CN1353615A - Compositions of A-beta peptide and processes for producing same - Google Patents

Abstract

The invention is directed to compositions comprising solubilized A beta peptide or suspension of A beta peptide and to processes for producing the same by adjusting the pH sufficient to effect the solubilization, and sterile filtration thereof, to producing method of the compositions and methods of treating and preventing Alzheimer's disease with the obtained compositions.

Description

Composition of Aβ peptide and its preparation method

The technical field of the present invention

The present invention generally relates to pharmaceutical compositions comprising proteins useful in eliciting antibody responses in mammals. More particularly, the present invention relates to a pharmaceutical composition comprising an effective amount of amyloid beta peptide capable of eliciting an immune response in a mammal and a pharmaceutically acceptable diluent. The diluent is preferably a sterile injectable aqueous phase.

Background technology related to the present invention

Amyloid beta peptide, also known as A-beta or Aβ peptide, is a cleavage product of amyloid precursor protein (APP). It is a major component of the amyloid plaques in the brains of mammals that are common and hallmark of Alzheimer's disease. Due to the diversity of proteases on APP, the Aβ peptide is a variable-length peptide chain containing 39-43 amino acids.

Some mutations in the APP protein are associated with the development of Alzheimer's disease. For example, Goate et al. "Nature" (Nature) 349, 704 (1991) (mutation of valine ⁷¹⁷ to isoleucine (valine ⁷¹⁷ to isoleucine)); Chartier Harlan et al. "Nature" (Nature) 353, 844 (1991 ) ( ^{valine 717} to glycine (valine ⁷¹⁷ to glycine)) ^; Murrell et al. ); Mullan et al., "Nature Genet." (Nature Genet.) 1,345 (1992) (double mutation makes lysine ⁵⁹⁵ , aspartic acid ⁵⁹⁶ mutated into methionine ⁵⁹⁵ , leucine ⁵⁹⁶ (a double mutation changing lysne ⁵⁹⁵ -methionine ⁵⁹⁶ to asparagines ⁵⁹⁵ -leucine ⁵⁹⁶ )). It is generally believed that these mutations cause Alzheimer's disease by enhancing or altering the processing from APP to Aβ peptide, especially the processing of APP leads to an increase in the amount of Aβ42 and Aβ43. Mutations in other genes, such as early aging genes PS1 and PS2, are considered to indirectly affect the processing of APP and increase the amount of Aβ42 and Aβ43 (Hardy, TINS20, 154 (1997). Observations show that Aβ peptides, especially Aβ42 The pathogenic factor of Alzheimer's disease.In the brain, Aβ peptide aggregates to form amyloid deposits, including polypeptides forming fibrils through the β-sheet structure.

The impetus for recent therapeutic research on the treatment or prevention of Alzheimer's disease has focused on preventing or slowing down the production of Aβ peptide in the brain, either preventing its post-release process, or preventing the deposition to form amyloid plaques. A particularly important therapeutic approach to the application of the present invention is the use of the Aβ peptide to elicit an immune response in the body to counteract its effects. For example, PCT Publication No. WO99/27944, incorporated herein by reference.

The present invention relates to a novel and unexpected method for carrying out the invention disclosed in PCT Publication No. WO99/27944. In particular, the invention encompasses the administration of specific formulations of the long chain form of the Aβ peptide to a patient to elicit an immune response. However, in the prior art, the long-chain form of Aβ peptide is difficult to dissolve in conventional formulation systems.

In particular, Hilbich et al. "Journal of Molecular Biology" (J. Mol. Biol.), 218(1), pP.149-64, reported that although the Aβ1-43 peptide is soluble to some extent in pure water, However, the addition of ionic components, such as buffers, or salts, or organic solvents, can cause the peptide to precipitate out of solution in the form of amorphous aggregates. For example, Hilbich found that phosphate buffered saline ("PBS", in this example containing 135 mM NaCl, 3 mM KCl, 8 mM Na ₂ HPO ₄ ·H ₂ O, and 2 mM KH ₂ PO ₄ , pH 7.5) , which renders 90-94% of the peptides in the composition insoluble. PBS is a commonly used carrier for injection compositions, and its ion concentration and pH level are similar to those in living systems. 5 mM NaCl caused precipitation of 42-50% of the peptides. (The cited literature is the same as before, p.153, Table2). Peptide solutions in pure water were hypotonic and the pH of these solutions was determined to be 5.5 by Hilbich (same author). Normally, the pH of human blood is around 7.4. Dyrks et al. reported that Aβ peptide is insoluble under physiological conditions. Dyrks, T., Weidemann, A., Multhaup, G, et al., European Molecular Biology Journal (EMBOJ) 7, p. 949-57 (1988).

The conformation of the Aβ peptide in solution can be determined by circular dichroism (C.D.). Hilbich et al. (same authors) report the study of the conformation of A[beta] peptides and fragments using circular dichroism. In addition, M. Manning's book "Protein Structure and Stability Assessment by Circular Dichrosim Spectroscopy" (Protein Structure and Stability Assessment by Circular Dichrosim Spectroscopy) describes the stability and specificity of biological enzymes. Himmel, M.E. and Georgiou, G., ACS Symposium Series 516 (1993) p.36. References cited therein, as well as those cited by Manning, are hereby incorporated by reference.

Kline et al., U.S. Patent No. 5,851,996 ('996 patent) and No. 5,753,624 ('624 patent), describe the administration of very small amounts (10 ^-2 mg or less) of Aβ peptides or fragments, with liquid or solid carriers Under the oral administration, such as phenyl salt solution as a carrier. The '996 patent states that Aβ peptides exist in various structural forms (col. 2, line 13) and can be used to treat Alzheimer's disease. The patent does not specifically define the meanings of various structural forms, and does not specify its characteristics except for the 28 amino acid fragments used in the examples. The '996 patent states that Aβ peptide doses range from 10-10 to 10-2 mg (Vol. 8, pp. 442-43).

From the above point of view, the prior art has demonstrated difficulties in solubilizing and keeping A[beta] peptides soluble. Furthermore, the low solubility of the long-chain form of A[beta] peptide makes it difficult to achieve standardized sterilization thereof. Most standard sterilization methods are incompatible with peptide agents, such as radiation, autoclaving, and chemical sterilization techniques (eg, with ethylene oxide and glutaraldehyde), which can cause peptide degradation. Therefore, filtration of peptides can be chosen for Aβ peptide sterilization. However, the insolubility of Aβ peptides can cause clogging of the filter membranes, preventing the recovery and commercial production of sufficient Aβ peptides.

Purpose of the invention

The present invention relates to a surprising and unexpected discovery that a high-concentration aqueous solution of Aβ peptide can be prepared by adjusting the acid/alkalinity of the aqueous solution to achieve a pH range suitable for dissolving Aβ peptide. A preferred pH range is about 8.5-12, preferably about 9-10.

The present invention has further found that a soluble solution of Aβ peptide can be sterilized by passing through a suitable microporous filter paper, at least 50% of Aβ peptide can be recovered after filter sterilization. Under favorable conditions, at least 70% of the filter-sterilized A[beta] peptide is recovered, preferably 90%. The sterile solution can be made into a pharmaceutical composition containing sufficient Aβ peptide, which can cause immune response when administered to mammals. Preferably, parenteral administration can be performed using a suspension composition.

Thus, in one aspect of the composition invention, after sterile filtration, the pH of the composition is adjusted to physiologically appropriate conditions to form a suspension containing at least 0.1 mg/ml Aβ peptide. The composition can be used for parenteral administration. The pH of the suspension composition is about 5-7, preferably 5.5-6.5. A more preferred composition comprises sufficient amounts of QS-21 and Aβ peptide together to form a visibly clear sterile suspension.

It is a further discovery of the present invention that soluble sterile A[beta] peptide solutions can be processed by lyophilization to provide lyophilized formulations comprising A[beta] peptide. These compositions can be reconstituted at an appropriate time to provide an aqueous formulation comprising A[beta] peptide.

In another aspect of the composition, the invention relates to an aqueous solution containing at least 0.01 mg/ml Aβ peptide, wherein the aqueous solution maintains a pH suitable for dissolution of the Aβ peptide. Preferably, the solution can maintain a suitable pH value after using an effective amount of a pharmaceutically acceptable buffer.

In yet another aspect of the composition, the present invention relates to a sterile aqueous solution comprising at least 0.01 mg/ml Aβ peptide, wherein the aqueous solution maintains a pH suitable for dissolution of the Aβ peptide. Preferably, the solution can maintain a suitable pH value after using an effective amount of a pharmaceutically acceptable buffer.

In another aspect, the present invention relates to lyophilization of a composition comprising Aβ peptide, prepared by the following steps:

a) freezing a sterile aqueous solution comprising at least 0.01 mg/ml Aβ peptide, wherein the aqueous solution maintains a pH suitable for dissolution of the Aβ peptide; and

b) freeze-drying the frozen composition prepared in a) above.

Preferably, the compositions of the invention comprise the long chain form of the A[beta] peptide (as described below). More preferably, the composition comprises a pharmaceutically acceptable buffer selected from amino acids, salts, and derivatives thereof, pharmaceutically acceptable alkalizing agents, alkali metal hydroxides, and ammonium hydroxide, organic and inorganic acids and salts; and mixtures of the foregoing.

In another aspect of the composition, the present invention relates to a composition comprising an aqueous solution of at least 0.01 mg/ml Aβ peptide, wherein the aqueous solution maintains a pH suitable for dissolution of the Aβ peptide, wherein the Aβ peptide is substantially in a random coil conformation .

The composition of the present invention can be made into a pharmaceutical form suitable for administration to mammals suffering from Alzheimer's disease, or for prophylactic administration to persons at risk of suffering from the disease. The compositional aspect of the invention relates to pharmaceutical compositions wherein the Aβ peptide is a soluble random coil, or a stable aqueous suspension of at least 0.01 mg/ml Aβ peptide, or in lyophilized form, which can be is sterile and can be used for parenteral administration.

In one aspect of the process, the invention relates to a process for the preparation of a sterile composition of a long chain form of Aβ peptide comprising:

Adjust the pH value of the aqueous solution so that it can dissolve the Aβ peptide;

A certain amount of Aβ peptide is dissolved in the solution so that it can reach a concentration that can obtain mammalian immunity;

filtering the resulting solution through a membrane of uniform pore size capable of excluding bacteria allowing substantially all of the Aβ peptide to pass through the membrane; and

For solutions containing 0.01 mg/ml or more of A[beta] peptide, the pH of the resulting solution is optionally adjusted to about 5-7, resulting in a suspension of the peptide.

In another aspect of the process, the present invention relates to a method of preventing and treating Alzheimer's disease in a mammal comprising administering to the mammal a sufficient amount of a sterile aqueous solution comprising at least 0.05 mg/ ml of Aβ peptide to elicit an immune response in mammals.

More preferably, the A[beta] peptide used in the filtration treatment of the invention is substantially in random coil conformation.

Brief description of the drawings

Figure 1 is the average residue ellipticity measurements represented by circular dichroism spectra as a function of wavelength for two different Aβ42 peptide solutions. The dashed line indicates the absorbance value at pH 6, attributed to the β-sheet structure of the molecule. The solid line is the absorbance of the Aβ42 peptide solution at pH 9, indicating the random coil conformation of the peptide.

Figure 2 is a graph of Aβ42 peptide in solution corresponding to the amount of dissolved peptide calculated by peak area, determined by reversed-phase high performance liquid chromatography, demonstrating the solubility of Aβ peptide 42.

Detailed description of the invention

The present invention relates to compositions using aqueous solutions containing therapeutically effective concentrations of Aβ peptides and methods for their use. Before discussing the present invention in detail, the terms used are defined as follows.

definition:

The term "substantial identity" means that the sequences of two peptides are at least 65% identical when aligned optimally, as in the programs GAP or BESTFIT using the default gap weight, preferably At least 80%-90% sequence identity, preferably at least 95% or more sequence identity (eg, 99% or more sequence identity). Preferably, residue positions that differ are due to conservative amino acid substitutions.

For sequence comparison, typically one sequence is employed as a reference sequence, to which test sequences are compared. A suitable reference sequence is the human A[beta] peptide sequence, in particular the 42 amino acid sequence described below. Other suitable types are truncated forms, such as A[beta]39; or extended forms, such as A[beta]43 (with an extra threonine at the C-terminus). When using a sequence comparison algorithm, test and reference sequences are entered into a computer, sequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. According to the specified program parameters, the sequence comparison algorithm is used to calculate the percent identity of the test sequence and the reference sequence.

Optimal comparative sequence alignment, such as Smith & Waterman's "Advanced Applied Mathematics" (adv.Appl.Math.2: 482 (1981)) local homology algorithm, Needleman and Wunsch "Journal of Molecular Biology", (J.Mol .Biol.48: 443 (1970)), Pearson and Lipman, "Proceedings of the National Academy of Sciences" (Pro.Nat'l.AcadSci.USA) 85: 2444 (1988) similarity algorithm research; Applicable computer processing of these algorithms (GAP, BESTFIT, FASTA, and TFASTA of the Gemics Software Package of Wisconsin, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visualization Test (cf. generally Ausubel et al., supra). An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, described by Altschul et al., J. Mol. Biol. 215:403-410 (1990). BLAST analysis software is available from the National Center for Biotechnology Information (http: ∥www.ncbi.nlm.nih.gov/). Although specific parameters can be used, default program parameters can generally be used for sequence comparisons. For amino acid sequences, BLASTP can use the default 3W (wordlength), 10E (expectation), and BLOSUM62 scoring matrix (see Henicoff and Henikoff "Proceedings of the National Academy of Sciences of the United States" (Pro.Nat'l.Acad Sci.USA) 89: 10915 (1989)).

To classify amino acid substitutions as conservative and non-conservative, amino acids are divided into the following groups: Group 1 (hydrophobic side chains): norleucine, methionine, alanine, valine, leucine , isoleucine; the second group (neutral hydrophilic side chain): cysteine, serine, threonine; the third group (acidic side chain): aspartic acid, glutamic acid; the fourth group (Basic side chains): Asparagine, Glutamine, Histidine, Lysine, Arginine; Group V (residues affecting side chain orientation): Glycine, Proline; and Group VI (aromatic side chains): tryptophan, tyrosine, and phenylalanine. Conservative substitutions occur between amino acids of the same group. Non-conservative substitutions occur between members of one group and members of another group.

APP ⁶⁹⁵ , APP ⁷⁵¹ , and APP ⁷⁷⁰ refer to the polypeptide chains of 695, 751, and 770 amino acid residues encoded by the human APP gene, respectively. See Kang et al., Nature 325, 773 (1987); Ponte et al., Nature 331, 525 (1988), and Kitaguchi et al., Nature 331, 530 (1988). The amino acids of the human amyloid precursor protein (APP) are numbered by the sequence of the APP770 isoform.

In the present invention and literature, the weight of A[beta] peptide represents about 70%-80% A[beta] peptide and about 15%-30% salt and moisture. This is determined by amino acid analysis and/or nitrogen analysis. For example, if the peptide composition is calculated for 0.1 mg of Aβ42 peptide, it means that it contains 0.075 mg of Aβ42 peptide and 0.025 mg of water and salt; 0.6 mg of Aβ40 means that it contains 0.45 mg of Aβ40 peptide and 0.15 mg of water and salt ; 2.0mg of Aβ42 means that it contains 1.5mg of Aβ42 peptide and 0.5mg of water and salts.

The Aβ peptides involved in the present invention are those Aβ peptide fragments which can form a β-sheet conformation and which can elicit an immune response when administered alone or in combination with an adjuvant to a mammal. Methods for determining the conformation of beta sheets are well known to those skilled in the art, such as the cyclodichromatic method mentioned herein. Immunogenicity can be determined as described in the Biological Activity section of the Examples below.

"Long-chain form of Aβ peptide" includes Aβ38, Aβ39, Aβ40, Aβ41, Aβ42, and Aβ43 in any natural state, and substantially identical peptide sequences, preferably in the form existing in the human body. Aβ39, Aβ40, Aβ41, Aβ42, and Aβ43 refer to those Aβ peptides containing 1-39, 1-40, 1-41, 1-42, 1-43 amino acid residues, respectively, which excise the C-terminal amino acid of the peptide chain respectively . Therefore, Aβ41, Aβ40, and Aβ39 are different from Aβ42 in that alanine, alanine-isoleucine, and alanine-isoleucine-valine are missing from their C-termini respectively, which can be obtained from the following Aβ seen in the peptide sequence. Aβ43 differs from Aβ42 by the presence of a threonine residue at its C-terminus. The sequences of these peptides and their relationship to APP are described in Hardy et al. TINS 20, 155 Fig. 1 (1997).

The sequence of Aβ42: H ₂ N-aspartic acid-alanine-glutamine-phenylalanine-arginine-histidine-aspartic acid-serine-glycine-tyrosine-glutamic acid -Valine-Histidine-Histidine-Glutamine-Lysine-Leucine-Valine-Phenylalanine-Phenylalanine-Alanine-Glutamic Acid-Aspartic Acid Acid-Valine-Glycine-Serine-Asparagine-Lysine-Glycine-Alanine-Isoleucine-Isoleucine-Glycine-Leucine-Methionine-Valine-Glycine -Glycine-Valine-Valine-Isoleucine-Alanine-OH.

"Aβ peptide in long chain form" also includes analogues thereof. Analogs include allelic, species, and mutagenic genes. Typically, analogs differ from naturally occurring peptides at one or a few positions, often by conservative substitutions. Typically an analog will have at least 80% to 90% sequence identity to a naturally occurring peptide. Some analogs also include unnatural amino acids or N- or C-terminal modifications. Examples of unnatural amino acids are α-amino acids, α-disubstituted amino acids, N-terminal alkylated amino acids, lactic acid, 4-hydroxyproline, and γ-carboxyglutamic acid, ε-N,N,N-trimethyl Lysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methyl-histidine, 5-hydroxylysine, ω- N-methylarginine.

The use of A[beta] peptides in the compositions and processes of the invention can range from about 0.05 mg/ml to an upper limit solubility of about 2.0 mg/ml (see Figure 2). Suitable peptides range from about 0.1 to about 0.8 mg/ml, preferably range from about 0.3 to about 0.6 mg/ml.

The insoluble form of the Aβ peptide was reported to be found in amyloid plaques in a β-sheet conformation. Soto, C. et al., Neuroscience Letters, 186(2-3), pp. 115-118, (1995). In addition, Simmons, L.K., etc. "Molec.Pharmacol.", 45(3)pp.372-379(1994), reported that the conformation of the β sheet is related to the neurotoxicity of the peptide, while the random coil form has only Very little or no toxicity. From the above it will be appreciated that the methods and compositions of the present invention can use the random coil form of the A[beta] peptide. Applicants demonstrate that the random coil form is capable of eliciting an immune response in experimental mammals. The random coil form is most suitable for microfiltration processing.

"Random coil" refers to the open-chain conformation of the Aβ peptide. The random coil is the secondary conformation of the peptide backbone, rather than the orderly formation of a regular conformation, such as the conformation of α-helix or β-sheet. Random coil is the disordered interaction of hydrophobic base disordered folding and hydrogen bonding, rather than regular arrangement. Random coils can still form some peptide backbone folds or partial order, however these features are random and dynamic and thus not typical among all random coils. In the random coil conformation, the peptide exhibits excellent solubility and filterability. The α-helix or β-sheet conformation are well known peptide conformations in the art. For example, α-helices and β-sheets are described in Lehninger, Biochemistry ( ^2nd Ed., Worth Publishers, 1975)) pp. 128-9, incorporated herein by reference.

The random coil conformation of the A[beta] peptide can be determined by circular dichroism ("CD") and is easily distinguished from the [beta] sheet conformation. The circular dichroism method shows that the random coil has a strong negative band at 190 and 200nm, and almost no signal can be seen at wavelengths greater than 215nm. If there is a very weak positive signal at longer wavelengths, it is centered at 220-225nm. This can be seen in Figure 1. On the other hand, the β-sheet conformation exhibits a sharp and strong positive band at 200 nm. For a review of CD spectroscopy for the elucidation of peptide secondary structures, please refer to the Manning reference cited above.

When there is more than 50% random coil in the Aβ peptide, its conformation is in the random coil conformation. Preferably it is more than 70% or 80% random coil, preferably more than 85% or 90% of the Aβ peptide conformation is random coil.

"Compositions for parenteral administration" are those compositions which are sterile and suitable for direct administration to the human body, either in the form of injection or infusion, that is to say, through a Mode of administration for systemic protection. The other way is to enter the human body through the skin, mucous membrane or digestive and respiratory system. Therefore, the sterilization of parenteral administration compositions is very necessary.

"Infusion", as a method of drug administration, is a method in which the fluid of a drug solution enters the bloodstream in a relatively long period of time, basically continuously and at a slow rate. And "injection" is the rapid delivery of a dose of solution or suspension.

Intravenous (IV), intramuscular (IM), intraperitoneal (IP), subcutaneous (SC) and intrasternal injections are all routes of administration for parenteral ingredients. These methods describe in anatomical terms the part of the body where the injection or infusion is administered. This requires that the ingredients of this invention have a pH value under physiological conditions when acting on the human body through the above-mentioned pathways, and the specific value depends on the judgment of specific patients and doctors. Higher pH solutions (pH>8) are most suitable for slow intravenous infusion or drip.

To understand "buffer" and "buffer" in the context of the present invention, it is useful to review an acid-base titration curve, which has a relatively flat region extending to 1.0 pH units on either side of the titration midpoint. At the midpoint of the titration there are equal amounts of proton donor and proton acceptor (ie acid and base). In this region, when a small amount of H ⁺ or OH ^- is added, the pH value will only change slightly. In this region the conjugate acid-base pair acts as a buffer system, which prevents the pH from changing when H ⁺ or OH ^- is added. If the pH value is outside this region, the buffering capacity of the buffer will be weakened. The buffering capacity of a buffer is greatest at its pH value at the midpoint of the titration curve, that is, it contains equal concentrations of proton donor and proton acceptor, and the pH value at this time is equal to its pK' (dissociation constant ). The preparation of the buffer is described in detail in "Data for Biochemical Research" (Rex, MC, Oxford Science Publications, 1995).

There are many physiological mechanisms that occur in the body to maintain the pH of the blood within the narrow range of 7.35-7.45. Some of these buffer mechanisms are based on the carbonic acid monophosphate homeostasis system, and many others involve amino acids and proteins. For example, the pH of tears is maintained at 7.4 by proteins that act as buffers. Single amino acids are also useful buffer systems, and their titration curves are more complex because there are both proton acceptors and proton donors in the same molecule. The molecule is amphoteric, that is, it has both positively and negatively charged sites within the same molecule. As shown below, amino acids can act as a buffer when either H ⁺ or ^OH- is added.

In order to have a "pharmaceutical buffer system" in the present invention, the buffer used widely includes conjugated acid-base pairs and some acid-base compounds, which can adjust and maintain the Aβ peptide solution at the required pH level. The present invention has a pH of 8.5 or higher during dissolution/filtration, and a pH lower than 4 on the other hand.

The components of the buffer may be selected from amino acids, salts and their derivatives; pharmaceutically acceptable alkalinizing agents, alkali metal hydroxides, ammonium hydroxide, organic and inorganic acids, salts; and some mixtures of the above-mentioned substances . The concentration of each component in the buffer solution should be sufficient to achieve and maintain the required pH value, and its concentration should be determined according to its acidity and alkalinity. Effective concentrations can be determined by methods known in the art, such as the use of pH meters and titration techniques.

The composition used in the embodiment of the present invention has the substance of hydroxide class, comprises the hydroxide of alkali metal and ammonium hydroxide, and the basifying agent of pharmacy includes but not limited to trishydroxymethylaminomethane (tris), boric acid Sodium (Na ₂ B ₄ O ₇ ) and disodium citrate, amino acids, salts, esters or amides of amino acids and some simple derivatives (such as N-acetylated derivatives of amino acids). Glycine (such as sodium glycinate), arginine and lysine, and sodium and ammonium hydroxide are particularly suitable as buffers.

Pharmaceutically acceptable acids used in the practice of the methods of the present invention include, but are not limited to, hydrochloric acid, phosphoric acid, citric acid, acetic acid, maleic acid, malic acid, and succinic acid, and the like. Alternatively, these acids can be used to titrate the pH of the base solution to a lower, more physiologically acceptable level to obtain a peptide suspension.

On the contrary, the various basic compounds mentioned above can be used to titrate the filtered solution with low pH value to make it more physiologically acceptable, and also to obtain suspensions of peptides.

Mixtures of pH adjusters are also contemplated. Conjugate acid-base pairs of the aforementioned salt forms (eg, ammonium acetate) can also be used in the buffers of the present invention. Such a conjugated buffer system is prepared by titrating a basic solution of ammonium hydroxide with acetic acid until a near-neutral ammonium acetate solution is formed.

As used herein, the term "tonicity modifier" refers to various factors that form osmolarity. Tonicity modifiers used in embodiments of the present invention include (but are not limited to) sugars, such as mannitol, sucrose, glucose, and salts, such as NaCl, KCl, and the like. The consumption of tonicity adjusting agent should make the osmotic pressure of solution be lower than 350 mOsm/kg, preferably can reach 250-350 mOsm/kg (mOsm/kg), and preferably remain in the scope of 280-320 mOsm/kg Inside. It is worth mentioning that some charged compounds that act as buffer components can also have an effect on tonicity. Therefore, before adding a tonicity regulator to further adjust the tonicity of the solution, the tonicity of the Aβ peptide buffer solution should be determined first.

Some chelating agents can be selected for use in the composition and production process of the present invention. Preferred examples of the chelating agent used include ethylenediaminetetraacetic acid (EDTA) and its salts (such as sodium salt), these salts are in the concentration range of 0.05-50mM, the effect is better when the concentration is 0.05-10mM, and the preferred concentration is 0.1 -5mM. Other known chelating agents (or release agents) such as polyvinyl alcohol may also be used.

Optionally, some surfactants or detergents such as polysorbates (such as Tween®) or 4-(1,1,4,4-tetramethylbutylene may also be included in the composition. base) phenoxypolyethoxyethanol (4-(1,1,4,4-tetramethylbutyl)phenyloxypolyethoxyehthanols) (Triton®), or polyethylenepolypropylene glycols (polyethylenepropylene glycols) (Pluronics®). The content of surfactant can be 0.005-1%, preferably 0.02-0.75%. A more suitable polysorbate is PS-80, which is available under the trade name Tween(R) 80.

Wetting agent is also one of the adjuvants used in the present invention. Polyethylene glycol, such as PEG3350, can bind to Aβ peptide in the aqueous phase and stabilize its hydrophilic part, thereby regulating the association of Aβ peptide particles and their solubility. The content of the wetting agent is preferably 0.5-5% (w/v (expressing the grams of effective components in 100 ml of solution, percent by weight in unit volume)).

Pharmaceutically acceptable sugars (such as sucrose, glucose, maltose or lactose) or sugar alcohols (such as mannitol, xylitol, sorbitol) had no effect on the pharmaceutical action of the active ingredient. In one aspect of the present invention, the sugar or sugar alcohol used has a molecular weight of less than 500 and is easy to disperse or dissolve in water, so as to be suitable for the present invention. Sugars and sugar alcohols employed in embodiments of the present invention include xylitol, mannitol, sorbitol, arabinose, ribose, xylose, glucose, mannose, galactose, sucrose, lactose, and the like. These may be used alone or in combination of two or more. A preferred sugar is mannitol, especially for use in lyophilized compositions; sucrose is more suitable for solution compositions.

It was also found that the use of a QS-21 adjuvant can interact with the suspended protein of the composition of the present invention to form a transparent and clear suspension. During this required interaction, the peptides exist in a β-sheet conformation and are suspended in solution as very small particles, which can increase the stability of the suspension and facilitate the improvement of the immunogenicity of the composition. potency. Similar to the mode of action of QS-21 on Aβ peptide, DPPC (dipalmitoylphosphatidylcholine) in the prior art can be used to react with Aβ peptide to produce a suspension containing tiny particles, so it can also be used as an adjuvant. to form a transparent clear suspension. You can also choose other adjuvants to mix with QS-21.

Freeze-drying is a commonly used technique in the pharmaceutical process to maintain the stability of peptides during and after freeze-drying. Lyophilization in amino acids can stabilize proteins or peptides, and the use of sugars as matrices is also well known to those skilled in the art. Refer to Lueckel B., etc. "Formulations of sugars with amino acids or mannitol-Influence of concentration ratio on the properties of the freeze-concentrate and the freeze-drying" lyophilzate), "Advances in Pharmaceutical Technology" (PHARM.DEV.TECHNOL.) 3(3) pp.325-336 (1998). The Royal Pharmaceutical Society of GB Symp: 'New Analytical Approaches to the Characterization of Biotechnology Procucts'. June 1996 Lueckel B. et al., "A strategy for optimizing the lyophilization of biotechnolofical products", "Pharmaceutical Science" (UK) 3 (PHARM.SCI.(UK) 3) (1 ) pp. 3-8 (1997). The above two documents are hereby cited in their entirety as references.

The term "pharmaceutically acceptable" modifies any component which does not have any deleterious or adverse effect on the host in which it is administered in the context in which it is administered. "Pharmaceutical diluent" and "pharmaceutical adjuvant" means any component that maintains or does not alter the activity of the active compound and has no toxic or adverse effect on the subject to which it is For administration, such as non-toxic pH adjusters, buffers, tonicity adjusters, or chelating agents and the like.

A composition or method "comprises" one or more of the listed ingredients as well as ingredients not specifically listed. For example, a composition containing Aβ peptide includes not only the Aβ peptide in the listed composition, but also other components not listed in the composition containing Aβ peptide.

Other descriptions are as follows: Abbreviation Description ℃ Degree Celsius cc Cubic centimeter CD Circular dichroism pH log[H ⁺ ], hydrogen ion concentration and acidity and alkalinity in a solution

The dissociation constant of an acid is related to pH:

Pk '+log [(H+receptor) ÷ (H+supply)] PS80 polyshan peorteol 80 or TWEEN80

and ethylene oxide copolymer); Merck Index (Merck

INDEX) Specialized NO.7559 (11th Edition) μM Micron MIN MIN Mlml Mlm N Positive Concentration -Meaning the Moore concentration of the Moore concentration of the solution, with Moore/liter meter MMSO dimoso EDTA ethimine tethamine tetramium tetrams Acetic acid, usually its sodium salt Tris Tris Tris (Merck Index)

Index) Topic No.9684 (11th Edition) RP HPLC Reversed-Phase High Pressure Liquid Chromatography RPM Revolutions per minute CFA/IFA Freund's Complete Adjuvant/Freund's Incomplete Adjuvant (Chang et al.,

  "Advanced Drug Delivery System Review" (Acvance Drug

Delivery Reviews) 32, 173-186 (1998)) MPL 3-oxo-deacylated monophosphoryl lipid A (MPL ^™ ) (see

GB2220211). QS21 is extracted from the bark of the Quillaja japonica tree in South America

    from triterpene saponins (refer to Kensil et al., "Vaccine Design

Vaccine Design: Adjuvants and Adjuvant Research

(Subunit and Adjuvant Approach)

(Powell & Newman, Plenum Press, New York,

                             ; U.S. Patent No. 5,057, 540).

(Stimulon ^TM QS-21) FIO Information For Information Only

Filtration is a process in which a liquid flows through a filter membrane with a uniform pore size, and impurities are excluded according to their size. Although micron-sized particles can be removed by non-membrane materials or fibrous media materials, only filters with precise pore sizes can quantitatively exclude smaller particles. In this way, the bacteria can be quantitatively removed by the filter membrane when passing through the micro-filter, so as to achieve the effect of sterilization. In the past, when purifying Aβ peptides, the peptides were often insoluble or aggregated into clusters, which would block the membrane pores with particles, or/and the recovery rate of the peptides in filtration was very low, so commercial-scale filtration could not be performed.

More suitable is a filter that can exclude particles above 0.2 microns.

Filter membranes are made uniformly on substrates and are classified according to the pore size of the membrane surface. This surface-type filter membrane can retain particles either in the membrane or on the membrane. A class of different pore sizes that allow particles smaller than that to pass through while leaving particles larger than the pore size on the membrane. These filters with a certain pore size have a certain load limit (that is, when the filter is full of excluded particles, a small amount of excluded substances will pass as the pressure of the filtrate rises), and they are all optional for microbial control. devices, "sterile grade" surface type membrane filters are well known in the pharmaceutical industry.

In all filtration applications, the permeability of the filter media is influenced by the chemical, molecular, electrostatic, etc. properties of the filtrate. However, the hydrophilic microfilter used in the present invention is stable in both high pH and low pH environments required by the experimental method, and can accurately remove unwanted particulate matter without being clogged by it. Those of ordinary skill in the art will be able to select and use hydrophilic microfilters. Using some commercially available product descriptions or URLs can help us choose the desired filter, such as the following URL: http:∥millispider.millipore.com/corporate/sitemap.nsf/catalogs (from May 1999).

The filter selected in the example of the present invention has: Millipore Durapore ®, also known as Millex GV (Millipore Company, headquartered in Bedford, Massachusetts), is polyvinylidene fluoride (polyvinylidene fouoride), a kind of hydrophilic polymer , has good stability and low protein affinity, and its pore size is 0.22 μm; MillexGN ^TM , a hydrophilic nylon material, has a pore size of 0.2 μm; Millex Gp ^TM , a hydrophilic surface-modified Polyethersulfone (polyethersulfone) polymer with a pore size of 0.22 μm. Durapore remains stable at pH 9-9.5, making it more suitable.

The filtered Aβ peptide should be fully recovered, and the recovery rate of more than 50% can be considered as relatively complete recovery, such as the recovery rate of the filtered Aβ peptide is greater than 80%, it is better, preferably greater than 90%.

method

The method of the present invention involves preparing an aqueous solution of Aβ peptide at a minimum concentration of 0.01 mg/ml. Preparation of such compositions involves adjusting the pH of the aqueous solution to dissolve the Aβ peptide and achieve the desired concentration (0.1-2.0 mg/ml).

The traditional method is used to adjust the pH value of the aqueous solution, by adding acid or base to achieve the required pH value. Preferably the amount of acid or base is pharmaceutically acceptable. To maintain the pH of the solution during storage, pharmaceutically acceptable buffers are permitted in the composition. Selection of an appropriate buffer according to the desired pH is well known in the art.

Preferably, the pH of the aqueous solution is adjusted to about 2-4 or 8.5-12, at which pH the A[beta] peptide has surprisingly been found to dissolve readily.

The low pH value (approximately pH 2-4) that needs during the operation of the present invention can be realized by selecting hydrohalic acid (such as hydrochloric acid, hydrobromic acid), phosphoric acid, citric acid, acetic acid and other pharmaceutically acceptable acids. Selection of suitable acids is well known to those skilled in the art.

The high pH value (about 8.5-12) that needs in the operation process of the present invention can be by selecting some pharmaceutically acceptable alkalis for use, for example, alkali metal, ammonium hydroxide (i.e. NaOH, NH OH) etc. to improve the pH value of the solution to reach Require. It is better to use a buffer solution to stabilize the pH value between 8.5-10 under high pH value conditions, and preferably control the pH value between 9-10.

Regardless of whether the pH is adjusted first or later, it is necessary to add a specified amount of Aβ peptide. Of course, in order to dissolve the peptide immediately, it is preferable to adjust the pH before adding the Aβ peptide. Additionally, slight stirring and heating may be used to speed up dissolution.

Any optional additional components, such as pharmaceutically acceptable buffers, tonicity agents, adjuvants, etc., can be added at a convenient time, either before or after adding the Aβ peptide.

Once the required aqueous solution is prepared, it can be sterilized filtered or/and freeze-dried according to the well-known operating procedures in the art, and the specific procedures are shown in the following examples.

The following solutions are the preferred buffer system for solubilizing Aβ peptides and achieving the required concentration range (0.6-2.0 mg/ml):

Preferred amino acid composition:

10 mM sodium glycinate, pH 9.0, 9.5 or 10.0 and/or 0.02-1.0% (w/v)

Polysorbate 80 (PS-80)

Optionally contain one or more tonicity regulators to facilitate parenteral administration;

10mM L-arginine-HCl, or 10mM L-lysine, both pH 9.0

or 10.0 and/or polysorbate 80 (PS-80) with 0.02-1.0% (w/v)

Optionally contain one or more tonicity regulators to facilitate parenteral administration;

The composition of the present invention is suitable for cryopreservation to prevent degradation or aggregation of peptides. The preferred storage temperature is 2 to 8°C.

The following examples illustrate the implementation of the present invention. These examples are only for the purpose of facilitating understanding and cannot limit the scope of the present invention.

Detailed description of the preferred embodiment

The solubility of embodiment 1 peptide

Peptides and Reagents

The trifluoroacetate salt of Aβ42 was obtained from American Peptide Company with batch numbers M05503T1 and M10028T1, and was used without any modification. Other salts are available and have been used successfully in the present invention. See Table 3 for examples of alternative Aβ peptide counterion salts.

All acids, bases, and buffer solutions are prepared with reagents that meet the experimental requirements, and are stored after sterile filtration for convenience. Polysorbate 80 (polysorbate 80) (also known as Tween 80, PS80) preparation: according to w/v (weight per unit volume), use a standard low peroxide PS80 solution (10% low peroxide from Aldrich-Sigma Company) Peroxide polysorbate monooleate), diluted to prepare a concentration of 4% mother liquor.

Peptide Dissolution:

500-700 μg of Aβ peptide was weighed and dissolved in an appropriate amount of buffer to prepare an Aβ42 solution with a final concentration of 0.6, 0.8, or 1.0 mg/ml. Peptides were weighed directly into 4 ml Wheaton glass vials with screw caps. Add the appropriate amount of buffer and stir gently for about 15 to 30 minutes. Solutions were visually inspected and solubility was assessed using the following criteria: (+) = poorly soluble, cloudy suspension; (++) = clear suspension with insoluble particles; (+++) = clear solution.

Table 1. Visual Solubility of Solutions Buffer properties Aβ42 target concentration (mg/ml) Visual solubility 0.01NaOH 1.0, 0.8 +++ 0.01 equivalent NH ₄ OH 1.0, 0.8 +++ 50mM sodium glycinate, pH9.0 1.0 +++ 10mM sodium glycinate, pH9.0 1.0, 0.8, 0.6 +++ 10mM sodium glycinate, pH9.5 0.6 +++ 10mM sodium glycinate, pH10.0 1.0, 0.8 +++ 10mM sodium glycinate, pH9.0, 0.02% PS80 1.0 +++ 10mM sodium glycinate, pH9.5, 0.1% PS80, 5% sucrose 0.6 +++ 10mM sodium glycinate, pH9.5, 4% mannitol, 1% sucrose 0.6 +++ 10mM Sodium Glycinate, pH 9.5, 4% Mannitol 0.6 +++ 10mM sodium glycinate, pH10.0, 0.02% PS80 1.0 +++ 10mM L-arginine hydrochloride, pH9.0 0.6 +++ 10mM L-arginine hydrochloride, pH10.0 0.8 +++ 10mM Sodium L-lysine, pH9.0 0.8, 0.6 +++ 10mM Sodium L-lysine, pH10.0 0.8 +++ 10mM Sodium Lysinate, pH 9.5, 4% Mannitol 0.6 +++ 10mM ammonium acetate, pH9.0 1.0 +++ 10mM ammonium acetate, pH9.0, 0.02% PS80 1.0 +++ 50mM Tris-HCl buffer, pH10.0, EDTA (0.5mM), 0.02% PS80 1.0 ++ 10mM sodium borate, pH9.0 1.0 +++ 10mM Sodium N-acetyl D-glutamate, pH9.0 0.8 +++ 10mM glycine hydrochloride, pH3.0 1.0 ++ 0.01 N HCl 1.0 +++ 0.01 mol/L phosphoric acid 1.0 +++

continued from previous page Buffer properties Aβ42 target concentration (mg/ml) Visual solubility Dimethylsulfoxide (DMSO) (pure) 0.6 +++ 10mM sodium glycinate, pH8.0 1.0 ++ 10mM L-arginine hydrochloride, pH9.0 0.8, 1.0 ++ 10mM sodium ammonium bicarbonate, pH9.0 0.8 ++ 10mM sodium ammonium carbonate, pH9.0 0.8 ++ 50mM Tris-HCl buffer, pH10.0, EDTA (0.5mM) 1.0 ++ 10mM sodium borate, pH10.0 1.0 ++ 10mM L-arginine hydrochloride, pH8.0 1.0 + 10mM ammonium acetate, pH8.0 1.0 + 10mM ammonium acetate, pH8.0, 0.02% PS80 1.0 + 50mM Tris-HCl buffer, pH8.0, pH9.0, or pH10.0 1.0 + 50mM Tris-HCl buffer, pH8.0 or pH9.0, EDTA (0.5mM) 1.0 + 50mM Tris-HCl buffer, pH8.0 or pH9.0, EDTA (0.5mM), 0.02% PS80 1.0 + 50mM or 100mM NaCl, pH 9.0 1.0 + 50mM sodium phosphate, pH8.0, pH9.0, or pH10.0 1.0 + 50mM sodium glycinate, pH8.0 or pH10.0 1.0 + 10mM N-Acetyl Dext-Glucosamine Sodium, pH 9.0 or pH 10.0 0.8 + 10mM Sodium N-acetyl D-glutamate, pH10.0 0.8 + 10mM sodium citrate, pH3.0 or pH4.0 1.0 + 10mM sodium acetate, pH4.0 1.0 +

Solutions marked "+++" indicate clear solutions at the target concentration. The pH of the buffer was varied in the range of 9-10. Solutions of inorganic solutes such as NaOH and _NH4OH are basic, while HCl and phosphoric acid are strongly acidic. At pH greater than 9, the solubility of the peptide is 0.6-1.0 mg/ml. Additives such as polysorbate 80, sucrose, mannitol, and ethylenediaminetetraacetic acid (EDTA) do not affect the solubility of the peptides and can assist in increasing the tonicity and recovery of the peptides after filtration, or as Chelating agent. Acidic solutions (pH 4 or lower) can also dissolve Aβ42 in the concentration range of 0.6-1.0 mg/ml. Solutions marked "++" indicate partial dissolution of the peptide at the target concentration. The solubility of the peptide may be lower than the concentration detected. Solutions marked "+" indicate that the peptide was not sufficiently dissolved at the target concentration. Example 2 Solubility of Aβ Peptide in Buffer

Aβ42 (0.6, 1.0, 1.5, 2.0, 3.0, and 3.5 mg/ml) was dissolved in 10 mM sodium glycinate buffer, pH 9.0. Each solution was centrifuged in a long benchtop centrifuge (greater than 10,000 rpm, about 10 minutes), and observed that the peptide concentration reached 2.0-3.5 mg/ml as a turbid suspension (the solution with a concentration of 0.6-1.5 mg/ml was clear ).

Aliquots of supernatants from each solution were analyzed by reversed-phase high-performance liquid chromatography (RP HPLC).

Table 2 is a table of peptide peak areas, and the data are plotted to show the solubility curve of Aβ42 in sodium glycinate buffer at pH 9.0.

Table 2 Solubility Limits, Aβ42 Peptide, Trifluoroacetate (TFA) Aβ42mg/ml (10mM glycine, pH9.0) HPLC peak area 0.6 3613553 1 5921792 1.5 8850393 2 9213446

In addition, the trifluoroacetate, ammonium, hydrochloride, and sodium salt forms of the Aβ42 peptide can be purified. These salts were dissolved in different buffers, and the concentration of Aβ42 peptide was adjusted to 0.45 mg/ml after correction for the counter ion contribution of each salt. After filtration, the recovery rate of the peptide was determined by comparing the peak area of reversed-phase high performance liquid chromatography of the peptide before and after filtration. All salts were soluble and filterable at a concentration of 0.45 mg/ml and the results are listed in Table 3.

Table 3. Solubility of various salts of Aβ42 peptide 0.45mg/ml Aβ42 Recovery rate(%) Ammonium salt solution containing 1mM NH ₄ OH 70 Ammonium salt solution containing 2mM NH ₄ OH 106 Ammonium salt solution containing 10mM sodium glycinate, pH9.0 115 Ammonium salt solution containing 10mM sodium glycinate, 5% sucrose, pH9.0 96 Ammonium salt solution containing 10mM sodium glycinate, pH9.5 101 Ammonium salt solution containing 10mM sodium glycinate, pH10.0 106 Trifluoroacetate solution containing 10 mM sodium glycinate, pH 9.0 101 Hydrochloride solution containing 10mM sodium glycinate, pH9.0 101 Sodium salt solution containing 10mM sodium glycinate, pH9.0 100 Example 3 Recovery of Solubilized Peptides Through Typical Hydrophilic Filters

Experimental syringe-type filters (filter diameter 25 mm, filter area 3.9 cm ² ) were as follows: Millex GV, 0.22 uM: a hydrophilic polyvinylidene fluoride (PVDF, Durapore®) membrane with low protein binding properties; Millex GN, 0.20 uM: a hydrophilic nylon membrane with low protein binding properties; and Millex GP, 0.22 uM: a hydrophilic surface engineered polyvinylsulfone (PES) with low protein binding properties.

The above filters are representative of various types of hydrophilic microfilters that are commercially available. Filtration studies were performed through the dissolution system as follows:

1. 0.01 NH ₄ OH solution containing 0.6 mg/ml Aβ42

2. A solution of 10 mM sodium glycinate containing 0.6 mg/ml Aβ42, pH 9.0

3. A solution of 10 mM sodium lysine containing 0.6 mg/ml Aβ42, pH 9.0

4. 10mM arginine hydrochloride solution containing 0.6mg/ml Aβ42, pH 9.0

Each filter filters approximately 2 ml of each Aβ42 solution. The concentration of peptides was detected by reversed-phase high-performance liquid chromatography (RP HPLC). The recovery rate was determined by comparing the peak areas of the peptides before and after microfiltration, and the results are shown in Table 3 and Table 4 below.

Table 3 - Filtration Recovery Solution components 0.6mg/ml Aβ dissolved in: Filter Model (Recovery Rate%) Millex GV MillexGN MillexGP 10mM sodium glycinate 99.1 97.2 98.0 10mM arginine hydrochloride 91.9 86.6 92.5 10mM sodium lysine 95.0 94.7 97.4 0.01 equivalent NH ₄ OH 102.2 104.8 105.1 Example 4 Filtration of Aβ42 dissolved in sodium glycinate and sodium lysine buffer with and without excipients

In these studies at a concentration of 0.6 mg/ml Aβ42 was dissolved in 10 mM sodium glycinate solution containing 0.1% polysorbate 80 (PS80), 0.9% NaCl, or 0.1% PS80, 0.9% NaCl, 5% A mixture of sucrose, 1% sucrose and/or 4% mannitol. Take about 2-5ml of each solution and filter through Millex GV. Aliquot supernatants were centrifuged at > 10,000 rpm for approximately 3 minutes in a long benchtop microcentrifuge. The recovery was determined by comparing the peak areas of the peptides before and after microfiltration in reversed-phase high-performance liquid chromatography.

The filtration recoveries of table 4 peptides in various buffer solutions Solution components 0.6mg/ml Aβ42 dissolved in: recovery percentage 10mM sodium glycinate, pH9.0 104.5% 10mM sodium glycinate, pH9.0, 0.1% PS80 106.2% 10mM sodium glycinate, pH9.0, 5% sucrose 115.4% 10mM Sodium Glycinate, 4% Mannitol, pH9.5 103.0% 10mM sodium glycinate, pH9.5, 4% mannitol, 1% sucrose 98.0% 10mM sodium glycinate, pH 9.0, 0.9% NaCl 76.7% 10mM sodium glycinate, pH9.0, 0.1% PS80, 0.9% NaCl 65.0% 10mM Lysine/Citrate, pH 9.5 93.2% 10mM Lysine/Citrate, 4% Mannitol, pH 9.5 80.0%

Millex GV filter, as a preferred filtration device for filtering Aβ peptide solution, has the main advantages of high recovery of peptides and can be used in commercial processes. Peptide solutions containing 0.9% NaCl were visually observed to be insufficiently dissolved, resulting in lower recoveries detected by reversed-phase HPLC. To increase the solubility of the peptide in the presence of inorganic salts such as NaCl, etc., preferably after microfiltration of the peptide in buffer, a sterile tonicity modifier solution is added.

On the other hand, carbohydrates acting as tonicity modifiers exhibit different solubilizing activities and may facilitate the state maintenance of peptide suspensions. This property, also known as hydration increasing order, hydrates the peptide chain into a thermodynamically more stable αβ-sheet conformation.

The pH range of the filterable operation of Aβ peptide is about 8.5-12, and the pH value in the range of 9-10 has good recovery. MillexGV membranes with a pore size of 0.2 μm are often used in manufacturing filtration.

In order to obtain Aβ peptide preparations suitable for clinical application, meeting physiological requirements, stable and biologically active, the main steps in the preparation of the preparation include sterilizing and filtering the peptide preferably in the pH range of 8.5-10. Embodiment 5 dissolving liquid preparation

test methods

Three Aβ42 peptides were supplied by American Peptide Corporation (APC). Formulation validation of the APC peptide, revision of chemical and biological characterization, and replica production of Aβ42 were performed by California Peptide Research.

Formulation instructions are described in detail in Sections 1, 2 and 3 below.

Stability Analysis:

A detailed description of the formulations follows below. Peptides from different formulations were dispensed into separate vials, stored at 2-8°C for several months, and analyzed periodically. Formulation appearance, pH, peptide concentration and purity were regularly monitored during the stability studies. And reversed-phase high performance liquid chromatography was used to determine the concentration and area percent purity of Aβ peptide. The reversed-phase high-performance liquid chromatography has a polymeric reversed-phase column, which is eluted with ammonium bicarbonate (or tris (tris))/acetonitrile gradient, and detected at 220nm. Peptide concentration was determined by standard control; purity was determined by the percentage of Aβ peptide calculated by quadratic integration of the entire chromatogram.

Characteristic description

The structural properties of Aβ42 in the dissolved state were determined by circular dichroism, as shown in Figure 1.

biological activity

Inject Swiss Webster mice (4-8 mice per group) with different formulations of Aβ42 and adjuvants such as CFA/IFA, MPL (Corixa Immunochemicals) or QS 21 (Aquila Pharmaceuticals), according to the antigens indicated in professional studies / adjuvant concentration injection. Unless otherwise specified, injections were generally given every two weeks, that is, at weeks 0, 2, and 4. Mice were bled after injection at 2 and 4 weeks. Serum was used for standard cross-enzyme-linked immunosorbent assay (ELISA), using Aβ42 as antigen and horseradish peroxidase-linked goat anti-mouse immunoglobulin as labeled antibody. The titers of antibodies in each animal in each group were represented by some data scatter points or geometric mean, and the results were compared with the titers of antibodies produced using aggregated Aβ42/CFA/IFA as a control immunogen. The titer results are shown in Tables 8 and 10.

1. Mixed solution (preparation)

Aβ peptide was dissolved at a concentration of 0.6 mg/ml in a buffer containing 10 mM sodium glycinate, pH 9-9.5, and filtered through Millex GV with a pore size of 0.2 μm. Each 0.5ml of the mixed solution is filled into a 2cc vial (Gensia P/N X34-113-002) and sealed with a gray butyl synthetic rubber stopper (Gensia P/N X66-113-030) . Store vials at 2-8°C.

2. Stability test

The concentration and purity of Aβ42 in the mixed solution were monitored by reversed-phase high-performance liquid chromatography. The sample solution was analyzed directly or after microcentrifugation. The table below (Table 5) presents the analytical data for the fully dissolved formulations. There was no significant difference in the results of samples that were centrifuged or not centrifuged prior to analysis, indicating continued solubility of the peptide at the current concentration. The area percentage purity of the peptide was compared with the standard control: no obvious peptide degradation phenomenon was found. The dissolved formulation is stable for three months when stored at 2-8°C.

Table 5. Results after three months of storage 0.6mg/ml Aβ42 dissolved in: Exterior pH Area percent purity Aβ concentration (mg/ml) Reference standard none none 68% none 10mM sodium glycinate, pH9.0 clear solution 8.6 69% 0.65

3. Characteristic description

The detection of the circular dichroism characteristic of the Aβ peptide preparation shows that the peptide is in the state of random coils in the solution, and has a characteristic negative ellipticity absorption at 189-205 nanometers.

biological activity

A solubilized preparation of Aβ42 at pH 9 was injected into Swiss Webster mice with adjuvants to increase antibody titers. Injection of 50 μg MPL or 25 μg QS21 adjuvant together with 33 μg Aβ42 every two weeks (week 0, 2, 4 weeks) elicited a significant immune titer response compared to the control group.

Embodiment 6 liquid suspension preparation

Glycine/Acetate Formulations

Aβ peptide was dissolved in 10 mM sodium glycinate at 0.6 mg/ml, or in a pH 9.0-9.5 buffer containing other excipients. After the peptide solution was filtered with Millex GV, the pH value was adjusted to about 6 with 0.1M acetic acid to obtain a peptide suspension. 0.5 ml of the mixed solution was filled into a 2cc vial (Gensia P/N X34-113-002) and sealed with a gray butyl synthetic rubber stopper (Gensia P/N X66-113-030). Store vials at 2-8°C.

Glycine/Citrate Preparations

A 0.6 mg/ml Aβ peptide preparation was prepared in a 10 mM sodium glycinate solution containing 0.1% PS-80, or 5% sucrose (25 ml). The peptide solution was filtered through Millex GV and titrated to about pH 6.0 with 0.1 M citric acid. Optionally 0.9% NaCl can be added to the glycine/citrate/PS 80 mixture after titration to pH 6. 0.5 ml of the mixed solution was filled into a 2cc vial (Gensia P/NX34-113-002) and sealed with a gray butyl synthetic rubber stopper (Gensia P/NX66-113-030). Store vials at 2-8°C.

The glycine/citrate formulation was formulated as a buffering solution at pH 6. Several 100 ml 0.6 mg/ml Aβ peptides were formulated with 10 mM sodium glycinate, and 5% sucrose, or a pH 9 formulation also containing 0.1% PS 80. A 0.1 mg/ml Aβ42 formulation was also prepared in 10 mM sodium glycinate containing 5% sucrose or 0.1% PS80. The peptide solution was filtered with Millex GV before pH adjustment. The pH value was adjusted to 6 with 1M sodium citrate pH 5.5, and the sodium citrate concentration in the buffer was 10 mM and 20 mM. 0.5 ml of the mixed solution was filled into a 2 cc vial (Gensia P/N X34-113-002), capped with a gray butyl synthetic rubber stopper, and sealed with aluminum. Store vials at 2-8°C.

stability test

The concentration and purity of Aβ42 in the preparations were monitored by reversed-phase high-performance liquid chromatography. Before analysis, the preparation of the peptide was diluted with equal volume of 0.01 N NaOH solution containing 2% sodium dodecylsulfonate (SDS), and then heated at 100°C for 1 minute to re-solubilize the peptide, and then measure Aβ42 Total concentration of peptide in suspension. An alternative method for measuring the concentration of dissolved A[beta]42 is to place the test sample in a microcentrifuge and centrifuge at a speed of 10,000 rpm or more for 3-5 minutes. An equivalent volume of this resolubilized peptide solution or supernatant was analyzed by reverse phase high performance liquid chromatography. During the research process, the conditions of the reversed-phase high-performance liquid chromatography column were constantly explored to improve the resolution of the determination and more accurate quantification. Therefore, the area percentage purity of the peptide is compared with the standard control used in the analysis, and the chromatograms at each time point are compared to determine the degree of degradation of the peptide. Both appearance and pH were monitored during the stability study.

Table 6 lists data for several Aβ42 suspension formulations. All formulations were visually observed as suspensions. These formulations were adjusted to pH 6 with various acids listed in the table. Depending on the role of the different excipients, the suspension of the peptides formed immediately or took two weeks or more to form. Adding NaCl to the peptide formulation or replacing citrate with acetate accelerated the formation of the suspension.

The concentration of peptide in all formulations was a target concentration of 0.6 mg/ml. The area integral percentage purity of the peptide was equal to the standard control used in the analysis. No degradation and loss of the peptide was found when stored at 2-8°C for 3 months. The appearance and pH of this formulation are the same as the data listed in Table 1.

9% NaCl and 5% sucrose provide physiological tonicity for parenteral administration. Also the pH of the suspension is within the pH range required for injection.

Table 6 Liquid Suspension Preparations Preparation composition Exterior pH Purity percentage Aβ42(mg/ml) 0.6mg/ml Aβ dissolved in: Total concentration of peptide Supernatant T=0 T = 2 weeks 10mM sodium glycinate/sodium acetate, pH6 suspension 5.9 70% 0.54 0.23 .04 10mM sodium glycinate/sodium acetate, pH6, 5% sucrose suspension 5.9 68% 0.65 0.21 .04 10mM sodium glycinate/sodium acetate, pH6, 0.1% PS80, 5% sucrose suspension 6 67% 0.62 0.43 .08 10mM sodium glycinate/sodium acetate, pH6, 0.1% PS80, 0.9% NaCl suspension 5.9 68% 0.55 0.04 .04 10mM sodium glycinate/sodium acetate, pH6, 0.9% NaCl suspension 6 70% 0.53 0.26 .05 10mM Sodium Glycinate/Sodium Citrate, pH6, 0.1% PS80, 5% Sucrose suspension Untested 77% 0.63 less than 0.01 Untested 10mM Sodium Glycinate/Sodium Citrate, pH6, 0.1% PS80, 0.9% NaCl suspension Untested 71% 0.63 0.02 Untested

The formulations in Table 7 had a buffering capacity of 10 mM or 20 mM provided by citrate, allowing the solutions to maintain a pH of 6 during formulation. These formulations form suspensions quickly. According to the own characteristic of Aβ peptide, in the process of forming the suspension system, the structure change of the peptide mainly occurs in its internal sterilization core. Compared with titration, the use of buffers of known concentration can make the operation in the sterile room easier to handle and improve the reproducibility of the experiment.

Table 7. Buffered Suspensions 0.6mg/ml Aβ42 dissolved in: Exterior pH Purity percentage Total concentration of peptide mg/ml 10mM Sodium Citrate, 10mM Glycine, pH6.0, 0.1% PS80, 5% Sucrose suspension 6.0 81% 0.66 20mM Sodium Citrate, 10mM Glycine, pH6.0, 0.1% PS80, 5% Sucrose suspension 5.9 83% 0.69 20mM Sodium Citrate, 10mM Glycine, pH 6.0, 5% Sucrose suspension 6.0 81% 0.67 0.1mg/ml Aβ42 dissolved in: 20mM Sodium Citrate, 10mM Glycine, pH6.0, 0.1% PS80, 5% Sucrose suspension 5.9 82% 0.2 20mM Sodium Citrate, 10mM Glycine, pH 6.0, 5% Sucrose suspension 6.0 81% 0.09

The composition of these preparations is further increased. The trifluoroacetate or acetate of Aβ42 peptide was dissolved in 10 mM sodium glycinate solution, pH 9-9.5. The salt concentration was adjusted so that the concentration of Aβ42 was 0.45 mg/ml. PS80 concentrations varying from 0.02% to 0.5% were dissolved in the peptide solution before filtration. Likewise, the addition of sucrose and NaCl can be before or after acid addition. Adjust the pH of the peptide solution with citric or hydrochloric acid, as listed in the table below. All formulations formed a suspension and the concentration of Aβ42 peptide reached the expected value. Table 7b suspension formulation 0.45mg/ml Aβ42 dissolved in: 10mM sodium glycinate, 20mM citrate, 0.1% PS80, pH6.0 10mM sodium glycinate, 20mM citrate, 0.5% PS80, pH6.0 10mM sodium glycinate, 20mM citrate, 154mM NaCl, pH6.0 10mM sodium glycinate, 20mM citrate, 154mM NaCl, 0.1% PS80, pH6.0 10mM sodium glycinate, 154mM NaCl, pH6.0 (hydrochloric acid) 10mM sodium glycinate, 154mMNaCl, 0.1% PS80, pH6.0 (hydrochloric acid)

feature description

Circular dichroism and Fourier transform infrared detection of Aβ42 suspension at pH 6 indicated that the peptide configuration in suspension was β-sheet. This configuration is the same as that of the suspended peptide in a solution at a concentration of 0.6 mg/ml, regardless of whether buffers (citrate, acetate, phosphate) and excipients (sucrose, NaCl) are added to the formulation . Adding PS-80 to this preparation can make the suspension system more evenly dispersed.

biological activity

The antibody titer was increased after co-injection of Aβ peptide suspension and adjuvant into Swiss Webster mice. As shown in Table 8, biweekly injections (0, 2, 4 weeks) will elicit a titer response of sufficient magnitude compared to the control group.

Table 8. Antibody Responses to Suspension Formulations Peptide preparation 0.6mg/ml Aβ peptide in: Peptide (μg) Adjuvant (μg) Geometric mean antibody titer (second blood draw) ^** 10mM sodium glycinate/sodium acetate, pH6, 5% sucrose 33μg 50 μg MPL ^* ≈7000 10mM sodium glycinate/sodium acetate, pH6, 5% sucrose 33μg 25 μg QS21 ≈10,000 10mM sodium glycinate/sodium acetate, pH6, 0.1% PS-80, 5% sucrose 33μg 50 μg MPL ^* ≈10,000 10mM sodium glycinate/sodium acetate, pH6, 0.1% PS-80, 5% sucrose 33μg 25 μg QS21 ≈7,000 10mM sodium citrate or sodium acetate, pH6, 0.1% PS-80, 5% sucrose 33μg 50 μg MPL ^* ≈10,000 10mM sodium citrate/sodium acetate, pH6, 0.1% PS-80, 5% sucrose 33μg 25 μg QS21 ≈8,000 10mM sodium citrate/sodium acetate, pH6, 0.9% NaCl 33μg 50 μg MPL ^* ≈12,000 10mM sodium citrate or sodium acetate, pH6, 0.9% NaCl 33μg 25 μg QS21 ≈14,000 Control: Calif. Peptide Lot No. MF0639 33μg CFA/IFA ≈1,200

^* MPL preparations contain triethanolamine

^** The sample titer value is calculated based on 50% maximum absorbance Example 7 Lyophilized preparation

preparation

0.6mg/ml Aβ42 peptide, 10mM glycine salt, or lysine buffer, and add mannitol or four components of mannitol and sucrose, and filter and sterilize through Millex GV filter. One formulation, 0.6 mg/ml of Aβ42 peptide and 10 mM glycine sodium salt at pH 9.5, added with 4% mannitol, was filled into stoppered vials without titrating to physiological pH. The remaining three solutions (lysine/citrate/4% mannitol; glycinate/citrate/4% mannitol; glycinate/HCl/4% mannitol/1% sucrose) were subsequently treated with lemon Titrate to pH 7.5 with acid or HCl. Then take 0.5ml of the three preparations and put them into 2cc glass vials (Gensia P/N X34-113-002), and gently stopper them with gray butyl synthetic rubber freeze-dried stoppers (Gensia P/N X66-113- 030).

freeze-dried

The formulation was lyophilized in a programmable Virtis lyophilizer (supplied by Gensia Sicor). Mannitol is selected as the main matrix component. To obtain mannitol crystals, the formulation was frozen in a thermal cycle (annealing), followed by primary and secondary product drying.

Stability Analysis

The concentration and purity of the lyophilized formulations were monitored by reverse-phase high-performance liquid chromatography. Each formulation was reconstituted with 1.0 ml of water for injection (WFI). Analyzes were performed immediately after reconstitution (with or without centrifugation in a long benchtop microcentrifuge at > 10,000 rpm for 3-5 minutes), or after reconstitution as described above.

The following table (Table 9) lists the concentrations of the four Aβ42 peptide preparations after lyophilization and reconstitution. As shown by the data, these formulations were all soluble. Samples were analyzed in two formats: reconstituted formulations, and reconstituted formulations re-solubilized from peptide suspensions. No significant difference was seen between the reconstituted formulation and the total peptide solution (resolubilized sample). In the stability test of Example 5, as determined by reverse-phase high-performance liquid chromatography, no significant degradation and loss of the peptide occurred during storage at 2-8 degrees Celsius for three months.

Table 9. Lyophilized formulations Preparation 0.6mg/ml Aβ42 peptide dissolved in Exterior pH purity% mg/ml Aβ42 Reconstitute peptide solution resolubilized peptide 10mM Sodium Glycinate, pH 9.5, 4% Mannitol clear solution 8.2 82% 0.59 0.61 10mM Sodium Glycinate/Sodium Citrate, pH 7.5, 4% Mannitol clear solution 7.4 82% 0.57 0.60 10mM Sodium Lysinate/Sodium Citrate, pH 7.5, 4% Mannitol clear solution 7.6 81% 0.57 0.55 10mM Sodium Glycinate/HCl, pH7.5, 4% Mannitol, 1% Sucrose clear solution 7.4 81% 0.56 0.59

Characteristic description

Circular dichroism and FTIR analysis of the lyophilized preparation of the Aβ42 peptide at pH 7.5 showed that the peptide chain maintained a random coil conformation during both lyophilization and reconstitution. The same observation was made for the Aβ42 peptide preparation in sodium glycinate solution at pH 9, which was also soluble, filterable and assumed a random coil conformation in solution.

biological activity

Containing 0.6mg/ml Aβ42 peptide, 10mM sodium glycinate/HCl/4% mannitol/1% sucrose pH7.5 Aβ42 peptide lyophilized preparation was selected as a representative lyophilized preparation. When mixed with any of MPL and QS21, this product can induce a certain level of antibody titer in Swiss Webster mice. Table 10 lists the geometric mean titers compared to the control group.

Table 10 Antibody responses to lyophilized preparations Peptide preparation 0.6mg/ml Aβ42 peptide dissolved in: μg peptide μg excipient Geometric mean antibody titer (second blood draw) ^** 10mM Sodium Glycinate/HCl, pH7.5, 4% Mannitol, 1% Sucrose 33μg 50 μg MPL ^* ≈14,000 10mM Sodium Glycinate/HCl, pH7.5, 4% Mannitol, 1% Sucrose 33μg 25 μg QS21 ≈3,000 Control: Calif. Peptide lot number: MF0639 33μg CFA/IFA ≈1,200

^* MPL preparations contain triethanolamine

^** The sample titer value is calculated based on 50% of the maximum absorbance. Example 8 is scaled up using GMP production standards.

Peptide suspensions were scaled up to a 1.5 liter scale and produced and packaged by a contracted GMP pharmaceutical manufacturer. Encapsulation was performed at two concentrations: 0.6 mg/ml and 0.1 mg/ml. Both concentrations of product were successfully scaled up, encapsulated, and stable for two months at 2-8°C and 25°C.

Aβ peptide was dissolved in 10 mM sodium glycinate buffer at pH 9, containing 5% sucrose, at any concentration of 0.6 mg/ml or 0.1 mg/ml. The peptide solution was sterilized by filtration through a 0.2 micron Millipak20 microporous sterilizing filter, and then entered the sterile center. Reverse-phase HPLC showed no appreciable loss of peptide during filtration. Prepare a 1M sodium citrate buffer solution with a pH of 5.5, and let it also pass through a 0.2 micron Millipak20 microporous sterile filter and enter the sterile center. The peptide solution was weighed, and an appropriate amount of sodium citrate buffer was added to prepare a 20 mM citric acid, pH 6 preparation.

At a concentration of 0.6 mg/ml, the peptide formed a suspension immediately after the addition of citrate buffer; the pH during this process was measured to be 6.4. The peptide suspension was filled into 2cc borosilicate glass vials at 1.2ml each with constant stirring and closed with West 4416 stoppers and seals. Solutions at a concentration of 0.1 mg/ml in glass vials remained clear for several hours before forming a suspension the next day (also clear at the time of addition)

Table 11 lists the data about the Aβ42 peptide suspension at 0.6 mg/ml:

Table 11 Suspension of Aβ peptide 6mg/ml

Add 10mM glycine, 20mM citrate, 5% sucrose, pH 6 test Specifications result Exterior Colorless fuzzy suspension, impurity particles up to standard pass pH 5.5-6.5 6.3 concentration 0.5-0.7mg/ml 0.64mg/ml Area Purity Percentage (HPLC) FIO 84.4% Volume, ml FIO 1.13ml Overall Sterilization Status FIO no growth Container Terminal Sterilization Status no growth pass bacterial endotoxin FIO Less than 2 units/ml Example 9

Aβ1-42 peptide buffer suspension with QS-21 adjuvant

A single vial formulation of Aβ1-42 peptide combined with adjuvant was studied with QS-21, a triterpene saponin with immune stimulatory activity, and was surprisingly found to form a visually clear suspension of the peptide.

1. Single vial formulation of Aβ1-42 peptide/QS-21

Lyophilized QS-21 was dissolved in 10 mM sodium glycinate and Aβ1-42 peptide solution at pH 9. In each of the following examples, lyophilized QS-21 was dissolved in 10 mM glycine (Gly) Aβ1-42 peptide solution (trifluoroacetic acid (TSF) salt, 0.45 mg/ml) at pH 9. Addition of citrate buffer (Cit) rapidly adjusted the pH of the fraction containing dissolved QS-21 to 6.0 and formed a visually clear suspension. The clarity of the resulting suspension was measured with a spectrophotometer set at a wavelength of 405 nm. The results imply that the size of the particles in the peptide suspension is smaller than the wavelength of the reflected light. These formulations maintained stability for three months under storage conditions at 2-8 degrees Celsius under full stability monitoring. The Aβ1-42 peptide contained in all formulations was found to exist mainly in the β-sheet conformation. All formulations showed no turbidity when measured with a spectrophotometer at 405 nm. 0.45mg/ml Aβ1-42 peptide, 0.2mg/ml QS-21, 10mM glycine, 20mM citrate, 5% sucrose, pH 6 preparations were titered in mice, resulting in high titer degree of response.

Table 12A Formulation Exterior 0.45mg/ml Aβ1-42 peptide, 0.2mg/ml QS-21, 10mM glycine, 20mM citrate, 5% sucrose, pH 6.01 clarification 0.45mg/ml Aβ1-42 peptide, 0.1mg/ml QS-21, 10mM glycine, 20mM citrate, 5% sucrose, pH 6.0 clarification 0.225mg/ml Aβ1-42 peptide, 0.3mg/ml QS-21, 10mM glycine, 20mM citrate, 5% sucrose, pH 6.0 clarification 0.225mg/ml Aβ1-42 peptide, 0.15mg/ml QS-21, 10mM glycine, 20mM citrate, 5% sucrose, pH 6.0 clarification

1 found a high titer response in mice

2. Titration of Aβ42 peptide with QS-21

Aβ42 peptide (2 mg/ml) was dissolved in 10 mM glycine solution at pH 9.5. The pH was adjusted to 9.5 with 1 N of sodium hydroxide. The Aβ42 peptide solution was then filtered through a Millex GV filter. QS-21 (5 mg/ml) was dissolved in 10 mM sodium citrate solution at pH 6.0 and filtered through Millex GV filter. Mix the Aβ42 peptide and QS-21 solution of the aliquot sample to the required ratio, mix well, and then dilute to the following table with 10mM glycine solution with pH 9.5 and 1M citrate solution with pH 5.2 final concentration.

Table 12B. Initial AN1792/QS-21 co-constitutive formulations The preparation formula is in 10mM glycine, 20mM citrate, pH6.0 solution Exterior Absorbance 405nm 1.0mg/ml Aβ42 peptide, 1.0mg/ml QS-21 clarification 0.018 1.0mg/ml Aβ42 peptide, 0.5mg/ml QS-21 Opaque (+) 0.013 1.0mg/ml Aβ42 peptide, 0.25mg/ml QS-21 opaque (++) 0.030 1.0mg/ml Aβ42 peptide, 0.125mg/ml QS-21 Opaque (+++) 0.175 1.0mg/ml Aβ42 peptide, 0.063mg/ml QS-21 Opaque (+++) 0.308 1.0mg/ml Aβ42 peptide, 0.03 1mg/ml QS-21 Opaque (+++) 0.315 1.0mg/ml Aβ42 peptide, 0.016mg/ml QS-21 Opaque (+++) 0.268 1.0mg/ml Aβ42 peptide, 0.0mg/ml QS-21 Opaque (+++) 0.213 0.5mg/ml Aβ42 peptide, 1.0mg/ml QS-21 clarification 0.005 0.5mg/ml Aβ42 peptide, 0.5mg/ml QS-21 clarification 0.006 0.5mg/ml Aβ42 peptide, 0.25mg/ml QS-21 clarification 0.005 0.5mg/ml Aβ42 peptide, 0.125mg/ml QS-21 Opaque (+) 0.021 0.5mg/ml Aβ42 peptide, 0.063mg/ml QS-21 opaque (++) 0.148 0.5mg/ml Aβ42 peptide, 0.031mg/ml QS-21 Opaque (+++) 0.172 0.5mg/ml Aβ42 peptide, 0.016mg/ml QS-21 Opaque (+++) 0.162 0.5mg/ml Aβ42 peptide, 0.0mg/ml QS-21 none none 0.3mg/ml Aβ42 peptide, 1.0mg/ml QS-21 clarification 0.004 0.3mg/ml Aβ42 peptide, 0.5mg/ml QS-21 clarification 0.004 0.3mg/ml Aβ42 peptide, 0.25mg/ml QS-21 clarification 0.006 0.3mg/ml Aβ42 peptide, 0.125mg/ml QS-21 clarification 0.005 0.3mg/ml Aβ42 peptide, 0.063mg/ml QS-21 Opaque (+) 0.013 0.3mg/ml Aβ42 peptide, 0.031mg/ml QS-21 Opaque (+) 0.077 0.3mg/ml Aβ42 peptide, 0.016mg/ml QS-21 Opaque (+) 0.068 0.3mg/ml Aβ42 peptide, 0.0mg/ml QS-21 opaque (++) 0.046 0.1mg/ml Aβ42 peptide, 1.0mg/ml QS-21 clarification 0.004 0.1mg/ml Aβ42 peptide, 0.5mg/ml QS-21 clarification 0.002 0.1mg/ml Aβ42 peptide, 0.25mg/ml QS-21 clarification 0.002 0.1mg/ml Aβ42 peptide, 0.125mg/ml QS-21 clarification 0.001 0.1mg/ml Aβ42 peptide, 0.063mg/ml QS-21 clarification 0.002 0.1mg/ml Aβ42 peptide, 0.031mg/ml QS-21 clarification 0.003 0.1mg/ml Aβ42 peptide, 0.016mg/ml QS-21 clarification 0.004 0.1mg/ml Aβ42 peptide, 0.0mg/ml QS-21 clarification 0.008

3. Other preparations of Aβ42 peptide and QS-21

The chloride salt of the Aβ42 peptide was dissolved at a concentration of 1 mg/ml in a 10 mM glycinate solution at pH 9.0-9.5 with or without 5% sucrose, 0.1% PS-80 or 0.4% PS-80. Peptide solutions were filter sterilized through Millex GV syringe filters. Freeze-dried QS-21 was dissolved in 10 mM citrate solution at pH 6.0 at a concentration of 5 mg/ml, and then sterilized by filtration through a Millex GV syringe filter.

Appropriate volumes of Aβ42 peptide and QS-21 solution were mixed to prepare the solutions with the final concentrations of Aβ42 peptide and QS-21 listed in Table 12C. Finally, the pH was finally adjusted to 6 with 1 M citrate buffer at pH 5.4, at which point the concentration of citrate buffer was 20 mM. The visual appearance of the formulations was evaluated from clear to cloudy (+ to +++). Adjusting the concentration and ratio of Aβ42 peptide to QS-21 can adjust the visual appearance of the formulation. In addition, sucrose and surfactants can be used as additional excipients of the preparation. Table 12C. Supplementary formulations of Aβ42 peptides and QS-21 Formulation Exterior QS-21(mg/ml) Aβ42(mg/ml) Ratio of Aβ42/QS-21 excipient free 5% sucrose 0.1% PS-80 0.4% PS-80 0.05 0.05 1.0 clarification clarification clarification clarification 0.1 0.1 1.0 clarification clarification clarification clarification 0.2 0.2 1.0 clarification clarification clarification Cloudy (+) 0.1 0.23 2.3 Cloudy (+) Cloudy (+) Cloudy (+) Cloudy (+) 0.2 0.23 1.1 clarification clarification clarification Cloudy (+) 0.3 0.23 0.8 clarification clarification clarification Cloudy (+) 0.1 0.45 4.5 Cloudy (++) Cloudy (++) clarification Cloudy (+) 0.2 0.45 2.3 Cloudy (++) Cloudy (++) Cloudy (+) Cloudy (+) 0.3 0.45 1.5 clarification clarification clarification Cloudy (+)

Example 10 C.D. Spectroscopy Determination and Interpretation of Aβ42 Peptide Preparations

Circular dichroism data were obtained on an Aviv 62-DS polarimeter (Lakewood, NJ). Properly prepare the sample and load it into a tension-free quartz chamber with an optical path length of 1 mm for the determination of near-ultraviolet and far-ultraviolet spectra, respectively. The sample holder is at a steady 25 degrees Celsius. Values are taken at intervals of 0.5nm in units of 4 seconds on average. In the far ultraviolet region (wavelength 180-250nm), the signal of the peptide backbone dominates the spectrum, allowing an estimate of the secondary structure to be derived. The analysis in the near-UV region (1250-350nm) yields a completely different message: the main signal in this region comes from the aromatic side chains (phenylalanine, tyrosine and tryptophan). The characteristics and intensities of the signals show that the extension and directionality of the side chains at each position are related to the peptide backbone. Because the number of chromophores is smaller than that of amine groups, and these chromophores are distributed throughout the molecule, they can provide some information about the local structure.

Like the numerous modifications and changes made to the present invention in the above embodiments, those skilled in the art can also make various changes. Accordingly, the invention is limited only by the appended claims. Various changes without departing from the spirit and essence of the present invention are within the scope of the claims of the present invention.

Claims (47)

CLAIMS 1. A composition comprising an aqueous solution of Aβ peptide at a concentration of at least 0.01 mg/ml, wherein said aqueous solution maintains a pH for sufficient dissolution of said Aβ peptide. 2. The composition according to claim 1, wherein an appropriate pH value is maintained by adding an effective amount of a pharmaceutically acceptable buffer to said solution. 3. A composition comprising a sterilized aqueous solution of Aβ peptide at a concentration of at least 0.01 mg/ml, wherein said aqueous solution maintains a pH sufficient to dissolve said Aβ peptide. 4. The composition according to claim 3, wherein said pH is maintained by adding an effective amount of a pharmaceutically acceptable buffer to said solution. 5. The composition according to claim 1 or 3, wherein said A[beta] peptide is a long chain form of A[beta] peptide. 6. The composition according to claim 1 or 3, wherein said A[beta] peptide is A[beta]42. 7. The composition of claim 1 or 3, wherein said pH range is about 8.5 to 12. 8. The composition according to claim 7, wherein said pH range is about 9 to 10. 9. The composition according to claim 2 or 4, wherein the pharmaceutically acceptable buffer is selected from amino acids, salts, and derivatives thereof, pharmaceutically acceptable alkalizing agents, alkali metal hydroxides, and hydrogen Ammonium oxide, organic or inorganic acids and their salts, and mixtures of the above-mentioned substances. 10. The composition according to claim 9, wherein the pharmaceutically acceptable buffer is glycine (sodium glycinate), or arginine (arginine hydrochloride). 11. A lyophilized composition of Aβ peptide prepared by the following steps: a) freezing a sterilized aqueous solution of Aβ peptide at a concentration of at least 0.01 mg/ml, wherein said aqueous solution is maintained at a pH to dissolve said Aβ peptide; and b) Freeze-drying the frozen composition prepared in step a) above. 12. The composition of claim 11, wherein the A[beta] peptide is a long chain form of the A[beta] peptide. 13. The composition according to claim 11, wherein said A[beta] peptide is A[beta]42. 14. The composition of claim 11, wherein an effective amount of a pharmaceutically acceptable buffer is added to said solution to maintain said pH. 15. The composition according to claim 14, wherein said pharmaceutical buffer is selected from amino acids, salts, and derivatives thereof; pharmaceutical alkalinizing agents, alkali metal hydroxides, and ammonium hydroxide, organic or Inorganic acids, and salts thereof; and mixtures of the foregoing. 16. The composition of claim 1, 3, or 11, wherein the A[beta] peptide is substantially in a random coil state. 17. The composition of claim 1, 3, or 11, wherein said A[beta] peptide is present at a concentration of about 0.05 mg/ml to 2.0 mg/ml. 18. The composition according to claim 1, 3, or 11, wherein the composition further contains pharmaceutical adjuvants. 19. The composition according to claim 18, wherein said adjuvant is selected from Freund's incomplete adjuvant, MPL, QS-21, and alum. 20. A composition comprising a sterilized suspension of A[beta] peptide at a pH of about 5 to 7 at a concentration of at least 0.1 mg/ml. 21. The composition according to claim 20, wherein said peptide suspension also contains an effective amount of a pharmaceutically acceptable buffer. 22. The composition according to claim 20 or 21, wherein said A[beta] peptide is a long chain form of A[beta] peptide. 23. The composition according to claim 20 or 21, wherein said A[beta] peptide is A[beta]42. 24. The composition according to claim 21, wherein the pharmaceutically acceptable buffer is selected from the group consisting of amino acids, salts, and derivatives thereof; pharmaceutically acceptable alkalizing agents, alkali metal hydroxides, and ammonium hydroxide, organic or inorganic Acids, and their salts; and mixtures of the foregoing. 25. The composition of claim 20 comprising A[beta] peptide at a concentration of about 0.1 to 0.8 mg/ml, 10 mM glycine, and an acid sufficient to adjust the pH to about 5.5-6.5. 26. The composition according to claim 24 or 25, wherein said composition further comprises one or more excipients selected from tonicity adjusting agents, surfactants, and wetting agents . 27. The composition according to claim 24, wherein said composition further comprises a pharmaceutically acceptable adjuvant. 28. The composition according to claim 26, wherein said composition further comprises a pharmaceutically acceptable adjuvant. 29. The composition according to claim 28, wherein said adjuvant is selected from Freund's incomplete adjuvant, MPL, QS-21, and alum. 30. The composition according to claim 28, comprising Aβ peptide at a concentration of about 0.1 to 1.0 mg/ml, dissolved in 10 mM glycine at a pH of about 6, and QS-21 at a concentration of at least 0.1 mg/ml , in an amount effective to form a visually clear suspension. 31. A method for the preparation of a long-chain form of an Aβ peptide comprising: adjusting the pH of the aqueous solution to dissolve the Aβ peptide described therein; adding a sufficient amount of Aβ peptide to the aqueous solution to elicit an immune response in the mammal; and The resulting solution is filtered through a filter membrane with uniform pores, the size of which can both filter out bacteria and pass through a sufficient amount of substantially all species of the Aβ peptide. 32. The method of claim 31, wherein said filtering employs a hydrophilic polymer membrane having a uniform pore size of about 0.22 microns. 33. The method of claim 31, wherein the recovery of A[beta] peptides after filtration exceeds 50%. 34. The method of claim 31, wherein said pre-filter solution contains at least one diluent selected from pharmaceutically acceptable buffers at a concentration of about 5 mM to 45 mM. 35. The method of claim 34, wherein said prefilter solution contains a tonicity modifier in an amount of about 0.9%-6.0% (w/v). 36. The method of claim 34, wherein said pre-filter solution contains a surfactant at about 0.02%-1.0% (w/v). 37. The method of claim 34, wherein said prefiltration solution contains a chelating agent at a concentration of about 0.1 mM to 1.0 mM. 38. The method of claim 34, 35, 36, or 37, wherein the pH of the filtered sterile solution is adjusted to about 5-7 to form the peptide suspension. 39. A method for preventing or treating Alzheimer's disease in a mammal, comprising administering to said mammal an effective amount of a sterilized aqueous solution composition containing Aβ peptide at a concentration of at least 0.05 mg/ml, with An immune response is induced in the animal, wherein the aqueous solution maintains a certain pH value to dissolve the Aβ peptide in sufficient amount. 40. A method of eliciting an antibody response to Aβ peptide in a mammal in need thereof, comprising: Antibacterial composition of long-chain form Aβ peptide for parenteral administration of immunizing doses. 41. The method according to claim 39 or 40, wherein the method further comprises adding pharmaceutical adjuvants individually or in combination to the antibacterial composition. 42. The method according to claim 39 or 40, wherein the antimicrobial composition is the composition according to claim 30. 43. A composition comprising Aβ peptide at a concentration of at least 0.1 mg/ml, and an effective amount of QS-21 to form a visually clear suspension having a pH in the range of 5 to 7. 44. A composition comprising A[beta] peptide at a concentration of at least 0.1 mg/ml and an effective amount of DPPC at a pH in the range of 5 to 7 to form a visually clear suspension. 45. Use of a bactericidal composition containing long-chain form of Aβ peptide in the preparation of a medicament for inducing an antibody response to Aβ peptide. 46. The use of a bactericidal aqueous composition containing Aβ peptide in the preparation of a medicament for preventing or treating Alzheimer's disease. 47. The use according to claim 45 or 46, wherein the medicament further comprises a pharmaceutical adjuvant.

Images