WO2008075072A2

WO2008075072A2 - Formulation

Info

Publication number: WO2008075072A2
Application number: PCT/GB2007/004923
Authority: WO
Inventors: Paul Alexander Bateman; Howard James Orman; Charles Richard Wedge
Original assignee: Oxford Biosensors Limited
Priority date: 2006-12-21
Filing date: 2007-12-20
Publication date: 2008-06-26
Also published as: EP2121961A2; GB0625671D0; CA2679089A1; WO2008075072A3

Abstract

The present invention relates to the stabilisation of an enzyme formulation for use in a sensor such as a biosensor.

Description

Formulation

A biosensor is an analytical tool combining a biochemical recognition component or sensing element with a physical transducer. A biosensor has wide application in fields as diverse as personal health monitoring, environmental screening and monitoring, bioprocess monitoring and within the food and beverage industry. Biosensors offer the convenience and facility of distributed measurement ie the potential ability to take the assay to the point of concern or care. A properly designed and manufactured biosensor may be conveniently mass-produced.

The biological sensing element may be an enzyme, antibody, DNA sequence or microorganism which serves (for example) to catalyze selectively a reaction or facilitate a binding event. The selectivity allows for the operation of the biosensor in a complex sample matrix {eg a body fluid). The transducer converts the biochemical event into a measurable signal thereby providing the means for detecting it.

Enzyme based biosensors are widely used in the detection of analytes in clinical, environmental, agricultural and biotechnological applications. They offer specificity, sensitivity and operate under mild conditions. Analytes that can be measured in clinical assays of fluids of the human body include (for example) glucose, lactate, cholesterol, bilirubin and amino acids. Levels of these analytes in biological fluids (such as blood) are important for the diagnosis and monitoring of diseases.

Sensors which generally exploit enzyme based systems are provided as either point-of-care or over-the-counter devices. They can be used to test fresh, unmodified, whole blood finger prick samples in order to determine the concentrations of total cholesterol, triglycerides, HDL and LDL within (for example) 1 to 5 minutes of adding the sample to a device. There is, however, a significant disadvantage associated with enzyme-based-biosensors. The enzyme component is often unstable. This can lead to degeneration of the enzyme during storage and inconsistent results when analysing a biological material.

An enzyme is a protein having a primary, secondary and tertiary structure. The primary structure is based on the sequence of amino acids which form the backbone of the enzyme. The peptide bonds which hold the amino acids together are generally stable under normal biological conditions. The secondary and tertiary structures are formed by folding the enzyme into a complex three dimensional structure. These structures (in particular the tertiary structure) can be disrupted quite easily. Once the secondary and tertiary structures have been disrupted, the enzyme will generally lose its biological function. There have been considerable efforts to develop methods for stabilising enzymes in order to permit them to be stored for extended periods of time. These methods include immobilisation, chemical modification by cross-linking, polymer grafting or substitution, entrapment in a polymer matrix, storage in solution at low temperature and freeze drying. Freeze drying (which is also known as lyophilisation) permits long term storage of sensitive materials such as proteins During the freeze drying process, the protein is reduced to a dehydrated form for storage in a suitable environment. Freeze drying works by freezing a protein solution (usually an aqueous solution) and then removing the water by sublimation at reduced pressure. Once dehydration is complete, the protein is left as a powder or 'cake'. In a typical freeze drying process, a sample of aqueous protein solution is placed in a flask which is immersed in a cryogenic material (such as liquid nitrogen) until the solution is frozen. The flask containing the frozen material is attached to an appropriate manifold and freeze- drying occurs by applying a vacuum to effect sublimation.

The structure of the cake is important in allowing the material to be reconstituted. If the cake has small pores, the removal of water during the freeze-drying process can be impeded. As a result, the drying process is incomplete and the cake has a high moisture content. If the cake is formed with large pores, the drying process is more efficient and the cake has a low moisture content. A low moisture content is essential to prevent any undesirable chemical reaction/denaturation of the stabilised material.

During the initial drying stage, a vacuum is applied and in some cases heat is transferred to the frozen mass resulting in sublimation. Generally, freeze drying is used to remove water from a solution or formulation. As sublimation occurs, water vapour passes from the frozen mass through to a freeze drying chamber. As the temperature increases, there is a higher saturated vapour pressure which results in an increased rate of drying. This results in a shortened freeze drying cycle. The upper limit on the drying temperatures during this stage ensures that the temperature of the product is maintained below the system collapse temperature.

Collapse is associated with a decreased surface area of dried formulation, reduction in volume and may also be associated with increasing the constitution time. In the event that the freeze dried material collapses, solvent which has not been removed can become trapped. This may reduce undesirably the stability of the final product and have an adverse impact on its performance.

The collapse temperature is the temperature at which the material softens to the point of not being able to support its own structure. In general, as the level of solvent is reduced via sublimation, the collapse temperature increases. In most systems which contain a protein, the onset of this temperature is not well defined and can occur over a range of temperatures. A material that will sustain this higher structural stability at a higher temperature may therefore allow faster processing. Components of the mixture may, therefore, impart stability during the freeze drying process in addition to stabilising the protein during subsequent storage.

A possible drawback of freeze drying is the inability to redissolve the protein. This usually indicates that some denaturing has occurred during freeze drying. It is known to obviate or mitigate this by including a stabilising agent which is not itself removed during the freeze drying process and serves to stabilise the protein. A stabilising agent is usually added to the formulation to improve the stability of the protein by reducing denaturation, aggregation, deamidation or oxidization during freeze drying and storage. The stabilising agent can be in the form of an excipient. An excipient is an inactive material that is specifically added to a pharmaceutical to perform a number of functions. However, the main function is to aid the stability of the formulation such that it has a desired shelf life and bioavailability. An excipient may be used with small pharmaceutical molecules or in biotherapeutic formulations. In a small molecule formulation, the excipient is most often used to aid tablet formation by (for example) acting as a lubricant. In a biotherapeutic application, an excipient may be generally used to improve stability. Excipients which are typically used in biotherapeutic formulations are sugars, polyols, amino acids and polymers. For example, a saccharide such as sucrose can act as a cryoprotectant, a lyoprotectant and a tonicity modifier. Sucrose is a non-reducing sugar which can act as a stabilising agent. It is believed that such sugars are able to form hydrogen bonds with the protein structure preventing denaturation. This is known as water replacement. Another postulated mechanism is that the stabilising agent is excluded from the protein surface and destabilises the unfolded state thereby forcing the protein into the folded state.

A bulking agent is often included in a freeze drying formulation to generally enhance the formation of the solid cake and improve its structure. A freeze-dried protein formulation generally includes a stabilising agent, a crystalline bulking agent to improve the freeze drying characteristics and stability of the cake and a surfactant to provide further stabilisation and inhibition of protein loss by adsorption to surfaces. However, it is believed that bulking agents may reduce the stability of the protein. Typical bulking agents include glycine and mannitol. Mannitol is a naturally occurring carbohydrate. Glycine is a non-polar amino acid. Under certain conditions, some bulking agents (such as mannitol and glycine) form crystals during the freeze drying process. This can result in destabilisation of the active agent, collapse of the product cake or can impact on the structure of the cake by releasing water into the amorphous phase. The protein is generally in an amorphous phase.

The choice of bulking agent can determine the final physical properties (eg ease of dissolution) of a freeze dried product. A salt such as KCl and NaCl for instance forms a crystalline structure on freezing allowing large ice crystals to form which can rapidly sublime and leave a large porous dried structure that dissolves very quickly. However, physiochemical properties may be altered by the formation of eutectic phases with other excipients. A sugar such as sucrose can be used as a stabiliser and a bulking agent in its own right. However whilst the formulation will be stable to denaturation, the amorphous cake produced may be slow to dissolve. However, the quick dissolving, large pore cakes achieved by excipient bulking agents such as KCl can dissolve very quickly but are not commonly used as the main excipient due to the poor stability/ activity retention of the formulations. It is known in pharmaceuticals that adding a salt excipient to a formulation with a sugar as a bulking agent results in poor freeze drying.

The present invention is based on the suprising recognition that when a protein, a salt and a sugar are combined in an appropriate ratio, a viable freeze dried formulation is achieved with high collapse temperature and good cake structure whilst retaining protein activity. More particularly a desirable combination of attributes of two excipients is attainable without compromising the attributes of either one. According to a first aspect of the present invention there is provided the use of a stabilised freeze dried formulation in a biosensor wherein the formulation comprises one or more enzymes, a salt and a saccharide or polyol.

By exhibiting one or more useful properties such as stability, good cake formation or good dissolvability, the freeze dried formulation has advantages in biosensing which are exploited in the use according to the invention.

The freeze dried formulation may be dissolvable.

The freeze dried formulation may be porous. For example, the freeze-dried formulation may have a sponge or foam-like appearance. The freeze-dried formulation may have an extended network of pores and a low moisture content.

Typically the freeze dried formulation forms a cake. Preferably the cake is white. Particularly preferably the cake has a cake brightness (measured for example in accordance with Example 2 hereinafter) which exceeds 180, more preferably exceeds 190. The cake may have a cake brightness in the range 150-230.

Typically the freeze dried formulation has a collapse temperature of -24⁰C or higher. Preferably the freeze dried formulation has a collapse temperature of -22⁰C or higher, particularly preferably -2O⁰C or higher. A freeze dried formulation with a collapse temperature of -2O⁰C or higher advantageously gives a bright cake with good dissolution properties.

The freeze dried formulation may have a collapse temperature in the range -40 ⁰C to -15⁰C.

The freeze dried formulation may have a dissolution rate of 60s or less, preferably 50s or less, particularly preferably 35s or less.

The (or each) enzyme may be an oxidoreductase or hydrolase. Preferably the (or each) enzyme is a lipase, an esterase, an oxidase (eg a NADH oxidase), a dehydrogenase, a reductase or a kinase.

The (or each) enzyme may be selected from the group consisting of cholesterol esterase, glucose oxidase, cholesterol oxidase, glycerol-3 -phosphate oxidase, lactate oxidase, glycerol dehydrogenase, cholesterol dehydrogenase, lactate dehydrogenase, putidaredoxin reductase, diaphorase and glycerol kinase.

Preferably the (or each) enzyme is selected from the group consisting of cholesterol esterase, glycerol dehydrogenase, cholesterol dehydrogenase, putidaredoxin reductase, lipase and diaphorase.

Particularly preferably the (or each) enzyme is selected from the group consisting of cholesterol esterase, cholesterol dehydrogenase and putidaredoxin reductase.

Particularly preferably the (or each) enzyme is selected from the group consisting of glycerol dehydrogenase, lipase and diaphorase. In a preferred embodiment of the use of the invention, the freeze-dried formulation comprises an enzyme, a salt and a saccharide.

The saccharide may be a monosaccharide, disaccharide, trisaccharide, mucopolysaccharide, oligosaccharide or polysaccharide. The monosaccharide may be (for example) an aldohexose such as (for example) mannose or glucose. The disaccharide may be (for example) sucrose, lactose, maltose or trehalose. The trisaccharide may be (for example) raffinose. The mucopolysaccharide may be (for example) heparin.

Preferably the saccharide is a monosaccharide or disaccharide, particularly preferably a disaccharide.

The saccharide may be selected from the group consisting of raffinose, heparin, inositol (eg myo-inositol), mannose, glucose, sucrose, lactose, maltose or trehalose. Preferably the saccharide is sucrose.

In an embodiment of the use of the invention, the formulation comprises an enzyme, a salt and a polyol.

The polyol may be selected from the group consisting of mannitol, lactitol and erythritol (eg penta-erythritol).

The salt may be a simple salt (eg a simple inorganic salt). The salt may be a salt of a monovalent or polyvalent (eg divalent or trivalent) ion (eg metal ion). Preferably the salt is a salt of a monovalent ion (eg metal ion).

The salt may be an alkali or alkaline earth metal salt, a transition metal salt or an ammonium salt. Preferably the salt is an alkali or alkaline earth metal salt or an ammonium salt. Particularly preferably the salt is an alkali metal (eg lithium, sodium or potassium) or ammonium salt.

The salt may be a halide. Preferably the salt is a chloride or bromide. Particularly preferably the salt is a chloride.

The salt may be selected from the group consisting of ammomium chloride, lithium chloride, sodium chloride and potassium chloride. Preferably the salt is potassium chloride or ammonium chloride.

The weight of salt may be in the range 5 to 15% (w/v), preferably 7 to 13% (w/v), particularly preferably 8 to 10% (w/v) (eg about 9% (w/v)).

The weight of saccharide may be in the range 0.3 to 3% (w/v), preferably 0.5 to 1.5% (w/v), particularly preferably 0.8 to 1.2% (w/v) (eg about 1% (w/v)).

The weight ratio of saccharide : salt may be in the range 9:1 to 1 :200. Typically the weight of salt exceeds the weight of saccharide. Preferably the weight ratio of saccharide : salt is in the range 1 :2 to 1 :150, particularly preferably 1 :3 to 1 :20, more preferably about 1 :9. The total weight of saccharide and salt may be in the range 15 to 60% (w/w). Preferably the total weight of saccharide and salt is in the range 25 to 35%, particularly preferably 27 to 32%, more preferably about 29%.

The total weight of protein (including the (or each) enzyme) may be in the range 2 to 40% (w/w). Preferably the total weight of protein (including the (or each) enzyme) is in the range 15 to 25%, particularly preferably 20 to 22%.

The weight ratio of the total weight of saccharide and salt: the total weight of protein (including the (or each) enzyme) may be in the range 0.5:1 to 20:1. Preferably the weight ratio of the total weight of saccharide and salt: the total weight of protein (including the (or each) enzyme) is in the range 0.7:1 to 3:1, particularly preferably 1 : 1 to 2: 1 , more preferably about 1.5 : 1. In the context of a pharmaceutical formulation, it is normal to have an amount of excipient which significantly exceeds the amount of protein. It is therefore surprising to find that in preferred embodiments, the advantages associated with the invention are exhibited at an amount of excipient (ie saccharide and salt) which is less than or not significantly greater than the amount of protein (including the (or each) enzyme). Without wishing to be bound by theory, it is thought that this may be associated with "pure" saccharide portions of the formulation having low solubility.

The formulation may further comprise a protein, co-factor, buffer solution, surfactant, excipient or redox mediator.

The redox mediator may be an electron transfer agent for carrying electrons between an analyte or an analyte-reduced or analyte-oxidized enzyme, co-factor or other redox active species and an electrode (either directly or via one or more additional electron transfer agents). A ruthenium complex is a preferred redox mediator.

The co-factor may be a NAD analogue such as acetyl pyridine adenine dinucleotide, 3-acetyl pyridine hypoxanthine dinucleotide, nicotinamide guanine dinucleotide, nicotinamide hypoxanthine dinucleotide, nicotinic acid adenine dinucleotide, nicotinamide dinucleotide or thionicotinamide dinucleotide.

The buffer solution may be Tris, HEPBS, CAPS, CHES or TABS.

The surfactant may be a bile salt (eg cholic acid, taurocholic acid, glycocholic acid, lithocholic acid or deoxycholic acid), CHAPS (3-[(3-cholamidopropyl)- dimethylammino]propane), CHAPSO, BIGCHAP, deoxy BIGCHAP or a pryranoside (eg octylglucopyranoside).

According to a second aspect of the present invention there is provided a biosensor consisting essentially (eg consisting) of a biochemical substrate containing an analyte, a detector for detecting the presence (eg amount) of or changes in (eg changes in the amount of) the analyte and a freeze-dried formulation as hereinbefore defined. The biochemical substrate may be a biological fluid such as a bodily fluid (or a derivative thereof) in which the analyte may be a measurable chemical or biochemical substance such as an enzyme, an antibody, a DNA sequence or a microorganism. The biochemical substrate may be for example blood, urine, interstitial fluid, plasma, dermal fluid, sweat or tears.

Preferably the analyte is triglyceride or cholesterol.

The biosensor may be adapted to cause the generation of a measurable signal that may be correlated to the presence or an amount or concentration (or changes in amount or concentration) of the analyte. The measurable signal may be an electrode potential, fluorescence, absorption spectroscopy, luminescence, light scattering, NMR, IR, mass spectroscopy, heat change or a piezo-electric change. The measurable signal may be detected by the detector and correlated with the presence (eg amount) of or changes in (eg changes in the amount of) the analyte.

The biosensor may be an electrochemical biosensor configured to detect the presence of or measure the concentration or amount of an analyte in a sample via electrochemical oxidation or reduction reactions.

The biosensor may comprise a biosensing element such as a biosensing strip with a plurality (eg four) reagent wells and a common reference. Each well may have its own tubular micro-band working electrode. The (or each) well may be equipped with the same (or a different) freeze-dried formulation to permit single or multi- analyte sensing.

The biosensor may take the form of a microelectrode. Typically the microelectrode has a working microelectrode and a reference electrode (eg a pseudo or true reference electrode). The microelectrode may include a counter electrode.

The working electrode is typically made of palladium, platinum, gold or carbon. The counter electrode is typically made of carbon, Ag/AgCl, AgZAg₂SO₄, palladium, gold, platinum, Cu/CuSO₄, Hg/HgO, Hg/HgCl₂, Hg/HgSO₄ or Zn/ZnSO₄. In a preferred microelectrode, the working electrode is in a well of a receptacle forming said microelectrode. Examples of microelectrodes on which the biosensor of the present invention may be based are those disclosed in WO-A-2003/097860.

According to a third aspect of the present invention there is provided a formulation comprising an enzyme, a saccharide and a salt, wherein the enzyme is selected from the group consisting of a lipase, an esterase, an oxidase, a dehydrogenase, a NADH oxidase or a kinase.

The formulation according to the third aspect of the invention may be freeze- dried. The formulation according to the third aspect of the invention may be porous.

According to a fourth aspect of the present invention there is provided a method for stabilising an enzyme comprising the steps of: a) preparing a formulation as hereinbefore defined (eg according to the third aspect of the invention) in solution; b) freezing the solution; and c) freeze-drying the solution in order to obtain a stabilised freeze-dried formulation.

Steps (b) and (c) may be carried out in batches (eg in a batch free drier) or continuously (eg in a continuous freeze drier). A suitable system is described in WO- A-2007/066132.

Steps (b) and (c) may be carried out in in situ in a biosensing element such as a biosensing strip or card. For example, step (b) may be preceded by: al) pre-loading the formulation into a biosensing element.

In a preferred embodiment, step (b) is preceded by: al) obtaining a biosensing element, wherein the biosensing element comprises a plurality of biosensing wells each capable of acting as a microelectrode; a2) loading the formulation portionwise into the wells of the biosensing element.

Alternatively the method may further comprise: d) loading the freeze-dried formulation into a biosensing element.

The biosensing element may be a strip or card.

Particularly preferably, the method further comprises: (d) sealing the wells of the biosensing element.

According to a fifth aspect of the present invention there is provided use of a combination of a sugar and a salt to stabilise a freeze-dried formulation containing one or more lipase enzymes.

In this aspect of the invention, the sugar may be a disaccharide. The disaccharide may be selected from the group consisting of sucrose, lactose, maltose and trehalose.

In this aspect of the invention, the salt may be selected from the group consisting of lithium chloride, sodium chloride and potassium chloride.

In this aspect of the invention, the ratio of saccharide: salt may be in the range 1 :3 to 1 : 100. The ratio of saccharide can be in the range 1 :9 to 1 :20.

According to a sixth aspect of the invention there is provided use of a stabilised freeze dried formulation for biosensing wherein the formulation comprises one or more enzymes, a salt and a saccharide or polyol.

According to a seventh aspect of the invention there is provided a method of biosensing comprising: exposing a freeze-dried formulation as hereinbefore defined to a biochemical substrate containing an analyte in the presence of a detector for detecting the presence (eg amount) of or changes in (eg changes in the amount of) the analyte. Embodiments of the invention will now be described by way of example only with reference to the accompanying Figures in which:

Figures 1 - 3 show electrochemical results obtained using a 9%KCl/l%sucrose (w/v) freeze-dried formulation in a triglyceride sensor in accordance with the present invention (Figure 1 stability: t=70s; Figure 2 stability: t=l 12s; Figure 3 stability:

Gradients);

Figures 4 - 6 show electrochemical results obtained using a freeze-dried formulation in a triglyceride sensor without the KCl/sucrose stabiliser of the present invention

(Figure 4 stability: t=70s; Figure 5 stability: t=112s; Figure 6 stability: Gradients);

Figure 7 shows currents obtained for batch freeze dried triglyceride biosensors of the invention;

Figure 8 illustrates three wells containing a formulation in accordance with the present invention and a well containing a formulation containing glycine for comparison;

Figure 9 shows the UV/Vis reaction in the presence of two different excipient combinations;

Figure 10 shows the currents obtained for batch freeze dried triglyceride biosensors of the invention;

Figure 11 shows the collapse temperature of different excipient combinations for a total cholesterol formulation;

Figures 12 and 13 show how the collapse temperature varies with variations in the ratio of salt and sugar; and

Figure 14 illustrates calibration data for a triglyceride formulation.

The results illustrated in Figures 1-3 and 4-6 show that the presence of potassium chloride (KCl) and/or sucrose in the triglyceride solution 'activates' the three enzymes contained therein and leads to a larger electrochemical signal. In addition, the presence of KCl provides a rigid matrix of solute with associated air pores required to create a hydrophillic cake following the freeze drying process. Although not wishing to be bound by theory, it is believed that the KCl is acting as an excipient.

The use of KCl/sucrose prevents the shift in pH that was present when glycine was used as the excipient. The enzyme in the formulation of the present invention tends to favour a basic pH so any shift in the acidic direction would result in a movement away from optimum performance. In addition, the use of KCl/sucrose raises the collapse temperature of the formulation making it much easier to freeze dry both in the batch and the quasi-continuous freeze drier systems (as disclosed in WO- A-2007/066132).

With regard to freeze drying, the new combination of excipients and stabilisers elevates the collapse temperature of the cake from -35°C (when glycine was used) to - 28⁰C.

EXAMPLE 1 - A triglyceride sensor

A 0.1M HEPBS buffer adjusted to pH9 ±0.1 with 1OM KOH was acoustically mixed with NH₄Cl, KCl, CHAPS and sucrose. The buffer was acoustically mixed into solution with Ru (NHs)₆Cl₃, diaphorase reductase, lipase, GIyDH and TNAD. The approximate concentrations in the final mix were:

0.1 M HEPBS (pH 9.0)

10 mM Ammonium Choride

9% w/v KCl

1% w/v CHAPS

1% w/v sucrose

80 mM Ru (NH₃)₆C1₃

17.6 mM TNAD

6.5 mg/ml Diaphorase

100 mg/ml Lipase

45 mg/ml GIyDH

The final mixture was freeze-dried immediately either in a batch freeze drier (A) or a continuous freeze drier (B). The drying procedure for each freeze-drier was as follows:

Batch Freeze Drier (A)

T=0mins -3O⁰C at lOOOmbar

T=45mins -37.5⁰C at lOOOmbar

T=90mins -37.5⁰C vacuum applied

T=108mins -37.5⁰C at 4xlθΛnbar

T= 180mins -37.5⁰C at 4x 10^~2mbar heating starts

T=300mins 22⁰C at 4x 10^"2mbar

The product was removed from the freezer drier at any time thereafter. The product was then stored under dry N₂.

Continuous Freeze Drier (B)

T=0mins -58⁰C at lOOOmbar

T=5mins card transferred 25°C max pump applied

(2xlO^"2mbar achieved after 5mins)

T=I Omins 25⁰C backfill with dry N₂

T=10.5mins card transferred 25⁰C at lOOOmbar

T=10.1mins card ejected

The product was removed to a dry air area.

After freeze drying, each card (each of which comprises a number of sensors) was constructed with membranes and sealed in a foil pouch with desiccant.

A comparison of the freeze-dried formulation of the present invention with a formulation where KCl/sucrose was replaced with glycine was carried out. The results are detailed below.

Cake Quality | 3

A rating of 1 indicates a poor cake structure. The structure of the frozen liquid was not retained in the freeze dried product leading to a small or no pore structure. This led to slow dissolution and relatively high residual moisture.

A rating of 2 indicates an average cake structure. The frozen liquid structure was partially retained in the freeze dried product leading to a slightly porous cake structure with small pores.

A rating of 3 indicates a good cake structure. The frozen liquid structure was completely retained leading to a porous cake structure with numerous open pores. This led to fast re-dissolution and low moisture content.

An illustration of the above results is shown in Figure 8, wherein

1 is the cake formed using a formulation having a KChsucrose ratio of 10:1,

2 is the cake formed using a formulation having a KCl:sucrose ratio of 9:1,

3 is the cake formed using a formulation containing glycine and

4 is the cake formed using a formulation having a NaCl:sucrose ratio of 8:2.

The cakes formed using KCl or NaCl and sucrose had a similar light appearance denoting a good porous structure with a desired porosity. The cake formed by the formulation containing glycine had a poor porous structure where the size of the pores was very small resulting in a dark appearance.

The structure of the cake can be assessed by measuring the pore size or the compressive strength of the cake or by microscopy.

EXAMPLE 2 - Formulations and Testing Protocols Electrode sheets

Standard sheets: Screen printed electrodes with laser drilled wells with hydrophobic mesh backing. Electrodes are standard electrodes available from Oxford Biosensors as disclosed in WO-A-03/56319

Standard Total Cholesterol sensor (TC)

0.1 M Tris buffer pH 9.0 l% Ecotine

1% Myo-Inositol

80 mM Ruthenium Hexaamine trichloride

9 mM thionicotinamide dinucletotide (TNAD)

5% CHAPS

5% Deoxy-bigCHAPS

3ml/ml Cholesterol esterase

4 mg/ml Putidaredoxin reductase

66 mg/ml cholesterol dehydrogenase x% salt y% sugar

Unless otherwise stated, x is 9% and y is 1%.

Standard Triglyceride Sensor (TRG)

Approximate concentrations in final mix:

0. IM HEPBS Buffer pH 9

1OmM NH₄Cl

1% CHAPS

1 % sucrose

8OmM Ru(III)(NH₃)₆Cl₃

17.6mM thio-nicotinamide dinucleotide (TNAD)

9%KC1

45 mg/ml GIyDH

6.6 mg/ml Diaphorase

100 mg/ml Lipase

Production of biosensor

Using an electronic pipette, 0.4μl of enzyme solution was dispensed into the four wells of the electrode strip. This was completed for all the strips on a card. The card was then freeze dried either using the continuous freeze drier or the batch freeze drier (the protocol is given below)

Cake Production Using the Continuous Freeze Drier

The dispensed sheet was loaded into the continuous freeze drier and the following protocol was followed:

The freeze dried sensors were stored in a low relative humidity environment. Cake Production Using the Batch Freeze Drier

The card was placed in the freeze drier at atmospheric pressure and the freezing plates set to -3O⁰C. Once the card was loaded, the cycle was initiated and the temperature decreased reaching the minimum temperature of -37.5⁰C in 43 minutes. After a further 50 minutes, a vacuum was applied and the minimum vacuum of 4x10^" ²mbar was achieved after an additional 23 minutes. The vacuum was applied for 1.5hrs after which the temperature in the chamber was increased at a rate of 0.5°C/min to +22⁰C. After the card had been at 22⁰C under vacuum for 0.5hrs, it was removed. To remove the card, the chamber was filled with dry nitrogen until atmospheric pressure was reached. The card was retrieved and immediately transferred to a dry environment for further processing.

Measurement of Collapse Temperature

The collapse temperature of a solution was measured using a Linkam FDCS 196 freeze-drying microscope. An electronic pipette was used to dispense 2μL of the test sample onto the freeze drying stage of the microscope. It was covered by a glass lid supported by a shim after which the temperature of the stage was cycled as follows:

As its temperature was lowered to -5O⁰C, the sample froze. One minute after the sample had reached -5O⁰C, the vacuum was applied (the pressure was set to O.Olmbar). The sublimation front then progressed into the sample as the temperature increased. Approaching the collapse temperature, the sublimation front/bulk boundary showed the onset of collapse. The collapse temperature was measured when the structure at ihis interface started to melt.

Studies of Changes in Formulation

(A) Varying the Sugar to Salt Ratio Using the standard total cholesterol mixture, the ratio of sugar to salt was varied but the total amount of excipient in the formulation remained constant at 10% (w/v). The results are given in Table 1 (see samples A to H).

(B) Varying the Sugar

Using the standard total cholesterol mixture, the sugar to salt ratio was fixed at 9: 1 and the total amount of excipient in the formulation remained unchanged at 10% (w/v). The sugar was a mono-, di- or tri-saccharide, polyol or polysaccharide and the results are given in Table 1 (see samples I to Q).

(C) Varying the Salt

The sugar to salt ratio was fixed at 9: 1 and the total amount of excipient in the formulation remained unchanged at 10% (w/v). A number of salts were tested. Mg salt gave too low a collapse temperature to be useable. Copper salt precipitated and could not be used. K, NH₄ and Sr salts gave a cake with the standard total cholesterol formulation. K and Na gave a cake with the standard triglyceride formulation. In testing the salts for use in the invention, it became clear that the collapse temperature of the salt is important in determining whether or not is obtained a cake that is suitable for biosensing application. The results are given in Table 1 (see samples R to T3).

(D) Varying the Protein to Excipient Ratio

The ratio of excipient to protein was varied either by adjusting the amount of excipient in the formulation or by reducing the amount of protein in the formulation. The results are shown in table 1 (see samples U to Y).

Pictures of the cakes were taken with an Optical Gauging Product (model CNC 500 Smartscope) using the same lighting and magnification each time. This ensures an accurate and reproducible measurement of the cake brightness. A digital picture was produced and stored. It is made of pixels. The brightness of each pixel making up the image was encoded over 8bits: 2⁸=256 possible nuances (0 is black and 255 is pure white). The portion of the image which corresponds to the cake was analysed to get the average brightness value of the pixels that make up the image of the cake. This value is the cake brightness (see table 1).

In table 1 , the dissolution quality is expressed as a rating of 1 to 5 according to table 2:

Figure 11 shows the collapse temperature of different excipient combinations for a total cholesterol enzyme formulation mixture. Figure 12 shows how the collapse temperature varies with different ratios of salt and sugar (in this case KCl and lactose) in water. Figure 13 shows how the collapse temperature varies with different ratios of salt and sugar (in this case KCl and sucrose) in water.

EXAMPLE 3 - Lvophilised Serum: Predicted Currents

Triglyceride strips were manufactured using the standard triglyceride formulation and the batch freeze drier and stored at different temperatures (4⁰C at (•) 1.7 and (A)3.5 mM and 3O⁰C at (*)1.7 and (4)3.5 niM as illustrated in Figure 7) and tested every month over a 12 month period. The tests were completed with different plasma samples and the comparison was made by standardising the currents obtained experimentally to a particular concentration assuming a linear relationship between current and concentration. The currents given in Figure 7 are those obtained 98 seconds after the plasma had been applied to the strip.

EXAMPLE 4 - UV/Vis Data

The excipients 8%NaCl/2%Sucrose and 8%KCl/2%Sucrose in a triglyceride formulation were tested in UV/Vis using 1/16^th of the enzyme concentrations used in the electrochemical assay and for a triglyceride concentration of 2.36 mM. The results were similar to those obtained with the freeze dried electrochemical sensors (see Figure 9 where \ = 8 % NaCl and 2% sucrose and ■ = 8% KCl and 2 %sucrose). This suggests that the use of salt and sugar would also be viable in the production of optical biosensors.

EXAMPLE 5

Currents were obtained for batch freeze dried triglyceride sensors based on the standard triglyceride biosensor formulation but where the excipient is either (B) 8% KC1:2% sucrose or ( \) 8%NaCl:2% sucrose. The currents were obtained 56 seconds after the addition of plasma samples to the biosensor (see Figure 10).

EXAMPLE 6

Triglyceride calibration was carried out using electrodes manufactured by the continuous freeze drying process with the standard triglyceride formulation subject to the following modifications: ammonium chloride was removed, sucrose was replaced by 1% sodium heparin and the amount of lipase was reduced to 50 mg /ml.

The results are shown in Figure 14 for currents obtained 140 seconds after the application of the sample to the strip.

* Precipitated therefore did not progress onto freeze drying **These formulations were not tested using the OGP therefore there is no brightness data Table 1 : Effect of varying different parameters in the enzyme mixture

Claims

1. Use of a stabilised freeze dried formulation in a biosensor wherein the formulation comprises one or more enzymes, a salt and a saccharide or polyol.

2. Use as claimed in claim 1 wherein the freeze dried formulation has a cake brightness which exceeds 180.

3. Use as claimed in claim 1 or 2 wherein the freeze dried formulation has a collapse temperature of -22⁰C or higher.

4. Use as claimed in claim 3 wherein the freeze dried formulation has a collapse temperature of -2O⁰C or higher.

5. Use as claimed in claim 1 or 2 wherein the freeze dried formulation has a collapse temperature in the range -40⁰C to -15⁰C.

6. Use as claimed in any preceding claim wherein the (or each) enzyme is an oxidoreductase or hydrolase.

7. Use as claimed in any preceding claim wherein the (or each) enzyme is a lipase, an esterase, an oxidase, a dehydrogenase, a reductase or a kinase.

8. Use as claimed in any preceding claim wherein the (or each) enzyme is selected from the group consisting of cholesterol esterase, glucose oxidase, cholesterol oxidase, glycerol-3-phosphate oxidase, lactate oxidase, glycerol dehydrogenase, cholesterol dehydrogenase, lactate dehydrogenase, putidaredoxin reductase, diaphorase and glycerol kinase.

9. Use as claimed in any preceding claim wherein the (or each) enzyme is selected from the group consisting of cholesterol esterase, glycerol dehydrogenase, cholesterol dehydrogenase, putidaredoxin reductase, lipase and diaphorase.

10. Use as claimed in any preceding claim wherein the (or each) enzyme is selected from the group consisting of cholesterol esterase, cholesterol dehydrogenase and putidaredoxin reductase.

1 1. Use as claimed in any preceding claim wherein the (or each) enzyme is selected from the group consisting of glycerol dehydrogenase, lipase and diaphorase.

12. Use as claimed in any preceding claim wherein the formulation comprises an enzyme, a salt and a saccharide.

13. Use as claimed in any preceding claim wherein the saccharide is a monosaccharide or disaccharide.

14. Use as claimed in any preceding claim wherein the saccharide is selected from the group consisting of raffinose, heparin, inositol, mannose, glucose, sucrose, lactose, maltose and trehalose.

15. Use as claimed in any preceding claim wherein the saccharide is sucrose.

16. Use as claimed in any of claims 1 to 1 1 wherein the formulation comprises an enzyme, a salt and a polyol.

17. Use as claimed in claim 16 wherein the polyol is selected from the group consisting of mannitol, lactitol and erythritol.

18. Use as claimed in any preceding claim wherein the salt is a simple salt

19. Use as claimed in any preceding claim wherein the salt is a salt of a monovalent or polyvalent ion.

20. Use as claimed in any preceding claim wherein the salt is a salt of a monovalent ion.

21. Use as claimed in any preceding claim wherein the salt is an alkali or alkaline earth metal salt or an ammonium salt.

22. Use as claimed in any preceding claim wherein the salt is a chloride.

23. Use as claimed in any preceding claim wherein the salt is selected from the group consisting of ammomium chloride, lithium chloride, sodium chloride and potassium chloride.

24. Use as claimed in any preceding claim wherein the salt is potassium chloride or ammonium chloride.

25. Use as claimed in any preceding claim wherein the weight ratio of saccharide : salt is in the range 1 :2 to 1 :150.

26. Use as claimed in any preceding claim wherein the weight ratio of saccharide : salt is in the range 1 :3 to 1 :20.

27. Use as claimed in any preceding claim wherein the total weight of saccharide and salt is in the range 25 to 35% (w/w).

28. Use as claimed in any preceding claim wherein the total weight of saccharide and salt is in the range 27 to 32% (w/w).

29. Use as claimed in any preceding claim wherein the total weight of protein is in the range 15 to 25% (w/w).

30. Use as claimed in any preceding claim wherein the total weight of protein is in the range 20 to 22% (w/w).

31. Use as claimed in any preceding claim wherein the weight ratio of the total weight of saccharide and salt: the total weight of protein is in the range 0.7: 1 to 3:1.

32. Use as claimed in any preceding claim wherein the weight ratio of the total weight of saccharide and salt: the total weight of protein is in the range 1 :1 to 2:1.

33. A biosensor consisting essentially of a biochemical substrate containing an analyte, a detector for detecting the presence of or changes in the analyte and a freeze- dried formulation as defined in any preceding claim.

34. A biosensor as claimed in claim 33 wherein the analyte is triglyceride or cholesterol.

35. A formulation comprising an enzyme, a saccharide and a salt, wherein the enzyme is selected from the group consisting of a lipase, an esterase, an oxidase, a dehydrogenase, a NADH oxidase or a kinase.

36. A method for stabilising an enzyme comprising the steps of: a) preparing a formulation as defined in any of claims 1 to 35 in solution; b) freezing the solution; and c) freeze-drying the solution in order to obtain a stabilised freeze-dried formulation.

37. A method as claimed in claim 36 wherein step (b) is preceded by:

(al) obtaining a biosensing element, wherein the biosensing element comprises a plurality of biosensing wells each capable of acting as a micro electrode; and

(a2) loading the formulation portionwise into the wells of the biosensing element.

38. A method as claimed in claim 36 further comprising: d) loading the freeze-dried formulation into a biosensing element.

39. Use of a combination of a sugar and a salt to stabilise a freeze-dried formulation containing one or more lipase enzymes.

40. Use of a stabilised freeze dried formulation for biosensing wherein the formulation comprises one or more enzymes, a salt and a saccharide or polyol.

41. A method of biosensing comprising: exposing a freeze-dried formulation as defined in any of claims 1 to 32 or 35 to a biochemical substrate containing an analyte in the presence of a detector for detecting the presence of or changes in the analyte.