzyme activity was effectively preserved intact through the spray drying procedure and subsequent storage.
Example 1 was repeated with an air inlet temperature of 130� C. This produced an air temperature of 60� C. at the entry to the cyclone separator. After 138 days storage, enzyme activity was 112% of the control value.
Example 1 was repeated with two variations. 50 mg of LDH was added to 200 ml of the phosphate buffer/Ficoll solution (at 4� C.). Spray drying gave a dry powder containing 6.25 mg LDH/g powder. Air inlet temperature was 150� C. This produced an air temperature of 70� C. at the entry to the cyclone separator.
After 138 days storage enzyme activity was 117% of the control value.
The active material was alcohol oxidase from Provesta Enzymes. The glass forming substance employed was Ficoll 400 DL (Pharmacia, Reg. Trade Mark) as used in Example 1.
8 g of Ficoll was added to 200 ml of 0.1 M phosphate buffer pH 7.5 and stirred at ambient temperature until a clear solution was obtained. This solution was then cooled to, and stored at, 4� C. until use. All solutions were used within 72 hr. 100 μg (100 units) of alcohol oxidase was added to 200 ml of the phosphate buffer/Ficoll solution (at 4� C.). The resulting solution was then passed through a spray-drier (Drytec pilot scale drier) to give a dry powder calculated to contain 0.0125 U alcohol oxidase/g powder. An air inlet temperature of 150� C. was employed; this produced an air temperature of 70� C. at the entry to the cyclone separator.
The dried material was divided into 2 g portions and placed into vials. The vials were sealed under a normal atmosphere and stored at ambient temperature (fluctuating between 10 and 35� C.). Portions of powder were periodically removed and the vials resealed. Assays were performed prior to drying and following drying.
As a comparison freeze-dried samples were prepared using principles described in the literature, (F Franks, Cryo-Letters 11, 93-110). 300 mg of Ficoll was dissolved in 20 ml of 0.1 M phosphate buffer pH 7. 1000 units of alcohol oxidase was added to the solution. The solution was divided into 0.2 ml portions in ten 5 ml vials. These were frozen to −30� C. in a small laboratory freeze-drier. A vacuum of 1�10−1 mbar was applied and the samples dried for 24 hr. The vacuum was reduced to its minimum setting of 5�10−2 mbar and the temperature raised at 5� C./hr to 30� C. After holding the sample at this temperature for two hours the vials were removed and sealed with Bakelite screw-caps. Each vial was calculated to contain 1 unit of enzyme, assuming no activity had been lost during processing. Enzyme activity was assayed before and after freeze drying.
As a further comparison, samples of solution similar to those which were freeze dried, were dried in stationary vials, as described in our published European application EP-A-383569. Enzyme activity was assayed before and after drying.
Assays of enzyme activity were performed as follows: Dried powder was dissolved in phosphate buffer to give a test solution calculated to be 0.1 U per ml (for the control sample a portion of the solution to be dried was taken and serially diluted to 0.1 U per ml).
A stock solution was prepared containing:
16 mg 2,2′-azino-di(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)
2 ml absolute ethanol
1 ml of 1 mg/ml horseradish peroxidase in distilled water
All made up to 100 ml with 0.1 M potassium phosphate buffer, pH 7.5.
Another stock solution was:
0.1 M potassium phosphate buffer, pH 7.5.
2.5 ml of the ABTS stock solution was pipetted into a 3 ml cuvette, 25 μl of the enzyme solution with an expected activity of 0.1 U alcohol oxidase per ml was added. The cuvette was closed and its contents mixed by inversion. The cuvette was placed in a spectrophotometer and the absorbance change over 3 min at 390 nm recorded.
The absorbance change per minute was determined and multiplied by 3.06 (the extinction coefficient plus the cuvette dilution factor) to give the concentration of enzyme in the solution. Activity retention was calculated by dividing the measured value by 0.1 (the expected activity in the solution) and multiplying by 100 to give a percentage.
It was found that after drying the activity as a percentage of that before drying was:
Freeze drying
Drying in stationary vial
Spray drying
Suppliers' catalogues show that commercial freeze drying of alcohol oxidase reduces activity to 25%.
The spray dried and freeze dried alcohol oxidase were stored at 35� C. and activity was assessed at intervals. The freeze dried material lost all activity in 20 days. After 30 days the spray dried material retained 90% of its activity assayed directly after spray drying.
Example 1 was repeated, replacing Ficoll with Byco A which is a cold water soluble protein obtained from gelatin by enzymic hydrolysis. It has a Tg value of 126� C. Byco A is available from Croda Colloids Ltd, Widnes, Cheshire, England.
As in Example 1 enzyme activity was measured both directly following drying and after a period of storage. The measured values of enzyme activity were 88% directly following drying and 113% after 103 days storage at ambient temperature.
As in Example 1, the active material was lactate dehydrogenase (LDH) type XI and the glass forming substance was Ficoll 400 DL.
10 g of Ficoll was added to 100 ml of 0.01 M phosphate buffer pH 7 and stirred at ambient temperature until a clear solution was obtained. This solution was then cooled to, and stored at, 4� C. until use. All solutions were used within 72 hr. 10 mg of LDH was added to 200 ml of the phosphate buffer/Ficoll solution (at 4� C.). The solution was then passed through a spray-drier (Lab-Plant SD-04) to give a dry powder containing 1 mg LDH/g powder. An air inlet temperature of 170� C. was employed; this produced an air outlet temperature of 75� C. The dried material was sealed in a collection bottle in a normal atmosphere. The samples were stored at ambient temperature (fluctuating between 10 and 35� C.). Portions of powder were periodically removed and assays performed as for Example 1.
Enzyme activities were:
period (days)
This example describes converting biological cells into a storable composition by a method embodying the invention and then recovering the cells after varying periods of time.
The amount of aqueous solution used in recovery of cells was chosen such that, if there was survival of every cell, the concentration of cells in the recovered suspension would be the same as in the initial suspension before drying. The concentrations of viable cells in these suspensions, before drying and after recovery, were assayed by using a standard quantity of the suspension to make an agar plate. The plate is then incubated and the number of growing colonies was counted.
5 g Ficoll 400 DL (Pharmacia, Reg Trade Mark) was dissolved in 100 ml growth medium. 5 ml Lactobacillus bulgaricus cells in growth medium were added to the Ficoll solution to give a cell suspension of approximately 1�107 cells/ml. The suspension was then spray dried, using a laboratory scale spray drier, (Lab-Plant SD-04), with input temperature 190� C. and output temperature 104� C., and collected as a dry powder. The powder was immediately dispensed into several small vials and the vials were capped and stored at 4� C.
Viability testing:
Before drying (control):
0.5 ml cell suspension was transferred to a sterile tube containing 9.5 ml sterile growth medium. Serial dilutions were made in the manner of Miles and Misra. 1 ml cell suspension from an appropriate dilution was transferred to a 9 cm sterile petri dish and mixed with 10 ml molten growth medium at approximately 37� C. The agar plate was then allowed to set and incubated for 48 h at 35� C. The colony forming units were then counted.
After drying:
0.5 ml growth medium was added to 0.049 g dried powder (calculated weight of solids in 0.5 ml original cell suspension) and left to stand at room temperature for 30 min to ensure full rehydration. The assay was continued as described above and the number of colony forming units was compared to the number found in the sample assayed before drying.
Further dried samples were assayed at intervals.
Cfu's in
% of
Storage time
10−5 dilution
Before drying
Directly after
2 weeks
4 weeks
5 g Dextrin 10 (maltodextrin, ex Fluka) was dissolved in 100 ml 10% skimmed milk solution. 5 ml Lactobacillus bulgaricus cells in growth medium were then added to the solution to give a cell suspension of approximately 1�107 cells/ml. The suspension was then spray dried, using a laboratory scale spray drier, (Lab-Plant SD-04), with input temperature 160� C. and output temperature 89� C., and collected as a dry powder. The powder was immediately dispensed into several small vials and the vials were capped and stored at 4� C.
Viability testing was carried out as in the previous Example. For testing after drying 0.5 ml growth medium was added to 0.076 g dried powder as the calculated weight of solids in 0.5 ml of the original cell suspension.
Cfu's in
% of
Storage time
10−5 dilution
Before drying
Directly after
2 weeks
4 weeks
The moisture content of the spray dried powder was measured by a Karl Fischer coulometric method and found to be 5.2%.
5 g raffinose was dissolved in 100 ml growth medium. 5 ml Lactobacillus bulgaricus cells in growth medium were added to the solution to give a cell suspension of approximately 1�107 cells/ml. The suspension was then spray dried, using a laboratory scale spray drier, (Lab-Plant SD-04), with input temperature 190� C. and output temperature 108� C., and collected as a dry powder. The powder was immediately dispensed into several small vials and the vials were capped and stored at 4� C.
Viability testing was carried out as in Example 7.
Cfu's in
% of
Storage time
10−5 dilution
Before drying
Directly after
2 weeks
4 weeks
8 weeks
5 g sodium glutamate was dissolved in 100 ml growth medium. 5 ml Lactobacillus bulgaricus cells in growth medium were added to the solution to give a cell suspension of approximately 1�107 cells/ml. The suspension was then spray dried, using a laboratory scale spray drier, (Lab-Plant SD-04), with input temperature 190� C. and output temperature 114� C., and collected as a dry powder. The powder was immediately dispensed into several small vials and the vials were capped and stored at 4� C.
Viability testing was carried out as in Example 7.
Cfu's in
% of
Storage time
10−5 dilution
Before drying
Directly after
2 weeks
4 weeks
8 weeks
|Cited Patent
|Filing date
|Publication date
|Apr 16, 1872
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|Nov 17, 1970
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|Kraftco Corp
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|American Potato Co
|Preparation of dehydrated mashed potatoes
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|Polak's Frutal Works, Inc.
|Process of encapsulating an oil and product produced thereby
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|Process for improved ruminant feed supplements
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|Warner-Lambert Company
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|Jun 19, 1985
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|Publication date
|Aug 23, 2000
|Jun 15, 2004
|Advanced Inhalation Research, Inc.
|Formulation for spray-drying large porous particles
|Jul 22, 2002
|Sep 5, 2006
|Brigham Young University
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|Alkermes, Inc.
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|Novartis Ag
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|Biomatrica, Inc.
|Integration of sample storage and sample management for life science
|Jul 30, 2008
|Jul 14, 2015
|Biomatrica, Inc.
|Integration of sample storage and sample management for life science
|Jul 26, 2011
|Jun 28, 2016
|Biomatrica, Inc.
|Compositions for stabilizing DNA and RNA in blood and other biological samples during shipping and storage at ambient temperatures
|Apr 8, 2014
|Aug 23, 2016
|Novartis Ag
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|Sep 13, 2016
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|May 5, 2010
|Jan 31, 2017
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|Aug 8, 2017
|Biomatrica, Inc.
|Formulations and methods for stabilizing PCR reagents
|Nov 20, 2002
|Jul 10, 2003
|Advanced Inhalation Research, Inc.
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|Nov 20, 2002
|Jul 10, 2003
|Advanced Inhalation Research Inc.
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|Apr 28, 2003
|Jan 29, 2004
|Advanced Inhalation Research, Inc.
|Modulation of release from dry powder formulations
|Apr 28, 2003
|Jan 6, 2005
|Advanced Inhalation Research, Inc.
|Modulation of release from dry powder formulations
|Apr 8, 2005
|Dec 15, 2005
|Biomatrica, Inc.
|Integration of sample storage and sample management for life science
|Jul 7, 2005
|Jan 5, 2006
|Edwards David A
|Stable spray-dried protein formulations
|Apr 27, 2005
|Feb 23, 2006
|Bennett David B
|Methotrexate compositions
|Dec 1, 2005
|May 11, 2006
|Biomatrica, Inc.
|Integration of sample storage and sample management for life science
|Dec 5, 2006
|May 10, 2007
|Batycky Richard P
|Particulate compositions for pulmonary delivery
|Oct 22, 2007
|Jul 24, 2008
|Biomatrica, Inc.
|Integration of sample storage and sample management for life science
|Sep 24, 2007
|Sep 18, 2008
|Advanced Inhalation Research, Inc.
|Particles for inhalation having rapid release properties
|Jul 30, 2008
|Dec 11, 2008
|Judy Muller-Cohn
|Integration of sample storage and sample management for life science
|U.S. Classification
|435/260, 159/4.01, 530/345, 424/93.45, 424/93.4, 435/188, 424/93.1
|International Classification
|Cooperative Classification
|European Classification
|Feb 25, 2003
Owner name: NEKTAR THERAPEUTICS, CALIFORNIA
Free format text: CHANGE OF NAME;ASSIGNOR:INHALE THERAPEUTIC SYSTEMS, INC.;REEL/FRAME:013525/0753
Effective date: 20030113
|Dec 28, 2005
|Fee payment
Year of fee payment: 4
|Jan 7, 2009
Owner name: NOVARTIS PHARMA AG, SWITZERLAND
Free format text: ASSIGNMENT OF PATENT RIGHTS;ASSIGNOR:NEKTAR THERAPEUTICS;REEL/FRAME:022071/0001
Effective date: 20081231
Owner name: NOVARTIS PHARMA AG,SWITZERLAND
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Effective date: 20081231
|Dec 30, 2009
|Fee payment
Year of fee payment: 8
|Mar 7, 2014
|Maintenance fee reminder mailed
|Jul 30, 2014
|Lapse for failure to pay maintenance fees
|Sep 16, 2014
|Expired due to failure to pay maintenance fee
Effective date: 20140730


text:
St. Louis, MO (PressExposure) June 23, 2009 -- Many young professionals are under the impression that living in the Clayton area is out of their reach. However, there are plenty of very nice and affordable apartments in the 63105 zip code that are well within the 20-somethingâs budget.
âClayton is known for its stately neighborhoods, tree-lined streets and excellent school district,â said Steve Barron, President of Philip H. Barron Realty. âAs a result, many people think that living in Clayton isnât affordable. However, there are many reasonably priced apartments in the Moorlands, just a five minute walk from the heart of the downtown business district. Clayton neighborhoods such as the Moorlands and Clayshire offer Clayton luxury at extremely reasonable prices.â
The Moorlands neighborhood is renowned for unique architecture and old world charm. Within walking distance of Claytonâs restaurants, offices and nightlife, the Moorlands is a popular neighborhood for professionals and families.
Also in Clayton is the Clayshire neighborhood, located next to the St Louis Galleria and within a short walk of Shaw Park and the hub of Clayton. This neighborhoodâs convenient location is adjacent to large corporate offices such as Enterprise Rent-a-Car and Southwest Bank.
âClayton is a perfect location for both young professionals and families. The shopping, dining and nightlife canât be matched. Easy access to the major highways and MetroLink stations make it extremely convenient to get almost anywhere in the metropolitan area. Where else in St. Louis can you walk to work, restaurants, and a premiere shopping center all from your home?â said Steve Barron.
Philip H. Barron Realty recognized the need for affordable housing in Clayton and has restored over 100 apartments in the Moorlands and Clayshire neighborhoods to meet these needs. Ranging from efficient studios to large two bedroom apartments, Philip H. Barron Realty can meet a wide variety of budgets for individuals looking to live alone or with a roommate.
âOur apartments are ideal for young professionals, families and students alike. Because of its close proximity to Washington University many students prefer to live in the Moorlands because it is quieter and safer than the traditional student neighborhoods right next to campus,â said Steve Barron.
Philip H. Barron Realty prides itself on a portfolio of unique Clayton apartment buildings that are clean, desirable and well maintained. With gleaming hardwood floors and plenty of sunlight, renters choosing to make the Moorlands and Clayshire home with Barron Realty will enjoy all the amenities of Clayton without breaking the bank.
PressRelease Distribution By PressReleasePoint
Contact: Meg Fullenkamp Barron Realty.com 1722 Olive Street St. Louis, MO 63103 314-725-1949 email@example.com http://barronrealty.com

subset: commoncrawl


text:
Thalia Getaway Villa - self contained luxury
Villa, Murray Bridge
Rated 5 out of 5 stars 2 reviews
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From $231 per night {{quote.pricing.message}} Total {{quote.pricing.price | format:'currency':'short'}} for
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The Villa
Thalia Getaway is private self-contained luxury accommodation for couples / families, and can accommodate up to 6 people with two Queen size beds and two single beds. Located 8 km out of Murray Bridge towards Mannum on the Adelaide side of the Murray River in South Australia's Riverland Region.
Within walking distance of the river.
The Villa is on 6 acres surrounded by 100 year old peppercorn trees, pines, and gums, it offers an unusual retreat from modern lifestyle.
Thalia Getaway offers a blend of country living with the luxury of modern comforts. The house has been extensively renovated with modern kitchen, Luxury bathroom With double bath, reverse cycle ducted air conditioning and wood fires, but still retaining its charm of yesterday.
A generous cooked and continental breakfast is provided for your first morning, along with a complimentary bottle of Langhorne Creek wine for your arrival.
Entertainment:- 44' TV with DVD player, iPod / I phone dock player, in house DVD's books.
Cooking:- Well equipped Country kitchen, with electric oven / cook top. Microwave oven, fridge freezer, and out door barbecue.
Linen:- Linen supplied with luxury Alpac quilts and electric blankets.
Comfort:- Reverse cycle ducted air conditioning, slow combustion wood fire, double bath, and rainforest shower.
Laundry:- Washing machine, and ironing board
Other:- Under cover parking
Custom:- 6 acre private property for your exclusive enjoyment, 2 double bed rooms with Colonial Queen size beds, One double bedroom with 2 single beds, Lounge room. dinning room, Family kitchen area, Out door setting with barbecue.
Strictly:- Non Smoking
Not recommended for Children under 10
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text:
Plum Branch South Carolina in Mccormick county.
Plum Branch, South Carolina is located in Mccormick county within the zipcode 29845. With a total of 0 golf courses in Plum Branch, it lies at an elevation of 531 feet above sea level. The current date and time in Plum Branch South Carolina is November 21, 2017, 12:24 am. Add a course for free by clicking here or contact us here.


text: Big fan of cots. Quick to breakdown, great sleep on any surface, no pole for the gods to break. Been running the basic Coleman cot the last few years. Been good, get 2 years out of a $50 cot. Easy to breakdown and long enough for a 6'2" guy and a pillow. Perfect for steelheading. Looking to do some boat fishing, long drift, streamer fishing this fall. Hoping to find something under 25 lbs and $150. Any ideas?


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Contact Info / Websites
- XxDarkSonicxX
All 3 movie Reviews
Monkey Island Flash Movie
Rated 4.5 / 5 stars
Haha lol
Veeeeery funny movie xD Watched the german version. I can watch it every time and laugh everytime XD
"Oh dieses Brett ist locker, ich kann flüchten und nehmen Bananen mit. Hallo Affe hier noch mehr Bananen. Er folgt mir. Hier Affe, zieh an dem Hebel und bleib da für immer hengen. Danke!" XD
Zu geil XD
Sonic: Nazo Unleashed Pt1
Rated 3.5 / 5 stars
It is not bad...
Yeah, pretty good movie. But it is a little bit TOO Dragon Ball Z like. Look, they'll become stronger and stronger and stronger and stronger and stronger and then they will defeat the enemy. It's kinda too DBZ styled.
Many words little sense... It is a good movie, with good style (for a flash movie. Hate the Sonic X style XD) etc. Everybode told it already ^^"
Super Mario bros Z intro
Rated 4.5 / 5 stars
Hi, I like your Mario Bros Z movies. But I hate the readings xD Anyway. Really good battles and really good Opening.
But I know that I recognise those scenes of this movie. Do you have taken scenes from other openings? It's just a question. Maybe I am wrong, I don't know.


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A motorist was arrested early today after he drove a car through the front of CNN Center in downtown Atlanta, police said.
The Mercedes-Benz convertible crashed through the glass facade on the Marietta Street side of the building shortly after 4 a.m.
No injuries were reported, but Atlanta police spokesman John Chafee said the car's driver was arrested.
"Officers found the driver in possession of marijuana," Chafee said, adding that the man admitted to officers that he had smoked marijuana earlier.
He said Gerlmy Javon Todd, 22, of Gainesville, Fla., was charged with DUI, reckless driving and possession of marijuana. He has an extensive record in Florida, mostly drug-related offenses, records show.
Dominique Ayers said she is in town for a conference, and was in her hotel room when she heard a "loud boom."
"There was a car in the lobby of the hotel. Not what I'm expecting at 4 o'clock in the morning."
— Atlanta Journal-Constitution photographer John Spink contributed to this article.


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- Please select a date to see the proper prices!
- Meal can be requested on the spot
Best offer
50.31 € / person / night, no meal
Description of the accommodation:
Rooms, services The guests of Sao Salvador Hoteis e Convencoes Salvador may stay in 304 nicely furnished, comfortable rooms in Salvador. You are also welcome at the hotel for a fine local meal in the restaurant. For those arriving by car parking is available nearby. The bar can be an ideal place for a pleasant evening talk or a meeting. In the safe you may secure your valuables. At the hotel you may go online via wireless Internet connection (WiFi). An exact and detailed list of equipment in the rooms and of the services provided by Sao Salvador Hoteis e Convencoes Salvador you will find on the information sheet below, including any applicable additional fees.
Payment Credit card guarantee or advance bank card payment may be required for the reservation, depending on the chosen room, date and price.
Arrival, opening hours The earliest time of check-in is 14:00, the latest time of check-out is 12:00. The personnel can speak Portuguese - Portugal and English languages.
Sao Salvador Hoteis e Convencoes Salvador rooms Rooms
- Please select a date to see the proper prices!
Accommodation services
Free WiFi in shared places
- Finnish sauna
- Pool (Outdoor - seasonally open)
Bar, Elevator, Safe deposit box
Own restaurant
Hairdryer, Mini-bar


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The Legacy Dragonfly Flame from Michael Kelly puts boutique tone and playability within reach. The hand-carved, solid spruce top and AAAA flame maple back and sides are key, offering a full, resonant sound. The unique dragonfly vine inlay is carefully crafted with real pearl and abalone, all on a radiused fingerboard. A scooped fingerboard provides improved access, and top-of-the-line Grover tuners get you tuned up quickly and keep you in tune all night long.
What started as a simple string and pedal sales catalog has grown exponentially over the past thirty years. The early days were a time where catalogs didn't have much of a presence in the industry, but what began as a college dorm room operation grew rapidly. In 1986 we moved to a full product offering and 64-page catalog, which over the years has grown to 162 pages. Join the AMS family and get your free catalog now!
Copyright © 1996-2017 American Musical Supply, Inc. All rights reserved. American Musical Supply reserves the right to correct pricing and/or product specifications in the event of typographical errors found in our print catalog or on our website. If a product is listed at an incorrect price due to such errors or because of inaccurate information received from a supplier, American Musical Supply shall have the right to refuse or cancel any orders placed, whether or not the order has been confirmed and your credit card charged. If your credit card has already been charged for the purchase and your order is canceled, American Musical Supply will issue a credit to your credit card account in the amount of the charge.


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- Please select a date to see the proper prices!
- Meal can be requested on the spot
Best offer
39 € / person / night, no meal
Description of the Accommodation
Rooms, services Hotel Cristallo Brescia is offering 23 tastefully furnished rooms to those seeking accommodation in Brescia. Since the hotel has an own restaurant, you may also try fine local meals on the spot. For guests arriving by car parking possibility is available nearby. You may also rent a car to discover all must-sees of the area. If you wish to spend your leisure time actively, you may also take the nearby opportunities to rent a bike, play bowling, billiard and go water skiing. Upon preliminary notice airport transfer can be requested. For meetings and nice evening chats the local bar will provide an ideal location. In the safe your valuables may be the safely secured. You can browse the Internet via wireless connection (WiFi) available at the hotel. The exact list of services available at Hotel Cristallo Brescia (including those for an extra fee) and the exact list of equipment in a room can be found on the information sheet below.
Payment Advance payment by credit card or providing credit card guarantee may be necessary for finalizing the reservation, depending on the chosen date, room and price.
Arrival, opening hours The earliest time to check in at the hotel is 14:00, the latest time to check out is 11:00. Italian and English languages are spoken by the personnel.
Hotel Cristallo Brescia rooms Rooms
- Please select a date to see the proper prices!
Accommodation services
Parking (7 €/night)
Free WiFi in shared places
Relaxation: Billiard, Bowling, Bike rental at the accommodation
Car rental nearby, Water skiing nearby
Default services: Pet friendly, Bar, Safe deposit box, Room service
Airport transfer
Own restaurant
Hairdryer, Desk in the room, Mini-bar


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Bosch Home Tool Kit at 36% Offer
- Rs.4,675.00
Product Description
Drilling a hole or a minute repair task at your home no longer needs a professional assistance. It can be easily done with the help of a simple drill machine or other required hand tools. If you are looking for a toolkit that comprises a drill machine and other hand tools that can assist you in drilling a hole or carrying out a repair work, you must check out the GSB 10 RE Professional tool kit from the store of Bosch
Bosch Home Tool Kit in Snapdeal
How to get this deal:-
- Click here for the offer page
- Add product into the cart
- Login or Register
- Add your shipping details
- Make the final payment
Posted on 04/10/2016


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Chapter 24 . Section 4: From Isolationism to War. Neutrality Acts. 1935- banned the US from providing weapons to nations at war 1936- banned loans to such nations. 1937- permitted trade with fighting nations in nonmilitary goods, as long as they paid cash & transported the cargo Cash & carry
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Section 4: From Isolationism to War


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The Bottom Line / Don't Mention the Y Word
When transferring from one company to another becomes quick and easy, we can expect the companies to do much more to achieve higher yields.
Nitzan, a Holocaust survivor, a mother of two, and a professional with an above average salary, sat down with an insurance agent last week for her regular consultation. The agent clearly and succinctly outlined the policies and what they entailed - how much she should save for her old age and how much to take out in a lump sum at age 65, which plan would be best in terms of tax, and how much risk she should take to allow her children to live decently.
The agent was fine. As opposed to others Nitzan had come across in the past, this one didn't confuse her or suggest umpteen alternatives, but concentrated instead on two or three clear options. But when it came to the question of yield - the rate of return on the insurance policies over several years - the agent found himself somewhat short of words, and had nothing constructive to add. He found it difficult to explain why he was selling policies from the Phoenix and Migdal insurance companies and not Menorah, which is known to have the highest returns in the market.
When Nitzan pushed, the agent mumbled something about Menorah not providing the service, and its insurance package not including a particular risk coverage - but on whether having a higher yield means Nitzan would be several thousand shekels better off when she retires, or whether, on transferring her policy, Menorah would continue to reap the market's highest returns, he had nothing to say.
It is possible though to assume that the commissions from those companies whose policies he sells call the tune, and that determines what goes into his basket of goods.
And this is one of the major problems with insurance agents today. They can talk for hours on looking after your children and what happens to you after you're 65; and they are experts in diagnosing hypochondria in their clients and pushing just the right policy that covers all ills. When they raise an eyebrow and wonder if that's all you are prepared to leave your dependents, and "maybe you could slightly raise your risk component," you feel pressured. You feel like throttling them. But then that's their profession, the line of work for an insurance agent.
But on one thing - and it's not such a small thing - they have nothing to say: Which company knows how to invest their life insurance money best of all, and why they are not moving your money to that company.
It's not that they don't know. They are fully aware of which ones stand out and which are lagging behind, but the agent doesn't want the client to start choosing which company to buy their policy from, because this is what grants the agents their power - at least until now. But the reforms in the capital market and in the insurance and banking sectors will change the picture. Agents will become one of two kinds: consultants or marketers.
The marketer will market policies from two companies and will offer you the best of the two. The consultant will have to provide the best advice on all the products in the market. Both will have to acquire financial knowledge and offer their clients information on yields and not just on policy components and tax matters. And when transferring from one company to another becomes quick and easy, we can expect the companies to do much more to achieve higher yields.
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It has been boiling hot over here, and rain has been long overdue!! Please give us some rain - but not too much!
This week's Greeny Crafter is Mezzy of Recycled, Purposed, Renewed.
Tell us a little about yourself.
Hmmm, an interesting question. I kind of have a dual life. By day I work in the banking industry which is very conservative, structured and precise. A big part of my role finding solutions for complex problems. Once the working day is done I step out of my cloak of conservatism and morph into my creative being, which is a refashioner. These two existences may seem poles apart, but surprisingly enough they are quite similar. Refashioning too is all about problem solving. You start with something that has outlived it's use or is no longer appropriate for the task at hand and then you recreate it into something else. The steps in between to achieve a final outcome.....are what makes both my working and refashioning life interesting.
|Loving the cuff jeans!
How did you start doing refashioning / upcycling?
I was a thrifter before I was a refashioner. This resourcefulness all began when I first got married. We moved to the country, built our own house, had two babies and lived on one income. I realised early on that we could live a comfortable frugal life by purchasing other people's offcasts. Thrifting for me has also been about collecting and preserving history, but that is a whole other story.
Thrifting gets into your pysche and just emanates from the inside out.
The natural progression of thrifting was refashioning. When I first got married there were no pc's and certainly no You Tube tutorials. I bought a basic sewing machine and taught myself to sew. Outgrown t-shirts were refashioned into underwear for my daughters. Trousers that could not be lengthened were made into summer shorts. Tank tops would be refreshed by having vintage fabric added to them which would turn them into a dress. It was a very creative time of my life and then we had to move back to the city. My sewing machine was packed up and for ten years it sat in storage. In January of this year, my daughter announced that she was going to drive around Australia in a converted combi van and that I was going to make the curtains. My sewing machine finally got to see the light of day again and after the curtains there was no stopping us.
All my projects are created for a purpose so each and everyone of them is dear to me. However I do have two projects that are my most meaningful. The first one is my "Flowers for Lauren" Necklace. Lauren was an amazing spirit who came into my life when a long term friendship had ended abruptly. I was not in a mind frame to start a new friendship but Lauren made it her business to become my friend and ingrained herself into my life. I was devastated when after a sudden and short battle with cancer, Lauren passed away. "Flowers for Lauren" necklace was created for her birthday and in her memory.
My second meaningful project is the "Adrift @ Sea" Cuff. I spend hours walking along the beach, collecting sea glass and other washed up treasures. I find this past-time meditative, peaceful and soothing. The sea glass is beautiful when it simmers and shines in the wet sand. Therefore it is ironic that it begins it's life with the careless tossing of a glass bottle into the ocean. Why are we so wasteful? Why are we so willing to pollute our oceans and destroy marine life? Someone elses lack of care becomes my nature's jewel to admire and behold. The "Adrift @ Sea" Cuff turns an ugly careless act into something of calmness and beauty.
Where do you get your inspiration?
Inspiration is anywhere and everywhere. It is not just one thing. My inspiration starts with a seed which is then watered, nurtured and allowed to grow. What I mean by this is that I collect and buy things that capture my eye. Ninety percent of the time, I do not have an end project in mind. It is as simple as starting off with a small piece of amazing vintage fabric that I find in a dusty corner of a bursting to the brim thrift store. I buy it, put it aside and just look admiringly at it. Eventually the fabric will inspire me and an idea will come to mind.
A big thank you to Mezzy for contributing this. Looking forward to seeing more of your work :-)


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You might also get further travel health guidance from travel vaccination for Guinea
The travel advice info for Guinea for travelers from %%hostcountry%% on this web site does not substitute your doctor's advice. Details about travel disease risk in Guinea and travel vaccination immunisation for Guinea are drawn from the centres for Disease Control websites globally and relates to known health risks in Guinea.
The travel health information for Guinea provided does not contain any information associated with disease breakouts, which may take place frequently and with little warning.
Speak to your doctor before you visit Guinea for the most current travel health and travel immunisation information.
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