Aluminum compounds for use in therapeutic agents and vaccines

Abstract

The invention relates to means and methods for preparing an aqueous composition comprising aluminum and a protein. The invention further relates to aqueous compositions comprising a protein and an aluminum salt, said composition comprising less than 350 ppb of heavy metal based on the weight of the aqueous composition.

Description

Aluminum compounds for use in therapeutic agents and vaccines The invention relates to the fields of pharmaceuticals and vaccines. More specifically, the invention relates to the field of compounds and compositions that are co-administered with the drug and / or antigen. Aluminum compounds (also referred to herein as "aluminum"), including aluminum phosphate (AlPO4), aluminum hydroxide (Al(OH)3), and other aluminum-precipitated vaccines, are currently the most widely used adjuvants in human and veterinary vaccines. Adjuvants are frequently referred to as "alum" in the literature. Aluminum adjuvants have been used in routine vaccination for over half a century. They induce early, high-titer, and long-lasting protective immunity. Over the years, billions of doses of vaccines containing aluminum adjuvants have been administered. Their safety and efficacy have made them the most popular vaccine adjuvants to date. In general, aluminum adjuvants are considered safe when used according to current vaccination schedules. In human vaccination, aluminum adjuvants have historically been used in vaccines against tetanus, diphtheria, pertussis, and poliomyelitis as part of routine childhood immunization programs. Aluminum adjuvants have also been introduced in vaccines against hepatitis A and hepatitis B viruses and against Japanese encephalitis virus (also referred to herein as "JEV"). Other aluminum-adsorbed vaccines, such as those against anthrax, are available for special risk groups. In veterinary medicine, aluminum adjuvants have been used in numerous vaccine formulations against viral and bacterial diseases, and in attempts to develop antiparasitic vaccines. Adjuvants typically serve to bring the antigen—the substance that stimulates a specific protective immune response—into contact with the immune system and influence the type and quality of immunity produced (magnitude and duration). Adjuvants can also reduce the toxicity of certain antigens and provide solubility to some vaccine components. Studies have shown that many vaccines containing aluminum elicit larger and longer-lasting antibody responses than comparable vaccines without the adjuvant. The benefit of adjuvants is generally observed during the initial immunization series rather than with booster doses. There are three general types of adjuvants that contain aluminum: Aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate (collectively often referred to as "Alum") The effectiveness of each salt as an adjuvant depends on the characteristics of the specific vaccine and how the manufacturer prepares it. To function as an adjuvant, the antigen typically adsorbs to the aluminum; that is, it binds with the aluminum salt to retain the antigen at the injection site. Not all vaccines contain aluminum salts. Sometimes an adjuvant may not have been necessary, or a different adjuvant may have been selected. Examples of commercial vaccines that do not contain aluminum salts include the inactivated polio vaccine (IPV), the measles, mumps, and rubella (MMR) vaccine, the varicella vaccine, the meningococcal conjugate vaccine (MCV4), and influenza vaccines. The fact that commercial vaccines do not contain aluminum salts does not usually mean that an aluminum salt is ineffective. It simply means that, for some reason, a different adjuvant was selected. Examples of U.S.-licensed childhood vaccines containing aluminum adjuvants include: DTP (diphtheria, tetanus, and pertussis vaccine); DTaP (diphtheria, tetanus, and acellular pertussis vaccine); some, but not all, Hib (Haemophilus influenzae type b) conjugate vaccines; pneumococcal conjugate vaccine; hepatitis B vaccines; hepatitis A vaccines; human papillomavirus vaccine; anthrax vaccine; and rabies vaccine. Aluminum is a very abundant element in our environment. It is found in many foods we eat, many personal hygiene products we apply to our skin (deodorants, for example), and many medications we take. Various government agencies establish guidelines for exposure to potentially toxic substances. These guidelines are called "minimum risk levels": the maximum amount to which one can be exposed over time, usually daily, without expected harm. The U.S. Agency for Toxic Substances and Disease Registry (ATSDR) estimated these levels for infants by taking into account the amount of aluminum (for example, in the form of salt) that a child would eat and receive through vaccine injections. The body burden of aluminum from both sources is below the minimal risk level, except transiently after vaccinations; since 50–70% of injected aluminum is excreted within 24 hours, this is not believed to have any adverse effects. Aluminum hydroxide and aluminum phosphate adjuvants are generally prepared by exposing aqueous solutions of aluminum ions to typically slightly alkaline conditions in a well-defined and controlled chemical environment. Various soluble aluminum salts can be used for the production of aluminum hydroxide. Anions present at the time of precipitation may coprecipitate (for a review, see Lindblad, EB (2004) Immunol. and Cell Biol. Vol. 82: 497-505). Aluminum salts are also used in the manufacture and formulation of medicines. For example, factor VIII is purified from plasma cryoprecipitate. The precipitate is solubilized, adsorbed onto aluminum hydroxide, and then treated to inactivate lipid-enveloped viruses. After several other processing steps, the concentrate is used to treat patients with hemophilia A (Burnouf T, (1991) Vox Sang. Vol 60: pp. 8-15). Estey et al. (2009. Journal of Pharmaceutical Sciences, 98 (9): 2994-3012) describes the evaluation of the chemical degradation of a trivalent recombinant protein vaccine against botulinum neurotoxin by LysC peptide mapping and MALDI-TOF mass spectrometry. Aspects and embodiments of the present invention are set forth in the claims. The present invention demonstrates that the stability of a biological product in a composition that also includes an aluminum salt is not always the same. The present invention, for example, shows that the stability of a protein component (e.g., as such or within a complex such as, for example, a virus or other pathogen) in the context of an aqueous composition that also includes an aluminum salt depends on the heavy metal content. To estimate a priori whether the protein will be stable in this composition, the present invention establishes that it is necessary to determine the residual heavy metal content in the composition (otherwise, the aqueous composition comprising a protein risks degrading over time; in particular, the invention anticipates that this risk is considerable when the residual heavy metal content exceeds 350 ppb (i.e., approximately 350 ng per ml) in said aqueous composition).Furthermore, the invention also revealed that this residual heavy metal content cannot be easily removed from the aluminum compound. To this end, the description provides a method for preparing an aqueous composition comprising aluminum and a protein, said method comprising. - combine an aluminum salt, said protein and water to produce said aqueous composition and - Determine the level of a heavy metal in the aqueous composition and / or the aluminum salt. Compositions comprising less than 350 ppb of heavy metal based on the weight of the aqueous composition can be stored in a liquid phase at a temperature between 0 and 30 degrees Celsius for at least 1 month, such as, for example, 20 months at 2-8 °C. The protein component of such a composition is stable for at least 1 month in said liquid phase. Compositions comprising more than 350 ppb of heavy metal based on the weight of the aqueous composition cannot be stored for a prolonged period under conditions such that the protein component in said composition changes in at least one aspect during the stated time period. Therefore, one milliliter or one gram of aqueous composition preferably contains no more than 350 nanograms of heavy metal. The aqueous composition preferably comprises between 0.1 mg / ml and 2.5 mg / ml of aluminum.The average dose of aluminum per administration is preferably no more than 1.25 milligrams (mg). In a particularly preferred embodiment, the dose of aluminum per administration is no more than 0.25 mg. A dose typically comprises between 0.5 and 1 mL of the aqueous composition. In a further aspect of the present invention, it has been shown that the stability of a biological product in a composition comprising an aluminum salt and a reactive compound is not always the same. The present invention, for example, shows that the stability of a protein component (e.g., as such or within a complex such as, for example, a virus or other pathogen) in the context of an aqueous composition also comprising an aluminum salt and a reactive compound such as, for example, a sulfite, depends critically on the heavy metal content. To estimate a priori whether the protein component (such as, for example, the protein component within a complex, such as, for example, a virus particle; herein also referred to simply as a protein) will be stable in this composition, it is necessary to determine the heavy metal content in the composition.To this end, the description provides a method for preparing an aqueous composition comprising aluminum and a protein, the method comprising said method. - combine an aluminum salt, said protein and water to produce said aqueous composition and - Determine the level of a heavy metal in the aqueous composition and / or the aluminum salt. Compositions comprising less than 350 ppb of heavy metal based on the weight of the aqueous composition can be stored in a liquid phase at a temperature between 0 and 30 degrees Celsius for at least 1 month, such as, for example, 20 months at 2-8 °C. The protein component in such a composition is stable for at least 1 month in such a liquid phase, such as, for example, 20 months at 2-8 °C. Compositions comprising more than 350 ppb of heavy metal based on the weight of the aqueous composition cannot be stored for a prolonged period under conditions such that the protein component in said composition changes in at least one aspect during the stated time period. Therefore, one milliliter or one gram of aqueous composition preferably contains no more than 350 nanograms of heavy metal.The aqueous composition preferably comprises between 0.1 mg / ml (milligrams per milliliter) and 2.5 mg / ml of aluminum. The average dose of aluminum per administration is preferably not more than 1.25 milligrams (mg). In a particularly preferred embodiment, the dose of aluminum per administration is not more than 0.25 mg of aluminum. A dose normally comprises between 0.5 and 1 ml of the aqueous composition. An aqueous composition comprising a protein, an aluminum salt, and optionally a reactive compound, said composition comprising less than 350 ppb of heavy metal based on the weight of the aqueous composition, is also referred to herein as "an aqueous composition comprising a protein as described" or "a composition comprising a protein as described." The aluminum content of the aqueous composition is generally approximately 0.1 mg / ml to 2.5 mg / ml, with the average vaccine comprising approximately 0.5 to 1.5 mg / ml of aluminum adjuvant. For this purpose, the description provides an aqueous composition, an aqueous pharmaceutical composition, or a vaccine comprising aluminum, a reactive compound, and a protein, containing between 5 mcg / ml and 50 mg / ml of aluminum and comprising no more than 700 ppm of a heavy metal relative to the aluminum content (grams / grams). In this context, 700 ppm of a heavy metal is equivalent to 700 mcg of heavy metal per gram of aluminum. In a preferred embodiment, the aqueous composition, aqueous pharmaceutical composition, or vaccine comprising aluminum, a reactive compound, and a protein, comprises between 5 mcg / ml and 50 mg / ml of aluminum and comprises no more than 450 ppm of a heavy metal relative to the aluminum content (grams / grams). In this context, 450 ppm of a heavy metal is equivalent to 450 mcg of heavy metal per gram of aluminum. In a preferred embodiment, the aqueous composition, aqueous pharmaceutical composition, or vaccine comprising aluminum, a reactive compound, and a protein, comprises between 5 mcg / ml and 50 mg / ml of aluminum and comprises no more than 700 ppm of Fe relative to the aluminum content (grams / grams). Preferably, the Fe content is less than 420 ppm of Fe relative to the aluminum content.Preferably, the Fe content is less than 350 ppm, preferably less than 100 ppm, and more preferably less than 50 ppm compared to the aluminum content (1 ppm = 1 mcg / gram of Al). In a particularly preferred embodiment, the Fe content of the aqueous composition, aqueous pharmaceutical product, or vaccine is less than 420 ppm compared to the aluminum content. In a preferred embodiment, the aqueous composition, aqueous pharmaceutical product, or vaccine comprises more than 10 ppm of Fe compared to the aluminum content. In a preferred embodiment, the aqueous composition, aqueous pharmaceutical composition, or vaccine comprising aluminum, a reactive compound, and a protein, comprises between 5 mcg / ml and 50 mg / ml of aluminum and comprises no more than 35 ppm of Ni compared to the aluminum content (grams / grams). Preferably, the Ni content is less than 18 ppm of Ni compared to the aluminum content. Preferably, the Ni content is less than 9 ppm, preferably less than 3 ppm, and more preferably less than 1 ppm compared to the aluminum content (1 ppm = 1 mcg / gram of Al). In a particularly preferred embodiment, the Ni content of the aqueous composition, aqueous pharmaceutical composition, or vaccine is less than 18 ppm compared to the aluminum content. In a preferred embodiment, the aqueous composition, aqueous pharmaceutical composition, or vaccine comprises at least 200 ppb of Ni compared to the aluminum content. In a preferred embodiment, the aqueous composition, aqueous pharmaceutical composition, or vaccine comprising aluminum, a reactive compound, and a protein, comprises between 5 mcg / ml and 50 mg / ml of aluminum and comprises no more than 5 ppm of copper (Cu) relative to the aluminum content (grams / grams). Preferably, the copper content is less than 2.5 ppm relative to the aluminum content. Preferably, the copper content is less than 1 ppm, preferably less than 0.5 ppm, and more preferably less than 0.25 ppm relative to the aluminum content (1 ppm = 1 mcg / gram of Al). In a particularly preferred embodiment, the copper content of the aqueous composition, aqueous pharmaceutical product, or vaccine is less than 2.5 ppm relative to the aluminum content.In a preferred embodiment, the aqueous composition, aqueous pharmaceutical composition or vaccine comprises at least 50 ppb of Cu compared to the aluminum content. In a particularly preferred embodiment, the Fe content of the aqueous composition, aqueous pharmaceutical product, or vaccine is less than 420 ppm compared to the aluminum content; the Ni content of the aqueous composition, aqueous pharmaceutical product, or vaccine is less than 18 ppm compared to the aluminum content; and the Cu content of the aqueous composition, aqueous pharmaceutical product, or vaccine is less than 2.5 ppm compared to the aluminum content. In a preferred embodiment, the aqueous composition, aqueous pharmaceutical product, or vaccine comprises more than 10 ppm of Fe compared to the aluminum content, at least 200 ppb of Ni compared to the aluminum content, and at least 50 ppb of Cu compared to the aluminum content. In a method for preparing an aqueous composition, an aqueous pharmaceutical composition, or a vaccine comprising aluminum, a reactive compound, and a protein as described, or a method for preparing or selecting an aluminum salt according to the invention, or a method for preparing a clinical-grade aluminum salt precipitate as described, the heavy metal content relative to the aluminum content is preferably as indicated above herein. Therefore, the description provides a method for preparing an aqueous composition comprising aluminum, a reactive compound, and a protein, said method comprising - prepare or select an aluminum salt that is capable of providing an aqueous composition having less than 350 ppb of heavy metal based on the weight of the aqueous composition and - combine said aluminum salt, said reactive compound, said protein and water to produce said aqueous composition, whereby the aqueous composition comprises between 5 mcg / ml and 50 mg / ml of aluminum, preferably between 0.1 mg / ml and 2.5 mg / ml; and not more than 700 ppm of heavy metal and preferably not more than 450 ppm of heavy metal compared to the aluminum content; wherein said aqueous composition comprises not more than 420 ppm of Fe, preferably not more than 100 ppm, compared to the aluminum content; not more than 18 ppm, preferably not more than 3 ppm, of Ni compared to the aluminum content; and / or not more than 2.5 ppm of Cu, preferably not more than 0.5 ppm, compared to the aluminum content.In one example, such aqueous composition comprises no more than 700 ppm of heavy metal and preferably no more than 450 ppm of heavy metal compared to the aluminum content; no more than 420 ppm of Fe, preferably no more than 100 ppm, compared to the aluminum content; no more than 18 ppm, preferably no more than 3 ppm of Ni compared to the aluminum content; and no more than 2.5 ppm of Cu, preferably no more than 0.5 ppm, compared to the aluminum content. It has been observed that the aluminum component is a significant source of the heavy metal in the aqueous composition. Therefore, one way to control the amount of heavy metal in the aqueous composition is to control the amount of heavy metal in the aluminum source used to generate the aqueous composition. Therefore, the description further provides a method for preparing an aqueous composition comprising aluminum and a protein, said method comprising - prepare or select an aluminum salt solution (such as, for example, 10 mg / ml of aluminum hydroxide liquid (such as, for example, alhydrogel® 2% from Brenntag Biosector, catalog number 843261) which in the final protein formulation comprises no more than 350 ppb of heavy metal based on the weight of the aqueous composition (for example, for alhydrogel® 2% and a final amount of 0.25 mg of aluminum hydroxide in the aqueous composition of 0.5 ml (= dose), the selected or prepared aluminum salt solution must not contain more than 7 micrograms of heavy metals per milliliter of the alhydrogel® 2% solution, approximately 7 ppm heavy metal content), and - combine said aluminum salt solution, said protein, water and, optionally, a reactive compound to produce said aqueous composition (with no more than 350 ppb of heavy metal in the aqueous composition). For example, the aluminum salt solution, e.g., aluminum hydroxide liquid (used as a component to be mixed with to produce the final aqueous composition), must not have a heavy metal content exceeding 7 ppm (provided as an example herein where 10 mg / ml of aluminum hydroxide liquid will be diluted to 0.5 mg / ml to yield a heavy metal content of approximately 350 ppb with respect to the aqueous composition, assuming 1 ppm = approximately 1 mg / ml) on a weight basis of the aluminum hydroxide liquid. The acceptable heavy metal content limit may also be expressed with respect to the weight of the aluminum salt, such as aluminum hydroxide solution (also referred to as the "starting aluminum compound").The acceptable heavy metal content in this example cannot exceed 7 micrograms of heavy metals per gram of aluminum hydroxide solution (i.e., approximately 7 ppm), i.e., the alhydrogel® 2% solution. As stated earlier in this document, the aqueous composition comprising the protein (as, for example, if used as a vaccine) preferably comprises between 0.1 and 2.5 mg / ml of the aluminum compound. However, the concentration of heavy metals in the aqueous composition, depending on the composition, must not exceed 350 ppb, i.e., approximately 350 ng per ml of the final composition, and therefore the selection or preparation of the starting aluminum compound must be carried out accordingly.In order to further illustrate the selection of a suitable starting aluminum compound solution (e.g., in the form of a concentrated solution (see above alhydrogel® 2% = 10 mg / ml)), it is shown that a 10 mg / ml aluminum compound solution of aluminum hydroxide having approximately 7 ppm of heavy metal impurities corresponds to a concentration of heavy metals in the protein composition when the aluminum concentration is approximately 0.1 mg / ml of approximately 70 nanograms / ml or 70 ppb (well below the 350 ppb provided as the limit for heavy metal content as taught in the invention).A concentration of 2.5 mg / ml of aluminum hydroxide in the final aqueous composition, starting from a 10 mg / ml aluminum hydroxide solution containing approximately 7 ppm of heavy metal impurities, will result in a heavy metal content in the aqueous protein composition corresponding to a heavy metal concentration of approximately 1.75 micrograms / ml or approximately 1,750 ppm (well above the 350 ppb limit provided for heavy metal content as taught in the invention). Therefore, the 10 mg / ml aluminum hydroxide solution in this case (the final aluminum hydroxide content being 2.5 mg / ml) should not contain more than 1.4 ppm of heavy metal impurities. A method for preparing an aqueous composition comprising a protein preferably further comprises packaging aliquots of said aqueous composition having less than 350 ppb of heavy metal based on the weight of the aqueous composition in separate airtight storage containers. The protein in the airtight storage containers is stable and can be stored for at least three months, such as, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months, preferably 20 or 24 months, more preferably 20 months, at a temperature of between 2 and 8 °C. Without being bound by theory, the degradation of antigens in aqueous compositions, such as the immunogenic composition comprising heavy metal ions present in an aluminum salt, such as aluminum hydroxide, could be explained by an underlying degradation pathway involving free radicals, such as sulfite free radicals. The formation of free radicals can be catalyzed by heavy metal ions present in, for example, aluminum hydroxide, and this effect (in the case of a specific amount of heavy metal as indicated in the invention) could be the underlying root cause mechanism for stability problems, as identified as part of the inventive contribution.The experimental part of this application shows in great detail the evidence of this root cause for the Japanese encephalitis vaccine (also known as "JEV") and presents a similar demonstration for a polypeptide composition with a simple aluminum adjuvant comprising a reactive compound such as sulfite. It is therefore evident that a similar reaction can also occur in another aqueous composition comprising aluminum (with a high heavy metal content, for example, exceeding 350 ppb based on the weight of the composition), protein, and possibly a reactive component such as sulfite and / or other radical-forming particles. Heavy metal-catalyzed oxidation is a degradation pathway that results in the covalent modification of proteins.The altered physicochemical properties of the oxidized / modified protein or antigen can result in the loss of biological activity (Li et al., 1995; Mayo et al., 2003; Stadtmann, 1990). The following reaction schemes (which may possibly occur in the JEV product as described in the experimental part) are indicative of the compositions described, comprising in addition to the protein and heavy metal, also sulfite and / or another reactive compound such as, for example, sulfite and / or formaldehyde. Sodium metabisulfite (Na₂S₂O₅) in solution dissolves in bisulfite (S₂O₅²⁻). In an alkaline solution, the equilibrium of bisulfite is towards sulfite (SO₃²⁻), and in an acidic solution, towards H₂SO₃ / SO₂. After dissolving Na₂S₂O₅ in water, it hydrolyzes to NaHSO₃ as follows. Na2S2O5 or 2 NaHSO3 At neutral pH, the following equilibrium can be assumed: HSO3- or H+ + SO32- pKa=7.2 This means that at pH 7 the equilibrium shifts towards HSO3- and under more basic conditions (for example, pH 8) towards SO32-. Formaldehyde forms a bisulfite adduct during neutralization according to the following equation: CH2O + HSO3- ^ CH2 (OH) (SO3) - After the bisulfate is depleted, the reaction proceeds until equilibrium is reached as follows: CH2O + SO32- + H2O ^ CH2 (OH) (SO3) - + OH- Formaldehyde and sulfite react with each other; however, formaldehyde and sulfite have been found to still be present in equilibrium in the JEV vaccine and can be detected in the following range (n=49): According to the literature (Ranguelova et al., 2010), transition metal ions catalyze the auto-oxidation of (bi) sulfite through the formation of the sulfur trioxide radical anion (•SO 3): Mn+ + SO32" ^ M (n-1) + + •SO^ where M can be copper (Cu2+) , iron (Fe3+) , oxyvanadium (VO2+) , manganese (Mn2+) , nickel (Ni2+) or chromate anion (CrO42-) (Alipazaga et al. 2004; Berglund et al. 1993; Brandt and Elding 1998; Lima et al. 2002; Shi 1994) . It was demonstrated that these sulfite radicals are highly reactive and can oxidize various substances, such as ascorbate, hydroquinone, and histidine (Huie et al., 1985). Neta and Huie (1985) published a review of the chemistry of sulfite free radicals. The authors also showed that radical formation can be catalyzed by photoionization of sulfite as follows: SO32- + hv ^ *SO3- + e- Light-catalyzed radical formation could also explain the observed differences in potency and ELISA results between unlabeled, bare syringes (used for release assays and reference purposes) and fully packaged samples from the final vaccine batch of the JEV product. The fully packaged samples are completely protected from light, whereas the unlabeled syringes may be exposed to light during storage and handling. An important reaction of the sulfite radical in auto-oxidation systems is with molecular oxygen to form a peroxyl radical, which is much more reactive: •SO3- + O2 ^ *SO5- The solubility of O2 in water at 0 °C and 20 °C is 0.4 mM and 0.25 mM, respectively. Assuming that the solubility of O2 in a composition of the described range is within a similar range, a considerable amount of oxygen is present to form the peroxyl radical. This radical is a much stronger oxidant compared to SO3- and can oxidize certain substrates that are not bound by SO3- at all and that, in fact, can form radicals that oxidize sulfite ions. In such cases, when the redox potential of the substrate is intermediate between those of SO3- and SOs-, a reaction chain is likely to develop in the presence of O2 following the general pattern: •SO3- + O2 ^ ^SO5- •SO5- + X ^ SO52- + •X* •X+ + SO32- ^ X + ^SO3- The mediation of a substrate X can enhance the chain reaction of sulfite oxidation by oxygen. The one-electron reduction of ^SO5- yields HSO5- (peroxymonosulfate), a very strong oxidant capable of oxidizing many organic compounds (Lambeth et al., 1973; Ito and Kawanashi, 1991). Peroxymonosulfate is also a precursor to the sulfate anion radical ^SO4-. •SO5- + HSO3- ^ ^SO4-. + HSO4- The ^SO4- radical is a very strong oxidant, almost as strong as the hydroxyl radical (•OH) , and is very likely to oxidize other biomolecules by oxidation of one electron. In a preferred embodiment, the composition comprising a protein as described is a therapeutic or immunogenic composition, such as a vaccine. Therapeutic compositions are administered to individuals, such as humans or animals. Particularly important for such compositions is that the protein within the composition still retains its therapeutic effect at the time of administration to the individual. Protein degradation or changes in protein structure may result in the protein losing its therapeutic activity. Similarly, degradation or structural changes in the immunogenic composition will also lead to a reduction in the composition's efficacy in inducing and / or enhancing an immune response in an individual. An immunogenic composition is preferably administered to an individual to counteract or prevent a viral or bacterial infection.The protein contained within the aqueous immunogenic composition may be a single protein or a multimeric protein or part of a complex comprising such a protein (e.g., such as part of a virus or a cell, e.g., a bacterial cell). In a preferred embodiment, said protein or protein complex comprises a live attenuated, inactivated, or mutated bacterium or virus, or an immunogenic viral or bacterial protein or an immunogenic portion thereof (e.g., an immunogenic peptide), a divided bacterium or virus, or whole bacterial cells. If said immunogenic composition is administered to provide protection against a viral or bacterial infection, degradation of the protein may result in the loss of the protective capacity of the immunogenic composition. As used herein, a "live attenuated" virus or bacterium is a virus or bacterium that is less pathogenic than the wild-type virus or bacterium but has retained immunogenic properties.As used herein, an "inactivated" virus or bacterium refers to a virus or bacterium that has been inactivated so that it is no longer infectious, while its immunogenic properties have been at least partially retained. An inactivated virus or bacterium may be in the form of whole, inactivated viruses or bacterial cells. However, inactivation may result in the alteration of viruses or bacterial cells. Therefore, the inactivated virus or bacterium may also be in an altered form. As used herein, a "divided" virus or bacterium refers to a virus or bacterium that has been altered using, for example, a detergent. The aqueous composition described may include a reactive compound, such as formaldehyde, which is typically present to inactivate a virus or bacterium. An additional or alternative reactive compound, such as sulfite, may also be present to inactivate any residual formaldehyde in the aqueous solution. As detailed above, heavy metals are thought to catalyze the oxidation of (bi)sulfite and the formation of sulfite radicals present in the aqueous composition, which in turn can induce the degradation of the protein present in the composition. Therefore, compositions comprising inactivated viruses or bacteria, or immunogenic parts thereof, are likely to include one or more reactive compounds.Therefore, the absence of heavy metals or their presence below the levels identified by the present invention is particularly relevant when the aqueous composition described comprises an inactivated virus or bacterium, or an immunogenic portion thereof. In a particularly preferred embodiment, an immunogenic composition according to the description therefore comprises an inactivated virus. In a preferred embodiment, the aqueous composition, preferably the immunogenic composition, according to the description comprises inactivated viruses or bacteria. Said virus or bacteria is inactivated, for example, by a reactive compound such as formaldehyde as described herein. In another preferred embodiment, the aqueous composition, immunogenic composition or vaccine as described comprises a toxoid. As used herein, a "toxoid" refers to an inactivated bacterial toxin, such as an exotoxin, resulting in reduced or suppressed toxicity while at least partially retaining immunogenic properties. Such a toxin is inactivated, for example, by a reactive compound such as formaldehyde, as described herein. Examples of toxoids present in an immunogenic composition as described herein include, but are not limited to, diphtheria, tetanus, and botulism toxins. The term "immunogenic viral or bacterial protein" refers to a viral or bacterial protein capable of eliciting an immune response. The term "immunogenic portion," as used herein, refers to a portion of a viral or bacterial protein capable of eliciting an immune response. Preferably, the elicited immune response recognizes both the portion and the entire protein. Therefore, an aqueous composition comprising a protein as described is an immunogenic composition. Such a composition is preferably a therapeutic and / or prophylactic composition, such as a vaccine. A vaccine, which is an aqueous composition comprising a protein as described, is also provided. An "immunogenic composition" is defined herein as a composition capable of eliciting an immune response when administered to an individual. The immune response elicited may be humoral, cellular, or a combination thereof, and includes, but is not limited to, the production of antibodies, B lymphocytes such as activated B lymphocytes, and T lymphocytes such as activated T lymphocytes. An immune response as used herein is preferably specifically directed to one or more immunogens within a composition comprising a protein as described.An immunogenic composition of the present description may be administered to an individual by any technique known in the art, including, but not limited to, intramuscular (IM), intradermal (ID), subcutaneous (SC), intracranial (IC), intraperitoneal (IP), or intravenous (IV) injection, transdermal administration, oral, intranasal, or rectal administration, and combinations thereof; intramuscular (IM), intradermal (ID), subcutaneous (SC), intracranial (IC), intraperitoneal (IP), or intravenous (IV) injection are preferred. In a preferred embodiment, an immunogenic composition comprising a protein as described is used to trigger an immune response that may be useful in a chronic setting (such as cancer treatment) or prophylactically (such as a typical vaccine). It is preferred that the immunogenic composition be used as a vaccine, i.e., for prophylactic use. In this embodiment, aluminum is typically present as an adjuvant. An "adjuvant," as used herein, refers to a pharmacological or immunological agent that modifies the effect of other agents, such as an immunological agent that enhances the antigenic response. Adjuvants typically serve to bring the antigen—the substance that stimulates a specific protective immune response—into contact with the immune system and influence the type of immunity produced, as well as the quality of the immune response (magnitude or duration); decrease the toxicity of certain antigens; and provide solubility to some vaccine components. An "individual" is defined herein as a human being or an animal. Individuals include, but are not limited to, chickens, ducks, geese, turkeys, swans, emus, guinea fowl, and pheasants, humans, pigs, ferrets, seals, rabbits, cats, dogs, and horses. In a preferred embodiment of the description, an individual is a mammal, preferably a human being. The "micro" in microgram or microliter or other unit is sometimes called mc, the symbol for the letter u. A value of 3 mcgram is therefore 3 micrograms, 3 pl is 3 microliters, and 3 ugram is 3 micrograms. An aluminum adjuvant is frequently prepared by the controlled exposure of an aqueous solution of aluminum ions to alkaline conditions (for a review, see Lindblad, EB (2004) Immunol. and Cell Biol. Vol. 82: 497-505). In the present invention, it has been found for the JEV product (see the experimental part) that a large amount of the heavy metal in this aqueous aluminum ion solution ends up in the aluminum salt precipitate for the aluminum adjuvant. It has further been found that the amount of heavy metal in the aluminum precipitate affects the stability of the vaccine during vaccine storage. The amount of heavy metal present in the aluminum salt can be controlled by determining the amount of heavy metal in the salt, but also, and preferably, by controlling the amount of heavy metal in the aqueous aluminum ion solution. Therefore, the description further provides a method for preparing a clinical-grade aluminum salt precipitate for incorporation into a medicament and / or vaccine, said method comprising preparing an aqueous aluminum ion solution and precipitating said aluminum ions in said solution.and determining the level of a heavy metal in the solution and / or the aluminum salt precipitate, preferably wherein said solution and / or the aluminum salt precipitate comprises an amount that results in less than 350 ppb of heavy metal in the final composition, for example, when resuspended in the final composition. In a preferred embodiment, the description provides a method for preparing a clinical-grade aluminum salt precipitate for incorporation into a medicament and / or vaccine, said method comprising preparing an aqueous solution of aluminum ions and precipitating said aluminum ions in said solution, and determining the level of a heavy metal in the solution and / or the aluminum salt precipitate, wherein the precipitate is selected that is capable of providing an aqueous composition comprising between 5 mcg / ml and 50 mg / ml of aluminum, preferably between 0.1 mg / ml and 2.5 mg / ml.and comprising no more than 700 ppm of heavy metal and preferably no more than 450 ppm of heavy metal compared to the aluminum content, wherein said composition comprises no more than 420 ppm of Fe, preferably no more than 100 ppm, compared to the aluminum content, no more than 18 ppm, preferably no more than 3 ppm, of Ni compared to the aluminum content, and / or no more than 2.5 ppm of Cu, preferably no more than 0.5 ppm, compared to the aluminum content. In a preferred embodiment, said precipitate comprises no more than 700 ppm of heavy metal and preferably no more than 450 ppm of heavy metal compared to the aluminum content, no more than 420 ppm of Fe, preferably no more than 100 ppm compared to the aluminum content, no more than 18 ppm, preferably no more than 3 ppm, of Ni compared to the aluminum content, and no more than 2.5 ppm of Cu, preferably no more than 0.5 ppm.compared to the aluminum content. A pharmaceutical composition comprising a protein as described is also provided, optionally further comprising a pharmaceutically acceptable carrier and / or diluent. "A pharmaceutically acceptable diluent," as used herein, is defined as any solution, substance, or combination thereof that has no biological activity or undesirable activity, meaning that it can be administered to an individual along with other components of an immunological composition without causing a substantial adverse reaction. Examples of suitable carriers include, for instance, California limpet hemocyanin (KLH), serum albumin (e.g., BSA or RSA), and ovalbumin. In a preferred embodiment, such a suitable carrier comprises a solution, such as saline solution. In one aspect, a method according to the invention is used to extend the shelf life or storage life of an aqueous composition comprising a protein as described. As used herein, the term "shelf life" is defined as the period of time that a composition comprising a protein as described can be stored without becoming unsuitable for use, for example, due to protein degradation (e.g., it is within the potency specification of the composition, e.g., vaccine, as required by the regulatory agency that approved or will approve the vaccine). During the storage of aqueous compositions as described herein, protein degradation can occur, particularly when a certain level (as described herein) of heavy metals is exceeded. Degradation generally increases over time when such aqueous compositions are stored.Now that protein degradation has been found to be reduced in an aqueous composition comprising, in addition to the protein, an aluminum salt, if the composition contains less than 350 ppb of the heavy metal based on the weight of the aqueous composition, it has become possible to counteract protein degradation in aqueous compositions. By counteracting protein degradation using a method according to the invention, the stability of the protein within the composition is increased, and the shelf life of aqueous compositions comprising the protein is extended. An aqueous composition according to the present description offers the advantage of being stable and not undergoing protein degradation over an extended period.Said aqueous composition comprising a protein as described is stable for at least one month at elevated temperature such as, for example, 20 or 37 °C, preferably for at least three months at elevated temperature such as, for example, 20 or 37 °C. An aqueous composition comprising a protein as described is preferably stored at a temperature between 0°C and 20°C to contribute to a longer shelf life, more preferably between 2°C and 15°C, more preferably between 2°C and 10°C, and much more preferably between 2°C and 8°C. The shelf life at a temperature between 2°C and 8°C is preferably stable for at least three months, such as, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months, preferably 20 or 24 months, and more preferably 20 months at a temperature between 2°C and 8°C. Therefore, an aqueous composition comprising a protein as described is provided, which has a shelf life of approximately 12 to 24 months.The description further provides an aqueous solution as described that has been stored for at least one month, preferably for at least two months, more preferably for at least three months, more preferably for at least six months.An aqueous composition preferably comprises a protein and an aluminum salt, said composition comprising less than 350 ppb of heavy metal based on the weight of the aqueous composition, between 5 mcg / ml and 50 mg / ml of aluminum, preferably between 0.1 mg / ml and 2.5 mg / ml, and not more than 700 ppm of heavy metal and preferably not more than 450 ppm of heavy metal compared to the aluminum content, wherein said composition comprises not more than 420 ppm of Fe, preferably not more than 100 ppm, compared to the aluminum content, not more than 18 ppm, preferably not more than 3 ppm, of Ni compared to the aluminum content, and / or not more than 2.5 ppm of Cu, preferably not more than 0.5 ppm, compared to the aluminum content.In a preferred embodiment, said composition comprises no more than 700 ppm, more than 100 ppm, compared to the aluminum content, no more than 18 ppm, preferably no more than 3 ppm, of Ni compared to the aluminum content, and no more than 2.5 ppm of Cu, preferably no more than 0.5 ppm, compared to the aluminum content. As used herein, "a stable protein composition" means that, compared to a starting composition, no more than 50%, preferably no more than 40%, even more preferably no more than 30%, even more preferably no more than 20%, even more preferably no more than 10%, and even more preferably no more than 5% of the protein in that composition is degraded. "Degraded" in this context refers to any detectable modification of the protein compared to the protein in the starting composition. For example, a decrease in the detection of the protein with a monoclonal antibody, such as an antibody that recognizes a neutralizing epitope, is adequate and can be measured by any method known in the art, such as ELISA (see Example 4).The detected level can be compared, for example, to the level detected with a specific polyclonal for the same protein, such that it represents the total amount of protein (changed or unchanged). A method for extending the shelf life of an aqueous composition comprising a protein and an aluminum salt is also provided. This method comprises selecting and / or preparing an aluminum salt resulting in an aqueous composition with a heavy metal content of less than 350 ppb based on the weight of the aqueous composition, and combining the aluminum salt, the protein, and water to produce the aqueous composition. Furthermore, a method is provided for improving the reproducibility of the shelf life of aqueous compositions comprising a protein and an aluminum salt. This method comprises obtaining at least two different aluminum salt preparations, determining the amount of at least one heavy metal in these aluminum salt preparations, selecting from these aluminum salt preparations those comprising less than 350 ppb of the at least one heavy metal, and combining the aluminum salt from these selected preparations with the protein and water to produce the aqueous compositions.Preferably, a composition is selected comprising between 5 mcg / ml and 50 mg / ml of aluminum, preferably between 0.1 mg / ml and 2.5 mg / ml, and comprising no more than 700 ppm of heavy metal and preferably no more than 450 ppm of heavy metal compared to the aluminum content, wherein said composition comprises no more than 420 ppm of Fe, preferably no more than 100 ppm, compared to the aluminum content; no more than 18 ppm, preferably no more than 3 ppm, of Ni compared to the aluminum content; and / or no more than 2.5 ppm of Cu, preferably no more than 0.5 ppm, compared to the aluminum content.More preferably, said composition comprises not more than 700 ppm of heavy metal and preferably not more than 450 ppm of heavy metal compared to the aluminum content, wherein said composition comprises not more than 420 ppm of Fe, preferably not more than 100 ppm, compared to the aluminum content; not more than 18 ppm, preferably not more than 3 ppm, of Ni compared to the aluminum content; and not more than 2.5 ppm of Cu, preferably not more than 0.5 ppm, compared to the aluminum content. In another aspect, the description provides a method for analyzing the storage stability of a composition comprising aluminum and a therapeutic or prophylactic compound, said method comprising combining into a composition a predetermined amount of therapeutic product or vaccine and a predetermined amount of an aluminum salt compound, said method further comprising storing said composition for at least 2 weeks, preferably at least 4 weeks and preferably at least one month, at a temperature above 20 °C, preferably at a temperature of approximately 22 °C and determining the stability and / or the amount of protein, preferably therapeutic or prophylactic compound, in said composition.As demonstrated in Examples 1 and 2, a temperature of 22 °C, which is higher than normal storage conditions or approximately 2–8 °C higher, results in accelerated protein degradation in an aqueous composition comprising protein, an aluminum salt, and more than 350 ppb of that heavy metal by weight relative to that composition. Therefore, a temperature of approximately 22 °C and a storage duration of at least 2 weeks, preferably at least 4 weeks, are suitable for determining the storage stability of aqueous compositions comprising a protein as described. Storage stability can be determined by any method known in the art.The storage stability of an aqueous composition comprising a protein as described is preferably analyzed by determining the storage stability of that protein, preferably by determining a storage-sensitive epitope within that protein. For example, as described in Examples 1 and 2, the stability of a protein, preferably an antigen, is determined by determining the ratio of intact storage-sensitive epitope, such as the intact antigenic epitope content (e.g., an epitope of a neutralizing epitope), to the total protein content. "Intact storage-sensitive epitope" or "intact antigenic epitope," as used herein, means that no degradation has occurred within that epitope.The intact antigenic epitope content is measured, for example, by determining the protein bound to a monoclonal antibody specifically directed against that epitope in, for example, an ELISA. The total protein content is measured, for example, by determining the protein bound to a polyclonal antibody that targets various epitopes within the protein, for example, using an ELISA. The relative specific epitope content can then be expressed as the ratio of the total antigen content determined by monoclonal antibody binding to the total antigen content determined by polyclonal antibody binding. A high ratio indicates a high antigenic epitope content, and a low ratio indicates a low antigenic epitope content.A low ratio measured for an aqueous composition after storage at at least 20 °C, preferably 22 °C, for at least 2 weeks, preferably 4 weeks, compared to the ratio measured for that aqueous composition before storage, indicates that structural changes have occurred within the antigenic epitope. These structural changes within the antigenic epitope indicate reduced storage stability of the aqueous composition. The heavy metal content of an aqueous composition prepared according to the description is less than 350 ppb based on the weight of the aqueous composition. Generally, the aqueous composition with the lowest heavy metal content is more preferably less than 300 ppb, more preferably less than 275 ppb, more preferably less than 250 ppb, and more preferably less than 235 ppb based on the weight of the aqueous composition. As used herein, the term "heavy metal" refers to the total number of elements that exhibit metallic properties and includes transition metals, metalloids, lanthanides, and actinides. Transition metals are elements whose atoms have an incomplete d subshell, or that can give rise to cations with an incomplete d subshell, and include zinc, molybdenum, cadmium, scandium, titanium, technetium, palladium, vanadium, chromium, manganese, iron, cobalt, rhodium, hafnium, copper, nickel, yttrium, niobium, zirconium, rougenium, silver, tantalum, rhenium, tungsten, osmium, meitnerium, platinum, iridium, mercury, bohrium, seaborgium, and hassium. The metalloids are boron (B), silicon (Si), germanium (Ge), arsenic (As), antimony (Sb), tellurium (Te), polonium (Po).The lanthanides are the fifteen metallic chemical elements with atomic numbers from 57 to 71: lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium. The actinides are the fifteen metallic chemical elements with atomic numbers from 89 to 103: actinium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, and lawrencium. The heavy metal is preferably a metal from the d-block of the periodic table. The heavy metal is preferably a metal from group 3–12 of the periodic table, which includes the lanthanides and actinides. In a particularly preferred embodiment, the heavy metal is a metal from the D-block, or group 3-12 of the periodic table, excluding the lanthanides and actinides. Preferably, this heavy metal is an element selected from among the transition metals.In another preferred embodiment, the heavy metal is selected from Cu, Ni, W, Co, Os, Ru, Cd, Ag, Fe, V, Cr, Pb, Rb, and Mo. In another preferred embodiment, said heavy metal is a metal having a molar mass between 21 and 83, preferably said heavy metal is a D-block metal of the periodic table with a molecular mass between 21 and 83, more preferably from Cu, Ni, W, Co, Os, Ru, Cd, Ag, Fe, V, Cr, and Mo. Preferably, said heavy metal is a D-block metal of the periodic table with a molecular mass between 21-30 and 39-48, more preferably from Cu, Ni, Co, Ru, Cd, Ag, Fe, V, Cr, and Mo. In a further preferred aspect, the heavy metal is selected from the heavy metals Cu, Ni and Fe. As demonstrated in the Examples, Lot 4230 of Aluminum Hydroxide (Alum) was identified as a significant contributor to antigen degradation in the JEV vaccine FVL09L37. Example 3 shows that this Lot of Alum comprises at least the following metals: Cu, Ni, W, Co, Os, Ru, Cd, Ag, Fe, and V. Higher levels of Fe, Ni, and Cu ions were observed in Lot 4230 of Alum compared to other lots investigated. Lot 4230 was the only lot in which residual Cu ions were detected. Therefore, this heavy metal is preferably selected from the group consisting of Cu, Ni, W, Co, Os, Ru, Cd, Ag, Fe, and V, most preferably Fe, Ni, and Cu. The amount of heavy metal in the aqueous composition is preferably less than 350 ppb based on the weight of the aqueous composition. Preferably, the amount of heavy metal in the aqueous composition is less than 250 ppb, preferably less than 225 ppb, more preferably less than 200 ppb, more preferably less than 150 ppb, more preferably less than 100 ppb, more preferably less than 50 ppb, and more preferably less than 25 ppb based on the weight of the aqueous composition. The amount of heavy metal in an aqueous composition of the description has been defined earlier herein. This amount is normally for the total of the specified heavy metals, or for the heavy metals Fe, Cr, and Ni, or a combination thereof, which constitute the principal heavy metals by weight in the aqueous composition of the description. Different maximum amounts may be preferred for specific heavy metals.For example, it is preferred that the amount of Fe in the aqueous composition of the description be less than 350 ppb based on the weight of the aqueous composition. In a preferred embodiment, the amount of Fe is less than 250 ppb, preferably less than 210 ppb based on the weight of the aqueous composition. There is strong evidence that many of the pro-inflammatory effects of aluminum adjuvants are mediated through the formation of reactive oxygen species (ROS). Aluminum can, under physiological conditions, promote the reduction of Fe(III) to Fe(II) and the oxidation of the latter. Therefore, the combination of Fe and Al in the adjuvant will enhance the formation and activities of ROS (Exley, C (2010). Trends in Immunol. Vol. 31: pp. 103-109). In the present invention, it has been found that Fe can be present in an aqueous composition of the description without significantly affecting the storage stability of the composition. In this embodiment of the description, it is preferred that the aqueous composition of the description comprise between 5 ppb and 250 ppb of Fe based on the weight of the aqueous composition.At these quantities, the formation of ROS during storage due to the presence of this amount of Fe (if any ROS are present) does not significantly affect the storage stability of the aqueous composition as defined elsewhere in this document. However, the quantities are sufficient to allow for pro-inflammatory effects following in vivo administration of the vaccine. In the present invention, it has been found that the presence of the heavy metal Cu seriously affects the storage stability of the described composition. The amount of Cu in a protein-containing composition or method for producing the described protein composition is preferably less than 25 ppb. Preferably, less than 5 ppb, more preferably less than 1 ppb, and more preferably less than 0.2 ppb based on the weight of the aqueous composition. In a particularly preferred embodiment, an aqueous composition of the described composition therefore comprises less than 3 ppb of Cu based on the weight of the aqueous composition. Preferably, less than 2.5 ppb. The amount of Cu in a composition or method of the described composition is, in a particularly preferred embodiment, less than 1.25 ppb based on the weight of the aqueous composition.In a particularly preferred embodiment, said aqueous composition comprises Cu at a level that is below the detection limit of the method for the detection of copper as described in the Examples. In the present invention, it has been found that the heavy metal Ni particularly affects the storage stability of the described composition. The amount of Ni in a described composition or method is preferably less than 200 ppb. Preferably less than 40 ppb, preferably less than 9 ppb, and preferably less than 2 ppb based on the weight of the aqueous composition. In a particularly preferred embodiment, an aqueous composition of the description therefore comprises less than 40 ppb of Ni based on the weight of the aqueous composition. Preferably less than 30 ppb, more preferably less than 20 ppb, and more preferably less than 15 ppb of Ni based on the weight of the aqueous composition. In a particularly preferred embodiment, said aqueous composition comprises Ni at a level that is below the detection limit of the method for detecting nickel as described in the Examples. The heavy metal may be present in its electronically neutral form or it may be ionized. Normally and preferably, the heavy metal is present in its ionic form in an aqueous composition as described. The metal content of a composition can be determined in various ways. In one respect, a method as described involves determining the level of a heavy metal in an aqueous composition and / or the aluminum salt present in that aqueous composition. Methods for measuring the level of one or more heavy metals in an aqueous solution are known in the art. Examples of such methods include mass spectrometry, such as inductively coupled plasma mass spectrometry (ICP-MS), flame atomic absorption spectrometry (F-AAs), and / or graphite furnace atomic absorption spectrometry (GF-AAS). Example 3 describes an example of an assay that can be used to determine the heavy metal content. The assay involves treating a sample of an aqueous solution containing an aluminum salt with concentrated HNO3 under heat until a clear solution is obtained. The clear solution can then be diluted and further analyzed, for example, by ICP-MS, F-AAS, and / or GF-AAS, to determine the presence and content of metal ions, including Pb, Cd, Cr, Co, Fe, Cu, Ni, Ag, W, and Al. Examples 1 and 2 demonstrate that the JEV antigen exhibits greater stability at pH 7.5–8 compared to pH 7. In the examples, antigen stability is expressed as the monoclonal / polyclonal ELISA ratio. The monoclonal antibody used (clone 52-2-5) was shown to recognize a neutralizing epitope in the Japanese encephalitis vaccine (JEV). The relative specific epitope content can be expressed as the ratio of total antigen content determined by monoclonal ELISA to total antigen content determined by polyclonal ELISA. While not explicitly linked to the theory, the effect of greater antigen stability at pH 7.5–8 can be explained based on the underlying assumed complex reaction chemistry of sulfites.The pH could influence the reaction conditions related to the sulfite / formaldehyde reaction equilibrium and the surface charge of certain protein / amino acid side chains accessible to modification. The pH can affect oxidation by directly influencing the redox potentials of amino acid residues and oxidizing agents, such as free radicals. Therefore, in one embodiment, a method according to the description comprises buffering said aqueous composition at a pH between 7.5 and 8.5. Various aluminum salts are being used in compositions for administration to an individual. The aluminum adjuvant typically contains an aluminum oxide or sulfate, or a combination thereof. In a preferred embodiment, the aluminum salt comprises aluminum oxide (AhO3), aluminum hydroxide (Al(OH)3), or aluminum phosphate (AlPO4). In a preferred embodiment, the aqueous composition described further comprises a reactive compound. Typically, though not necessarily, the reactive compound is present as a result of manipulation of the aqueous composition, for example, to treat or inactivate an infectious agent, if present in the composition. The reactive compound may also be present for another reason. Sulfite, for example, is sometimes present to inactivate any residual formaldehyde in the aqueous solution. Formaldehyde is a chemical commonly used to inactivate infectious agents. The reactive compound is preferably a redox active compound, a radical-forming compound, and / or a stabilizing compound. In a preferred embodiment, the aqueous composition described comprises formaldehyde, ethanol, chloroform, trichloroethylene, acetone, Triton-X-100, deoxycholate, diethyl pyrocarbonate, sulfite, Na2S2O5, beta-propriolactone, polysorbate such as Tween 20®, Tween 80®, O2, phenol, pluronic-type copolymers, or a combination thereof. Sulfite is preferably present in an amount between 0.1 mM and 5 mM, or preferably between 0.5 and 2 mM. Formaldehyde is preferably present in an amount between 0.1 mM and 5 mM, more preferably between 0.5 mM and 2 mM. Oxygen is preferably present in an amount equivalent to the solubility of O₂ at the measured temperature; O₂ is preferably present in an amount between 10 and 250 µM when measured at 20°C. When measured at 0°C, O₂ is preferably present in an amount between 10 and 400 µM. A stabilizing compound is preferably present in an amount between 10 and 400 µM. Similarly, a redox-active compound is present in an amount between 0.1 mM and 5 mM, or preferably between 0.5 and 2 mM. A radical-forming compound is preferably present in an amount between 0.1 mM and 5 mM, or preferably between 0.5-2 mM.In this context, and for the sake of clarity, it is important to point out that the redox active compound, the radical-forming compound, and / or the stabilizing compound are consumed in the production of a radical, whereas the heavy metal mentioned earlier in this document acts as a catalyst in the production of a radical and is not consumed as such. Therefore, the redox active compound, the radical-forming compound, and / or the stabilizing compound is not a heavy metal. The total amount of redox-active compound, radical-forming compound, and / or stabilizing compound, although small in absolute quantities, may still be significant relative to the antigen or protein in the aqueous composition described. The antigen / protein is preferably present in an amount between 0.1 nmol and 1 µmol, more preferably between 1 nmol and 100 nmol. The concentration of protein, preferably a therapeutic or vaccine protein, in an aqueous composition comprising a protein as described is preferably between 1 ng / ml and 10 mg / ml, preferably between 10 ng / ml and 1 mg / ml, more preferably between 100 ng / ml and 100 mcg / ml, such as between 1 mcg / ml and 100 mcg / ml. The concentration is preferably at least 1 ng / ml to ensure that the therapeutic or vaccine protein is at a concentration sufficient to exert its therapeutic effect when administered to an individual. However, the concentration should preferably not exceed 10 mg / ml to prevent or reduce the occurrence of possible side effects associated with the administration of said protein to an individual. In particular, the concentration of viral protein in an aqueous composition as described comprising JEV is preferably between 0.01 pg / ml and 1 mg / ml, more preferably between 0.1 mcg / ml and 100 mcg / ml.In an example embodiment of the description, an aqueous composition as described comprises approximately 10 mcg / ml of JEV. The dose of a single administration of an aqueous composition comprising a protein, preferably a therapeutic or vaccine protein, as described, is preferably between 0.1 ml and 10 ml, preferably between 0.5 ml and 5 ml, such as 0.5 ml, 1 ml, 1.5 ml, 2 ml, 2.5 ml, because such a dose permits convenient administration to an individual, such as a human being. The aqueous composition as described may further comprise a nucleic acid molecule. The nucleic acid may be administered for therapeutic purposes. In that case, the aqueous composition as described preferably comprises a nucleic acid molecule, such as a plasmid comprising the nucleic acid sequence encoding an antigen or antigens, or a virus, or bacterium, or an immunogenic portion thereof, against which an immune response is sought. The incorporation of said nucleic acid molecule is based on the in situ production of the target antigen, virus, bacterium, or immunogenic portion. In another preferred embodiment, the aqueous composition as described comprises a nucleic acid in the form of an antisense RNA, RNAi, or an RNAi mimetic.In another embodiment, the aqueous composition as described comprises the nucleic acid in the form of an infectious agent as such, a virus, or a modified virus, as is the case in many gene therapy approaches. The nucleic acid concentration in the aqueous composition, immunogenic composition, or vaccine as described is preferably in the range of approximately 1 ng / ml to approximately 10 mg / ml, preferably from approximately 0.1 mcg / ml to approximately 1 mg / ml, and more preferably from approximately 1 mcg / ml to approximately 100 mcg / ml. The appropriate dosage will depend on the individual receiving the composition and the size of the nucleic acid sequences present in the composition. An aqueous composition as described may further comprise a polysaccharide and / or an oligosaccharide, preferably the polysaccharide and / or oligosaccharide capsule of encapsulated bacteria against which an immune response is sought. Examples of polysaccharides and oligosaccharides that may be present in an aqueous composition as described include pneumococcal polysaccharides, meningococcal polysaccharides, Haemophilus influenzae type b polysaccharide, group B streptococcal polysaccharides, Salmonella typhi VI polysaccharide, polysaccharides or oligosaccharides derived from group A streptococcus, staphylococci, Neisseria meningitidis, Klebsiella pneumoniae, enterococci, E. coli, Pseudomonas aeruginosa, and Bacillus anthracis. Said polysaccharide and / or oligosaccharide may be present in the aqueous composition as described, or alternatively, the polysaccharide and / or oligosaccharide may be conjugated with a protein.The concentration of polysaccharide and / or oligosaccharide in the aqueous composition, immunogenic composition, or vaccine as described is preferably in the range of approximately 10 ng / ml to approximately 500 mcg / ml, more preferably from approximately 0.1 mcg / ml to approximately 500 mcg / ml, or more preferably from approximately 1 mcg / ml to approximately 50 mcg / ml. The appropriate dosage will depend on the individual to whom the composition is administered. A method according to the invention is preferably used to increase the stability of an immunogenic composition, preferably a vaccine, comprising an aluminum salt-based adjuvant. Examples of such vaccines include those directed against infection by Bacillus anthracis (which causes anthrax), Cor and nebacterium diphtheriae (which causes diphtheria), Clostridium tetani (which causes tetanus), pseudomonas such as Pseudomonas aeruginosa, staphylococci such as Staphylococcus aureus or Staphylococcus epidermidis, Haemophilus influenzae type B (Hib) bacteria, poliovirus, hepatitis A virus, hepatitis B virus, human papillomavirus, influenza virus, Japanese encephalitis virus, rotavirus, Rickettsia bacteria (which cause typhus), yellow fever virus, varicella-zoster virus, meningococcus, or combinations thereof, such as DTP (diphtheria, tetanus, polio).Therefore, an aqueous composition comprising a protein as described preferably comprises a protein that is a viral or bacterial protein, preferably a protein from Bacillus anthracis, Corynebacterium diphtheriae, Clostridium tetani, Haemophilus influenzae type B (Hib) bacteria, poliovirus, hepatitis A virus, hepatitis B virus, human papillomavirus, influenza virus, Japanese encephalitis virus, rotavirus, Rickettsia bacteria, yellow fever virus, varicella-zoster virus, and / or meningococcus. In a preferred embodiment, said protein contained in a composition comprising a protein as described is a viral protein from a virus of the Flaviviridae family, preferably from a Japanese encephalitis virus (JEV).As demonstrated in the Examples, the stability of aqueous compositions comprising a JEV protein, an aluminum salt, and less than 350 ppb of heavy metal based on the weight of the aqueous composition, and in particular where the amount of Cu is less than 3 ppb based on the weight of the aqueous composition, is increased compared to aqueous compositions comprising more than 350 ppb of heavy metal and more than 3 ppb of Cu. Therefore, a method according to the invention is particularly suitable for increasing the stability of an aqueous composition comprising a JEV protein. Tables 25 and 26 provide a list of example aluminum-based vaccines for both human and veterinary use. A method particularly suitable for increasing the stability of a vaccine listed in Table 25 and / or Table 26 is also described.Therefore, a vaccine listed in Table 25 and / or Table 26 is preferably prepared using a method of the invention. In another embodiment or preferred aspect of the description, the protein contained in a composition comprising a protein as described is a bacterial protein from a bacterium of the Pseudomonas family, preferably Pseudomonas aeruginosa. As demonstrated in the Examples, the stability of aqueous compositions comprising Pseudomonas aeruginosa fusion protein (SEQ ID NO: 1) and an aluminum salt is reduced when more than 350 ppb of heavy metal is present, based on the weight of the aqueous composition. An aqueous composition comprising a protein as described is particularly suitable for use as a vaccine or immunogenic composition. For example, such compositions are particularly useful for immunizing an individual to treat or prevent a viral or bacterial infection. In one embodiment, the invention therefore provides a method for treating an individual comprising obtaining an immunogenic aqueous composition comprising a protein and an aluminum salt, said aluminum salt having less than 350 ppb of heavy metal based on the weight of the aqueous composition, preferably less than 3 ppb of Cu, and administering the immunogenic aqueous composition to an individual in need.A method is also provided for the prophylactic treatment of an individual, comprising obtaining an immunogenic aqueous composition comprising a protein and an aluminum salt, said aluminum salt having less than 350 ppb of heavy metal based on the weight of the aqueous composition, preferably less than 3 ppb of copper, and administering the immunogenic aqueous composition to an individual in need. A method is further provided for inducing and / or enhancing an immune response to an antigen in an individual, said method comprising obtaining an aqueous composition comprising a protein comprising said antigen and an aluminum salt, said aluminum salt having less than 350 ppb of heavy metal based on the weight of the aqueous composition, preferably less than 3 ppb of copper, and administering the aqueous composition to an individual in need.In another aspect, the invention provides a method for immunizing an individual comprising administering to said individual at least two immunogenic compositions with an interval of at least two weeks between each administration, wherein each of said at least two immunogenic compositions comprises the same antigen, and wherein at least one of said immunogenic compositions further comprises an aluminum salt having less than 350 ppb of heavy metal based on the weight of the aqueous composition, preferably less than 3 ppb of Cu, and administering the aqueous immunogenic composition to an individual in need. Nucleic acid compositions are sometimes also co-administered with aluminum. Therefore, for the purposes of the present description, it is possible to replace "protein" in an aqueous composition of the description with nucleic acid.Therefore, in one embodiment, the description provides a method for preparing an aqueous composition comprising aluminum and a nucleic acid, said method comprising. - combine an aluminum salt, said nucleic acid and water to produce said aqueous composition and - determine the level of a heavy metal in the aqueous composition and / or the aluminum salt. The description also provides a method for preparing an aqueous composition comprising aluminum and a nucleic acid, said method comprising - Prepare or select an aluminum salt containing less than 350 ppb of heavy metal based on the weight of the final aqueous composition, preferably less than 3 ppb of Cu, and administer the immunogenic aqueous composition to an individual in need and - combine said aluminum salt, said nucleic acid and water to produce said aqueous composition. In a preferred embodiment, these methods further comprise buffering the aqueous composition to a pH between 7.5 and 8.5. In a particularly preferred embodiment, these methods further comprise packaging aliquots of the aqueous composition containing less than 350 ppb of heavy metal, based on the weight of the aqueous composition, into separate airtight storage containers. The nucleic acid can be administered for therapeutic purposes, for example, in the form of an antisense RNA, RNAi, or an RNAi mimetic. The nucleic acid can also be administered in the form of an infectious agent, typically a virus or a modified virus, as is the case in many gene therapy approaches. In that case, the nucleic acid is enclosed in a protein-containing particle.Therefore, the description further provides an aqueous composition comprising a nucleic acid and an aluminum salt, said composition comprising less than 350 ppb of heavy metal based on the weight of the aqueous composition. The nucleic acid concentration in the aqueous composition, immunogenic composition, or vaccine as described is preferably in the range of approximately 1 ng / ml to approximately 10 mg / ml, preferably from approximately 0.1 mcg / ml to approximately 1 mg / ml, and more preferably from approximately 1 mcg / ml to approximately 100 mcg / ml. The appropriate dosage will depend on the individual to whom the composition is administered and the size of the nucleic acid sequences present in the composition. Compositions comprising polysaccharide, oligosaccharide, or polysaccharide-polypeptide conjugates are sometimes also administered with aluminum. Therefore, for the purposes of this description, "protein" in an aqueous composition of the description may be replaced with polysaccharide, oligosaccharide, or polysaccharide-polypeptide conjugate, or a combination thereof. Such polysaccharide or oligosaccharide is preferably the polysaccharide or oligosaccharide from the capsule of encapsulated bacteria against which an immune response is sought.Examples of polysaccharides and oligosaccharides that may be present in an aqueous composition as described include pneumococcal polysaccharides, meningococcal polysaccharides, Haemophilus influenzae type b polysaccharide, group B streptococcal polysaccharides, Salmonella typhi VI polysaccharide, polysaccharides or oligosaccharides derived from group A streptococcus, staphylococci, Neisseria meningitidis, Klebsiella pneumoniae, enterococci, E. coli, Pseudomonas aeruginosa, and Bacillus anthracis. Such polysaccharide and / or oligosaccharide may be present in the aqueous composition as described, or alternatively, the polysaccharide and / or oligosaccharide may be conjugated to a protein. Accordingly, in one embodiment, the description provides a method for preparing an aqueous composition comprising aluminum and a polysaccharide or oligosaccharide, the method comprising said method. - combining an aluminum salt, said polysaccharide or oligosaccharide and water to produce said aqueous composition and - determine the level of a heavy metal in the aqueous composition and / or the aluminum salt. The description also provides a method for preparing an aqueous composition comprising aluminum and a polysaccharide or oligosaccharide, said method comprising - Prepare or select an aluminum salt containing less than 350 ppb of heavy metal based on the weight of the final aqueous composition, preferably less than 3 ppb of Cu, and administer the immunogenic aqueous composition to an individual in need and - combine said aluminum salt, said polysaccharide or oligosaccharide and water to produce said aqueous composition. The concentration of polysaccharide and / or oligosaccharide in the aqueous composition, immunogenic composition, or vaccine as described is preferably in the range of approximately 10 ng / ml to approximately 500 pg / ml, more preferably from approximately 0.1 mcg / ml to approximately 500 mcg / ml, or more preferably from approximately 1 mcg / ml to approximately 50 mcg / ml. The appropriate dosage will depend on the individual to whom the composition is administered. The polysaccharide-polypeptide conjugate as described herein comprises at least one polysaccharide and at least one polypeptide. The polysaccharide as described is preferably a bacterial capsular polysaccharide. The capsular polysaccharides can be prepared using conventional techniques known to those skilled in the art. In one embodiment, the polysaccharide is a capsular polysaccharide of S. pneumoniae. In another embodiment, the S. pneumoniae capsular polysaccharide is selected from the group consisting of serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 1OA, HA, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F, representing the 23 pneumococcal serotypes that cause the vast majority of pneumococcal diseases in all age groups, out of the more than 90 serotypes known to date. The term "polysaccharide," as used herein, refers to polysaccharides and / or oligosaccharides. Polysaccharides are isolated from bacteria and can be depolymerized to a preferred size range by known methods (see, for example, EP 497524 and EP 497525). Oligosaccharides have a low number of repeating units (typically 5 to 30 repeating units) and are usually hydrolyzed polysaccharides. The capsular polysaccharides of Streptococcus pneumoniae comprise repeating oligosaccharide units that can contain up to eight sugar residues. For a review of the oligosaccharide units for key serotypes of Streptococcus pneumoniae, see Jones et al., An. Acad. Bras. Cienc, 2005, 77(2):293–324. In one embodiment, a capsular saccharide antigen may be a full-length polysaccharide; however, in others, it may be an oligosaccharide unit, or a saccharide chain of shorter-than-native length composed of repeating oligosaccharide units. Full-length polysaccharides can be "sized" or "depolymerized," i.e., their size can be reduced by various methods known in the art (as described above). The term "depolymerization" includes partial depolymerization. Subsequently, the depolymerization of the polysaccharides may be followed by an activation step before conjugation with a carrier polypeptide. "Activation" refers to the chemical treatment of the polysaccharide to provide chemical groups capable of reacting with the carrier polypeptide. Suitable methods for this are known in the art. "Polypeptide" or "protein" means any chain of amino acids having at least 10 and preferably at least 100 amino acids linked together by peptide bonds, irrespective of post-translational modification. Suitable polypeptide carriers include diphtheria toxin, diphtheria toxoid, CRM 197, tetanus toxoid, pertussis toxoid, E. coli lT, E. coli ST, exotoxin A, outer membrane complex c (OMPC), porin, transferrin-binding protein, pneumolysis, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), ovalbumin, California limpet hemocyanin (KLH), bovine serum albumin (BSA), or purified protein derivative of tuberculin (PPD). The carrier polypeptides are preferably non-toxic and non-reactogenic polypeptides that can be obtained in sufficient quantity and purity. In a particularly preferred embodiment, the carrier polypeptide comprises a tetanus toxoid.In another preferred embodiment, the carrier polypeptide comprises a derivative of any of the carrier polypeptides mentioned above, for example, a subunit or a mutated version of E. coli LT, such as LT or the LT subunit A (LTA) having an amino acid substitution at position aa 192 (for example, LTG 192, LTT 192, LTS 192, LTA 192), Lt K 63 LTR 72, or other mutants as described, for example, in documents WO 98 / 42375, WO 02 / 64162, US 4,761,372, US 5,308,835. The polypeptides may also contain elongations at the carboxy or amino end of the polypeptide that facilitate interaction with the polycationic compound(s) or the immunostimulatory compound(s). In addition, polypeptides can also be derivatized to include molecules that enhance antigen presentation and targeting of antigens to antigen-presenting cells. The polypeptide can be activated before conjugation. The nature and size of the saccharide, the nature and size of the protein or polypeptide, the ratio of saccharide to protein / polypeptide, as well as other factors and conditions for the preparation of a conjugate according to the present description may be determined by an expert, as described, for example, in Robbins et al., JAMA, 1996, 276 (14): 1181-5. For example, for a pneumococcal polysaccharide and a tetanus toxoid, a preferred ratio is approximately 2:1. A "conjugate" is a compound in which the polysaccharide is covalently linked to a carrier polypeptide. Many conjugation reactions known in the prior art have been used to covalently link polysaccharides to polypeptides to produce a polysaccharide-polypeptide conjugate.Three of the most commonly employed methods include: 1) reductive amination, in which the aldehyde or ketone group in one reaction component reacts with the amino or hydrazide group in the other component, and the resulting CN double bond is subsequently reduced to a CN single bond by a reducing agent; 2) cyanylation conjugation, in which the polysaccharide is activated by cyanogen bromide (CNBr) or l-cyano-4-dimethylammoniumpyridinium tetrafluoroborate (CDAP) to introduce a cyanate group into the hydroxyl group, which forms a covalent bond with the amino or hydrazide group upon addition of the protein component; and 3) a carbodiimide reaction, in which carbodiimide activates the carboxyl group in one conjugation reaction component, and the activated carbonyl group reacts with the amino or hydrazide group in the other component.These reactions are also frequently used to activate the components of the conjugate before the conjugation reaction. The polysaccharide can be conjugated to the polypeptide directly or through a linker. Linking through a linker group can be carried out using any known procedure, for example, the procedures described in US patents 4,882,317 and 4,695,624. Suitable linkers include carbonyl, adipic acid, B-propionamido (document WO 00 / 10599), nitrophenylethylamine (Gever et al., Med. Microbiol. Immunol, 1979, 165:171-288), haloacyl halides (document US 4,057,685), glycosidic linkages (documents US 4,673,574; US 4,761,283; US 4,808,700), 6-aminocaproic acid (document US 4,459,286), ADH (document US 4,965,338) and C4 to Ci2 moieties (document US 4,663,160). After the polysaccharide is conjugated to the carrier polypeptide, the polysaccharide-polypeptide conjugate can be purified (enriched with respect to the amount of polysaccharide-polypeptide conjugate) using a variety of techniques known in the art. One objective of the purification step is to remove the unbound polysaccharide and / or polypeptide from the polysaccharide-polypeptide conjugate. Purification methods include, for example, ultrafiltration in the presence of ammonium sulfate, size exclusion chromatography, density gradient centrifugation, and hydrophobic interaction chromatography. Aluminum adjuvants, such as aluminum hydroxide and aluminum phosphate, are generally prepared by exposing aqueous solutions of aluminum ions to typically slightly alkaline conditions in a well-defined and controlled chemical environment. Various soluble aluminum salts can be used to produce aluminum hydroxide. Anions present at the time of precipitation may coprecipitate with the aluminum hydroxide or sulfate. After precipitation of the ammonium salt by pH shift using, for example, NaOH, it is not possible to remove any heavy metals present in the aluminum adjuvant. Even thorough washing does not result in a sufficient reduction of the heavy metal content.Therefore, it is important to control heavy metals before the precipitation of the aluminum salt. This is achieved by selecting appropriate raw materials and controlling process conditions. For example, if ammonia alum and aluminum sulfate or other aluminum sources are in solution, no metal should be added, such as other salts like CuSO3. Alternatively, any heavy metals present in the solution can be removed before the precipitation of the aluminum adjuvant. This can be accomplished, for example, through crystallization or ion exchange (cations), which are well-known methods in the art. Ion exchange refers to a process by which (metal) ions in solution are transferred to a solid matrix, which, in turn, releases ions of a different type but the same polarity into the solution.Therefore, the ions in solution are replaced by different ions originally present in the solid matrix. Metal crystallization refers to the precipitation of insoluble metal crystals from metal ions in solution. Therefore, in a preferred embodiment, the description provides a method for preparing an aqueous composition comprising aluminum, a reactive compound, and a protein, the method comprising said method. - prepare an aluminum salt that is capable of providing an aqueous composition having less than 350 ppb of heavy metal based on the weight of the aqueous composition and - combine said aluminum salt, said reactive compound, said protein and water to produce said aqueous composition, Therefore, said aluminum salt is prepared by preparing an aqueous solution of aluminum ions, removing heavy metals from said aqueous solution such as by crystallization or ion exchange, preferably cation exchange, and precipitating said aluminum ions from said solution, preferably using a base. In addition, a method is provided for preparing a clinical-grade aluminum salt precipitate for incorporation into a drug and / or vaccine, said method comprising preparing an aqueous solution of aluminum ions, removing heavy metals from said aqueous solution such as by crystallization or ion exchange, preferably cation exchange, and precipitating said aluminum ions from said solution, and determining the level of a heavy metal in the solution and / or the aluminum salt precipitate, wherein the precipitate is selected that is capable of providing an aqueous composition comprising less than 350 ppb of heavy metal based on the weight of the aqueous composition. The preparation of aluminum hydroxide from raw materials is described, among others, in documents CN101734698 and WO98 / 14401. The description further provides a method for preparing an aqueous pharmaceutical or vaccine composition comprising aluminum, a reactive compound, and a protein, said method comprising - select an aluminum salt that is capable of providing an aqueous composition that has less than 350 ppb of heavy metal based on the weight of the aqueous composition and - combining said aluminum salt, said reactive compound, said protein and water to produce said aqueous composition (i) having less than 350 ppb of heavy metal based on the weight of the aqueous composition and (ii) comprising between 5 pg / ml and 50 mg / ml of aluminum; where the reactive compound is selected from the group consisting of a redox active compound, a radical-forming compound, a stabilizing compound, and a combination of any of the same. Preferably, the method further comprises buffering said aqueous composition to a pH of between 6.5 and 8.5. Preferably, the method further comprises packaging aliquots of said aqueous composition having less than 350 ppb of heavy metal based on the weight of the aqueous composition in separate airtight storage containers. The description further provides a method for selecting a clinical-grade aluminum salt precipitate for incorporation into a drug and / or vaccine, said method comprising preparing an aqueous solution of aluminum ions and precipitating said aluminum ions from said solution, and determining the level of a heavy metal in the solution and / or the aluminum salt precipitate, wherein the precipitate is selected that is capable of providing an aqueous composition comprising (i) less than 350 ppb of heavy metal based on the weight of the aqueous composition and (ii) between 5 pg / ml and 50 mg / ml of aluminum.An aqueous pharmaceutical or vaccine composition comprising a protein, a reactive compound, and an aluminum salt is also provided, said composition comprising (i) less than 350 ppb of heavy metal based on the weight of the aqueous composition and (ii) between 5 pg / ml and 50 mg / ml of aluminum, wherein the reactive compound is selected from the group consisting of a redox active compound, a radical-forming compound, a stabilizing compound, and a combination thereof. In a preferred embodiment, said heavy metal is selected from Cu, Ni, W, Co, Os, Ru, Cd, Ag, Fe, V, Cr, Pb, Rb, and Mo. In a particularly preferred embodiment, the heavy metal is selected from Cu or Ni. The heavy metal is preferably present in ionic form. The aluminum salt is preferably aluminum hydroxide (Al(OH)3) or aluminum phosphate (AlPO4).The aluminum salt is preferably aluminum hydroxide (Al(OH)3). The reactive compound is preferably selected from the group consisting of formaldehyde, ethanol, chloroform, trichloroethylene, acetone, 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, deoxycholate, diethyl pyrocarbonate, sulfite, Na2S2Os, beta-propriolactone, polysorbate such as polyethylene glycol sorbitan monolaurate, polyethylene glycol sorbitan monooleate, O2, phenol, pluronic-type copolymers, and a combination of any of the same. The aqueous vaccine pharmaceutical composition preferably comprises between 50 pg / ml and 5 mg / ml of aluminum. The aqueous or vaccine pharmaceutical composition preferably comprises between 5 ppb and 250 ppb of Fe based on the weight of the aqueous composition. The aqueous pharmaceutical or vaccine composition preferably comprises less than 3 ppb of Cu based on the weight of the aqueous composition.The aqueous pharmaceutical composition or vaccine preferably comprises less than 40 ppb of Ni based on the weight of the aqueous composition. The protein in said pharmaceutical composition or vaccine preferably comprises less than 40 ppb of Ni based on the weight of the aqueous composition. The protein in said aqueous pharmaceutical composition or vaccine is preferably a therapeutic agent and / or a vaccine. The protein is preferably a viral or bacterial protein. The viral protein is preferably a protein from the Japanese encephalitis virus or a protein from the bacterium Pseudomonas aeruginosa. The protein is preferably a protein within a formaldehyde-inactivated virus particle. The aqueous pharmaceutical composition or vaccine preferably further comprises sulfite. The description further provides a vaccine comprising an aqueous vaccine composition as described. Where in this document an interval between the values ​​X and Y is indicated, the interval includes the values ​​X and Y. The description further provides a method for preparing an aqueous composition comprising aluminum, a reactive compound, and a protein, said method comprising - prepare or select an aluminum salt that is capable of providing an aqueous composition that has less than 450 ppm of heavy metal based on the weight of aluminum (grams / grams) and - combining said aluminum salt, said reactive compound, said protein, and water to produce said aqueous composition. Preferably, the Fe content of the aqueous composition is less than 700 ppm based on the weight of aluminum in the composition; the Ni content is less than 18 ppm based on the weight of aluminum in the composition; or the Cu content is less than 2.5 ppm based on the weight of aluminum in the aqueous composition, or a combination thereof. Preferably, the method further comprises buffering said aqueous composition to a pH between 6.5 and 8.5. Preferably, the method further comprises packaging aliquots of said aqueous composition having less than 450 ppm of heavy metal based on the weight of aluminum in the composition into separate airtight storage containers. The description further provides a method for preparing a clinical-grade aluminum salt precipitate for incorporation into a drug and / or vaccine, said method comprising preparing an aqueous solution of aluminum ions and precipitating said aluminum ions from said solution, and determining the level of a heavy metal in the solution and / or the aluminum salt precipitate, wherein the precipitate is selected that is capable of providing an aqueous composition comprising less than 450 ppm of heavy metal based on the weight of the aluminum ions (grams / grams) in the solution. An aqueous composition comprising a protein and an aluminum salt is also provided, said composition comprising less than 450 ppm of heavy metal based on the weight of the aluminum in the composition. The aqueous composition has preferably been stored at temperatures above 20 °C for at least 1 month. The heavy metal is preferably selected from Cu, Ni, W, Co, Os, Ru, Cd, Ag, Fe, V, Cr, and Mo. Preferably, the heavy metal is selected from Cu, Ni, W, Co, Os, Ru, Cd, Ag, Fe, and V. In a particularly preferred embodiment, the heavy metal is selected from Cu or Ni. Preferably, the heavy metal is Cu. The invention is further explained in the following examples. References Alipazaga MV, Moreno RGM, Coichev N. 2004. Synergistic effect of Ni (II) and Co (II) ions on the sulphite induced autoxidation of Cu (II) / tetraglycine complex. Dalton Trans 13:2036-2040. Arunee Wittayanukulluk, Dongping Jiang, Fred E. Regnier, Stanley L. Hem, "Effect of microenvironment pH of aluminum hydroxide adjuvant on the chemical stability of adsorbed antigen", Vaccine 22 (2004) 1172-1176 Brandt C, Elding LI. 1998. Role of chromium and vanadium in the atmospheric oxidation of sulfur (IV) . Atmos Environ 32 (4) :797-800. Exley, C (2010) . Trends in Immunol. Vol. 31: págs. 103-109. Ito, Kimiko y Kawanashi, Shosuke. Site-specific fragmentation and modification of Albumin by sulphite in presence of metal ions or peroxidase / H2O2: Role of Sulphate radical. Biochem and Biophys Res Comm., 1991, 176, 1306-1312 Huie R.E., Neta P. One-electron redox reaction in aqueous solutions of sulphite with hydroquinone and other hydroxyphenols. J. Phys. Chem., 1985, 89 (18) , 3918-3921 Kalina Ranguelova, Marcelo G. Bonini y Ronald P. Mason: (Bi) sulphite Oxidation by Copper, Zinc-Superoxide Dismutase: Sulphite- Derived, Radical-Initiated Protein Radical Formation. Environmental Health Perspectives 2010, 118 (7) , 970-975 Lambeth D.O., Palmer G. The kinetics and mechanism of reduction of electron transfer proteins and other compounds of biological interest by dithionite. J. Biochem. Chem. 1973, 248, 6095-6103 Li S, Schoneich C, Borchardt RT. Chemical instability of protein pharmaceuticals: Mechanisms of oxidation and strategies for stabilization. Biotechnol Bioeng. 5 de diciembre de 1995;48 (5) :490-500 Lindblad, EB (2004) Immunol. y Cell Biol. Vol. 82: 497-505. Lima S, Bonifacio RL, Azzellini GC, Coichev N. 2002. Ruthenium (II) tris (bipyridyl) ion as a luminescent probe for oxygen uptake on the catalyzed oxidation of HSO3-. Talanta 56:547-556. Mayo JC, Tan DX, Sainz RM, Natarajan M, Lopez-Burillo S, Reiter RJ. Protection against oxidative protein damage induced by metal-catalyzed reaction or alkylperoxyl radicals: comparative effects of melatonin and other antioxidants. Biochim Biophys Acta. 17 de marzo de 2003;1620 (1-3) :139-50. Neta P., Huie RE: Free Radical Chemistr y of Sulphite. Environmental Health Perspectives 1985, 64, 209-217 Shi X. 1994. Generation of •SOa- and OH radicals in SO32- reactions with inorganic environmental pollutants and its implications to SO32- toxicity. J Inorg Biochem 56 (3) :155-165. Stadtman ER. Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. Free Radic Biol Med. 1990;9 (4) :315-25. Breve descripción de los dibujos Figura 1: Perfiles de elución por RP-HPLC del extracto de tapón de clorobutilo (diluido 1:4) y JEV09L37 SN. Figure 2: HPLC SEC elution profiles of PS (2 mg / ml) before and after trypsin excision. Figure 3: HPLC SEC elution profiles of trypsin-treated PS and degraded PS as present in NIV11A74. Note that the elution profiles were normalized to a similar peak height to allow for better comparison. Figure 4: DOE evaluation of the monoclonal / polyclonal ELISA ratio using Pareto chart analysis and main effects plots (4 weeks at 22 °C). Figure 5: DOE evaluation of the monoclonal / polyclonal ELISA ratio using Pareto chart analysis and main effects plots (8 weeks at 22 °C). Figure 6: Contour plot of the estimated response Figure 7: Residual graph of the estimated response Figure 8: ELISA ratio (monoclonal / polyclonal) for JEV formulations at pH 7 in the presence of Ni, Cu and Cr. Samples were stored at 22 °C for 5 weeks. Figure 9: Summary of results obtained after 7 weeks at 22 °C. Raw data for the relationship as a function of pH and metal ion type are shown, as well as the combined results for each parameter. Figure 10: Mean ratio of drug product (DP) formulations prepared with different lots of alum. Samples were stored for 6 weeks at 22°C. Error bars represent the 95% confidence interval calculated based on the pooled standard deviation. Examples from left to right: Alum 3877, Alum 4074, Alum 4230 non-GI, Alum 4230 GI, Alum 4470, Alum 4563, Alum 4621, Alum 4074-4230 mixture. Figure 11: Particle size distribution of Alhydrogel® samples. Figure 12: Titration curves of Alhydrogel® in PBS. Non-irradiated AIOH (RQCS0890) , AA AIOH GI (RQCS1200) , AIOH GI (RQCS1342) , AIOH GI (RQCS0448) Figure 13: Overview of the Alhydrogel® batches tested. The total concentration of contaminating metal ions in ng / ml and the proportion of the main metal ions Fe, Cr, and Ni are shown. Figure 14: Amino acid sequence of Ala-(His)6-OprF190-342-OprI21-83 (SEQ ID NO: 1.- in this document also referred to as "protein A". Examples Example 1 It was previously identified that Lot 4230 of aluminum hydroxide (alum) significantly contributes to the degradation of the antigen in FVL09L37. This particular lot of alum exhibited a much higher residual metal ion content compared to other lots of alum used in the formulation of the inactivated JEV antigen. This example demonstrates further studies conducted to identify the underlying root cause mechanism and the influence of metal ions on the JEV degradation pathway. A design of experiments (DOE) was implemented to determine the influence of individual parameters on antigen stability. The parameters tested in a full 25 factorial DOE were • Aluminum Hydroxide Lot 4230 vs. Aluminum Hydroxide Lot 4074 • Presence of excess protamine sulfate fragments • Presence of leachable materials from the chlorobutyl rubber stopper • pH range 7 to 8 • Residual formaldehyde content Lot 4230 of alum contains significantly higher levels of residual metal ion impurities compared to other lots of alum used in the JEV formulation. A design of experiments (DOE) was selected to further investigate the potential root cause mechanism and the interaction of parameters that could ultimately lead to product degradation. In factorial designs, multiple factors are investigated simultaneously during the trial. As in single-factor designs, both qualitative and quantitative factors can be considered. The objective of these designs is to identify the factors that have a significant effect on the response, as well as to investigate the effect of interactions (depending on the experimental design used). Predictions can also be made when quantitative factors are present, but caution is advised since certain designs are very limited in their choice of predictive model.For information on DOE in general, see (Siebertz, Karl; van Bebber, David, Hochkirchen, Thomas: Statistische Versuchsplanung: Design of Experiments (DoE). Editor: Springer Berlin Heidelberg; 1st edition (2010), ISBN-10: 3642054927). 1.1 DOE Study Design 1.1.1 Definition of Parameters and Levels for DOE Design The following parameters and levels were taken into consideration to design an appropriate DOE experiment: • Residual metal ion content of alum: Aluminum hydroxide lots 4230 and 4074 were selected as representative of the two quality extremes with respect to the residual metal ion content of aluminum hydroxide. The midpoint was a 50 / 50% mixture of both alum lots. Initial analysis of the remaining metal ion impurities in the 2% aluminum hydroxide stock solution by ICP-MS showed significant differences in the Cr, Fe, Ni, and Cu ion content between these two lots (see Table 1). • Protamine sulfate fragments: Protamine sulfate (PS) fragments are present in low quantities (<5 pg / ml) in the final vaccine batch. It was investigated whether PS fragments could contribute to modification of the virus surface (e.g., interaction / covalent binding with virus surface proteins) in combination with alum and other factors used in this study. Therefore, a stock solution of PS fragments was prepared by trypsin digestion followed by heat inactivation and ultrafiltration using a 5 kDa membrane for protease inactivation and enzyme removal. This stock solution was used to add additional PS fragments to the respective formulations at the high level of 50 pg / ml. No additional PS fragments were added to the low-level samples, and the actual level in the formulations was <5 pg / ml as determined by HPLC analysis. • pH: The lower and upper pH levels in the formulations were 7 and 8 with the center point at pH 7.5. • Syringe plunger leachables / extractables: Syringe plungers (made of black chlorobutyl PH701 / 50) currently used in the container closure system. It was tested whether chlorobutyl rubber leachates in the formulation could contribute to antigen modification. Therefore, a stock solution of leachables was prepared and used for addition experiments. The high level of added leachables in the formulation was estimated to be, on average, 1.4 times higher compared to the commercial Final Vaccine Batch (FVL). Due to the harsh extraction conditions, additional peaks were detected that were not present in the FVL samples. Therefore, the added formulations represent the "worst-case scenario" with respect to leachables and extractables. The formulations at the low level did not contain chlorobutyl rubber leachables. • Residual formaldehyde: For low-level formulations, no additional formaldehyde was added to the formulation samples. The lowest level was the residual formaldehyde still present in the diluted NIV sample after inactivation / neutralization, and the two-fold dilution was in the range of approximately 37 ppm (recalculated from the GMP analytical certificate of commercial DS release). For the high level, an additional 40 ppm of formaldehyde was added to the corresponding formulation (final total content approximately 77 ppm). It was tested whether the residual formaldehyde, in combination with a higher level of metal ions present in Alum 4230 and other potential factors, could further react with the virus, resulting in hypercrosslinking of surface proteins and loss of relevant epitopes. Determination of other residual impurities related to the process: Residual formaldehyde, sulfite, and sucrose in the final formulations were estimated based on GMP certificates for the commercial active ingredient JEV11A74. The results were recalculated using the actual two-fold dilution from NIV to DS used in the DOE experiments. Residual Sulfite: The residual sulfite concentration was constant in all formulations at approximately 93 ppm. Residual sucrose: The concentration of residual sucrose was constant in all formulations at approximately 1% v / w. 1.1.2 DOE Design These five factors were combined into a 25-DOE plan, resulting in a total of 34 experiments, including two center points, with the following basic design. DOE planning and evaluation were performed using appropriate software (Statgraphics Plus 3.0). Base design: 25 Factorial Number of experimental factors: 5 Number of blocks: 1 Number of responses: 1 Number of center points per block: 2 Number of executions: 34 Degrees of freedom for error: 18 Randomized: Yes Continuous factors1* Low High Units pH 7.0 8.0 Yeah Alum2) 0, 0 100, 0 % of Alum 4230 Yeah Added PS fragments3 0 50 pg / ml No Added leachables4) 0 1, 4 relative content compared to FVL No added formaldehyde5) 0 40 Ppm No Unit Answers Desorbed antigen UA / ml ELISA (monoclonal, polyclonal) 1) Continuous means that a central point (mid-value of high and low levels) is present in the study design. No central point means that only high and low levels are present in the study design. 2) Low level (0%) means that the formulation was prepared with Alum Lot 4074. High level (100%) means that the formulation was prepared with Alum Lot 4230. For center point formulations, an equal (50 / 50%) mixture of both Alum lots was used. 3) Since PS is present in the NIV used for the preparation of drug samples, the actual concentration of PS in the formulations without additions was <5 pg / ml and ~50-55 pg / ml for the formulations added with PS. 4) No leachables were assumed in formulations without additives since the samples were prepared / stored in low-junction Eppendorf tubes. The total leachable content of the chlorobutyl rubber syringe plunger in the additive samples was approximately 1.4 times higher compared to FVL. 5) The actual formaldehyde concentration in DP samples without additions was approximately 37 ppm, the total formaldehyde concentration in the added samples was approximately 77 ppm. 2 Definitions and abbreviations AcCN Acetonitrile DOE Design of Experiments DS Active ingredient FVL Final vaccine batch HPLC High-performance liquid chromatography ICP-MS Inductively coupled plasma-mass spectrometry PS Protamine sulfate RP Reverse Phase SEC Size exclusion chromatography SN Supernatant TFA Trifluoroacetic acid s / without 3 Materials and Methods 3.1 DO Studies 3.1.1 Materials • Syringe plunger caps: PH701 / 50 / C black Sil67002-1051 (obtained from West, Order No. 2116) • 100 ml glass bottle (Schott) Teflon-coated screw cap • Aluminum foil • HQ Water • Electric bain-marie (IKA, HBR 4 digital) • 2 ml Eppendorf LoBind tubes (Eppendorf, Cat. No. 0030 108.132) • Speed ​​Vac (Christ, RVC-2-25) • HPLC Vials, Clear Glass, 900 pl, Chromacol (VWR, Cat. No.: 548-1124) • HPLC vials, PP, 900 pl, (Agilent, Item No. 5182-0567) • HPLC vial stoppers, pre-cut (VWR, Cat. No.: 548-1260) • Falcon 15 ml tubes (Greiner, Cat. No. 188724) • Alum lot 4470 (RQCS 1342) ; Alum lot 4230 (RQCS 1200) • 10xPBS (Gibco, order number 14200-091) • Parafilm • Waters Atlantis T3 column; particle diameter 3 pm; column diameter / length 2.1 x 100 mm (Order No. 186003718; Batch 0107372331) • Acetonitrile (Merck, Cat. No. 1.13358.2500) • TFA (Sigma, Order No. 302031 • Dionex 3000 HPLC System • SR-3000 Solvent Rack • Pump UltiMate-3000, low-pressure gradient analytical pump • WPS-3000 TSL Autosampler, analytical autosampler - temperature controlled • TCC-3200 column compartment, temperature controlled • PDA-Detector PDA-3000 • 37% formaldehyde solution (Merck, Cat. No. 1.040031000) • Protamine sulfate (Intercell Biomedical Ltd, Batch No. 086056) • Ultrafilatration device (Amicon® Ultra 3 kDa) (Millipore, Cat. No. UFC900324) • Infors HT Incubator Multitron Standard Incubator (InforsAG) • Trypsin (Sigma, Order No.: T0303) 3.1.2 Procedure for preparing syringe plunger removables Syringe plungers (made of black chlorobutyl PH701 / 50) currently used in the West (Germany) vessel closure system were obtained. A stock solution of leachables was prepared by heat-treating the syringe plunger in water (90 °C / 2 h) followed by concentration in a speed-vac. The relative content of leachables in this stock solution was estimated by RP-HPLC using a C18 column (Atlantis T3 column) and compared with the supernatant of FVL JEV09L37. Extraction method A 100 ml Schott glass bottle with a Teflon-coated screw cap and a piece of aluminum foil was washed with hot water and thoroughly rinsed with HQ water. Thirty stoppers were loaded into the bottle, and 30 ml of HQ water was added. The bottle was closed with the aluminum foil, fitted between the bottle and the screw cap, and additionally sealed with Parafilm. The bottle was heated in a water bath at 90 °C for 2 hours and allowed to cool to room temperature. The extract was transferred to 14 low-junction Eppendorf tubes (a total of 28 ml of extract was recovered). Twelve vials (a total of 24 ml) were concentrated in a Speed ​​Vac for approximately 44 hours and pooled into a Falcon tube to obtain 6 ml of 4x concentrated stopper extract. A control sample containing 30 ml of HQ water without a stopper was prepared in the same manner to assess for any potential contamination. RP-HPLC C18 Method The leachables were separated using a C18 RP-HPLC column (Atlantis T3) operated at 40 °C and 0.25 mL / min. Solvent A was 0.1% TFA in H₂O, and solvent B was 0.1% TFA in AcCN. Separation was performed using a linear gradient ranging from 0 to 95% B over 30 min. Detection was carried out at 214 nm, 254 nm, and 280 nm. The total relative concentration of the concentrated plug extract was estimated to be 80 times higher compared to the peaks detected in the supernatant of the Final Vaccine Batch (FVL SN; obtained by removing alum particles by centrifugation at 5000 g / 5 min) at 254 nm. Therefore, a total relative content of 80 U / ml (Arbitrary Units U) was assigned to the stock solution, while the total relative concentration of leachables in FVL SN was set at 1 U / ml. For the DOE studies, the stock solution was diluted 16 times in the respective formulations, yielding approximately 5 U / ml of total extractables. 3.1.3 Preparation of protamine sulfate fragments A stock solution of PS fragments was prepared by digesting a PS solution (2 mg / ml in PBS) with trypsin (200 ng / ml for 60 min at 37 °C). Subsequently, the enzyme was inactivated by heat (90 °C for 10 min), followed by ultrafiltration using a 3 kDa membrane (Amicon® Ultra centrifugal filter). Due to membrane shearing, trypsin remained in the retained portion, while the PS fragments were present in the permeate fraction. Complete enzyme inactivation was assessed by adding 500 pg / ml of full-length PS to an aliquot of the obtained PS fragment, followed by incubation at 37 °C for 18 h. No degradation of full-length PS was observed, indicating complete trypsin inactivation / removal. Degradation was monitored by PS-HPLC SEC. 3.1.4 DOE Plan Samples were prepared according to the pipetting scheme shown in Table 2. NIV lot JEV11A74, obtained from a commercial production batch, was used as the starting sample. NIV was diluted 2x to DS using PBS buffer followed by pH adjustment. Five-mL aliquots were drawn and adjuvanted with the corresponding alum lot 4230, 4074, or a 50 / 50% mixture of both. The final amount of alum stock solution (2% Al₂O₃) added was 500 pg / mL of aluminum (0.1% Al₂O₃). Each 5-mL formulation was divided into two 2.5-mL portions using Eppendorf Lo-bind tubes. One aliquot was stored at 2-8 °C, another aliquot was stored at 22 ± 1 °C (Infors HT Incubator) with gentle stirring (20 rpm). 3.2 Inactivated JEV ELISA (based on polyclonal antibodies) Alum antigen desorption and ELISA analysis were performed using polyclonal sheep anti-JEV antibodies to coat 96-well ELISA plates as described in Example 4. 3.3 Inactivated JEV ELISA (based on monoclonal antibodies) A JEV ELISA based on monoclonal antibodies (mAbs) was developed. The assay is primarily based on the "polyclonal JEV ELISA" assay format, using only one anti-JEV monoclonal antibody (clone 52-2-5) for coating. The mab 52-2-5 employed was shown to be specific for JEV and to recognize a neutralizing epitope. The mab clone 52-2-5 was obtained by subcutaneous immunization of BALB / c mice with the commercially available vaccine lot JEV08J14B. Spleen cells from the mice were fused with myeloma cells. Individual clones were selected and subcloned from the resulting hybridoma cells. The clones were screened negatively for bovine serum albumin, protamine sulfate, and an extract from the JEV vaccine production cell line (Vero cells). A positive screening was performed against Neutralized Inactivated Virus (NIV) of vaccine lot JEV08M20.For screening, microtiter plates were coated with the relevant antigen and reacted with supernatant from cultures of the selected clones. For detection, a polyclonal goat anti-mouse antibody conjugated to alkaline phosphatase was used. The Mab clone 52-2-5 was shown to recognize a neutralizing epitope in domain III of the JEV envelope (E) protein containing Ser331 and Asp332 (Lin C.-W. and Wu W.-C. J Virol. 2003;77(4):2600-6). The binding of the mab to the indicated neutralizing epitope is determined, for example, as described in Lin and Wu (2003) by site-directed mutagenesis of domain III at position 331 (e.g., S^-R), and / or by alanine mutations at or near position 331 of domain III, e.g., of Ser 331 and Asp332 residues, followed by immunoblotting to determine the binding of the mab to the mutated proteins.The negative binding results indicate that the mab epitope is the neutralizing epitope identified by Lin and Wu (2003). The neutralizing characteristic of the epitope leads to the assumption that the epitope may be important for the antigen to trigger a protective immune response. JEV samples were analyzed using both polyclonal and monoclonal ELISA assays. The relative specific epitope content can be expressed as the ratio of the total antigen content determined by monoclonal ELISA (clone 52-2-5) to the total antigen content determined by polyclonal ELISA. Any difference in this ratio may indicate differences in the specific 52-2-5 epitope content. Results close to 1 would correspond to a high epitope content, and results close to 0 would correspond to a relatively low epitope content. A low ratio indicates the presence of structural changes in the neutralizing epitope. During the development of this "mAb ELISA", differences were detected between vaccine batches, which could be correlated with the potency results of these batches. 3.4 SEC-HPLC of protamine sulfate PS (full length) and its fragments were analyzed by size exclusion HPLC (SEC-HPLC) using a Superdex Peptide 10 / 300 GL, 10 * 300 mm, 13 pm (GE Healthcare) using 0.1% (v / v) trifluoroacetic acid (TFA) in 30% acetinitrile (CAN) as the mobile phase at a flow rate of 0.6 ml / min. Samples containing PS were prepared in duplicate, i.e., diluted with mobile phase before injection. 4 Results 4.1 Analysis of plug leachates used to add to experiments Figure 1 shows RP-HPLC elution profiles of the concentrated stock solution obtained after heat extraction of plugs compared to FVL SN. A similar peak pattern was observed for both samples. Due to the harsh extraction conditions, additional peaks were detected in the concentrate that were not present in the FVL samples or were present only at a much lower relative concentration. Therefore, the added formulations represent the worst-case scenario with respect to leachables and extractables. The total relative concentration of individual peaks in the concentrated extract and the added formulation compared to FVL SN is summarized in Table 3. The total amount of leachables in the stock solution was calculated as the sum of all detected peaks and expressed in arbitrary units as 67 U / ml.Since the stock solution was diluted 16 times in the respective formulation, the resulting total leachable content was estimated at 4.2 U / ml. This corresponds to an average increase of 1.4 times compared to the supernatant of FVL JEV09L37 (3.0 U / ml). 4.2 Analysis of protamine sulfate fragments The PS fragments obtained after cleavage of full-length PS by trypsin are shown in Figure 2. Similar peak profiles of trypsin-treated PS and already degraded PS present in NIV11A74 were obtained by HPLC (see Figure 3). 4.3 DOE Evaluation The formulations prepared for this DOE were analyzed after 4 and 8 weeks of incubation under accelerated conditions (22 °C). It was assumed that any degradation reaction would be accelerated when stored at a higher temperature compared to normal storage conditions (2–8 °C). However, the samples are still stored at 2–8 °C and will be analyzed at a later time (approximately 4–6 months). The initial analysis of the samples stored at 22 °C for 4 and 8 weeks is shown in Table 4. 4.3.1 Evaluation of DOE after 4 weeks at 22 °C Statistical evaluation of the DOE matrix results obtained after 4 weeks at 22 °C showed that the 52-2-5 specific epitope content (expressed as the ratio of desorbed antigen analyzed by monoclonal / polyclonal ELISA) was statistically significantly influenced (95% confidence level, see Table 5) by the following factors: • lower content of specific epitope 52-2-5 in the presence of lot 4230 of Alum • lower content of specific epitope 52-2-5 at a pH less than 7 • Higher content of specific epitope 52-2-5 with higher formaldehyde concentration The presence of higher concentrations of PS fragments and chlorobutyl rubber leachables showed no influence on the content of specific epitopes. No second-order or higher interactions were detected between individual parameters. The ANOVA table divides the variability in the "4-week relationship" into separate parts for each effect. It then tests the statistical significance of each effect by comparing the mean square to an estimate of the experimental error. In this case, three effects (alum, pH, formaldehyde) have p-values ​​less than 0.05, indicating that they are significantly different from zero at the 95.0% confidence level. The R-squared statistic indicates that the fitted model explains 74.85% of the variability in the 4-week relationship. The adjusted R-squared statistic, which is more suitable for comparing models with different numbers of independent variables, is 51.27%. The standard error of the estimate shows that the standard deviation of the residual values ​​is 0.063. The mean absolute error (MAE) of 0.0353 is the average value of the residual values.The Durbin-Watson (DW) statistic tests the residual values ​​to determine if there is any significant correlation based on the order in which they occur in the data file. Since the DW value is greater than 1.4, there is probably no significant autocorrelation in the residual values. The effects are also shown using standardized Pareto charts and main effects plots, as shown in Figure 4. 4.3.2 Evaluation of DOE after 8 weeks at 22 °C The statistical evaluation of the DOE matrix results obtained after 8 weeks at 22 °C was similar to the results obtained after 4 weeks. The evaluation shows that the 52-2-5 specific epitope content (expressed as the ratio of desorbed antigen analyzed by monoclonal / polyclonal ELISA) was statistically significantly influenced (95% confidence level, see Table 6) by the following factors: • lower content of specific epitope 52-2-5 in the presence of lot 4230 of Alum • lower content of specific epitope 52-2-5 at a pH less than 7 The presence of higher concentrations of PS fragments, chlorobutyl rubber leachables, and formaldehyde showed no influence on the content of specific epitopes. Note that the p-value for formaldehyde (0.08) is quite close to being statistically significant. No second-order or higher interactions were detected between individual parameters. The ANOVA table divides the variability in the "8-week relationship" into separate parts for each effect. It then tests the statistical significance of each effect by comparing the mean square to an estimate of the experimental error. In this case, two effects (Alum and pH) have p-values ​​less than 0.05, indicating that they are significantly different from zero at the 95.0% confidence level. The R-squared statistic indicates that the fitted model explains 75.9% of the variability in the "8-week relationship." The adjusted R-squared statistic, which is more suitable for comparing models with different numbers of independent variables, is 53.3%. The standard error of the estimate shows that the standard deviation of the residual values ​​is 0.095. The mean absolute error (MAE) of 0.057 is the average value of the residual values.The effects are also shown using standardized Pareto charts and main effects charts, as shown in Figure 5. A regression analysis was also performed on the fitted data, and the calculated regression coefficients are shown in Table 7. The regression equation fitted to the data, including pH, alum, and formaldehyde, is shown below. The equation of the fitted model is: "8-week ratio" = 0.0228125 + 0.113125*pH - 0.00185625*Alum + 0.0315625*Formaldehyde where the values ​​of the variables are specified in their original units, except for the categorical factors which take the values ​​-1 for the low level and +1 for the high level. The contour of the estimated response and the plot of residual values ​​are shown in Figure 6 and Figure 7. The relationship increases when the relative content of Lot 4230 Alum decreases and the pH increases. Table 8 contains information on the "8-week ratio" values ​​generated using the fitted model. The table includes: (1) the observed value of "8-week relationship" (2) the predicted value of "8-week relationship" using the fitted model (3) 95.0% confidence limits for the mean response As shown, the experimental results are well predicted by the regression model. 5. Summary Of the parameters tested, Alum lot 4230 was shown to contribute significantly to antigen degradation, as analyzed by monoclonal / polyclonal ELISA under accelerated conditions (22 °C, assay time points of 4 and 8 weeks). The DOE results obtained after 4 and 8 weeks at 22 °C show that Alum 4230 is the most significant factor in antigen degradation, as detected by the monoclonal / polyclonal ELISA ratio. Formulations prepared with Alum 4074 (which has much higher purity with respect to residual metal ions) generally show a much higher specific epitope content. Formaldehyde and pH also contributed to antigen stability, but to a lesser extent. The effect of increased antigen stability in samples formulated with Alum 4230 at a higher formaldehyde level was well demonstrated (e.g., samples 19 and 29). However, the influence of formaldehyde was less pronounced after a prolonged storage period (8 weeks at 22 °C). Improved antigen stability was observed at pH 8 compared to pH 7. Protamine sulfate and chlorobutyl rubber stopper leachables did not contribute to antigen degradation. Example 2 In previous studies (see Example 1), lot 4230 of aluminum hydroxide was identified as a significant contributing factor to the antigen degradation observed in FVL09L37. This particular lot of aluminum hydroxide (alum) exhibited a much higher residual metal ion content compared to other lots of alum used in the formulation of the inactivated JEV antigen. This Example summarizes additional studies conducted to evaluate the influence of metal ions on the stability of the inactivated JEV. Addition studies were performed with the antigen present in either an inactivated neutralized virus (NIV) solution or a drug suspension (DP) after formulation of the antigen with aluminum hydroxide. 1. Study Description It was previously demonstrated that lot 4230 of alum contains significantly higher levels of residual metal ion impurities compared to other lots of alum used for JEV formulation (see also Example 3). Further studies were conducted to evaluate the influence of metal ions on the stability and potential surface modification of JEV. The inactivated antigen was present in either the neutralized inactivated virus (NIV) solution or the drug suspension (DP) after further dilution of NIV and formulation with aluminum hydroxide. In another set of experiments, different lots of alum covering a wide range of residual metal ion content were used and formulated with a single defined lot of NIV. All of these formulations still contained residual formaldehyde and bisulfite at concentrations representative of the commercial product.The stock solution of metallic salts was dissolved in water and added to the samples at the desired final concentration. 2 Definitions and abbreviations. AcCN Acetonitrile ANOVA Analysis of variance DOE Design of Experiments DP Medication DS Active ingredient FBV Final bulk vaccine FVL Final vaccine batch GI Irradiated with gamma rays HPLC High-performance liquid chromatography Fisher's LSD Least Significant Difference mAb Monoclonal antibody NIV Virus inactivated neutralized PS Protamine sulfate RP Reverse Phase SEC Size exclusion chromatography SN Supernatant TFA Trifluoroacetic acid s / without 3 Materials and Methods 3.1 Materials Iron(II) chloride tetrahydrate (Sigma, Order No. 44939) Iron(III) chloride hexahydrate (Sigma, Order No. 31232) Nickel(II) sulfate hexahydrate (Sigma, Order No. N4882) Cobalt(II) chloride hexahydrate (Sigma, Order No. 31277) Copper(II) chloride dehydrate (Sigma, Order No. 807483) Zinc sulfate heptahydrate (Sigma, Order No. 24750) Chromium(III) hexahydrate (AlfaAesar, order no. 42114) Disodium salt dehydration of ethylenediaminetetraacetic acid (EDTA) (Sigma, E5134) Bidet water. (Fresenius Kabi, Art. No. 0712221 / 01 A) 10xPBS (Gibco, order number 14200-091) 37% formaldehyde solution (Merck, Cat. No. 1.040031000) Protamine sulfate (Intercell Biomedical Ltd, Batch No. 086056) 2 ml Eppendorf LoBind Tubes (Eppendorf, Cat. No. 0030 108.132) Falcon 15 ml tubes (Greiner, Cat. No. 188724) Infors HT Incubator Multitron Standard Incubator (InforsAG) 0.2pm Mini Kleenpak 25mm Filter (Pall) The NIV11A74 and final bulk vaccine (FBV, formulated with Alum lot 4539) JEV 11D87 from commercial production cycles were obtained from Intercell Biomedical (Livingston, UK) and stored at 2-8 °C until further processing Stock solutions of metallic salts in water (final concentration 1 mM) were prepared for the addition experiments and stored at 2-8 °C until use. Aluminum hydroxide (2% Al2O3, Brenntag Biosector) samples were obtained from Intercell Biomedical or purchased directly from Brenntag. Alum samples were stored at 2–8 °C. The following alum lots were used in this study: 4470, 4563, 4621, 3877, 4230 (non-gamma-irradiated and gamma-irradiated). 3.2 Preparation of metal stock solutions 3.2.1 Iron (II) stock solution A 20 mM iron (II) stock solution was prepared by dissolving 397 mg of iron (II) chloride tetrahydrate in 100 ml of double-distilled water. 3.2.2 Iron (III) stock solution A 20 mM iron (III) stock solution was prepared by dissolving 540 mg of iron (III) chloride hexahydrate in 100 ml of double-distilled water. 3.2.3 Nickel (II) stock solution A 20 mM nickel (II) stock solution was prepared by dissolving 525 mg of nickel (II) sulfate hexahydrate in 100 ml of double-distilled water. 3.2.4 Cobalt (II) stock solution A 20 mM cobalt(II) stock solution was prepared by dissolving 476 mg of cobalt(II) chloride hexahydrate in 100 ml of double-distilled water. 3.2.5 Copper (II) stock solution A 20 mM copper(II) stock solution was prepared by dissolving 341 mg of copper(II) chloride dihydrate in 100 ml of double-distilled water. 3.2.6 Zinc stock solution A 20 mM zinc stock solution was prepared by dissolving 575 mg of zinc sulfate heptahydrate in 100 ml of double-distilled water. 3.2.7 Chromium (III) stock solution A 20 mM Chromium (III) stock solution was prepared by dissolving 533 mg of Chromium (III) chloride hexahydrate in 100 ml of double-distilled water. 3.3 Preparation of work solutions Metal ion working solutions (final concentration of 1 mM unless otherwise stated) were prepared by diluting metal ion stock solutions with double-distilled water and sterile filtration through a 0.2 pm syringe filter. 3.4 Formulation Preparation All formulations were prepared under sterile conditions. NIV and FBV obtained from commercial production batches were adjusted to the desired pH and added with aliquots of metal stock solution. All samples were stored in plastic tubes unless otherwise noted. In all formulations using alum, the final Al content was 500 pg / ml, corresponding to 0.1% Al₂O₃. It should be noted that the metal ions, especially iron(II), iron(III), and, to some extent, Cr(III), formed a precipitate with the phosphate ions present in the buffer, resulting in partial coprecipitation of the inactivated virus, as indicated by the low recovery observed by size exclusion high-performance liquid chromatography (SEC-HPLC). 3.4.1 Experiment 20110913 (NIV): NIV formulation at different concentrations of Ni(II), Cu(II), Cr(III) metal ions with or without the presence of PS fragments NIV 11A74 was adjusted to pH 7 and pH 8, followed by the addition of metal ions (Ni(II), Cu(II), Cr(III)) to a final concentration of 100 / 500 / 1000 ng / ml. All formulations were stored in low-binding Eppendorf tubes at 22 °C. Aliquots of all formulations were also prepared in the presence of protamine sulfate fragments (50 pg / ml). This was done to evaluate any effect of the PS fragments on the stability of JEV in the presence of metals. The preparation of protamine sulfate (PS) fragments is described in Example 1. Samples were prepared on the same day (see Table 9) and analyzed three weeks later. All samples were analyzed by SEC-HPLC, but only the pH 8 samples (Nos. 21–40) were analyzed by ELISA. 3.4.2 Experiment 20110913 (DP) : Formulation of DP at different concentrations of metal ions of Ni (II) , Cu (II) , Cr (III) In this study, FBV 11D87 (formulated with Lot 4539 of alum) was used. The FBV was adjusted to pH 7 and pH 8 and supplemented with Ni(II) / Cu(II) / Cr(III) at 100, 500, and 1000 ng / ml to evaluate any effects dependent on metal ion concentration / pH. Table 10 shows the experimental design for this experiment. All formulations were stored in Falcon tubes at 2–8 °C and 22 °C. Samples stored at 22 °C were analyzed by SEC-HPLC and ELISA after 5 weeks. 3.4.3 Experiment 20110812-DP added with metal Final bulk vaccine 11D87 (formulated with alum lot 4539) was obtained from a commercial production run and used in this study. Residual formaldehyde in the desiccant (DS) was analyzed as 28.1 ppm, and residual sulfites were 92.2 ppm. The actual content in the desiccant (DP) can be considered to be within the same range. FBV JEV11D87 was adjusted to pH 7.0 / 7.4 / 7.8 and supplemented with 500 ng / ml (final concentration) of Fe(II), Fe(III), Ni(II), Co(II), Cu(II), and Zn(II). A metal ion mixture formulation containing all individual metal ions together in solution was also prepared. Subsequently, formulations were prepared with Cr(III), and Cr(III) was not included in the metal ion mixture. The control formulations were only adjusted to the desired pH, but no metals were added. All formulations (No. 1-24) were prepared on the same day and stored in Falcon tubes at 2-8 °C and 22 °C. Additional Cr(III)-enhanced samples (Nos. 25–27) were prepared by taking aliquots of the control samples stored at 2–8 °C and adding Cr(III) to a final concentration of 500 ng / mL. The formulations were stored only under accelerated conditions (22 °C). Table 11 shows the experimental setup for this experiment. All samples stored at 22 °C were analyzed by ELISA (monoclonal and polyclonal) after 4 and 7 weeks. 3.4.4 Experiment 20110819: DP formulation using various batches of alum Addition studies, as described above, can provide initial evidence of the potential instability of the formulated antigen in the presence of certain metals, but they may not be entirely representative of the actual conditions under which the metals present in aluminum hydroxide are incorporated into the three-dimensional structure of the gel, resulting in varying local concentrations and orientation / accessibility. To overcome these limitations, an initial study was initiated to simulate real-world conditions. A single batch of NIV (11A74) obtained from a commercial production run was formulated with various batches of alum produced by Brenntag, covering a wide range of residual metals. 4.75 ml of NIV were mixed with 0.25 ml of alum (2%) in Falcon tubes. The final aluminum hydroxide concentration was 500 pg / ml (0.1% Al₂O₃).The formulated vaccine samples were stored at 2–8 °C and under accelerated conditions at 22 °C. All of these alum lots contained residual metal ions at varying concentrations. Alum lot 4230 had the highest levels of Fe, Cu, Ni, and V (see Example 3). Note that ICP-MS cannot specify the valences of metal ions. A mixed alum sample containing equal amounts of lots 4230 and 4074 was also prepared to achieve an “intermediate” level for Ni(II) and Cu(II). The samples were analyzed after 6 weeks of storage at 22 °C. The residual amounts of formaldehyde and sulfite, estimated by recalculating the available DS analysis results corrected by the NIV-to-DS dilution factor, were 76 ppm for formaldehyde and 192 ppm for sulfite, respectively. 3.1 Desorption of hydroxide antigen from aluminum for analysis by SEC-MALLS Viral particles were desorbed from aluminum hydroxide. Approximately 625 ml of DP (8 °C, 5 min, 3300 x g) were centrifuged, and the supernatant was discarded unless otherwise noted or analyzed by JEV-SEC-MALLS to detect the concentration of unbound antigen. Viral particles were desorbed by resuspending the aluminum hydroxide particles in 62.5 ml of 0.8 M potassium phosphate buffer (pH 8) containing BSA (50 pg / ml). BSA was added to the desorption buffer for SEC-MALLS analysis to minimize losses due to nonspecific antigen adsorption. After shaking (500 rpm) the aluminum hydroxide particles for 10 minutes at room temperature, the particles were removed by centrifugation and the supernatant was collected in an Eppendorf LoBind tube and the desorption procedure was repeated on the remaining sample.The pooled desorbed antigen (concentrated sample ~5 times; final volume 125 pl; starting volume ~625 pl) was then further analyzed by SEC-MALLS. 3.2 HPLC SEC-MALLS Method The desorbed antigen was analyzed using SEC-MALLS. In summary, after desorption of the antigen from aluminum hydroxide, 100 lp of the clustered desorbed material (concentrated ~5-fold) was loaded onto a SEC Superose 610 / 300 GL column. 1* PBS + 250 mM NaCl was used as the mobile phase. Ultraviolet (UV) signals at 214 nm and MALLS signals from viral particles were recorded and analyzed using the Chromeleon and ASTRA software packages. 3.3 Inactivated JEV ELISA (based on polyclonal antibodies) Alum antigen desorption and ELISA analysis were performed using polyclonal sheep anti-JEV antibodies to coat 96-well ELISA plates as described in Example 4. 3.4 Inactivated JEV ELISA (based on monoclonal antibodies) During the course of this research trial, a JEV ELISA based on monoclonal antibodies (mAbs) was developed. The assay is primarily based on the "polyclonal JEV ELISA" assay format, using only one anti-JEV monoclonal antibody (clone 52-2-5) for coating and the polyclonal antibody for detection. The mab 52-2-5 employed was shown to be specific for JEV and to recognize a neutralizing epitope. The 52-2-5 mab clone was obtained by subcutaneous immunization of BALB / c mice with the commercially available vaccine lot JEV08J14B. Spleen cells from the mice were fused with myeloma cells. From the resulting hybridoma cells, individual clones were selected and subcloned. The clones were negatively screened against bovine serum albumin, protamine sulfate, and an extract from the JE vaccine production cell line (Vero cells).A positive screening test against Neutralized Inactivated Virus (NIV) was performed on vaccine lot JEV08M20. For screening, microtiter plates were coated with the relevant antigen and reacted with supernatant from cultures of selected clones. A polyclonal goat anti-mouse antibody conjugated to alkaline phosphatase was used for detection. Mab clone 52-2-5 was shown to recognize a neutralizing epitope in domain III of the JEV envelope (E) protein containing Ser331 and Asp332 (Lin C.-W. and Wu W.-C. J Virol. 2003;77(4):2600-6).The binding of the mab to the indicated neutralizing epitope was determined, for example, as described in Lin and Wu (2003), by site-directed mutagenesis at position 331 of domain III (e.g., S^-R), and / or by alanine mutations at or near position 331 of domain III, e.g., at Ser331 and Asp332, followed by immunoblotting to determine the binding of the mab to the mutated proteins. Negative binding results indicated that the mab epitope was the neutralizing epitope identified by Lin and Wu (2003). The neutralizing characteristic of the epitope led to the assumption that the epitope may be important for the antigen to elicit a protective immune response. JEV samples were analyzed using both polyclonal and monoclonal ELISA assays.The relative specific epitope content can be expressed as the ratio of the total antigen content determined by monoclonal ELISA (clone 52-2-5) to the total antigen content determined by polyclonal ELISA. Any difference in this ratio may indicate differences in the 52-2-5 specific epitope content. Results close to 1 would correspond to a high epitope content, and results close to 0 would correspond to a relatively low epitope content. A low ratio indicates the presence of structural changes in the neutralizing epitope. During the development of this "mAb ELISA", differences were detected between vaccine batches, which could be correlated with the potency results of these batches. 3.5 Statistical evaluation Statistical evaluation was performed using Statgraphic Plus 3.0. 4 Results 4.1 Experiment 20110913 (NIV): NIV formulation at different concentrations of Ni(II), Cu(II), Cr(III) metal ions with or without the presence of PS fragments The SEC-HPLC results of NIV formulations (pH 7 and pH 8) containing metal ions [Ni(II), Cu(II), Cr(III)] with and without PS fragments are summarized in Table 12. The SEC-HPLC results show that antigen recovery from most samples was >80%. Some samples (No. 7, No. 36, No. 38) showed slightly reduced recoveries in the 70–80% range. It should be noted that the actual virus content is quite low, and the precision of the HPLC results can be estimated at approximately ±20%. Since for samples No. 36 and No. 38 the recoveries for the following formulations (No. 37, No. 39) at the next level of individual metal ion content were again higher, these differences may be due to assay variability and were not considered significant.Based on the results obtained, it was not possible to clarify the influence of metal ions on the recovery of soluble inactivated JEV. However, SEC-HPLC only provides information on the soluble virus content, but not on any possible surface modification. Only the formulations prepared at pH 8 were also analyzed by ELISA (duplicate analysis). The monoclonal / polyclonal ELISA ratio was calculated and can be used for comparison of results. Analysis of samples by ELISA (see Table 13) shows no significant influence of the tested metals on the degradation of inactivated JEV at pH 8 after three weeks at 22 °C. There may be a tendency for the ratio to decrease in the presence of Cu(II), but overall, a three-week incubation period at 22 °C does not appear to be sufficient to detect significant degradation.As also shown in the DOE experiment (Example 1), inactivated JEV appears to have greater stability at pH 8 when stored under accelerated conditions at 22 °C, which would also contribute to the lack of observed significant effects. This experiment also demonstrated that PS fragments have no influence on JEV stability. This is also highly consistent with the DOE results. NIV 1-20 samples formulated at pH 7 showed a significant reduction in monoclonal epitope content in the presence of Cu(II). At the highest concentration analyzed (1000 ng / ml), the ratio was close to zero, indicating significant structural changes in the antigen. 4.2 Experiment 20110913 (DP) : DP formulation with different concentrations of metal ions of Ni (II) , Cu (II) , Cr (III) The analysis of the desorbed JEV antigen is summarized in Table 14 (SEC-HPLC) and Table 15 (ELISA). Antigen recoveries for all samples determined by SEC-HPLC were >80% after 5 weeks at 22 °C, indicating no significant influence of the assayed metal ions on desorption recovery. As shown in Figure 8, there is a trend toward a decrease in the ratio as analyzed by ELISA in the presence of Cu(II) and Cr(III) at pH 7. The formulations at pH 8 appear to be more stable. 4.3 Experiment 20110812 (DP) : DP added with metal ions In this experiment, FBV11D87 was used as the starting material. The pH of the formulation was adjusted to a narrower range (pH 7, 0, 7, 4, 7, 8), and additional metal ions were added, each at 500 ng / ml (final concentration). The metal ion mixture contained all individual metal ions except Cr(III) in separate formulations (each metal at 500 ng / ml). The ELISA results obtained after 4 and 7 weeks at 22 °C are summarized in Table 16. The results are also shown graphically in Figure 9. A statistical evaluation of the stability of samples stored at 22 °C for 7 weeks was performed. ANOVA (analysis of variance) showed significant effects of parameters (pH and metal type) on antigen stability, expressed as the monoclonal / polyclonal ELISA ratio (see Table 17). The ANOVA table decomposes the variability of the ratio into contributions due to various factors. Since Type III sums of squares were chosen, the contribution of each factor is measured by removing the effects of all other factors. P-values ​​test the statistical significance of each factor. Because the P-values ​​for pH and metal ion type are less than 0.05, these factors have a statistically significant effect on the ratio at the 95.0% confidence level. Table 18 presents a multiple comparison procedure used to determine the significance of the observed differences between the means. A significant effect on the ratio was observed for Cu(II) and the metal mixture compared to the control formulations without additives. The lower half of the results shows the estimated difference between each pair of means. An asterisk is placed next to seven pairs, indicating that these pairs show statistically significant differences at the 95.0% confidence level. At the top of the page, three homogeneous groups are identified using columns of X's. Within each column, the levels containing X's form a group of means within which there are no statistically significant differences. The method currently used to discriminate between the means is Fisher's Least Significant Difference (LSD) procedure.With this method, there is a 5.0% risk of calling each pair of means significantly different when the actual difference is equal to 0. A significant effect on the ratio was observed for Cu(II) and the metal ion mixture. The influence of other metal ions may become significant over longer storage periods. The metal ion mixture contained the highest total concentration and could represent the worst-case scenario. However, it was concluded that several metal ions present in the alum could contribute to the degradation, each to a different extent. These results further support the proposed root cause of metal ion-catalyzed antigen degradation. It should be noted that the addition experiment may not fully simulate the actual conditions of residual metal ion impurities present in Alum 4230. Metal ions are incorporated into the alum structure, and their local concentration and orientation may differ from the metal ions used in the addition experiments.It is also known that metal ions (e.g., Fe) have low solubility in the presence of phosphate ions (PO43-). Therefore, the actual concentration of soluble metal ions and the contribution of metals present as metal-phosphate complexes to JEV degradation are unknown. 4.4 Experiment 20110819: Preparation of DP samples with different batches of Alum Addition studies, as described above, can provide initial evidence of the potential instability of the formulated antigen in the presence of some metal ions, but they may not be entirely representative of the actual conditions under which these metal ions present in aluminum hydroxide are expected to incorporate into the three-dimensional structure of the aluminum hydroxide gel, resulting in varying local concentrations and orientation / accessibility. To overcome these limitations, an initial study was initiated to simulate real-world conditions. A single NIV (11A74) was formulated using various alum batches obtained from Brenntag, covering a wide range of residual metals. The formulated vaccine samples were stored at 2–8 °C and under accelerated conditions at 22 °C. All of these alum batches contained residual metal ions at varying levels.Lot 4230 had the highest levels of Fe, Cu, Ni, and V (see Table 19). Note that the actual metal ion content in the formulated product is only 1 / 20 of the concentration in the alum stock solution (2%). Note that ICP-MS cannot specify the valences of metal ions. The analysis of the desorbed antigen by ELISA of samples stored at 22 °C for 6 weeks is shown in Table 20. The pooled standard deviation was calculated from all samples (pooled ~0.075) as a measure of experimental uncertainty. Mean values ​​for the ratio and 95% confidence intervals (calculated based on pooled) were plotted against the individual formulations (see Figure 10). Samples formulated with Alum 4230 showed a trend toward a lower ratio compared to the other samples. However, the differences were not large enough to show statistically significant differences between the various formulations. 5 Summary Metal ions were shown to contribute to the degradation of inactivated JEV under accelerated storage conditions (22°C). In the addition studies, a higher concentration of residual metal ions (range 100–1000 ng / ml) was used than that present in FVL formulated with alum lot 4230 (e.g., Fe ~310 ng / ml; Cr ~64 ng / ml; Ni ~52 ng / ml). The copper content in FVL can only be estimated at 3 ng / ml based on ICP-MS data from a 2% alum stock solution, since the LDD is 25 ng / ml. Higher metal concentrations and storage temperatures were chosen to increase the rate of any potential degradation reactions. In fact, for FVL JEV09L37, potency loss occurred after 11 months of storage at 2–8°C. It was also shown that metals can form insoluble complexes with phosphate ions, making it difficult to estimate the actual levels of metals present. In the addition experiments, ELISA results showed statistically significant structural changes on the virus surface in as little as 4 weeks at 22 °C in the presence of metal ions. ELISA ratios for formulations containing Cu(II) and a metal ion mixture (containing Fe(II), Fe(III), Co(II), Cu(II), and Zn(II)) were shown to be statistically significantly lower compared to the control formulation without additions. Cu(II) was also found in lot 4230 of Alum (2% stock solution) at 64 ng / ml, corresponding to ~3 ng / ml in FVL. In all other lots of Alum (2%), the Cu(II) content was <25 ng / ml (below the limit of detection). For antigen formulation experiments using different batches of Alum, a longer storage time (>6 weeks at 22 °C) is required under accelerated conditions.There is a trend showing that formulations prepared with Alum 4230 exhibited lower ELISA ratios compared to other batches. The slower degradation rate compared to the added formulation could be contributing to a lower metal ion content in commercial batches of Alum. It was also observed that the antigen shows greater stability at pH 7.5–8 compared to pH 7, and that the PS fragments do not contribute to any degradation reaction. These results are in good agreement with the DOE results described in Example 1. Example 3 As part of the out-of-specification investigation related to FVL JEV09L37, the lot of aluminum hydroxide used (lot 4230) was determined to be the most likely root cause of the observed power loss. The aluminum hydroxide (referred to as alum during the JE-PIV manufacturing process) is sourced from Brenntag Biosector as an autoclaved suspension called "Alhydrogel® Aluminum Hydroxide Gel Adjuvant." Each lot is radiation sterilized before use in the JEV production process. The appearance, metal ion content, and physical properties of several different lots of Alhydrogel® were analyzed. 1. Introduction 1.1 Aluminum hydroxide Brenntag Biosector's Alhydrogel® has a specific aluminum content of 10 mg / ml, which translates to 2% Al₂O₃ and 3% Al(OH)₃. Other specifications include nitrogen (max. 0.005%), free sulfate (max. 0.05%), total sulfate (max. 0.1%), and pH (6.5 ± 0.5). It has a shelf life of 26 months when stored at room temperature. 1.2 Generation of Aluminum Hydroxide Alhydrogel® 2% (called Aluminum Hydroxide) is manufactured by Brenntag (CAS No. 21645-51-2). 1.3 Use of aluminum hydroxide batches in the manufacture of JEV For the production of commercial batches of JEV vaccines, a total of 5 different batches of Brenntag Alhydrogel® 2% have been used so far. 2 Definitions and abbreviations Aluminum hydroxide solution Alhydrogel 2% (also called Alum) DS / DP Active ingredient / Medicine ESG Environmental Scientists Group F-AAS Flame Atomic Absorption Spectrometry FVL Final Vaccine Batch GF-AAS Graphite Furnace Atomic Absorption Spectrometry ICP-MS Inductively Coupled Plasma-Mass Spectrometry JEV Japanese encephalitis virus JE-PIV Purified inactivated Japanese encephalitis virus LDC Limit of quantification P&TD Patch and technical development PSD Particle Size Distribution PZC Zero Load Point QCI Quality Control Immunology 3 Materials and Methods 3.1 Alhydrogel® Batches Alhydrogel® 2% batches: 3877, 4074, 4187, 4230, 4414, 4470, 4539, 4563, 4587, 4621 (not all of the listed Alum batches were used in the JE-PIV formulation) Alhydrogel® 2% batches washed 7 times: 4577, 4580, 4596 (from Brenntag, not typical of the 2% Alum received for formulation) 3.2 PSD Measurements of Alhydrogel® The particle size distribution (PSD) of aluminum hydroxide was analyzed using a Malvern Mastersizer 2000 |jP system with a 20 mL sample cell. The bulk substance Alhydrogel® 2% was diluted 1:20 in water, and 1 mL was added to the sample cell. Therefore, the final dilution of the sample in the sample cell was 400 times (0.005% aluminum hydroxide). 3.3 Measurements of the zeta potential of Alhydrogel® The zeta potential and point of zero charge (PZC) were measured using a Malvern Zetasizer ZS system equipped with an MPT-2 autotitrator. The bulk substance Alhydrogel® 2% was diluted 1:20 in PBS and equilibrated overnight at room temperature. To record the charge titration curve, the pH was adjusted using 100 mM HCl and 100 mM NaOH solutions. The PZC was determined by extrapolating the point of zero charge on the titration graph (intercept of the titration curve and the x-axis). The point of zero charge corresponds to the pH value at which the sample surface has no net charge. 3.4 Analysis of metal ions in aluminum hydroxide The selected metal ions were analyzed by inductively coupled plasma mass spectrometry (ICP-MS), flame atomic absorption spectrometry (F-AAS), and graphite furnace atomic absorption spectrometry (GF-AAS) at the Medical Laboratory in Bremen, Germany. In summary, samples containing aluminum hydroxide were treated with concentrated HNO3 under heat until a clear solution was obtained. The clear solution was then diluted and further analyzed. The presence and concentration of the following metal ions were determined: Pb, Cd, Cr, Co, Fe, Cu, Ni, Ag, W, and Al. Depending on the sample dilution, the limit of quantification (LOQ) ranged from 5 to 25 ng / ml. In addition, ESG (UK) performed a semi-quantitative screening of 70 elements using a combination of ICP-MS (Agilent 7500ce) and ICP-AES (Perkin Elmer Optima 4300DV), which were calibrated using certified standards. Element screening is a screening method and is not as sensitive as trace metal analysis for selected metals, as performed by Medical Laborator and Bremen. However, this screening provides a good overview of the presence and levels of certain metals. 4 Results 4.1 Determination of the particle size distribution of Alhydrogel® Table 21 summarizes the PSD data from two sub-batches of each of Alhydrogel® batches 4230 and 4740. The distribution results are shown in Figure 11. The mean particle size was ~2–4 µm, with smaller (<1 µm) and larger (>20 µm) particle populations present in all four samples. The four Alhydrogel® samples tested showed no significant differences in mean particle size distribution. 4.2 Zeta potential measurements Two sub-batches from each of Alhydrogel® batches 4230 and 4740 (2% stock solution diluted 20-fold in PBS and equilibrated overnight at room temperature before analysis) were analyzed to determine the zero-charge point. Table 22 summarizes the zero-charge point (ZCP) results for the four samples, which showed very similar ZCP values ​​in PBS buffer. The titration curves are shown in Figure 12. No differences in the titration curves or ZCP values ​​were observed among the four samples analyzed. 4.3 Determination of the residual metal ion content in Alhydrogel® batches The current limits for Fe in 2% aluminum hydroxide solutions according to Ph. Eur. are 15 ppm (15 pg / ml) and a total maximum of 20 ppm (20 pg / ml) for other heavy metals (such as Pb). However, a concentration of 15 ppm of Fe would correspond to 0.27 mM Fe in solution. Considering that even small amounts of residual metal ions can catalyze a variety of protein degradation reactions (e.g., oxidation and protease activation) and that metals remain stable in solution, differences in metal ion content between batches of aluminum hydroxide could lead to differences in antigen stability over time. The concentrations of various metal ions in commercially available aluminum hydroxide batches were analyzed using ICP-MS. The results of these analyses are summarized in Table 23. Batches 4074, 4230, 4470, 4414, and 4539 were used in the production of commercial JEV batches. Since a 2% Alhydrogel® stock solution corresponds to an Al concentration of 10 mg / ml, the Al content in the different samples can be used as a reference for the results obtained for the other metal ions. In fact, an average aluminum content of 10.3 mg / ml was measured, demonstrating the accuracy and reproducibility of the method. When different batches of Alhydrogel® were compared, significant variations in the amount of contaminating metal ions were observed. The most notable contaminating metal ions were Fe, Cr, and Ni, which were detected in all batches. In addition, batch 4230 contained detectable amounts of Cu that were below the LDC in all other batches. However, it should be noted that none of these metals were detected in quantities close to the Alhydrogel® specifications mentioned above. For example, the highest concentration of iron found in lot 4230 was 5.6 pg / ml, or approximately 40% of the permitted concentration. An "improved" Alhydrogel® is washed seven times with water during the purification stage instead of only four times for conventional Alhydrogel®. To test whether these additional washing stages would result in reduced metal ion contamination, three different batches (4580, 4596, and 4577) were analyzed. The results are included in Table 23. No difference in metal ions was observed compared to conventional-grade Alhydrogel®, suggesting that either the metal ions are strongly bound to the surface of the aluminum hydroxide particles or they actually coprecipitate during the production process. Figure 13 shows a comparison of the different batches of Alhydrogel® analyzed. The total content of contaminating metal ions for all the contaminant elements tested is shown, with the absolute proportions of the three main metals, Fe, Cr, and Ni, represented in different colors. As can be seen, batch 4074 has very few contaminating metal ions compared to most of the other batches analyzed. Only batch 3877 showed similar batch contamination, while batch 4230 shows by far the highest contamination of all the batches analyzed during this investigation. During the research trials, a large variation in metal ion content was observed among different batches of Alhydrogel® (see Figure 13). To test whether these contaminating metal ions were located in the aluminum hydroxide fraction or in the supernatant, Batch 4230 was separated into a supernatant and a sediment fraction (see Table 24). As can be seen, less than 2% of the metal ions could be detected in the supernatant, indicating that all the contaminating metal ions are bound to the surface of the aluminum hydroxide particle or within the particle structures. Therefore, it can be estimated that the local concentration of metal ions is at least 50-100 times higher, since the solid volume fraction (volume of Alum granules after centrifugation) of 0.1% Al2O3 (corresponding to 0.5 mg / ml of Al) used in the JEV vaccine formulation is approximately10-20 pl per 1000 pl of FVL. 5 Summary Alhydrogel® is used at a final concentration of 0.1% as an adjuvant in the current formulation of the JEV vaccine. During an investigation into an out-of-specification (OOS) potency result for the production of FVL JEV09L37, an evaluation of the Alhydrogel® production process was initiated. A total of 13 different batches of Alhydrogel® were analyzed for the presence of contaminating metal ions that could reduce protein stability. Large variations in the concentration of several metal ions were observed for different batches of Alhydrogel®. When the raw materials were analyzed, these contaminants were found to be present at the same concentration as those found within the Alhydrogel. Higher levels of Fe, Ni, and Cu ions were observed in Alhydrogel® lot 4230 compared to the other lots investigated. Lot 4230 was the only lot in which residual Cu ions were detected. This lot 4230 was used for the formulation of FVL JEV09L37. When the supernatant and insoluble fraction of a batch of Alhydrogel® were analyzed, these contaminating metal ions could only be found in the precipitate, indicating that these ions are attached to the surface of the aluminum hydroxide particle or are actually part of the particle. Although macroscopically and in composition different from other batches of Alhydrogel® used for the production of JEV, batch 4230 met all the requirements detailed by the Ph. Eur. In addition, the physical characterization (particle size distribution and zero charge point) showed no differences between batch 4230 and other batches of Alhydrogel® that did not show these high metal ion contaminations. Example 4 1.1. Materials, Equipment and Methods 1.2. Equipment Analytical balance (readability of 0.1 mg; for example, Mettler Toledo XP205DR / M) Precision balance (0.1 g readability; e.g., Mettler Toledo, Model No. XS6002S Delta Range) 0.22 pm filter units (e.g., Stericup Cat. No. SCGPV01RE) or 0.2 pm filter system (e.g., Steriflip Millipore 50 ml) Freezer (-20 °C) and Ultra-low temperature freezer (-80 °C) Refrigerator (+2 to 8 °C) Magnetic stirrer (e.g., KIKA Labortechnik RCT basic) and magnetic stir bars Microplate washer: for example, BioTek ELx405 Microplate reader: for example, BioTek Synergy 2 and Gen5 Secure software Microplate incubator (37 °C) Microtiter sealing tape (e.g., Thermo Electron 9503130) Multichannel pipettes and tips (e.g., Eppendorf Research Pro 50-1200 pl, Eppendorf Research, 10-100 pl) pH meter (e.g., WTW ino Lab Series, Terminal 740 and pH / Cond. 740) Pipettes and tips (e.g., Eppendorf Research, 0, 5-10 pl, 2-20 pl, 20-200 pl, 100-1000 pl, 500-5000 pl) Micropipette (e.g., IBS Biosciences Pipetboy) 15 ml PP tubes (e.g., Sarstedt 62.515.006) or 50 ml PP tubes (e.g., Greiner 227261) 50 ml reagent container (e.g., Corning Incorporated 4870) Serological pipettes (e.g., Falcon, 2 ml, 5 ml, 10 ml, 25 ml, 50 ml) Titertube Microtubes: Bulk (BioRad 223-9391) Vortex mixer (for example, VWR Analog Vortex Mixer, model No. 945304) Eppendorf LoBind tubes of 1.5 ml or 2.0 ml (Cat. No. 0030 108.116, Cat. No. 0030 108.132, respectively) 96-well microplate (F96 Cert. Maxisorp Nunc-Immunoplates) For the analysis of DP samples, in addition: Benchtop centrifuge (e.g., Beckman Coulter, Microfuge 16 Centrifuge, Cat. No. A46473) Orbital shaker (e.g., Eppendorfer Thermomixer compact) 50ml PP tubes (e.g., Greiner 227261) 1.3. Reagents PBS 10x (e.g., Gibco, Cat. No. 14200-083) Tween 20 (for example, Sigma Cat. No. P7949) 2 M sulfuric acid (volumetric solution, e.g., Fisher, Cat. No. J / 8410 / 17) Deionized water, e.g., (Milli-Q, 18, 2 O) Sodium carbonate - bicarbonate capsules (e.g., Sigma, Cat. No. C3041) Hydrochloric acid (HCl) 1 mol / l (e.g., Merck, Cat. No. 1.09057.1000) Sodium hydroxide (NaOH) 1 mol / l (e.g., Merck, Cat. No. 1.09132.1000) Glycerol (e.g., Sigma) For the analysis of DP samples in addition: Potassium hydrogen phosphate trihydrate (e.g., Sigma, Cat. No. P5504) Potassium dihydrogen phosphate (e.g., VWR, AnalaR Normapur, Cat. No. 26936.260) Albumin, bovine serum (BSA), ELISA grade (e.g., Sigma, Cat. No. A3059) TMB substrate (e.g., BioFX, TMBW-1000-01) Donkey anti-rabbit IgG HRP conjugate (Jackson Immuno Research, Cat. No. 711-035-152) Reconstitution: The contents of one vial (0.4 mg) are reconstituted in 0.5 ml of deionized water and mixed thoroughly until completely dissolved. Add 0.5 ml of glycerol and mix further until homogeneous. Aliquots are stored at -20 °C until use. Inactivated JEV Reference Standard (Intercell Biomedical Ltd.) Purified sheep anti-JEV (Intercell Biomedical Ltd.) Purified rabbit anti-JEV (Intercell Biomedical Ltd.) 1.4. Solutions a) 0.05 M carbonate buffer at pH 9.6 (used for coating ELISA plates) For 100 ml of buffer, dissolve one bicarbonate / carbonate buffer capsule in 100 ml of deionized water. Check the pH and adjust to 9.6 ± 0.1 with HCl or NaOH if necessary. Use only on the day of preparation. Keep the ELISA coating buffer at room temperature on the day of use and then discard. b) ELISA wash buffer and part of the sample block / diluent (PBS-T) Prepare approximately 1 liter per plate used. Dilute 10x PBS stock solution 1+9 in deionized water, mix well, and check the pH (7.4 ± 0.1). Adjust with 1 M HCl or 1 M NaOH as needed. Add 0.05% (v / v) Tween20 and mix well. for example, ELISA wash buffer (PBS-T) [1 l]: 100 ml PBS 10x 900 ml deionized water Mix well, check / adjust the pH (7.4 + / - 0.1). 0.5 ml of Tween20 Mix well. Use only on the day of preparation; keep the ELISA wash swab at room temperature during the day of use and then discard. c) Blocking solution: 5% BSA in PBS-T Prepare approximately 25 ml for each plate. Measure the required amount of PBS-T into a clean glass bottle using a serological pipette. Add a clean magnetic stir bar. Weigh the required amount of BSA, add it to the surface of the PBS-T, and gently mix with a magnetic stir bar until all the BSA has dissolved. Filter the solution using a 0.2 µm filter (either a Steriflip filter system or a syringe filter). For example, blocking solution [100 ml] 5 g BSA 100 ml PBS-T Use only on the day of preparation; keep the blocking solution at room temperature during the day of use and then discard. d) Sample diluent: 1% BSA in PBS-T Prepare as above, but with 1 g of BSA per 100 ml of PBS-T; approximately 25 ml per plate is required. For example, sample diluent [100 ml] 1 g BSA 100 ml PBS-T Use only on the day of preparation; keep the sample diluent at room temperature during the day of use and then discard. For the analysis of DP samples, in addition: e) PBS 1x Prepare 1 part of PBS 10x with 9 parts of deionized water for example, PBS 1x [100 ml] 10 ml of PBS 10x 90 ml of deionized water Use only on the day of preparation; maintain PBS 1x at TA during the day of use, then discard. f) 20x ELISA buffer Weigh an appropriate amount of BSA into a suitable container to prepare a 20x solution. Add the appropriate volume of 1x PBS. Add Tween20 to a final concentration of 0.05%. Mix on a magnetic stirrer until the BSA is completely dissolved. Filter the solution through a 0.2 µm filter (using a Steriflip filter system or a syringe filter) into a sterile container (and divide into aliquots as needed). for example, 20x ELISA buffer [25 ml] 5 g of BSA 25 ml of PBS 1x 12.5 pl of Tween20 The solution can be stored at 2-8 °C for 1 week. g) The 2x ELISA buffer is prepared by diluting the 20x ELISA Buffer with 1x PBS (1 part of 20x ELISA Buffer and 9 parts of 1x Pb S). For example, ELISA Buffer 2x [20 ml] 2 ml ELISA 20x buffer 18 ml of PBS 1x Use only on the day of preparation; keep the 2x ELISA buffer at room temperature during the day of use and then discard. h) Desorption buffer Potassium phosphate stock solution: Prepare a 3x potassium phosphate stock solution (2.4 M) by dissolving the appropriate volume of dipotassium phosphate trihydrate and potassium dihydrogen phosphate in deionized water. Place on a magnetic stirrer and, once dissolved, make up to the required volume. Verify that the pH of the solution is 8.0 ± 0.1. Filter through a 0.2 µm filter. for example, 3x stock solution of Potassium Phosphate (2.4 M) [50 ml] 23,963 g Dipotassium phosphate trihydrate 2,041 g Potassium dihydrogen phosphate Fill to 50 ml with deionized water Store at +2°-8°C for up to 1 month. Prepare a working-strength desorption buffer (0.8 M potassium phosphate buffer containing 1% BSA and 0.05% Tween20) by adding the appropriate volume of potassium phosphate stock solution (2.4 M), Tween20, and BSA to the required volume of deionized water. Mix thoroughly and use on the day of preparation. For example, working-strength desorption buffer [15 mL] 5 ml Potassium phosphate (2.4 M) 7, 5 pl Tween20 0.15 g BSA 10 ml Deionized water Keep the working strength desorption tampon at TA throughout the day of use. 1.5. Test samples and antibodies Test samples: or Active ingredient and / or VIN (various batches) or Samples of JEV vaccine (final bulk vaccine and final vaccine lot) Inactivated JEV reference standard (neutralized inactivated virus - NIV) (Intercell Biomedical Ltd.) Polyclonal antibodies: or Coating antibody: Purified sheep anti-JEV (Intercell Biomedical Ltd.) or Primary detection antibody: Purified rabbit anti-JEV (Intercell Biomedical Ltd.) Secondary conjugated antibody: Donkey anti-rabbit HRP conjugate (Jackson Immuno Research Cat. No. 711-035-152) 2 Procedure 2.1. Plate coating or Label the plate with the plate number, date, and analyst. Prepare a fresh 0.05 M carbonate buffer (pH 9.6) on the day of plate coating. Allow approximately 12 ml for each coated plate. Remove the required number of aliquots of the coating antibody from the freezer and allow to thaw at room temperature. Prepare a dilution of purified sheep anti-JEV antibody in carbonate buffer. Mix thoroughly by inverting the tube. or Using the multichannel pipette, apply 100 pl / well to a 96-well Maxisorp plate 15 min after antibody dilution preparation. or Cover with microtiter sealing tape and incubate for 17 to 72 hours at +2-8 °C. 2.2. Washing or Remove the plate from the refrigerator and let it warm to room temperature. Wash the plate(s) with the Microtiter plate washer three times using the appropriate wash program (300 pl per well, three times, final dispensing). After that, remove any remaining wash buffer by decanting. Invert the plate and tap it against a clean paper towel. Do not allow the microtiter plate to dry between washing and reagent addition. 2.3. Blocking or Prepare a 5% (w / v) BSA blocking solution in PBS-T as above. Apply 200 pl of blocking solution per well, cover the plate or plates with a cover plate and incubate at 37 °C for 1 hour + / - 10 min. 2.4. Preparation of standard curve dilutions Remove the NIV reference standard from the freezer, allow it to thaw at room temperature, and mix thoroughly. Prepare a 1 AU / ml stock dilution of the current reference standard; use at least 20 ml of NIV reference standard for the dilution. For example, NIV Predilution reference pattern: Concentration: 235 UA / ml (lot No. 03 / 2009) To prepare a working standard solution of 1 AU / ml, dilute it from 1 to 235 in sample diluent: 4680 pl sample diluent 20 pl NIV reference pattern or Then, prepare the following working standard solutions from the 1 AU / ml predilution: 0.8 UA / ml, 0.6 UA / ml, 0.4 UA / ml, 0.2 UA / ml, 0.1 UA / ml and 0.05 UA / ml in sample diluent. 2.5. Quality control samples a) Quality control (QC) samples (e.g., at 0.75, 0.30 and 0.18 AU / ml) should be prepared from the predilution of the new NIV reference standard at the time of the assay and then discarded after use. b) These controls are part of the system suitability criteria and allow monitoring of test performance over time. 2.6. Preparation of test samples Preparation of active ingredient Test samples of active ingredients are received for testing at unknown concentrations. These will be tested in triplicate at six dilutions. The dilutions will be performed independently within the range of the standard curve, for example, a preliminary dilution of 1:15 or another suitable dilution, followed by six dilutions with sample buffer. NIV sample preparation NIV samples will be received for analysis at unknown concentrations and pre-diluted within the range of the standard curve (e.g., 1 in 30 or other suitable dilution) and then diluted six times in the same manner as DS (drug substance) samples. Preparation of drug supernatant a) For the analysis of bulk drug product (DP) samples, mix the sample thoroughly by vortexing. Transfer exactly 1 ml to a 1.5 ml Eppendorf LoBind tube. For analysis of samples from finished product containers, transfer the contents of two syringes (0.6 ml per syringe) from the same batch into a 1.5 ml Eppendorf LoBind tube. Thoroughly mix the contents of the tube by inversion to ensure homogeneity of the DP and transfer exactly 1 ml to a new 1.5 ml Eppendorf LoBind tube. b) Centrifuge tubes containing 1 ml of DP each at 3300 x g for 5 minutes. c) For each sample, pipette 25 pl of 20x ELISA buffer into a new Eppendorf LoBind tube. d) Carefully remove 475 µl of the supernatant without disturbing the alum pellet and transfer it to the tube containing the 20x ELISA buffer. Mix gently by inversion. Centrifuge again for 2 min at 16,000xg. Store the sample at +2–8 °C before analysis. NOTE: DP supernatant samples prepared in this manner must be measured pure in triplicate on the inactivated JEV ELISA. e) Carefully remove as much residual supernatant as possible from the centrifuged tube using a 10-200 pl pipette without disturbing the alum sediment and discard the supernatant. f) The sediment obtained is subjected to the Desorption procedure as described below. Drug desorption procedure a) Add 158 pl of working strength desorption buffer to each pellet remaining in the LoBind tube. b) Resuspend the pellet by pipetting up and down several times to ensure complete pellet resuspension and sample homogenization. c) Incubate the samples for 10 min at TA in an orbital shaker at 500 rpm. d) After incubation, centrifuge the samples at 3300xg for 5 minutes. e) For each sample, pipette 250 ml of 2x ELISA buffer into a new Eppendorf LoBind tube. f) Carefully remove 83.3 pl from the top of the supernatant containing the desorbed product without disturbing the precipitate and transfer it to the tube containing the 2x ELISA buffer. Remove the remaining supernatant using a 20-200 pl pipette without disturbing the precipitate and discard the supernatant. g) Add another 158 pl of working strength desorption buffer to each sediment. h) Perform two more desorption cycles (three in total) by pooling the three 83.3 ml of desorbed material with 250 ml of ELISA buffer in the appropriate tube. After the last step, the remaining pellet can be discarded. i) The final concentration of viral antigen in the desorbed pool(s) should now be the same as the original 1 ml of DP from which it was desorbed. Therefore, the concentration of inactivated JEV antigen measured in the desorbed pool can be directly related to that of the original DP. Note: Analyze the desorbed samples by ELISA on the day of desorption. j) Dilution of desorbed DP samples: These will be tested in six dilutions in triplicate. An appropriate pre-dilution will be made within the range of the standard curve, for example, 1 in 15 (100 pl to 1400 pl of diluent) or another suitable dilution, then six 1 in 15 dilutions will be made independently using sample diluent. 2.7. Sample loading and plate plan Prepare samples and patterns before analysis. After blocking, wash the plate using the plate washer with the JEV ELISA program. Then, remove any remaining wash buffer by decanting. Invert the plate and tap it against a clean paper towel. Do not allow the microtiter plate to dry between washing and reagent addition. Add 100 pl / well of standards / controls / samples and cover with a cover plate and incubate for 1 hour + / - 10 min at 37 °C. Add 100 ml of sample diluent to all wells not required for the assay. 2.8. Preparation of the primary antibody Remove the required aliquots of the primary antibody from the freezer and allow to thaw at room temperature. Prepare rabbit anti-JEV antibody in sample diluent at an appropriate dilution no more than 15 minutes before use. After sample incubation, wash the plate using a plate washer with the JEV ELISA program. Remove any remaining wash buffer by decanting. Invert the plate and tap it against a clean paper towel. Do not allow the microtiter plate to dry between washing and reagent addition. Add 100 pl / well of diluted primary antibody, cover with a cover plate, and incubate for 1 hour ± 10 minutes at 37°C. 2.9. Preparation of secondary antibody conjugate Remove the required amount of aliquots of the secondary antibody conjugate from the freezer and allow to thaw at room temperature. Prepare a dilution of Rabbit Donkey anti-HRP antibody in sample diluent no more than 15 minutes before use; for example, for a 1:10,000 dilution, prepare a 1:100 predilution and then a second 1:100 dilution. After incubating the antibody first, wash the plate using a plate washer and the JEV ELISA program. Remove any remaining wash buffer by decanting. Invert the plate and tap it against a clean paper towel. Do not allow the microtiter plate to dry between washing and reagent addition. Add 100 pl / well of diluted secondary antibody conjugate, cover with a cover plate, and incubate for 1 hour ± 10 minutes at 37°C. 2.10. Substrate incubation Once the conjugate has been added, remove the TMB from the refrigerator at 2–8 °C. Pipette the required volume (12 mL of TMB per plate) into a 50 mL centrifuge tube using a serological pipette. Allow the TMB to reach room temperature in the dark. After conjugate incubation, wash the plate three times using the plate washer and the JEV ELISA program. Remove any remaining wash buffer by decanting. Invert the plate and tap it against a clean paper towel. Do not allow the microtiter plate to dry between washing and reagent addition. Add 100 l / well of TMB and develop the plate in the dark at room temperature for 10 minutes. 2.11. Stopping and reading After 10 minutes of incubation with TMB, stop development by adding 100 pl / well of 2 M sulfuric acid. Read the plate at 450 nm (reference filter 630 nm) within 10 minutes of stopping using the BioTek reader and Gen5 Secure software. 2.12. Data Analysis NIV / DS data analysis: The Gen5Secure software will be used to calculate the % recovery of the QCs, concentration x dilutions, average concentration of the samples corrected by dilutions, and this value multiplied by 1.05 to correct for the addition of ELISA buffer. DP data analysis: The Gen5Secure software will be used to calculate the % recovery of the QC, the concentration x dilutions, and the average concentration of the sample dilutions. For PD supernatant samples: If the concentration of the supernatant sample is below the assay's LIDC (i.e., 0.05 UA / ml), then the supernatant sample should be recorded as <0.05 UA / ml If the concentration of the supernatant sample is within the assay's LDC (i.e., from 0.05 UA / ml to 1.25 UA / ml), the concentration value for the supernatant sample is recorded. If the supernatant sample concentration is above the assay's upper limit of concentration (ULDC) (i.e., 1.25 AU / ml), the drug supernatant preparation must be repeated. The supernatant sample will be retested by performing an appropriate predilution within the range of the standard curve, followed by 6 sample dilutions. (It is not necessary to repeat the desorbed drug sample.) The average concentration for dilutions that are within the assay's ULDC (0.05 AU / ml to 1.25 AU / ml) will be the recorded concentration value for the supernatant sample, provided that the system is suitable and at least 4 of the 6 sample dilutions are within the ULDC. 2.13. Test acceptance criteria a) The correlation coefficient of the calibration curve must be > 0.980. b) % CV <15% for standards and samples (except DP supernatant) for the four highest concentrations of the dilutions, % CV <15% for controls c) Individual blank DOs must be < 0.2. d) Test controls must be within the specified defined limits (for freshly prepared controls, 2 of 3 QC must have observed concentrations within ± 30% of nominal values; OR the levels established during QC qualification) in order to pass the test. e) The validity of the test will be recorded on the printed copy of Gen5. If the board does not meet the defined acceptance criteria, the test is considered invalid. 2.14. Data notification LEVEL a) Antigen content The reported value for inactivated UA / ml is the mean of the concentrations (within the LDC of 0.04 to 1.25 UA / ml) calculated for single-sample dilutions, corrected by the respective dilution factors, and the mean multiplied by a correction factor of 1.05 to account for 5% of the 20x ELISA buffer volume added to each sample at collection. Antigen content will be recorded on the Gen5 printout. DS: a) Identity If the absorbances of the samples at the lowest dilution (highest concentration) are greater than 3 standard deviations above the mean blank value, the result will be reported as positive. b) Antigen content The reported value for inactivated UA / ml is the mean of the concentrations (within the LDC of 0.04 to 1.25 UA / ml) calculated for single-sample dilutions, corrected by the respective dilution factors, and the mean multiplied by a correction factor of 1.05 to account for the 5% volume of 20x ELISA buffer added to each sample at collection. Antigen content will be recorded on the Gen5 printout. Desorbed DP: a) Identity: If the absorbances of the samples at the lowest dilution (highest concentration) are greater than 3 standard deviations above the mean blank value, the result will be reported as positive. b) Antigen content The reported value for inactivated UA / ml is the mean of the concentrations (within the LDC of 0.05 to 0.8 UA / ml) calculated for single-sample dilutions corrected by the respective dilution factors. The antigen content will be recorded on the Gen5 printout. DP supernatant (degree of adsorption / degree of non-adsorption): The degree of adsorption is reported with respect to the drug substance formulated with aluminum hydroxide after filtration. a) For the calculation of the reported value, the reported antigen content (UA / ml) for the respective DS sample (post-filtration), corrected for dilution with aluminum hydroxide (5%), will be set at 100%, and the percentage of the concentration measured in the supernatant (corrected for the addition of 5% ELISA buffer) will be calculated relative to that. The reportable value will be the difference between 100% and the percentage calculated for the supernatant. Results will be reported to two decimal places. DP supernatant (AU / ml) * 1.05 Degree of adsorption (%) = 100% - ------------------------------------------------- * 100% DS (UA / ml) * 0.95 The degree of non-adsorption will be calculated as detailed below and the results will be reported to 2 decimal places: DP supernatant (AU / ml) * 1.05 Degree of non-adsorption (%) = .......................................................... * 100 % DS (UA / ml) *0.95 b) If the pure supernatant does not contain any measurable antigen (i.e., the observed supernatant concentration is less than the LIDC, where LIDC = 0.05 AU / ml), the LIDC will be used to calculate the result. The result in this case is reported as "greater than x%". For example, if the DS sample is measured as 12.00 AU / ml and no signal was measured in the supernatant, with the LIDC of 0.05 AU / ml, the amount in the supernatant is <0.05 * 1.05 = <0.0525 AU / ml. The amount of DS after buffer correction is 12.00*0.95 = 11.40 UA / ml, and the reported result for the degree of adsorption is < 100-0.0525 / 11.4*100 = > 99.54%. The degree of non-adsorption (i.e., 10.- the % degree of adsorption) will also be reported. Example 5 Introduction: In order to further investigate the mode of action leading to product instability / loss of potency of the JEV vaccine, Ala-(His)6-OprF190-342-OprI21-83 (SEQ ID n O: 1, Figure 14.- also referred to herein as "protein A") was used in a preliminary screening assay incorporating alhydrogel batches with different metal contents and additions with copper and sulfite ions. Material: • Copper(II) chloride dihydrate (Sigma, Order No. 807483) • 10xPBS (Gibco, order number 14200-091) • Falcon 15 ml tubes (Greiner, Cat. No. 188724) • Infors HT Incubator Multitron Standard Incubator (InforsAG) • Bidet water. (Fresenius Kabi, Art. No. 0712221 / 01 A) Preparation of stock solutions: • Copper(II) stock solution • A 20 mM copper (II) stock solution was prepared by dissolving 341 mg of copper (II) chloride dihydrate in 100 ml of double-distilled water. • Sodium metabisulfite stock solution A 200 mM sodium metabisulfite stock solution was prepared by dissolving 1.52 g of sodium metabisulfite in 35 mL of PBS. The pH of this solution was adjusted to 7.3 with NaOH, and the volume was brought up to 40 mL with PBS. The solution was then filtered through a 0.2 µm syringe filter. Preparation of work solutions Working solutions were prepared by diluting metal stock solutions with double-distilled water and sterile filtration through a 0.2p syringe filter (Mini Kleenpak 25 mm-Pall). Preparation of buffer solutions • / PBS + NaCl at 0.9% A 1x PBS buffer solution was prepared by diluting 10x PBS 1:10 with double-distilled water. The pH of this buffer solution was 7.5. PBS buffer solutions adjusted to pH 7.3 and 8.0 were prepared by adjusting the pH with HCl or NaOH, respectively. 9 g of NaCl were dissolved in 333 ml of pH 7.3 or pH 8.0 buffer solution and then brought to 1000 ml with double-distilled water, followed by filtration through a 0.2 µm bottle filter. Sample preparation: Protein A formulations and different batches of alhydrogel (Batch 4230 and Batch 4074) were prepared in / PBS + 0.9% NaCl at two different pH values ​​and were supplemented with sulfite according to the following scheme: Samples 1, 4, 7, and 0 were stored at 4 °C (reference samples). All other samples were incubated at 37 °C for 96 hours. After 96 hours, all samples underwent a desorption procedure to separate the antigen from the hydrogel. The desorbed antigen was analyzed by PCR. Results: The results showed severe degradation of the Protein A antigen in the presence of sulfite. The degradation was more pronounced in the samples formulated with alhydrogel containing higher levels of metallic impurities. Table 1. Metal ion content in batches 4230 and 4074 of aluminum hydroxide analyzed by ICP-MS. *Calculated residual metal content Table 2: DOE pipetting scheme Table 2 continued Table 2 continued Table 2 continued Table 3: Comparison of leachables from plug extract and JEV09L37 SN. All peaks with an area > 0.lmUA.min are included in this table. Table 4: DOE results obtained after 4 and 8 weeks at 22 °C. Antigen was desorbed from alum and analyzed by ELISA (monoclonal and polyclonal). * (x-fold increase compared to FVL) Table 4 continued Table 5: Analysis of variance for the monoclonal / polyclonal ELISA ratio after 4 weeks of storage at 22 °C Analysis of Variance for the 4-Week Relationship Source Sum of squares df Mean square F ratio PA value: pH 0.02205 1 0.02205 5.48 0.0326 B: Alum 0, 117612 1 0, 117612 29, 20 0, 0001 C: PS Fragments 0, 0008 1 0, 0008 0, 20 0, 6618 D: Extractables 0, 0008 1 0, 0008 0, 20 0, 6618 E: Formol 0.0242 1 0.0242 6.01 0.0261 AB 0.0001125 1 0.0001125 0.03 0.8694 AC 0.00045 1 0.00045 0.11 0.7425 AD 0, 01125 1 0, 01125 2, 79 0, 1141 AE 0.00405 1 0.00405 1.01 0.3309 BC 0.0000125 1 0.0000125 0.00 0.9563 DB 0, 0010125 1 0, 0010125 0, 25 0, 6229 BE 0, 0006125 1 0, 0006125 0, 15 0, 7017 CD 0.0008 1 0.0008 0.20 0.6618 EC 0, 0008 1 0, 0008 0, 20 0, 6618 FROM 0, 0072 1 0, 0072 1, 79 0, 1999 Total error 0, 0644375 16 0, 00402734 Total (corr.) 0, 2562 31 R squared = 74.8488 percent R square (adjusted for df) = 51.2695 percent Standard error of Est. = 0.0634614 Mean absolute error = 0.0352734 Durbin-Watson statistic = 1.47556 Table 6: Analysis of variance for the monoclonal / polyclonal ELISA ratio after storage at 22 °C for 8 weeks. Analysis of Variance for the 8-week Relationship Source Sum of squares df Mean square F ratio PA value: pH 0.102378 1 0.102378 11.19 0.0041 B: Alum 0, 275653 1 0, 275653 30, 13 0, 0000 C: PS Fragments 0, 00300312 1 0, 00300312 0, 33 0, 5747 D: Extractables 0, 0108781 1 0, 0108781 1, 19 0, 2917 E: Formol 0.0318781 1 0.0318781 3.48 0.0804 AB 0, 00137813 1 0, 00137813 0, 15 0, 7031 AC 0.00382812 1 0.00382812 0.42 0.5269 AD 0, 00137813 1 0, 00137813 0, 15 0, 7031 AE 0, 0166531 1 0, 0166531 1, 82 0, 1961 BC 0.000253125 1 0.000253125 0.03 0.8700 DB 0, 00382813 1 0, 00382813 0, 42 0, 5269 BE 0, 00195313 1 0, 00195313 0, 21 0, 6503 CD 0, 00195313 1 0, 00195313 0, 21 0, 6503 EC 0.000153125 1 0.000153125 0.02 0.8987 FROM 0, 00525313 1 0, 00525313 0, 57 0, 4596 Total error 0, 1464 16 0, 00915 Total (corr.) 0, 606822 31 R squared = 75.8743 percent R square (adjusted for df) = 53.2565 percent Standard error of Est. = 0.0956556 Mean absolute error = 0.0571484 Durbin-Watson statistic = 0.888586 Table 7: Regression analysis for "8-week relationship" including pH, Alum and Formaldehyde. Regression coefficients for the 8-week relationship Constant = 0.0228125 A: pH = 0.113125 B: A lum bre = -0, 00185625 E: Formaldehyde = 0.0315625 Table 8: Estimation of "8-week relationship" results generated using the adjusted model. Estimated Results for the 8-Week Relationship Observed Ajusted Lower 95.0% LC Upper 95.0% LC Row Value Value for Mean for Mean 1 0.58 0.5975 0.536766 0.658234 2 0, 81 0, 783125 0, 722391 0, 843859 3 0, 9 0, 84625 0, 785516 0, 906984 4 0, 92 0, 89625 0, 835516 0, 956984 6 0, 99 0, 84625 0, 785516 0, 906984 7 0, 67 0, 5975 0, 536766 0, 658234 8 0, 99 0, 89625 0, 835516 0, 956984 9 0, 84 0, 710625 0, 649891 0, 771359 10 0, 78 0, 710625 0, 649891 0, 771359 11 0, 98 0, 959375 0, 898641 1, 02011 12 0, 59 0, 5975 0, 536766 0, 658234 13 1, 03 0, 959375 0, 898641 1, 02011 14 0, 78 0, 660625 0, 599891 0, 721359 15 0, 94 0, 89625 0, 835516 0, 956984 16 0, 81 0, 77375 0, 713016 0, 834484 17 0, 82 0, 783125 0, 722391 0, 843859 18 0, 88 0, 84625 0, 785516 0, 906984 19 0, 82 0, 77375 0, 713016 0, 834484 20 0, 82 0, 959375 0, 898641 1, 02011 21 0, 82 0, 959375 0, 898641 1, 02011 22 0, 63 0, 710625 0, 649891 0, 771359 23 0, 74 0, 84625 0, 785516 0, 906984 24 0, 66 0, 660625 0, 599891 0, 721359 25 0, 67 0, 783125 0, 722391 0, 843859 26 0, 76 0, 783125 0, 722391 0, 843859 27 0, 64 0, 710625 0, 649891 0, 771359 28 0, 74 0, 77375 0, 713016 0, 834484 29 0, 73 0, 77375 0, 713016 0, 834484 30 0, 44 0, 5975 0, 536766 0, 658234 32 0, 87 0, 89625 0, 835516 0, 956984 33 0, 67 0, 660625 0, 599891 0, 721359 34 0, 59 0, 660625 0, 599891 0, 721359 Table 9: Plan for experiment 20110913 (NIV) Pipetting plan Mother Sun [mM] Total volume: 2000 M Ni (II) SO4 1 NIV Material: 11A74 Cu(II)Cl2 1 Table 9 continued Table 9 continued PM [g / mol] other additives: Neither 58, 7 Mother Sun: C 63.6 PS frag 2 mg / ml Cr 52.0 Table 9 continued Table 10: Plan for experiment 20110913 (DP) Pipetting plan Mother solution [mM] Total volume: 10000 Hl Ni (II) SO4 1 Cu(II)Cl2 1 DP:11D87 bulk Cr(III)Cl3 1 Table 10 Continued PM [g / mol] other additives: Neither 58, 7 Mother Sun: C 63, 6 PS frag 2 mg / ml Cr 52, 0 Table 11: Plan for experiment 20110812-DP added with metal ions Pipetting plan Mother sol [mM] PM [g / mol] FVL ng / ml pM Total volume: 30000 pl Fe (II) Cl3 1 Fe 55. 9 282 5. 049 DP: 11D87 bulk Fe (III) Cl3 1 Fe 55. 9 282 5. 045 Ni (II) SO4 1 Ni 58. 7 41 0,698 Co (II) Cl2 1 Co 58.9 0.33 0.006 Cu (II) Cl2 1 Cu 63.6 3 0.047 Zn (II) SO4 1 Zn 65.4 Cr (III) Cl3 1 Cr 52, 0 Table 11 continued Sample Volume [pl] No. Name NIV dii Buffer Fe (II) CI3 Fe (III) CI3 Ni (II) SO4 Co (II) CI2 Cu (II) CI2 Zn (II) SO4 Total 1 DP_Fe (II) 27750 1981 269 0 0 0 0 0 2020 III_F 1982 0 26 0 0 0 0 0 30000 3 DP_Ni (II) 27750 1981 0 0 26 0 0 0 30000 4 DP_Co (II) 27750 1994 0 0 5 0 20 0 0 0 0 0 0 0 1995 0 0 0 0 25 0 30000 6 DP_Zn_p 27750 2014 0 0 0 0 0 23 30000 7 DP_mezcl 27750 698 26 26 20H a 25 ciones 30 DPp 7 27750 2250 0 0 0 0 0 0 30000 9 DP_Fe (II) _pH7, 4 7, 4 27750 1981 26 0 0 0 0 0 30000 25 DP_p7H (III0, .4) _ 1982 0 26 0 0 0 0 30000 11 DP_Ni (II) _pH7, 4 7, 4 27750 1981 0 0 26 0 0 0 30000 12 DP_Co (II) _pH7, 0 7, 4 1 2 7 25 0 0 30000 13 DP_Cu (II) _pH7, 4 7, 4 27750 1995 0 0 0 0 255 0 30000 Table 11 continued Table 12: Antigen recovery determined by SEC-HPLC from ex. 20110913 (NIV). For pH 7 and pH 8, recoveries are based on NIV control samples without additives, no. 1 and no. 21, respectively. Samples were stored at 22 °C for 3 weeks. Samples labeled "na" were not analyzed due to sample prioritization. Results eration (%) 28 NIV_CR (III) _100_pH8_22 °C 4, 213 100, 0 Table 13: ELISA results of experiment 20110913 (NIV) obtained after 3 weeks at pH 8 (sample 21-40) and 7 weeks at pH 7 (sample 1-20) at 22 °C. Samples labeled "na" were not analyzed due to sample prioritization. 20110913 added with metal NIV 22 °C 7 weeks at pH 8 1st analysis 2nd analysis on 32 NIV_Ni (II) _100_PS addition_pH8_22 °C 18, 729 19, 250 1, 028 nano Table 14: Antigen recoveries after 5 weeks at 22 °C from desorbed JEV obtained by SEC-HPLC. Recoveries were based on DP control samples without additions stored at pH 7 or pH 8 Table 15: ELISA results of desorbed JEV antigen after 5 weeks at 22 °C Table 16: ELISA results of desorbed JEV antigen after 4 and 7 weeks of storage at 22 °C. Samples labeled "na" were not analyzed due to sample prioritization. Table 17: ANOVA for stability samples stored at 22 °C for 7 weeks. Analysis of Variance for Relationship - Type III Sums of Squares Source Sum of squares df Mean square F ratio P value MAIN EFFECTS R: metal type 0. 139643 8 0. 0174554 3. 00 0. 0293 B: pH 0. 0462028 2 0. 0231014 3. 97 0. 0398 RESIDUAL 0. 0931046 16 0. 00581904 TOTAL (CORRECTED) 0.27895 26 All F relationships are based on the residual mean squared error. Table 18: Multiple interval assay for the ratio by metal ion type. 1=Fe (II) ; 2=Fe (III) ; 3=Ni (II) ; 4=Co (II) ; 5=Cu (II) ; 6=Zn (II) ; 7=Cr (III) ; 8=Mixture[1-6]; 9=no added control Multiple interval tests for the ratio by metal type Method: LSD 95.0 percent Metal type Average LS count Homogeneous groups 8 3 0, 666087 X 5 3 0, 770168 XX 2 3 0, 793725 XXX 7 3 0, 807248 XX 4 3 0, 844388 XX 6 3 0, 853867 XX 1 3 0, 878563 XX 3 3 0, 887505 XX 9 3 0, 919926 X Contrast Difference + / - Limits 1-2 0, 084838 0, 132037 1-3 -0, 0089421 0, 132037 1-4 0, 0341754 0, 132037 1-5 0, 108395 0, 132037 1-6 0, 0246961 0, 132037 1-7 0, 0713155 0, 132037 1-8 *0, 212476 0, 132037 1-9 -0, 0413627 0, 132037 2-3 -0, 0937801 0, 132037 2-4 -0, 0506626 0, 132037 2-5 0, 0235569 0, 132037 2-6 -0, 0601419 0, 132037 2-7 -0, 0135225 0, 132037 2-8 0, 127638 0, 132037 2-9 -0, 126201 0, 132037 3-4 0, 0431175 0, 132037 3-5 0, 117337 0, 132037 3-6 0, 0336382 0, 132037 3-7 0, 0802576 0, 132037 3-8 *0, 221418 0, 132037 3-9 -0, 0324206 0, 132037 4-5 0, 0742195 0, 132037 4-6 -0, 00947935 0, 132037 4-7 0, 0371401 0, 132037 4-8 *0, 1783 0, 132037 4-9 -0, 0755381 0, 132037 5-6 -0, 0836988 0, 132037 5-7 -0, 0370794 0, 132037 5-8 0, 104081 0, 132037 5-9 *-0, 149758 0, 132037 6-7 0, 0466195 0, 132037 6-8 *0, 18778 0, 132037 6-9 -0, 0660588 0, 132037 7-8 *0, 14116 0, 132037 7-9 -0, 112678 0, 132037 8-9 *-0, 253838 0, 132037 * indicates a statistically significant difference Table 19: ICP-MS results of residual metal ion impurities present in various batches of Alum (2%) Residual metal content (ng / ml) Alum Batch (2%) Cr Fe Ni Cu V Co 4074 19, 8 266 14, 8 <25 <5 <5 *Calculated residual metal content based on Alum batches 4230 and 4074 **non-GI: not irradiated with gamma rays ***GI: irradiated with gamma rays Table 20: Summary of metal ion content and analysis of DP samples formulated with various lots of alum. Samples were analyzed in duplicate by ELISA, and the monoclonal / polyclonal ELISA ratio is reported. Formulations were stored at 22 °C for 6 weeks. *The interval is the absolute difference between the 1st and 2nd analysis. Table 21: PSD analysis results of the Alhydrogel® stock solution (2%) in water. d (0, 1) : 10% of all measured particles have a diameter below this value d (0, 5) : 50% of all measured particles have a diameter below this value d (0, 9) : 90% of all measured particles have a diameter below this value Darkening: amount of laser light reduction per sample; corresponds to the concentration of the sample in the measuring chamber Table 22: Results of the Alhydrogel® titration curves for the determination of POZ. The samples were analyzed in PBS (1:20 dilution). Table 23: Summary of metal ion analysis for various aluminum hydroxide stock solutions; Note: A 2% stock solution *Results below the LDC were not used in the average calculation; **The average cannot be calculated due to the results below the LDC Table 24: Analysis of the aluminum hydroxide gel fraction and supernatant (Batch 4230) to detect contaminating metal ions shows that the metal ions are found in the gel, not in the supernatant Table 25: Aluminum-based vaccines for human use Table 26: Aluminum-based vaccines for veterinary use

Claims

1. A method for preparing an aqueous composition comprising aluminum, a reactive compound, and a protein, wherein said method comprises: - selecting an aluminum salt capable of providing an aqueous composition having less than 350 ppb of total heavy metal and less than 2.5 ppb of Cu based on the weight of the aqueous composition; and - combining said aluminum salt, said reactive compound, and said protein within formaldehyde-inactivated virus particles and water to produce said aqueous composition having less than 350 ppb of total heavy metal and less than 2.5 ppb of Cu based on the weight of the aqueous composition; and wherein the reactive compound is a redox-active compound, a radical-forming compound, and / or a stabilizing compound.

2. A method according to claim 1, further comprising buffering said aqueous composition to a pH between 6.5 and 8.

5. 3.A method according to claim 1 or claim 2, wherein the aqueous composition comprises no more than 5 ppm of Cu compared to the aluminum content.

4. A method according to any one of claims 1 to 3, wherein the aqueous composition comprises less than 1.25 ppb of Cu based on the weight of the aqueous composition.

5. A method according to any one of claims 1 to 4, further comprising storing the aqueous composition for at least three months at a temperature between 2 °C and 8 °C. 6.A method according to claim 5, wherein protein degradation during storage is counteracted such that: (i) the aqueous composition is stable for at least three months at a temperature between 2°C and 8°C; (ii) the aqueous composition has a shelf life of 20 or 24 months; (iii) the aqueous composition can be stored for 20 or 24 months without becoming unsuitable for use due to protein degradation; (iv) a vaccine comprising the aqueous composition remains within potency specification after 20 or 24 months of storage; or (v) no more than 30% of the protein in the composition degrades after 20 or 24 months of storage.

7. A method according to any one of claims 1 to 6, wherein the selected aluminum salt comprises less than 700 ppm of heavy metal, on a weight basis relative to the aluminum content. 8.A method according to any one of claims 1 to 7, wherein the aluminum salt is aluminum hydroxide (Al(OH)3) or aluminum phosphate (AlPO4), preferably wherein the aluminum salt is aluminum hydroxide (Al(OH)3).

9. A method according to any one of claims 1 to 8, comprising between 5 pg / ml and 50 mg / ml of aluminum, preferably comprising between 50 pg / ml and 5 mg / ml of aluminum.

10. A method according to any one of claims 1 to 9, wherein the reactive compound is selected from the group consisting of formaldehyde, ethanol, chloroform, trichloroethylene, acetone, 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxyethanol, deoxycholate, diethyl pyrocarbonate, sulfite, Na2S2O5, beta-propriolactone, polysorbate (optionally Polysorbate 20 or Polysorbate 80), O2, phenol, pluronic-type copolymers, and a combination thereof, preferably wherein the reactive compound comprises sulfite. 11.A method according to any one of claims 1 to 10, wherein the protein is a viral protein from a virus of the Flaviviridae family, preferably wherein said virus of the Flaviviridae family is a Japanese encephalitis virus.

12. A method according to any one of claims 1 to 11, further comprising determining the heavy metal content in the aqueous composition and / or the aluminum salt. 13.Use of an aluminum salt comprising less than 700 ppm of heavy metal on a weight basis with respect to the aluminum content, to prepare an aqueous composition or vaccine, wherein the aqueous composition or vaccine comprises a protein, a reactive compound, and an aluminum salt, said composition comprising less than 350 ppb of heavy metal and less than 2.5 ppb of Cu on a weight basis of the aqueous composition, wherein the reactive compound is a redox-active compound, a radical-forming compound, and / or a stabilizing compound, and wherein said protein is a protein within formaldehyde-inactivated viral particles. 14.Use of an aluminum salt comprising less than 700 ppm of heavy metal on a weight basis with respect to the aluminum content, for (i) counteracting the degradation of a protein in an aqueous composition or vaccine; and / or (ii) extending the shelf life of an aqueous composition or vaccine; wherein the aqueous composition or vaccine is obtained by the method of any of claims 2 to 12 or comprises a protein, a reactive compound, and an aluminum salt, said composition comprising less than 350 ppb of heavy metal and less than 2.5 ppb of Cu on a weight basis of the aqueous composition, wherein the reactive compound is a redox active compound, a radical-forming compound, and / or a stabilizing compound, and wherein said protein is a protein within formaldehyde-inactivated viral particles. 15.Use according to claim 13 or claim 14, wherein the aqueous composition comprises not more than 5 ppm of Cu compared to the aluminum content.

16. Use according to claim 15, wherein the aqueous composition comprises not more than 2.5 ppm of Cu compared to the aluminum content.

17. Use according to any of claims 13 to 16, wherein the aluminum salt is aluminum hydroxide (Al(OH)3).

18. Use according to any of claims 13 to 17, wherein the aqueous composition or vaccine comprises between 0.1 and 2.5 mg / ml of aluminum, preferably wherein the aqueous composition or vaccine has an aluminum content of 0.5 mg / ml.

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