Patent Abstract:
derivatives are synthesized of starting materials , usually polysaccharides , having sialic acid at the reducing terminal end , in which the reducing terminal unit is transformed into an aldehyde group . where the polysaccharide has a sialic acid unit at the non - reducing end it may be passivated , for instance by converting into hydroxyl - substituted moiety . the derivatives may be reacted with substrates , for instance containing amine or hydrazine groups , to form non - cross - linked polysialylated compounds . the substrates may , for instance , be therapeutically useful drugs peptides or proteins or drug delivery systems .

Detailed Description:
ammonium carbonate , ethylene glycol , polyethylene glycol ( 8 kda ), sodium cyanoborohydride (& gt ; 98 % pure ), sodium meta - periodate and molecular weight markers were obtained from sigma chemical laboratory , uk . the colominic acid used , linear α -( 2 → 8 )- linked e . coli k1 polysialic acids ( 22 . 7 kda average , high polydispersity 1 . 34 , 39 kda p . d . 1 . 4 ; 11 kda , p . d . 1 . 27 ) was from camida , ireland , radioactive iodide ( na 125 i ) was purchased from amersham , uk . other materials included 2 , 4 dinitrophenyl hydrazine ( aldrich chemical company , uk ), dialysis tubing ( 3 . 5 kda and 10 kda cut off limits ; medicell international limited , uk ), sepharose sp hitrap , pd - 10 columns ( pharmacia , uk ), tris - glycine polyacrylamide gels ( 4 - 20 % and 16 %), tris - glycine sodium dodecylsulphate running buffer and loading buffer ( novex , uk ). deionised water was obtained from an elgastat option 4 water purification unit ( elga limited , uk ). all reagents used were of analytical grade . a plate reader ( dynex technologies , uk ) was used for spectrophotometric determinations in protein or ca assays . cd 1 outbred mice ( 8 - 9 weeks old ; 29 - 35 g body weight ) were purchased from charles river ( uk ) and acclimatized for at least one week prior to their use quantitative estimation of polysialic acids ( as sialic acid ) with the resorcinol reagent was carried out by the resorcinol method [ svennerholm , 1957 ] as described elsewhere [ gregoriadis et al ., 1993 ; fernandes and gregoriadis , 1996 , 1997 ]. fab ( protein ) was measured by the bca colorimetric method . covalent psa - protein conjugates generated by reductive amination with sodium cyanoborohydride using the natural form of polysialic acid ( colominic acid , ca ) from e . coli , via its weakly reactive reducing end . ca = colominic acid ; cao = oxidised colominic acid as in fernandes and gregoriadis , 1996 ; jain , et al ., 2003 . sodium cyanoborohydride ( nacnbh 3 ) was used at a concentration of 4 mg ml − 1 . the results are shown in table 1 . the molar ratios in column 1 are the ratio of starting ca ( o ) to protein . ( n = 3 , ± standard deviation ). freshly prepared 0 . 1 m sodium metaperiodate ( naio 4 ) solution was mixed with ca ( 100 mg ca / ml naio 4 ) at 20 ° c . and the reaction mixture was stirred magnetically for 15 min in the dark . a two - fold volume of ethylene glycol was then added to the reaction mixture to expend excess naio 4 and the mixture left to stir at 20 ° c . for a further 30 min . the oxidised colominic acid was dialysed ( 3 . 5 kda molecular weight cut off dialysis tubing ) extensively ( 24 h ) against a 0 . 01 % ammonium carbonate buffer ( ph 7 . 4 ) at 4 ° c . ultrafiltration ( over molecular weight cut off 3 . 5 kda ) was used to concentrate the cao solution from the dialysis tubing . following concentration to required volume , the filterate was lyophilized and stored at − 40 ° c . until further use . oxidised colominic acid ( cao ; 22 . 7 kda ) was reduced in presence of sodium borohydride . freshly prepared 0 . 15 mm sodium borohydride ( nabh 4 ; in 0 . 1m naoh diluted to ph 8 - 8 . 5 by diluting with dilute h 2 so 4 solution ) was mixed with cao ( 100 mg ca / ml ) at 20 ° c . and the reaction mixture was stirred for up to 2 h in the dark . the ph was brought down to 7 by the completion of the reaction . the oxidised / reduced colominic acid ( caor ) was dialysed ( 3 . 5 kda molecular weight cut dialysis tubing ) against 0 . 01 % ammonium carbonate buffer ph ( 7 ) at 4 ° c . ultracentrifugation was used to concentrate the caor solution from the dialysis tubing . the filtrate was lyophilized and stored at 4 ° c . until further required . the determination of any aldehyde content was determined as described under ‘ determination of ca oxidation ’. after confirmation of no aldehyde content the oxidised / reduced colominic acid ( caor ) was again oxidised as reported under activation of colominic acid except caor was incubated with periodate solution for longer time ( up to 1 h ). the degree of oxidation in the caoro product was measured on lyophilized powder obtained from this stage as well . qualitative estimation of the degree of colominic acid oxidation was carried out with 2 , 4 dinitrophenylhydrazine ( 2 , 4 - dnph ), which yields sparingly soluble 2 , 4 dinitrophenyl - hydrazones on interaction with carbonyl compounds . non - oxidised ( ca ), oxidised ( cao ), reduced ( caor ) and re - oxidised ( caoro ) ( 5 mg each ), were added to the 2 , 4 - dnph reagent ( 1 . 0 ml ), the solutions were shaken and then allowed to stand at 31 ° c . until a crystalline precipitate was observed [ shriner et . al ., 1980 ]. the degree ( quantitative ) of ca oxidation was measured with a method [ park and johnson , 1949 ] based on the reduction of ferricyanide ions in alkaline solution to ferric ferrocyanide ( persian blue ), which is then measured at 630 nm . in this instance , glucose was used as a standard . colominic acid samples ( ca , cao , caor and caoro ) were dissolved in nano 3 ( 0 . 2m ), ch 3 cn ( 10 %; 5 mg / ml ) and were chromatographed on over 2 × gmpw xl columns with detection by refractive index ( gpc system : ve1121 gpc solvent pump , ve3580 ri detector and collation with trisec 3 software ( viscotek europe ltd ). samples ( 5 mg / ml ) were filtered over 0 . 45 μm nylon membrane and run at 0 . 7 cm / min with 0 . 2m nano 3 and ch 3 cn ( 10 %) as the mobile phase . colominic acid ( ca ), a polysialic acid , is a linear alpha - 2 , 8 - linked homopolymer of n - acetylneuraminic acid ( neu5ac ) residues ( fig1 a ). periodate , however , is a powerful oxidizing agent and although selective [ fleury and lange , 1932 ] for carbohydrates containing hydroxyl groups on adjacent carbon atoms , it can cause time - dependent cleavage to the internal neu5ac residues . therefore , in the present work exposure of colominic acids to oxidation was limited to 15 - 60 min using 100 mm periodate at room temperature [ lifely et . al ., 1981 ]. moreover , as periodate decomposes on exposure to light to produce more reactive species [ dyer , 1956 ], reaction mixtures were kept in the dark . the integrity of the internal alpha - 2 , 8 linked neu5ac residues post periodate and borohydride treatment was analysed by gel permeation chromatography and the chromatographs obtained for the oxidised ( cao ), oxidised reduced ( caor ), double oxidised ( caoro ) materials were compared with that of native ca . it was found ( fig6 ) that oxidized ( 15 minutes ) ( cao ) ( 6b ), reduced ( caor ) ( 6c ), double oxidised ( 1 hr ) ( caoro ) ( 6d ) and native ( 6a ) ca exhibit almost identical elution profiles , with no evidence that the successive oxidation and reduction steps give rise to significant fragmentation of the polymer chain . the small peaks are indicative of buffer salts . quantitative measurement of the oxidation state of ca was performed by ferricyanide ion reduction in alkaline solution to ferrocyanide ( prussian blue ) [ park and johnson , 1949 ] using glucose as a standard [ results are shown in table 2 ]. table 2 shows that the oxidized colominic acid was found to have a greater than stoichiometric (& gt ; 100 %) amount of reducing agent , i . e . 112 mol % of apparent aldehyde content comprising the combined reducing power of the reducing end hemiketal and the introduced aldehyde ( at the other end ). no reactivity was seen in caor demonstrating that the neutralisation of both the aldehyde and the hemiketal of cao had been successfully accomplished by borohydride reduction . after the second cycle of periodate oxidation , the aldehyde content of the polymer was restored to 95 % in caoro ( within experimental error of 10 %) demonstrating the successful introduction of a new aldehyde moiety at the reducing end . the results of quantitative assay of colominic acid intermediates in the double oxidation process using ferricyanide ( table 2 ) were consistent with the results of qualitative tests performed with 2 , 4 dinitrophenylhydrazine which gave a faint yellow precipitate with the native ca , and intense orange colour with the aldehyde containing forms of the polymer , resulting in an intense orange precipitate after ten minutes of reaction at room temperature . fab was dissolved in 0 . 15 m pbs ( ph 7 . 4 ) and covalently linked to different colominic acids ( ca , cao , caor and caoro ) via reductive amination in the presence of sodium cyanoborohydride ( nacnbh 3 ). colominic acid from each step of the synthesis ( starting material and products of each of examples 1a to c ) together with fab in a ca : fab molar ratios ( 100 : 1 ) were reacted in 0 . 15 m pbs ( ph 7 . 4 ; 2 ml ) containing sodium cyanoborohydride ( 4 mg / ml ) in sealed vessels with magnetic stirring at 35 ± 2 ° c . in an oven . the mixtures was then subjected to ammonium sulphate (( nh 4 ) 2 so 4 ) precipitation by adding the salt slowly whilst continuously stirring , to achieve 70 % w / v saturation . the samples , stirred for 1 h at 4 ° c ., were centrifuged for 15 min ( 5000 × g ) and the pellets containing polysialylated fab suspended in a saturated solution of ( nh 4 ) 2 so 4 and centrifuged again for 15 min ( 5000 × g ). the precipitates recovered were redissolved in 1 ml 0 . 15m na phosphate buffer supplemented with 0 . 9 % nacl ( ph 7 . 4 ; pbs ) and dialysed extensively ( 24 h ) at 4 ° c . against the same pbs . the dialysates were then assayed for sialic acid and fab content and the conjugation yield was expressed in terms of ca : fab molar ratio . controls included subjecting the native protein to the conjugation procedure in the presence of non - oxidised ca or in the absence of ca , under the conditions described . stirring was kept to a minimum to avoid concomitant denaturation of the protein . polysialylated fab was further characterised by size exclusion chromatography , ion exchange chromatography and sds - page . zero ( control ) and 48 h samples ( 0 . 5 ml ) from the reaction mixtures were subjected to ion exchange chromatography ( iec ) on a sepharose sp cation exchange column ( 1 ml ; flow rate 1 ml / min ; binding / washing buffer 50 mm sodium phosphate , ph 4 . 0 ; elution buffer , 50 mm sodium phosphate buffer , ph 4 . 0 containing 1m sodium chloride ). the columns were washed , eluted and the eluent fractions were assayed for ca and protein ( fab ) content . pd - 10 columns were used for desalting samples before applying to column . sds - page ( minigel , vertical gel unit , model vgt 1 , power supply model consort e132 ; vwr , uk ) was employed to detect changes in the molecular size of fab upon polysialylation . sds - page of fab and its conjugates ( with ca , cao , caor and caoro ) of 0 ( control ) and 48 h samples from the reaction mixtures as well as a process control ( non oxidised ca ), was carried out using a 4 - 20 % polyacrylamide gel . the samples were calibrated against a wide range of molecular weight markers . in previous experiments [ jain et . al ., 2003 ; gregoriadis , 2001 ] with other proteins it was found that optimal ca : fab ( derived from sheep igg ) molar conjugation yields required a temperature of 35 ± 2 ° c . in 0 . 15 m pbs buffer at ph 6 - 9 for 48 h . the imine ( schiff base ) species formed under these conditions between the polymer aldehyde and protein was successfully reduced with nacnbh 3 to form a stable secondary amine [ fernandes and gregoriadis , 1996 ; 1997 ]. exposure of protein to periodate - oxidised natural ca generates a metastable schiff &# 39 ; s base ca - protein adduct ( as reported for the polysialylation of catalase ) [ fernandes and gregoriadis , 1996 ]. likewise , in the reaction of oxidised forms of ca with fab , we first created a metastable schiff &# 39 ; s base adduct , by incubation of the oxidised polymer with fab for 48 h at 37 ° c . which was then consolidated by selective reduction ( reductive amination ) with nacnbh 3 ( which reduces the schiff &# 39 ; s base imine structure , but not the aldehyde moiety of the polymer ). in order to characterise the protein reactivity of the various ca intermediates of the ‘ double oxidation method ’ fab was subjected to reductive amination in the presence of natural ca ( ca ), ca oxidized ( cao ), ca oxidised - reduced ( caor ) and ca ‘ double oxidised ’( caoro ). for these studies 22 . 7 kda psa was used , at ca : fab molar ratio of ( 100 : 1 ). after 48 h of incubation in the presence of nacnbh 3 , fab conjugates were isolated from reaction mixtures by precipitation with ammonium sulphate ( as described in the “ examples ”) and the results expressed in terms of ca : fab molar ratios in the resulting conjugates ( table 3 ). it is evident from table 3 that when natural , non - oxidized ca ( in the presence of cyanoborohydride ) was used , a significant but low level of conjugation was observed ( resulting in a 0 . 21 : 1 , ca : fab molar ratio ) via reaction with the hemiacetal group of ca at its reducing end . formation of the ca - fab conjugates was further confirmed by the co - precipitation of the two moieties on addition of ( nh 4 ) 2 so 4 ( ca as such does not precipitate in the presence of the salt ). evidence of conjugation was also confirmed by ion exchange chromatography ( iec , not shown ) and polyacrylamide gel electrophoresis ( sds - page ; fig7 ). for ion - exchange chromatography , polysialylated fab obtained by ( nh 4 ) 2 so 4 precipitation was redissolved in sodium phosphate buffer ( 50 mm , ph 4 . 0 ) and subjected to iec using a sepharose sp hitrap column ( cation exchange ). in contrast with results indicating complete resolution of ca ( in the wash ) and fab ( in eluted fractions ), both ca and fab from the 48 h reaction samples co - eluted in the wash fractions , demonstrating the presence of ca - fab conjugate . fig7 describes the analysis of the antibody fab conjugates described above . these data confirm that the molecular weight distributions of the two conjugates are very similar ( as expected , since the byproducts obtained from the asymmetrically bifunctional ca make up only a small percentage of the total population of molecules ). it is also evident from fig7 that whether fab conjugates were prepared from asymmetrically bifunctional ca ( i . e . periodate oxidised natural ca ) or from monofunctional psa , that conjugates of a wide molecular weight distribution , elevated from the molecular weight of uderivatised fab control , were created . this is consistent with the known polydispersity of the natural polymer reported in our previous published works . fig7 also confirms that reductive amination with monofunctional ca gives rise to an fab conjugate with comparable yield to that of the earlier method based on periodate oxidised natural ca ( described in fig1 ). it is also evident from fig7 that only trace amounts of underivatised fab remained in each conjugate sample . the trace amounts of remaining fab were removed from these conjugates by ion exchange chromatography prior to in vivo studies ( example 3 below ). samples of sheep igg fab fragment or conjugates with cao or caoro were radiolabelled with i 125 as follows : 10 % by volume of each of these samples was removed (˜ 100 μl ) and placed into fresh iodo - gen tubes . a 20 μl sample of pbs containing 200 mci of 125 i ( as nai ) was added to the protein or conjugate and the tubes were capped and allowed to incubate at room temperature for 10 min . the contents of the tubes were then transferred to 500 μl centrifugal filters ( 3 . 5 kda m . w . cut off ) and the samples spun at 6 , 500 rpm in a microcentrifuge . the eluent was discarded and the volume in the retentate ( above the membrane ) made up to 500 μl . this process was repeated a further 5 times after which the radioactivity above ( protein ) and below ( free iodine ) the membrane for a 5 μl sample was assessed using a packard cobra gamma counter . if the counts due to free i 125 were less than 5 % of those in the conjugated fraction , no further purification was carried out . if the free i 125 was & gt ; 5 % the purification cycle was repeated and the samples re - assessed . cd1 mice ( 29 - 35 g body weight ) were dosed with 40 μg ( 100 μl volume in pbs ) of protein per mouse (˜ 1 . 6 mg / kg ) by the i . v . route ( tail vein ) as a single injection and 50 μl samples of blood were then taken ( using heparinised graduated capillaries ) at time intervals from a different tail vein and added into 500 μl pbs . the last bleed recorded was a total bleed in order to allow sufficient counts . samples were then centrifuged at 3000 rpm for 10 minutes and recorded supernatant removed and placed in gamma counter tubes . samples were counted along with representative samples of the injected protein in a packard cobra ii auto gamma counter . recorded counts were expressed as a percentage of the original dose injected . samples of radio - iodinated fab , and cao and caoro fab conjugates , and injected intravenously into mice to monitor half - life in the blood circulation . fig8 shows the pharmacokinetics of native fab vs fab - colominic acid conjugates prepared by the original method ( using cao ) and by the new double - oxidation method ( using caoro ). these results demonstrate that cao - fab and caoro - fab gave rise to marked and significantly longer residence times in the circulation , than was the case for underivatised fab , giving rise to increases of 6 . 28 fold and 5 . 28 fold ( respectively ) in auc values compared to native fab . the caoro synthesised in example 1c above was reacted with 5 molar equivalents of n -[ β - maleimidopropionic acid ] hydrazide in 0 . 1m sodium acetate for 2 h at 37 ° c . the product hydrazone was precipitated in ethanol , resuspended in sodium acetate and precipitated again in ethanol , redissolved in water and freeze - dried . the product is useful for site - specific conjugation to the thiol groups of cysteine moieties in proteins and peptides . the monofunctional polysialic acid aldehyde derivative could also be reacted with a linking compound having a hydrazide moiety and a n - maleimide moiety to form a stable hydrazone having an active maleimide functionality useful for reacting with a thiol group . fractionation of colominic acid by ion exchange chromatography ( ca , 22 . 7 kda , pd 1 . 34 ) an xk50 column ( amersham biosciences , uk ) was packed with 900 ml sepharose q ff ( amersham biosciences ) and equilibrated with 3 column volumes of wash buffer ( 20 mm triethanolamine ; ph 7 . 4 ) at a flow rate of 50 ml / min . ca ( 25 grams in 200 ml wash buffer ) was loaded on column at 50 ml per minute via a syringe port . this was followed by washing the column with 1 . 5 column volumes ( 1350 ml ) of washing buffer . the bound ca was eluted with 1 . 5 column volumes of different elution buffers ( triethanolamine buffer , 20 mm ph 7 . 4 , with 0 mm to 475 mm nacl in 25 mm nacl steps ) and finally with 1000 mm nacl in the same buffer to remove all residual ca and other residues ( if any ). the samples were concentrated to 20 ml by high pressure ultra filtration over a 5 kda membrane ( vivascience , uk ). these samples were buffer exchanged into deionised water by repeated ultra filtration at 4 ° c . the samples were analysed for average molecular weight and other parameters by gpc ( as reported in example 1e ) and native page ( stained with alcian blue ). the following samples were fractionated using an identical wash and gradient system on a smaller scale ( up to 75 ml matrix ; 0 . 2 - 3 gram of colominic acid ): colominic acid ( ca , 22 . 7 kda , pd 1 . 34 ; ca , 39 kda , pd = 1 . 4 ), colominic acid - aldehyde ( cao , 22 . 7 kda , pd 1 . 34 ), monofunctional colominic acid ( caoro , 22 . 7 kda ; pd 1 . 34 ), colominic acid - amine ( ca - nh2 , 22 . 7 kda , pd 1 . 34 ), colominic acid maleimide ( cam , as per example 4 and the m . w . of ca produced monitored throughout ). narrow fractions of ca produced using above procedure were oxidised with 10 mm periodate and analysed by gel permeation chromatography ( gpc ) and native page for gross alteration to the polymer . colominic acid and its derivatives ( 22 . 7 kda ) were successfully fractionated into various narrow species with a polydispersity less than 1 . 1 with m . w . averages of up to 46 kda with different % of populations . fig9 and 10 ; table 4 show the results of separating the 22 . 7 kda material at a scale of 75 ml . fig9 is the gpc result and fig1 is a native page . this process was scalable from 1 ml to 900 ml of matrix with the fractionation profile almost identical at each scale ( not all results shown ). the fractionation of larger polymer ( ca , 39 kda , pd 1 . 4 ) produced species up to 90 kda . this process can successfully be used for the fractionation of even large batches of the polymer . fig1 shows the native page results for the 3 ca samples as supplied and for fractions separated by ion - exchange analysed as in table 4 . the page results show that the ion exchange fractions are narrowly dispersed . this is consistent with the gpc data shown in fig1 which shows the results for 3 of the fractions separated from the 22 . 7 kda ca . the retention volumes are shown in table 5 . the 22 . 7 kda material is separated on a larger scale . using gpc the fractions from ion exchange are analysed . the following fractions shown in table 6 ( see fig1 ) were recovered . all narrow fractions were successfully oxidised with 10 mm periodate and samples taken from different stages of the production process and analysed by gpc and native page showed no change in the molecular weight and polydispersity the data for some of the samples are shown in fig1 . differential ethanol precipitation was used to precipitate different chain lengths of colominic acid . differential ethanol precipitation showed that smaller cas required more ethanol ( etoh ). broad p . d . 22 . 7 kda polymer was precipitated with 70 % etoh giving a yield & gt ; 80 % of product polymer . a concentration of 80 % etoh was required to precipitate & gt ; 80 % of a lower mw 6 . 5 kda ( pd & lt ; 1 . 1 ). this process also removes any salt contaminating the product . samples of 22 . 7 kda were purified by ultrafiltration over different molecular weight cut off membranes ( 5 , 10 , 30 , 50 , and 100 kda ). in all cases retentate was examined by gpc and native page . samples of 22 . 7 kda were purified by ultrafiltration over different molecular weight cut off membranes showed that there was a decrease in polydispersity of the polymer and a shift towards higher molecular weight with increase in membrane cut off ( fig1 ). combined methods and ion pair chromatography can also be for fractionation of the polymers . synthesis of growth hormone ( gh )- colominic acid conjugates ( broad and narrow dispersed ) colominic acid - oxidised ( cao ; 22 . 7 kda )) and narrow dispersed - colominic acid - oxidised ( ncao ; 27 . 7 kda pd = 1 . 09 ; 40 . 9 kda . pd = 1 . 02 ) prepared in reference example 2 . 2 was used for the preparation of gh conjugates . growth hormone was dissolved in 0 . 15 m pbs ( ph 7 . 4 ) and covalently linked to different colominic acids ( cao and ncao ). different cas ( 22 . 7 kda , cao ; 27 . 7 kda & amp ; 40 . 9 kda , nca )) were individually added to gh ( 2 mg ) in a ca : gh molar ratios ( 12 . 5 : 1 ), sodium cyanoborohydride was added to a final concentration of 4 mg / ml . the reaction mixtures were sealed and stirred magnetically for 24 h at 35 ± 2 ° c . the mixtures were then subjected to ammonium sulphate (( nh 4 ) 2 so 4 ) precipitation by adding the salt slowly whilst continuously stirring , to achieve 70 % w / v saturation , stirred for 1 h at 4 ° c ., then spun ( 5000 × g ) for 15 min and the pellets resuspended in a saturated solution of ( nh 4 ) 2 so 4 and spun again for 15 min ( 5000 × g ). the precipitates recovered were redissolved in 1 ml pbs ph 7 . 4 and dialysed extensively ( 24 h ) at 4 ° c . against the same buffer . controls included subjecting the native protein to the conjugation procedure in the presence of non - oxidised ca or in the absence of ca . shaking was kept to a minimum to avoid concomitant denaturation of the protein . polysialylated gh was characterised by sds - page . the polysialylated gh was passed through anion exchange chromatography as described in reference example 2 and the product fractions subjected to sds page . the results ( fig1 ) show that in control well ( with gh ) the migration of the sample is similar to that for fresh gh . in the conjugate lanes there are shifts in the bands which typically indicates an increase in mass indicative of a polysialylated - gh . the band width was significantly narrowed down in case of conjugates with narrow dispersed polymer in comparison to conjugates with broad dispersed polymers . further , gh conjugates ( with broad dispersed polymer ) were separated into different species by anion exchange chromatography ( fig1 ). activated polysialic acid ( colominic acid - oxidised ( cao )) and monofunctional polysialic acid ( colominic acid - oxidised - reduced - oxidised ( caoro )) prepared in example 1 was used for the preparation of rh - insulin conjugates . insulin was dissolved in a minimum volume of 15 mm hcl followed by dilution with 0 . 15 m pbs ( ph 7 . 4 ) and covalently linked to different colominic acids ( ca , cao and monofunctional caoro ). colominic acid ( 22 . 7 kda ) together with insulin ( 2 mg ) in a ca : insulin molar ratios ( 25 : 1 ) were reacted for 48 h in 0 . 15 m pbs ( ph 7 . 4 ; 2 ml ) containing sodium cyanoborohydride ( 4 mg / ml ) in sealed vessels with magnetic stirring at 35 ± 2 ° c . in an incubator . the mixtures was then subjected to ammonium sulphate (( nh 4 ) 2 so 4 ) precipitation by adding the salt slowly whilst continuously stirring , to achieve 70 % w / v saturation . the samples were stirred for 1 h at 4 ° c ., then spun ( 5000 × g ) for 15 min and the pellets suspended in a saturated solution of ( nh 4 ) 2 so 4 and centrifuged again for 15 min ( 5000 × g ). the precipitates recovered were redissolved in 1 ml 0 . 15m na phosphate buffer supplemented with 0 . 9 % nacl ( ph 7 . 4 ; pbs ) and dialysed extensively ( 24 h ) at 4 ° c . against the same pbs . the dialysates were then assayed for sialic acid and protein content and the conjugation yield was expressed in terms of ca : insulin molar ratio ( as per example 1 ). controls included subjecting the native protein to the conjugation procedure in the presence of non - oxidised ca or in the absence of ca , under the conditions described . shaking was kept to a minimum to avoid concomitant denaturation of the protein . polysialylated insulin was further characterised by ion exchange chromatography and sds - page . results are expressed in terms of ca : insulin molar ratios in the resulting conjugates ( table 7 ). it is evident from table 7 that when natural , non - oxidized ca ( in the presence of cyanoborohydride ) was used , a significant but low level of conjugation was observed ( resulting in a 0 . 07 : 1 , ca : insulin molar ratio ) via reaction with the hemiacetal group of ca at its reducing end . formation of the ca - insulin conjugates was further confirmed by the co - precipitation of the two moieties on addition of ( nh 4 ) 2 so 4 ( ca as such does not precipitate in the presence of the salt ). evidence of conjugation was also confirmed by ion exchange chromatography ( iec ) and polyacrylamide gel electrophoresis ( sds - page ). insulin and polysialylated insulin constructs of example 6 were tested for their ability to reduce blood glucose level in normal female t / o outbred mice ( 22 - 24 gram body weight ). animals were divided into groups of five , injected subcutaneously ( s . c .) with insulin ( 0 . 3 units per mouse in 0 . 9 % sodium chloride or with the same protein equivalence of polysialylated insulin ) and glucose levels in blood samples were measured at time intervals using a glucose assay kit ( accu - chek advantage , roche , uk ). the pharmacological activity of polysialylated insulin constructs was compared with that of intact insulin in normal mice injected subcutaneously and bled at time intervals . the blood glucose levels of the mice for the 3 insulins are shown in fig1 . the data points show the average of 5 samples and the error bars are the s . e . m . values . results in fig1 clearly show that polysialylated insulins ( prepared by original method ( using cao ) and by the new double - oxidation method ( using monofunctional caoro )) exerted a more prolonged reduction of blood glucose levels . thus , whereas glucose levels attained nadir values at 0 . 75 hours to return to normal levels two hours after treatment with intact insulin , glucose levels in mice treated with the polysialylated peptide , although also lowest at 0 . 75 h , returned to normal values at 6 hours . these results demonstrate that cao - 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