EP0892600A1

EP0892600A1 - Storage of materials

Info

Publication number: EP0892600A1
Application number: EP97916530A
Authority: EP
Inventors: Felix Franks; Anthony David Auffret; Barry John Aldous
Original assignee: Inhale Therapeutics Systems Inc
Current assignee: Nektar Therapeutics
Priority date: 1996-04-12
Filing date: 1997-04-10
Publication date: 1999-01-27
Also published as: AU732700B2; WO1997038578A1; AU2515297A; JP2000514783A; CA2251333A1; GB9607636D0

Abstract

Blood plasma is preserved by drying to an amorphous state, with a glass transition temperature of at least 15 °C. The glass transition temperature of dried plasma is higher than would be predicted. Consequently, although a carrier material may be mixed with the plasma and included in the dried glassy composition, the weight of plasma solids can exceed the weight of any carrier material. Because admixed carrier can be absent or in low amount, the dried composition can be a unit dose requiring only rehydration prior to transfusion.

Description

STORAGE OF MATERIALS

This invention relates to the storage of materials, more specifically blood plasma. In pending United States application 08/241,457 and corresponding European application 92305769.9 published as EP-A-520748 it is explained that unstable biological materials can be placed in a stable form by spray drying them in the presence of an appropriate carrier substance which, when dried leads to a composition in the form of a glassy or rubbery amorphous state. The disclosure of these documents is incorporated herein by reference.

It is mentioned that "the_material which is to be stored may occur in a form which already incorporates a suitable carrier substance, "and" 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."

Whole blood, such as given by a human blood donor, is routinely separated into a fraction which contains erythrocytes and a fraction which is blood plasma without erythrocytes. Both of these materials can be used for transfusion into a patient. It is also possible to fractionate the blood plasma further, to isolate specific components, and use the required component in therapy.

Blood plasma cannot be stored at room temperature but can be frozen and stored indefinitely as the frozen material. Many hospitals have facilities for storing such frozen blood plasma.

However, such facilities are not available in some countries. Also, if substantial quantities of blood plasma are required for medical treatment following a natural disaster or some other emergency, the transport of the blood plasma in a frozen state presents a substantial problem.

We have now. demonstrated that the procedure of the above US and European patent applications can be applied to the drying of blood plasma; and have demonstrated that when this is done a number of important constituents of blood plasma are found to be preserved in stable form. Moreover, if the plasma is spray dried without addition of carrier substance, relying on constituents of the blood plasma to provide the amorphous carrier material in the dried product, the preservation of a number of components is as good as when a separate carrier material is added.

Accordingly, there is now provided dried blood plasma. This will be a composition, in the form of an amorphous glass or rubber (as to have the appearance of a solid) containing the solids of blood plasma.

This composition is preferably in the form of an amorphous powder and may have a glass transition temperature of at least 15^*C, better at least 20 ^*C or above.

The composition may possibly contain an added carrier material, mixed with the plasma solids. However, we have found that such an added carrier may be omitted, or may be present in an amount which is less than the weight of added plasma. Even when some carrier is added, the composition preferably contains at least 75% by weight plasma solids, better at least 80 or 90%.

Surprisingly, the glass transition temperature of blood plasma dried to a low moisture content and without added carrier, is higher than would be predicted from the glass transition temperature of dried human albumin, even though albumin is a major constituent of the solutes in blood plasma.

This of course signifies that drying to achieve a glassy state is easier than would have been foreseen. Consequently a preferred form of product is blood plasma, dried without addition of extra carrier substance, and having a glass transition temperature of at least 20 "C, better at least 25^*C or at least 30°C.

Also, since admixed carrier may be absent or in low amounts, the composition may be such as to require minimal processing before transfusion.

The blood plasma may be packaged in individual quantities of such a size as will give an appropriate volume for transfusion when rehydrated. Such a quantity may well be such as will require between 50 and- 1000 ml, more likely between 100 and 500 ml of water for rehydration to the original concentration found in blood plasma. The published value for the mean solids content of plasma is 8.6% by weight. Consequently, such an individual dose may contain plasma solids in a quantity between 4.3 and 86 grams, more likely between 8.6 and 43 grams.

In another aspect there is now provided a method of rendering blood plasma suitable for storage which comprises spraying blood plasma into a hot gas stream, thereby drying the blood plasma to particles which are in a glassy or rubbery amorphous state and separating these particles from the gas stream.

The Amorphous State

A glass is defined as an undercooled liquid with a very high viscosity, that is to say at least 10 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 T_g, 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.

Many hydrophilic materials, either water-soluble or water-swellable, 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 T_g which depend on the molecular weight and on molecular complexity of the substance concerned. T is depressed by the addition of plasticisers . Water is a universal plasticiser for all such hydrophilic materials. Therefore, the glass/rubber transition temperature is adjustable by the addition of water or an aqueous solution. Starting Material

The blood plasma may be obtained by conventional techniques for the separation of blood plasma from whole blood. Because of fears of virus infection, it is nowadays conventional that blood plasma is subjected to a treatment to inactivate any viruses present. (This may entail exposure of the blood plasma to solvent and to surfactant . ) Blood plasma which has been virus-inactivated in this way may be used.

The blood plasma may be "whole" plasma in that it contains all the water-soluble constituents of blood (except gases) in their natural proportions relative to each other, although with their concentrations changed by the separation of erythrocytes and possibly by dilution with anticoagulant solution.

The blood plasma may, if desired, be stored temporarily in the frozen state prior to drying. This may be found to be more convenient than carrying out the drying operation on freshly obtained blood plasma.

If no additional carrier substance is added, at least 90% by weight of the dried plasma, preferably at least 95%, will be solutes from natural plasma.

Added Carrier Substance

It is an advantageous feature that there is no necessity to add a separate carrier material to blood plasma prior to spray drying. However such a substance may be added if desired. As discussed in the US and European applications mentioned above, it is desirable that any carrier substance should be a material which is hydrophilic so that it is either water-soluble or water-swellable and is such that it can exist in an amorphous state.

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 T_g 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. 6_0 1841

(1988) ) . Water depresses T and for these carbohydrates the depression of T_g by small amounts of moisture is approximately 6°C for each percent of moisture added. We have determined the T 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 further group of glass forming substances which may in particular be employed are sugar copolymers described in US Patent 3 300 474 and sold by Pharmacia under the Registered Trade Mark "Ficoll". This US 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 . T 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.

Processing

Processing begins with either liquid blood plasma or a mixture of such liquid blood plasma and an added carrier substance.

The next step is a spray drying operation in which the aqueous mixture is sprayed into a hot gas stream. The gas employed will generally be air but it could be some other gas such as nitrogen. If the blood plasma will eventually be used in transfusion, the gas used to provide the hot gas stream should be sterile. Apparatus for the filtration of gas to render it sterile is commercially available and is routinely used in the pharmaceutical industry.

We have found that the spray drying of blood plasma can be carried out effectively by spray drying the blood plasma (or mixture of blood plasma and added carrier) into a hot gas stream with a temperature in the range of 80 ^*C to 250^*C, better 125^*C to 250^*C.

A laboratory scale spray dryer may well dry blood plasma to an amorphous powder with a small residual moisture content such that the amorphous powder displays a glass transition temperature in the range from -20^*C to 20 ^*C. When such an apparatus is used, the addition of a carrier substance may make it easier to obtain a higher value of T_g in the spray drying operation. For this purpose it will be desirable that the added carrier substance is a glass forming material which, on its own, has a glass transition temperature of at least 40"C, better at least 50 ^*C. While there is no theoretical upper limit on the glass transition temperature for an added carrier material, in practice suitable materials have values of T_g below 250^*C. Consequently if a carrier substance is added to the blood plasma it will frequently be the case that the glass transition temperature of this material when pure and amorphous lies in a range from 50 to 200^*C.

Larger scale spray drying apparatus may well be able to dry blood plasma directly to a product with a T value of at least 15^*C or 20^*C even without any separate addition of carrier material to the blood plasma.

Apparatus to carry out spray drying on a fairly small scale is available from various manufacturers. One is Drytec Ltd, Tonbridge, Kent, England 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.

Storage

The spray dried blood plasma can be stored for a long time, provided the storage temperature is below the T_g of the amorphous dried plasma.

If τ_ of the dried plasma is sufficient high, storage can be at room temperature. However, if T_g is close to or below room temperature it may be necessary or desirable.to refrigerate the dried plasma in cooled or refrigerated storage, especially if storage is for a prolonged period. This is less convenient but nevertheless requires less refrigeration capacity than does the storage of frozen plasma.

Notably, if blood plasma is dried to an amorphous state with a glass transition temperature below 15^*C it may be desired to keep it in refrigerated storage, at a temperature below 5^*C, to keep it below the glass transition temperature of the material.

If a composition is heated above its T 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 is allowed to rise above T for a limited time, such as during transportation.

If a composition is maintained slightly above its T (and therefore in a rubbery condition) the storage life will still be considerable, although not as long as when the storage temperature is below T .

Further Drying

A possibility, which may avoid a need for cold storage, is that the material obtained by spray drying could be subjected to a further drying operation. Because most of the water will have been removed in the spray drying step the volume of material to be subjected to the further drying operation will be small compared with the original volume of blood plasma. Further drying could, most conveniently, be carried out under vacuum with moderate heating.

Recovering from Storage

When required for use the dried blood plasma can be reconstituted by the addition of water or possibly aqueous saline solution. It will generally be desired that the water which is used is sterile so that if the spray drying was also carried out under sterile conditions the reconstituted blood plasma is in a sterile condition for use in transfusion. Apparatus for the purification of water to a sterile state is of course readily available. The Drawing

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 spray and the hot air stream travel co- currently down the main chamber 10.

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.

Example 1

A model experiment was carried out to investigate the glass transition temperature which might be achievable on drying of blood plasma.

Albumin constitutes the largest single protein component (3-5% w/v) of plasma; it was therefore used as a model substance. An aqueous solution, containing 299 mg/ml of human albumin, was diluted with distilled water to give a solution that contained 125 mg/ml of the protein. Portions of 2ml were pipetted into vials and frozen to - 37 ^*C in a freeze-drier. The temperature was maintained constant and the pressure was reduced to 0.21 mbar. The ice was completely sublimed during a period of 14 hours. The pressure was then reduced to 0.12 mbar and secondary drying was performed by ramping the temperature at a rate of 5 deg/hour to 30'C. The product was maintained at that temperature for 2 hours.

T was then determined by differential scanning calorimetry and found to be 48°C. The moisture content, by Karl Fischer determination, was 1.4% by weight. Example 2

One Unit (200ml) of frozen, virus-inactivated human plasma was thawed. Of the thawed plasma, 5ml was refrozen, to serve as control for subsequent assay. The remaining 195ml was added to 34ml distilled water and spray dried, using a Lab Plant Spray Drier, Model SD-04, the construction of which as shown in the drawing. The liquid plasma was pumped into the main chamber at a rate of 425 ml/h, being injected through a nozzle of 0.5mm diameter under a pressure of <2 bar. The hot air was introduced into the chamber in a co-current manner, at a rate of 62 m³/h and a temperature of 200^*C. The outlet air had a temperature of 81^*C. The collected material was a straw- coloured, free-flowing powder. The yield of dried plasma collected was 72.5% of the amount predicted from the published mean solids content of plasma (8.6% by weight) . It was stored in a plasma bottle at -40 °C for two months, before carrying out assay as described below.

Example 3

One Unit (200ml) of frozen, virus-inactivated human plasma was thawed. Of the thawed plasma, 5ml was refrozen, to serve as control for subsequent assay. The remaining 195ml was added to 34ml distilled water and spray dried, using a Lab Plant Spray Drier, Model SD-04, as follows: The liquid plasma was pumped into the chamber at a rate of 350 ml/h, being injected through a nozzle of 0.5mm diameter under a pressure of <2 bar. The hot air was introduced into the chamber in a co-current manner, at a rate of 64 m³/h and a temperature of 150'C. The outlet air had a temperature of 81^*C. The collected material was a pale yellow, free-flowing powder. The yield of dried plasma collected was 79.8% of the amount predicted. It was stored in a plasma bottle at -40^*C for two months, before carrying out assay as described below.

Example 4

One Unit (200ml) of frozen, virus-inactivated human plasma was thawed. Of the thawed plasma, 5ml was refrozen, to serve as control for subsequent assay. The remaining 195ml were spray dried, using a Lab Plant Spray Drier, Model SD-05, fitted with a scrubber to remove any entrained material from the exhaust gas. The liquid plasma was pumped into the main drying chamber at a rate of 175 ml/h, being injected through a nozzle of 0.5mm diameter under a pressure of <2 bar. The hot air was introduced into the chamber in a co-current manner, at a rate of 52 m³/h and a temperature of 200^*C. The outlet air had a temperature of 91°C. The collected material was a straw coloured, free- flowing powder. The yield of dried plasma collected was 68%.

Example 5

One Unit (200ml) of frozen, virus-inactivated human plasma was thawed. Of the thawed plasma, 5ml was refrozen, to serve as control for subsequent assay. The remaining 195ml was added to 34ml distilled water which contained lOg sucrose of food grade and spray dried, using a Lab Plant Spray Drier, Model SD-04, as follows: The liquid mixture was pumped into the chamber at a rate of 425 ml/h, being injected through a nozzle of 0.5mm diameter under a pressure of <2 bar. The hot air was introduced into the chamber in a co-current manner, at a rate of 62 m³/h and a temperature of 200'C. The outlet air had a temperature of 82"C. The collected material was a straw coloured, free- flowing powder. The yield of dried plasma collected was 74.3%. It was stored in a plasma bottle at -40'C for two months, before assay.

Quality Assays and Physical Properties of Dried Plasma

Assays were performed on the frozen/thawed control and the reconstituted materials as described by Hellstern et al . , Vox Sanguinis, Vol. 63, pp. 178-185 (1992) . The dried plasma was reconstituted by adding distilled water to give the same solids content as in the frozen control material. Aliquots were taken and analysed for the following: time for reconstitution, Factors V, VII, VIII, fibrinogen, pH of reconstituted plasma, and the activated partial thromboplastin time (APTT) . Factor VIII was determined by two independent methods: a one-stage clotting assay and by a chromogenic substrate assay. The assay results are in Table 1 below. The glass temperature of the dried product was determined by differential scanning calorimetry (Perkin-Elmer DSC-2) and the water content by a coulometric Karl Fischer (Mitsubishi) method. These results are given in Table 2 below.

Examples 6 and 7

Samples of dried plasma, obtained in Examples 2 and 5 respectively, were subjected to further drying in a vacuum oven connected to a cold trap, as follows: The powdered plasma was spread on a petrie dish and heated for 5 days at 50^*C-60'C and a pressure of 2 mbar. Glass temperatures and moisture contents were determined, as described above. The results are included in Table 2.

v

TABLE 1 o t o QUALITY ASSAYS OF DRIED PLASMA PREPARATIONS

Time to Factor V Factor VII Factor VIII Factor III PTT Fibrinogen pH reconstitute U/ml U/ml one stage chromagenic sec mg/ml U/ml U/ml

Frozen

Control Ex2 N/A 0.6 1.0 0.6 0.6 34 2.1 7.6

Dried Ex2 <20min 0.6 0.9 0.6 0.5 46 2.0 8.2 πϊ

UJ Frozen

3 Control Ex3 N/A 0.7 1.1 0.6 0.7 33 2.5 7.6

& Dried Ex3 <20min 0.6 0.9 0.6 0.5 46 2.1 8.2

Dried Ex5 <20min 0.6 1.0 0.7 0.5 43 2.0 8.3

O 09 so

TABLE 2

GLASS TRANSITIONS AND MOISTURE CONTENTS

re

ro O

73 c Dried plasma (Ex 3) -2.8 13.5

K>

Dried plasma (Ex 4) 26 5.3

Dried plasma + sucrose (Ex 5) 30 5.9

after secondary drying (Ex 7) 80 1 4

Samples of blood plasma dried by procedures similar to Examples 2 to 5 were also taken (on dry ice) to a different laboratory, stored there for 5 months at -lOO'C, and analysed for the content of Factor VIII. The amount present was found to be slightly greater than the amount in control samples of blood plasma which had been stored frozen without drying.

Claims

1. A composition which is an amorphous glass, comprising the solids of blood plasma, with a glass transition temperature of at least 15^*C.

2. A composition according to claim 1- which includes an added carrier material which is in an amorphous state and which is present in an amount by weight which is less than the amount of plasma solids.

3. A composition according to claim 1, wherein at least 90% by weight is blood plasma solids .

4. A composition according to any one of the preceding claims with a glass transition temperature of at least 20^*C.

5. A package containing an individual dose of a composition according to any one of the preceding claims wherein the weight of plasma solids in the dose is in the range from 8.6 to 43 grams.

6. A method of rendering blood plasma suitable for storage which comprises spraying blood plasma into a hot gas stream, thereby drying the blood plasma to particles which are in a glassy or rubbery amorphous state, separating these particles from the gas stream and optionally further drying the particles so as to form an amorphous glassy composition with a glass transition temperature of at leaεt 15 ^*C.

7. A method according to claim 6 including mixing a carrier material with the blood plasma prior to spray drying.

8. A method according to claim 7 wherein the carrier solids are less than the amount by weight of solids in the blood plasma.