AU726684B2 - Spray-drying haemoglobin - Google Patents

Description

WO97/10268 PCTINL96/00355 Title: Spray-drying haemoglobin.

Field of the 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 of 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.

Backaround of the 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 0 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 it 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 WO 97/10268 PCT/NL96/00355 2 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 (Chaillot, 1981), spray-drying (Franks et al., 1992, Labrude et al., 1989) or spraygranulation. 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-dry 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% 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 For a physiologically acceptable formulation, a maximal sucrose/haemoglobin ratio of about 0.44 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 major 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 WO 97/10268 PCT/NL96/00355 3 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 pm and should not exceed 100 per ml for particles greater than 5.0 pm. 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 mosm/l, 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 WO 97/10268 PCTINL96/00355 4 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 with 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-l, 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/l, preferably in the range between 250 and 350 mosm/l, more WO 97/10268 PCT/NL96/00355 preferably between 280-300 mosm/l (Henry et al 1974).

also depending on the therapeutic use.

The maximum methaemoglobin concentration that can be allowed is 15% of the total haemoglobin concentration.

Preferably the methaemoglobin concentration should be below 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 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 WO 97/10268 PCT/NL96/00355 6 approximately 50 mM and 300mM, preferably 130mM. For dextran 1 this amount will vary between approximately 30mM and 100mM, preferably 70mM and for human serum albumin between approximately 15u1M and 1.5mM, 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 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 SEP 20 '00 02:44PM SPRUSON FERGUSON 92615486P. P.6 7 lowerclcd to Corm a Fluidised bed and the grkinulationl prmiess strirts, In tlhis way) haLeinjoglohinl itself i.S Used as the granulate starter.

The samie miethod as aboVe ISsed, but( nov, a binding aLieti.1 uSCLd tO granulate the dried hu~nmoglobin based portiCcs. IIn this waV tlhe part1-iCles inISIde the fluidised bed ire agglonieratad.

A separate starting matcrial is used to create a Iluidised bed. Tlhe product 1o 110 dried is directly sprayvd intuo the fluidized bed and is g~ranot.10d immelldiately.

Accrdigly threis provided in a first Cembodilcnlt 01, thle presenit invenltion ;I ehIOdLL I'm- preparint. a dried reconstitutable haemglotybin Corripositioll having a long s~jcf' F i Fe. which coiposi LiOnl CUn h10 recOni tISO d tLo a SOlutionl havinig aI Colloid osmotic prL1sre f bout I 0-30(0 mbar and/or an osmolarity of' abhout 150f-00) miosm! I whecreby ill Said mnethod a solution comlprising hacimoglobinl is providcd. wvhich solution is dIivided iilto smakll droplets., which droplets are dried to a solid particle form he- I sirean mof' gas.

:uIdRIC condiionIS Whlerein said gas is inert.

hdAccording to a second emnbodimnii of'li the resent invenition there Is provided a ilmelho for preparing a dried recons'titutable haemioglobin composition having a long shelf fifFe, which composition can be reconstituted to a solution having It colloid osmotio pressure of about 20-40 inbar aad/or anl osmolarity of'about 250-350 mosri/l. whereby inl said method thle solution is divided into small droplets, which droplets are dried to a solid partaicle form by a streamn of gas, under conditions whercin said gas is inert.

Apparatus to carry out spray-granulation on a tairly small scale are available fromi various mrnatux-ers. One is He~inen GmbH, Varcl, Giermiany who inartufacturvs .I S. small scalo batch-drier. Another- manufacturer is Niro iAeronatic.8. tBubendort., S witzerland who manufactures a small-scale sprny-drier/grantulator. Process plats to 2 i ar ut spray-granulation on a larger scale are also available.

lFigure I (appendix 1) shows a schematic diagramn of' the spray-granulator. In this upparatus the drying gas is drawn in by a blower I and passcs over an electric henter Tlhen the gas is blown upwards into thle drying chamber passing it sieve Tlhu aqIUeouS mixture to be sprayed is drawn up From a supply vessel by meants of a .W peristaltic metering pump and delivered to a spray nozzle which discharges thle MaLCLoS mixturo as a fine spray (10) into the streams of hot drying gas comning From the heiter The stream of hot drying gas arld the product are counter current.

The spray droplets arc dried to a solid powder florm ais they pass down inside the dryVing chamber Before thle spray droplets are completely dried, they make contact (RAiIBZ.Z]03363 .do~rmirr 20/09 '00 WED 15:43 [TX/RX NO 5968] SEP. 2000 12:56 SPRUSON FERGUSON NO. 1866 P. 6 withi the material in the fluidised bed Here the granulation process takes place by wetting the surface oft1he particles in the fluidised bed. This causes the particlcs to fuse, or whien a binding agent is used, the particles arm "glued" together by agglomeration. In this way, after multiple steps, a granulate .9~ 99.9 9*99 9* .9 9 r..

*9 9 9 I RALIBZZIO33rildocurnr 15/09 '00 FRI 13:53 [TX/RX NO 5915] WO 97/10268 PCT/NL96/00355 8 is formed. The gas stream leaves the drying chamber passing.

the filter To separate the outgoing gas from the dried material, a filter is placed in front of 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 spraydrying 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 invention this inlet temperature of the gas stream will generally exceed 80 0 C, will usually be approximately 100 0 C and may well lie in a range from 100 0 C up to 150 0 C. The drying gas will typically 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 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 0 300. 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 2pm and less than 100 particles per ml having a size of greater than 5 pxm.

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 physiologically acceptable formulation.

With respect to the use in emergency situations, as stated herein before, it is important that reconstitution of WO 97/10268 PCT/NL96/00355 9 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 Tetramethylbutyl)phenyl]-w-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/l, preferably between about 250-350 mosm/l, more preferably between about 280-300 mosm/l. 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.

WO 97/10268 PCT/NL96/00355 Examples Example 1 Preparation of anaerobically spray-granulated deoxyhaemoglobin A 0.22 pm filtered solution of modified haemoglobin (polyHbXl, CLB) in Water For Injection, containing 150 gram haemoglobin and 68 gram sucrose (Merck) per litre is spraygranulated by the use of a Heinen batch drier, type CWT RFS. The haemoglobin solution is deoxygenated before use (Capiox 350 oxygenator, Terumo) and the drying gas is nitrogen (medical grade, Air Products).

The inlet temperature of the drying gas is set to 100 0

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 0 After 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 spraygranulated, the inlet temperature of the drying gas is set to 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 resulting in a 10% haemoglobin solution with 133 mM sucrose. The final product so obtained has substantially not WO 97/10268 PCT/NL96/00355 11 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 2 Preparation of anaerobically spray-granulated stabilized deoxyhaemoglobin 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 resulting in a 10% haemoglobin solution with 133 mM sucrose. 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 3 Preparation of anaerobically spray-granulated deoxyhaemoglobin, 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 in the final reconstituted solution when 111.1 of 150 g/l 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 resulting in a haemoglobin solution with 133 mM sucrose and 102 mM sodium chloride. The final product so obtained has WO 97/10268 PCT/NL96/00355 12 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 deoxyhaemoglobin A 0.22 pm 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 Bichi 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 inlet temperature of the drying gas is set to 100 0

C.

The solution is sprayed into the drying chamber with a flow rate of approximately 3 ml/min. The temperature of the emerging gas is approximately 70 0 C. When all of the haemoglobin solution has been spray-dried, the inlet temperature of the drying gas is set to 20 0 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 resulting in a 10% 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 0 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 of storage at 4 0

C.

WO 97/10268 PCT/NL96/00355 13 Example Preparation of anaerobically spray-dried deoxyhaemoglobin with the use of a wetting agent.

The procedure is similar to that of example 4 with the exception that the haemoglobin composition is Hemolink (Hemosol, Etobicoke, Canada) and 37.5 p. 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 resulting in a 10% haemoglobin solution with 133 mM 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 of anaerobically spray-dried deoxyhaemoglobin and granulated with the use of a binder agent.

To a 0.22pj 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 RFS. The drying gas is nitrogen (medical grade, Air products). The inlet temperature of the drying gas is set to 100 0 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 0 C. When all of the haemoglobin has been spray-dried, the gas flow is lowered in order to obtain a fluidised bed. To granulate the spray-dried haemoglobin, a binder solution, containing 1 M sodium chloride, is sprayed on top of the fluidised bed. The temperature of the emerging gas is kept at approximately 50 0

°C

WO 97/10268 PCTINL96/00355 14 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 inlet temperature of the drying gas is set to 20 0 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 resulting in a 10% 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 7 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 spraygranulated, the inlet temperature of the drying gas is set to 0 C to cool the haemoglobin granulates. The cooled powder is aseptically removed from the granulator and filled in 100.0 WO 97/10268 PCT/NL96/00355 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 resulting in a haemoglobin solution with 133 mM sucrose. Within minutes 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 0 C and 20 0 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 0

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 deoxyhaemoglobin.

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 resulting in a 10% haemoglobin solution with 133 mM sucrose. The final product so obtained has substantially not altered physicalchemical properties if compared with the starting material.

WO 97/10268 PCT/NL96/00355 16 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 resulting in a 10% 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 increased.

WO 97/10268 PCT/NL96/00355 17 Literature British Pharmacopoeia, Volume II. London, 1980.

Chaillot, P. Labrude, C. Vigneron and D. Simatos.

'Freeze-drying of haemoglobin solutions without adjuvant and in presence of glucose, tris and I-alanine.' American Journal of haematology 10, 1981. p319-326.

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

Henry, D.C. Cannon, J.W. Winkelman Clinical Chemistry, 2nd edition, New York: Harper Row Publishers Inc.

1974.

Labrude, M. Rasolomanana, C. Vigneron, C. Thirion and B.

Chaillot. 'Protective effect of sucrose on spray-drying of oxyhaemoglobin.' Journal of Pharmaceutical Science. vol.78, no.3, 1989. p.223-229.

Wade, A. 'Martindale: The extra Pharmacopoeia', 27th edition. London: The pharmaceutical press, 1979.

Claims (28)

1. 4. A method according to claimn 3 whereby, a stabilising additive is sucrose, :LIUMx-0an- I or human serum albumin. A method according to any one of' the foregoino claimis whereby thec SolIution comprising haenmoglabin further contains at least one electron quenIChling
2. 0006. A method according to claim 5 whlereby a electron quenching additive is ascorbic acid or human serum albuminl. A method according to any one of' the fObegoing claims whereby the ***:Solut60ios comprising haemoglobin further contains a wetting agent.
3. 8. A method according to claim 7 wh-ereby a wetting agent is TWecil (polyoxyethylezne sorbitan mlonooleaat) or l'ritonl (alIpli a4 4 1. 11, VIetramilmtylb~ityl)lilhCI1yll(i)-hydroxylelYt (oxy-1 .2-ethancdiyl). 20/09 '00 WED 15:43 [TX/RX NO 5968]
4. 9. A method according to any one of the foregoing claims which further comprises granulation of the drying or dried particles. A method according to claim 9 whereby at least one starter material is used in the granulation process. S11. A method according to claim 10 whereby a starter material is sodium chloride or human serum albumin.
5. 12. A method according to any one of claims 9-11, whereby at least one binding agent is used to granulate the dried particles.
6. 13. A method according to claim 12 whereby the binding agent is sodium I0 chloride, sucrose, dextran-1, or sodium lactate.
7. 14. A method according to any one of the foregoing claims whereby the droplets are provided by forcing the solution comprising haemoglobin through a spray nozzle. A method according to any one of the foregoing claims whereby the 15 droplets are provided as a stream, which stream is in counter current with the stream of inert gas.
8. 16. A method according to any one of the foregoing claims whereby the inert gas is first contacted with the droplets at a temperature of at least 80 0 C.
9. 17. A method according to claim 15 whereby the temperature of the gas is 20 between about 100-150°C.
10. 18. A method according to any one of the foregoing claims whereby the gas s nitrogen or air.
11. 19. A method according to any one of claims I to 17 which is carried out under oxygen free conditions.
12. 20. A method according to any one of the foregoing claims, whereby the solid particles are separated from the stream of inert gas by a filter.
13. 21. A dried haemoglobin composition obtainable by a method according to any one of the foregoing claims.
14. 22. A dried haemoglobin composition obtainable by a method according to any one of claims 1 to 20, comprising less than 15% methaemoglobin.
15. 23. A dried haemoglobin composition obtainable by a method according to any one of claims 1 to 20, comprising less than 5% methaemoglobin.
16. 24. A dried haemoglobin composition according to any one of claims 21 to 23, which upon reconstitution results in a solution having [R:\LIBZZ]03363.doc:mrr less than 1000 particles per ml having a size greater than 2.tm and less than 100 particles per ml having a size greater than 51am. A dried haemoglobin composition according to any one of claims 21 to 24 being in the form of a granulate.
17. 26. A method for producing an immediately transfusable haemoglobin solution, whereby a dried haemoglobin composition according to any one of claims 21 to is provided, optionally stored, said composition being reconstituted with Water For Injection or an aqueous solution resulting in a physiologically acceptable solution.
18. 27. A method according to claim 26 whereby the solvent comprises a I wetting agent.
19. 28. A method according to claim 27 whereby a wetting agent is lecithin S.*(phosphatidylcholine), Tween (polyoxyethylene sorbitan nonooleaat) or Triton (alpha [4- (1.1.3,3-Tetramethylbutyl)phenyl]-o-hydroxypely (oxy-1,2-ethanediyl).
20. 29. A reconstituted haemoglobin solution obtainable by a method according 5i to any one of claims 26 to 28. A reconstituted haemoglobin solution according to claim 29 having an osmolarity of between 150-600 mosm/1, a colloid osmotic pressure between about 10-300 mbar, less than 1000 particles per ml having a size greater than 2um and less than 100 particles per ml having a size greater than 5ptm and whereby less than 15% of the 20 haemoglobin is methaemoglobin.
21. 31. A reconstituted haemoglobin solution according to claim 29 or claim wherein the osmolarity is between 280-300 mosm/1.
22. 32. A reconstituted haemoglobin solution according to any one of claims 29 to 31 wherein the colloid osmotic pressure is between 20-40 mbar.
23. 33. A reconstituted haemoglobin solution according to any one of claims 29 to 32 wherein less than 5% 0 of the haemoglobin is methaemoglobin.
24. 34. A spray-granulated haemoglobin composition comprising haemoglobin dried by a stream of inert gas. A method for preparing a dried reconstitutable haemoglobin composition having a long shelf life, substantially as hereinbefore described with reference to any one of the Examples.
25. 36. A dried haemoglobin composition, substantially as hereinbefore described with reference to any one of the Examples. [R:\LIBZZ]03364.doc:nmrr
26. 37. A method for producing an immediately transfusable haemoglobin solution, substantially as hereinbefore described with reference to any one of the Examples.
27. 38. A reconstituted haemoglobin solution, substantially as hereinbefore described with reference to any one of the Examples.
28. 39. A spray-granulated haemoglobin composition, substantially as hereinbefore described with reference to any one of the Examples. Dated 15 September, 2000 Staat der Nederlanden, de Minister van Defensie, voor deze: het il hoofd van de afdeling Militair Geneeskundig Beleid S Patent Attorneys for the Applicant/Nominated Person 15 SPRUSON FERGUSON S *o S [R\L I BZZ]03 364. doc: mrr