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WO2002007769A1 - A method of stabilisation and compositions for use therein - Google Patents

A method of stabilisation and compositions for use therein Download PDF

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Publication number
WO2002007769A1
WO2002007769A1 PCT/AU2001/000912 AU0100912W WO0207769A1 WO 2002007769 A1 WO2002007769 A1 WO 2002007769A1 AU 0100912 W AU0100912 W AU 0100912W WO 0207769 A1 WO0207769 A1 WO 0207769A1
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WIPO (PCT)
Prior art keywords
protein
composition
derivative
thioglycollic acid
divalent metal
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PCT/AU2001/000912
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French (fr)
Inventor
Michael Kerin Mcnamara
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Csl Limited
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Publication date
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Priority to AU2001276162A priority Critical patent/AU2001276162A1/en
Publication of WO2002007769A1 publication Critical patent/WO2002007769A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the present invention relates to a method of stabilising a protein or derivative thereof and agents for use therein. More particularly, the present invention provides a method of stabilising the protein portion of a liquid composition. The method of the present invention now facilitates, inter alia, the generation of improved protein based vaccine formulations.
  • thiomersal As a result of studies where thiomersal has been replaced by another preservative (2-phenoxyethanol) or where thiomersal is omitted it has been recognised that thiomersal also functions as a stabiliser of influenza haemagglutinin (HA) - the major protective antigen in the influenza vaccine.
  • HA haemagglutinin
  • thiomersal breaks down in aqueous solution to thiosalicylic acid and ethyl mercuric chloride. The latter compound is a toxic product related to a well known neurotoxic agent methyl mercury.
  • proteins can be stabilised by either monothioglycerol in the presence of divalent metal ions or thioglycollic acid but not by other thio(sulphydryl) reagents or other antioxidants.
  • thioglycollates can function as stabilisers of proteins stored at temperatures above that of freezing and therefore provides a realistic alternative for stabilising protein compositions, such as vaccines, which are preferably stored in a liquid state at temperatures of 2°C-8°C.
  • One aspect of the present invention is directed to a method of stabilising a protein or a derivative thereof in a liquid composition, said method comprising incorporating into said composition an effective amount of:
  • a method of stabilising an immunogen or a derivative, homologue, analogue, equivalent or mimetic thereof in a liquid composition comprising, incorporating into said composition an effective amount of:
  • said immunogen is influenza haemmaglutinin, pertussis toxin, diphtheria toxin, influenza neuraminidase, gpl20 of HIV, virus-like particles (NLPs) derived from HPN and HepB or derivatives, homologues, analogues, mutants, equivalents or mimetics thereof.
  • NLPs virus-like particles
  • a method of stabilising a protein or a derivative thereof in a liquid composition comprising incorporating into said composition an effective amount of:
  • composition is stored preferably at 1°C - 10°C and more preferably at 2°C - 8°C.
  • said formulation is stored preferably at 1°C - 10°C and more preferably at 2°C - 8°C.
  • said immunogen is an influenza immunogen and, more particularly, influenza haemagglutinin or influenza neuraminidase.
  • said immunogen is pertussis toxin, diphtheria toxin, gpl20 of HIN, virus-like particles derived from HPN and HepB.
  • the present invention is directed to the use of an effective amount of:
  • Still yet another further aspect of the present invention provides a protein or derivative thereof in a liquid composition, which protein is stable, said composition comprising a stabilising effective amount of:
  • Figure 1 is a graphical representation showing stabilisation of influenza B/Harbin HA by thioglycollic acid over six months at 6°C.
  • Figure 2 is a graphical representation showing stabilisation of influenza A/Beijing HA by thioglycollic acid over six months at 6°C.
  • Figure 3 is a graphical representation showing stabilisation of influenza A/Sydney HA by thioglycollic acid over six months at 6°C.
  • the present invention is predicated, in part, on the determination that both monothioglycerol in the presence of divalent metal ions and thioglycollic acid can function as stabilisers of proteins and, in particular, as stabilisers of proteins which are stored in a liquid environment at temperatures of greater than 0°C.
  • This determination has facilitated the development of methodology and agents for stabilising proteins, and in particular those stored in a liquid state such as vaccines, without the need to utilise the stabilisation capacity of thiomersal which gives rise to a toxic by-product.
  • one aspect of the present invention is directed to a method of stabilising a protein or a derivative thereof in a liquid composition, said method comprising incorporating into said composition an effective amount of:
  • stabilising or “stabilise” should be understood as a reference to effectively maintaining the tertiary conformation of the subject protein.
  • By “effectively” maintaining is meant that the tertiary conformation, to the extent that is required for the subject protein to at least partially achieve its purpose, is preserved.
  • stabilisation of that protein within the context of the present invention will have been achieved if a tertiary conformation of the epitope is sufficiently maintained in respect of a sufficient proportion of the protein molecules comprising a given formulation such that an effective level of immunogenicity in respect of the formulation is retained.
  • each and every protein molecule comprising a given formulation is stabilised (e.g. stabilisation of 70% of the protein molecules of a given formulation may be sufficient to achieve the requisite degree of immunogenicity).
  • protein should be understood to encompass peptides, polypeptides and proteins.
  • the protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • Reference hereinafter to a "protein” includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydates or other peptides, polypeptides or proteins.
  • the subject protein is an immunogen or derivative, homologue, analogue, mutant, equivalent or mimetic thereof.
  • immunogen is meant that the protein comprises at least one epitope.
  • the immunogen is a hapten it may be necessary to couple the hapten to a proteinaceous or non-proteinaceous carrier in order to effect antigenicity. Accordingly, such a hapten-carrier complex should be understood to fall within the definition of "protein” as detailed above.
  • An immunogen may take any suitable form. For example, to the extent that the immunogen is a viral immunogen it may take the form of live attenuated virus, inactivated whole virus, split inactivated virus or a subunit vaccine.
  • immunogens which may be the subject of stabilisation according to the method of the present invention include, but are not limited to, influenza haemmaglutinin, pertussis toxin, diphtheria toxin, influenza neuraminidase, gpl20 of HIN, virus-like particles (NLPs) derived from HPN and HepB, or derivatives, homologues, analogues, mutants, equivalents or mimetics thereof.
  • the present invention therefore more particularly provides a method of stabilising an immunogen or a derivative, homologue, analogue, equivalent or mimetic thereof in a liquid composition, said method comprising, incorporating into said composition an effective amount of:
  • said immunogen is influenza haemmaglutinin, pertussis toxin, diphtheria toxin, influenza neuraminidase, gpl20 of HIN, virus-like particles (NLPs) derived from HPN and HepB, or derivatives, homologues, analogues, mutants, equivalents or mimetics thereof.
  • thioglycollic acid should be understood as a reference to any form of thioglycollic acid or derivative, analogue or chemical equivalent thereof.
  • the subject thioglycollic acid may be utilised in either its liquid form or thioglycollic acid salts may be utilised.
  • Thioglycollic acid is a molecule of the chemical formula HSCH 2 COOH, although it should be understood that the use of derivatives, analogues or chemical equivalents is also encompassed within the scope of the invention. It should also be understood that thioglycollic acid is known by alternative terminology including “thioglycollate” (which is used particularly in relation to its salts) and "mercaptoacetic acid".
  • Monothioglycerol is a molecule of the chemical formula CH 2 (OH)CH(OH)CH 2 SH, although it should be understood that the use of derivatives, analogues or chemical equivalents is also encompassed within the scope of the invention.
  • Derivatives of the proteins herein defined include fragments, parts, portions, variants, analogues and mimetics from natural, synthetic or recombinant sources including fusion proteins. Parts or fragments include, for example, active regions of the subject molecule. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence.
  • substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.
  • An example of substitutional amino acid variants are conservative amino acid substitutions.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valitie, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
  • Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins.
  • Derivatives of thioglycollic acid or monothioglycerol should be understood to extend to all forms of thioglycollic acid or monothioglycerol including, for example, their salts.
  • Chemical and functional equivalents and analogues of the subject proteins or thioglycollic acid or monothioglycerol should be understood as molecules exhibiting any one or more of the functional activities of these molecules and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.
  • Derivatives of the subject proteins include fragments having particular epitopes or parts of the entire molecule fused to peptides, polypeptides or other proteinaceous or non- proteinaceous molecules.
  • Analogues of the proteins contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trimtrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • Table 1 A list of unnatural amino acids contemplated herein is shown in Table 1.
  • Non-conventional Code Non-conventional Code amino acid amino acid ⁇ -aminobutyric acid Abu L-N-methylalanine Nmala ⁇ -amino- ⁇ -methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile
  • D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval
  • D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
  • divalent metal ion should be understood in its broadest context and includes reference to a divalent electrically charged metal atoms.
  • the subject charge may be either positive or negative although the change is preferably positive. Since some ions are known to shift from one valency to another in certain circumstances, it should therefore be understood that the subject ion will satisfy the definition of being a "divalent” ion provided that the ion is at least transiently divalent while in the presence of monothioglycerol or thioglycollic acid. For example, the ion may exhibit a divalency only at the time of initial incorporation. Alternatively, it may become divalent only after incorporation.
  • the selection of divalent metal ions suitable for use in the present invention may require consideration of their acceptability for human or animal injection. Accordingly, ions exhibiting a degree of toxicity, such as Hg ++ , may be unsuitable for use in certain circumstances.
  • the divalent metal ion is one which is a trace element and even more preferably Mg "1-1" , Mn" " , Fe ++ , Co** or NX.
  • the antioxidant thioglycollic acid stabilises proteins by its ability to maintain disulphide bonds in their reduced state.
  • previous understanding in relation to the functioning of thioglycollic acid has centred on its use as a stabiliser under freezing conditions.
  • thioglycollic acid has been found to act as a protective agent (and not as a stabiliser) against the effects of freezing (for example, as per DMSO) (Bailey et al, 1986).
  • freezing for example, as per DMSO
  • thioglycollic acid does in fact stabilise proteins in terms of maintaining their tertiary conformation and therefore, in particular, provides a valuable and previously unidentified tool for maintaining the immunogenieity of vaccine formulations.
  • This finding is rendered more surprising by the observation that neither thiosalicylate (derived from the breakdown of thiomersal) nor other related thio compounds appear active as stabilisers in the liquid state.
  • reference to a "liquid" composition should be understood as a reference to the composition within which the protein component is dissolved or otherwise suspended, which composition is in a state which is neither solid (for example, frozen) or gaseous.
  • thioglycollic acid may be optionally formulated in the presence of divalent metal ions.
  • the present invention is directed to the stabilisation of a protein in a liquid composition which composition is maintained at 1°C - 10°C and even more preferably 2°C - 8°C.
  • a method of stabilising a protein or a derivative thereof in a liquid composition comprising incorporating into said composition an effective amount of:
  • composition is stored preferably at 1°C - 10°C and more preferably at 2°C - 8°C.
  • said protein is an immunogen or a derivative thereof.
  • a method of stabilising an immunogen in a liquid vaccine formulation comprising incorporating into said formulation an effective amount of:
  • said formulation is stored preferably at 1°C - 10°C and more preferably at 2°C - 8°C.
  • said immunogen is an influenza immunogen and, more particularly, influenza haemagglutinin or influenza neuraminidase.
  • said immunogen is pertussis toxin, diphtheria toxin, gpl20 of HIN, virus-like particles derived from HPN and HepB.
  • references to "incorporating" monothioglycerol or thioglycollic acid into a protein containing liquid composition should be understood in its broadest sense to include any form of incorporation.
  • the subject composition may be completely formulated prior to addition of monothioglycerol or thioglycollic acid, for example where monothioglycerol or thioglycollic acid is added as a last step.
  • the monothioglycerol or thioglycollic acid may be added to the subject composition during formulation of the composition itself (for example, a liquid composition comprising monothioglycerol or thioglycollic acid may be formulated prior to addition of the protein - this may be necessary, for example, where a protein is particularly unstable).
  • the monothioglycerol or thioglycollic acid may be incorporated pursuant to a single step or multiple step protocol.
  • monothioglycerol or thioglycollic acid may be incorporated at a time point subsequently to formulation of the composition.
  • references to monothioglycerol or thioglycollic acid being "in the presence of divalent metal ions should be understood as a reference to any type of formulation of monothioglycerol or thioglycollic acid with divalent metal ions.
  • the subject ions may become linked, bound or otherwise associated with the monothioglycerol, thioglycollic acid and/or protein, such as via the generation of covalent bonds or any other interactive bonding mechanism.
  • the subject ions may remain in an unassociated form.
  • Formulation of monothioglycerol or thioglycollic acid with the divalent metal ions may be achieved by any suitable means.
  • thioglycollic acid or monothioglycerol may be incorporated with the monothioglycerol or thioglycollic acid prior to incorporation of these stabilisers with the subject protein or it may be incorporated directly with the protein formulation either prior to or subsequently to incorporation of the monothioglycerol or thioglycollic acid.
  • the subject incorporation may occur as a single step or multi-step protocol.
  • an “effective amount” means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition of the individual to be treated, the taxonomic group of individual to be treated, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • the present invention is directed to the use of an effective amount of:
  • the present invention is directed to the use of an effective amount of:
  • said protein is an immunogen and even more preferably said liquid composition is a vaccine formulation.
  • said immunogen is an influenza immunogen and still more preferably influenza haemagglutinin.
  • Yet another aspect of the present invention provides: (i) monothioglycerol in the presence of divalent metal ions; or
  • said composition is maintained at 1°C - 10°C and even more preferably at 2°C - 8°C.
  • said protein is an immunogen and said liquid composition is a vaccine formulation.
  • said immunogen is an influenza immunogen and, more particularly, influenza haemagglutinin.
  • the development of the method of the present invention now facilitates the formulation of protein containing liquid compositions, in particular vaccine formulations, which can be stably stored at temperatures above those of freezing and, in particular at 2°C - 8°C.
  • the present invention provides a protein or derivative thereof in a liquid composition, which protein is stable, said composition comprising a stabilising effective amount of:
  • said composition is stored at 1°C - 10°C and more preferably at 2°C - 8°C.
  • said protein is an immunogen and even more preferably said liquid composition is a vaccine formulation.
  • said immunogen is influenza immunogen and, more particularly, influenza haemagglutinin.
  • influenza virus - A Beijing 262/95 (H1N1 strain), A/Sydney 5/97 (H3N2 strain) and B/Harbin 7/94 (B strain) were used to formulate trivalent vaccines.
  • the viruses for all strains were grown in the allantoic fluid of embryonated chicken eggs then purified by centrifugation on sucrose gradients and inactivated with ⁇ propiolactone.
  • the inactivated viruses were then split into their component proteins using taurodeoxycholate, which was subsequently removed by dialtrafiltration against phosphate buffered saline pH 7.2. These split, inactivated virus antigens are known as antigen concentrates or process pools.
  • the materials for the vaccine formulation buffers and the additives, thiomersal and L- cysteine hydrochloride were all of pharmaceutically acceptable grades.
  • Zinc chloride (ACS reagent), L-ascorbic acid, ⁇ -monothioglycerol and thioglycolic acid (sodium salt, minimum 99% purity) and thiosalicylic acid (Ultra grade, minimum 95% purity) were supplied by Sigma.
  • Water for injection (WFI) was used to prepare all buffers. The bulk trivalent vaccines were dispensed by hand into syringes under aseptic environmental conditions.
  • the formulation buffer used to prepare the influenza vaccines contained 0.14 M NaCl, 3.47 mM disodium hydrogen phosphate anhydrous, 1.53 mM sodium dihydrogen phosphate dihydrate pH 7.2.
  • trivalent vaccines were formulated (33 ⁇ g /mL for each strain) using this buffer with 0, 0.001 or 0.01% w/v thiomersal.
  • the formulations also contained 0.0014% w/v calcium chloride.
  • a trivalent vaccine formulated using the calcium containing buffer with 0.0039% w/v thiosalicylic acid (no thiomersal) was also prepared.
  • Thiosalicylic acid is the non- mercury containing breakdown product of thiomersal, potentially responsible for the stabilisation of HA in the influenza vaccine. Details of the buffers used are summarised in Table 2.
  • the vaccines were dispensed (0.5 mL) into syringes fitted with elastomeric stoppers under aseptic environmental conditions.
  • the stability of haemagglutinin was monitored using the SRD assay at each time point. Appearance and pH were also monitored at each time point.
  • the vaccine was stored under the following conditions:
  • Sterility was also monitored at time zero and at the end of the trial at 37° and 6 ⁇ 2°C. Twenty syringes were used for each sterility test.
  • Trivalent vaccines were formulated (33 ⁇ g/mL for each strain) with buffer containing 0.14M NaCl, 5 mM phosphate, 0.0014% w/v calcium chloride and the pharmaceutically accepted excipients, including thiomersal (as a control) at the concentration shown in Table 3 at pH 7.2 and 7.7.
  • the vaccines were dispensed (0.5 mL) into syringes fitted with elastomeric stoppers under aseptic environmental conditions. The stability of haemagglutinin was monitored using the SRD assay at each time point. Appearance and pH are also monitored at each time point.
  • the vaccine was stored under the following conditions: a. 0, 1, 2 and 4 weeks at 37°C and b. 0, 3, 6 and 12 months at 6 ⁇ 2°C.
  • the pseudo first order rate constant for the rate of degradation of HA was obtained from the slope of the line of best fit of the linear regression of -log [HA] versus time of storage (days).
  • the value for the rate constants for each of the strains used is given in Tables 4 & 5.
  • Table 2 Details of buffers used to formulate the polyvalent vaccines for assessing the effect of thiomersal and thiosalicylate on the stability of influenza haemagglutinin. All of the buffers contain 0.14 M NaCl, 3.47 mM disodium hydrogen phosphate anhydrous, 1.53 mM sodium dihydrogen phosphate dihydrate and 0.0014 % w/v calcium chloride.
  • Table 3 Details of the buffers used to formulate the polyvalent vaccines containing pharmaceutically accepted excipients. All of the buffers contain 0.14 M NaCl, 3.47 mM disodium hydrogen phosphate anhydrous, 1.53 mM sodium dihydrogen phosphate dihydrate and 0.0414% w/v calcium chloride.

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Abstract

The present invention relates to a method of stabilising a protein or derivative thereof and agents for use therein. More particularly, the present invention provides a method of stabilising the protein portion of a liquid composition. The method of the present invention now facilitates, inter alia, the generation of improved protein based vaccine formulations.

Description

A METHOD OF STABILISATION AND COMPOSITIONS FOR USE THEREIN
FIELD OF THE INVENTION
The present invention relates to a method of stabilising a protein or derivative thereof and agents for use therein. More particularly, the present invention provides a method of stabilising the protein portion of a liquid composition. The method of the present invention now facilitates, inter alia, the generation of improved protein based vaccine formulations.
BACKGROUND OF THE-INVENTION
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.
Influenza vaccines and indeed a considerable number of human (including childhood) and animal vaccines can contain 0.01% thiomersal as a preservative. As a result of studies where thiomersal has been replaced by another preservative (2-phenoxyethanol) or where thiomersal is omitted it has been recognised that thiomersal also functions as a stabiliser of influenza haemagglutinin (HA) - the major protective antigen in the influenza vaccine. However, a review of the chemistry of thiomersal has revealed that thiomersal breaks down in aqueous solution to thiosalicylic acid and ethyl mercuric chloride. The latter compound is a toxic product related to a well known neurotoxic agent methyl mercury. In fact, this has lead regulatory authorities to raise concern over the accumulative effect of mercury preservatives, like thiomersal especially, in vaccines administered to infants less than 6 months old. Accordingly, thiomersal is no longer accepted as a component of vaccines. Its previous use as a vaccine preservative was, in fact, directed to its use as an antimicrobial preservative. In this regard, it provides sterility assurance with respect to the product in which it was incorporated. However, in addition to problems associated with toxicity of the thiomersal breakdown products, antimicrobial preservatives are no longer acceptable in single dose vaccines and therapeutic proteins. This has now lead regulatory agencies such as the FDA, TGA and EMEA to issue requests to manufacturers to develop and present plans for the removal of thiomersal from their products. It is expected that these directives will also extend to manufacturers of veterinary vaccines.
Accordingly, in light of the dual role attributable to thiomersal, being its antimicrobial and stabilisation properties, there is a need to develop alternative methodology for achieving these objectives. In terms of the sterility requirement, it has been determined that the use of a sterile filtration step can replace thiomersal to the extent that sterility assurance is required. However, to date there has been no suitable alternative identified to act as a stabilising agent.
In work leading up to the present invention, the inventors have determined that proteins can be stabilised by either monothioglycerol in the presence of divalent metal ions or thioglycollic acid but not by other thio(sulphydryl) reagents or other antioxidants. In particular, it has been surprisingly determined that thioglycollates can function as stabilisers of proteins stored at temperatures above that of freezing and therefore provides a realistic alternative for stabilising protein compositions, such as vaccines, which are preferably stored in a liquid state at temperatures of 2°C-8°C. SUMMARY OF THE INVENTION
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
One aspect of the present invention is directed to a method of stabilising a protein or a derivative thereof in a liquid composition, said method comprising incorporating into said composition an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or chemical equivalent thereof for a time and under conditions sufficient to stabilise said protein.
In another aspect there is provided a method of stabilising an immunogen or a derivative, homologue, analogue, equivalent or mimetic thereof in a liquid composition, said method comprising, incorporating into said composition an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or chemical equivalent thereof for a time and under conditions sufficient to stabilise said protein.
Preferably, said immunogen is influenza haemmaglutinin, pertussis toxin, diphtheria toxin, influenza neuraminidase, gpl20 of HIV, virus-like particles (NLPs) derived from HPN and HepB or derivatives, homologues, analogues, mutants, equivalents or mimetics thereof.
In yet another aspect there is provided a method of stabilising a protein or a derivative thereof in a liquid composition, said method comprising incorporating into said composition an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or chemical equivalent thereof for a time and under conditions sufficient to stabilise said protein, wherein said composition is stored preferably at 1°C - 10°C and more preferably at 2°C - 8°C.
In still another aspect there is provided a method of stabilising an immunogen in a liquid vaccine formulation, said method comprising incorporating into said formulation an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or chemical equivalent thereof for a time and under conditions sufficient to stabilise said immunogen, wherein said formulation is stored preferably at 1°C - 10°C and more preferably at 2°C - 8°C.
In a most preferred embodiment, said immunogen is an influenza immunogen and, more particularly, influenza haemagglutinin or influenza neuraminidase.
In another most preferred embodiment, said immunogen is pertussis toxin, diphtheria toxin, gpl20 of HIN, virus-like particles derived from HPN and HepB. In still yet another aspect, the present invention is directed to the use of an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or equivalent thereof to stabilise a protein or derivative thereof in a liquid composition.
In a further aspect the present invention is directed to the use of an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or equivalent thereof to stabilise a protein or derivative thereof in a liquid composition wherein said composition is stored preferably at 1°C - 10°C and more preferably at 2°C - 8°C.
In another further aspect there is provided the use of an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or equivalent thereof to stabilise an immunogen or derivative thereof in a liquid vaccine formulation, wherein said formulation is stored preferably at 1 °C - 10°C and more preferably at 2°C - 8°C. Yet another further aspect of the present invention provides:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or equivalent thereof for use in the stabilisation of a protein or derivative in a liquid composition.
Still yet another further aspect of the present invention provides a protein or derivative thereof in a liquid composition, which protein is stable, said composition comprising a stabilising effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or derivative, analogue or equivalent thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graphical representation showing stabilisation of influenza B/Harbin HA by thioglycollic acid over six months at 6°C.
Figure 2 is a graphical representation showing stabilisation of influenza A/Beijing HA by thioglycollic acid over six months at 6°C.
Figure 3 is a graphical representation showing stabilisation of influenza A/Sydney HA by thioglycollic acid over six months at 6°C.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is predicated, in part, on the determination that both monothioglycerol in the presence of divalent metal ions and thioglycollic acid can function as stabilisers of proteins and, in particular, as stabilisers of proteins which are stored in a liquid environment at temperatures of greater than 0°C. This determination has facilitated the development of methodology and agents for stabilising proteins, and in particular those stored in a liquid state such as vaccines, without the need to utilise the stabilisation capacity of thiomersal which gives rise to a toxic by-product.
Accordingly, one aspect of the present invention is directed to a method of stabilising a protein or a derivative thereof in a liquid composition, said method comprising incorporating into said composition an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or chemical equivalent thereof for a time and under conditions sufficient to stabilise said protein.
Reference to "stabilising" or "stabilise" should be understood as a reference to effectively maintaining the tertiary conformation of the subject protein. By "effectively" maintaining is meant that the tertiary conformation, to the extent that is required for the subject protein to at least partially achieve its purpose, is preserved. For example, where the subject protein comprises an epitope and is therefore utilised as part of a vaccine formulation, "stabilisation" of that protein within the context of the present invention will have been achieved if a tertiary conformation of the epitope is sufficiently maintained in respect of a sufficient proportion of the protein molecules comprising a given formulation such that an effective level of immunogenicity in respect of the formulation is retained. Accordingly, it should be understood that it may not be necessary to retain the tertiary conformation of the protein molecule in its entirety (for example, with respect to a vaccine only certain epitopic region(s) of the protein are of importance). Nor may it necessarily be required that each and every protein molecule comprising a given formulation is stabilised (e.g. stabilisation of 70% of the protein molecules of a given formulation may be sufficient to achieve the requisite degree of immunogenicity).
The term "protein" should be understood to encompass peptides, polypeptides and proteins. The protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins. Reference hereinafter to a "protein" includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydates or other peptides, polypeptides or proteins.
Preferably, the subject protein is an immunogen or derivative, homologue, analogue, mutant, equivalent or mimetic thereof. By "immunogen" is meant that the protein comprises at least one epitope. In this regard, if the immunogen is a hapten it may be necessary to couple the hapten to a proteinaceous or non-proteinaceous carrier in order to effect antigenicity. Accordingly, such a hapten-carrier complex should be understood to fall within the definition of "protein" as detailed above. An immunogen may take any suitable form. For example, to the extent that the immunogen is a viral immunogen it may take the form of live attenuated virus, inactivated whole virus, split inactivated virus or a subunit vaccine. Examples of immunogens which may be the subject of stabilisation according to the method of the present invention include, but are not limited to, influenza haemmaglutinin, pertussis toxin, diphtheria toxin, influenza neuraminidase, gpl20 of HIN, virus-like particles (NLPs) derived from HPN and HepB, or derivatives, homologues, analogues, mutants, equivalents or mimetics thereof.
The present invention therefore more particularly provides a method of stabilising an immunogen or a derivative, homologue, analogue, equivalent or mimetic thereof in a liquid composition, said method comprising, incorporating into said composition an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or chemical equivalent thereof for a time and under conditions sufficient to stabilise said protein.
Preferably, said immunogen is influenza haemmaglutinin, pertussis toxin, diphtheria toxin, influenza neuraminidase, gpl20 of HIN, virus-like particles (NLPs) derived from HPN and HepB, or derivatives, homologues, analogues, mutants, equivalents or mimetics thereof.
Reference to "thioglycollic acid" should be understood as a reference to any form of thioglycollic acid or derivative, analogue or chemical equivalent thereof. For example, the subject thioglycollic acid may be utilised in either its liquid form or thioglycollic acid salts may be utilised. Thioglycollic acid is a molecule of the chemical formula HSCH2COOH, although it should be understood that the use of derivatives, analogues or chemical equivalents is also encompassed within the scope of the invention. It should also be understood that thioglycollic acid is known by alternative terminology including "thioglycollate" (which is used particularly in relation to its salts) and "mercaptoacetic acid".
Reference to "monothioglycerol" should be understood as a reference to any form of monothioglycerol or derivative, analogue or chemical equivalent thereof.
Monothioglycerol is a molecule of the chemical formula CH2(OH)CH(OH)CH2SH, although it should be understood that the use of derivatives, analogues or chemical equivalents is also encompassed within the scope of the invention.
"Derivatives" of the proteins herein defined include fragments, parts, portions, variants, analogues and mimetics from natural, synthetic or recombinant sources including fusion proteins. Parts or fragments include, for example, active regions of the subject molecule. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. An example of substitutional amino acid variants are conservative amino acid substitutions. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valitie, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins.
"Derivatives" of thioglycollic acid or monothioglycerol should be understood to extend to all forms of thioglycollic acid or monothioglycerol including, for example, their salts.
Chemical and functional equivalents and analogues of the subject proteins or thioglycollic acid or monothioglycerol should be understood as molecules exhibiting any one or more of the functional activities of these molecules and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.
Derivatives of the subject proteins include fragments having particular epitopes or parts of the entire molecule fused to peptides, polypeptides or other proteinaceous or non- proteinaceous molecules.
Analogues of the proteins contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trimtrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate. Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 1.
TABLE 1
Non-conventional Code Non-conventional Code amino acid amino acid α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile
D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys
D-aspartic acid Dasp L-N-methylmethionine Nmmet
D-cysteine Dcys L-N-methylnorleucine Nmnle
D-glutamine Dgln L-N-methylnorvaline Nmnva
D-glutamic acid Dglu L-N-methylornithine Nmorn
D-histidine Dhis L-N-methylphenylalanine Nmphe
D-isoleucine Dile L-N-methylproline Nmpro
D-leucine Dleu L-N-methylserine Nmser
D-lysine Dlys L-N-methylthreonine Nmthr
D-methionine Dmet L-N-methyltryptophan Nmtrp
D-ornithine Dorn L-N-methyltyrosine Nmtyr
D-phenylalanine Dphe L-N-methylvaline Nmval
D-proline Dpro L-N-methylethylglycine Nmetg
D-serine Dser L-N-methyl-t-buty glycine Nmtbug
D-threonine Dthr L-norleucine Nle
D-tryptophan Dtrp L-norvaline Nva
D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib
D-valine Dval α-methyl- -aminobutyrate Mgabu
D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen
D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap
D-α-methylaspartate Dmasp α-methylpenicillamine Mpen
D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-α-methylhistidine Dmhis N-(3 -aminopropyl)glycine Norn
D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu
D-α-methylleucine Dmleu α-napthylalanine Anap
D-α-methyllysine Dmlys N-benzylglycine Nphe
D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln
D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-α-methylserine Dmser N-cyclobutylglycine Ncbut
D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-α-methylvaline Dmval N-cylcododecylglycine Ncdod
D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct
D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe
D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(l-hydroxyethyl)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-( 1 -methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(jp-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys
L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-α-methylalanine Mala
L-α-methylarginine Marg L-α-methylasparagine Masn
L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug
L-α-methylcysteine Mcys L-methylethylglycine Metg
L-α-methylglutamine Mgln L-α-methylglutamate Mglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe
L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet
L-α-methylleucine Mleu L-α-methyllysine Mlys
L-α-methylmethionine Mmet L-α-methylnorleucine Mnle
L-α-methylnorvaline Mnva L-α-methylornithine Morn L-α-methylphenylalanine Mphe L-α-methylproline Mpro
L-α-methylserine Mser L-α-methylthreonine Mthr
L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr
L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe
N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine
1 -carboxy- 1 -(2,2-diphenyl-Nmbc ethylamino)cyclopropane
Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH )n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety.
Reference to "divalent metal ion" should be understood in its broadest context and includes reference to a divalent electrically charged metal atoms. The subject charge may be either positive or negative although the change is preferably positive. Since some ions are known to shift from one valency to another in certain circumstances, it should therefore be understood that the subject ion will satisfy the definition of being a "divalent" ion provided that the ion is at least transiently divalent while in the presence of monothioglycerol or thioglycollic acid. For example, the ion may exhibit a divalency only at the time of initial incorporation. Alternatively, it may become divalent only after incorporation. Without limiting the present invention in any way, the selection of divalent metal ions suitable for use in the present invention may require consideration of their acceptability for human or animal injection. Accordingly, ions exhibiting a degree of toxicity, such as Hg++, may be unsuitable for use in certain circumstances. Preferably, the divalent metal ion is one which is a trace element and even more preferably Mg"1-1", Mn"", Fe++, Co** or NX.
Without limiting the present invention to any one theory or mode of action, the antioxidant thioglycollic acid stabilises proteins by its ability to maintain disulphide bonds in their reduced state. However, previous understanding in relation to the functioning of thioglycollic acid has centred on its use as a stabiliser under freezing conditions. In this context and at -70°C, thioglycollic acid has been found to act as a protective agent (and not as a stabiliser) against the effects of freezing (for example, as per DMSO) (Bailey et al, 1986). However, in a non-freezing thermal environment, it was found not to provide any benefits. The inventors have surprisingly determined that in a liquid environment, thioglycollic acid does in fact stabilise proteins in terms of maintaining their tertiary conformation and therefore, in particular, provides a valuable and previously unidentified tool for maintaining the immunogenieity of vaccine formulations. This finding is rendered more surprising by the observation that neither thiosalicylate (derived from the breakdown of thiomersal) nor other related thio compounds appear active as stabilisers in the liquid state. In the context of the present invention, therefore, reference to a "liquid" composition should be understood as a reference to the composition within which the protein component is dissolved or otherwise suspended, which composition is in a state which is neither solid (for example, frozen) or gaseous. It should be understood that thioglycollic acid may be optionally formulated in the presence of divalent metal ions.
With respect to vaccine formulations, in particular, it is generally not appropriate and highly inconvenient to store such formulations in a frozen state. Further, it is a well known principle of protein chemistry that freeze/thawing some proteins results in their denaturation. In the context of vaccines, such an effect is of particular concern since it can lead to the conformational modification of certain epitopes required for the protein component's immunogenicity thereby rendering the vaccine essentially useless. Accordingly, in a particularly preferred embodiment the present invention is directed to the stabilisation of a protein in a liquid composition which composition is maintained at 1°C - 10°C and even more preferably 2°C - 8°C.
Accordingly, there is provided a method of stabilising a protein or a derivative thereof in a liquid composition, said method comprising incorporating into said composition an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or chemical equivalent thereof for a time and under conditions sufficient to stabilise said protein, wherein said composition is stored preferably at 1°C - 10°C and more preferably at 2°C - 8°C.
In a still more preferred embodiment, said protein is an immunogen or a derivative thereof.
According to a most preferred embodiment, there is provided a method of stabilising an immunogen in a liquid vaccine formulation, said method comprising incorporating into said formulation an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or chemical equivalent thereof for a time and under conditions sufficient to stabilise said immunogen, wherein said formulation is stored preferably at 1°C - 10°C and more preferably at 2°C - 8°C.
In a most preferred embodiment, said immunogen is an influenza immunogen and, more particularly, influenza haemagglutinin or influenza neuraminidase.
In another most preferred embodiment, said immunogen is pertussis toxin, diphtheria toxin, gpl20 of HIN, virus-like particles derived from HPN and HepB.
Reference to "incorporating" monothioglycerol or thioglycollic acid into a protein containing liquid composition should be understood in its broadest sense to include any form of incorporation. For example, the subject composition may be completely formulated prior to addition of monothioglycerol or thioglycollic acid, for example where monothioglycerol or thioglycollic acid is added as a last step. Alternatively, the monothioglycerol or thioglycollic acid may be added to the subject composition during formulation of the composition itself (for example, a liquid composition comprising monothioglycerol or thioglycollic acid may be formulated prior to addition of the protein - this may be necessary, for example, where a protein is particularly unstable). The monothioglycerol or thioglycollic acid may be incorporated pursuant to a single step or multiple step protocol. In yet another alternative, monothioglycerol or thioglycollic acid may be incorporated at a time point subsequently to formulation of the composition. For example, it may be known that a given protein survives a single freeze-thaw step. Accordingly, it may be desirable to manufacture the protein composition, freeze it i r a purpose such as a extended storage and incorporate the monothioglycerol or thioglycollic acid only upon thawing of the subject composition for the purpose of maintaining the composition's stability during its on-going storage in a liquid state.
Reference to monothioglycerol or thioglycollic acid being "in the presence of divalent metal ions should be understood as a reference to any type of formulation of monothioglycerol or thioglycollic acid with divalent metal ions. For example, the subject ions may become linked, bound or otherwise associated with the monothioglycerol, thioglycollic acid and/or protein, such as via the generation of covalent bonds or any other interactive bonding mechanism. Alternatively, the subject ions may remain in an unassociated form. Formulation of monothioglycerol or thioglycollic acid with the divalent metal ions may be achieved by any suitable means. For example, it may be achieved by means analogous to those described in relation to the incorporation of thioglycollic acid or monothioglycerol into a protein containing liquid composition. That is, it may be incorporated with the monothioglycerol or thioglycollic acid prior to incorporation of these stabilisers with the subject protein or it may be incorporated directly with the protein formulation either prior to or subsequently to incorporation of the monothioglycerol or thioglycollic acid. Further, the subject incorporation may occur as a single step or multi-step protocol.
An "effective amount" means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition of the individual to be treated, the taxonomic group of individual to be treated, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
In another aspect, the present invention is directed to the use of an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid or a derivative, analogue or equivalent thereof to stabilise a protein or derivative thereof in a liquid composition.
Preferably, the present invention is directed to the use of an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or equivalent thereof to stabilise a protein or derivative thereof in a liquid composition wherein said composition is stored preferably at 1°C - 10°C and more preferably at 2°C - 8°C.
In yet another preferred embodiment said protein is an immunogen and even more preferably said liquid composition is a vaccine formulation.
According to this most preferred embodiment there is provided the use of an effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or equivalent thereof to stabilise an immunogen or derivative thereof in a liquid vaccine formulation, wherein said formulation is stored preferably at 1 °C - 10°C and more preferably at 2°C - 8°C.
In a most preferred embodiment said immunogen is an influenza immunogen and still more preferably influenza haemagglutinin.
Yet another aspect of the present invention provides: (i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or a derivative, analogue or equivalent thereof for use in the stabilisation of a protein or derivative in a liquid composition.
Preferably, said composition is maintained at 1°C - 10°C and even more preferably at 2°C - 8°C.
Still more preferably said protein is an immunogen and said liquid composition is a vaccine formulation.
In a most preferred embodiment said immunogen is an influenza immunogen and, more particularly, influenza haemagglutinin.
The development of the method of the present invention now facilitates the formulation of protein containing liquid compositions, in particular vaccine formulations, which can be stably stored at temperatures above those of freezing and, in particular at 2°C - 8°C.
Accordingly, in another aspect the present invention provides a protein or derivative thereof in a liquid composition, which protein is stable, said composition comprising a stabilising effective amount of:
(i) monothioglycerol in the presence of divalent metal ions; or
(ii) thioglycollic acid
or derivative, analogue or equivalent thereof.
Preferably, said composition is stored at 1°C - 10°C and more preferably at 2°C - 8°C. Still more preferably, said protein is an immunogen and even more preferably said liquid composition is a vaccine formulation.
In a most preferred embodiment, said immunogen is influenza immunogen and, more particularly, influenza haemagglutinin.
Further features of the present invention are more fully described in the following non- limiting examples.
EXAMPLE 1 INVESTIGATING EFFECT OF ALTERNATIVE EXCIPIENTS ON STABILITY
OF INFLUENZA HAEMAGGLUTININ IN FLUVAX®
MATERIALS AND METHODS Materials
In this study three strains of influenza virus - A Beijing 262/95 (H1N1 strain), A/Sydney 5/97 (H3N2 strain) and B/Harbin 7/94 (B strain) were used to formulate trivalent vaccines. The viruses for all strains were grown in the allantoic fluid of embryonated chicken eggs then purified by centrifugation on sucrose gradients and inactivated with β propiolactone. The inactivated viruses were then split into their component proteins using taurodeoxycholate, which was subsequently removed by dialtrafiltration against phosphate buffered saline pH 7.2. These split, inactivated virus antigens are known as antigen concentrates or process pools.
The materials for the vaccine formulation buffers and the additives, thiomersal and L- cysteine hydrochloride were all of pharmaceutically acceptable grades. Zinc chloride (ACS reagent), L-ascorbic acid, α-monothioglycerol and thioglycolic acid (sodium salt, minimum 99% purity) and thiosalicylic acid (Ultra grade, minimum 95% purity) were supplied by Sigma. Water for injection (WFI) was used to prepare all buffers. The bulk trivalent vaccines were dispensed by hand into syringes under aseptic environmental conditions.
Methods
(i) Investigating the effect of thiomersal on stability
The formulation buffer used to prepare the influenza vaccines contained 0.14 M NaCl, 3.47 mM disodium hydrogen phosphate anhydrous, 1.53 mM sodium dihydrogen phosphate dihydrate pH 7.2. To investigate the effect of thiomersal on the stability of the influenza haemagglutinin, trivalent vaccines were formulated (33μg /mL for each strain) using this buffer with 0, 0.001 or 0.01% w/v thiomersal. The formulations also contained 0.0014% w/v calcium chloride. A trivalent vaccine formulated using the calcium containing buffer with 0.0039% w/v thiosalicylic acid (no thiomersal) was also prepared. Thiosalicylic acid is the non- mercury containing breakdown product of thiomersal, potentially responsible for the stabilisation of HA in the influenza vaccine. Details of the buffers used are summarised in Table 2.
The vaccines were dispensed (0.5 mL) into syringes fitted with elastomeric stoppers under aseptic environmental conditions. The stability of haemagglutinin was monitored using the SRD assay at each time point. Appearance and pH were also monitored at each time point. The vaccine was stored under the following conditions:
a.. 0, 1 , 2 and 4 weeks at 37°C and b. 0, 3, 6, 9 and 12 months at 6 ± 2°C.
Sterility was also monitored at time zero and at the end of the trial at 37° and 6 ± 2°C. Twenty syringes were used for each sterility test.
(ii) Investigating the effect of alternative excipients on stability
Trivalent vaccines were formulated (33μg/mL for each strain) with buffer containing 0.14M NaCl, 5 mM phosphate, 0.0014% w/v calcium chloride and the pharmaceutically accepted excipients, including thiomersal (as a control) at the concentration shown in Table 3 at pH 7.2 and 7.7. The vaccines were dispensed (0.5 mL) into syringes fitted with elastomeric stoppers under aseptic environmental conditions. The stability of haemagglutinin was monitored using the SRD assay at each time point. Appearance and pH are also monitored at each time point. The vaccine was stored under the following conditions: a. 0, 1, 2 and 4 weeks at 37°C and b. 0, 3, 6 and 12 months at 6 ± 2°C.
Ten syringes were used at each time point for SRD testing and for pH / appearance. Sterility was monitored at time zero and at the end of the trials at 37°C and 6 ± 2°C.
Twenty syringes were used for each sterility test.
RESULTS
Degradation of HA
(i) Results at 37°C
The pseudo first order rate constant for the rate of degradation of HA was obtained from the slope of the line of best fit of the linear regression of -log [HA] versus time of storage (days). The value for the rate constants for each of the strains used is given in Tables 4 & 5.
The data in Table 4 shows that formulations containing thiomersal have small rate constants for the degradation of HA indicating little degradation of HA occurs whereas formulations where thiomersal is omitted have large rate constants which indicates that substantial HA degradation occurs. These results show that thiomersal is a stabiliser of influenza HA and that omitting thiomersal from the vaccine leads to an increase in the rate of HA degradation. This data also shows that thiosalicylic acid (a potential antioxidant stabiliser of influenza HA and a natural breakdown product of thiomersal in solution) does not stabilise influenza HA.
The data in Table 5 shows that only formulations containing thioglycollic acid (either in the presence or absence of zinc chloride and at either pH) and monothioglycerol in the presence of zinc chloride have similar low rate constants to those observed for formulations containing 0.01% of thiomersal at pH 7.2 or pH 7.7. Of the excipients tested, only these formulations acted as a stabiliser of HA. Surprisingly, formulations containing either L-cysteine or ascorbic acid (a non- sulphydryl antioxidant) were degraded at a markedly greater rate than those containing either thiomersal thioglycollic acid, or monothioglycerol in the presence of zinc chloride.
(ii) Results at 6°C
Thiomersal studies show that thioglycollic acid or thiomersal formulations are both stable at 6°C for 6 months (Figures 1-3).
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
Table 2 : Details of buffers used to formulate the polyvalent vaccines for assessing the effect of thiomersal and thiosalicylate on the stability of influenza haemagglutinin. All of the buffers contain 0.14 M NaCl, 3.47 mM disodium hydrogen phosphate anhydrous, 1.53 mM sodium dihydrogen phosphate dihydrate and 0.0014 % w/v calcium chloride.
Figure imgf000029_0001
Table 3: Details of the buffers used to formulate the polyvalent vaccines containing pharmaceutically accepted excipients. All of the buffers contain 0.14 M NaCl, 3.47 mM disodium hydrogen phosphate anhydrous, 1.53 mM sodium dihydrogen phosphate dihydrate and 0.0414% w/v calcium chloride.
Figure imgf000030_0001
obtained from Merck Index "Monothioglycl = monthioglycerol bThioglyc = thioglycolic acid (sodium salt) Table 4: Effect of Thiomersal on HA Stability
Figure imgf000031_0001
*Results based on fitting data over whole 25 day period.
** Other results were significantly non-linear and data is based on the slope from time zero to first time point (11 days)
Table 5: Effect of Alternative Stabilisers on HA Stability
Figure imgf000032_0001
*Results based on fitting data over whole 28 day period.
** Other results were non-linear and k is based on the slope from time zero to first point (11 days)
BIBLIOGRAPHY
Bailey, Marsha L. and Grogan, W. McLean, (1996) "Protein kinase-mediated Activation of Temperature-labile and temperature-stable Cholesteryl Ester Hydrolases in the Rat Testis" J Biol Chem, vol 261 pp7717-7722
The Merck Index - An Encyclopedia of Chemicals, Drugs and Biologicals, Edited by S. Budavari, M. J. ONeil, A. Smith and P.E. Heckelman, 11th Edition, Merck and Co., Inc., Rahway, USA, 1989.

Claims

CLAIMS:
1. A method of stabilising a protein or a derivative thereof in a liquid composition, said method comprising incoφorating into said composition an effective amount of monothioglycerol in the presence of divalent metal ions or a derivative, analogue or chemical equivalent thereof for a time and under conditions sufficient to stabilise said protein.
2. A method of stabilising a protein or a derivative thereof in a liquid composition, said method comprising incoφorating into said composition an effective amount of thioglycollic acid or a derivative, analogue or chemical equivalent thereof for a time and under conditions sufficient to stabilise said protein.
3. The method according to claim 2 wherein said thioglycollic acid is a thioglycollic acid salt.
4. The method according to claim 2 or 3 wherein said thioglycollic acid is incoφorated into said composition in the presence of divalent metal ions.
5. The method according to claim 1 or 4 wherein said divalent metal ion is Mg*"1", Mn++, Fe++, Co++ αrNi++.
6. The method according to any one of claims 1 -5 wherein said protein is an immunogen.
7. The method according to claim 6 wherein said immunogen is influenza haemmaglutinin, pertussis toxin, diphtheria toxin, influenza neuraminidase, gpl20 of HIV, virus-like particles derived from HPV or HepB, or derivatives, homologues, analogues, mutants, equivalents or mimetics thereof.
8. The method according to any one of claims 1-7 wherein said composition is stored at 1°C-10°C.
9. The method according to claim 8 wherein said composition is stored at 2°C-8°C.
10. Use of an effective amount of monothioglycerol in the presence of divalent metal ions or a derivative, analogue or equivalent thereof to stabilise a protein or derivative thereof in a liquid composition.
11. Use of an effective amount of thioglycollic acid or a derivative, analogue or equivalent thereof to stabilise a protein or derivative thereof in a liquid composition.
12. Use according to claim 11 wherein said thioglycollic acid is a thioglycollic acid salt.
13. Use according to claim 11 or 12 wherein said thioglycollic acid is incoφorated into said composition in the presence of divalent metal ions.
14. Use according to claim 10 or 13 wherein said divalent metal ion is Mg++, Mn *, Fe^ OX orNi^.
15. Use according to any one of claims 10-14 wherein said protein is an immunogen.
16. Use according to claim 15 wherein said immunogen is influenza haemmaglutinin, pertussis toxin, diphtheria toxin, influenza neuraminidase, gpl20 of HIN, virus-like particles derived from HPN or HepB, or derivatives, homologues, analogues, mutants, equivalents or mimetics thereof.
17. Use according to any one of claims 10-16 wherein said composition is stored at 1°C-10°C.
18. Use according to claim 17 wherein said composition is stored at 2°C-8°C.
19. A protein or derivative thereof in a liquid composition, which protein is stable, said composition comprising a stabilising effective amount of monothioglycerol in the presence of divalent metal ions or derivatives, analogues or equivalent thereof.
20. A protein or derivative thereof in a liquid composition, which protein is stable, said composition comprising a stabilising effective amount of thioglycollic acid in the presence of divalent metal ions or derivatives, analogues or equivalent thereof.
21. A protein according to claim 20 wherein said thioglycollic acid is a thioglycollic acid salt.
22. A protein according to claim 20 or 21 wherein said thioglycollic acid is incoφorated into said composition in the presence of divalent metal ions.
23. A protein according to claim 19 or 22 wherein said divalent metal ion is Mg -1", Mn^ Fe^ αX orNX.
24. A protein according to any one of claims 19-23 wherein said protein is an immunogen.
25. A protein according to claim 24 said immunogen is influenza haemmaglutinin, pertussis toxin, diphtheria toxin, influenza neuraminidase, gpl20 of HIN, virus-like particles derived from HPN or HepB, or derivatives, homologues, analogues, mutants, equivalents or mimetics thereof.
26. A protein according to any one of claims 19-26 wherein said composition is stored at 1°C-10°C.
27. A protein according to claim 26 said composition is stored at 2°C-8°C.
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