Title of Invention


Abstract The present invention relates to water soluble particles of less than 50 flm comprising a coprecipitant core such as herein described with a dehydrated biological macromolecule as herein described coated thereon. The present invention also relates to a method of preparing water-soluble particles and a method of isolating a biological macromolecule from an aqueous solution using the water soluble particles.
Full Text

The present invention relates to water soluble particles comprising a biological macromolecule and a method of isolating a biological inacromolacule from an aqueous solution with simultaneous dehydration of the protein, to provide protein biological macroiuolecule particles- The present invention also relates to water fusible organic solvents comprising the protein precipitated therein. The present invention may find particular application in preparing enaymes for use as biocatalysts; preparation of therapeutic proteins for use in pharmaceutical formulations; production of cleansing agents comprising enzymes; production of paints, varnishes, coatings, films and the like comprising proteins which impart protective and/or antifouling properties; production of films, polymers, inks, coatings, electrodes and/or optical materials comprising proteins for diagnostic kits and/or biosensor applications; use of proteins for studies of molecular recognition, molecular binding and inhibitor binding in non-aqueous media; and preparation of protein based food additives. Additionally the precipitated biological macromolecule may thereafter be dissolved in organic solvents for use in at least some of the aforementioned applications as well as in solid phase chemistry such as in the preparation of catalysts for attachment, cleavage and/or modification of compounds bound to an insoluble support.

Proteins are used in a great variety of applications-However, generally speaking, for therapeutic purposes it is necessary to have a preparation of protein substantially free from impurities for use. There are many ways purification may be achieved such as by differential centrifugation, selective precipitation, solvent extraction and chromatographic processes. Additionally it is often desirable to dehydrate or dry the protein prior to use, that is remove water from the protein, in order to facilitate handling and/or improve shelf life.
Typically proteins may be dehydrated by freeze drying, vacuum drying or air drying techniques commonly known in the art. However these techniques suffer from a number of disadvantages. For example, the drying processes are not generally very quick and can be extremely expensive-Moreover, even freeze-drying may lead, particularly in the case of enzymes and fragile proteins, to a decrease in protein function. In order to preserve protein function additional stabilising excipients are often added. However, addition of stabilising excipients may in itself be undesirable particularly, for example, from a regulatory point of view for proteins to be used therapeutically.
US 5,198,353 discloses a method of preparing a stabilised enzyme dispersion. There is described a method of co precipitating a polymer and an enzyme from an aqueous solution in order to produce a finely dispersed enzyme for use in aqueous based liquid detergents. The polymer and enzyme are precipitated by the addition of either salts or

organic solvents. When using an organic solvent as the precipitant it is disclosed that the organic solvent is added to the aqueous protein/polymer solution slowly with vigorous stirring in order to precipitate the protein* However the method and the amount of organic solvent added is such that there is not extensive and rapid dehydration of the protein.
US 5,589’167 and US 5’753,219 disclose recipient stabilization of polypeptides treated with organic solvents. Polios such as trehalose are disclosed as stabilising dry or aqueous polypeptides treated with organic solvents. However, there is no suggestion that the polios could be used to coprecipitate with the protein on addition to an organic solvent or the relevance/importance of dehydrating the protein.
Randen et al (J, Pham, Pharmacia.’ 1988, 4_a, 761 -766) describes the co precipitation of enzymes with water soluble starch as an alternative to freeze-drying. Starch of molecular weights 12 700 and 100 000 is disclosed as a co precipitant of krill proteases when mixed with an organic solvent of acetone, ethanol or isopropanol. The particles produced after precipitation are described as irregular needles with low density with a size in the range of 200 -7 0 O/am • After drying the particles had to be further processed by milling or grinding to obtain a more uniform size distribution.

In a later paper citing the Randen et al paper, Busts et al (J. Chem. Tech. Biotechnol. , 1996, ‘, 193 - 199) describe the use of additional polymeric compounds for use as coprecipitants. The polymeric compounds disclosed are hydrolysed collagen, casein and maltodextrins PSM 10 (12,100 Mw) and PSM'100 (100,000 Mw).
It is amongst the objects of the present invention to provide a rapid process for isolating a protein from an aqueous solution wherein the protein is simultaneously dehydrated.
It is a further object of an embodiment of the present invention to provide bioactive molecule coated particles, such protein/nucleic acid coated micro-crystals•
In one aspect the present invention provides a method of preparing water soluble particles comprising the steps of:
a) preparing an aqueous solution comprising a coprecipitant and a biological macromolecule;
b) rapidly admixing the biological macromolecule/coprecipitant solution with an excess of a water miscible organic solvent such that the coprecipitant and bioactive molecule immediately coprecipitate from solution forming said particles; and
c) isolating said particles from the organic solvent-
It is to be understood that the term "biological macromolecule" refers to a protein, peptide, polypeptide or the like, or nucleic acid such as DNA or RNA. Hereinafter

reference to the biological macromolecule will generally be made by reference to a protein- However, it should be understood that such reference may also be equated with the other aforementioned biological macro molecules.
The term crystal-shaped is intended to mean a three-dimensional shape comprising planar surfaces and is thus distinguishable from generally spherical or spheroid
It is to be understood that the term "coprecipitant" refers to the compound which precipitates out of solution with the protein when added to the organic solvent and that term "coprecipitate" when used as a noun, refers to a bioactive molecule-coprecipitant complex.
The protein to be isolated from the aqueous solution may be any protein or mixture of proteins. Typical obtains include enzymes such as subtilisin, chymotrypsin axe proteases; blood proteins such as albumin, fibrinogen, thrombin and blood factors; and therapeutic proteins such ‘ ". insulin, antibodies, blood and transport proteins, proteins, lipoproteins, lipoproteins, hormones p . interferon’s.
The coprecipitant may be provided as a solid, for as a powder, which is to be dissolved in the a -roués. solution. Alternatively the coprecipitant may be i: solution or suspension prior to dissolving in the aqueous solution. Typically the coprecipitant may be provided as a substantially saturated or highly co-’cent rated solution.

The coprecipitant must be sufficiently soluble in the aqueous solution such that a suitable weight fraction can be obtained relative to the protein in solution. Desirably the coprecipitant should have a very much lower solubility in the chosen solvent than in the aqueous solution. Moreover, if well defined particles are required the coprecipitant should form crystals and coprecipitants with high melting points are therefore preferred* The concentration of coprecipitant required is a function of the amount of protein in the solution and the molecular mass of the protein. Generally speaking the solution prior to precipitation comprises a high molar ratio of coprecipitant to protein. Typically the coprecipitant: protein molar ratio may be greater than 50, preferably greater than 2 00, more preferably greater than 4 00.
Preferably the solid form of the coprecipitant (which may exist as a hydrate) should absorb very little water when exposed to humid environments. The coprecipitant should preferably have very low solubility in the organic solvent used for the co precipitation.
The coprecipitant should also be chosen such that little or substantially none of the protein is denatured thereby,
Coprecipitants which may display at least some of the above desirable properties may be selected from: inorganic salts, for example, potassium soleplate and pot-.Nassau chloride;

sugars, polysaccharides, carbohydrates, polyols, and
derivatives thereof, for example trehalose, typically with
a ::;molecular weight of less than 10,000 Da;
a. ..no-acids such as glycine and arginine;
a’\d-base buffers, for example, potassium hydrogen
phosphate, MOPS and POPSO;
7w(termini compounds for example, butanes;
volcanic salts, for example choline and sodium benzoate;
corh’ounds containing multiple basic groups, such as
FpQvmidine and salts thereof;
c’fo'‘ounds containing multiple acidic groups, such as citric
Ci’CvcV and salts thereof;
bi V’ salts;
vat over soluble dyes;
Tudor or ionic polymers; and
darn or ionic dendrites.
The protein-coprecipitant solution is admixed with a V'ctlc’ miscible organic solvent or water miscible mixture of Sol V'‘nts, preferably one where the solvent or solvent is fully miscible* It should be noted that the proteins-coprecipitant solution is preferably added to the ex
- IC's and conveniently less than 5% v/v. In this manner the organic solvent should preferably initially contain 1-sr than 10% v/v water or be substantially dry, but may not necessarily be completely dry- Suitable organic s(y 1 include methanol, ethanol, propane, acetonitrile trr’hydrofuran and acetone. In certain instances the solvent may be pre-saturated with the protein ‘pi’ or coprecipitate to ensure than on addition of the p 1r -us solution the two components precipitate out

r ::idual solvent to leave a solvent free dry protein-coated pOJlticles precipitate.
It has advantageously been found that the precipitated pYOtein-coated particles may be stored in the organic sotvont and that the protein displays extremely good r’ -ention of activity and stability over an extended period ot time* Moreover, since the precipitated protein is typtr’’lly stored in the organic solvent, it will therefore bC r'‘‘istant to attack by bacteria, thus increasing its s\oT'aqe lifetime-
If necessary, the precipitated protein-coated pCC’'ir cles may be further dehydrated by further washing with f’TCSVi organic solvent.
The precipitated protein may be redissolved in an ac ,eous solution prior to use. Alternatively the prfCi pitated protein may be dissolved directly into an oy*a’r:.c solvent. This may be achieved for example using an o2’a.nc soluble ion-pairing agent, non-covalent binding of ar hi nhilic compounds such as non-ionic detergents or cr’Rl nt attachment of organic soluble groups such as PEG, Ir ‘ hain alkyl chains, dendritic molecules or polymers.
revious wisdom has taught where ion pairing agents h- -een used to solubilise enzymes in organic solvents, th 't the protein be in aqueous solution when the ion pp 7 takes place. The present method however allows ion pe g to take place under very low water conditions. Th t should be noted has several potential advantages: tc • xample, interfacial protein denaturation may not

occur; electrostatic and/or polar interactions may be stronrar; direct solubilisation into polar solvents is possiMe; water sensitive ion pairing agents can be used; mixtu: IS of different ion pairing agents can be used; the protein ionisation state can be controlled with solid-state acid-v-’.se buffers that do not interfere with the ion pairi’l process; the process can be carried out at contr'‘ lied water activity; no lyophilisation steps are required and the solubilisation process requires only simplf’ equipment and is easy to scale up.
'‘ le method described herein may also allow organic solub’ 3 components present in the aqueous solution to be separ’"ed from the protein- For example a buffer such as Tris v'hich in its free base form is soluble in an organic solve- t: like ethanol may be separated from the protein durin’ precipitation. However, it may be necessary to conver : all the buffer to the free base by the addition of anoth* r organic soluble base to the aqueous solution or organ’ ■: solvent- Thus the present invention also discloses a me-" lod of removing undesirable components from the prote 1, such that the undesirable components are not copr’’ pitated with the protein and so remain dissolved in the r ranic phase. This may be achieved by the inclusion of ad tives, such as acids, bases, ion-pairing agents and chelr ■ ng agents in the aqueous or organic solvent prior to prot’ 1 precipitation.

'he present invention may be used for a great many appld'‘ations. For example, enzyme-coprecipitant particles may b '. used as biocatalysts, particularly for reactions in low ater systems, organic solvents and supercritical fluic -,
''he good retention of catalytically active enzyme stru-" :ure within the fine, dry enzyme-coprecipitate part: ‘les provide significant advantages for biocatalysis in I'W water systems, organic solvents and supercritical fluic ' when compared with lyophilised powders, Appl:’ ‘ations include biocatalysis in the organic synthesis of ine chemicals and pharmaceutical intermediates, agrc’ lemicals, detergents, fats, emulsifiers, food-stuffs, vita" ins, sweetners, flavours and perfumes, monomers and poly" ‘rs and modification of synthetic and natural poly’ ‘rs- Other applications include combinatorial bior-talysis for use in for example identification of new leari compounds, enzyme catalysed solid-solid synthesis, pept le synthesis and high temperature and low temperature bior tialysis- In addition biocatalysts in enzyme-cop’ 'ipitate particles can be used for the degradation of che’ -zals and polymers including those found in toxic wa’’ , chemical and biological weapons, domestic and ix\’' "-rial waste and waste for natural sources. Enzyme ca"*"' ‘sed processes often have the advantage over chemical me’ is of imparting regiospecificity, enantiospecificity an’’ tereospecif icity.

Additionally the present method allows the preparation of iierapeutic bioactive itiolecules for pharmaceutical forn’ lations,
:he method produces fine dry particles containing pro’ 'n and a coprecipitant. Thus, in a further aspect the prer nt invention provides water soluble crystal-shaped par*" "les of less than 50 fim comprising a coprecipitant and dehi ated biological macromolecule located at or close to an r ‘er surface of the particle.
‘t is to be understood that the terra "dehydrated bio’ rical macromolecule" refers to a biological mac’ olecule substantially unassociated with water and the ter’ coprecipitant" is as previously defined.
Ypically, the dehydrated biological macromolecule is loc ‘d at or near the surface of the coprecipitant. Ger lly speaking, the biological macromolecule retains a nat" '. or near native configuration when dehydrated ie. it is t irreversibly denatured. For example, if the bio" 'ical macromolecule is an enzyme then it is to be exT '‘d that the enzyme retains most of its activity when ker n solvent and/or reconstituted in aqueous media,
iditionally in the dehydrated state enzymes and other bio’ alysts are able to efficiently catalyse reactions unr= low water conditions such as in organic solvents, Thr- tention of native conformation on dehydration can be prc> for example by carrying out an active site titration in r ow water organic solvent.

Preferably, the co-precipitant within the particles is cry alline. The crystalline precipitant provides a dense cor vith the dehydrated protein located at or close to the pai cle surface. This minimises diffusion limitations so the *:he dehydrated state of the biological tnacromolecule is ‘sily accessible for example to solvent, reagents, su’ ‘‘ates, stabilisers or modifiers.
'"Generally, the size of the particles is less than 10 ‘m 1 is, for example, less than 5-1 fim.
■typically the particles within a coprecipitate have
fa"' -' uniform dimensions and exhibit a particular regular
cry 1-shape such as for example cubes, rhomboids, plates
anc’ ‘edles- The crystal-shape of the coprecipitate varies
with both coprecipitant and protein. The flat surfaces
ex’ ted by crystal-shaped particles make them well suited
f o"‘ ‘Tirrying out scanning probe microscopy and surface
to’ microscopy such as for example atomic force
mic ‘.copy- Using such techniques the dehydrated protein can -i imaged on the surface of the particles and its di* ■ ■bution, organisation and structure can be probed. Th" can be used to probe the tertiary and quaternary st’ ure of proteins such as membrane proteins where it is difi ult to obtain structures by x-ray crystallography, t is to be understood that the coprecipitate crystals ca- ‘. produced using a wide variety of coprecipitants as pr ‘usly described.

Generally these particles can be redissolved rapidly in aqueous solution or easily form suspensions and can be reproducibly dispensed for example using pipettas (by hand or automated) and so are attractive as a starting point for formulating proteins for medical applications* if a therapeutic protein is employed the particles can be used in the production of many different types of drug formulations including tablets, creams, powders, gels ‘ foams, aerosols, suspens ions, tapes and patches, The bioactive molecule coated particles may be particularly suited for trihsport across mucosal surfaces and may therefore be suitable for administration via inhalation* The dimensions of the particles maice them particularly suited for pulmonary administration via inhalation into the lower alveolar regions of the lungs where absorption into the blood-stream is most efficient. For this application particles in the range 0,5 microns to 5 microns are most desirable. This may require mixing ot the protein-coprecipitant particles with additional excipients to act for example as fillers, bulKers and/or binders. The particles can be used as the starting point for further manipulations including encapsulation into natural and synthetic polymers for the production of beads; films,

The present method also allows the production of cleansing agents containing enzymes.
hs aforementioned, th’ method produces fine dry particles containing protein and a coprecipitant that can be redissolved rapidly in aqueous solution and are thus also attractive for the production of cleansing agents that contain enzymes. En«ymes can be incorporated into tablets’ creams, powders, gels, foams, aerosols and suspensions to be used for cleansing. This may require mixing of the protein-coprecipitant particles with additional excipients to act for' example as fillers, bulkers and binders. Examples include a) preparation of tablets containing enzymes such as proteases or peroxidases for cleaning contact lenses and b) preparation of tablets, powders or suspensions containing enzymes such as proteases, lipases or cellulases to include in washing powders for fabrics or dish washers- The particles can be used as the starting point for further manipulations including encapsulation into natural and synthetic polymers. Coatings can be applied to the surfaces of the particles to alter their solubility, processability and dispersability. Coatings are useful for altering the surface properties of the particles and to change their behaviour in solvents or on resuspension in water.
The method may be used in production of paints, varnishes, coatings and films containing proteins to impart protective or antifouling properties.


The fine protein-coprecipitant particles can be dispersed in a carrier medium in a similar way to that employed for pigments for the production of paints, varnishes, coatings and films. if ensymes such as proteases, lipases or cellulases are used the resultant coatings :uay have antifouling properties preventing the attachment of live biological organisms such as bacteria, yeasts, fungi, micro-organisms and molluscs*
The production of films, polymers, inks, coatings, electrodes and optical materials containing proteins for diagnostic kits and biosensor applications may also be achieved using the present method-
The fine protein-coprecipitant particles can be dispersed into a carrier medium such as a paint or ink and used to produce films or coatings on test strips, electrodes or optical materials. These can then be used as the active element in diagnostic kits and biosensor applications.
In addition the use of protein’-coprecipitant particles prepared according to the present invention may be used for studies of molecular recognition, molecular binding’ molecular imprinting and inhibitor binding in non aqueous media«
The protein retains native like structure in the protein-coprecipitant particles and enaymes retain high catalytic activity • The precipitates can therefore be used for quantitative studies of molecular recognition, molecular binding and inhibitor binding in non-aqueous

media. This can be used for the improvement of inhibitor and substrate design for applications in for example medicine, vetinary science and agriculture.
Moreover protein-coprecipitant particles of the present invention may be as protein based food additives*
The precipitation solvent and coprecipitants used can be chosen to be non-toxic for ingestion or inhalation by humans or animals and so the method can be used for rapid and cheap production of dry protein based food additives or pharmaceuticals.
The present invention will now be further described by way of example only and with reference to the accompanying figures which show;
Figure 1 is a typical image obtained by transmission electron microscopy of protein-coprecipitant particles isolated by the method of the present invention;
Figure 2 is a high-magnification image of the protein’ coprecipitant: particles illustrated in Figure 1;
Figure 3 shows subtilisin precipitated into 1-PrOH with no salt present;
Figure 4 shows subtilisin coprecipitated into l-PrOH containing 2 6% HgO;
Figure 5 shows the effect of l-*PrOH addition to aqueous phase solution of subtilisin and K’SO’;
Figure 6 shows AFM images of sutotilisin coated crystals of K2S04;
Figure 7 showe AFM images of the surface of a single
crystal of K2S04 in the absence of subtilisin;

Figure 8 shows AFM images of the surface of a single crystal of K2SO4 coated with subtilisin; and
Figure 9 shows the effect of various amino acid co-precipitants, on the activity of subtilisin.
Example 1- Preparation of subtilisin
Subtilisin Carlsberg (type VIII; bacterial’ from bacillus lichenforicnis, crystallised and lyophilised was obtained from Sigina, Poole, U.K.)- 2 mg of subtilisin (as received) was dissolved in 50 (il buffer (Tris, 10 mM, pH 7.8,) to which, 150 ‘l of saturated solution of a coprecipitant, potassiui:n sulphate, KaSo’’ (l2 0gl'‘) was added. The final concentration of protein in the solution was 0-37 mM and the molar ratio of KaSO*; enzyme in the precipitate was approxiinately 1400 corresponding to --11% by weight subtilisin-
200 lil of the coprecipitant-enayme solution was pipetted, immediately after preparation, into 3 ml of propanol contained in a 7 ml glass vial. The solution was pipetted using a Gilson micropipette in approxiirtately 4 x 50 lil portions while agitating with an orbital shaker, shaking at approximately 100 rpm. The addition of the aqueous solution to the dry organic solvent results in immediate co-precipitation of both the K2SO4 and protein. The vial containing the very fine dispersion of coprecipitant-enzyme solid was capped and shaken for a further 15 min- at an increased speed of 8 00 rpm; the water content of the resultant mixture was approximately 6.25%

v/v. The vial was removed from the shaker and the precipitate allowed to settle. After the precipitate settled ("30 min), the supernatant was removed, leaving behind approximately loo /zl of the original solvent, (Settling can be speeded by gentle centrifugation for approximately i minute, A further 3 ml of the solvent was added and the mixture shaken for 15 min on the orbital shaker resulting in a final water content of approximately 0.2% v/v* The mixture was left to settle or centrifuged and most of the solvent removed to leave the salt-enzyme precipitate suspended in approximately lOO ‘1 of solvent. The suspension can be stored as it is or further treated depending upon the application.
Potassium chloride, Kcl, (saturated solution/ 281.5 gl"‘) was also tested as a coprecipitant following the same procedure as described above for KjSO’, Using the same concentration of enzyme and same volume of saturated salt solution results in a molar salt:enzyme ratio of -7500 corresponding to -5% subtilisin by weight. It is found that for precipitation into acetonitrile (CHiCN) the KCl-enzyme mixture was not suitable as it forms a two-liguid phase mix-
Ainino acids as co-precipitants
Glycine I'‘'‘sine ar’’inine and clutsmic -’cid W'‘r’ obuained from Aidrich u.K* and tested as coprecipitantts.

■ „ IB MH»P II"
_ - _ «
■* I ,
4 lag subtiiisin in ioo ixl saturateu aoiuuxovi or ariurio-
.’r -« —J J «* ■« /*T ‘ ‘r* ‘ »‘* ■% ‘ ,t’ ‘4 T T’ W" ‘‘
Precipitated samples were also prepared with D(+) trehalose (a-D-glucopyranosyl"OC--D-'glucopyranoside) obtained froHi Sigma (poole, U.K-) as the coprecipitant, The trehalose was dissolved in distilled water to saturation (-76 g 1"*)/ and the preparation carried out in an identical manner/ to that described above* The final molar ratio of sugar:protein was 406 corresponding to 15% by wt subtiiisin.
General appearance and properties of coprecipitant’ subtiiisin precipitates
A very fine white precipitate forms im’cnediately upon addition of the protein-coprecipitant solution to the organic solvent: individual particles are extremely small and take some time to settle in the solvent. The si’e of the particles is visibly different from coprecipitant precipitated without protein present (for KaSO’, KCl and trehalose) which, in this case, are largar. If a protein solution containing no coprecipitant is added to solvent again the particle morphology is very different: a stringy white precipitate forms- Precipitated K2S04-subtiiisin particles show no obvious change of morphology or

aggregation over several weeks when left in the solvent*
The KsSO’-subtilisin coprecipitate o’n be easily re-
dissolved in aqueous solution/ pH 7.s, or distilled water
for assays in aqueous solution. Dissolution can be
achieved by either dissolution from a small volume of propanoi solution (typically less than 50 iil of l-propanol) into 1 ml of buffer, pH 7,8 or by drying the precipitate and redissolution into aqueous.
With the amino-acids as coprecipitants in all cases a fine white precipitate was obtained. Electron microscopy showed that with glycine needle shaped particles were obtained.
Example 2 ‘ Testing of various organic solvents
The solvents so far tested for the precipitation are shown in Table 1. They were all obtained from Aldrich, Co, and were of analytical/spectrophotoxnetric grade (99+%) ,
TabXe 1. Water content of solvents used for precipitation. Water level was assayed by Karl Fischer autOTuatic water titration using a Metrohra 684F Coulometer (Metrohm, Switzerland).

Measuring the bioactivity of coprecipitant-ensyme preparations in various organic solvents
It is well known that serine proteases such as subtilisin Carlsberg, or a*chymotrypsin exhibit catalytic activity when suspended in organic solvents. This type of system can therefore be used as a convenient measure of how the bioactivity of a protein is affected by the dehydration process. By assaying, under identical conditions, a range of en’yime-coprecipitant precipitates isolated from different solvents it was possible to determine what solvent and coprecipitant resulted in the least protein denaturation* In addition the results could be compared to those obtained with freeze dried enzyme powders* The enzyroe-coprecipitant suspensions prepared as described above were rinsed once with the assay solvent to remove residual precipitation solvent then assayed as described below. The results of the experiments are shown in Tables 2 and 3,
The assays of catalytic activity were carried out in two different solvents, (CH’CN and n-hexane) , containing a controlled amount of water- Substrates were; N-acetyl-L-phenylalanine ethyl ester (10 mM) and l-propanol (l M)-With CHaCN as the reaction solvent W-acetyl’L-tyrosine ethyl ester (10 mM) was the chosen substrate and 1 H 1-propanol as before. Enzyme concentration was 1 mg/ml» Typically, the reaction vial contained 2 ml of solvent in a 4 ml screw-cap vial v/ith teflon liner. The reaction vials were shalcen for the duration of the experiment on an orbital

shaJcer at approximately 250 rpin. Periodically SO ‘l of the solvent mix was removed and diluted into the appropriate solvent (450 ‘1). These vials were then stored at -4*C for gas chromatographic (G’c.) analysis at a later date.
Table 2. Catalytic activity of subtilisin Carlsberg preparations in dry n-hexane. The precipitations were carried out as in section 2.0 but using subtilisin dissolved in water with no buffer is present.

From Table 2, it can be seen that generally, using KjSOi as a coprecipitant results in higher catalytic activity in /j-’hexane’ than that found using KCl. When K2SO4 was used at a concentration 5x lower than saturation reduced activity was observed. Additionally, as mentioned

previously KCX-enzyitie (aq) when precipitated into acetonitrile (CH3CN) forms a two-phase mix. In nearly all cases the coprecipitant-ensyme precipitate showed superior bioactivity than lyophilised powder.
TaUDle 3. Catalytic activity of preparations of subtilisin Carlsberg and chyiaotrypsin in acetonitrile containing 0.5% water. The enzymes were precipitated with buffer present as described in Example l.

It can be seen from Table 3 that the coprecipitant-enayme precipitates are much more active in AcN than lyophilised powders indicating much better retention of the bioactivQ conformation.

Activity assay, of amino-acic’ precipitances: The precipitate from l eppendorf (0,67 mg enzyme) prepared as described'previously w’s used for each enzyme assay. Activity was measured by HPLC following the transesterification of N-acetyl-L-tyrosine ethyl ester (lo mM) and 1-propanol (1 M) with acetonitrile/1% H’o as solvent.
Figure 9 shows the effect of various amino acid co-precipitants, on the activity of subtiliain in comparison to K3SO4. Arginine led to an increased initial rate, whereas glycine and lysine increased the final conversion after 3 hours slightly. With glutamic acid the transformation was much slower and with lypohilised enzyme less than 1% conversion was observed. These results are generally as expected because amino-acids can act as solid-state acid-base buffers in organic solvents- Lysine and glycine are able to mop up protons produced by hydrolysis by-product. Glutamic acid will increase the protonatation state of subtilisin so that it becomes less catalytically active.
Example 3 - R’4issQ’T’1:i activity in aqueous golution
Precipitated KsSO’-subtilisin could be fully and rapidly redissolved in buffer indicating no irreversible denaturation had occurred during dehydration. The activity of subtilisin Carlsberg in aqueous solution was assayed using the following procedure: Assays were carried out

using p-nitrophenyl acetate (97%, Aldrich, Poole, U,K,) which releases the chroiuophoric nitrophenol when hydrolysed- The reaction rate was monitored by U-V spectrophotometry, detection wavelength (X)- 400 nm- A 1 ml quarts cell, contained 200 /xl of a 3 mM solution of p-nitrophenyl acetate (97%), Aldrich, U.K.); 800 /il of tris buffer, pH 7.8 and an aliquot (20 ‘1) of the KaSO ‘ subtilisin, re-dissolved into buffer solution (1 mg/iml) .
K2S04-subtilisin precipitate left suspended in propanol for 72 hrs was found to have retained 100% activity when re-dieaolved back into aqueous. Similarly upon air drying, for two days KaSO’-subtilisin dissolved back into water immediately and was found to be 100% active, A qualitative test of activity with p-nitrophenyl acetate also showed that after 3 weeks of storage over PaOs, (room temp) the KaSO’-subtilisin could be easily redissolved in buffer solution, pH 7.S, and remained catalytically active.
Examplflf 4 - Active site titration of precipitated enzyme in
Samples of c.a* 2 mg subtilisin Carlsberg and c.a. 18 mg potassium sulphate dissolved in 200 /’l 2. 5mM Tris buffer, pH 7,8, were coprecipitated into 3 ml propanol containing 1% water using the method described in Example 1- On settling of th’ particles the ma;]ority of solvent was decanted off and the samples were rinsed once with 3 ml of the same solvent• Half the samples were then incubated with a 10 mM solution of the active site titrant

phenylmethane sulfonyl fluoride (PMSF) in 3 ml propanol for X hour. Most of the titrant mixture was decanted from the incubated samples and they were rinsed three times with 3 ml aliquots of pure propanoic The catalytic activity of the PMSF treated and non’treated samples were measured in aqueous solution using the standard assay described in Example 3, The results were then compared to those of non-precipitated subtilisin Carlsberg. The assays showed that the normal precipitated enzyme retained >95% activity while that treated with PMSF exhibited 90% of the subtilisin molecules in the precipitate retain a biologically active conformation following the dehydration and precipitation process.
Example 5 - Transmission electron microscopy
Aliquots of a standard protein-coprecipitant particles of subtilisin Carlsberg/K2S04 suspended in propanol were dropped onto carbon coated electron microscope grids* The samples were air dried and then examined using a Jeol JEM 1200EX transmission electron microscope (Jeol Tokyo,

Figures i and 2 show typical images obtained. It can be see that the protein-coprecipitant forms regular shaped crystals. From the scale bars (SOOnm and 2Q0nm respectively) the protein-coprecipitant particles are observed to have dimensions generally less than 2 microns. In the higher magnification image a thin surface coating can be observed on the crystals. It is believed that this layer consists of layers of the protein which is excluded from the crystal lattice during the crystallisation process* In the absence of any protein’ similar shaped but larger crystals are obtained via the precipitation procedure.
Figure 3 shows the agglomerates of protein which are formed when subtilisin is precipitated without salt* This is easily compared to the protein-coated crystals {see Figure 4) obtained when subtilisin is coprecipitated with K2SO4 in l"PrOH, As can be seen in Figure 5 if 1-PrOH is added to an aqueous solution of subtilisin and K’SO’ different structures are formed with protein strands being attached between salt crystals (ie* protein is not coated on the crystals) .

Example fi—* surface microscopy of the eQprecipitqtg obtained from a mixture of aubtiiisi’ and K.SQ’
It was found that coprecipitation of a niixture of subtilisin and KaS04 in the manner described in Example i provided regular crystals with large flat surfaces as shown above by electron microscopy. This makes them well suited for study by scanning force microscopy (SPM) which can b’ used to study the detailed topography of surfaces, if the underlying surface is flat scanning force microscopy techniques can also be used to study molecules located on a surface. In this study a Digital Nanoscope atomic force microscope was used to examine the coprecipitate using tapping-mode amplitude-phase distance measurements. Figure 6 shows an image of a collection of crystals taken with a scan size of 6 /’m x 6 ‘m and a 2-height of 1.5 /im. It can be seen that the crystals have fairly uniform dimensions and exhibit regular tablet-like shapes with flat planar surfaces. At this scale images of the crystals formed by KzSOi precipitated without protein present were similar. Higher resolution images, were then obtained of parts of faces of individual crystal precipitated in the absence and presence of protein. Figure 7 shows a representative image of a 400 nm x 400 nm area of a crystal obtained in the absence of protein. It can be seen from the s-axis range of 4 nm that the surface is quite featureless and fairly flat.

Figure 8 shows a representative image of a 500 nm x 500 nm area of a crystal obtained by coprecipitation of the salt with protein. It can be seen immediately from the increased s-height range of 15 nm that the surface is much rougher* Closer inspection shows that the surface is coated with a layer of protein particles of nanometre dimensions.


Estample 7 ‘ Precipitation of Insulin
Insulin from bovine pancreas was obtained from Sigma, UK (Product Number 1-5500).
2 mg insulin was dissolved in 200 ‘1 HCl (o.oio M) , and the pH increased by adding 333 ‘1 of NaOH (0*010 M). The insulin solution was raii’ed with 150 ‘1 saturated K2SO4 solution and precipitated into 5*317 ml PrOH containing 1.3% HgO. The obtained suspension was centrifuged, and washed once with l-PrOH/l.3% H2O. The fine particles were essentially of the same appearance as obtained with subtilisin (see Example l) .
Circular The following samples were measured on a JASCO J-600 spectropolarimeter under PC control* insulin from bottle insulin co-precipitated with K2S04 as described above.

The spectra obtained were very similar both to each other and to a literature spectruiu showing that insulin substantially retains its native structure following precipitation and redissolution.
Example 8 - pr’pipit:q1:ion of PWft
DNA-genoiuic, ultrapure from calf thymus, with average molecular weight - 8,6 MDa corresponding to approximately 13 Kbase pairs was bought from Sigma,
0.5 unit of DNA was dissolved in 100 ‘1 and mixed with 300 Ml Qf a saturated KaSO* solution. This was added to 4.5 ml of 1-PrOH (previously dried over jnolecular sieves) resulting in immediate formation of a fine precipitate* The suspension was shaken at 600 rpm for 2 mins, allowed to settle, and centrifuged in eppendorfs at eooorpm. The PROH supernatant was removed and the precipitate redissolved in 1 ml of 10 mM Tria’HCl buffer (pH 7.S) containing 1 mM EDTA and ImM NaCl*
The up spectrum of the precipitate was compared with a sample of the original DNA dissolved at the same concentration of 0.5 unit/ml in l ml of lo mM Tris-HCl buffer (pH 7.8) containing 1 mM EDTA and imams NAACP.
From bottle as received from Sigma: Abs at 260 nm -0.421, Abs at 280 nm = 0-219.

1. Water soluble particles of less than 50 comprising a coprecipitant core such
as herein described with a dehydrated biological macromolecule as herein
described coated thereon.
2. Water soluble particles according to claim 1, wherein the coprecipitant is partially or substantially crystalline,
3. Water soluble particles according to any one of the preceding claims, wherein the dehydrated biological macromolecule is selected from peptides, polypeptides, proteins and nucleic acid,
4. Water soluble particles according to any one of the preceding claims, having a diameter less than 10 jim.
5. Water soluble particles according to any one of the preceding claims, wherein the coprecipitant is selected from inorganic salts, preferably, potassium sulphate and potassium chloride; sugars, carbohydrates, polyols and derivatives thereof preferably, trehalose, typically with a molecular weight of less than 10,000 Da; amino-acids, preferably, glycine and arginine; acid-base buffers, preferably, potassium hydrogen phosphate, MOPS and POPSO; zwitterionic compounds, preferably, betaines; organic salts, preferably, choline and sodium benzoate; compounds containing multiple basic groups, preferably, sperm dine and salts

thereof; compounds containing multiple acidic groups, preferably, citric acid and salts thereof; bile salts; and water soluble dye.
6. A method of preparing water soluble particles comprising a coprecipitant core with a dehydrated biological macromolecule coated thereon comprising the steps of: a) preparing an aqueous solution comprising a coprecipitant and a biological macromolecule;
b) admixing the biological macromolecule/coprecipitant solution with an excess of a water miscible organic solvent such that the coprecipitant and bioactive molecule immediately coprecipitate from solution forming said particles; and
c) isolating said particles from the organic solvent.
7. The method according to claim 6, wherein the water soluble particles are formed directly and are less than 50 |im.
8. The method according to any one of claims 6 or 7, wherein the aqueous solution comprising the coprecipitant and the biological macromolecule is prepared by dissolving the coprecipitant in an aqueous solution comprising the biological macromolecule.
9. The method according to any one of claims 6 or 8, wherein the biological macromolecule/coprecipitant solution is added to the water miscible organic solvent.

10. The method according to anyone of claims 6 to 9, wherein the
coprecipitantibiological macromolecule molar ratio is greater than 50.
11. The method according to anyone of claims 6 to 10, wherein the coprecipitant is
selected from inorganic salts, preferably, potassium sulphate and potassium
chloride;sugars, carbohydrates, polyols, and derivatives thereof, preferably,
trehalose, typically with a molecular weight of less than 10,000 Da; amino-acids,
preferably, glycine and arginine; acid-base buffers, preferably, potassium hydrogen
phosphate, MOPS and POPSO; zwitterionic compounds, preferably, betaines;
organic salts, preferably choline and sodium benzoate; compounds containing
multiple basic groups, preferably, spermidine and salts thereof; bile salts; and
water soluble dyes.
12. The method according to anyone of claims 6 to 11, wherein the organic solvent
is selected from methanol, ethanol, propanol, acetonitrile tetrahydrofuran and
13. Particles obtainable by the process according to any one of claims 6 to 12.
14. Water soluble particles substantially as herein above descried with reference
to the accompanying drawings.



in-pct-2001-1574-che-claims filed.pdf

in-pct-2001-1574-che-claims granted.pdf






in-pct-2001-1574-che-form 1.pdf

in-pct-2001-1574-che-form 26.pdf

in-pct-2001-1574-che-form 3.pdf

in-pct-2001-1574-che-form 5.pdf

in-pct-2001-1574-che-other document.pdf


Patent Number 212792
Indian Patent Application Number IN/PCT/2001/1574/CHE
PG Journal Number 07/2008
Publication Date 15-Feb-2008
Grant Date 17-Dec-2007
Date of Filing 12-Nov-2001
Applicant Address McCance Building, 16 Richmond Street, Glasgow G1 1XQ
# Inventor's Name Inventor's Address
1 MOORE, Barry, Douglas Top Flat, 36 Queen Mary Avenue, Crosshill, Glasgow G42 8DT
2 PARKER, Marie, Claire Top Flat, 36 Queen Mary Avenue, Crosshill, Glasgow G42 8DT
3 HALLING, Peter, James 2/2 34 Montague Street, Glasgow G4 9HX
4 PARTRIDGE, Joann 38 Edgehill Road, Glasgow G11 7JD
PCT International Classification Number C07K 01/00
PCT International Application Number PCT/GB00/01854
PCT International Filing date 2000-05-15
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 9910975.3 1999-05-13 U.K.