US20130164296A1 - Excipients for Stabilising Viral Particles, Polypeptides or Biological Material - Google Patents

Excipients for Stabilising Viral Particles, Polypeptides or Biological Material Download PDF

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US20130164296A1
US20130164296A1 US13/637,913 US201113637913A US2013164296A1 US 20130164296 A1 US20130164296 A1 US 20130164296A1 US 201113637913 A US201113637913 A US 201113637913A US 2013164296 A1 US2013164296 A1 US 2013164296A1
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virus
solution
alkyl
antibody
ester
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Jeffrey Drew
David Woodward
John Bainbridge
Amanda Corteyn
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Stabilitech Ltd
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Stabilitech Ltd
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Priority claimed from GBGB1005517.6A external-priority patent/GB201005517D0/en
Priority claimed from GBGB1005521.8A external-priority patent/GB201005521D0/en
Priority claimed from GBGB1017648.5A external-priority patent/GB201017648D0/en
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Assigned to STABILITECH LTD. reassignment STABILITECH LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CORTEYN, AMANDA, BAINBRIDGE, JOHN, DREW, JEFFREY, WOODWARD, DAVID
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    • 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
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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Definitions

  • the present invention is concerned with storage-stable formulations of viruses and polypeptides.
  • Some biological molecules are sufficiently stable that they can be isolated, purified and then stored in solution at room temperature. However, this is not possible for many materials and techniques involving storage at low temperature, addition of stabilisers, freeze-drying, vacuum formation and air-drying have been tried to ensure shelf preservation. Despite the availability of these techniques, some biological materials still show unsatisfactory levels of stability during storage and some techniques lead to added cost and inconvenience. For example, refrigerated transportation and storage is expensive. Further, refrigerated transport is often not available for the transport of medicines such as vaccines in countries in the developing world.
  • Freeze drying of biopharmaceuticals involves freezing solutions or suspensions of thermosensitive biomaterials, followed by primary and secondary drying. The technique is based on sublimation of water at subzero temperature under vacuum without the solution melting. Freeze-drying represents a key step for manufacturing solid protein and vaccine pharmaceuticals. The rate of water vapour diffusion from the frozen biomaterial is very low and therefore the process is time-consuming. Additionally, both the freezing and drying stages introduce stresses that are capable of unfolding or denaturing proteins.
  • Proteins are molecules with defined primary, secondary, tertiary and in some instances quaternary structures. The structure plays an important role in giving a protein its specific biological function. Unfortunately, the structural complexity of biological pharmaceuticals such as proteins makes them susceptible to various processes that result in structural and functional instability. Conformational integrity and functional groups must be protected from degradation
  • Instability can be a consequence of a variety of covalent and non-covalent reactions or modifications in solution.
  • Degradation is generally classified into two main categories: firstly physical degradation or non-covalent pathway degradation and secondly the covalent degradation pathway.
  • Proteins can degrade via physical processes such as interfacial adsorption and aggregation which can significantly reduce a protein drug's potency and stability.
  • a second consequence is that unfolding mediated by adsorption at an interface can often be an initiating step for irreversible aggregation of the protein in solution. Exposure of the protein's core at a hydrophobic surface can result in adsorption as a consequence of agitation, temperature or pH induced stresses; all of which can lead to aggregation.
  • Proteins may be subject to chemical modification such as oxidation, isomerisation, hydrolysis, disulfide scrambling, beta elimination, deamidation, and adduct formation.
  • the principal hydrolytic mechanisms of degradation include peptide bond hydrolysis, deamidation of asparagine and glutamine and the isomerisation of aspartic acid.
  • a common feature of the hydrolytic degradation pathway is that one significant formulation variable, with respect of the rates of the reactions is the pH.
  • composition of components in a biopharmaceutical formulation can affect the extent of protein degradation.
  • the method of formulation of a biopharmaceutical also can impact the ease and frequency of administration.
  • proteins are freeze dried (lyophilised) to provide stable formulations of the proteins.
  • a bulking agent is often present in the formulations.
  • the freeze dried formulations are distributed and stored in dried form, typically as a powder, in a sealed vial, ampoule or syringe.
  • WO 97/04801 describes stable lyophilised formulations of anti-IgE antibodies which have to be reconstituted immediately prior to use.
  • WO-A-2006/0850082 reports a desiccated or preserved product comprising a sugar, a charged material such as a histone protein and a dessication- or thermo-sensitive biological component.
  • the sugar forms an amorphous solid matrix.
  • the histone may have immunological consequences if the preserved biological component is administered to a human or animal.
  • WO 2008/114021 describes a method for preserving viral particles.
  • the method comprises drying an aqueous solution of one or more sugars, a polyethyleneimine and the viral particles to form an amorphous solid matrix comprising the viral particles.
  • the aqueous solution contains the polyethyleneimine at a concentration of 15 ⁇ M or less based on the number-average molar mass (M n ) of the polyethyleneimine and the sugar concentration or, if more than one sugar is present, total sugar concentration is greater than 0.1M.
  • WO 2010/035001 describes a method for preserving a polypeptide in which an aqueous solution of the polypeptide is dried, for example freeze dried, in the presence of one or more sugars and a polyethyleneimine (PEI).
  • the resulting dried composition is typically provided as a stable dry powder in a sealed vial, ampoule or syringe.
  • a solution is reconstituted from the powder in order to administer the polypeptide to a patient e.g. by injection.
  • aqueous solutions of viral particles or polypeptides can be provided by use of certain excipients and optionally one, two or more sugars. These formulations retain long term stability. They can be prepared without a drying or freeze drying step. They circumvent the need to reconstitute a solution from a freeze dried powder prior to use. It has also been found that these excipients and optionally one, two or more sugars can preserve viral particles or polypeptides during manufacture. Further, it has been found that these excipients and optionally one, two or more sugars can preserve samples taken from a human or animal.
  • the present invention provides a sterile pharmaceutically acceptable aqueous solution, typically suitable for parenteral administration, which solution is provided in a sealed container and comprises:
  • the present invention also provides a sterile pharmaceutically acceptable aqueous solution, which solution comprises:
  • a process for the preparation of a pharmaceutically acceptable aqueous solution of viral particles or polypeptide comprises: (a) providing a solution of the viral particles or physiologically active polypeptide, an excipient of the invention and, optionally, one or more sugars; and (b) removing the excipient;
  • an excipient of the invention and, optionally, one or more sugars to preserve viral particles or a polypeptide during manufacture of a pharmaceutically acceptable aqueous solution of said virus or polypeptide;
  • a process for preserving a sample taken from a human or animal comprising providing an aqueous solution of (i) said sample, (ii) an excipient of the invention, and (iii) optionally one or more sugars.
  • a process for obtaining and preserving a sample from a human or animal comprising (a) obtaining the sample from the human or animal, and (b) preparing an aqueous solution of said sample, an excipient of the invention, and optionally one or more sugars;
  • an aqueous solution which comprises (i) a sample taken from a human or animal, (ii) an excipient of the invention, and (iii) optionally one or more sugars;
  • an excipient of the invention and, optionally, one or more sugars to preserve a sample taken from a human or animal;
  • an excipient of the invention and, optionally, one or more sugars to preserve a solution comprising viral particles, prior to freeze-drying of said solution.
  • FIG. 1 shows the results of an experiment evaluating whether excipients enhance adenovirus stability in a liquid at room temperature for 4 hours prior to freeze drying.
  • Suc sucrose.
  • Raf raffinose.
  • Statistical analysis was carried out using a one-way ANOVA followed by a Bonfferoni post test. P value summary, ** designates P ⁇ 0.01. Error bars show standard error of the mean (n 3).
  • FIG. 2 shows the results of an experiment investigating liquid stability of adenovirus following heat challenge at 37° C. for one week.
  • FIG. 3 shows the results of an experiment investigating the effect of excipients in stabilising influenza hemagglutinin (HA) in liquid form.
  • HA hemagglutinin
  • FIG. 4 a shows the effect of test formulations on the recovered activity of formulations of adenovirus held at 4° C. for one week.
  • Grey and white bars represent test formulations.
  • Figures on the x-axis refer to concentration in M.
  • Black bars represent control samples.
  • FIG. 4 b shows the effect of test formulations on the recovered activity of formulations of adenovirus containing sugars (1M sucrose, 100 mM raffinose) held at 4° C. for one week.
  • Grey and white bars represent test formulations.
  • FIG. 4 c shows the effect of test formulations on the recovered activity of formulations of adenovirus containing no sugars held at 37° C. for one week.
  • Grey and white bars represent test formulations.
  • FIG. 4 d shows the effect of test formulations on the recovered activity of formulations of adenovirus containing sugars (1M sucrose, 100 mM raffinose), held at 37° C. for one week.
  • Grey and white bars represent test formulations.
  • FIG. 5 shows TNF- ⁇ neutralisation by anti T (anti-TNF- ⁇ antibody). Samples were assayed following 10 days' incubation at room temperature.
  • FIG. 6 shows the results of an investigation into the effect of excipients on the stabilisation of IgG in liquid formulation.
  • Bars represent percentage purity of IgG in novel excipient formulations relative to PBS only formulations of each comparable thermal treatment (thus PBS only purity adjusted to 100%) from Example 6.
  • Represented treatments comprise sugars only (white), DMG only (grey) and DMG and sugars (black), all collected on day 1 of the experiment.
  • FIG. 7 shows the average monomer peak on day 1, 5 and 31 for the formulations prepared in Example 6 stored at 4° C.
  • FIG. 8 shows the average monomer peak on day 1, 5 and 31 for the formulations prepared in Example 6 stored at 37° C.
  • FIG. 9 shows the results obtained in Example 7 in which the ability of eleven formulations to stabilise adenovirus against thermal challenge was assessed following 7 days at 37° C.
  • FIG. 10 shows the results obtained in Example 8 in which the ability of eleven formulations to stabilise MVA against thermal challenge at 37° C. for 7 days was assessed.
  • FIG. 11 shows a 3D representation of the design space in Example 9. Spheres represent formulations within the design space that were tested. This design is a Doehlert RSM design.
  • FIG. 12 summarises statistics for the model used to represent the branched PEI (P-Bra) data in Example 9
  • FIG. 13 shows the terms retained in the model in Example 9 after fine tuning.
  • FIG. 14 plots the surface response of the predicted recovered viral titre in formulations of P-Bra and sucrose at three different levels of raffinose using the model in Example 9.
  • FIG. 15 shows a screen capture of settings and outputs from the optimum predictions based on the model of the data in Example 9 generated using Monte-Carlo simulations.
  • FIG. 16 shows a representation of the design space in Example 10. Numbered circles represent formulations within the design space that are tested. This design is a CCF RSM design. Numbers in circles refer to sample I.D.s in Table 7.
  • FIG. 17 summarises the statistics of the model used to represent the data in Example 10.
  • FIG. 18 shows terms retained in the model after fine-tuning in Example 10. Error bars not crossing the origin indicate a significant factor at the 95% C.I.
  • FIG. 19 is a surface response plot of the predicted recovered viral titre in formulations of TMG and mannitol in Example 10.
  • FIG. 20 is a screen capture of settings and outputs from the optimum predictions based on the model of the data in Example 10, generated using Monte-Carlo simulations. Highlighted formulation (line 4) is the optimum identified.
  • FIG. 21 shows a 3D representation of the design space in Example 11. Spheres represent formulations within the design space that are tested. This design is a Doehlert RSM design.
  • FIG. 22 summarises the statistics of the model used to represent the data in Example 11.
  • FIG. 23 shows terms retained in the model after fine tuning in Example 11. Error bars not crossing the origin indicate a significant factor at the 90% C.I.
  • FIG. 24 shows a contour plot of the model describing the recovery of adenovirus formulated in MSM, sucrose, and raffinose and thermo-challenged at +37° C. for 1 week in Example 11.
  • Raffinose is not shown as a variable as it had no effect on titre, and was thus eliminated from the model.
  • the response shown is recovered viral titre as a percentage of the positive control (starting titre).
  • FIG. 25 shows a screen capture of settings and outputs from the optimum predictions based on the model of the data in Example 11, generated using Monte-Carlo simulations.
  • FIG. 26 shows a 3D representation of the design space in Example 12. Spheres represent formulations within the design space that are tested. This design is a Doehlert RSM design.
  • FIG. 27 summarises statistics of the model used to represent the data in Example 12.
  • FIG. 28 shows terms retained in the model in Example 12 after fine tuning. Error bars not crossing the origin indicate a significant factor at the 95% C.I.
  • FIG. 30 shows a screen capture of settings and outputs from the optimum predictions based on the model of the data in Example 12, generated using Monte-Carlo simulations.
  • the predicted optima highlighted is sucrose concentration of 0.5M, DMG concentration 0.4M and raffinose concentration of 272.5 mM.
  • FIG. 31 shows an optimum region plot using the model derived from Example 12
  • FIG. 31A is a contour plot where a cross marks the predicted optimum. Colouring indicates level of variable.
  • FIG. 31B is an graph highlighting region of model where predicted recovered viral activity is greater than or equal to that input.
  • FIG. 32 shows the recovered viral activity from various formulations after 6 months storage at +4° C. in Example 13.
  • FIG. 33 shows recovered viral activity for the ‘best’ formulation in Example 13 comprising 1M sucrose, 100 mM raffinose and 0.7M DMG at each time point and thermal challenge.
  • FIG. 34 shows reduction in recovered viral activity over time at +37° C. thermal challenge in various formulations in Example 13.
  • FIG. 35 shows a representation of the design space in Example 14. Numbered circles represent formulations within the design space that were tested. This design is a CCF RSM design.
  • FIG. 36 summarises the statistics of the model used to represent the data in Example 14.
  • FIG. 37 shows terms retained in the model in Example 14 after fine tuning. Error bars not crossing the origin indicate a significant factor at the 95% C.I.
  • FIG. 38 shows a contour plot of the predicted recovered viral titre in formulations of DMG and mannitol in Example 14.
  • FIG. 40 shows the residual F(ab′)2 activity (at 2 ⁇ g.ml) remaining in Example 15 at 24 hours, 5 days and 7 days following thermal challenge at +56° C.
  • FIG. 41 shows the residual F(ab′)2 activity (at 0.5 ug.ml) remaining at various time points in Example 16 after 14 days thermal challenge; 1 day at +40° C. and 13 days at +56° C.
  • FIG. 42 shows a y-normalised superposition of the standards trace acquired in Example 17 before sample injection and the first injection of the untouched positive control sample.
  • the FAb elutes just before the third weight marker, giving it an estimated hydrodynamic weight of more than 44 kDa. This value is consistent with a monovalent FAb.
  • FIGS. 43 to 45 show a superpositions of seven HPLC traces in Example 17 corresponding to the first injection of each condition.
  • the large peaks at 13 minutes (labelled b) in FIG. 44 are due to excipient whilst the smaller peak at ten minutes (labelled a) is due to the FAb.
  • a black rectangle highlights the area that is expanded and shown in FIG. 45 .
  • FIG. 46 shows a series of integrated HPLC traces in Example 17 as follows: FIG. 46A : Condition 1: Untouched FAb (positive control); FIG. 46B : Condition 2: FAb after 130 h at 56° C. in PBS (negative control); FIG. 46C : Condition 3: FAb after 130 h at 56° C. in SR mix; FIG. 46D : Condition 4: FAb after 130 h at 56° C. in SR mix & low (0.1M) DMG; FIG. 46E : Condition 5: FAb after 130 h at 56° C. in SR mix and high (1.0M) DMG; FIG. 46F : Condition 6: FAb after 130 h at 56° C. in SR mix & low (0.1M) TMG; and FIG. 46G : Condition 7: FAb after 130 h at 56° C. in SR mix & high (1.0M) TMG.
  • FIG. 46A Condition 1: Untouched FAb (positive control); FIG. 46B : Condition 2:
  • FIG. 47 summarises purity (light grey) and monomer retention (dark grey) parameters for each of the seven conditions in Example 17. All samples were at 167 ⁇ g/mL. Untouched was the non-heat-challenged positive control. All other samples were heat-challenged at 56° C. (hence 56C) for 130 h. Square brackets indicate sample composition.
  • Stable aqueous solutions of viral particles or polypeptides are provided according to the invention.
  • the solutions are sterile pharmaceutically acceptable liquids that can be administered to a patient without having to be reconstituted from e.g. a dried powder immediately prior to use.
  • the present invention relates to the preservation of viral particles by a N-alkylated glycine derivative or a salt or ester thereof, and a sulfone compound of formula (IIC).
  • the N-alkylated glycine derivative and the sulfone compound can interact synergistically to stabilise the viral particles in a liquid setting.
  • the solutions may take the form of small-volume parenterals of 100 ml or less or large-volume parenterals of 100 ml or more.
  • the solutions are sterile pharmaceutically acceptable liquids that can be administered to a patient without having to be reconstituted from e.g. a dried powder immediately prior to use.
  • the solutions are capable of exhibiting long term storage stability. They can therefore be stored for 6 to 18 months or longer in a refrigerator, i.e. at temperatures of from 2 to 8° C. In some instances, the solutions can be stored at room temperature for such periods of time.
  • the solutions thus possess sufficient stability to enable them to be manufactured in a factory, distributed e.g. to pharmaceutical wholesalers and pharmacies, and stored prior to use without an unacceptable level of degradation occurring.
  • the solutions are provided as clear liquids.
  • the solutions are usually colourless. They may additionally comprise a physiologically acceptable buffer and/or a tonicity adjustment agent and/or a preservative.
  • the solutions may thus be isotonic.
  • the solutions are sealed in an appropriate container in a vial, ampoule, syringe, cartridge, flexible bag or glass bottle. They are thus manufactured in ready-to-use form in a factory. They have not therefore been reconstituted from a solid composition such as a lyophilisate immediately prior to use.
  • the excipients of the invention can additionally preserve virus particles or polypeptides during manufacture of solutions of said virus particles or polypeptides. Further, the excipients of the invention can preserve solutions of samples taken from a human or animal.
  • the viral particles used in the present invention may be whole viruses such as live viruses, killed viruses, live attenuated viruses, inactivated viruses such as chemically inactivated viruses or virulent or non-virulent viruses.
  • a live virus is capable of infecting and replicating within the host cell.
  • a killed virus is inactivated and is unable to replicate within the host cell.
  • the particles may be virus-like particles (VLPs) or nucleocapsids.
  • the virus may be infectious to prokaryotic or eukaryotic cells.
  • the virus may be a human or animal virus.
  • the viral particle may be, or may be derived from, a dsDNA virus, a ssDNA virus, a dsRNA virus, a (+)ssRNA virus, a ( ⁇ )ssRNA virus, a ssRNA-RT virus or a dsDNA-RT virus.
  • the viral particle can be, or can be derived from, a virus of the following families:
  • the viral particle can be or can be derived from an Adenoviridae, Orthomyxoviridae, Paramyxoviridae, Parvoviridae, Picornaviridae or Poxviridae virus.
  • the viral particle can be or can be derived from an adenovirus, vaccinia virus, influenza virus, or measles virus.
  • VLPs include viral proteins derived from the structural proteins of a virus, but lack viral nucleic acid. When overexpressed, these viral structural proteins spontaneously self-assemble into particles. VLPs are replication incompetent. In some embodiments, the VLPs are viral proteins embedded within a lipid bilayer. Examples of VLPs includes phage-derived VLPs, human papillomavirus (HPV) L1 major capsid protein VLPs, Norwalk virus capsid protein VLPs and VLPs assembled from influenza virus structural proteins such as M1 protein, HA hemagglutinin protein and N1 neuraminidase protein.
  • HPV human papillomavirus
  • Viral particles can be prepared using standard techniques well known to those skilled in the art.
  • a virus may be prepared by infecting cultured host cells with the virus strain that is to be used, allowing infection to progress such that the virus replicates in the cultured cells and can be released by standard methods known in the art for harvesting and purifying viruses.
  • polypeptide such as a physiologically active polypeptide is suitable for use in the invention.
  • the polypeptide may be a small peptide of less than 15 amino acids such as 6 to 14 amino acids (e.g. oxytocin, cyclosporin), a larger peptide of between 15 and 50 amino acids (e.g. calcitonin, growth hormone releasing hormone 1-29 (GHRH)), a small protein of between 50 and 250 amino acids in length (e.g. insulin, human growth hormone), a larger protein of greater than 250 amino acids in length or a multisubunit protein comprising a complex of two or more polypeptide chains.
  • the polypeptide may be a peptide hormone, growth factor or cytokine.
  • polypeptide may be an antigen-binding polypeptide, receptor inhibitor, ligand mimic or receptor blocking agent.
  • the polypeptide is in substantially pure form. It may thus be an isolated polypeptide.
  • the polypeptide may be isolated following recombinant production.
  • the polypeptide may be a hormone selected from a growth hormone (GH), prolactin (PRL), a human placental lactogen (hPL), a gonadotrophin (e.g. lutenising hormone, follicle stimulating hormone), a thyroid stimulating hormone (TSH), a member of the pro-opiomelanocortin (POMC) family, vasopressin and oxytocin, a natriuretic hormone, parathyroid hormone (PTH), calcitonin, insulin, a glucagon, somatostatin and a gastrointestinal hormone.
  • GH growth hormone
  • PRL prolactin
  • hPL human placental lactogen
  • a gonadotrophin e.g. lutenising hormone, follicle stimulating hormone
  • TSH thyroid stimulating hormone
  • POMC pro-opiomelanocortin
  • vasopressin and oxytocin a natriuretic hormone
  • PTH parathyroid hormone
  • the polypeptide may be a Tachykinin peptide (e.g. Substance P, Kassinin, Neurokinin A, Eledoisin, Neurokinin B), a vasoactive intestinal peptide (e.g. VIP (Vasoactive Intestinal Peptide; PHM27), PACAP (Pituitary Adenylate Cyclase Activating Peptide), Peptide PHI 27 (Peptide Histidine Isoleucine 27), GHRH 1-24 (Growth Hormone Releasing Hormone 1-24), Glucagon, Secretin), a pancreatic polypeptide-related peptide (e.g.
  • NPY neuropeptide Y
  • PYY Peptide YY
  • APP vian Pancreatic Polypeptide
  • PPY Pancreatic Polypeptide
  • an opioid peptide e.g. Proopiomelanocortin (POMC) peptides, Enkephalin pentapeptides, Prodynorphin peptide, a calcitonin peptide (e.g. Calcitonin, Amylin, AGG01) or another peptide (e.g. B-type Natriuretic Peptide (BNP)).
  • POMC Proopiomelanocortin
  • BNP B-type Natriuretic Peptide
  • the polypeptide may be a growth factor selected from a member of the epidermal growth factor (EGF) family, platelet-derived growth factor family (PDGF), fibroblast growth factor family (FGF), Transforming Growth Factors- ⁇ family (TGFs- ⁇ , Transforming Growth Factor- ⁇ (TGF- ⁇ ), Erythropoietin (Epo), Insulin-Like Growth Factor-I (IGF-I), Insulin-Like Growth Factor-II (IGF-II).
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor family
  • FGF fibroblast growth factor family
  • TGFs- ⁇ Transforming Growth Factors- ⁇ family
  • TGF- ⁇ Transforming Growth Factor- ⁇
  • Epo Erythropoietin
  • IGF-I Insulin-Like Growth Factor-I
  • IGF-II Insulin-Like Growth Factor-II
  • the growth factor is a Transforming growth factor beta (TGF- ⁇ ), a Nerve growth factor (NGF), a Neurotrophin, a Platelet-derived growth factor (PDGF), Erythropoietin (EPO), Thrombopoietin (TPO), Myostatin (GDF-8), a Growth differentiation factor-9 (GDF9), Acidic fibroblast growth factor (aFGF or FGF-1), Basic fibroblast growth factor (bFGF or FGF-2), Epidermal growth factor (EGF) or a Hepatocyte growth factor (HGF).
  • TGF- ⁇ Transforming growth factor beta
  • NGF Nerve growth factor
  • NGF Nerve growth factor
  • Neurotrophin a Platelet-derived growth factor
  • EPO Erythropoietin
  • TPO Thrombopoietin
  • GDF-8 Myostatin
  • GDF-9 Growth differentiation factor-9
  • Acidic fibroblast growth factor aFGF or FGF-1
  • Basic fibroblast growth factor
  • the polypeptide may be a cytokine selected from Interleukin-1 (IL-1), Interleukin-2 (IL-2), Interleukin-6 (IL-6) Interleukin-8 (IL-8), Tumor Necrosis Factor- ⁇ (TNF- ⁇ ), Tumor Necrosis Factor- ⁇ (TNF- ⁇ ), Interferon- ⁇ (INF- ⁇ ) and a Colony Stimulating Factor (CSF).
  • IL-1 Interleukin-1
  • IL-2 Interleukin-2
  • IL-6 Interleukin-6
  • IL-8 Interleukin-8
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • INF- ⁇ Interferon- ⁇
  • CSF Colony Stimulating Factor
  • the cytokine is a Granulocyte-colony stimulating factor (G-CSF) or a Granulocyte-macrophage colony stimulating factor (GM-CSF).
  • the polypeptide may be a blood-clotting factor such as Factor VIII, Factor V, von Willebrand factor or coagulation factor III.
  • An antibody for use in the invention may either be a whole antibody or an antigen- or ligand-binding fragment thereof.
  • the antibody is an immunoglobulin (Ig) monomer, dimer, tetramer, pentamer, or other oligomer.
  • Each antibody monomer may comprise four polypeptide chains (for example, a conventional antibody consisting of two identical heavy chains and two identical light chains).
  • each antibody monomer consists of two polypeptide chains (for example, a heavy chain antibody consisting of two identical heavy chains).
  • the antibody can be any class or isotype of antibody (for example IgG, IgM, IgA, IgD or IgE) or any subclass of antibody (for example IgG subclasses IgG1, IgG2, IgG3, IgG4 or IgA subclasses IgA1 or IgA2).
  • the antibody is an IgG such as an IgG1, IgG2 or IgG4 antibody.
  • the antibody is an IgG1 or IgG2 antibody.
  • the antibody or antigen-binding fragment is of mammalian origin.
  • the antibody may thus be a primate, human, rodent (e.g. mouse or rat), rabbit, ovine, porcine, equine or camelidae antibody or antibody fragment.
  • the antibody or antibody fragment may be of shark or chicken origin.
  • the antibody may be a monoclonal or polyclonal antibody.
  • Monoclonal antibodies are obtained from a population of substantially homogenous antibodies that are directed against a single determinant on the antigen.
  • a population of polyclonal antibodies comprises a mixture of antibodies directed against different epitopes.
  • the antigen-binding fragment can be any fragment of an antibody which retains antigen- or ligand-binding ability, for example a Fab, F(Ab′) 2 , Fv, disulphide-linked Fv, single chain Fv (scFv), disulphide-linked scFv, diabody, linear antibody, domain antibody or multispecific antibody.
  • Such fragments comprise one or more antigen or ligand binding sites.
  • the antigen- or ligand-binding fragment comprises four framework regions (e.g. FR1, FR2, FR3 and FR4) and three complementarity-determining regions (e.g. CDR1, CDR2 and CDR3).
  • the antibody or binding fragment may be a monospecific, bispecific or multispecific antibody.
  • a multispecific antibody has binding specificity for at least one, at least two, at least three, at least four or more different epitopes, antigens or ligands
  • a bispecific antibody is able to bind to two different epitopes, antigens or ligands.
  • a bispecific antibody may comprise two pairs of V H and V L , each V H /V L pair binding to a single antigen or epitope.
  • Methods for preparing bispecific antibodies are known in the art, for example involving coexpression of two immunoglobulin heavy chain-light chain pairs, fusion of antibody variable domains with the desired binding specificities to immunoglobulin constant domain sequences, or chemical linkage of antibody fragments.
  • the bispecific antibody “diabody” comprises a heavy chain variable domain connected to a light chain variable domain in the same polypeptide chain (V H -V L ).
  • Diabodies can be generated using a linker (e.g. a peptide linker) that is too short to allow pairing between the two domains on the same chain, so that the domains are forced to pair with the complementary domains of another chain and create a dimeric molecule with two antigen- or ligand-binding sites.
  • a suitable scFv antibody fragment may comprise V H and V L domains of an antibody wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains, which enables the scFv to form the desired structure for antigen binding.
  • a domain antibody for use in the methods of the invention may essentially consist of a light chain variable domain (e.g. a V L ) or of a heavy chain variable domain (e.g. a V H ).
  • the heavy chain variable domain may be derived from a conventional four-chain antibody or from a heavy chain antibody (e.g. a camelidae V HH ).
  • the whole antibody or fragment thereof may be associated with other moieties, such as linkers, which may be used to join together two or more fragments or antibodies.
  • linkers may be chemical linkers or can be present in the form of a fusion protein with a fragment or whole antibody. The linkers may thus be used to join together whole antibodies or fragments, which have the same or different binding specificities.
  • the antibody or antigen- or ligand-binding fragment is linked to a further moiety such as a toxin, therapeutic drug (e.g. chemotherapeutic drug), radioisotope, liposome or prodrug-activating enzyme.
  • a further moiety such as a toxin, therapeutic drug (e.g. chemotherapeutic drug), radioisotope, liposome or prodrug-activating enzyme.
  • therapeutic drug e.g. chemotherapeutic drug
  • radioisotope e.g. chemotherapeutic drug
  • liposome e.g., liposome or prodrug-activating enzyme
  • the antibody or antigen- or ligand-binding fragment may be linked to one or more small molecule toxins (e.g. calicheamicin, maytansine, trichothene and CC1065) or an enzymatically active toxin or fragment thereof (e.g. diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa , ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, curcin, crotin, gelonin, mitogellin, restrictocin, phenomycin, enomycin or tricothecenes).
  • small toxins e.g. calicheamicin, maytansine, trichothene and CC1065
  • an enzymatically active toxin or fragment thereof e.g. diphtheria toxin, exotoxin A chain from Pse
  • Radioisotopes suitable for linking to the antibody or antigen-binding fragments include, but are not limited to Tc 99 , At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 and P 32 .
  • the antibody or antigen- or ligand-binding fragment may be linked for example, to a prodrug-activating enzyme that converts or is capable of converting a prodrug to an active anti-cancer drug.
  • a prodrug-activating enzyme that converts or is capable of converting a prodrug to an active anti-cancer drug.
  • alkaline phosphatase can be used to convert phosphate-containing prodrugs into free drugs
  • arylsufatase may be used to convert sulfate-containing prodrugs into free drugs
  • cytosine deaminase may be used to convert non-toxic 5-fluorocytosine into the anti-cancer drug 5-fluorouracil
  • proteases such as serratia protease, thermolysin, subtilisin, carboxypeptidases and cathepsins are useful for converting peptide-containing prodrugs into free drugs.
  • the enzyme may be a nitroreductase which has been identified as useful in the metabolism of a number of prodrugs in anti-cancer gene therapy.
  • antibodies or antigen- or ligand-binding fragments with enzymatic activity can be used to convert prodrugs into free active drugs.
  • a suitable chemotherapeutic agent may include, but is not limited to an alkylating agent such as thiotepa and cyclosphosphamide; an alkyl sulfonate such as busulfan, improsulfan and piposulfan; an aziridine such as benzodopa, carboquone, meturedopa and uredopa; a nitrogen mustard such as chlorambucil, chlornaphazine, ifosfamide, melphalan; a nitrosurea such as carmustin and fotemustine; an anti-metabolite such as methotrexate and 5-fluorouracil (5-FU); a folic acid analogue such as denopterin and pteropterin; a purine analogue such as fludarabine and thiamiprine; a pyrimidine analogue such as ancitabine, azacitidine, carmofur and doxifluridine; a taxoid
  • the antibody or antibody fragment may be PEGylated.
  • one or more polyethylene glycol molecules may be covalently attached to the antibody molecule or antibody fragment molecule.
  • From one to three polyethylene glycol molecules may be covalently attached to each antibody molecule or antibody fragment molecule.
  • PEGylation is predominantly used to reduce the immunogenicity of an antibody or antibody fragment and/or increase the circulating half-life of the antibody or antibody fragment.
  • the antibody or antigen- or ligand-binding fragment is a chimeric antibody or fragment thereof comprising sequence from different natural antibodies.
  • the chimeric antibody or antibody fragment may comprise a portion of the heavy and/or light chain identical or homologous to corresponding sequences in antibodies of a particular species or antibody class, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies of another species or antibody class.
  • the chimeric antibody or antibody fragment comprises a chimera of mouse and human antibody components.
  • Humanized forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a suitable humanized antibody or antibody fragment may comprise for example, immunoglobulin in which residues from a hypervariable region (e.g. derived from a CDR) of the recipient antibody or antigen- or ligand-binding fragment are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity and/or capacity.
  • donor antibody such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity and/or capacity.
  • some framework region residues of the human immunoglobulin may be replaced by corresponding non-human residues.
  • human antibodies or antigen-binding fragments can be generated.
  • transgenic animals e.g. mice
  • transgenic animals e.g. mice
  • homozygous deletion of the antibody heavy-chain joining region (J H ) gene in chimeric and germ-line mutant mice can result in complete inhibition of endogenous antibody production.
  • Human germ-line immunoglobulin genes can be transferred to such germ-line mutant mice to result in the production of human antibodies upon antigen challenge.
  • a human antibody or antigen-binding fragment can also be generated in vitro using the phage display technique.
  • an antibody or antigen- or ligand-binding fragment capable of binding any target antigen is suitable for use in the methods of the present invention.
  • the antibody or antibody fragment may be capable of binding to an antigen or ligand associated with an autoimmune disorder (e.g. Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and myasthenia gravis), an antigen or ligand associated with a cancer or an inflammatory state, an antigen associated with osteoporosis, an antigen associated with Alzheimer's disease, or a bacterial or viral antigen.
  • an autoimmune disorder e.g. Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and myasthenia gravis
  • an antigen or ligand associated with a cancer or an inflammatory state e.g. Type I diabetes, multiple sclerosis, rheum
  • the target to which an antibody or antigen- or ligand-binding fragment may bind can be a CD antigen, growth factor, growth factor receptor, cell surface receptor such as an apoptosis receptor, a protein kinase or an oncoprotein.
  • the antibody or antigen-binding fragment may thus be capable of binding to tumour necrosis factor ⁇ (TNF- ⁇ ), interleukin-2 (IL-2), interleukin-6 (IL-6), glycoprotein IIb/IIIa, CD33, CD52, CD20, CD11a, CD3, RSV F protein, HER2/neu (erbB2) receptor, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), anti-TRAILR2 (anti-tumour necrosis factor-related apoptosis-inducing ligand receptor 2), complement system protein C5, ⁇ 4 integrin or IgE.
  • TNF- ⁇ tumour necrosis factor ⁇
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • glycoprotein IIb/IIIa CD33
  • CD52 interleukin-2
  • IL-6 interleukin-6
  • glycoprotein IIb/IIIa CD33
  • CD52 interleukin-2
  • IL-6 interleukin-6
  • the antibody or antigen-binding fragment may be an antibody or antibody fragment capable of binding to epithelial cell adhesion molecule (EpCAM), mucin-1 (MUC1/Can-Ag), EGFR, CD20, carcinoembryonic antigen (CEA), HER2, CD22, CD33, Lewis Y and prostate-specific membrane antigen (PMSA).
  • EpCAM epithelial cell adhesion molecule
  • MUC1/Can-Ag mucin-1
  • EGFR epithelial cell adhesion molecule
  • CD20 carcinoembryonic antigen
  • HER2 HER2, CD22, CD33
  • Lewis Y prostate-specific membrane antigen
  • Suitable monoclonal antibodies include, but are not limited to: infliximab (chimeric antibody, anti-TNF ⁇ ), adalimumab (human antibody, anti-TNF ⁇ ), basiliximab (chimeric antibody, anti-IL-2), abciximab (chimeric antibody, anti-GpIIb/IIIa), daclizumab (humanized antibody, anti-IL-2), gemtuzumab (humanized antibody, anti-CD33), alemtuzumab (humanized antibody, anti-CD52), edrecolomab (murine Ig2a, anti-EpCAM), rituximab (chimeric antibody, anti-CD20), palivizumab (humanized antibody, RSV target), trastuzumab (humanized antibody, anti-HER2/neu(erbB2) receptor), bevacizumab (humanized antibody, anti-VEGF), cetuximab (chimeric antibody, anti-EGFR), eculizumab (humanized antibody,
  • Suitable monoclonal antibodies may be obtained for example, by the hybridoma method (e.g. as first described by Kohler et al Nature 256:495 (1975)), by recombinant DNA methods and/or following isolation from phage or other antibody libraries.
  • the hybridoma technique involves immunisation of a host animal (e.g. mouse, rat or monkey) with a desired immunogen to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunogen.
  • lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
  • An antibody or antibody fragment can also be isolated from antibody phage libraries as an alternative to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.
  • phage display may be used to identify antigen- or ligand-binding fragments for use in the methods of the invention.
  • phage display for the high-throughput screening of antigen-antibody or ligand-antibody binding interactions
  • antibody fragments displayed on phage coat proteins can be isolated from a phage display library.
  • a phage that displays an antibody capable of binding that antigen or ligand will remain on the support while others can be removed by washing.
  • Phage eluted in the final selection can be used to infect a suitable bacterial host from which phagemids can be collected and the relevant DNA sequence excised and sequenced to identify the relevant antigen- or ligand-binding fragment.
  • Polyclonal antiserum containing the desired antibodies is isolated from animals using techniques well known in the art. Animals such as sheep, rabbits or goats may be used for example, for the generation of antibodies against an antigen of interest by the injection of this antigen (immunogen) into the animal, sometimes after multiple injections. After collection of antiserum, antibodies may be purified using immunosorbent purification or other techniques known in the art.
  • the antibody or antigen- or ligand-binding fragment used in the method of the invention may be produced recombinantly from naturally occurring nucleotide sequences or synthetic sequences.
  • sequences may for example be isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), nucleotide sequences isolated from a library (e.g. an expression library), nucleotide sequences prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known, e.g. mismatch PCR), nucleotide sequence prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis.
  • a suitable naturally occurring template e.g. DNA or RNA isolated from a cell
  • nucleotide sequences isolated from a library e.g. an expression library
  • nucleotide sequences prepared by introducing mutations into a naturally occurring nucleotide sequence using any suitable technique known, e
  • affinity maturation for example, starting from synthetic, random or naturally occurring immunoglobulin sequences
  • CDR grafting for example, CDR grafting
  • veneering for example, veneering
  • combining fragments derived from different immunoglobulin sequences and other techniques for engineering immunoglobulin sequences may also be used.
  • nucleotide sequences of interest may be used in vitro or in vivo in the production of an antibody or antigen-binding fragment for use in the invention, in accordance with techniques well known to those skilled in the art.
  • the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning or for expression.
  • the vector components generally including, but is not limited to one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • Suitable host cells for cloning or expressing the DNA in the vectors are prokaryote, yeast, or higher eukaryote cells such as E. coli and mammalian cells such as CHO cells.
  • Suitable host cells for the expression of glycosylated antibody are derived from multi-cellular organisms. Host cells are transformed with the expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the antibody can be produced intracellularly or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris of either host cells or lysed cells, is removed, for example by centrifugation or ultra filtration. Where the antibody is secreted into the medium, supernatants from expression systems are generally first concentrated using a commercially available protein concentration filter.
  • the antibody composition prepared from the cells can be purified using, for example, hydyoxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography.
  • the purified antibodies may then be isolated and optionally made into antigen- or ligand-binding fragments and/or derivatised.
  • Any protein enzyme is suitable for use in the invention.
  • Such an enzyme comprises an active site and is capable of binding a substrate.
  • the enzyme may be a monomer consisting of one polypeptide chain.
  • the enzyme may be a dimer, tetramer or oligomer consisting of multiple polypeptide chains.
  • the dimer, tetramer or oligomer may be a homo- or hetero-dimer, tetramer or oligomer respectively.
  • the enzyme may need to form an aggregate (e.g. a dimer, tetramer or oligomer) before full biological activity or enzyme function is conferred.
  • the enzyme may be an allosteric enzyme, an apoenzyme or a holoenzyme.
  • the enzyme may be conjugated to another moiety (e.g. a ligand, antibody, carbohydrate, effector molecule, or protein fusion partner) and/or bound to one or more cofactors (e.g. coenzyme or prosthetic group).
  • another moiety e.g. a ligand, antibody, carbohydrate, effector molecule, or protein fusion partner
  • cofactors e.g. coenzyme or prosthetic group
  • the moiety to which the enzyme is conjugated may include lectin, avidin, a metabolite, a hormone, a nucleotide sequence, a steroid, a glycoprotein, a glycolipid, or any derivative of these components.
  • Cofactors include inorganic compounds (e.g. metal irons such as iron, manganese, cobalt, copper, zinc, selenium, molybdenum) or organic compounds (e.g. flavin or heme).
  • Suitable coenzymes include riboflavin, thiamine, folic acid which may carry hydride iron (H ⁇ ) carried by NAD or NADP + , the acetyl group carried by coenzyme A, formyl, methenyl or methyl groups carried by folic acid and the methyl group carried by S-adenosyl methionine.
  • the enzyme may be PEGylated especially if the enzyme is a therapeutic enzyme that is administered to a patient.
  • one or more polyethylene glycol molecules may be covalently attached to the enzyme molecule. From one to three polyethylene glycol molecules may be covalently attached to each enzyme molecule.
  • PEGylation is predominantly used to reduce the immunogenicity of an enzyme and/or increase the circulating half-life of the enzyme.
  • a suitable enzyme includes any enzyme classified under the International Union of Biochemistry and Molecular Biology Enzyme classification system of EC numbers including an oxidoreductase (EC 1), a transferase (EC 2), a hydrolase (EC 3), a lyase (EC 4), an isomerase (EC 5) or a ligase (EC 6).
  • EC 1 oxidoreductase
  • EC 2 transferase
  • hydrolase EC 3
  • a lyase EC 4
  • an isomerase EC 5
  • ligase ligase
  • An enzyme that is specific for any type of substrate is suitable for use in the present invention.
  • a suitable enzyme includes a ⁇ -galactosidase, ⁇ -galactosidase, luciferase, serine proteinase, endopeptidase (e.g. cysteine endopeptidase), caspase, chymase, chymotrypsin, endopeptidase, granzyme, papain, pancreatic elastase, oryzin, plasmin, renin, subtilisin, thrombin, trypsin, tryptase, urokinase, amylase (e.g.
  • ⁇ -amylase xylanase, lipase, transglutaminase, cell-wall-degrading enzyme, glucanase (e.g. ⁇ -glucanase), glucoamylase, coagulating enzyme, milk protein hydrolysate, cell-wall degrading enzyme, blood coagulating enzyme, hementin, lysozyme, fibre-degrading enzyme, phytase, cellulase, hemicellulase, polymerase, protease, mannanase or glucoamylase.
  • glucanase e.g. ⁇ -glucanase
  • glucoamylase glucoamylase
  • coagulating enzyme milk protein hydrolysate
  • cell-wall degrading enzyme e.g. hementin, lysozyme
  • fibre-degrading enzyme phytase, cellulase, hemicellulase
  • An enzyme preserved according to the invention may thus be a therapeutic enzyme that is used to treat a disease or other medical condition, an enzyme used in industry for the production of bulk products such as glucose or fructose, in food processing and food analysis, in laundry and automatic dishwashing detergents, in the textile, pulp, paper and animal feed industries, as a catalyst in synthesis or fine chemicals, in diagnostic applications such as in clinical diagnosis, in biosensors or in genetic engineering.
  • Therapeutic enzymes to which the present invention can be applied include:
  • Enzymes for use in food applications such as baking include amylases, xylanases, oxidoreductases, lipases, proteases and transglutaminase.
  • Enzymes for use in fruit juice production and fruit processing include cell-wall-degrading enzymes.
  • Enzymes for use in brewing include bacterial ⁇ -amylase, ⁇ -glucanase and glucoamylase in mashing, fungal ⁇ -amylase in fermentation and cysteine endopeptidase in post fermentation.
  • Enzymes for use in dairy applications include coagulating enzymes, lipase, lysozyme, milk protein hydrolysates, transglutaninase, and ⁇ -galactosidase.
  • Enzymes for use in detergent compositions include proteases, amylases, lipases, cellulases and mannanase.
  • Enzymes for use in animal feed include fibre-degrading enzymes, phytases, proteases and amylases.
  • Enzymes for use in pulp and paper processing include cellulases and hemicellulases.
  • the enzyme may alternatively be an enzyme used in research and development applications.
  • luciferases may be used for real-time imaging of gene expression in cell cultures, individual cells and whole organisms. Further, luciferases may be used as reporter proteins in molecular studies, for example to test the activity of transcription from specific promoters in cells transfected with luciferase. Enzymes may also be used in drug design for example in the testing of enzyme inhibitors in the laboratory. Further, enzymes may be used in biosensors (for example, a blood glucose biosensor using glucose oxidase).
  • the luciferase enzyme may be a firefly, beetle or railroad worm luciferase, or a derivative thereof.
  • the luciferase may be derived from a North American firefly ( Phorinus pyralis ), Luciola cruciata (japanese firefly), Luciola lateralis (japanese firefly), Luciola mingelica (russian firefly), Beneckea hanegi (marine bacterial luciferase), Pyrophorus plagiophthalamus (click beetle), Pyrocelia miyako (firefly) Ragophthalamus ohbai (railroad worm), Pyrearinus termitilluminans (click beetle), Phrixothrix hirtus (railroad worm), Phrixothrix vivianii, Hotaria parvula and Photuris pensilvanica , and mutated variants thereof.
  • ⁇ -galactosidase or ⁇ -galactosidase is derived from bacteria (such as Escherichia coli ), a mammal (such as human, mouse, rat) or other eukaryote.
  • the enzyme may be a naturally-occurring enzyme or a synthetic enzyme. Such enzymes may be derived from a host animal, plant or a microorganism.
  • Microbial strains used in the production of enzymes may be native strains or mutant strains that are derived from native strains by serial culture and selection, or mutagenesis and selection using recombinant DNA techniques.
  • the microorganism may be a fungus e.g. Thermomyces acermonium, Aspergillus, Penicillium, Mucor, Neurospora and Trichoderma .
  • Yeasts such as Saccharomyces cereviseae or Pishia pastoris may also be used in the production of enzymes for use in the methods of the present invention.
  • a synthetic enzyme may be derived using protein-engineering techniques well known in the art such as rational design, directed evolution and DNA shuffling.
  • Host organisms may be transformed with a nucleotide sequence encoding a desired enzyme and cultured under conditions conducive to the production of the enzyme and which facilitate recovery of the enzyme from the cells and/or culture medium.
  • a vaccine immunogen suitable for use in the invention includes any immunogenic component of a vaccine.
  • the vaccine immunogen comprises an antigen that can elicit an immune response in an individual when used as a vaccine against a particular disease or medical condition.
  • the vaccine immunogen may be provided by itself prior to formulation of a vaccine preparation or it may be provided as part of a vaccine preparation.
  • the vaccine immunogen may be a subunit vaccine, a conjugate useful as a vaccine or a toxoid.
  • the vaccine immunogen may be a protein, bacterial-specific protein, mucoprotein, glycoprotein, peptide, lipoprotein, polysaccharide, peptidoglycan, nucleoprotein or fusion protein.
  • the vaccine immunogen may be derived from a microorganism (such as a bacterium, virus, fungi), a protozoan, a tumour, a malignant cell, a plant, an animal, a human, or an allergen.
  • the vaccine immunogen is preferably not a viral particle.
  • the vaccine immunogen is preferably not a whole virus or virion, virus-like particle (VLP) or virus nucleocapsid. The preservation of such viral particles is described in WO 2008/114021.
  • the vaccine immunogen may be synthetic, for example as derived using recombinant DNA techniques.
  • the immunogen may be a disease-related antigen such as a pathogen-related antigen, tumour-related antigen, allergy-related antigen, neural defect-related antigen, cardiovascular disease antigen, rheumatoid arthritis-related antigen.
  • the pathogen from which the vaccine immunogen is derived may include human papilloma viruses (HPV), HIV, HSV2/HSV1, influenza virus (types A, B and C), para influenza virus, polio virus, RSV virus, rhinoviruses, rotaviruses, hepaptitis A virus, norwalk virus, enteroviruses, astroviruses, measles virus, mumps virus, varicella-zoster virus, cytomegalovirus, epstein-barr virus, adenoviruses, rubella virus, human T-cell lymphoma type I virus (HTLV-I), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, poxvirus, vaccinia virus, Salmonella, Neisseria, Borrelia, Clamydia, Bordetella such as Bordetella pertussis, Plasmodium, Coxoplasma, Pneumococcus,
  • the vaccine may further be used to provide a suitable immune response against numerous veterinary diseases, such as foot and mouth disease (including serotypes O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1), coronavirus, bluetongue, feline leukaemia virus, avian influenza, hendra and nipah virus, pestivirus, canine parvovirus and, bovine viral diarrhoea virus.
  • foot and mouth disease including serotypes O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1
  • coronavirus including serotypes O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1
  • coronavirus including serotypes O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1
  • coronavirus including serotypes O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1
  • coronavirus including serotypes O, A, C, SAT-1
  • Tumor-associated antigens include for example, melanoma-associated antigens, mammary cancer-associated antigens, colorectal cancer-associated antigens or prostate cancer-associated antigens
  • An allergen-related antigen includes any allergen antigen suitable for use in a vaccine to suppress an allergic reaction in an individual to which the vaccine is administered (e.g. antigens derived from pollen, dust mites, insects, food allergens, dust, poisons, parasites).
  • a suitable subunit vaccine immunogen includes any immunogenic subunit of a protein, lipoprotein or glycoprotein derived from a microorganism (for example a virus or bacteria).
  • the subunit vaccine immunogen may be derived from a disease-related antigen such as a tumour related protein.
  • the subunit vaccine immunogen may be a naturally occurring molecule or a synthetic protein subunit.
  • the vaccine immunogen may be a full-length viral or bacterial protein, glycoprotein or lipoprotein or a fragment of the full-length viral or bacterial protein, glycoprotein or lipoprotein.
  • a viral protein suitable as a subunit vaccine immunogen may be derived from a structural or non-structural viral protein.
  • a suitable viral subunit immunogen is capable of stimulating a subject's immune system even in the absence of other parts of the virus.
  • a suitable viral subunit vaccine immunogen includes a capsid protein, surface glycoprotein, envelope protein, hexon protein, fiber protein, coat protein or immunogenic fragment or derivative of such proteins or glycoproteins.
  • the viral subunit vaccine immunogen may consist of a surface protein of the Influenza A, B or C virus.
  • the vaccine immunogen may be a hemagglutinin (HA), neuraminidase (NA), nucleoprotein, M1, M2, NS1, NS2(NEP), PA, PB1, PB1-F2 and or PB2 protein, or an immunogenic derivative or fragment of any of these proteins.
  • the immunogen may be HA1, HA2, HA3, HA4, HA5, HA6, HA7, HA8, HA9, HA10, HA11, HA12, HA13, HA14, HA15 and/or HA16, any immunogenic fragment or derivative thereof and any combination of the HA proteins, fragments or derivatives.
  • the neuraminidase may be neuraminidase 1 (N1) or neuraminidase 2 (N2).
  • the viral subunit vaccine immunogen may be a hepatitis B virus viral envelope protein or a fragment or derivative thereof.
  • the subunit vaccine immunogen may be the hepatitis B surface antigen (HbsAg) or an immunogenic fragment or derivative thereof.
  • the bacterial subunit vaccine immunogen is a bacterial cell wall protein (e.g. flagellin, outer membrane protein, outer surface protein), a polysaccharide antigen (e.g. from Neisseria meningitis, Streptococcus pneumonia ), toxin or an immunogenic fragment or derivative of such proteins, polysaccharides or toxins.
  • a bacterial cell wall protein e.g. flagellin, outer membrane protein, outer surface protein
  • a polysaccharide antigen e.g. from Neisseria meningitis, Streptococcus pneumonia
  • toxin e.g. from Neisseria meningitis, Streptococcus pneumonia
  • Derivatives of naturally occurring proteins include proteins with the addition, substitution and/or deletion of one or more amino acids.
  • amino acid modifications can be generated using techniques known in the art, such as site-directed mutagenesis.
  • the subunit vaccine immunogen may be a fusion protein comprising a fusion protein partner linked with for example, a bacterial or viral protein or an immunogenic fragment or derivative thereof.
  • a suitable fusion protein partner may prevent the assembly of viral fusion proteins into multimeric forms after expression of the fusion protein.
  • the fusion protein partner may prevent the formation of virus-like structures that might spontaneously form if the viral protein was recombinantly expressed in the absence of the fusion protein partner.
  • a suitable fusion partner may also facilitate purification of the fusion protein, or enhance the recombinant expression of the fusion protein product.
  • the fusion protein may be maltose binding protein, poly-histidine segment capable of binding metal ions, antigens to which antibodies bind, S-Tag, glutathione-S-transferase, thioredoxin, beta-galactosidase, epitope tags, green fluorescent protein, streptavidin or dihydrofolate reductase.
  • a subunit vaccine immunogen may be prepared using techniques known in the art for the preparation of for example, isolated peptides, proteins, lipoproteins, or glycoproteins.
  • a gene encoding a recombinant protein of interest can be identified and isolated from a pathogen and expressed in E. coli or some other suitable host for mass production of proteins.
  • the protein of interest is then isolated and purified from the host cell (for example by purification using affinity chromatography).
  • the subunit may be purified from the viral particle after isolating the viral particle, or by recombinant DNA cloning and expression of the viral subunit protein in a suitable host cell.
  • a suitable host cell for preparing viral particles must be capable of being infected with the virus and of producing the desired viral antigens.
  • host cells may include microorganisms, cultured animal cells, transgenic plants or insect larvae.
  • Some proteins of interest may be secreted as a soluble protein from the host cell.
  • viral envelope or surface proteins such proteins may need to be solubilized with a detergent to extract them from the viral envelope, followed by phase separation in order to remove the detergent.
  • a subunit vaccine immunogen may be combined in the same preparation and preserved together with one, two three or more other subunit vaccine immunogens.
  • a toxoid is a toxin, for example derived from a pathogen, animal or plant, that is immunogenic but has been inactivated (for example by genetic mutation, chemical treatment or by conjugation to another moiety) to eliminate toxicity to the target subject.
  • the toxin may be for example, a protein, lipoprotein, polysaccharide, lipopolysaccharide or glycoprotein.
  • the toxoid may thus be an endotoxin or an exotoxin that has been toxoided.
  • the toxoid may be a toxoid derived from a bacterial toxin such as tetanus toxin, diphtheria toxin, pertussis toxin, botulinum toxin, C. difficile toxin, Cholera toxin, shiga toxin, anthrax toxin, bacterial cytolysins or pneumolysin and fragments or derivatives thereof.
  • the toxoid may therefore be tetanus toxoid, diphtheria toxoid or pertussis toxoid.
  • Other toxins from which a toxoid can be derived include poisons isolated from animals or plants, for example from Crotalis atrox .
  • the toxoid is derived from botulinum toxin or anthrax toxin.
  • the botulinum toxin may be derived from Clostridium botulinum of serotype A, B, C, D, E, F or G.
  • the vaccine immunogen derived from a botulinum toxin may be combined in the same preparation and preserved together with one or more other vaccine immunogens derived from a botulinum toxin (eg a combination of immunogens derived from botulinum serotypes A, B, C, D, E, F or G, such as for example A, B and E).
  • the anthrax toxin may be derived from a strain of Bacillus anthracis .
  • the toxoid may consist of one of more components of the anthrax toxin, or derivatives of such components, such as protective antigen (PA), the edema factor (EF) and the lethal factor (LF).
  • PA protective antigen
  • EF edema factor
  • LF lethal factor
  • PA protective antigen
  • the toxoid may be conjugated to another moiety, for example as a fusion protein, for use as a toxoid vaccine.
  • a suitable moiety in a conjugate toxoid includes a substance that aids purification of the toxoid (e.g histidine tag) or reduces toxicity to a target subject.
  • the toxoid may act as an adjuvant by increasing the immunogenicity of an antigen to which it is attached.
  • the B polysaccharide of Haemophilus influenzae may be combined with diptheria toxoid.
  • a vaccine immunogen may be combined in the same preparation and preserved together with one, two three or more vaccine immunogens.
  • a diphtheria toxoid may be preserved with tetanus toxoid and pertussis vaccine (DPT).
  • Diptheria toxoid may be preserved with just tetanus toxoid (DT), or diphtheria toxoid may be preserved with diphtheria toxoid, tetanus toxoid and acellular Pertussis (DTaP).
  • Toxin genes may be cloned and expressed in a suitable host cell. The toxin product is then purified and may be converted to toxoid chemically, for example using formalin or glutaraldehyde. Alternatively, a toxin gene may be engineered so that it encodes a toxin having reduced or no toxicity e.g. by addition, deletion and/or substitution of one or more amino acids. The modified toxin can then be expressed in a suitable host cell and isolated. The toxicity of toxin genes may also be inactivated by conjugation of toxin genes or fragments thereof to a further moiety (e.g. polysaccharide or polypeptide).
  • a further moiety e.g. polysaccharide or polypeptide
  • a conjugate vaccine immunogen may be a conjugate of an antigen (for example a polysaccharide or other hapten) to a carrier moiety (for example a peptide, polypeptide, lipoprotein, glycoprotein, mucoprotein or any immunostimulatory derivative or fragment thereof) that stimulates the immunogenicity of the antigen to which it is attached.
  • a carrier moiety for example a peptide, polypeptide, lipoprotein, glycoprotein, mucoprotein or any immunostimulatory derivative or fragment thereof
  • the conjugate vaccine immunogen may be a recombinant protein, recombinant lipoprotein or recombinant glycoprotein conjugated to an immunogen of interest (for example a polysaccharide).
  • the conjugate vaccine immunogen may be used in a vaccine against Streptococcus pneumonia, Haemophilus influenza , meningococcus (strains A, B, C, X, Y and W135) or pneumococcal strains.
  • the vaccine may be for example, the heptavalenti Pneumococcal CRM197 Conjugate Vaccine (PCV7), an MCV-4 or Haemophilus influenzae type b (Hib) vaccine.
  • a conjugate vaccine immunogen may be combined in the same preparation and preserved together with one, two three or more other conjugate vaccine immunogens.
  • conjugation may occur via a linker (e.g. B-propionamido, nitrophenyl-ethylamine, haloalkyl halides, glycosidic linkages).
  • a linker e.g. B-propionamido, nitrophenyl-ethylamine, haloalkyl halides, glycosidic linkages.
  • PEI is an aliphatic polyamine characterised by the repeating chemical units denoted as —(CH 2 —CH 2 —NH)—.
  • Reference to PEI herein includes a polyethyleneimine homopolymer or copolymer.
  • the polyethyleneimine copolymer may be a random or block copolymer.
  • PEI may consist of a copolymer of polyethyleneimine and another polymer such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the polyethyleneimine may be linear or branched.
  • Reference to PEI also includes derivatised forms of a polyethyleneimine.
  • a polyethyleneimine contains nitrogen atoms at various positions. Nitrogen atoms are present in terminal amino groups, e.g. R—NH 2 , and in internal groups such as groups interrupting an alkyl or alkylene group within the polymer structure, e.g. R—N(H)—R′, and at the intersection of a polymer branch, e.g. R—N(—R′)—R′′ wherein R, R′ and R′′ may be alkylene groups for example.
  • Alkyl or aryl groups may be linked to the nitrogen centres in addition to or instead of hydrogen atoms. Such alkyl and aryl groups may be substituted or unsubstituted.
  • An alkyl group would be typically a C 1 -C 4 alkyl group, e.g. methyl, ethyl, propyl, isopropyl, butyl, sec.butyl or tert.butyl.
  • the aryl group is typically phenyl.
  • the PEI may be a polyethyleneimine that has been covalently linked to a variety of other polymers such as polyethylene glycol.
  • Other modified versions of PEI have been generated and some are available commercially: branched PEI 25 kDa, jetPEI®, LMW-PEI 5.4 kDa, Pseudodendrimeric PEI, PEI-SS-PEI, PEI-SS-PEG, PEI-g-PEG, PEG-co-PEI, PEG-g-PEI, PEI-co-L lactamide-co-succinamide, PEI-co-N-(2-hydroxyethyl-ethylene imine), PEI-co-N-(2-hydroxypropyl)methacrylamide, PEI-g-PCL-block-PEG, PEI-SS-PHMPA, PEI-g-dextran 10 000 and PEI-g-transferrin-PEG, Pluronic85®/Pluronic123®-g-PEI.
  • PEI is available in a broad range of number-average molar masses (M n ) for example between 300 Da and 800 kDa.
  • the number-average molar mass is between 300 and 2000 Da, between 500 and 1500 Da, between 1000 and 1500 Da, between 10 and 100 kDa, between 20 and 100 kDa, between 30 and 100 kDa, between 40 and 100 kDa, between 50 and 100 kDa, between 60 and 100 kDa, between 50 and 70 kDa or between 55 and 65 kDa.
  • a relatively high M n PEI of approximately 60 kDa or a relatively low M n of 1200 Da is suitable.
  • the weight-average molar mass (M w ) of PEI is between 500 Da and 1000 kDa.
  • the M w of PEI is between 500 Da and 2000 Da, between 1000 Da and 1500 Da, or between 1 and 1000 kDa, between 100 and 1000 kDa, between 250 and 1000 kDa, between 500 and 1000 kDa, between 600 and 1000 kDa, between 750 and 1000 kDa, between 600 and 800 kDa, between 700 and 800 kDa.
  • a relatively high M w of approximately 750 kDa or a relatively low M w of approximately 1300 Da is suitable.
  • the weight-average molar mass (M w ) and number-average molar mass (M n ) of PEI can be determined by methods well known to those skilled in the art.
  • M w may be determined by light scattering, small angle neutron scattering (SANS), X-ray scattering or sedimentation velocity.
  • M n may be determined for example by gel permeation chromatography, viscometry (Mark-Houwink equation) and colligative methods such as vapour pressure osometry or end-group titration.
  • PEI branched, relatively high molecular weight form of PEI used herein with an M n of approximately 60 kDa and a M w of approximately 750 kDa is available commercially (Sigma P3143).
  • This PEI can be represented by the following formula:
  • PEI polystyrene-maleic anhydride copolymer
  • Aldrich 482595 a relatively low molecular weight form of PEI used herein is also available commercially (e.g. Aldrich 482595) which has a M w of 1300 Da and M n of 1200 Da.
  • the compounds of formula (I) and (II) may be present as a physiologically acceptable salt or ester thereof.
  • the salt is typically a salt with a physiologically acceptable acid and thus includes those formed with an inorganic acid such as hydrochloric or sulphuric acid or an organic acid such as citric, tartaric, malic, maleic, mandelic, fumaric or methanesulphonic acid.
  • the hydrochloride salt is preferred.
  • the ester is typically a C 1-6 alkyl ester, preferably a C 1-4 alkyl ester.
  • the ester may therefore be the methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl ester.
  • the ethyl ester is preferred.
  • a C 1-6 alkyl group is preferably a C 1-4 alkyl group.
  • Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.
  • the definitions of compounds of formula (I) and formula (II) also include compounds in which the carboxylate anion is protonated to give —COOH and the ammonium or sulfonium cation is associated with a pharmaceutically acceptable anion.
  • the compounds defined above may be used in any tautomeric or enantiomeric form.
  • R 1 represents hydrogen or C 1-6 alkyl and R 4 represents hydrogen.
  • R 2 represents hydrogen or C 1-6 alkyl.
  • R 1 represents hydrogen or C 1-6 alkyl
  • R 4 represents hydrogen and R 2 represents hydrogen or C 1 -1-6 alkyl. More preferably R 1 represents hydrogen or C 1-6 alkyl, R 4 represents hydrogen and R 2 represents C 1-6 alkyl.
  • the compound of formula (I) is an N—C 1-6 alkyl-, N,N-di(C 1-6 alkyl)- or N,N,N-tri(C 1-6 alkyl)-glycine or physiologically acceptable salt or ester thereof, more preferably an N,N-di(C 1-6 alkyl)- or N,N,N-tri(C 1-6 alkyl)-glycine or physiologically acceptable salt or ester thereof.
  • the alkyl group is typically a C 1-4 alkyl group.
  • Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.
  • Preferred compounds of formula (I) are N-methylglycine, N,N-dimethylglycine or N,N,N-trimethylglycine or physiologically acceptable salts or esters thereof.
  • N-Methyl-glycine is also called sarcosine.
  • N,N-Dimethylglycine is also termed dimethylglycine (DMG) or 2-(dimethylamino)-acetic acid.
  • N,N,N-trimethylglycine is termed trimethylglycine (TMG).
  • the compound of formula (I) is typically a glycine derivative of formula (IA) or a physiologically acceptable salt or ester thereof:
  • R 5 and R 6 independently represent C 1-6 alkyl, for example C 1-4 alkyl such as methyl or ethyl; and R 7 represents C 1-6 alkyl, for example C 1-4 alkyl such as methyl or ethyl, or —(CH 2 ) 2-5 NHC(O)(CH 2 ) 5-15 CH 3 .
  • Preferred compounds of formula (IA) are trimethylglycine (TMG) and cocamidopropyl betaine (CAPB) or physiologically acceptable salts or esters thereof. Trimethyglycine is preferred.
  • the compound of formula (I) is typically a proline derivative of formula (IB) or a physiologically acceptable salt or ester thereof:
  • R 8 and R 9 independently represent C 1-6 alkyl, for example C 1-4 alkyl such as methyl or ethyl.
  • the compound of formula (IB) is an S-proline derivative.
  • R 8 and R 9 both represent methyl; this compound is known as proline betaine.
  • S-proline betaine or physiologically acceptable salt or ester thereof is particularly preferred:
  • the compound of formula (I) is N,N-dimethylglycine or N,N,N-trimethylglycine or physiologically acceptable salt or ester thereof. Most preferably, the compound of formula (I) is N,N-dimethylglycine or physiologically acceptable salt or ester thereof.
  • R c is attached to the same carbon atom of the R c alkyl moiety.
  • R c is a C 2-4 or C 2-3 alkyl moiety.
  • the compound of formula (II) is typically a sulfone compound of formula (IIA) or a physiologically acceptable salt or ester thereof:
  • R c and R d independently represent C 1-6 alkyl, for example C 1-4 alkyl.
  • Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.
  • a preferred sulfone compound is methylsulfonylmethane (MSM), which is also known as dimethylsulfone (DMSO 2 ).
  • the compound of formula (II) is typically a compound of formula (IIB) or a physiologically acceptable salt or ester thereof:
  • R e and R f independently represent C 1-6 alkyl, for example C 1-4 alkyl such as methyl or ethyl
  • R g represents C 1-6 alkyl, for example C 1-4 alkyl such as methyl or ethyl, substituted with a carboxylate anion and with an amine (—NH 2 ) moiety.
  • the carboxylate and amine substituents are attached to the same carbon atom.
  • a preferred compound of formula (IIB) is S-methyl-L-methionine (SMM) or a physiologically acceptable salt or ester thereof.
  • the excipient may be an N-alkyl-, N,N-dialkyl- or N,N,N-trialkyl-glycine or a physiologically acceptable salt or ester thereof.
  • the alkyl group is typically a C 1-6 alkyl group such as a C 1-4 alkyl group.
  • Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.
  • Preferred glycine derivatives for use in the invention are N-methylglycine, N,N-dimethylglycine, N,N,N-trimethylglycine and physiologically acceptable salts and esters ester thereof.
  • N-Methyl-glycine is also called sarcosine.
  • N,N-Dimethylglycine is also termed dimethylglycine (DMG) or 2-(dimethylamino)-acetic acid.
  • DMG dimethylglycine
  • TMG trimethylglycine
  • TMG trimethylglycine
  • the salt is typically a salt with a physiologically acceptable acid and thus includes those formed with an inorganic acid such as hydrochloric or sulphuric acid or an organic acid such as citric, tartaric, malic, maleic, mandelic, fumaric or methanesulphonic acid.
  • the hydrochloride salt is preferred.
  • the ester is typically a C 1-6 alkyl ester, preferably a C 1-4 alkyl ester.
  • the ester may therefore be the methyl, ethyl, propyl, isopropyl, butyl, isobutyl or tert-butyl ester.
  • the ethyl ester is preferred.
  • the N-alkylated glycine derivative is an N—C 1-6 alkyl-, N,N-di(C 1-6 alkyl)- or N,N,N-tri(C 1-6 alkyl)-glycine.
  • the alkyl group is typically a C 1-4 alkyl group.
  • Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.
  • Preferred glycine derivatives for use in the invention are N-methylglycine, N,N-dimethylglycine, N,N,N-trimethylglycine.
  • N-Methyl-glycine is also called sarcosine.
  • N,N-Dimethylglycine is also termed dimethylglycine (DMG) or 2-(dimethylamino)-acetic acid.
  • N,N,N-trimethylglycine is termed trimethylglycine (TMG).
  • a physiologically acceptable salt or ester of a N-alkylated glycine derivative may be employed.
  • the sulfone compound is a compound of formula (IIC):
  • R a and R b independently represent C 1-6 alkyl, for example C 1-4 alkyl.
  • Preferred alkyl groups are selected from methyl, ethyl, propyl, isopropyl, butyl, isobutyl and tert-butyl. Methyl and ethyl are particularly preferred.
  • a preferred sulfone compound is methylsulfonylmethane (MSM), which is also known as dimethylsulfone (DMSO 2 ).
  • Sugars suitable for use in the present invention include reducing sugars such as glucose, fructose, glyceraldehydes, lactose, arabinose and maltose; and preferably non-reducing sugars such as sucrose and raffinose.
  • the sugar may be a monosaccharide, disaccharide, trisaccharide, or other oligosaccharides.
  • sugar alcohols include sugar alcohols.
  • Monosaccharides such as galactose and mannose; dissaccharides such as sucrose, lactose and maltose; trisaccharides such as raffinose; and tetrasaccharides such as stachyose are envisaged.
  • Trehalose, umbelliferose, verbascose, isomaltose, cellobiose, maltulose, turanose, melezitose and melibiose are also suitable for use in the present invention.
  • a suitable sugar alcohol is mannitol.
  • sucrose and raffinose are used.
  • Sucrose is a disaccharide of glucose and fructose.
  • Raffinose is a trisaccharide composed of galactose, fructose and glucose.
  • the aqueous solvent is generally water. Pure water such as water for injections is generally used. Alternatively, physiological saline may be used.
  • the aqueous solution may be buffered. Any suitable physiologically acceptable buffer may be used such as a phosphate buffer. Typically, the pH will be adjusted to from 4 to 9, preferably between 5 and 8 and especially from about pH 6.5 to 7.5. The exact pH will depend, for example, on the stability in aqueous solution of the viral particles.
  • the solutions of the present invention should be protected from microbial contamination and growth.
  • a preservative may therefore be present, for example in an amount of from 0.001 to 1% by weight.
  • pharmaceutically acceptable anti-microbial agents that can be used in the formulation include:
  • a tonicity adjustment agent is sometimes desirable to achieve isotonicity with body fluids resulting in reduced levels of irritancy on administration to a patient.
  • suitable tonicity adjustment agents are sodium chloride, dextrose and calcium chloride.
  • the isotonicity adjustment agent will desirably be added in a sufficient quantity to achieve this function.
  • the tonicity adjustment agent is present in an amount of between 0.1 and 10% by weight.
  • An adjuvant is generally present when a solution of the invention is used as a vaccine.
  • the adjuvant is used in order to increase potency of the vaccine and/or modulate humoral and cellular immune responses.
  • Suitable adjuvants include, but are not limited to, mineral salts (e.g., aluminium hydroxide (“alum”), aluminium phosphate, calcium phosphate), particulate adjuvants (e.g., virosomes, ISCOMS (structured complex of saponins and lipids)), microbial derivatives (e.g., MPL(monophosphoryl lipid A), CpG motifs, modified toxins including TLR adjuvants such as flagellin), plant derivatives (e.g., saponins (QS-21)) and endogenous immunostimulatory adjuvants (e.g., cytokines and any other substances that act as immunostimulating agents to enhance the effectiveness of the vaccine).
  • mineral salts e.g., aluminium hydroxide (“alum”), aluminium phosphate, calcium phosphate
  • particulate adjuvants e.g., virosomes, ISCOMS (structured complex of saponins and lipids
  • Solutions of the invention can be prepared by admixing the viral particles or polypeptide and other ingredients in any convenient order in the selected aqueous solvent.
  • the viral particles or polypeptide are provided in the required amount, for example in a unit dosage amount.
  • a pharmaceutically effective amount of the viral particles or polypeptide can thus be provided in the solution.
  • a preparation of the viral particles or polypeptide is admixed with an aqueous solution of the excipient(s) and optionally one or more sugars.
  • the components of the solution may be admixed under sterile conditions. Alternatively, the components of the solution may be first admixed and the resulting solution sterilised.
  • the excipient(s) and/or optional sugars may be added during manufacture of viral particles or polypeptides, so that viral particles or polypeptides are stabilised during manufacture as well as in the final product. In some cases, however, it may be desirable to remove the excipient(s) and/or optional sugars in a purification step prior to formulation of the final product.
  • the solution with which the viral particles or polypeptide are admixed may be buffered or the solution may be buffered after admixture with the viral particles. It may be a HEPES, phosphate-buffered, Tris-buffered or pure water solution.
  • the pH may be adjusted as desired. Typically, a solution will have a pH of from 4 to 9, preferably from 5 to 8 and especially about pH 6.5 to 7.5.
  • the excipient and, optionally, one or more sugars are present at concentrations which provide solutions of the required storage stability.
  • the excipient may be an excipient of the invention as herein defined. Suitable concentrations can be determined and optimised by routine experimentation. The concentrations used in a particular instance will depend on a number of factors including:
  • the excipient and sugar(s) can be present in amounts that result in synergy interactions between the excipient and the sugar(s).
  • synergistic interactions may arise between (a) sulfones such as MSM and raffinose, and (b) N,N-dialkylglcycines such as DMG and sucrose. Suitable concentrations can be determined and optimised by routine experimentation.
  • the concentration of PEI is in the range of 20 ⁇ M or less or preferably 15 ⁇ M or less based on M n .
  • the PEI concentration may be 10 ⁇ M or less based on M.
  • concentrations of PEI are particularly effective at preserving biological activity.
  • the PEI is provided at a concentration based on M n of less than 5 ⁇ M, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 100 nM, less than 40 nM, less than 25 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.25 nM, less than 0.1 nM, less than 0.075 nM, less than 0.05 nM, less than 0.025 Nm or less than 0.0025 nM.
  • the PEI concentration based on M n is 0.0025 nM or more, 0.025 nM or more, or 0.1 nM or more.
  • a suitable PEI concentration range based on M n is between 0.0025 nM and 5 ⁇ M, or between 0.025 and 200 nM. Further preferred concentration ranges are between 0.1 nM and 5 ⁇ M and between 0.1 nM and 200 nM.
  • the PEI concentration based on M w is less than 5 ⁇ M, less than 1 ⁇ M, less than 500 nM, less than 400 nM, less than 300 nM, less than 200 nM, less than 0.1 ⁇ M, less than 0.01 ⁇ M, less than 5 nM, less than 4 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.25 nM, less than 0.1 nM, less than 0.05 nM, less than 0.02 nM, less than 0.002 nM or less than 0.1 nM.
  • the PEI concentration based on M w is 0.00001 nM or more, 0.001 nM or more or 0.01 nM or more.
  • a suitable PEI concentration range based on M w is between 0.00001 and 20 nM, between 0.0001 and 20 nM or between 0.0001 and 5 nM.
  • the concentration of a compound of formula (I) or physiologically acceptable salt or ester thereof or compound of formula (II) or physiologically acceptable salt or ester thereof in the aqueous solution is generally in the range of from 0.001M to 2.5M and more especially from 0.01M to 2.5M.
  • the concentration range may be from 0.1M to 2.5M.
  • compositions of formula (I) or physiologically acceptable salt or ester thereof or compound of formula (II) or physiologically acceptable salt or ester thereof that is employed will depend on several factors including the viral particles or polypeptide; the particular compound of formula (I) or physiologically acceptable salt or ester thereof or compound of formula (II) or physiologically acceptable salt or ester thereof being used; whether one, two or more sugars are present and the identity of the sugar(s).
  • concentration of compound of formula (I) or physiologically acceptable salt or ester thereof or compound of formula (II) or physiologically acceptable salt or ester thereof that is employed will depend on several factors including the viral particles or polypeptide; the particular compound of formula (I) or physiologically acceptable salt or ester thereof or compound of formula (II) or physiologically acceptable salt or ester thereof being used; whether one, two or more sugars are present and the identity of the sugar(s).
  • the concentration of an N-alkyl-, N,N-dialkyl- or N,N,N-trialkyl-glycine or a physiologically acceptable salt or ester thereof in the aqueous solution is generally in the range of 0.1 mM to 3M or from 1 mM to 2M.
  • the concentration may be from 1 mM to 1.5M or from 5 mM to 1M.
  • Preferred concentrations are from 7 mM to 1.5M or from 0.07M to 0.7M.
  • N-alkyl-, N,N-dialkyl- or N,N,N-trialkyl-glycine or a physiologically acceptable salt or ester thereof that is employed will depend on a number of factors including the viral particles or polypeptide; whether one or more sugars is used and, if so, the particular type of sugar(s) used.
  • concentration of an N-alkyl-, N,N-dialkyl- or N,N,N-trialkyl-glycine or a physiologically acceptable salt or ester thereof will depend on a number of factors including the viral particles or polypeptide; whether one or more sugars is used and, if so, the particular type of sugar(s) used.
  • the components When the solution contains an N-alkylated glycine derivative or salt or ester thereof, a sulfone compound of formula (IIC) and, optionally, one or more sugars, the components are present at concentrations which provide solutions of the required storage stability. Suitable concentrations can be determined and optimised by routine experimentation. The N-alkylated glycine derivative or salt or ester thereof and the sulfone compound of formula (IIC) can thus be present in amounts that result in synergy. The concentrations used in a particular instance will depend on a number of factors including:
  • the concentration of sugar or the total concentration of sugars is at least 0.01M, typically up to saturation.
  • the sugar concentration is at least 0.1M, at least 0.2M or at least 0.5M up to saturation e.g. saturation at room temperature or up to 3M, 2.5M or 2M.
  • the sugar concentration may therefore range from, for example, 0.1M to 3M or 0.2M to 2M.
  • the sugar concentration or the total sugar concentration if more than one sugar is present may therefore range from 0.08M to 3M, from 0.15M to 2M or from 0.2M to 1M.
  • a suitable range is from 0.05 to 1M.
  • sucrose When more than one sugar is present in the solutions of the invention, preferably one of those sugars is sucrose.
  • the sucrose may be present at a concentration of from 0.05M, 0.1M, 0.25M or 0.5M up to saturation e.g. saturation at room temperature or up to 3M, 2.5M or 2M.
  • the ratio of the molar concentration of sucrose relative to the molar concentration of the other sugar(s) is typically from 1:1 to 20:1 such as from 5:1 to 15:1.
  • the ratio of molar concentrations of sucrose is typically from 1:1 to 20:1 such as from 5:1 to 15:1 and preferably about 10:1.
  • a solution of the invention may be adjusted as desired.
  • a solution will have a pH of from 4 to 9, preferably from 5 to 8 and especially about pH 6.5 to 7.5.
  • a solution of the invention is pyrogen-free.
  • the solution is thus sterilised.
  • a solution can be sterilised by passing it through a sterilising filter.
  • the sterilised solution can then be introduced into containers, such as vials, which are then hermetically sealed.
  • containers such as vials, which are then hermetically sealed.
  • sterilisation can take place e.g. by autoclaving after the solution has been sealed in a container.
  • the solution can thus be provided in a sealed vial, ampoule, syringe, cartridge, flexible bag or glass bottle.
  • SVP small volume parenteral
  • LVP large volume parenteral
  • the containers are vials with non-reactive stoppers.
  • the stopper may be TeflonTM-coated or -faced. Silicone rubber stoppers or other non-reactive stoppers are contemplated.
  • Cartridges, syringes, vials and ampoules are usually composed of Type I or II glass, or polypropylene.
  • Flexible bags are typically constructed with multilayered plastic. Stoppers and septa in cartridges, syringes, and vials are typically composed of elastomeric materials.
  • the input (medication) and output (administration) ports for flexible bags may be plastic and/or elastomeric materials.
  • An overwrap may be used with flexible bags to retard solvent loss and to protect the flexible packaging system from rough handling.
  • the solutions of the invention can be used as desired, depending upon the viral particles or polypeptide in solution.
  • the solution can be withdrawn from a sealed container e.g. by a syringe and injected into a patient by a suitable route.
  • the solution may thus be administered by subcutaneous, intramuscular, intravenous or intraperitoneal injection.
  • a solution may alternatively be administered by infusion.
  • the solution may be diluted prior to administration.
  • the excipient of the invention may be desirable to use during manufacturing of a solution of viral particles or polypeptides, in order that the viral particles or polypeptides are preserved or stabilised during the manufacturing process. This can increase the yield of the process.
  • the excipient of the invention will be retained in the solution of viral particles or polypeptides and thereby in the final product. This can be advantageous since the excipient of the invention will continue to stabilise the viral particles or polypeptides in the final product.
  • the solution of viral particles or polypeptides is typically sealed in a container, such as vial, ampoule, syringe, cartridge, flexible bag or glass bottle.
  • the solution is sterilised, for example by passing the solution through a sterilising filter, prior to introducing the solution into the container.
  • a sterilising filter for example by passing the solution through a sterilising filter, prior to introducing the solution into the container.
  • the concentration of the excipient of the invention is preferably as set out above under “Production of Solutions of the Invention” above.
  • the concentration of the sugar(s), where present, is also preferably as set out under “Production of Solutions of the Invention” above.
  • Samples taken from a human or animal can be preserved by an excipient of the invention and optionally one or more sugars.
  • an excipient of the invention When a sample is taken from a human or animal, it often necessary to transport that sample to another location where it can be assayed or tested. Degradation of the sample generally occurs during transport, even when the sample is frozen or refrigerated. This can lead to negative or poor results in assays and tests on the sample.
  • an excipient of the invention and optionally one or more sugars in a solution of a sample taken from a human or animal generally preserves the sample.
  • the invention is typically carried out in vitro on a sample obtained from the human or animal.
  • the sample typically comprises a body fluid of the human or animal.
  • the sample is preferably a blood, plasma, serum, urine, cerebrospinal fluid or joint fluid sample.
  • the sample is most preferably a blood sample. Samples taken from humans, such as human blood samples are preferred.
  • the sample may be carried on a swab.
  • the samples taken from a human or animal may be infectious or non-infectious. It is particularly preferable to preserve infectious samples comprising viral particles, since the viral particles are preserved by the excipient and optionally one or more sugars.
  • sample taken from a human or animal the excipient of the invention and optionally one or more sugars may be added to an aqueous solution in any convenient order.
  • excipient of the invention may be added to an aqueous solution in any convenient order.
  • sugars may be added to an aqueous solution in any convenient order.
  • the concentration of the excipient of the invention is preferably as set out above under “Production of Solutions of the Invention” above.
  • the concentration of the sugar(s), where present, is also preferably as set out under “Production of Solutions of the Invention” above.
  • the aqueous solution comprising the sample taken from a human or animal, an excipient of the invention and optionally one or more sugars is typically stored in a refrigerator or in a freezer.
  • the temperature of a refrigerator is typically 2 to 8° C., preferably 4 to 6° C., or for example about 4° C.
  • the temperature of a freezer is typically ⁇ 10 to ⁇ 80° C., preferably ⁇ 10 to ⁇ 30° C., for example about ⁇ 20° C.
  • aqueous solution comprising the sample taken from a human or animal, an excipient of the invention and optionally one or more sugars is typically stored in a sealed container, such as vial, ampoule, syringe, cartridge, flexible bag or glass bottle.
  • the sample can generally be tested or assayed without prior removal of the excipient or, where present, sugars.
  • Preservation in relation to viral particles refers to resistance of the viral particle to physical or chemical degradation and/or loss of biological activity.
  • Methods of assaying for viral activity such as infectivity and/or immunogenicity are well known to those skilled in the art and include but are not limited to growth of a virus in a cell culture, detection of virus-specific antibody in blood, ability to elicit T and/or B cell responses, detection of viral antigens, detection of virus encoded DNA or RNA, or observation of virus particles using a microscope.
  • a virus gives rise to morphological changes in the host cell, which can be measured to give an indication of viral activity. Detectable changes such as these in the host cell due to viral infection are known as cytopathic effect. Cytopathic effects may consist of cell rounding, disorientation, swelling or shrinking, death and detachment from the surface. Many viruses induce apoptosis (programmed cell death) in infected cells, measurable by techniques such as the TUNEL (Terminal uridine deoxynucleotidyl transferase dUTP nick end labelling) assay and other techniques well known to those skilled in the art.
  • TUNEL Terminal uridine deoxynucleotidyl transferase dUTP nick end labelling
  • Viruses may also affect the regulation of expression of the host cell genes and these genes can be analysed to give an indication of whether viral activity is present or not. Such techniques may involve the addition of reagents to the cell culture to complete an enzymatic or chemical reaction with a viral expression product.
  • the viral genome may be modified in order to enhance detection of viral infectivity.
  • the viral genome may be genetically modified to express a marker that can be readily detected by phase contrast microscopy, fluorescence microscopy or by radioimaging.
  • the marker may be an expressed fluorescent protein such as GFP (Green Fluorescent Protein) or an expressed enzyme that may be involved in a colourimetric or radiolabelling reaction.
  • the marker could also be a gene product that interrupts or inhibits a particular function of the cells being tested.
  • An assay for plaque-forming units can be used to measure viral infectivity and to indicate viral titre.
  • suitable host cells are grown on a flat surface until they form a monolayer of cells covering a plastic bottle or dish.
  • the selection of a particular host cell will depend on the type of virus. Examples of suitable host cells include but are not limited to CHO, BHK, MDCK, 10T1 ⁇ 2, WEHI cells, COS, BSC 1, BSC 40, BMT 10, VERO, WI38, MRCS, A549, HT1080, 293, B-50, 3T3, NIH3T3, HepG2, Saos-2, Huh7, HEK293 and HeLa cells. The monolayer of host cells is then infected with the viral particles.
  • a plaque is produced when a virus particle infects a cell, replicates, and then kills that cell.
  • a plaque refers to an area of cells in the monolayer which display a cytopathic effect, e.g. appearing round and darker than other cells under the microscope, or as white spots when visualized by eye; the plaque center may lack cells due to virus-induced lysis.
  • the newly replicated virus infects surrounding cells and they too are killed. This process may be repeated several times.
  • the cells are then stained with a dye such as methylene blue, which stains only living cells. The dead cells in the plaque do not stain and appear as unstained areas on a coloured background.
  • plaque is the result of infection of one cell by one virus followed by replication and spreading of that virus.
  • viruses that do not kill cells may not produce plaques.
  • a plaque refers to an area of cells in a monolayer which display a cytopathic effect, e.g. appearing round and darker than other cells under the microscope, or as white spots when visualized by eye; the plaque center may lack cells due to virus-induced lysis.
  • An indication of viral titre is given by measuring “plaque forming units” (PFU).
  • PFU plaque forming units
  • Levels of viral infectivity can be measured in a sample of biological material preserved according to the present invention and compared to control samples such as freshly harvested virus or samples subjected to desiccation and/or thermal variation without addition of the preservation mixture of the present invention.
  • viral particles of the invention such as viral proteins, VLPs, or some inactivated viruses do not have the ability to form plaques in the plaque assay.
  • preservation can be measured by other methods such as methods for determining immunogenicity which are well known to those skilled in the art.
  • in vivo and in vitro assays for measuring antibody or cell-mediated host immune responses are known in the art and suitable for use in the present invention.
  • an antibody based immune response may be measured by comparing the amount, avidity and isotype distribution of serum antibodies in an animal model, before and after immunization using the preserved viral particle of the invention.
  • the solutions of the invention can be used as desired.
  • the solution can be withdrawn from a sealed container e.g. by a syringe and injected into a patient by a suitable route.
  • the solution may thus be administered by subcutaneous, intramuscular, intravenous or intraperitoneal injection.
  • a solution may alternatively be administered by infusion.
  • the solution may be diluted prior to administration.
  • solutions of the present invention may find use as vaccines.
  • solutions containing whole killed virus, live attenuated virus, chemically inactivated virus, VLPs or live viral vectors are suitable for use as vaccines.
  • the viral particles may be used as antigens or to encode antigens such as viral proteins for the treatment or prevention of a number of conditions including but not limited to viral infection, sequelae of viral infection including but not limited to viral-induced toxicity, cancer and allergies.
  • antigens contain one or more epitopes that will stimulate a host's immune system to generate a humoral and/or cellular antigen-specific response.
  • a vaccine of the invention may be used to prevent or treat infection by viruses such as human papilloma viruses (HPV), HIV, HSV2/HSV1, influenza virus (types A, B and C), para influenza virus, polio virus, RSV virus, rhinoviruses, rotaviruses, hepaptitis A virus, norwalk virus, enteroviruses, astroviruses, measles virus, mumps virus, varicella-zoster virus, cytomegalovirus, epstein-barr virus, adenoviruses, rubella virus, human T-cell lymphoma type I virus (HTLV-I), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, poxvirus and vaccinia virus.
  • viruses such as human papilloma viruses (HPV), HIV, HSV2/HSV1, influenza virus (types A, B and C), para influenza virus, polio virus, RSV virus, rhinovirus
  • the vaccine may further be used to provide a suitable immune response against numerous veterinary diseases, such as foot and mouth disease (including serotypes O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1), coronavirus, bluetongue, feline leukaemia virus, avian influenza, hendra and nipah virus, pestivirus, canine parvovirus and bovine viral diarrhoea virus.
  • foot and mouth disease including serotypes O, A, C, SAT-1, SAT-2, SAT-3 and Asia-1
  • coronavirus coronavirus
  • bluetongue feline leukaemia virus
  • avian influenza avian influenza
  • hendra and nipah virus pestivirus
  • canine parvovirus bovine viral diarrhoea virus.
  • bovine viral diarrhoea virus bovine viral diarrhoea virus.
  • the vaccine is a subunit, conjugate or multivalent vaccine.
  • the potency of the vaccine can be measured using techniques well known to those skilled in the art.
  • the generation of a cellular or humoral immune response can be tested in an appropriate animal model by monitoring the generation of antibodies or immune cell responses to the vaccine.
  • the ability of vaccine samples to trigger an immune response may be compared with vaccines not subjected to the same preservation technique.
  • a virus or viral vector can be used according to the present invention to transfer a heterologous gene or other nucleic acid sequence to target cells.
  • the heterologous sequence i.e. transgene
  • the heterologous sequence encodes a protein or gene product which is capable of being expressed in the target cell.
  • Suitable transgenes include desirable reporter genes, therapeutic genes and genes encoding immunogenic polypeptides (for use as vaccines).
  • Gene therapy an approach for treatment or prevention of diseases associated with defective gene expression, involves the insertion of a therapeutic gene into cells, followed by expression and production of the required proteins. This approach enables replacement of damaged genes or inhibition of expression of undesired genes.
  • the virus or viral vector may be used in gene therapy to transfer a therapeutic transgene or gene encoding immunogenic polypeptides to a patient.
  • the viral particle is a live viral vector.
  • live viral vector is meant a live viral vector that is non-pathogenic or of low pathogenicity for the target species and in which has been inserted one or more genes encoding antigens that stimulate an immune response protective against other viruses or microorganisms, a reporter gene or a therapeutic protein.
  • nucleic acid is introduced into the viral vector in such a way that it is still able to replicate thereby expressing a polypeptide encoded by the inserted nucleic acid sequence and in the case of a vaccine, eliciting an immune response in the infected host animal.
  • the live viral vector is an attenuated live viral vector i.e. is modified to be less virulent (disease-causing) than wildtype virus.
  • recombinant viruses as potential vaccines involves the incorporation of specific genes from a pathogenic organism into the genome of a nonpathogenic or attenuated virus.
  • the recombinant virus can then infect specific eukaryotic cells either in vivo or in vitro, and cause them to express the recombinant protein.
  • Live viral vector vaccines derived by the insertion of genes encoding sequences from disease organisms may be preferred over live attenuated vaccines, inactivated vaccines, subunit or DNA approaches.
  • One of the most important safety features of live viral vectors is that the recipients may be immunized against specific antigens from pathogenic organisms without exposure to the disease agent itself.
  • Safety is further regulated by the selection of a viral vector that is either attenuated for the host or unable to replicate in the host although still able to express the heterologous antigen of interest.
  • a vaccine strain that has a history of safety in the target species offers an additional safety feature.
  • Several systems have been developed in which the vector is deleted of essential genes and preparation of the vaccine is carried out in cell systems that provide the missing function.
  • a variety of vectors such as retroviral, lentiviral, herpes virus, poxvirus, adenoviral and adeno-associated viral vectors can be used for the delivery of heterologous genes to target cells.
  • the heterologous gene of interest may be inserted into the viral vector.
  • the viral vectors of the invention may comprise for example a virus vector provided with an origin of replication, optionally a promoter for the expression of the heterologous gene and optionally a regulator of the promoter.
  • adenoviruses useful in the practice of the present invention can have deletions in the E1 and/or E3 and/or E4 region, or can otherwise be maximized for receiving heterologous DNA.
  • the viral vector may comprise a constitutive promoter such as a cytomegalovirus (CMV) promoter, SV40 large T antigen promoter, mouse mammary tumour virus LTR promoter, adenovirus major late promoter (MLP), the mouse mammary tumour virus LTR promoter, the SV40 early promoter, adenovirus promoters such as the adenovirus major late promoter (Ad MLP), HSV promoters (such as the HSV IE promoters), HPV promoters such as the HPV upstream regulatory region (URR) or rous sarcoma virus promoter together with other viral nucleic acid sequences operably linked to the heterologous gene of interest.
  • Tissue-specific or inducible promoters can also be used to control expression of the heterologous gene of interest. Promoters may also be selected to be compatible with the host cell for which expression is designed.
  • the viral vector may also comprise other transcriptional modulator elements such as enhancers.
  • Enhancers are broadly defined as a cis-acting agent, which when operably linked to a promoter/gene sequence, will increase transcription of that gene sequence. Enhancers can function from positions that are much further away from a sequence of interest than other expression control elements (e.g. promoters) and may operate when positioned in either orientation relative to the sequence of interest. Enhancers have been identified from a number of viral sources, including polyoma virus, BK virus, cytomegalovirus (CMV), adenovirus, simian virus 40 (SV40), Moloney sarcoma virus, bovine papilloma virus and Rous sarcoma virus.
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • Suitable enhancers include the SV40 early gene enhancer, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, and elements derived from human or murine CMV, for example, elements included in the CMV intron A sequence.
  • LTR long terminal repeat
  • the viral vector containing a heterologous gene of interest may then be preserved according to the method of the invention before storage, subjecting to further preservation techniques such as lyophilisation, or administration to a patient or host cell.
  • Nucleic acids encoding for polypeptides known to display antiviral activity immunomodulatory molecules such as cytokines (e.g. TNF-alpha, interleukins such as IL-6, and IL-2, interferons, colony stimulating factors such as GM-CSF), adjuvants and co-stimulatory and accessory molecules may be included in the viral vector of the invention.
  • immunomodulatory molecules such as cytokines (e.g. TNF-alpha, interleukins such as IL-6, and IL-2, interferons, colony stimulating factors such as GM-CSF)
  • cytokines e.g. TNF-alpha, interleukins such as IL-6, and IL-2, interferons, colony stimulating factors such as GM-CSF
  • adjuvants and co-stimulatory and accessory molecules may be included in the viral vector of the invention.
  • such polypeptides may be provided separately, for example in the preservation mixture of the invention or may be administrated simultaneously,
  • the preserved viral vector of the invention may be introduced into suitable host cells using a variety of viral techniques that are known in the art, such as for example infection with recombinant viral vectors such as retroviruses, herpes simplex virus and adenoviruses.
  • administration of the preserved viral vector of the invention containing a gene of interest is mediated by viral infection of a target cell.
  • a selected recombinant nucleic acid molecule can be inserted into a vector and packaged as retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • Retroviral vectors may be based upon the Moloney murine leukaemia virus (Mo-MLV).
  • Mo-MLV Moloney murine leukaemia virus
  • one or more of the viral genes are generally replaced with the gene of interest.
  • a number of adenovirus vectors are known.
  • Adenovirus subgroup C serotypes 2 and 5 are commonly used as vectors.
  • the adenovirus may be a human or non-human adenovirus.
  • the wild type adenovirus genome is approximately 35 kb of which up to 30 kb can be replaced with foreign DNA.
  • Adenovirus vectors may have the E1 and/or E3 gene inactivated. The missing gene(s) may then be supplied in trans either by a helper virus, plasmid or integrated into a helper cell genome. Adenovirus vectors may use an E2a temperature sensitive mutant or an E4 deletion. Minimal adenovirus vectors may contain only the inverted terminal repeats (ITRs) & a packaging sequence around the transgene, all the necessary viral genes being provided in trans by a helper virus.
  • ITRs inverted terminal repeats
  • Suitable adenoviral vectors thus include Ad4, Ad5, Ad7, Ad11, Ad14, Ad26, Ad35 and Ad36 vectors and simian adenovirus vectors, preferably Ad4, Ad5, Ad7, Ad35 and Ad36 vectors.
  • Ad5 is most commonly used.
  • Viral vectors may also be derived from the pox family of viruses, including vaccinia viruses and avian poxvirus such as fowlpox vaccines.
  • modified vaccinia virus Ankara is a strain of vaccinia virus which does not replicate in most cell types, including normal human tissues.
  • a recombinant MVA vector may therefore be used to deliver the polypeptide of the invention.
  • AAV adeno-associated virus
  • HSV herpes simplex virus
  • Solutions according to the present invention may be administered to a subject in vivo using a variety of known routes and techniques.
  • the solutions are suitable for parenteral administration.
  • the vaccines can be provided as an injectable solution, suspension or emulsion and administered via parenteral, subcutaneous, oral, epidermal, intradermal, intramuscular, interarterial, intraperitoneal, intravenous injection using a conventional needle and syringe, or using a liquid jet injection system.
  • Vaccines may be administered topically to skin or mucosal tissue, such as nasally, intratrachealy, intestinal, sublingually, rectally or vaginally, or provided as a finely divided spray suitable for respiratory or pulmonary administration.
  • HEK-293 cells (ECACC 85120602)
  • Adenovirus GFP (Vector Biolabs cat. 1060)
  • N,N-DMG (Sigma D1156, Lot 077K1856)
  • VWR Elcold ⁇ 45° C. freezer
  • VWR Forma 900 series ⁇ 80° C. freezer
  • Biotek Synergy HT microplate reader
  • the loading times of vials prior to freeze drying may impact viral recovery and vaccines efficacy as long fill times can increase variation of the batch.
  • Excipients were tested with sugars only or a combination of and sugars and PEI to see if they were able to protect virus during a standing period prior to freeze drying.
  • Samples were prepared in 2 ml glass vials in triplicate. Final sugar concentrations were 1M Suc 100 mM Raf. The final PEI concentration was 1 nM.
  • adenovirus expressing Green Fluorescent Protein (GFP) was added and left for 4 hours at room temperature. Following incubation adenovirus was added to the remaining vials.
  • Freeze drying was carried out using a Modulyo D Freeze Dryer for 3 days where the condenser was set to ⁇ 80° C. and the vacuum was 200 mTorr. Following completion of the freeze drying vials were stoppered.
  • Virus titre was calculated by infecting cells with the adenovirus expressing GFP.
  • 96 flat bottomed cell culture dishes (Jencons, UK) were seeded with HEK 293 cells (ECACC 85120602) at 10 5 cells per ml (100 ⁇ l per well) and maintained at 37° C. with 5% CO 2 .
  • vials containing the adenovirus plus excipient were reconstituted in 1 ml of Dulbecco's Minimum Essential Medium (DMEM) plus 5% Foetal Bovine Serum (FBS).
  • DMEM Dulbecco's Minimum Essential Medium
  • FBS Foetal Bovine Serum
  • the experiment in this Example investigated whether PEI and sugars enhance virus stability in a liquid prior to freeze drying.
  • a mixture of adenovirus, sugars and PEI or adenovirus and sugars were incubated for 4 hours prior to freeze drying and compared to samples prepared and immediately freeze dried.
  • virus titre was higher than in the virus only controls, there was a significant loss of virus titre following the 4 hour incubation compared to sugar excipients that were immediately freeze dried.
  • the experiment in this Example examined stability of adenovirus in an aqueous solution following heat challenge at 37° C. for 1 week.
  • PBS controls showed a significant loss in titre whereas sugars plus PEI at 0.26 ⁇ M showed good preservation of the adenovirus following heat challenge (see FIG. 2 ).
  • the upper limit of PEI concentrations appeared to be 2.6 ⁇ M as above this concentration no virus infectivity was seen.
  • Samples were diluted 1/20 to give HA concentration of 1 ⁇ g/ml in PBS. 50 ⁇ l volumes of each solution were used to coat 6 replicate wells of an ELISA plate (Nunc maxisorb) which was then incubated at 37° C. for 1 hour prior to washing ⁇ 3 in PBS.
  • a monospecific polyclonal sheep anti H1 Solomon Islands was diluted 1/200 in a blocking buffer comprising PBS/0.1% Tween20/5% non-fat dried milk powder (PBSTM). 50 ⁇ l volumes were added to each assay well and the plate was incubated at 37° C. for 1 hour. The plate was washed ⁇ 3 PBS and 50 ⁇ l of a 1/1000 dilution in PBSTM of a horse radish peroxidase conjugated polyclonal rabbit anti sheep immunoglobulins (Abcam) was added per well. The plate was incubated for a further 1 hour at 37° C. prior to washing ⁇ 4 with PBS.
  • PBSTM non-fat dried milk powder
  • the assay was developed by the addition of 50 ⁇ l/well of a substrate/chromogen solution comprising 0.4 ⁇ l/ml of a 30% H 2 O 2 solution (Sigma) and 0.4 mg/ml O-phenylenediamine (OPD) (Sigma) in 0.05M citrate/phosphate buffer pH 5.0. The plate was incubated at room temperature for 10 minutes. The reaction was stopped by the addition of 50 ⁇ l/well of 1M H 2 SO 4 . The plate was read on a Synergy HT microplate reader (Biotek) with a 490 nm interference filter.
  • the excipient composition shows similar results to the water composition (which is the recommended storage medium) after 5 days incubation, however, following a further months incubation at +4° C., recognition of the excipient composition is substantially better than that of the water composition with very little, if any, further deterioration. Further, there is little difference between the +4° C. maintained and ⁇ 20° C. freeze-thawed excipient compositions. The freeze-thawing of influenza virus HA is not recommended (and hence was not included for the water composition in this experiment) as aggregation is known to occur.
  • Recombinant adenovirus (Vector Biolabs) expressing enhanced GFP (Green Fluorescent Protein) under a CMV promoter was used for ease of detection during assay.
  • GFP Green Fluorescent Protein
  • glycinergic compounds and one thetin were each tested for efficacy as a preservative (of adenovirus) at a final concentration of 0.07-0.70M, both in co-formulations with sugars (1M Sucrose, 100 mM Raffinose) and in their absence.
  • the glycinergic compounds tested were Glycine, Sarcosine (mono-methyl glycine), DMG (Di-methyl glycine), TMG (Tri-Methyl glycine).
  • Thetin tested was SMM (S-methyl-methionine).
  • Virus was formulated with excipient mixtures in order to test their efficacy in preserving viral activity through a period of thermal challenge. Each mixture of excipients plus virus (see Table 2 below) was made up as a stock in PBS and 300 ⁇ l added to appropriately labelled 5 ml glass vials.
  • HEK 293, ECACC 85120602 Cells permissive to the Adenovirus (HEK 293, ECACC 85120602) were seeded into 96-well-flat-bottomed cell culture dishes (VWR, UK) at 10 5 cells per ml (100 ⁇ l per well) and maintained at 37° C. with 5% CO 2 . After achieving 90% confluence, vials containing the adenovirus plus excipient were removed from the fridge and 1 in 10, and 1 in 100 dilutions produced by serial dilution in DMEM. 100 ⁇ l of each of the resultant dilutions (1 in 10 and 1 in 100) was then added wells of the plate containing HEK 293 cells.
  • a further sample of adenovirus from the same source and with the same titre (on storage at ⁇ 80° C.) used in the excipient treatments, was thawed and used to produce a 1 in 10 dilution series (in DMEM). Dilutions ranging from 1 in 10 to 1 in 10 6 were also added to individual wells containing HEK 293s. At 48 hours, post inoculation the number of GFP (Green Fluorescent Protein) cells per well were counted using fluorescent microscopy, and this was subsequently converted to pfu/ml of the treated samples taking into account the volume applied and dilution of the inoculum.
  • GFP Green Fluorescent Protein
  • FIG. 4 a & b Recovered Viral Activity after 1 Week at 4° C.
  • FIG. 4 c & d (Glycinergics and SMM WITHOUT Added Sugar or WITH Added Sugar Respectively)
  • VWR Elcold ⁇ 45° C. freezer
  • VWR Forma 900 series ⁇ 80° C. freezer
  • Biotek Synergy HT microplate reader
  • Samples were prepared in 2 ml glass vials in triplicate. Final sugar concentrations were 1M Suc 100 mM Raf. 200 ⁇ g of rat anti TNF- ⁇ antibody in 1 ml of PBS was used as the neutralising antibody (lots 555790A and 47758B). Stocks were stored at 2-8° C. until use.
  • Solutions were diluted to give a range of sugar concentrations (see Table 3 below). Glass vials were left at room temperature for 10 days prior to carrying out an L929 TNF- ⁇ neutralisation assay (see below). Included in the assay was a fresh liquid stock used to indicate original antibody activity and a freeze thaw control.
  • L929 cells were purchased from HPA cultures (cat no. 85011425). A cell suspension at a density of 3.5 ⁇ 10 5 cells per ml was prepared in 2% FBS (fetal bovine serum) in RPMI medium. 100 ⁇ l of the cell suspension was added to each well in a 96 well plate and incubated overnight at 37° C., 5% CO 2 .
  • FBS fetal bovine serum
  • VWR Ceti inverted fluorescent microscope
  • Empower 2 software Waters
  • Forma 900 series ⁇ 80° C. freezer Thermofisher
  • HERA safe class II cabinet Thermo Fisher
  • KERN EW220-3NM balance VWR
  • Sheep IgG purified from adult sheep serum (Sigma) by sodium sulphate precipitation was used to investigate the efficacy of sugars, and DMG, as excipients in the liquid storage of immunoglobulins.
  • the sheep IgG was stored at 4° C. prior to use at a concentration of 46 ⁇ g/ ⁇ l (as determined by Bradford assay). Aliquots of IgG were diluted in PBS and a selection of novel excipients to a concentration of 4.6 ⁇ g/ ⁇ l in a volume of 300 ⁇ l.
  • the novel excipient component varied as shown in Table 4 below. Each formulation treatment was replicated 12 times.
  • the 60 ⁇ l subsamples were placed in maximum recovery vials in the separations unit and held at 4° C.
  • a size exclusion chromatography (SEC) column (TSK gel G3000 SWXL) and compatible guard column (TSK gel SWXL) were attached in series (guard first) to the separations unit and conditioned at 25° C. to the mobile phase (0.1M Sodium phosphate, 0.1M Sodium sulphate, pH6.8) with a flow rate of 1.0 ml/min.
  • the mobile phase 0.1M Sodium phosphate, 0.1M Sodium sulphate, pH6.8
  • Purity of the IgG was measured by calculating the area under the monomer peak as a percentage of total area under the identified peaks (monomer, aggregate and void).
  • IgG % purity of each treatment was then converted relative to the purity of the PBS sample from the same day and temperature set.
  • the percentage point change in IgG % purity between day 1 and day 5 was calculated by subtracting the mean of the former from the mean of the later for each excipient treatment.
  • FIGS. 7 and 8 show the area of the monomer peak for all formulations over time (day 1, 5, and 31) stored at 4° C. and 37° C. respectively. Area of the monomer peak is taken as an estimate of recovered monomeric IgG.
  • FIG. 3 shows that as early as day 1 formulations held at 4° C. exhibit differences in the area of their monomer peak. These differences could be mediated by excipient specific disaggregation. Treatments formulated with DMG and sugars have the largest monomer peak and hence most recovered monomeric IgG. Treatments formulated with sugars alone show the smallest monomer IgG and hence least recovered monomeric IgG. Samples formulated with PBS or DMG alone are not significantly different and are intermediate to these.
  • FIGS. 7 and 8 shows that treatments held at 37° C. all have a higher amount or recovered monomeric IgG than equivalent formulations held at 4° C. This is evidence of temperature mediated disaggregation.
  • Example 7 The following materials, equipment and techniques were employed unless stated otherwise in Example 7 and Example 8:
  • DMG Chemical Dimethylglycine
  • MSM Dimethylsulfone
  • MSM Dimethylsulfone
  • FBS Foetal Bovine Serum
  • PS Penicillin Streptomycin
  • SSC Saline Sodium Citrate
  • SZB90120 Sucrose
  • BHK-21 cell line (ECCAC CB2857)
  • HEK 293 (ECACC 85120602)
  • Other 5 ml glass vials (Adelphi Tubes VCD005)
  • 14 mm freeze-drying stoppers (Adelphi Tubes FDIA14WG/B)
  • 14 mm caps (Adelphi Tubes CWPP14)
  • HERA safe class II cabinet (Thermo Fisher, EQP# 011 & 012) DMIL LED Inverted Microscope (Leica, EQP#062) Binder CO 2 Incubator (Binder, EQP#014) Forma 900 series ⁇ 80° C. freezer (Thermofisher, EQP#015) ATL-84-1 Atlion Balance (Acculab, EQP#088) IP250 37° C. Incubator (LTE, EQP#016)
  • Recombinant Human Adenovirus Ad5 (Vector Biolabs) Expressing Enhanced GFP (Green Fluorescent Protein) under a CMV promoter, and with a titre (pre-freeze) of 6.7 ⁇ 10 5 pfu/ml in SSC, was removed from storage at ⁇ 80° C. and allowed to thaw. 50 ⁇ l aliquots were added to 5 ml glass vials. To these 500 virus samples was added 250 ⁇ l of a formulation mixture composed of DMG, MSM and optionally sucrose. Each formulation mixture was made up in SSC. The concentration of DMG, MSM and sucrose in each formulation after addition to the virus sample is shown in Table 5:
  • the vials were stoppered and capped (screw cap) before being placed at 37° C. for thermal challenge. Thermal challenge was for 7 days, after which all the vials were returned to 4° C. until it was practical to assay them.
  • 96 flat bottomed cell culture dishes (VWR, UK) were seeded with HEK 293 (ECACC 85120602) cells at 10 5 cells per ml (100 ⁇ l per well) and maintained at 37° C. with 5% CO 2 . After achieving 90% confluence, cells were inoculated.
  • Vials containing adenovirus plus excipient were reconstituted in 300 ⁇ l SSC.
  • a 1 in 10 dilution step was then taken by taking 20 ⁇ l from the reconstituted vial and adding to 180 ⁇ l of Dulbecco's Modified Eagle Medium (DMEM).
  • a further 1 in 100 dilution (of the original sample) was performed by taking 20 ⁇ l of the 1 in 10 dilution and adding it to 180 ⁇ l of DMEM.
  • 100 ⁇ l of each of the resultant dilution (1 in 10 and 1 in 100) was then added to wells of the plate containing HEK 293 cells.
  • a further sample of adenovirus from the same source and with the same titre (on storage at ⁇ 80° C.) used in the excipient treatments, was thawed and used to produce a 1 in 10 dilution series (in DMEM+10% FBS). Dilutions ranging from 1 in 10 to 1 in 10 6 were also added to individual wells containing HEK 293s. At 48 hours post inoculation, the number GFP (Green Fluorescent Protein) cells per well were counted using fluorescent microscopy, and this was subsequently converted to pfu/ml of the treated samples taking into account the volume applied and dilution of the inoculum.
  • GFP Green Fluorescent Protein
  • MVA was recovered from storage at ⁇ 80° C. and thawed. 50 ⁇ l aliquots were added to 5 ml glass vials. To these vials was added 250 ⁇ l of a formulation mixture listed in Table 1 above. The vials were stoppered and screw caps tightened to seal. The vials were immediately placed at 37° C. for thermal challenge. Thermal challenge was for 7 days, after which all the vials were returned to 4° C. until it was practical to assay them.
  • Assay plates (96 wells) were seeded with BHK-21 cells (100 ⁇ l per well, 10 5 cells/ml). Cells were diluted in DMEM supplemented with 10% FBS, and 1% PS. The plates were placed at +37° C., +5% CO 2 for 1 to 2 hours.
  • a dilution series of the formulated MVA samples was prepared (in the same growth media) ranging from 10 ⁇ 1 to 10 ⁇ 4 .
  • Each dilution series was prepared 4 times.
  • 35 ⁇ l of each dilution was applied to individual wells containing BHK-21 cells and the wells were topped up with a further 65 ⁇ l of media.
  • CPE cytopathic effect
  • Doehlert designs are response surface modelling designs constructed from regular simplexes. They are easily extendable in different directions and new factors can be added to an existing design. Unlike regular formulation designs non-significant factors can be eliminated from the analysis and so do not become a confounding factor.
  • Sucrose was tested between 0 and 1M.
  • Raffinose was tested over a range of 0 to 300 mM although the nature of the Doehlert design meant that tested levels did not include 0 mM. Instead the following ranges were tested: 27.5, 150.0, and 272.5 mM.
  • PEI was tested over a logarithmic range of from 0.04-4000 nM.
  • Recombinant Adenovirus expressing enhanced GFP under a CMV promoter with a titre (pre-freeze) of 6.7 ⁇ 10 5 pfu/ml in SSC, was removed from storage at ⁇ 80° C. and allowed to thaw. Subsequently, 50 ⁇ l aliquots of virus were added to 15, 5 ml, glass vials. To each vial 250 ⁇ l of an excipient blend was admixed. The excipient blend formulations mixed with virus are described in Table 6 and were made up in SSC.
  • the vials were stoppered and capped (screw cap) before being placed at +37° C. for 1 week of thermochallenge and later transferred to +4° C. until it was practical to assay them.
  • HEK 293 cells were prepared in 96 well flat bottomed cell culture dishes for inoculation by seeding at 10 5 cells per ml (100 ⁇ l per well) and maintained at 37° C. with 5% CO 2 . After 2 hours cells were inoculated as follows.
  • Thermo-challenged virus samples were diluted 1 in 10, and 1 in 100 in DMEM+10% FBS+1% PS. 100 ⁇ l of each of the resulting diluted virus samples were then added to individual wells of the assay plate. Additionally, a second aliquot of the original adenovirus in SSC was thawed from ⁇ 80° C. and a 10 fold dilution series (from 1 in 10 to 1 in 100,000) also prepared in DMEM+10% FBS+1% PS. The positive control dilution series was inoculated in duplicate to each 96 well plate used. After a further 48 hours, the number of GFP cells per well were counted using fluorescent microscopy.
  • FIG. 12 shows that the model based on the P ⁇ Bra+Suc+Raff data is a strong one.
  • R 2 (0.93) demonstrates a good fit, Q 2 (0.72) suggests a relatively strong predictive model.
  • FIG. 14 Surface response plots ( FIG. 14 ) illustrate the effects of sucrose and branched PEI (P-Bra) most clearly.
  • the peak on each graph represents the optimum formulation at each stated raffinose concentration (0, 150 and 300 mM).
  • the three graphs show that raffinose does little to alter the optimum P-Bra or sucrose concentrations but does alter the maximum achievable recovered viral activity.
  • a positive control had also been assayed alongside the test samples.
  • the virus used as a control was an additional aliquot of the same virus used in this assay that had been stored at ⁇ 80° C.
  • the assayed titre of this sample was 6.7 ⁇ 10 5 pfu/ml.
  • Monte-Carlo simulations were used to predict an optimum formulation.
  • the positive control was used as a target for optimisation since the model predicts some formulations would result in greater than 100% recovered viral activity (see below).
  • the optimum formulation identified herein was predicted to result in a recovered viral titre of 5.1 ⁇ 10 5 pfu/ml, or a loss of only 24% of viral activity.
  • CCF Central Composite Face-Centred
  • RBM Response Surface Modelling
  • MVA was recovered from storage at ⁇ 80° C. and thawed. 50 ⁇ l aliquots of the MVA were added to 15, 5 ml, glass vials. Subsequently, 50 ⁇ l aliquots of virus were added to 15, 5 ml, glass vials. To each vial 250 ⁇ l of an excipient blend was admixed. The excipient blend formulations once mixed with virus are described in Table 7 and were made up in SSC.
  • the vials were stoppered and capped (screw cap) before being placed at +37° C. for 1 week of thermochallenge and later placed at +4° C. until it was practical to assay them.
  • Assay plates (96 well) were seeded with BHK-21 cells (100 ⁇ l per well, 10 5 cells/ml). Cells were diluted in DMEM supplemented with 10% FBS, and 1% PS. The plates were placed at +37° C., +5% CO 2 for 1-2 hours.
  • a 10 fold dilution series of the formulated MVA samples was prepared (in the same growth media) ranging from 1 in 10 to 1 in 10,000. Each dilution series was prepared 5 times. 100 ⁇ l of each dilution was applied to individual wells containing BHK-21 cells (described above).
  • Sucrose, TMG and raffinose were all predicted to have 1 st order positive effects on viral recovery over the concentration range tested. Although, the raffinose effect was only significant at the 90% C.I. it was retained in the model as it improved the strength of the model and was required to preserve the model hierarchy. This was required because an interaction of TMG and raffinose was also predicted. Finally, a 2 nd order non-linear effect of sucrose was observed. See FIG. 18 for a summary of retained coefficients in the model.
  • FIG. 19 is of a 4D contour plot that illustrates the interactions clearly.
  • the optimum Sucrose concentration can be seen to be consistently between 0.6 and 0.8M, no other excipients significantly alter this. In general the higher the TMG concentration the greater the recovery of viral activity.
  • Monte-Carlo simulations point to the extreme of the tested range for an optimum (1M Sucrose, 1M TMG, 300 mM Raffinose). This suggests that the optimum formulation is not covered by the tested range. However, the simulations predict that formulations close to this optimum should yield recovered viral activity of 94% starting titre.
  • MODDE 9.0 (Umetrics) was used to generate a Doehlert experimental design (see FIG. 21 ), as described in Example 9.
  • MSM was tested at seven levels, whilst sucrose was tested at five and raffinose at three.
  • This model retains the ability to model for second order effects and interactions.
  • the design included three factors and three replicate centre-points resulting in fifteen test samples.
  • Sucrose was tested between 0 and 1M.
  • Raffinose was tested over a range of 0 to 300 mM although the nature of the Doehlert design meant that tested levels did not include 0 mM. Instead the following ranges were tested: 27.5, 150.0, and 272.5 mM.
  • MSM was tested over a linear range of 0 to 2M.
  • Recombinant Adenovirus expressing enhanced GFP under a CMV promoter with a titre (pre-freeze) of 6.7 ⁇ 10 5 pfu/ml in SSC, was removed from storage at ⁇ 80° C. and allowed to thaw. Subsequently, 50 ⁇ l aliquots of virus were added to 15, 5 ml, glass vials. To each vial 250 ⁇ l of an excipient blend was admixed. The excipient blend formulations once mixed with virus are described in Table 8 and were made up in SSC.
  • the vials were stoppered and capped (screw cap) before being placed at +37° C. for 1 week of thermochallenge and later placed at +4° C. until it was practical to assay them.
  • the adenovirus assay was as described in Example 9.
  • FIG. 23 shows the retained model coefficients after model fine tuning.
  • a 3D plot of the model (see FIG. 24 ) demonstrates that increasing sucrose concentration results in increased recovered viral activity.
  • the tested range here does not include the optimum sucrose concentration.
  • an intermediate MSM concentration ( ⁇ 1M) enhances the protective effect.
  • MODDE 9.0 (Umetrics) was used to generate a Doehlert experimental design (see FIG. 26 ), as described in Example 9. DMG was tested at seven levels, whilst sucrose was tested at five and raffinose three. This model retains the ability to model for second order effects and interactions. The design included three factors and three replicate centre-points resulting in fifteen test samples.
  • Sucrose was tested between 0 and 1M.
  • Raffinose was tested over a range of 0 to 300 mM although the nature of the Doehlert design meant that tested levels did not include 0 mM. Instead the following ranges were tested: 27.5, 150.0, and 272.5 mM.
  • DMG was tested over a linear range of 0 to 2M.
  • Recombinant Adenovirus expressing enhanced GFP under a CMV promoter with a titre (pre-freeze) of 6.7 ⁇ 10 5 pfu/ml in SSC, was removed from storage at ⁇ 80° C. and allowed to thaw. Subsequently, 50 ⁇ l aliquots of virus were added to 15, 5 ml, glass vials. To each vial 250 ⁇ l of an excipient blend was admixed. The excipient blend formulations once mixed with virus are described in Table 9 and were made up in SSC.
  • the vials were stoppered and capped (screw cap) before being placed at +37° C. for 1 week of thermochallenge and later placed at +4° C. until it was practical to assay them.
  • the adenovirus assay was as described in Example 9.
  • FIG. 29 shows that the optimum sucrose concentration is beyond that tested. However, it is unlikely the sucrose concentration would be significantly increased due to constraints on the osmolarity of the product. At some levels DMG enhances the protective effect of the formulation, and raffinose alters the optimum DMG concentration for this purpose.
  • Monte-Carlo simulations were used to predict an optimum formulation (see FIG. 30 ).
  • the program was set to maximise recovered viral activity to a limit of 4.3 ⁇ 10 5 pfu/ml (the titre of a positive control).
  • the predicted optimum formulation was 0.5M Sucrose, 0.4M DMG, 272 nM Raffinose and this was predicted to yield a titre of 3.6 ⁇ 10 5 pfu/ml or 84% of starting titre (based on the positive control).
  • FIG. 31 a shows an alternative way of looking at the data.
  • a contour plot shows DMG concentration plotted against sucrose concentration at a number of different raffinose concentrations. The plot shows the darker region (higher recovery of virus activity) moves down the Y-axis (DMG concentration) as raffinose is increased.
  • a black cross marks the predicted optimum formulation.
  • FIG. 31 b shows the region where recovery is predicted to be 100% or greater.
  • Recombinant adenovirus expressing enhanced GFP under a CMV promoter with a titre (after thawing) of 10.2 ⁇ 10 5 pfu/ml in PBS, was removed from storage at ⁇ 80° C. and allowed to thaw. Subsequently, 50 ⁇ l aliquots of virus were added to 15, 5 ml, glass vials. To each vial 250 ⁇ l of an excipient blend was admixed. The excipient blend formulations once mixed with virus are described in Table 10 and were made up in PBS.
  • “Best” 1M Sucrose, 100 mM Raffinose, 0.7M DMG in PBS.
  • the vials were stoppered and capped (screw cap) before being thermally challenged under the conditions set out in Table 11. At appropriate time points, an adenovirus assay was carried out as described in Example 9.
  • the detection threshold for this assay is 600 pfu/ml which equates to 0.03% recovered activity.
  • the DMG only treatment (“NE”) preserved only 65.1% or virus activity, but when used in concert with the sugars a recovered activity of 99.3% was observed. This finding is close to zero loss of adenovirus at +4° C. after 6 months.
  • thermo-challenges demonstrate that the “Best” formulation is more effective in stabilising the adenovirus than its constituent components.
  • CCF Central Composite Face-Centred
  • RBM Response Surface Modelling
  • MVA was recovered from storage at ⁇ 80° C. and thawed. Subsequently, 50 ⁇ l aliquots of virus were added to 15, 5 ml, glass vials. To each vial 250 ⁇ l of an excipient blend was admixed. The excipient blend formulations once mixed with virus are described in Table 12 below and were made up in SSC. The vials were stoppered under vacuum, and capped (screw cap) before being placed at +37° C. for 1 week of thermochallenge and later placed at +4° C. until it was practical to assay them.
  • Assay plates (96 well) were seeded with BHK-21 cells (1000 per well, 10 5 cells/ml). Cells were diluted in DMEM supplemented with 10% FBS, and 1% PS. The plates were placed at +37° C., +5% CO 2 for 1-2 hours.
  • a 10 fold dilution series of the formulated MVA samples was prepared (in the same growth media) ranging from 1 in 10 to 1 in 10,000. Each dilution series was prepared as 5 replicates. 100 ⁇ l of each dilution was applied to individual wells containing BHK-21 cells (described above).
  • the bivalent F(ab′)2 was thermally challenged in the presence of various concentrations of excipients and assayed at different points.
  • An ELISA assay was used to assess the residual F(ab′)2 activity—this was used to measure the extent of damage sustained.
  • Bivalent F(ab′)2 in PBS was removed from storage at ⁇ 80° C. and allowed to thaw at room temperature.
  • 900 ⁇ l of each formulation with an antibody concentration of 4 ⁇ g/ml was made up—this quantity is sufficient to assay three separate timepoints. See Table 13 for details of each formulation.
  • the activity of the Bivalent F(ab′)2 was assayed by ELISA.
  • Antigen (Rat IgG2b-kappa) diluted to 0.5 ⁇ g/ml in PBS was coated 100 ⁇ l/well in row A to G of a 96-well plate, as well as two extra wells in row H for the +4° C. control conditions. Normal mouse serum at a 1:400,000 dilution was also added to two wells of row H as a positive control. These controls were used to normalise data later. Plates were incubated for 18 hours at +4° C. then washed three times with PBS containing 0.05% Tween 20 (wash buffer).
  • Plates were dried by blotting onto a paper towel. This method of blotting was used in every wash step. Plates were blocked for 1.5 hours with PBS containing 5% skimmed milk powder and 0.05% Tween 20. Plates were washed three times with wash buffer before adding the samples.
  • the F(ab′)2 formulations were removed from incubator/fridge and 250 ⁇ l was removed from each. This was diluted 1:2 with wash buffer. Each diluted sample was added to the plate in duplicate and was diluted 2-fold down the plate (final concentrations ranging from 2 ⁇ g/ml to 0.0625 ⁇ g/ml). A condition with no bivalent F(ab′)2 was also included to measure the background signal. The plates were incubated at room temperature for 1.5 hours after which time the plates were washed five times with wash buffer.
  • a goat anti-human HRP conjugated antibody was diluted 1:5000 in wash buffer and 100 ⁇ l added to all the wells containing bivalent F(ab′)2.
  • Rabbit anti-mouse HRP conjugate was diluted 1:1000 in wash buffer and 100 ⁇ l added to the wells containing the normal mouse serum control. The plates were incubated at room temperature for 1.5 hours then washed five times with wash buffer.
  • TMB stabilised chromogen 100 ⁇ l was added to each well and was allowed to react for 10 minutes at room temperature, after which time 100 ⁇ l 200 mM sulphuric acid was added to stop the reaction.
  • the plates were read at 450 nm using Synergy HT Microplate reader.
  • the bivalent F(ab′)2 was thermally challenged in the presence of various concentrations of the excipients and assayed at different points (see FIG. 40 ). After 24 hours storage at +56° C. most samples maintained the majority of their F(ab′)2 activity (when compared to the control sample stored a +4° C.), however after 5 days samples formulated with low or no sugar, the residual F(ab′)2 activity dropped to between 21% and 33% when compared to the activity remaining after 24 hours. Samples which contain high sugar concentration retained at least 44% activity after 5 days storage at +56° C.—this was increased to 63% to 94% with the addition of PEI.
  • the final timepoint was taken at 7 days thermal challenge at +56° C.
  • the control sample had not lost any activity, as expected.
  • the samples which were formulated with low or no sugar had lost the majority of their F(ab′)2 activity. Samples which contained high sugar concentration maintained at least 27% of the 24 hour sample, this was increased to 79% when 10 ⁇ g/ml of PEI was added.
  • the bivalent F(ab′) 2 was thermally challenged in the presence of various concentrations of the excipients and assayed at different points.
  • An ELISA assay was used to assess the residual F(ab′) 2 activity—this was used to measure the extent of damage sustained.
  • Bivalent F(ab′) 2 in PBS was removed from storage at ⁇ 80° C. and allowed to thaw at room temperature.
  • 1050 ⁇ l of each formulation with an antibody concentration of 4 ⁇ g/ml was made up—this quantity is sufficient to assay four separate timepoints. See Table 14 for details of each formulation.
  • the positive and negative control samples were prepared as 167 ⁇ g FAb in PBS (positive control was prepared fresh immediately prior to HPLC analysis).
  • Sucrose-Raffinose mix-only control was prepared as 167 ⁇ g FAb in PBS with 0.15 M Sucrose and 0.015M Raffinose.
  • Test samples were prepared as 167 ⁇ g FAb in PBS with 0.15 M Sucrose and 0.015M Raffinose with one of the following 0.1M (low) or 1.0M (high) DMG or TMG. All samples except the positive control were subjected to a 130 h heat challenge at 56° C. This resulted in a total of seven samples.
  • the positive control was prepared as described above before all samples were subjected to centrifugation at 16.3 k ⁇ g for 5 minutes at room temperature to remove any insoluble matter. Supernatants were carefully decanted so as not to disturb any pellets. Decanted supernatants were then used for HPLC analysis as described below.
  • the sample chamber was kept at 5° C. and the column at 25° C. Samples were injected twice as blocks. GFC molecular weight standards (BioRAD #151-1901) were run before and after each block to ensure correct functioning of the HPLC setup.
  • Peak picking parameters for all peaks were as follows: Peak Match was set to Closest; Y Value was set to Area; Fit was set to Linear and Weighting was disabled.
  • the peak areas derived from the processing described above were used to generate Purity and Monomer Retention parameters for each condition.
  • Purity was defined as monomer peak area divided by total peak within each sample.
  • Monomer Retention was defined as monomer peak area divided by the monomer peak area in the positive control (non-heat-challenged) sample.
  • FIG. 42 a Y-normalised HPLC overlay trace of molecular weight standards (BioRAD, 151-1901; light grey) and untouched monovalent FAb (dark grey).
  • the molecular weights standards feature an initial void peak (as labelled), five standard components (numbered 1 to 5) and an unknown peak (as labelled).
  • the identity and sizes of the five standards components are as follows: (1) bovine thyroglobin—670 kDa; (2) bovine-globulin—158 kDa; (3) chicken ovalbumin—44 kDa; (4) horse myoglobin—17 kDa and (5) vitamin B 12 1.35 kDa.
  • the FAb peak elutes prior to the third standard indicating an hydrodynamic-equivalent size of greater than 44 kDa as would be expected for a monovalent FAb.
  • the FAb elutes just before the third weight marker, giving it an estimated hydrodynamic weight of more than 44 kDa. This value is consistent with a monovalent FAb.
  • FIGS. 43 to 45 all show a superposition of seven HPLC traces corresponding to the first injection of each condition.
  • the large peaks at 13 minutes (labelled b) in FIG. 43 are due to excipient whilst the smaller peak at ten minutes (labelled a) is due to the FAb.
  • a black rectangle highlights the area that is expanded and shown in FIG. 44 .
  • FIG. 43 is a full scale HPLC trace of all seven conditions described in the main text.
  • the small peak at 10 minutes (labelled a) is the Antibody fragment (FAb) peak.
  • the large peak at 13 minutes (labelled b) is due to excipient.
  • the dark box highlights the area expanded and shown in FIG. 44 .
  • FIG. 44 shows the same superposition of the first injection of all seven conditions as shown in FIG. 43 . However, the trace in FIG. 44 is terminated after 12 minutes.
  • FIG. 44 highlights the FAb peak and indicates that some but not all the samples contain a shoulder peak at the tail end of the monomer peak. The FAb peak itself is highlighted in annotated form in FIG. 45
  • FIG. 44 shows HPLC trace of all seven conditions described in the main text. The trace is shown up to 12 minutes (the Antibody Fragment (FAb) peak occurs at 10 minutes). Distinctions in the magnitude and shouldering of the peak can be seen between the seven conditions. The FAb peak is highlighted in FIG. 45 .
  • Fb Antibody Fragment
  • FIG. 45 is an annotated HPLC trace of all seven conditions described above. The trace is shown zoomed to highlight the Antibody Fragment (FAb) peak at 10 minutes. The identity of the each of the seven conditions is annotated on the Figure.
  • SR is sugar mix (0.15 M Sucrose and 0.015 M Raffinose)
  • DMG is Dimethyl Glycine
  • TMG is Trimethyl Glycine.
  • the suffix ‘lo’ refers to a low concentration of the excipient and is 0.1M.
  • the suffix ‘hi’ refers to a high concentration of the excipient and is 1.0 M.
  • FIG. 45 indicates that of all the heat-challenged samples, those with 1.0 M DMG or TMG (plus SR mix) produce a trace closest to the positive control (non-heat-challenged) sample.
  • FAb heat-challenged in PBS alone suffers the greatest loss and also experiences a marked increase in the shoulder peak.
  • the next lowest monomer height occurred with FAb challenged in SR alone.
  • SR mix plus 0.1M of either DMG or TMG provided medial protection that was better than SR mix alone but inferior to SR mix plus either 1.0M DMG (better) or 1.0M TMG (best).
  • HPLC processing method described above was used to integrate all seven HPLC traces as shown in FIG. 46 (only traces for one of two injections of each sample is shown). Arrows highlight both the baseline to baseline (triangle) and inflection change (diamond) peak events shown on the x-axis along the baseline.
  • FIG. 47 summarises purity (light grey) and monomer retention (dark grey) parameters for each of the seven conditions described above. All samples were at 167 ⁇ g/mL. Untouched was the non-heat-challenged positive control. All other samples were heat-challenged at 56° C. for 130 hours. Square brackets indicate sample composition:
  • FIG. 47 quantitatively mirrors the qualitative ordinal results from FIG. 45 and indicates that simply diluting the FAb into PBS prior to HC at 56° C. for 130 h causes a one third loss in purity and a two thirds loss in monomer content. These losses are somewhat reduced by incubation with SR mix (0.15M Sucrose and 0.015M Raffinose). Losses are further minimised by incubation with SR mix and 0.1M DMG or TMG.
  • Human blood samples were diluted with an equal volume of: a) PBS; b) 0.7M DMG; or c) 0.7M DMG/0.1M sucrose. After storage at 25° C. for 30 minutes, 5 ⁇ l of blood sample was mixed with 250 ul of Guava ⁇ Viacount ⁇ reagent in an eppendorf tube. The mixture was incubated at room temperature for 5 minutes. After incubation, the viability of the white cell fraction was assessed using the Guava PCA ⁇ cell analyser.

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