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  • C07K14/52—Cytokines; Lymphokines; Interferons
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Definitions

• the present invention relates to novel polysaccharide derivatives of insulin and methods for producing such derivatives.
• the derivatives are useful for improving the stability, pharmacokinetics and pharmacodynamics of insulin.
• Diabetes is a disorder of carbohydrate metabolism and results from insufficient production of, or reduced sensitivity to, insulin.
• Insulin is synthesized in the beta cells of the islets of Langerhans of the pancreas and is necessary for normal utilization of glucose by most cells in the body. In persons with diabetes, the normal ability to use glucose is inhibited, thereby increasing blood sugar levels (hyperglycem ia) .
• Type I insulin-dependent diabetes mellitus
• IDDM insulin-dependent diabetes mellitus
• IDDM insulin-dependent diabetes mellitus
• IDDM was formerly referred to as juvenile onset diabetes.
• IDDM insulin is not secreted by the pancreas and must be provided from an external source.
• Type Il adult-onset diabetes can ordinarily be controlled by diet although in some advanced cases insulin is required.
• the insulin molecule consists of two chains of amino acids linked by disulfide bonds (mw 5804).
• the [beta]-cells of the pancreatic islets secrete a single chain precursor of insulin, known as proinsulin.
• Proteolysis of proinsulin results in removal of four basic amino acids (numbers 31 , 32, 64 and 65 in the proinsulin chain: Arg, Arg, Lys, Arg respectively) and the connecting ("C") polypeptide.
• the A chain has glycine at the amino terminus
• the B chain has phenylalanine at the amino terminus.
• Insulin may exist as a monomer, dimer or a hexamer formed from three of the dimers.
• the hexamer is coordinated with two Zn 2+ atoms.
• Biological activity resides in the monomer.
• bovine and porcine insulin were used almost exclusively to treat diabetes in humans, numerous variations in insulin between species are known.
• Porcine insulin is most similar to human insulin, from which it differs only in having an alanine rather than threonine residue at the B-chain C-terminus. Despite these differences most mammalian insulin has comparable specific activity.
• animal extracts provided all insulin used for treatment of the disease.
• the advent of recombinant technology allows commercial scale manufacture of human insulin (e.g., Humulin(TM) insulin, commercially available from EIi Lilly and Company, Indianapolis, Ind.).
• PEG-insulin (Nektar Therapeutics).
• PEG is a neutral, water-soluble, nontoxic polymer comprising any number of repeating units of ethylene oxide. PEGylation is designed to increase the size of the active molecule and ultimately improve drug performance by optimising pharmacokinetics, increasing bioavailability, and decreasing immunogenicity and dosing frequency.
• the design and development of PEG-insulin is described further in WO2004091494. A variety of methods for producing PEGylated insulin derivatives are known. Davis et al. (U.S. Pat. No.
• N.sup..alpha.A1 , N.sup..epsilon.B29-bis-protected-insulin was reacted with an activated (e.g., acid chloride or isocyanate) PEG derivative with subsequent removal of the protecting groups using techniques common to peptide chemistry.
• activated e.g., acid chloride or isocyanate
• the inventors observed that the amino groups of GIyAI and LysB29 were more reactive than PheBI's amino group under alkaline reaction conditions. They determined their site-specific mPEG(1500)-B1 -insulin conjugates had a 100% insulin effect (calculated on a molar basis) on reduction of blood sugar levels in rabbits.
• PEG-insulin preparations (Caliceti et al., 1999; Uchio et al., 1999; Hinds et al., 2002 are either: 1) centered on the basic three-step protection/conjugation/deprotection schemes outlined above, 2) result in non-specific modification of the insulin molecule, or 3) do not produce the most effective conjugates, namely, PEG-B1 -insulins.
• Liu et al. (U.S. Pat. No. 6,323,311 B1) describe a useful method for the synthesis of PEG-B1 -insulin conjugates. This method is an extension of the Obermeier three-step protection/conjugation/deprotection scheme, but does not require the isolation of reaction intermediates between steps (i.e., one-pot synthesis). Thus, the insulin is protected at residues GIyAI and LysB29, immediately reacted with PEG, and subsequently deprotected before any isolation of species. The inventors claim that their one-pot reaction may yield up to 50% of the correct positional isomer (i.e. PEG-B1 -insulin) and 30% unreacted insulin that can be recycled for subsequent derivatization.
• the correct positional isomer i.e. PEG-B1 -insulin
• the invention in US2007083006 addresses the shortcomings of prior art methods for PEGylating insulins by providing a method for the simple preparation of highly pure insulin derivatives specifically PEGylated at the N-terminus of insulin's B-chain (PheB1) in a single-step.
• the present method employs specific conditions of pH control, use of a metal ion chelator and addition of organic solvent to enhance the relative reactivity of the PheB1 amino terminus to where it becomes the predominant site of PEGylation.
• the biological properties of polysialic acids particularly those of the alpha-2,8 linked homopolymeric polysialic acid, have been exploited to modify the pharmacokinetic properties of protein and low molecular weight drug molecules.
• Polysialic acid derivatisation gives rise to dramatic improvements in circulating half-life for a number of therapeutic proteins including catalase and asparaginase, and also allows such proteins to be used in the face of pre-existing antibodies raised as an undesirable (and sometimes inevitable) consequence of prior exposure to the therapeutic protein [Fernandes and Gregoriadis, 1996, 1997].
• the alpha- 2,8 linked polysialic acid offers an attractive alternative to PEG, being an immunologically invisible biodegradable polymer which is naturally part of the human body, and which degrades, via tissue neuraminidases, to sialic acid, a non-toxic saccharide.
• Figure 1 shows the oxidation of colominic acid (alpha-2,8 linked polysialic acid from E. coli) with sodium periodate to form a protein-reactive aldehyde at the non-reducing end; and
• Figure 2 shows the selective reduction of the Schiff's base with sodium cyanoborohydride to form a stable irreversible covalent bond with the protein amino group.
• Unintentional by-products may be generated during the conventional conjugation reactions described above by reaction of the colominic acid with side chains of amino acids, for instance. These may be sufficient to be troublesome in the manufacture of chemically defined conjugates required by regulatory authorities for therapeutic use in man and animals. It is not straightforward to purify the intended reaction product (for instance the monopolysialylated product) away from the various unintended products, since the physicochemical characteristics of most of the reaction products are similar. This means that techniques such as ion-exchange chromatography and gel-permeation chromatography (which separate on the basis of charge and size respectively) produce poor purification profiles.
• composition comprising a population of polysaccharide derivatives of a protein, wherein the protein is insulin or an insulin-like protein and the polysaccharide is anionic and comprises between 2 and 125 saccharide units, and wherein the population consists substantially only of N-terminal derivatives of the protein.
• insulin-like protein we mean a protein which has an activity equivalent to that of insulin. Insulin typically decreases blood glucose concentration. It also increases cell permeability to monosacchorides, amino acids and fatty acids, and accelerates glycolysis, the pentose phosphate cycle, and glycogen synthesis in the liver. Preferably, the insulin-like protein has at least 35% more preferably at least 50% of the activity of human insulin derived from Swissprot accession number P01308.
• Mutants of insulin which have the requisite activity may also be used.
• An "insulin-like" protein may also be referred to as an "insulin- homologue". Whether two sequences are homologous is routinely calculated using a percentage similarity or identity, terms that are well known in the art. Sequences should be compared to SEQ I. D. No. 1 which is the unprocessed precursor of human insulin. Residues 25-54 correspond to the insulin B chain and residues 90-110 correspond to the insulin A chain. Homologue sequences may also be compared to the active form of human insulin.
• Homologues preferably have 50% or greater similarity or identity at the nucleic acid or amino acid level, more preferably 60%, 70%, 80% or greater, more preferably 90% or greater, such as 95% or 99% identity or similarity at the nucleic acid or amino acid level.
• a number of programs are available to calculate similarity or identity; preferred programs are the BLASTn, BLASTp and BLASTx programs, run with default parameters, available at www.ncbi.nlm.nih.gov.
• the insulin may be natural, i.e. derived from a human or animal, or synthetic, for instance made by a recombinant method.
• insulin comprises two peptide chains.
• the insulin is derivatised with the polysaccharide at the N-terminus of its B chain.
• population we mean that there is more than one polysaccharide derivative in the composition.
• the derivatives may comprises the same or different numbers of saccharide units.
• the polydispersity of the polysaccharide in the composition is less than 1.3, more preferably less than 1.1.
• substantially all of the proteins are derivatised at the N- terminal only. Since there are two peptide chains in insulin there are two N terminal units. Preferably at least 85%, more preferably at least 90%, most preferably at least 95% of the B chains in the population are derivatised at the N- terminus with anionic polysaccharide. The N-terminus of the A chains need not be derivatised.
• the degree of derivatisation at the N-terminus may be determined using standard techniques in the art, such as peptide mapping or Edman Degradation.
• the polysaccharide has at least 2, more preferably at least 5, most preferably at least 10, for instance at least 50 saccharide units.
• the anionic polysaccharide is preferably selected from polysialic acid, heparin, hyaluronic acid and chondroitin sulphate.
• the polysaccharide is polysialic acid and consists substantially only of sialic acid units.
• the polysaccharide may have units other than sialic acid in the molecule. For instance, sialic acid units may alternate with other saccharide units.
• the polysaccharide consists substantially of units of sialic acid.
• the polysaccharide has a terminal sialic acid group, and as detailed above, is more preferably a polysialic acid, that is a polysaccharide comprising at least 2 sialic acid units joined to one another through ⁇ -2-8 or ⁇ -2-9 linkages.
• a suitable polysialic acid has a weight average molecular weight in the range 2 to 100 kDa, preferably in the range 1 to 35 kDa.
• the most preferred polysialic acid has a molecular weight in the range of 10-2OkDa, typically about 14kDa.
• the polysialic acid is derived from a bacterial source, for instance polysaccharide B of E. coli Kl, N. meningitidis, Maraxella liquefaciens or Pasteurella aeruginosa or K92 polysaccharide from E. coli K92 strain. It is most preferably colominic acid from E. coli K1.
• the anionic polysaccharide, preferably polysialic acid may be in the form of a salt or the free acid. It may be in a hydrolysed form, such that the molecular weight has been reduced following recovery from a bacterial source.
• the polysaccharide, preferably polysialic acid may be material having a wide spread of molecular weights such as having a polydispersity of more than 1.3, for instance as much as 2 or more.
• the polydispersity of molecular weight is less than 1.3 or 1.2, preferably less than 1.1 , for instance as low as 1.01.
• the compound of this invention is a polysialic acid derivative of insulin and comprises 2-125 sialic acid units. More typically, the compound comprises 10-80 sialic acid units, preferably 20-60 sialic acid units, most preferably 40-50 sialic acid units.
• the polysaccharide derivatives in the first aspect of this invention may be covalently-linked conjugates between the N-terminus of insulin and an anionic polysaccharide.
• Other means of association between the polysaccharide and the insulin include electrostatic attraction.
• covalent bonding is preferred.
• the covalent linkage may be an amide linkage between a carboxyl group and an amine group.
• Another linkage by which the insulin could be covalently bonded to the polysaccharide is via a Schiff base. Suitable groups for conjugating to amines are described further in WO 2006/016168.
• the polysaccharide may be a naturally occurring polysaccharide, or a derivative of a naturally occurring polysaccharide, for instance, a polysaccharide which has been derivatised by a reaction of one or more active groups on the saccharide residues, or which has been covalently linked to a derivatising group at the end of the polysaccharide chain.
• the polysaccharide may be linked to the insulin via either its reducing or non-reducing terminal unit.
• Methods for attaching polysaccharides to proteins are well known in the art and are described in more detail in WO 92/22331 and WO-A-0187922. The preferred methods in this invention are described in more detail below. Methods are also described in Figures 1 and 2 of this application.
• the polysaccharide may be linked to the insulin via its reducing and non- reducing terminal unit. This means that one polysaccharide chain may be linked to two insulin proteins, i.e. be derivatised at both its reducing and non-reducing end.
• the polysaccharide may be linked to the insulin peptide directly, i.e. as shown in Figures 1 and 2, or via a linker.
• Suitable linkers are derived from N- maleimide, vinylsulphone, N-iodoacetamide, orthopyridyl or N- hydroxysuccinimide-containing reagents.
• the linker may also be biostable or biodegradable and comprise, for instance, a polypeptide or a synthetic oligomer.
• the linker may be derived from a bifunctional moiety, as further described in WO
• a suitable bifunctional reagent is, for instance, Bis-NHS.
• the reagent may have general formula Z-R 1 -Z wherein each Z is a functional group and may be the same or different and R 1 is a bifunctional organic radical.
• R 1 is selected from the group consisting of alkanediyl, arylene, alkarylene, heteroarylene and alkylheteroarylene, any of which may substituted and/or interrupted by carbonyl, ester, sulfide, ether, amide and/or amine linkages. Particularly preferred is C 3 -C 6 alkanediyl. Most preferably, R 1 corresponds to the appropriate portion of the suitable bifunctional reagent
• a preferred polysaccharide derivative is of general formula (I) wherein m is at least one;
• HNB is derived from B-NH 2 which is the N-terminus of insulin or a insulin- like peptide;
• L is a bond, a linking group, or comprises a polypeptide or a synthetic oligomer;
• GIyO is an anionic saccharide unit; wherein the linking group, if present, is of general formula -Y-C(O)-R 1 - C(O)-; wherein Y is NR 2 or NR 2 -NR 2 and R 1 is a difunctional organic radical as defined above; and R 2 is H or Ci -6 alkyl.
• the insulin is linked to the non-reducing end of the polysaccharide.
• the terminal polysaccharide unit is a sialic acid unit.
• the other saccharide units in the polysaccharide are represented by GIyO and may be the same or different. Suitable saccharide units include heparin, hyaluronic acid or chondroitin sulphate.
• the group L is a bond.
• the group L may alternatively be derived from an N-maleimide, vinylsulphone, N-iodoacetamide, orthopyridyl or N-hydroxysuccinimide containing reagent.
• the reagent may have general formula Z-R 1 -Z as defined above.
• L is typically a group o o .
• compositions as defined above which is a pharmaceutical composition and further comprises one or more pharmaceutically acceptable excipients.
• the pharmaceutical composition may be in the form of an aqueous suspension.
• Aqueous suspensions contain the novel compounds in admixture with excipients suitable for the manufacture of aqueous suspensions.
• the pharmaceutical compositions may be in the form of a sterile injectable aqueous or homogeneous suspension. This suspension may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• compositions may be administered orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, intranasally, intradermal ⁇ , topically or intratracheal ⁇ for human or veterinary use.
• compositions may further comprise a formulation additive.
• formulation additive we mean an excipient which is capable of stabilising the insulin either internally or externally, as described in Wang et al (1999).
• the excipient may be a stabiliser, a solubilser or a metal ion.
• Suitable examples of formulation additives include one or more buffers, stabilisers, surfactants, salts, polymers, metal ions, sugars, polyols or amino acids. These may be used alone or in combination.
• Stabilisers typically act by destabilisation of the denatured state of a protein leading to increased Gibbs free energy change for unfolding of the protein.
• the stabiliser is preferably a sugar or a polyol, for example sucrose, sorbitol, trehalose, glycerol, mannitol, lactose and ethylene glycol.
• a stabilising buffer is sodium phosphate.
• the solubiliser is preferably a surfactant, preferably a non-ionic surfactant.
• Suitable examples include Tween 80, Tween 20, Tween 40, Pluoronic F68, Brij 35 and Triton X 100.
• the metal ion is preferably divalent. Suitable metal ions include Zn2+, Ni 2+ , Co 2+ , Sr 2+ , Cu 2+ , Ca 2+ , Mg 2+ and Fe 2+ .
• the formulation additive may also be a polymer selected from PSA, PEG or hydroxy-beta-cyclodextrin. Preservatives such as m-Cresol may also be used.
• Suitable amino acids and amino acid derivatives for use as the formulation additive include histidine, glycine, other similar amino acids and sodium aspartate.
• a further aspect of the invention is a compound as described above for use in therapy.
• phase "chemically reacted substantially only at the N-terminal amine” means that in a population of derivatives at least 85%, more preferably at least 90%, most preferably at least 95% of the protein is derivatised only at its N- terminal amine. Preferably, this is at the N-terminal amine of the B chain of insulin.
• the polysaccharide may also react with a modified form of insulin. For instance, one or more groups on the insulin may have undergone a chemical transformation, for instance, by reduction or oxidation. A reactive carbonyl may be generated in the place of the terminal amino group of insulin using oxidation conditions, for instance.
• Suitable polysaccharides for use in the method of this invention are as described previously for the novel compositions.
• the compounds of the invention may be manufactured by any of the suitable methods described in the prior art. For example, a typical method is described to our previous patent application WO 92/22331.
• the anionic polysaccharide has been activated before derivatisation to insulin. It may, for instance, have a reactive aldehyde group and the derivatisation reaction may be carried out under reducing conditions.
• the reactive aldehyde group may be produced by controlled oxidation of a hydroxyl group of the polysaccharide. Most preferably this reactive aldehyde is generated in a preliminary step, in which the polysaccharide is reacted under controlled oxidation conditions, for instance using sodium periodate, in aqueous solution.
• the oxidation is a chemical oxidation, although enzymes which are capable of carrying out this step may also be used.
• the reactive aldehyde group may be at the non-reducing end or reducing end of the polysaccharide.
• the insulin typically the N-terminus, may then react with the reactive aldehyde group to produce an adduct which, when reduced, produces the N-terminal derivative of insulin.
• the activation of the polysaccharide should preferably be carried out under conditions such that there is substantially no mid-chain cleavage of the backbone of the polysaccharide, that is substantially no molecular weight reduction.
• the oxidant is suitably perrhuthenate, or, preferably, periodate. Oxidation may be carried out with periodate at a concentration in the range 1mM to 1M, at a pH in the range 3 to 10, a temperature in the range 0 to 6O 0 C for a time in the range 1 min to 48 hours.
• Suitable reduction conditions for the derivatisation reaction may utilise hydrogen with catalysts or, preferably hydrides, such as borohydrides. These may be immobilised such as Amberlite (trade mark)-supported borohydride. Preferably alkali metal hydrides such as sodium borohydride is used as the reducing agent, at a concentration in the range 1 ⁇ M to 0.1 M, a pH in the range 4 to 10, a temperature in the range 0 to 60 0 C and a period in the range 1 min to 72 hours. The reaction conditions are selected such that pendant carboxyl groups on the starting material are not reduced.
• Other suitable reducing agents are cyanoborohydride under acidic conditions, e.g. polymer supported cyanoborohydride or alkali metal cyanoborohydride, L-ascorbic acid, sodium metabisulphite, L-selectride, triacetoxyborohydride etc.
• activated derivatives of polysaccharides may have utility in the present invention, including those with pendant functional groups such as NHS, as described in our earlier patent application WO 06/00540.
• the reactive aldehyde is at the reducing end of the polysaccharide and the non-reducing end has been passivated such that it does not react with pendant groups on the insulin.
• WO 2005/016973 describes polysialic acid derivatives that are useful for conjugation to proteins, particularly those which have free sulfhydryl drugs.
• the polysialic acid compound is reacted with a heterobifunctional reagent to introduce a pendant functional group for site-specific conjugation to sulfhydryl groups.
• the anionic polysaccharides used in the present invention may also be derivatised with a heterobifunctional reagent in this manner.
• the polysaccharide may be derivatised before it reacts with insulin. For instance, the polysaccharide may react with a bifunctional reagent.
• the polysaccharide may be subjected to a preliminary reaction step, in which a group selected from a primary amine group, a secondary amine group and a hydrazine is formed on the terminal saccharide, which is preferably sialic acid, followed by a reaction step in which this is reacted with a bifunctional reagent to form a reaction-intermediate, as further described in WO 2006/016168.
• the intermediate may then react with the insulin or insulin-like peptide.
• the bifunctional reagent may have general formula Z-R 1 -Z, as defined previously.
• the derivatisation reaction should be carried out in a first aqueous solution of acidic pH, and the resultant polysaccharide derivative should then be purified in a second aqueous solution of higher pH than the first aqueous solution.
• acidic pH we mean a pH less than 7.
• the pH of the first aqueous solution is in the range 4.0-6.5, preferably 4.0-6.0 and the pH of the second aqueous solution is in the range of 6.5-9.0, preferably 6.5-8.5 or 6.5-8.0.
• the low pH of the derivatisation reaction promotes selective derivatisation at the N-terminus of the protein rather than at any mid-chain sites.
• formulation additives promotes the formation of a selective, stable, polysaccharide insulin-derivative.
• the formulation additive may be selected from one or more buffers, stabilisers, surfactants, salts, polymers, metal ions, sugars, polyols or amino acids. These may be added to the reaction medium, or alternatively may be added to the final product composition, as a stabiliser.
• the formulation additive is sorbitol, trehalose or sucrose.
• the formulation additive is a non-ionic surfactant.
• the formulation additive may alternatively be a polymer selected from PSA, PEG or hydroxy-beta-cyclodextrin.
• the formulation additive is a divalent metal ion. Preferred divalent metal ions include Zn 2+ , Ni 2+ , Co 2+ , Sr 2+ or Fe 2+ .
• the formulation additive may be a buffer.
• the formulation additive is a buffer, it is sodium phosphate or sodium acetate.
• the purification of the polysaccharide derivative in the method of the present invention may be carried out using a variety of methods known in the art.
• suitable purification methods include HIC (hydrophobic interaction chromotography), SEC (size exclusion chromotography), HPLC (high performance liquid chromotography), and IEC (ion exchange chromotography).
• a population of polysialic acids having a wide molecular weight distribution may be fractionated into fractions with lower polydispersities, i.e. into fractions with differing average molecular weights.
• Fractionation is preferably performed by anion exchange chromatography, using for elution a suitable basic buffer, as described in our earlier patent applications WO 2005/016794 and WO 2005/03149.
• the fractionation method is suitable for a polysaccharide starting material as well as to the derivatives. The technique may thus be applied before or after the essential process steps of this invention.
• the resultant polysaccharide derivative of insulin has a polydispersity of less than 1.1
• the derivatisation of insulin in accordance with this invention results in increased half life, improved stability, reduced immunogenicity, and/or control of solubility and hence bioavailability and the pharmacokinetic properties of insulin.
• the new method is of particular value for creation of a monopolysialylated-insulin conjugates. The invention is illustrated by Examples 1-6 and by reference to the following drawings:
• Figure 1 shows the oxidation of colominic acid (alpha-2,8 linked polysialic acid from E. coli) with sodium periodate
• Figure 2 shows the selective reduction of the Schiff's base with sodium cyanoborohydride to form a stable irreversible covalent bond with the protein amino group
• Figure 3 is an SDS-PAGE of 22 kDa CAO-rh-lnsulin conjugates at different temperatures
• Figure 4 shows the effects of temperature on the degree of derivatisation
• Figure 5 is an SDS-PAGE of 27 kDa CAO-rh-lnsulin conjugations at different molar ratios
• Figure 6 shows the effects of molar ratio on the degree of derivatisation
• Figure 7 is an SDS-PAGE of 8 and 11 kDa CAO-rh-lnsulin conjugates
• Figure 8 is an SE-HPLC of 8 kDa CAO-rh-lnsulin formulations and insulin;
• Figure 9 shows the in vivo efficacy of 10 , 15 and 21.5 kDa CAO-insulin formulations;
• Figure 10 shows the experimental set-up of preparative HPLC with IEC or HIC for purifying a conjugate
• Figure 11 shows the purification of a CAO-insulin conjugate by Hydrophobic interaction chromatography over a HiT rap Butyl FF column
• Figure 12 shows the purification of a CAO-insulin conjugate from HIC peak 2, with 13 kDa CAO as example, by anion exchange chromatography over a Hitrap Q FF column column;
• Figure 13 is an Isoelectric focusing (IEF) gel of a 13 KDa and 27 KDa CAO-insulin conjugate;
• Figure 14 shows In vivo results of CAO-insulin conjugate in mice; and Figure 15 shows Edman amino acid degradation results. Examples
• Ultrafiltration over molecular weight cut off 3.5kDa was used to concentrate the CAO solution from the dialysis tubing. Following concentration to required volume, the filterate was lyophilized and stored at -40 0 C until further use. Alternatively, CAO was recovered from the reaction mixture by precipitation (twice) with ethanol.
• Non-oxidised (CA)/oxidised (CAO) were added to the 2,4-DNPH reagent (1.0ml), the solutions were shaken and then allowed to stand at 37°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 630nm. In this instance, glucose was used as a standard.
• Colominic acid samples (CA and CAO) were dissolved in NaNO3 (0.2M), CH3CN (10%; 5mg/ml) and were chromatographed on over 2x GMPWXL columns with detection by refractive index (GPC system: VE1121 GPC solvent pump, VE3580 Rl detector and collation with Trisec 3 software (Viscotek Europe Ltd). Samples (5mg/ml) were filtered over 0.45 ⁇ m nylon membrane and run at 0.7cm/min with 0.2M NaNO3 and CH3CN (10%) as the mobile phase. 5. Colominic Acid Stability
• PSA is an acid labile polymer and is stable for weeks around neutral pH (Fig. 3).
• the results in Fig. 3 show that at pH 6.0 and 7.4 CAO is stable for 8 days, at pH 5.0 there is slow degradation (after 48 hours 92% of initial MW), and at pH 4.0 there is slow degradation (after 48 hours 70% of initial MW).
• Polysialic acid is highly hydrophilic whereas PEG is an ampiphilic molecule in nature.
• Insulin 5804 Da was supplied as white solid. The insulin was dissolved by minimum 100 mM HCI, and then adjusted to the required the pH and placed on ice. The amount of CAO to be added for conjugation was calculated based on formula:
• CAO CAO was solubilised in 10 mM NaOAc, pH 6.0 gently vortexed the mixture until all the CAO has dissolved and then either filtered into a new container to remove any aggregated/precipitated material.
• Required amount of insulin protein solution was added to the CAO solution to give a 7.5 molar excess (small scale) and 5 (large scale) of CAO and gently mixed by keeping the reaction mixture on a gentle shaker at 4 ⁇ 1°C.
• 100 mg/ml NaCNBH3 solution was added in order to have 8 mg/ml in the final reaction mixture, gently mixed and pH of the final reaction mixture was checked, if necessary adjusted the pH to 6.0 with 0.5 M NaOH/HCI at 4 ⁇ 1oC.
• HIC HisTrap Butyl FF
• HIC buffer B 20 mM sodium phosphate + 0.8 M (NH4)2SO4, pH 7.4).
• Product with HIC buffer A (10 mM sodium phosphate buffer, pH 7.4) (rate 5 ml/min) was eluted and collected the fractions (1 column volume fraction; 6 column volume) and label (EI-Ex).
• IEC buffer A 35%
• IEC buffer B 65%
• gradient of 5CV & washing of 3CV 1 flow rate 0.25ml/min
• IEC buffer A 0%
• IEC buffer B 100%
• gradient of 5CV & washing of 3CV 1 flow rate 0.25ml/min
• the IEC fractions containing the purified conjugate are combined, washed to remove salt with buffer change of PBS buffer. Adjust pH after removing salt to 7.4. The solution is then concentrated at 4 ⁇ 1°C and the protein concentration analysed by UV spectroscopy (280nm). Conjugates were sterile filtered and samples taken for activity assay and for characterisation by SDS-PAGE and SE- HPLC. If required an aliquot was removed for a protein assay and CA assay. The remainder was stored at 4 ⁇ 1°C until further use and studied for physical stability by SE-HPLC.
• Insulin (5808 Da) was supplied as white solid. The insulin was dissolved by adding minimum quantitylOO mM HCI, and then adjusted to the required the pH and placed on ice. The amount of 14 kDa CA to be added for conjugation was calculated based on formula:
• Weight of 14 kDa CAO x (MW of CAO) x (Molar excess of CAO) (MW of protein)
• 14 kDa CAO required amount of 14 kDa CAO was weighed out. 14 kDa CAO was solubilised in 10 mM phosphate buffer, pH 6.0 (20% volume of the final reaction volume was used here), gently vortexed the mixture until all the 14 kDa CAO has dissolved and then either filtered into a new container to remove any aggregated/precipitated material. Required amount of insulin protein solution was added to the 14 kDa CAO solution to give a 7.5 molar excess (small scale) and 5 (large scale) of 14 kDa CAO and gently mixed by keeping the reaction mixture on a gentle shaker at 4 ⁇ 1°C.
• HIC fractions containing protein fractions are washed with IEC buffer A (20 mM phosphate buffer, pH 7.4). To remove ammonium sulphate if any in Vivaspin 20(MW: 5 Kd). Check pH and adjust if required to pH 7.4. Load on the IEC column previously equilibrated with IEC buffer A. The gradient system was applied in the following manner:
• IEC buffer A 68%
• IEC buffer B 32%
• gradient of 5 CV & washing of 3CV flow rate: 0.25ml/min
• IEC buffer A 35%
• IEC buffer B 65%
• gradient of 5CV & washing of 3CV flow rate: 0.25m l/m in
• IEC buffer A 0%, IEC buffer B: 100%, gradient of 5CV & washing of 3CV, flow rate : 0.25ml/min
• the IEC fractions containing the purified conjugate are combined, washed to remove salt with buffer change of PBS buffer. The pH is adjusted after removing salt to 7.4. The solution is then concentrated at 4 ⁇ 1°C and the protein concentration analysed by UV spectroscopy (280nm). Conjugate were sterile filtered and samples taken for activity assay and for characterisation by SDS- PAGE and SE-HPLC. If required an aliquot was removed for a protein assay and CA assay. The remainder was stored at 4 ⁇ 1°C until further use and studied for physical stability by SE-HPLC.
• Novex® IEF gel was used to determine differences in insulin and CAO- insulin conjugate isoelectric points. Samples was dissolved to a concentration of 0.5 mg/ml. 5 ul sample was diluted with 5 ul Novex IEF Sample Buffer pH 3-10 and then loaded the protein sample on the gel. 6.10 Stability Studies
• CA Colominic acid
• N- acetylneuraminic acid (Neu ⁇ Ac) residues was used. Exposure of colominic acids to oxidation was carried out for 15 min using 20 mM periodate at room temperature. The integrity of the internal alpha-2.8 linked Neu5Ac residues post periodate treatment was analysed by gel permeation chromatography and the chromatographs obtained for the oxidised (CAO), material was compared with that of native CA. It was found that oxidized and native CA exhibit almost identical elution profiles, with no evidence that the successive oxidation step give rise to significant fragmentation of the polymer chain.
• 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. It shows that the oxidized colominic acid was found to have a greater than stoichiometric (>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).
• Table 1 shows the effect of molar ratio on polysialylation.
• Table 2 shows a T-Test (statistical analysis, paired-test) of different chain CAO-insulin conjugates in vivo efficiency.
• Monodisperse CAO-insulin can be successfully conjugated and highly pure conjugates were purified by scale-up HIC and IEC.
• the purification efficiency was improved from the IEC and HIC combination set-up with preparative HPLC instrument as demonstrated in Figure 10 .
• the procedure to prepare and purify colominic acid (CA) conjugates of insulin in an N-terminally selective manner by conducting the reaction at a reduced pH (pH 6.0) and at 4+1 0 C is detailed above. This involves conjugation in the presence of sodium cyanoborohydride, followed by purification using hydrophobic interaction chromatography (HIC) to remove free CA ( Figure 11) followed by removal of insulin by ion-exchange chromatography (IEC) ( Figure 12) .
• HIC hydrophobic interaction chromatography
• the low pH was used to favour selective derivatisation at the N-terminus of insulin's B-chain (PheB1), and also in order to minimise aggregation of insulin during the reaction.
• the composition of the final reaction buffer was 1 mg/ml insulin, 8mg/ml NaCNBH3 and 5 molar excess CAO in 10 mM NaOAc at pH 6.0.
• Isoelectric focusing (IEF) gels of 13 Kda and 27 Kda CAO-insulin conjugates in Figure 13 show the conjugate of polysialylated insulin has no fixed isoelectric point (pi).
• SE-HPLC change of retention time of insulin-PSA as compared to insulin; also co-elution of both moieties
• ion exchange chromatography binding of conjugates on to the IEC column
• polyacrylamide gel electrophoresis SDS-PAGE; shifting of bands with high m.w. species.
• Insulin conjugates used in the in vivo efficiency (on CD-1 mice, average
• PSA conjugates were found to be active in the in vitro activity assay. In vivo efficacy study shows that PSA-insulin conjugates are vastly superior to insulin. References

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