WO1997010268A1

WO1997010268A1 - Spray-drying haemoglobin

Info

Publication number: WO1997010268A1
Application number: PCT/NL1996/000355
Authority: WO
Inventors: Jeroen Joost Valentijn Tahey; Henri Joseph Hubert Hens; Joachim Cornelis Bakker; Petrus Theodurus Maria Biessels; Willem Karel Bleeker
Original assignee: Staat Der Nederlanden, De Minister Van Defensie, Voor Deze: Het Hoofd Van De Afdeling Militair Geneeskundig Beleid
Priority date: 1995-09-11
Filing date: 1996-09-10
Publication date: 1997-03-20
Also published as: CA2229408A1; AU726684B2; JP2000505049A; AU7098996A; EP0862583A1

Abstract

The present invention relates to a method to obtain a dried haemoglobin product, that is stable during extended storage (over years) at ambient temperature (4-30 °C) and dissolves quickly (within 10 minutes, preferably within 5 minutes, more preferably within 1 minute), easily (without the use of special facilities) and completely (in a clear, dark red solution with minimal particulate contamination). The solution reconstituted from the dried haemoglobin has sufficient low levels of methaemoglobin to effectively function as an oxygen carring solution after reconstitution and upon parenteral administration to a patient. Besides the reconstituted product has a physiologically acceptable formulation (osmolarity: typically 250-350 mosm/1) and has colloid-osmotic activity. The drying process is applicable for haemoglobin, modified haemoglobin, recombinant haemoglobin and/or encapsulated haemoglobin preparations. The process to obtain the dried haemoglobin product is based on spray-granulation with addition of one or more protective additives. Besides, a carrier substance might be used to reduce particulate contamination. To prevent oxidation of haemoglobin, the process and storage can be performed under oxygen free conditions by the use of nitrogen or any other inert gas.

Description

O 97/10268 PC17NL96/00355

Title Spray-drying haemoglobin

Field ---£ i-ii-- invention

This invention relates to methods for obtaining a dried haemoglobin product with a long shelf life, which can be reconstituted quickly and easily, having sufficient low levels ot methaemoglobin to effectively function as an oxygen carrying solution upon administration to a patient The invention further relates to the dried compositions themselves and to the reconstituted product which has a physiologically acceptable formulation The haemoglobin based composition includes all preparations of haemoglobin, modified haemoglobin, recombinant haemoglobin and/or encapsulated haemoglobin

Backgrou d ol tii≤ invention

Several solutions of (chemically modified) haemoglobin are now in clinical evaluation for use as plasma expanders with oxygen transporting capacity These solutions may temporarily replace the function of erythrocytic haemoglobin when they are transfused into patients with a deficiency of oxygen transport capacity and a reduced blood volume

Especially from the army point of view, there is a growing need for a universally applicable blood-substitute which can also be stored for a long period (at least for about a year) at ambient temperature conditions (4 - 30°C) for use during, for instance, calamities A very important requirement for such products is that they can be reconstituted easily and quickly and that t can be administered to the patient without the use of special facilities

Storage of the haemoglobin solution must be carried out in such a way that characteristics, such as oxygen-carrying capacity, polymer- and ionic composition remain substantially unchanged The haemoglobin can be stored either as a solution or as a dried product A dried product can be achieved for instance by freeze-drying (ChaiHot 1981) spray-drvmg (Franks et al 1992, Labrude et al , 1989) or spray- granulation The advantages of a dried product over a solution are clearly the storage life (shelf life) and the storage volumes The advantages of spray-drying or spray-granulation over freeze-drying are shorter process times and less energy consumption Furthermore spray-dried/granulated materials generally take the form of homogeneous powders, which are less hygroscopic than those obtained by freeze-drying

Only a few attempts have been made to spray- ry haemoglobin, mainly because of the sensitivity of haemoglobin to temperature and oxidation Only Labrude et al (1989) carried out spray-dry experiments with oxyhaemoglobin They indicated that addition of protective compounds, such as sucrose, is needed to keep the methaemoglobin formation at low levels (an increase of less than 5% (w/w) of the total haemoglobin caused by the drying process) Labrude et al showed by means of electron spin resonance absorption that the structure of the haeme is not altered by desiccation and that the sucrose does not penetrate the haeme pocket Therefore, Labrude et al showed that it is possible to obtain a stable dried haemoglobin product with unaltered physical properties with the use of spray-drying

However, Labrude et al found an optimal sucrose concentration at a sucrose/haemoglobin ratio of 0 85 (w/w) For a physiologically acceptable formulation, a maximal sucrose/haemoglobin ratio of about 0 44 (w/w) is recommended The present invention provides a combination of features which makes it possible to lower the sucrose concentration to obtain this lower ratio

Another manor disadvantage of spray-dried haemoglobin is the poor solubility and high particulate contamination after reconstitution, which may disturb the blood circulation

According to the British Pharmacopoeia (1980), injectable preparations which are solutions when examined under suitable conditions of visibility, should be clear and practically free from particles. Additionally, where stated in individual monographs, solutions to be injected and which are supplied in containers of 100 ml or more should comply with the limit test for particulate matter In this test, the number of particles per 1 0 ml of the product is determined The average particle count, for the undiluted product, should not exceed 1000 per ml for particles greater than 2 0 μ and should not exceed 100 per ml for particles greater than 5 0 μm. Because until now no individual monograph for haemoglobin based compositions exists, no limits for particulate contamination for such products exists either Nevertheless, we think that haemoglobin based compositions should comply with a test for (large volume) parenteral injections The use of the granulation technique, with or without a binding agent, in combination with the process conditions reduces the particulate contamination to an acceptable level

The invention thus provides a method for preparing a dried reconstitutable haemoglobin composition having a long shelf life, which composition can be reconstituted to a solution having a colloid-osmotic pressure of about 20-40 mbar and an osmolarity of about 250-350 rrtosm/1, whereby in said method a solution comprising haemoglobin is provided, which solution is divided into small droplets, which droplets are dried to a solid particle form by a stream of inert gas. In said method the haemoglobin may be in any form, it may also be chemically modified, recombinant or encapsulated It is however important to protect the haemoglobin in whatever form of degradation Therefore the haemoglobin solution further contains at least one stabilising additive. The additive may be any one that protects the haemoglobin from said degradation and is compatible with administration to a patient Preferred stabilising additives are sucrose, dextran-1 or human serum albumin or combinations thereof As stated before, the dried compositions must be easily reconstitutable to solutions having physiologically acceptable properties especially regarding the osmotic properties of the reconstituted solution. Therefore the protective additives should be added in amounts that will lead to protection of the haemoglobin but also to physiologically acceptable properties upon reconstitution w th Water For Injection or aqueous solutions such as saline To ensure the quick and easy solubility of the dried product, the granulation step is included in the process of drying Granulation of the particles may be carried out by wetting the surface of the solid particles in a fluidised bed This causes the particles to fuse When a binding agent is used, the solid particles will agglomerate In this way the material of the binding agent forms bridges between the solid particles in the fluidised bed The binding agent should be very soluble in aqueous solutions and capable to "glue" the solid haemoglobin based particles together The binding agent should be compatible with administration to a patient and should be added in amounts that will lead to a functional and physiologically acceptable formulation Preferred binding agents are sodium chloride, sucrose, dextran-1, sodium lactate or compositions thereof These amounts can be readily determined by a person skilled in the art and depend among others on the final formulation of the composition

Also a carrier substance may be used in the granulation process to reduce particulate contamination The carrier substance should be very soluble in aqueous solutions, compatible with administration to a patient and added in amounts that will lead to a functional and physiologically acceptable formulation Preferred carrier substances are sodium chloride and human serum albumin These amounts can be readily determined by a person skilled in the art and depend among others on the final formulation of the composition.

For a physiologically acceptable formulation, the final composition should have a colloid osmotic activity 10-300 mbar, preferably an colloid osmotic pressure in a range between 20 and 40 mbar depending on the therapeutic use The osmolarity should be in a range between 150 and 600 mosm/1 preferably in the range between 250 and 350 mosm/1 , more preferably between 280-300 mosm/1 (Henry et al (ed), 1974) also depending on the therapeutic use.

The maximum methaemoglobin concentration that can be allowed is 15% (w/w) of the total haemoglobin concentration Preferably the methaemoglobin concentration should be below 5% of the total haemoglobin concentration

Until the present invention solutions of chemically modified haemoglobin are preserved by frozen storage or in a liquid state under oxygen free conditions The present invention describes a method to prepare dried granulated haemoglobin which can be stored for years at ambient temperatures. Moreover, spray-granulated powders according to the invention are expected to be instantaneously soluble Therefore they can be rapidly transformed into a parenteral solution without loss of the functional and physiological characteristics required for oxygen transport. It is the combination of the spray-granulation process and the formulation of the haemoglobin based composition that results in a product with the above named characteristics

The spray-drying and -granulation process for instance operate as follows.

The first stage is to provide a solution of the haemoglobin based composition formulated in Water For Injection This may be done by ultrafiltration or dialysis or any other suitable method. To shorten the process time of the drying process, a concentrated solution, of up to 20% (w/w) haemoglobin is preferred.

The second step is the addition of the desired protective additives The protective additive may be sucrose, dextran-1 or any other additive (e.g. sugars or amino acids) that protects the haemoglobin from degradation The protective additives should be added in an amount to be able to protect the haemoglobin from degradation and to preserve the physiological acceptability of the reconstituted product

These amounts can be readily determined by a person skilled in the art For sucrose this amount will vary between approximately 50 mM and 300mM, preferably 130mM. For dextran 1 this amount will vary between approximately 30mM and lOOmM, preferably 70mM and for human serum albumin between approximately 15μM and 1.5rr-M, preferably 0 15mM. These concentrations are all based on the final reconstituted formulation of approximately 6.4mM haemoglobin (16kD) and depend on the therapeutic use and the final formulation of the composition.

In addition, an electron quenching additive may be used to prevent oxidation of the haemoglobin caused by free radicals, e.g induced by (direct) light The electron quenching additive should be soluble in aqueous solutions, compatible with administration to a patient, and added in amounts that lead to a functional and physiologically acceptable formulation These amounts can be readily determined by a person skilled in the art and depend among others on the final formulation of the composition. Preferred electron quenching additives are ascorbic acid and human serum albumin, but may be any other additive that quenches free electrons (e.g. anti-oxidants) .

After addition of the protective compounds, the solution is preferably stabilised by exclusion of oxygen. For this purpose, the haemoglobin is deoxygenated by use of, for example, a hollow fibre system in combination with nitrogen, but any other inert gas may be used. Also the haemoglobin may be stabilised by saturation with a specific binding compound such as carbon monoxide .

The next step is the drying operation In this step, a spray-granulation process is performed to dry the product, preferably into granulates Three methods to be used to start the granulate formation are exemplified

1 In a first stage the main part of the aqueous haemoglobin mixture is spray-dried into a drying chamber by using a high flow of a drying gas. The spray-dried powder is for the largest part collected on a filter and no fluidised bed is formed In a second stage, the drying gas throughput is lowered to form a fluidised bed and the granulation process starts In this way haemoglobin itself is used as the granulate starter

2 The same method as above s used, but now a binding agent is used to granulate the dried haemoglobin based particles

In this way the particles side the fluidised bed are agglomerated

3 A separate starting material is used to create a fluidised bed The product to be dried is directly sprayed into the fluidized bed and is granulated immediately

Apparatus to carry out spray-granulation on a fairly small scale are available from various manufacturers One is Heinen GmbH, Varel, Germany who manufactures a small scale batch-drier. Another manufacturer is Niro Aeromatics, Bubendorf, Switzerland who manufactures a small-scale spray-drier/granulator Process plants to carry out spray- granulation on a larger scale are also available

Figure 1 (appendix 1) shows a schematic diagram of the spray-granulator In this apparatus the drying gas is drawn in by a blower (1) and passes over an electric heater (2) Then the gas is blown upwards into the drying chamber (3) passing a sieve (4) The aqueous mixture to be sprayed is drawn up from a supply vessel (5) by means of a peristaltic metering pump (6) and delivered to a spray nozzle (7) which discharges the aqueous mixture as a fine spray (10) into the streams of hot drying gas coming from the heater (2) The stream of hot drying gas and the product are counter current The spray droplets are dried to a solid powder form as they pass down mside the drying chamber (3) Before the spray droplets are completely dried, they make contact with the material in the fluidised bed (9) Here the granulation process takes place by wetting the surface of the particles in the fluidised bed This causes the particles to fuse or when a binding agent is used, the particles are "glued" together by agglomeration In this way, after multiple steps, a granulate is formed The gas stream leaves the drying chamber passing the filter (8) To separate the outgoing gas from the dried material, a filter is placed IP front ot the gas output Every few seconds, the filter is cleared from particle dust by an opposite gas stream The particles fall down into the fluidised bed and again participate place in the granulation process

A significant parameter in the operation of any spray- drying or -granulating apparatus is the temperature of the gas stream which is admitted to the drying chamber and into which the spray is delivered For the present mvention this inlet temperature of the gas stream will generally exceed 80°C will usually be approximatel 100°C and mav veil lie _.n a range from 100°C up to 150°C The drying gas will topically be nitrogen but could be some other gas

The methods described above will lead to novel spray-dried or spray-granulated haemoglobin compositions having properties which were not obtainable until the present invention The haemoglobin composition according to the invention comprises less than 15%, preferably less than 5% methaemoglobin The composition should be stable for periods in the order of many months, preferably for at least a year, when kept at temperatures in the range of 0 - 30° For actual prolonged storage, refrigerator temperatures should of course be chosen Upon reconstitution the dried product results in a solution containing less than 1000 particles per ml having a size greater than 2μm and less than 100 particles per ml having a size of greater than 5μm

Reconstitution, optionally after storage, from a composition produced by drying in accordance with this invention can be effected by adding the desired solution to a quantity of the composition The solvent may be Water For Injection or an infusion liquid, but the result has to be a physiologicallv acceptable formulation

With respect to the use in emergency situations, as stateα nerein before, _.t is important that reconstitution of the material is an easy process, without the need of any (electronical) equipment.The material has to dissolve almost instantaneously and completely. To prevent foaming, and thereby denaturation of the haemoglobin, the solution should not be shaken during reconstitution. To improve the solubility, a wetting agent may be used. The wetting agent may be lecithin (phosphatidylcholine), Tween (polyoxyethylene sorbitan monooleaat) , Triton (alpha [4-(l,l,3,3,- Tetramethylbutyl)phenyl] -ω-hydroxypely (oxy-1, 2-ethanediyl) or any other additive (eg. surfactants) which improve the solubility. The wetting agent should be compatible with administration to a patient and should be added in amounts that will lead to a physiologically acceptable formulation. These amounts can be readily determined by a person skilled in the art.

The reconstituted product according to the invention is an optically clear, dark red solution and has sufficient low levels of particulate contamination to be safe for use as a large volume parenteral injection. It has an osmolarity of between 150 and 600 mosm/1, preferably between about 250-350 mosm/1 , more preferably between about 280-300 mosm/1. Besides, it has a colloid osmotic pressure between 10 and 300 mbar, preferably between about 20-40mbar. The solution reconstituted from the dried haemoglobin has sufficient low levels of methaemoglobin to effectively function as an oxygen carrying solution after reconstitution and upon parenteral administration to a patient. The reconstituted product obtainable by a method according to this invention is of course also part of the invention.

The invention will now be illustrated in more detail in the following examples. E x a mp l e s

Example 1 Preparation of anaerobically spray-granulated deoxyhaemoglobm A 0.22 μm filtered solution of modified haemoglobin (polyHbXl, CLB) in Water For Injection, containing 150 gram haemoglobin and 68 gram sucrose (Merck) per litre is spray- granulated by the use of a He en batch drier, type CWT 3,5 RFS The haemoglobin solution is deoxygenated before use

(Capiox 350 oxygenator, Terumo) and the drying gas is nitrogen (medical grade, Air Products)

The mlet temperature of the drying gas is set to 100°C The gas flow is set high, so no fluidised bed is formed yet The haemoglobin solution is spray-dried into the drying chamber with a flow rate of approximately 50 ml/min The temperature of the emerging gas is approximately 55°C. After 80% of the solution has been spray-dried, the gas flow is lowered in order to obtain a fluidised bed The temperature of the emerging gas is kept at approximately 55°C by lowering the flow of the haemoglobin solution to approximately 20 ml/min. The spray-dried material becomes granulated by spraying on top of the fluidied bed.

When all of the haemoglobin solution has been spray- granulated, the inlet temperature of the drying gas is set to 20°C to cool the haemoglobin granulates The cooled powder is aseptically removed from the granulator and filled in 72.7 gram quantities (excl residual moisture) in sterilised glass bottles of 500 ml The removal of the haemoglobin granulates and the filling of the glass bottles is performed under anaerobic conditions The glass bottles are stoppered and capped.

To reconstitute the product, one unit (72 7 gram) of the dried product is dissolved in a 500 ml solution of lactated Ringer (Fresenius) and Water For Injection (50/50 (v/v)) resulting in a 10% (w/w) haemoglobin solution with 133 mM sucrose The final product so obtained has substantially not altered physical-chemical properties when compared with the raw material . Withm minutes after addition of the solvent the granulated product is dissolved and the solution is ready to use.

Example 2

Preparation of anaerobically spray-granulated stabilized deoxyhaemoglobm

The procedure is similar to that of example 1 with the exception that 15 gram human serum albumin (HSA, CLB) per litre is added to stabilize the haemoglobin solution. For each unit 77,7 gram of powder (excl. residual moisture) is filled into sterilised glass bottles of 500 ml. To reconstitute the product, one unit is dissolved in a 500 ml solution of lactated Ringer (Fresenius) and Water For Injection (50/50 (v/v) ) , resulting in a 10% (w/w) haemoglobin solution with 133 mM sucrose. The final product so obtained has substantially not altered physical-chemical properties when compared w th the raw material. Within minutes after addition of the solvent the granulated product is dissolved and the solution is ready to use .

Example 3

Preparation of anaerobically spray-granulated deoxyhaemoglobm, with the use of a starter material

The procedure is similar to that of example 1 with the exception that 1000 gram of sodium chloride (Merck) is used as starter material . This amount is equivalent to 6 g sodium chloride per litre m the final reconstituted solution when 111.1 of 150 g/1 haemoglobin is dried. For each unit, 75.7 gram of powder (excl. residual moisture) is filled into sterilised glass bottles of 500 ml. To reconstitute the product, one unit (75 7 gram powder) of the dried product is solved in a 500 ml solution of lactated Ringer without sodium chloride and Water For Injection (50/50 (v/v)), resulting in a 10% (w/w) haemoglobin solution with 133 mM sucrose and 102 mM sodium chloride. The final product so obtained has substantially not altered physical-chemical properties when compared with the raw material Within minutes after addition of the solvent the granulated product is dissolved and the solution is ready to use

Example 4

Preparation of anaerobically spray-dried deoxyhaemoglobm

A 0 22 μm filtered solution of modified haemoglobin (polyHbXl, CLB) in Water For Injection, containing 150 gram haemoglobin and 68 sucrose (Merck) per litre is spray-dried by the use of a Buchi Laboratory spray-drier type 190 The haemoglobin solution is deoxygenated before use (Capiox 350 oxygenator Terumo) and the drying gas is nitrogen (medical grade, Air Products) The mlet temperature of the drying gas is set to 100°C The solution is sprayed into the drying chamber ^ith a flow rate of approximately 3 ml/min The temperature of the emerging gas is approximately 70°C When all of the haemoglobin solution has been spray-dried the inlet temperature of the drying gas is set to 20°C to cool the haemoglobin product The cooled powder is removed from the drier and filled in 1 0 gram quantities (excl residual moisture) into sterilised glass vials of 20 ml The removal of the haemoglobin and the filling of the vials is performed under anaerobic conditions The vials are stoppered and capped and stability studies are performed with the dried haemoglobin

To reconstitute the product, one unit (1 0 gram) of the dried product is dissolved in a 6 9 ml solution of lactated Ringer (Fresenius) and Water For Injection (50/50 (v/v)) resulting in a 10% (w/w) haemoglobin solution with 133 mM sucrose The characteristics (oxygen affinity molecular weight distribution) of the haemoglobin proved to be unchanged after the drying process and storage at 4°C The methaemoglobin content increased by less than 1% of the total composition during the drying process and less than 10% of the total composition during the first 11 months ot storage at 4°C Example 5

Preparation ot anaerobically spray-dried deoxyhaemoglobm with the use ot a wetting agent The procedure is similar to that of example 4 with the exception that the haemoglobin composition is Hemol k (Hemosol, Etobicoke Canada) and 37 5 μl Tween-20 (Merck) per litre is added to the haemoglobin solution before drying

To reconstitute the product, one unit (1.0 gram, excl residual moisture) of dried product is solved in a 6 9 ml solution of lactated Ringer (Fresenius) and Water For Injection (50/50 ( //v)), resulting in a 10% (w/w) haemoglobin solut-on th 133 mil sucrose and 25 ppm Tween The time necessary to dissolve the dried product decreased with approximately 30% when compared with the product obtained according to example 4

Example 6

Preparation ot anaerobically spray-dried deoxyhaemoglobm and granulated with the use of a binder agent

To a 0 22μm filtered solution of modified haemoglobin (Hemolink Hemosol, Etobicoke, Canada) in Water For Injection, containing 150 gram haemoglobin per litre, 68 gram sucrose (MercK) per litre is added The haemoglobin solution is deoxygenated before use (Capiox 350 oxygenator, Terumo) The drying process is performed on a Heinen batch drier, type CWT 3,5 RFS The drying gas is nitrogen (medical grade, Air products) The mlet temperature of the drying gas is set to 100°C The gas flow is set high, so no fluidised bed is formed yet The haemoglobin solution is spray-dried into the drying chamber with a flow rate of approximately 50 ml/min The temperature of the emerging gas is approximately 50°C When all of the haemoglobin has oeen spray-dried, the gas flow is lowered in orαer to obtain a fluidised bed To granulate the spra'_ -dried haemoglobin a binder solution, containing 1 M sodium chloride is sprayed on top of the fluidised bed The temperature of tne emerging gas is kept at approximately 50°C by varying the rate of addition the binding solution The amount of sodium chloride used to granulate the haemoglobin is equal to an amount of 6 gram per 100 gram of haemoglobin (solid) When all of the haemoglobin solution has been granulated, the mlet temperature of the drying gas is set to 20°C to cool the haemoglobin granulates The cooled powder is aseptically removed from the granulator and filled in 75.7 gram quantities (excl residual moisture) into sterilised glass bottles of 500 ml The removal of the haemoglobin granulates and the filling of the glass bottles is performed under anaerobic conditions The glass bottles are stoppered and capped.

To reconstitute the product, one unit (75 7 gram of the dried product is dissolved in a 500 ml solution of lactated Ringer without sodium chloride and Water For Injection (50/50 (v/v/)), resulting in a 10% (w/w) haemoglobin solution with 133 mM sucrose and 102 mM sodium chloride The final product so obtained has substantially not altered physical-chemical properties when compared with the raw material Withm minutes after addition of the solvent the granulated product is dissolved and the solution is ready to use

Example ^~ι

Preparation of aerobically spray-granulated oxy-haemoglobin The procedure is similar to that of example 1 with the exception that the haemoglobin solution is formulated containing 150 gram of haemoglobin (polyHbXl, CLB) and 150 gram of sucrose (Merck) per litre, which results in a physiologically unacceptable sucrose concentration The solution is spray-granulated by the use a a Heinen

WT 1,4/16 LMR drier. The principle of this machine is comparable to that of the CWT 3 , 5 RFS batch drier mentioned before

When all of the haemoglobin solution has been spray- granulated, the let temperature of the drying gas is set to 20°C to cool the haemoglobin granulates The cooled powder is aseptically removed from the granulator and filled in 100 0 gram quantities (excl residual moisture) into sterilised glass bottles of 1000 ml The removal of the haemoglobin granulates and the filling of the glass bottles is performed under aerobic conditions The glass bottles are stoppered and capped

To reconstitute the product, one unit (100 gram) of the dried product is solved in a 500 ml solution of lactated Ringer and Water For Injection (50/50 (v/v)), resulting in a 10% (w/w) haemoglobin solution with 133 mM sucrose. Within mmutes after addition of the solvent the granulated product is dissolved and the solution is ready to use

During the drying process, the methaemoglobin content increased by approximately 5% of the total haemoglobin The characteristics (oxygen affinity, molecular weight distribution) of the haemoglobin proved to be substantially not altered during 24 months of storage at 4°C and 20°C At 4°C the methaemoglobin content increased by less than 6% of the total haemoglobin over the total period and less than 2% of the total haemoglobin during the last 23 months At 20°C, the methaemoglobin content increased by less than 15% of the total haemoglobin over the total period The stability of the haemoglobin proved to be even better when the complete process and storage is performed under anaerobic conditions Based on these and other data, we expect the granulated haemoglobin product to be stable for a period of at least 5 years

Example 8

Preparation of anaerobically spray-dried deoxyhaemoglob

The procedure is similar to that of example 4 with the exception that the haemoglobin is a pasteurised stroma free haemoglobin solution (sfHb, CLB) To reconstitute the product, one unit (1 0 gram powder;excl residual moisture) of the dried product is solved in a 6 9 ml solution of lactated Ringer and Water For Injection (50/50 (v/v)), resulting in a 10% (w/w) haemoglobin solution with 133 mM sucrose The final product so obtained has substantially not altered physical- chemical properties if compared with the starting material Example 9

Preparation of aerobically spray-granulated CO-stabilized haemoglobin The procedure is similar to that of example 1 with the exception that the haemoglobin solution is stabilised by saturation with carbon monoxide (Air Products) and the drying process is performed under aerobic conditions, the drying gas is fresh air To reconstitute the product, one unit (72.7 gram,excl residual moisture) of the dried product is solved in a 500 ml solution of lactated Ringer and Water For Injection (50/50 (v/v)), resulting in a 10% (w/w) haemoglobin solution with 133 mM sucrose The final product so obtained has substantially not altered physical-chemical properties if compared with the starting material, with exception to the HbCO-level, which has mcreased

Literature

British Pharmacopoeia, Volume II London, 1980.

Chaillot, B. , P Labrude, C Vigneron and D. Simatos 'Freeze-drying of haemoglobin solutions without adjuvant and in presence of glucose, tris and β-alanme. ' American Journal of haematology 10, 1981. p319-326.

Franks, F , R.H.M. Hatley and S.F. Mathias . 'European patent application: Storage of materials. ' Date of filing: 24-06- 1992

Henry, R.J., D C. Cannon, J.W. Winkelman (ed), Clinical

Chemistry, 2nd edition, New York: Harper & Row Publishers Inc.

1974

Labrude, P , M Rasolomanana, C Vigneron, C. Thirion and B. Chaillot 'Protective effect of sucrose on spray-drying of oxyhaemoglobm. ' Journal of Pharmaceutical Science, vol.78, no.3, 1989. p.223-229.

Wade, A (ed) 'Martmdale: The extra Pharmacopoeia' , 27th edition. London: The pharmaceutical press, 1979.

Claims

C l a im s

1 A method for preparing a dried reconstitutable haemoglobin composition having a long shelf life, which composition can be reconstituted to a solution having a colloid osmotic pressure of about 10-300 mbar and/or an osmolarity of about 150-600 mosm/1, whereby in said method a solution comprising haemoglobin is provided, which solution is divided into small droplets, which droplets are dried to a solid particle form by a stream of inert gas

2 A method for preparing a dried reconstitutable haemoglobin composition having a long shelf life, which composition can be reconstituted to a solution having a colloid osmotic pressure of about 20-40 mbar and/or an osmolarity of about 250-350 mosm/1, whereby the said method is divided into small droplets, which droplets are dried to a solid particle form by a stream of inert gas

3 A method according to claim 1 or 2 whereby the solution comprising haemoglobin further contains at least one stabilising additive.

4 A method according to claim 3 whereby a stabilising additive is sucrose, dextran-1 or human serum albumin

5 A method according to any of the foregoing claims whereby the solution comprising haemoglobin further contains at least one electron quenching additive

6 A method according to claim 5 whereby a electron quenching additive is ascorbic acid or human serum albumin

7 A method according to any of the foregoing claims whereby the solutions comprising haemoglobin further contains a wetting agent

8 A method according to claim 7 whereby a wetting agent s Tween (polyoxyethylene sorbitan monooleaat) or Triton (alpha

[4-(l,l,3,3, -TetramethyIbutyl) phenyl] -ω-hydroxypely (oxy-1, 2- ethanediyl)

9 A method according to any of the foregoing claims which further comprises granulation of the drying or dried particles

10 A method according to claim 9 whereby at least one starter material is used in the granulation process

11 A method according to claim 10 whereby a starter material is sodium chloride or human serum albumin

12 A method according to any one of claim 9-11, whereby at least one binding agent is used to granulate the dried particles

13 A method according to claim 12 whereby one binding agent is sodium chloride, sucrose, dextran-1, or sodium lactate

14 A method according to any of the foregoing claims whereby the droplets are provided by forcing the solution comprising haemoglobin through a spray nozzle

15 A method according to any of the foregoing claims whereby the droplets are provided as a stream, which stream is in counter current with the stream of inert gas

16 A method according to any of the foregoing claims whereby the inert gas is first contacted with the droplets at a temperature of at least 80°C

17 A method according to claim 15 whereby the temperature of the gas is between about 100-150°C

18 A method according to any of the foregoing claims whereby the gas is nitrogen or air

19 A method according to any of the foregoing claims which is carried out under oxygenfree conditions

20 A method according to any of the foregoing claims, whereby the solid particles are separated from the stream of inert gas by a filter

21 A dried haemoglobin composition obtainable by a method according to any of the foregoing claims

22 A dried haemoglobin composition according to claim 21, comprising less than 15%, preferably less than 5% methaemoglobin

23 A dried haemoglobin composition according to claim 21 or 22, /hich upon reconstitution results in a solution having less than 1000 particles per ml having a size greater than 2μm and less than 100 particles per ml having a size greater than 5μm.

24 A dried haemoglobin composition according to any one of claims 21-23 being in the form of a granulate.

25. A method for producing an immediately transfusable haemoglobin solution, whereby a dried haeomoglobm composition according to any one of claims 21-24 is provided, optionally stored, said composition being reconstituted with Water For Injection or an aqueous solution resulting in a physiologically acceptable solution.

26 A method according to claim 25 whereby the solvent comprises a wetting agent.

27. A method according to claim 26 whereby a wetting agent is lecithin (phosphatidylcholine), Tween (polyoxyethylene sorbitan monooleaat or Triton (alpha [4-(l, 1,3,3,- TetramethyIbutyl)phenyl] -ω-hydroxypely (oxt-1, 2-ethanediyl) .

28. A reconstituted haemoglobin solution obtainable by a method according to claim 25 or 27.

29. A reconstituted haemoglobin solution according to claim 28 having an osmolarity of between 150-600 mosm/1, preferably between 280-300 mosm/1 , a colloid osmotic pressure between about 10-300 mbar, preferably 20-40 mbar, less than 1000 particles per mi having a size greater than 2μm and less than 100 particles per ml having a size greater than 5 μm and whereby less than 15%, preferably less than 5% of the haemoglobin is methaemoglobin.

30. A spray-granulated haemoglobin composition.