Additives for protein formulations to improve thermal stability

Abstract

To provide methods for enhancing the thermal stability of liquid protein or peptide formulations, and stabilized formulations.SOLUTION: A method comprises the step of combining a protein or peptide solution with at least one ionic liquid selected from the group consisting of choline dihydrogenphosphate, a hydrated deep eutectic mixture of choline chloride and a sugar, sugar alcohol or polyol, selected from the group of glucose, sucrose, glycerol, and sorbitol, and 2-hydroxyethyl-trimethylammonium L-(+)-lactate, or a mixture of them, in a concentration suitable for measurably increasing the thermostability of protein or peptide formulation.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to protein formulations comprising excipients suitable for improving thermal stability against denaturation and deactivation. In particular, the present invention relates to additives for the thermal stabilization of vaccine formulations and to methods for the preparation of these formulations. [Background technology]

[0002] technical level Proteins or peptides are commercially used in various pharmaceutical forms, such as vaccines. Many of these include proteins and protein-based structures / complexes. The proteins are often produced by recombinant DNA technology or other means. The proteins utilized in vaccine formulations can be of bacterial or viral origin. The proteins can be classified into groups including, but not limited to, toxins, toxoids, or mutant proteins. Furthermore, vaccines can be derived from the bacterial or viral proteins / peptides in the form of epitope vaccines, chemically inactivated toxins (toxoids), glycoproteins, or conjugated proteins, in which the pharmacologic active substance is covalently bound to a carrier protein. These entities can include protein-protein conjugates (e.g., tetanus toxoid), fusion proteins, protein-polysaccharide conjugates, protein-nucleic acid conjugates, protein-DNA conjugates, protein-RNA conjugates, protein-small molecule conjugates, protein-dye conjugates, protein-alum adsorbates, luminescent or bioluminescent proteins, biotinylated proteins, etc. In addition, proteins form one of the building blocks of viral fragments.

[0003] From a chemical point of view, proteins are large organic molecules or macromolecules composed of amino acid residues covalently linked together by peptide bonds into linear, unbranched polypeptide chains with relative molecular weights ranging from several thousand to several million Da. The useful properties of proteins as drugs depend on the polypeptide chain adopting a unique three-dimensional folded conformation, i.e., the tertiary structure of the protein. This unique fold is the reason why proteins are highly selective about the molecules they recognize. However, the ability to maintain a unique three-dimensional structure is certainly one of the obstacles that make the use of polypeptides in human and animal diseases problematic.

[0004] Therefore, maintaining the potency of vaccines or protein particles against degradation is a major challenge in providing correct vaccination services, since it is well known that biological materials, including biological materials in solution, are prone to inactivation due to heat, oxidizing reagents, salts, etc.

[0005] Although viral particles, bacteria and other infectious agents used in vaccines may be easily inactivated after a short period at ambient temperature, low temperature storage is costly. Exposure to high temperatures even for short periods can lead to loss of vaccine activity, even at low temperatures, limiting vaccine use and increasing costs, thereby restricting vaccine distribution, especially in developing third world countries.

[0006] It is known from the literature that up to 80% of the costs of vaccination programs are due to cold chain issues (keeping the vaccines cold), meaning that it is essential to keep the vaccines cold and to maintain their effectiveness.

[0007] Typically, this requires that the vaccine be kept refrigerated at all times from production to administration, a major undertaking especially in remote areas of the developing world.

[0008] The high costs and risks associated with protecting vaccines from degradation, referred to as the cold chain problem, have been recognized by the World Health Organization as one of the most important challenges for the expansion of global vaccination programs. Thus, there are various approaches to stabilize vaccination formulations, and attempts have been made to solve this problem and to delay the degradation of viral particles by the addition of low-cost, biocompatible additives. For example, anionic gold nanoparticles (10 μg / ml) were used to increase the half-life of green fluorescent protein-expressing adenovirus from ~48 h to 21 days at 37 °C (7 to >30 days at room temperature). -8 ~10 -6 M) or polyethylene glycol (PEG, molecular weight 8,000 Da, 10 -7 ~10 -4It is proposed to improve the storage time of adenovirus type 5 by the addition of PEG and sucrose (M). Stabilizing effects are also known from sucrose, which is added in low concentrations. It was found that the addition of PEG and sucrose can maintain the immunogenicity in vivo for viruses stored at 37°C for 10 days. However, these stabilizing effects are not sufficient to achieve storage stability at high temperatures above 60°C. However, the latter is necessary for the safe use of vaccine formulations in tropical regions, so it is surprising that liquid protein formulations such as vaccine formulations are currently produced without any specific heat stabilizers or excipients that would provide heat stability to temperatures above 60°C. One reason for this, among others, is that the addition of additives to current vaccines usually means going through a costly approval process by health authorities again, which is commercially unattractive. It would therefore be desirable to stabilize vaccine formulations against inactivation at elevated temperatures by the addition of additives or excipients, preferably those already recognized as compatible ingredients for pharmaceutical compositions. One approach has been the addition of sucrose to vaccine formulations, as mentioned in a review by Pellicca, M. et al. [Pelliccia, M. et al. Additives for vaccine storage to improve thermal stability of adenoviruses from hours to months. Nat. Commun. 7, 13520 doi: 10.1038 / ncomms13520 (2016)]. Trehalose, glycerol, glucose, maltose and raffinose have also been suggested for this use, but the desired effect can only be found at high concentrations and not at temperatures higher than 40°C. PEG, the most effective heat-stabilizing additive, has proven unsuitable for vaccine formulations, as up to 50% of humans have an allergic reaction to it, as reported by Pellicca, M. et al.

[0009] In WO2012 / 125658A1, a process is provided for the production of a stabilized vaccine composition of an immunogen, particularly an unstable immunogen, where oral granules are aerosolized with an antigenic virus culture at a temperature of about 25° C. to about 50° C., preferably about 30° C., so that the immunogen coat dries onto the particles. The vaccine product contains particles having a moisture content of between about 0.1% w / w and 10% w / w to yield a stabilized vaccine composition.

[0010] In US 2013 / 209503 A1, freeze-dried and lyophilized vaccine compositions are stabilized by first providing a polysaccharide-protein conjugate that is stabilized by a combination of an amorphous excipient selected from the group consisting of sucrose, lactose, trehalose, dextrose, xylose, cellobiose, raffinose, isomaltose and cyclodextrin, and a buffer selected from the group consisting of a salt of citrate, histidine, phosphate, Tris, succinate, and acetate buffers.

[0011] In addition, there is a series of publications and patent literature that attempt to solve the higher temperature stability problems of corresponding proteinaceous formulations in a similar manner, but at the expense of the need for the injectable solution to be reconstituted prior to application. The latter, however, may be a significant problem in developing countries and in crisis areas. Here, an improvement in the stability of a protein formulation for even a few hours relative to the elevated ambient temperature would represent an improvement. Summary of the Invention

[0012] Objective of the invention Heat stability and cold chain interruptions are major obstacles in ensuring stable delivery of vaccine doses, especially to remote areas in poverty-stricken countries. Currently, vaccines are refrigerated during shipping, but 25% of all liquid and 50% of all lyophilized vaccine doses shipped to developing countries are lost because continued refrigeration cannot be guaranteed.

[0013] The main objective of the present invention is to provide a thermally stabilized vaccine formulation, which can preferably be made available with the same quality as comparable products and which is stable for long periods of time when stored at temperatures up to and exceeding 60°C.

[0014] A brief summary and subject matter of the invention An excipient from one of the classes of molecules listed below is added to the protein or peptide formulated in an aqueous buffer at an optimized concentration. The melting temperature T m is thereby substantially increased, so that high temperatures of up to 60° C. and above can be tolerated.

[0015] Another subject of the invention are the excipients disclosed below, which at suitable concentrations confer sufficient heat stabilization to vaccine formulations so that they can be stored at temperatures above 60° C. while maintaining their folded protein structure for a sufficiently long time.

[0016] The present invention delivers stabilized solutions of proteins at a relatively high concentration, advantageously about 2.0 mg / ml, which contain at least one stabilizing agent, which minimizes the degradation and inactivation of the contained proteins, which has a direct positive impact on maintaining their efficacy and allows these formulations of the present invention to be stored and even shipped to underdeveloped markets in tropical regions.

[0017] More specifically, the present invention relates to a method for the thermostabilization of liquid protein formulations as characterized in claim 1 and in particular embodiments as described in claims 2 to 16. The present invention also comprises the formulations prepared according to the method as characterized in claims 17 to 24 and as described in claims 1 to 16. A further embodiment of the present invention consists in a kit as described in claims 25 to 27 for the use of the stabilized protein formulations. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 presents a summary of the results of the added excipients showing best performance. [Diagram 2] FIG. 2 presents a summary of the results for the change in melting temperature conferred by each excipient under optimal conditions. [Figure 3a] Figure 3 shows the storage stability of anthracnose PA at 50°C, assayed by fluorescence spectroscopy. Figure 3a) (left) shows the folded fraction. [Figure 3b] Figure 3b) (right) shows the normalized transition temperatures. [Figure 4a] Figure 4 shows the storage stability of anthracnose PA at 55° C. Figure 4a) (left) shows the folded fractions. [Figure 4b] Figure 4b) (right) shows the normalized transition temperatures.

[0019] [Figure 5a] Figure 5 shows the stability of anthracnose PA at 60° C. Figure 5a) (left) shows the folded fraction. [Figure 5b] Figure 5b) (right) shows the normalized transition temperatures. [Figure 6] FIG. 6 shows the stability of CRM197 at 50° C. [Figure 7] FIG. 7 shows the storage stability of CRM197 at 55° C. [Figure 8] FIG. 8 in turn shows the storage stability of CRM197 at 60° C. [Figure 9]FIG. 9 shows the effect of excipient candidates on the mean unfolding temperature of LDH.

[0020] [Figure 10] FIG. 10 shows the shift in melting temperature, dTm, of LDH. [Figure 11] FIG. 11 shows the effect of lactulose, magnesium gluconate, polysucrose 400, mannitol, maltose and ammonium acetate on the mean unfolding temperature of LDH. [Figure 12] FIG. 12 shows the effect of hydroxy-proline, L-citrulline, L-ornitine, potassium dihydrogen phosphate, lactobionic acid, meglumine, ectoine, myo-inositol and methyl sulfonate on the mean unfolding temperature of lysozyme. [Figure 13] FIG. 13 shows the results of a second group of materials tested in the lysozyme:ionic liquid system. [Figure 14] FIG. 14 summarizes the results for the combination of potassium dihydrogen phosphate with lactobionic acid.

[0021] [Figure 15] FIG. 15 summarizes the results for the combination of potassium dihydrogen phosphate and 4-OH-proline. [Figure 16] FIG. 16 summarizes the results for the combination of potassium dihydrogen phosphate and choline chloride / sorbitol. [Figure 17] FIG. 17 summarizes the results of the addition of a combination of Choline Cl / sorbitol and lactobionic acid. [Figure 18] FIG. 18 summarizes the results of the combination of choline chloride / sorbitol and 4-OH-proline. [Figure 19] FIG. 19 shows the results for heat stabilizing anthracnose PA with sugar.

[0022] [Figure 20]FIG. 20 shows the results for heat stabilizing anthracnose PA with osmolytes. [Figure 21] FIG. 21 shows the results for heat stabilizing anthracnose PA using salts and ionic liquids. [Figure 22] FIG. 22 shows the results for heat stabilizing Td with sugars. [Figure 23] FIG. 23 shows the results for heat stabilizing Td with osmolytes. [Figure 24] FIG. 24 shows the results for thermally stabilised Td using ionic liquids.

[0023] [Diagram 25] FIG. 25 shows the results for the heat stabilization of CRM with sugars. [Figure 26] FIG. 26 shows the results for the thermostabilization of CRM with osmolytes. [Figure 27] FIG. 27 shows the results for the thermal stabilization of CRM using ionic liquids. [Figure 28] FIG. 28 shows the results for anthrax PA as an example. [Figure 29] FIG. 29: Tagg vs. Tm. [Diagram 30] FIG. 30 shows the transition temperatures that were determined. [Diagram 31] FIG. 31 provides a summary of all the results for each protein and excipient combination.

[0024] Detailed Description of the Invention and preferred embodiments The present invention relates to a method for obtaining liquid protein or peptide formulations, which are heat-stabilized and, if desired, directly usable formulations containing proteins, which may be produced by recombinant DNA technology or other means, protein-conjugates, i.e. protein-saccharides, protein-nucleotides, protein-RNA / DNA or protein-peptides, cross-linked proteins, such as formaldehyde-treated proteins, such as tetanus toxoid, as well as ultimately larger structures utilized in vaccine formulations or other proteins, which may be of bacterial or viral origin, viral protein subunits, enzymes, hormones or others. Consequently, in the following, whenever reference is made to protein formulations, it is always meant to also mean peptide formulations, unless specific formulations containing one or the other are explicitly mentioned.

[0025] The present invention also relates to stabilized formulations, in which the proteins maintain their folded, effective structure even when the temperature is raised to values ​​up to 60° C. The present invention also relates to such formulations that maintain their stability for up to 3 or even 4 days. Advantageously, stabilized protein formulations can be easily obtained by adding selected excipients in effective amounts to solutions, optionally buffered and having their pH adjusted to an optimal range for the protein contained. The formulation may also contain a reducing agent.

[0026] The liquid formulations of the invention are prepared containing the protein at a concentration ranging between 0.05 and 1.8 mg / ml. The thermostabilizing effect of the added excipients is evidenced by an increase in the melting temperature of the contained protein; at the same time, an increase in the protein melting temperature is an indication of increased thermostability of the protein in the formulation. By means of certain experiments, it was found that, on the one hand, depending on the type of protein and added excipient, different amounts of excipients have to be added to obtain the desired stabilization of the contained protein, and, on the other hand, it is also advantageous if the pH value in the stabilizing solution is adjusted depending on the contained protein. Depending on the excipient, concentrations ranging from 0.25 to 4 M are useful in the protein solution for the desired protein stabilization, minimizing degradation and activation during storage as well as exposure to temperatures higher than room temperature, in particular higher than 40° C. As previously described, reliable stabilization of the protein also depends inter alia on the pH of the solution. Depending on the protein, the optimal pH can range between pH 6 and 8, advantageously set with a suitable buffer.

[0027] The protein solution can then be stabilized against the deactivating effects of elevated temperatures. Depending on the added protein and excipients, a stabilization of up to 20°C can be determined by the following experiments. Only experiments were considered in which an increase in the melting temperature of the protein of at least 5°C could be determined by the addition of a specific excipient to improve the thermal stability.

[0028] For further use, the formulations stabilized in this way can be filled into bottles for further dosing, dilution or processing.However, they can also be formulated as vaccination solutions, which can be optionally finished as solutions for use in pharmaceutical injection vials, unless further processing or dilution is required.In yet another form of application, the protein solutions stabilized in this way can be provided in immunoassay test kits or preparations and bottled for analysis.Also, the storage formulations can be stabilized in this way and shipped worldwide for subsequent filling and finishing.

[0029] Kits containing a heat-stabilized protein preparation may also contain other container means containing necessary or convenient solutions for carrying out a method of use of the protein preparation. The container means may be made of glass, plastic or foil, and may be a vial, bottle, pouch, tube, bag, and the like. The kit may contain written information, such as procedural or analytical information for carrying out a method of use, such as procedural or analytical information for carrying out a method of use, such as the amount of reagent contained in the first container means. The container means may be another container means, for example a box or bag with written information.

[0030] Being liquids, in a further aspect of the invention, the pharmaceutical compositions of the invention may be preformulated for later finishing for injection into the body, hi an alternative aspect, the pharmaceutical compositions may be preformulated for introduction into small implantable pumps or other devices for therapeutic use.

[0031] Excipients suitable as heat stabilizers for use in the present invention can be selected from various types of organic molecules. The prerequisite for the use of these substances according to the present invention is that they are compatible with the protein or peptide and that they are physiologically tolerated without causing side effects or allergic reactions in both animal and human organisms when used at the correct concentrations.

[0032] Liquid formulations containing proteins may contain, in addition to the excipients added according to the invention, one or more additional compounds such as amino acids, proteins, chelating agents, buffers, salts, preservatives, stabilizers, antioxidants, emulsifiers, plasticizers or lubricants.

[0033] Peptides are organic chemical compounds that contain peptide bonds between amino acids. According to their number, oligopeptides are distinguished from some polypeptides that have many amino acids. Long polypeptide chains are also called proteins, especially those produced by protein biosynthesis. Due to this chemical relationship between peptides and proteins, when proteins are mentioned below, both classes of substances are meant.

[0034] In the literature, the expression "thermostable" is used frequently, but often in a misleading manner. In the literature, it usually means only that a protein solution can be stored at a slightly elevated temperature below 40° C. 40° C. - this is the midpoint of the unfolding transition (T m ) (For example, anthrax PAT m By increasing the storage temperature to - still significantly below 48°C (=48°C), the spontaneous unfolding rate of the protein is increased. In terms of equilibrium thermodynamics, there is a certain probability that the protein may spontaneously unfold at any time, however, the likelihood of such an unfolding event is temperature dependent. By increasing the storage temperature, the frequency of potential unfolding events is increased, and therefore, long-term storage of protein formulations can be evaluated during a shortened period of time.

[0035] Here, according to the present invention, the expression "thermostable" is used in the context that the protein or peptide remains exclusively in its native, unfolded form during storage at temperatures above 40°C, preferably above 50°C, in particular up to 60°C.

[0036] The present analytical efforts are directed to the thermostability by determining the thermodynamic transition midpoints of heat-stressed proteins. To this end, the effects of model molecules or of selected excipients are examined for their stabilizing effect on the structure of the model proteins at elevated temperatures (which means at temperatures above 40° C., preferably up to 60° C.). At the same time, the experiments analyze in which concentration range the heat-stabilizing excipients are effective.

[0037] By definition, dT >5°C m Excipients that lead to dT are good stabilizers. m If the dT is <5°C and >0°C, the excipient is defined as a poor stabilizer. m identifies the molecule as a protein destabilizing agent.

[0038] Transition midpoint T m was determined by fluorescence spectroscopy, which means by nano differential scanning fluorimetry (nanoDSF) using protein solutions containing proteins at concentrations between 0.05 and 1.8 mg / ml.

[0039] The prepared protein solution is heated at a heating rate of 1° C. / min, and the ratio of fluorescence emission at 350 / 330 nm is recorded and plotted versus temperature. Typically, the resulting curve resembles a Boltzmann function, with the inflection point of this curve being the mean unfolding temperature, T m From this curve, T m The change in m賦形剤 From T mネイティブ This is determined by subtracting dT m produces.

[0040] The expression "folded fraction" is used below to describe the amount of native protein in liquid formulations. Secondly, the expression "thermal denaturation midpoint" is also used in relation to protein stability, with a deviation of 10% in either direction being defined as acceptable. It is important to note that any change in transition midpoint, even to higher temperatures, is the result of either changes in protein structure or data quality. The latter can be influenced, for example, by protein aggregation or by a myriad of other effects. Thus, transition temperatures are sought to deviate less than 10% from the initial measurement.

[0041] As used herein, the term "sugar" refers to any of a group of relatively low molecular weight water-soluble carbohydrates. The term sugar includes reducing sugars (such as maltose), non-reducing sugars (such as sucrose), sugar alcohols (such as sorbitol) and sugar acids (such as lactobionic acid).

[0042] All ranges defined herein are intended to encompass the lower and upper limits of the range. The term "about" is meant to encompass the given range to a lesser extent. Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the method employed to determine the value.

[0043] The use of the words "a" or "an" in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0044] As used in this specification and the claim(s), the words "comprising" (and any form of "comprising" such as "comprise" and "comprises"), "having" (and any form of "having" such as "have" and "has"), "including" (and any form of "including" such as "includes" and "include") or "containing" (and any form of "containing" such as "contains" and "contain") are inclusive or open ended and do not exclude additional, unrecited elements or method steps.

[0045] It is also specifically understood that any numerical value set forth herein includes every value from the lower limit to the upper limit, i.e., every possible combination of numerical values ​​between the lowest and highest values ​​recited is intended to be expressly recited in this application. For example, if a range is recited as 1% to 20%, values ​​from 2% to 5%, 10%, 15%, or 3% to 4.5%, etc. are intended to be expressly recited in this application.

[0046] "Contacting" refers to the process of bringing at least two distinct species into contact such that they can interact or react.

[0047] "Excipient" generally refers to a compound or material added to ensure or increase the stability of a therapeutic agent, i.e., long-term stability.

[0048] A "stable" formulation or composition is one in which the biologically active material or protein essentially retains its physical and / or chemical stability and / or biological activity upon storage. Stability can be measured at a selected temperature for a selected period of time. Trend analysis can be used to estimate the expected shelf life before the material has actually been stored for that period of time.

[0049] "Pharmaceutically acceptable" refers to active agents, salts, and excipients that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and are effective for their intended use.

[0050] As already mentioned above, the protein solution of the present invention is adjusted to a specific pH. In order to maintain a stable pH, a suitable buffer is added to the protein solution.

[0051] The buffer may be added in the form of a suitable carrier liquid to dissolve and / or disperse the dissolved protein. The buffer is often selected from a pharma- ceutically acceptable buffer system. Preferred buffers are pharma-ceutically acceptable buffer systems with the ability to withstand changes in pH upon addition of acids, bases, inorganic compounds, organic compounds, or solvents or diluents. Buffer components such as phosphate or citrate are included to control the pH of the vaccine-containing solution as well as to adjust the osmolality of the solution. The buffer concentration may range from 5 mM to 2 M, with the pH of the solution being adjusted to a range of about pH 4 to about pH 10, preferably from about pH 6 to about pH 8.

[0052] Pharmaceutically acceptable buffers may be selected from the group consisting of potassium phosphate, sodium phosphate, sodium acetate, histidine, imidazole, sodium citrate, sodium succinate, HEPES, Tris, Bis-Tris, ammonium bicarbonate, and other carbonates. Buffers may include a pH ranging from about pH 4 to about pH 10, preferably from about pH 6 to pH 8, but also from about pH 6 to about pH 7.

[0053] When one or more types of sugars are added to the protein solution, the sugars are preferably selected from monomeric and / or dimeric molecules, and may in particular be selected from the group of glucose galactose, maltose, maltulose, melibiose, sucrose, trehalose, lactose, lactulose, fructose, lactose, mannitol, sorbitol, xylitol, cellobiose, palatinose, turanose, sophorose, nigerose, and combinations thereof. Most preferably, the sugars are selected from the group of maltose, melibiose, lactose, lactulose, cellobiose, maltulose, palatinose, turanose, sophorose, nigerose. The amount of sugar in the aqueous heat-stabilized solution may range from very low to fairly high concentrations. Thus, for stabilization, the selected sugar can be added in a relatively small amount, but often there is particularly effective heat stabilization of proteins when the sugar contained is in a relatively high concentration, i.e. when added to the solution in molar amounts. Thus, it has been found by experiment that particularly good stabilization results can be achieved when the selected excipient is added in an amount of 0.25-4 M / l protein solution.

[0054] The aqueous solution may further include pharma- ceutically acceptable surfactants, polymers, amino acids, and other pharma-ceutically acceptable excipients. Polymers may be included to stabilize proteins. These polymers may be added in amounts of 0.1% w / v or more. Surfactants may be added to reduce surface tension and displace protein molecules from surfaces. Surfactants may also increase the solubility of other formulation components. Surfactants may be included in amounts of 0.005% or more by weight of the protein-containing formulation.

[0055] Divalent cations and amino acids may be included to stabilize proteins and to adjust the pH and osmolality of the solution. The concentration of divalent cations may range from 0.1 mM to about 100 mM, and amino acids may be included at concentrations ranging from about 0.1% to about 1% (w / v).

[0056] In summary, this means that depending on what protein or peptide solution according to the invention is to be used and depending on the properties of the protein or peptide contained in the solution, different components of the final formulation may be added.

[0057] Preferably, according to the invention, the heat stabilizing excipient or combination of heat stabilizing excipients is selected from the following classes of molecules: 1) Osmolyte :Trimethylamine-N-oxide;betaine, 4-hydroxyproline;ornitine;citrulline;N-acetyl-serine;hydroxylectoine;myo-inositol, allo-inositol, L-chiro-inositol, D-chiro-inositol 2) Ionic Liquids : Choline dihydrogen phosphate; hydrated deep eutectic mixtures of choline chloride with sugars, sugar alcohols or polyols (e.g. glucose, sucrose, glycerol or sorbitol); 2-hydroxyethyl-trimethylammonium L-(+)-lactate, 3) SaltDihydrogen phosphate compounds such as potassium dihydrogen phosphate, calcium dihydrogen phosphate, or sodium dihydrogen phosphate, ammonium acetate, 4) Sugar : Maltose, melibiose, lactose, lactulose, cellobiose, maltulose, palatinose, turanose, sophorose, nigerose 5) Sugar acids and their salts : Lactobionic acid, calcium lactobionate, magnesium gluconate or calcium gluconate 6) Amino sugars : Meglumine 7) Sugar alcohols : Mannitol, maltitol, sorbitol,

[0058] The thermostabilizing excipients are added to the protein formulations in various concentrations. The protein concentrations are within 0.05 and 1.8 mg / ml. Increased thermostability can be detected starting with 100 mM excipient, however, best results are obtained in the 0.4-4 M regime. For the hydrated deep eutectic mixture of choline chloride with the second component, and for 2-hydroxyethyl-trimethylammonium L-(+)-lactate, the concentrations are expressed in volume percent. Increased thermostability can be detected starting with 15%, with best results obtained using a 95% solution.

[0059] The aqueous protein solutions of the present invention may be provided in primary containers such as in either glass or plastic / resin vials, or as foil pouch devices or any other suitable and compatible devices. Depending on the desired subsequent use, the solutions provided in this way may be in amounts from a few μl up to many liters.

[0060] The present invention provides a method in which proteins or peptides in solutions used in pharmacy and medicine can be protected against the destabilizing and inactivating effects of elevated temperatures, meaning that the proteins can be thermostabilized. In particular, this stabilizing effect concerns protection against the onset of protein unfolding and aggregation, which generally leads to inactivation and loss of protein activity / or drug efficacy. The method of the present invention is applicable to any protein, including viral protein subunits, or to vaccines that can be used in microgram amounts. The protein can be a protein as a vaccine component, but also an enzyme, a hormone, or other therapeutically or analytically used protein, such as an enzyme, an antibody, a nanobody or a monobody. Depending on the type of protein and its use, the solutions are provided in different quantities, packaging or devices, but also in combination with others required for the desired use composition. Consequently, they can be part of a bioassay or already present as a solution in an application or analytical or diagnostic kit, or a molecular biology kit containing the enzyme. This means that the protein can be included in vaccines and in diagnostic assays for the detection of pathogens in samples from subjects with a disease. The disease agent may be from an infection, a tumor, or an allergen or otherwise.

[0061] As mentioned above, the present invention relates to the thermostabilization of the corresponding protein formulations. Through a designed experimental program, the effect of selected additives on the thermostability was investigated using the sample proteins listed above.

[0062] In order to test the heat stabilization by addition of the suitable excipients listed above on as many different proteins as possible, experiments were carried out with the following sample proteins: Enzymes, in particular lactate dehydrogenase and lysozyme as model enzymes; and Vaccine proteins, in particular anthrax PA (anthrax protective antigen), tetanus toxoid, and diphtheria toxoid (CRM197) as model vaccines.

[0063] To demonstrate effective thermostabilization by the addition of one or more excipients, liquid formulations are prepared containing the protein at concentrations ranging between 0.05 and 1.8 mg / ml. To do so, stock solutions of the protein are prepared. Also, stock solutions of excipients are prepared in applicable buffers. The protein, excipients, and buffers, if necessary, are combined to produce the formulations described in the examples. As shown in the "Examples" section, the melting temperatures (T m ) were tested in the presence of the most efficient and most applicable thermostabilizers, since an increase in protein melting temperature is an indication of increased thermostability of the protein in the liquid formulation.

[0064] Denaturation or melting temperature, T m The determination of Tm can be carried out in a high-throughput manner on small volumes using differential scanning fluorimetry (NanoDSF: Prometheus.NT48 equipped with standard capillaries, NanoTemper Technologies GmbH). In addition, for some preparations, the Tm was determined using circular dichroism spectroscopy (Spectropolarimeter J-815 with 0.1 cm quartz Suprasil cuvette type 110-QS from Hellma, Jasco Deutschland GmbH) to obtain additional structural information about the proteins in the preparation.

[0065] Proteins and peptides generally fold into a stable three-dimensional structure that is stabilized by multiple weak interactions of residues within the polypeptide chain. By increasing the temperature of the formulation, the increased thermal motion and other factors cause the protein to denature. To quantify the thermal stability of a protein, a temperature called Tm is used.

[0066] T mrepresents the temperature of the equilibrium transition midpoint obtained here for the heat-induced denaturation of a model protein. Assuming a two-state transition from native to a non-native state (unfolded / partially unfolded), T m represents the temperature at which both states are equally populated. Measurement of the transition midpoint is sensitive to the kinetic stability of the material, and therefore only such values ​​obtained under equal conditions are comparable. All T m Values ​​were recorded at a heating rate of 1°C / min. T increased upon addition of excipients m Values ​​indicate and represent protein stabilization.

[0067] The denaturation of proteins can be measured by a variety of spectroscopic techniques that exploit the specific optical properties of proteins.

[0068] Differential scanning fluorimetry is a fluorescence-based method that exploits the specific emission properties of the natural proteinogenic amino acid tryptophan. The fluorescence emission of the indole side chain is solvatochromic due to its two distinct dipole axes. When buried inside a protein, which is a more lipophilic environment, tryptophan typically exhibits a maximum fluorescence emission around 328-330 nm. When a protein is denatured, the tryptophan residue becomes progressively less shielded from the surrounding water. Thus, the emission maximum of tryptophan approaches 350 nm. This spectral shift to longer wavelengths is referred to as a red shift. NanoDSF records this shift by recording the protein's emission at 330 nm and 350 nm and plotting the ratio vs. temperature. This typically results in a sigmoidal curve with a positive amplitude. Fluorescence spectroscopy primarily probes tertiary structure transitions. It should be noted that the fluorescence spectrum of a protein represents an overlay of all of the emission spectra of the amino acids contained therein, and therefore the emission characteristics of a particular protein may vary from the values ​​stated above.

[0069] CD spectroscopy exploits the differential absorption of circularly polarized light by chiral substances or large molecules with asymmetric structure, such as proteins. Depending on the wavelength regime used, CD spectroscopy can be used to probe the secondary or tertiary structure of proteins. Here, we use CD spectroscopy exclusively to obtain information about the secondary structure of proteins. Natively folded proteins exhibit very characteristic CD spectra based on their structural composition. Upon thermally induced unfolding, the differential absorption of circularly polarized light is reduced and the CD signal approaches zero. T m To determine the CD-signal at a particular wavelength, typically 222 nm, 218 nm, 208 nm or 196 nm, is plotted against temperature and then fitted to a sigmoidal function.

[0070] Regardless of the method used, there are certain characteristics of a protein that report on the structural features of the protein. If the transition midpoint is elevated upon addition of an excipient, then the added excipient stabilizes the protein. In addition, the steepness of the sigmoid curve can be modified by the addition of an excipient. The steepness of the curve reflects the kinetic stability of the protein. m describes at what temperature a protein will unfold, while the steepness of the unfolding curve indicates and represents how fast the transition occurs.

[0071] In the case of fluorescence spectroscopy, additional features can modify the behavior of the sigmoid curve. Instead of the red shift described so far, the curve can show a negative amplitude, which indicates a deviation of the fluorescence to lower wavelengths. This can be explained by protein aggregation. If all tryptophan residues are not buried within the protein, the emission of the natively folded protein can be at a wavelength greater than 330 nm. Upon unfolding, the tryptophan residues will be more exposed to water, however, upon aggregation, the residues are buried within the lipophilic core of the aggregate. Despite the negative amplitude, the sigmoid fit can still be used to determine the temperature that reflects the thermal stability of the protein.

[0072] Now, to evaluate which additives or excipients are optimal for heat stabilization of proteins and in what amounts they must be added to produce the desired effect, in a first step, test formulations with anthrax PA as a model vaccine were prepared and tested over a period of 3 days. The detailed formulation composition is outlined in Table 1 in the "Examples" chapter. The aim of these experiments is to find the most suitable excipient or mixture of excipients for heat stabilization over several days of protein compositions of different origins.

[0073] To date, because the stabilizer must be used at high concentration, such compositions cannot be used to prepare pre-filled syringes, because patients will probably not tolerate the stabilizer.A dilution step is therefore required prior to administration to patients.Another focus of these experiments is also to determine the highest possible tolerated level of stabilizer for use in in vitro assays.Based on the experimental results, protein concentration can be adjusted and vaccine stock solutions can be produced, both of which can provide heat protection for solute proteins, but can still be effectively administered to patients after dilution.

[0074] For example, it has been found that melibiose can be most likely tolerated by patients up to a concentration of 250 mM. From previous screening, however, it is known that at least 2.25 M melibiose is essential to stabilize anthrax PA at high temperatures. Thus, the protein concentration is increased by a factor of 2250 mM (stock) / 250 mM (final) = 9. However, it is essential to dilute this stabilized vaccine stock formulation at least 9-fold for patient use.

[0075] Following these considerations, test formulations are prepared to test for the model proteins listed above which excipients are optimal as physiologically acceptable additives for the most feasible heat stabilization at temperatures above 40° C., preferably above 60° C. From the available potential excipients that are recognized as acceptable to patients in liquid pharmaceutical formulations, lactulose and maltose have been initially selected as promising temperature-stabilizing additives, and the corresponding protein formulations have been prepared and tested with these additives. TMAO, choline dihydrogen phosphate, and melibiose were also examined under the same conditions, and the results were compared with those of lactulose and maltose.

[0076] First, samples are spiked with selected excipients that are suitably subjected to fluorescence spectroscopy to confirm that all protein samples are natively folded, and then to determine whether they are suitable for stabilizing proteins at elevated temperatures, including elevated protein levels (up to 1.8 mg / ml). Following these tests, incubations are carried out at specified temperatures, specifically at 4°C and in the range between 50-60°C, as well as at higher temperatures if appropriate. Tests have shown, inter alia, that when melibiose is added as a stabilizing additive to protein formulations, better thermostabilization generally occurs, as can be demonstrated by melting curves and by transition midpoint determinations, and that the formulations are generally stable at higher temperatures and have higher melting points.

[0077] To ascertain the heat stabilizing effect of various excipients over longer periods, small samples are taken after incubation periods of 6, 24, 32, 48 and 72 hours and the structural state and stability of the contained proteins are assessed using fluorescence spectroscopy.

[0078] To determine whether the tested protein-containing sample is still native or damaged, two parameters are now repeatedly monitored by fluorescence spectroscopy during the experimental period. The first parameter refers to the amount of damaged or unfolded protein in the liquid formulation. The second parameter refers to the thermal denaturation midpoint. (However, it is essential for the quality and reliability of the determined data that the deviation of the determined transition temperature is less than 10%. This deviation may be caused by various effects such as aggregation or other influences.)

[0079] Based on the experiments carried out, it has been found that for proteinaceous formulations, a significant improvement in temperature stability can be achieved by the addition of excipients selected according to the present invention. For example, in its native formulation, anthrax PA has an unfolding transition temperature of 48°C, which would not allow storage at or above 50°C. At this temperature, anthrax PA instantly denatures. However, experiments have shown that anthrax PA is stable up to 50°C when the formulation contains a suitable amount of TMAO or choline dihydrogen phosphate. Furthermore, melibiose, maltose, is suitable for stabilizing liquid anthrax PA formulations at 55°C for at least 3 days. When the storage temperature is increased to 60°C during storage, the formulation remains stable for at least 6 hours when formulated with melibiose.

[0080] However, comparable results are not always found in all cases. For example, the evaluation of the thermal stability of CRM197 in liquid formulations is somewhat different from that of the anthrax PA formulation. In its native formulation, CRM197 shows a biphasic unfolding transition when assayed by fluorescence spectroscopy. The initial transition concerns a localized loss of structure, while on a global scale the protein remains structurally intact. This is why different criteria must be applied to study the stability of CRM197. In this case, the conditions for two modifications are mandatory. First, the median unfolding temperature cannot be used as a readout, since conformational recombinations arise due to localized loss of structure, which cannot be stabilized by simply adding selected excipients. In addition, changes in the initial transition may affect the determination of the second unfolding temperature, leading to an erroneous assessment of the overall structural state based thereon. Secondly, the degree of damage done to the temperature-dependent region of CRM affects the fluorescence emission ratio from 350 nm to 330 nm. To evaluate the effect of the excipients examined here, all results are compared with those measured in preparations containing the protein in its native formulation incubated and denatured at about 50° C. Under the same conditions, CRM197 shows unfolding in its native formulation at a temperature of 53° C. By the addition of melibiose or maltose to the corresponding CRM197 preparation, unfolding is largely suppressed for at least 72 hours when the preparation is incubated at 50° C. At this temperature, but also at 55° C., the addition of melibiose has a stabilizing effect for up to 96 hours, while maltose stabilizes CRM197 only for 72 hours. Incubation at 60° C. reverses the ratio. The measured results show that CRM197 is stabilized by maltose at 60° C. for 48 hours, while melibiose stabilizes CRM only for a 24-hour incubation period at 60° C. Furthermore, TMAO, choline dihydrogen phosphate, and lactulose have been shown to be suitable stabilizers for CRM197.When incubated at 50°C, these excipients also stabilize for a period of at least 72 hours, while melibiose and maltose, as previously described, are still stabilized at 55°C and 60°C.

[0081] By adding TMAO, choline dihydrogen phosphate, lactulose, melibiose, and maltose to protein formulations, formulations containing protein at concentrations up to 1.8mg / ml can be heat-stabilized for 3 days.The protein can be a vaccine protein.It can be a protein such as anthrax PA (anthrax protective antigen) or CRM197 or similar proteins.The protein can be a heterologously expressed protein or an overexpressed protein.

[0082] The experiment identifies the best stabilization for liquid protein formulations containing anthrax PA or CRM as protein. The anthrax PA formulation contains 5mM HEPES as buffer, and 50mM NaCl. As stabilizing excipient, melibiose is contained at a concentration of 2.25M, and the pH value of the formulation is 7.5. The CRM formulation contains 20mM HEPES as buffer, and 150mM NaCl. As stabilizing excipient, melibiose is contained at a concentration of 2.4M, and the pH value of this formulation is 8.0.

[0083] As mentioned for the previous experiments and investigations with B. anthracis PA and CRM197, formulations with the enzymes lactate dehydrogenase and lysozyme as model proteins are also examined for thermostabilization. The excipients in Table 1 are added at suitable concentrations and examined for their possible thermostabilizing effects. In this context, further potential thermostabilizers are examined and selected from a group of ionic liquids, which are selected and prepared based on four biodegradable compounds and which can be regularly found in the human body: choline chloride is found in bile, while glucose, sucrose, sorbitol, and glycerol are natural metabolites in the respective sugar or fat pathways. In this regard, the investigations carried out here for the heat stabilization of the vaccine and enzyme proteins have found that, depending on the protein contained in the solution, the concentration of the added excipient must be adjusted to show an effect. It has also been found that the same excipient can show different effects depending on its concentration and on the protein type.

[0084] Surprisingly, based on the results found, it cannot be readily inferred from the fact that a selected excipient belongs to a certain substance class that it is also a good heat stabilizing additive for a specific protein in solution, since the substance in question provides good results with another protein. The outlined results of the study show that the excipients that gave the greatest change in the melting temperature of the tested proteins, and that showed the broadest applicability and were suitable as stabilizers for all three vaccine proteins tested, belong to very different substance groups. These excipients are TMAO, choline dihydrogen phosphate, melibiose, maltose and lactulose.

[0085] a) TMAO exhibits a thermostabilizing effect beginning at a concentration of 250 mM. Optimal stabilizing results are obtained at concentrations ranging from 1 to 4 M in solution. b) Choline dihydrogen phosphate exhibits a thermostabilizing effect beginning with a 1 molar solution. Optimal results are found for compositions where the concentration is in the range of 2-4M. c) Melibiose shows a thermostabilizing effect starting at 250 mM, with optimal results being found for compositions whose concentrations range between 1 and 2.5 M. d) Maltose shows a thermostabilizing effect beginning at 500 mM. Optimal results are found for compositions whose concentrations range between 1 M and 1.8 M. and e) Lactulose shows a heat stabilizing effect beginning at 250 mM. Optimal results are found for compositions whose concentrations range between 0.5M and 1.7M.

[0086] The corresponding results are shown and commented in the "Examples" section below.

[0087] Furthermore, formulations with the enzymes lactate dehydrogenase and lysozyme as model proteins are also investigated here.The above-mentioned excipients are added at suitable concentrations and examined for their possible thermostabilizing effects.Accordingly, experiments to stabilize lactate dehydrogenase have shown that 4-hydroxy-proline, L-ornithine, lactobionic acid and potassium dihydrogen phosphate are good thermostabilizers (meaning that the excipients increase the thermal transition midpoint of LDH by more than 5°C).

[0088] In addition to this, further potential thermostabilizers from the group of four biodegradable ionic liquids, which are ionic liquids, are selected and prepared based on compounds that can be regularly found in the human body: choline chloride is found in bile, while glucose, sucrose, sorbitol, and glycerol are natural metabolites in the respective sugar or fat pathways. The corresponding ionic liquids are prepared in three steps as hydrated deep eutectic mixtures. First, 0.5 moles of choline chloride are mixed with respective amounts of the two sugar components sorbitol and glucose, used in a 0.2 molar amount (ratio 5:2). Glycerol is used in a 0.5 molar amount (ratio 1:1). Sucrose is used in a 0.125 molar amount (ratio 4:1), and 20% w / w H2O is added. Here, studies have shown that choline lactate and hydrated deep eutectic mixtures of choline chloride with sorbitol, glucose, and sucrose are good heat stabilizers for lactate dehydrogenase.

[0089] 4-hydroxyproline, L-ornitine, potassium dihydrogen phosphate, lactobionic acid, and meglumine show the requisite heat stabilizing effect on lactate dehydrogenase (LDH). Excipients identified as suitable for heat stabilizing LDH are also used in experiments for heat stabilizing vaccine proteins. These screening experiments have shown that lactulose and maltose are good heat stabilizers in formulations with vaccine proteins.

[0090] In experiments involving the enzyme lysozyme, choline lactate and hydrated deep eutectic mixtures of choline chloride with sorbitol, glucose, and sucrose, respectively, were identified as good heat stabilizers.

[0091] However, since the thermostabilizing effect of the added excipients was not satisfactory in every case, additional tests were made if the synergistic effect of the two excipients used in combination would improve the thermostabilizing effect of the two molecules. In the following, salts and ionic liquids are combined with each other, as well as with osmolytes and sugar acids. All experiments are carried out with lactate dehydrogenase in the corresponding native formulation (50 mM sodium phosphate, 1 mM DTT (1,4-dithiothreitol), pH 7.6). The protein concentration is adjusted to a concentration of 0.05 mg / ml.

[0092] Here, it is found that potassium dihydrogen phosphate and lactobionic acid in combination induce similar levels of heat stability to LDH, as would be expected from individual components.The results suggest that when corresponding salts and sugar acids are compatible with each other in such protein solutions, their use will result in additive effects.This result is confirmed by the use of potassium dihydrogen phosphate in combination with 4-hydroxyl-proline, which induces similar levels of heat stabilizing effects on LDH.

[0093] The results using a combination of potassium dihydrogen phosphate with a hydrated deep eutectic mixture of choline chloride and sorbitol (5:2 molar ratio) are less clear and can be summarized as follows: The thermal stabilizing effect of salts and ionic liquids is - Compatible and additive when the salt is present in excess compared to the ionic liquid - When the ionic liquid is present in excess compared to the salt, it is incompatible but still thermostabilizing It behaves like this.

[0094] However, the effect of different combinations of lactobionic acid with a hydrated deep eutectic mixture of choline chloride and sorbitol (molar ratio 5:2) on the composition of the model protein lactate dehydrogenase is different. In this context, the following can be concluded about the interaction of ionic liquids with sugar acids in this model composition: - The interaction between ionic liquids and sugar acids is concentration dependent. The interaction between the ionic liquid and the sugar acid also depends on the ratio of ionic liquid to sugar acid. When a sufficiently large excess of ionic liquid is used, a synergistic effect of both excipients on the thermal stability of the protein is observed. The ionic liquid:sugar acid ratio required for such a synergistic effect increases with increasing ionic liquid fraction. - The sugar acid concentration must be a minimum of 0.25M in order to observe the synergistic effect of the two excipients.

[0095] In a model protein composition containing lactate dehydrogenase, the effect of different combinations of a hydrated deep eutectic mixture of choline chloride and sorbitol (5:2 molar ratio) with the amino acid 4-hydroxyl-proline had a synergistic thermostabilizing effect on LDH over the entire range of concentrations and ratios examined, resulting in thermostability 5% to 36% higher than that predicted for the two single components.

[0096] The effects of these combinations on vaccine proteins are similar.

[0097] In this third group of selected excipients, according to the definition, it is found that hydrated deep eutectic mixtures of potassium dihydrogen phosphate, choline dihydrogen phosphate, choline lactate, and choline chloride with sorbitol, glucose, and sucrose, respectively, can be classified as good heat stabilizers under some conditions.

[0098] After evaluation of all tests performed, it is found that:

[0099] 1. There are a number of excipients that can stabilize protein formulations against the deleterious effects of elevated temperatures. a. The stabilizing compound / molecule or excipient belongs to the group selected from 4-OH-proline; L-ornithine; lactobionic acid; potassium dihydrogen phosphate; 2-hydroxyethyl-trimethylammonium L-(+)-lactate; hydrated deep eutectic mixtures of choline chloride and a second component of either sorbitol or glucose or sucrose or glycerol; lactose; lactulose; maltose; melibiose; meglumine; choline dihydrogen phosphate; TMAO; betaine; hydroxylectoine; ectoine; myo-inositol; citrulline; ammonium acetate; magnesium gluconate; mannitol; tris(2-hydroxyethyl)methylammonium methylsulfate.

[0100] b. Particularly efficient heat stabilizers are: 4-OH-proline; L-ornithine; lactobionic acid; potassium dihydrogen phosphate; 2-hydroxyethyl-trimethylammonium L-(+)-lactate; hydrated deep eutectic mixtures of choline chloride with any of the following second components: sorbitol or glucose or sucrose; lactose, lactulose, maltose, melibiose, meglumine, choline dihydrogen phosphate, TMAO, betaine, hydroxylectoin. c. The most efficient and widely applicable excipients are TMAO, choline dihydrogen phosphate, lactulose, maltose, and melibiose.

[0101] 2. The required concentration ranges are different for each excipient and each individual excipient a) stabilize certain proteins; and b) Stabilize certain proteins above 5°C above which the protein is still effective The same is true for range.

[0102] 3. Experiments disclose that each of the excipients listed in Table 4 is capable of stabilizing proteins at temperatures above 60°C. 4. The excipient is compatible with a wide spectrum of buffers used in biology / biochemistry (HEPES, phosphate buffers, histidine buffers, citrate buffers).

[0103] 5. The excipients may be used in the pH range between 6 and 8. 6. The excipients are compatible with reducing agents (i.e., DTT). 7. The excipient is compatible with chloride salts. 8. Experiments show that melibiose and maltose are suitable for inhibiting protein aggregation.

[0104] 9. The excipients tested provide thermal stabilization by stabilizing the tertiary structure of the protein, by stabilizing the molten globule state of the protein, or by inhibiting aggregation of dissolved monomers. [A molten globule is a compact, partially folded protein conformation that has near-native compactness, substantial secondary structure, little detectable tertiary structure, and increased solvent-exposed hydrophobic surface area compared to the native state. (Anthony L Fink, University of California, Santa Cruz, California, USA, published online 2001)]

[0105] 10. The use of a combination of two excipients can have very different effects: a. Salts combined with sugar acids exhibit additive behavior: the resulting T m The change in is similar to the sum of the changes in unfolding temperature induced by the individual components. b. Salts combined with osmolytes exhibit additive behavior: the resulting T m The change in is similar to the sum of the changes in unfolding temperature induced by the individual components. c. Salts combined with osmolytes exhibit additive behavior: the resulting T m The change in is similar to the sum of the changes in unfolding temperature induced by the individual components. d. Salts combined with hydrated deep eutectic mixtures of choline chloride and sorbitol exhibit complex behavior: i. While exhibiting additive behavior, the salt is used in excess of the ionic liquid; ii. still thermally stabilizes, but is less efficient when ionic liquids are used in excess of salt than the sum of the two individual components;

[0106] e. Hydrated deep eutectic mixture of choline chloride and sorbitol combined with lactobionic acid: i. The interaction between ionic liquids and sugar acids is concentration dependent ii. The interaction between the ionic liquid and the sugar acid also depends on the ratio of ionic liquid to sugar acid. iii. When a sufficiently large excess of ionic liquid is used, we observe a synergistic effect of both excipients on the thermal stability of the protein. iv. The ratio of ionic liquid to sugar acid required for such a synergistic effect increases with increasing ionic liquid fraction. v. The sugar acid concentration must be at least 0.25M to observe the synergistic effect of the two excipients. f. Hydrated deep eutectic mixtures of choline chloride and sorbitol in combination with osmolytes / amino acids have a synergistic thermostabilizing effect on LDH leading to thermal stability 5%-36% greater than expected from the effects of the two individual components.

[0107] The results described above, but also the inhibition of aggregation by the addition of melibiose and maltose, were obtained by formulating a group of different proteins in the test solution in order to study the influence of the excipients tested so far. The proteins selected were of bacterial and animal origin. Among these proteins were an enzyme, a pore-forming agent, and two inactivated toxins. One toxin was inactivated by mutation, while the second one was inactivated by formaldehyde cross-linking.

[0108] Because these results have been obtained with such different proteins, it can be inferred from these results that the addition of these selected excipients will produce similar effects in formulations containing soluble proteins as the major component. An alternative conclusion to these results is also permissible: these effects also occur when additional components are covalently bound to the respective protein, for example when the protein is a protein-protein conjugate (e.g. tetanus toxoid), a fusion protein, a protein-polysaccharide conjugate, a protein-nucleic acid conjugate, a protein-DNA conjugate, a protein-RNA conjugate, a protein-dye conjugate, a protein-biotin conjugate or a protein-alum adsorbate.

[0109] As disclosed herein, the preparation of certain protein formulations can be carried out by the methods described above and as shown in the examples below.

[0110] This description, combined with the provided examples, will enable those skilled in the art to comprehensively practice the present invention.

[0111] The examples provided below to illustrate the invention will be commented in detail in relation to the results found in the following sections. These comments also serve to broadly describe the invention in the context of various protein formulations.

[0112] Without further comment, it is therefore assumed that a person skilled in the art will be able to utilize the above description to the broadest extent.

[0113] It is understood that if anything is unclear, the cited publications and patent documents known to those skilled in the art should be consulted, and accordingly, the cited documents are considered part of the disclosure content of this description and are incorporated herein by reference.

[0114] For a better understanding and to illustrate the invention, examples are provided below that are within the scope of protection of the invention, which also serve to illustrate possible variants.

[0115] Furthermore, it will be understood by those skilled in the art that, both in the examples provided and in the remaining description, the amounts of components present in the composition always add up only to 100% by weight or mole%, based on the composition as a whole, and cannot exceed this percentage, even if higher values ​​may result from the percentage ranges indicated and expressed. Unless otherwise stated, % data are therefore % by weight or mole%, except for the proportions indicated in the volume data.

[0116] example Here, model proteins such as lactate dehydrogenase (rabbit muscle) and lysozyme (hen egg, white) and vaccine proteins such as anthrax protective antigen (anthrax PA), tetanus toxoid and diphtheria toxoid (CRM197) are used to demonstrate effective heat stabilization by the addition of one or more of the excipients listed above. Liquid formulations are prepared containing the proteins at concentrations ranging between 0.05 and 1.8 mg / ml. Depicted below are the melting temperatures of these proteins in the presence of the most efficient and most applicable heat stabilizers tested. An increase in the melting temperature of the protein is an indication of increased heat stability of the protein in the formulation.

[0117] material and method Table 1 [Table 1-1] [Table 1-2] [Table 1-3]

[0118] 1) Osmolyte: example TMAO a) The buffer is prepared from buffering agents (sodium dihydrogen phosphate, HEPES, histidine) and adjusted to a pH of 7, 7.5, or 8. b) TMAO is dissolved in a buffer solution at a concentration of 4.2 M and the pH is readjusted to pH 7 or 7.5 or 8. c) Vaccine protein, anthrax protective antigen or CRM197 or tetanus toxoid, is added to the vehicle. Protein concentration is 0.1 mg / ml. Vehicle concentration is 4M. d) The melting temperatures of protein-excipient mixtures are measured by differential scanning fluorimetry and compared to excipient-free protein solutions. e) Results: TMAO The melting temperature of anthrax protective antigen is 20°C. The melting temperature of tetanus toxoid is 3°C. The melting temperature of diphtheria toxoid is 7°C. To increase.

[0119] 2) Ionic liquids: example Choline Dihydrogen Phosphate a) Choline dihydrogen phosphate is dissolved in water at a concentration of 4M and the pH is adjusted to pH 7, 7.5 or 8. b) Vaccine protein, anthrax protective antigen or CRM197 or tetanus toxoid, is added to the vehicle. Protein concentration is 0.1 mg / ml. Vehicle concentration is 3M. c) The melting temperatures of the protein-excipient mixes are measured by differential scanning fluorimetry and compared to excipient-free protein solutions. d) Result: Choline dihydrogen phosphate is The melting temperature of anthrax protective antigen is 19°C. The melting temperature of tetanus toxoid is 5°C. The melting temperature of diphtheria toxoid is 13°C. To increase.

[0120] 3) Salt: example Potassium dihydrogen phosphate a) A buffer solution is prepared containing a buffering agent (sodium dihydrogen phosphate, HEPES, histidine) and the pH is adjusted to a value of 7, 7.5 or 8. b) Potassium dihydrogen phosphate is dissolved in a buffer solution at a concentration of 1.2 M and the pH is readjusted to pH 7 or 7.5 or 8. c) Vaccine protein, anthrax protective antigen or CRM197 or tetanus toxoid, is added to the vehicle. Protein concentration is 0.1 mg / ml. Vehicle concentration is 1M. d) The melting temperatures of the protein-excipient mixes are measured by Differential Scanning Fluorimetry and compared to excipient-free protein solutions. e) Result: Potassium dihydrogen phosphate is The melting temperature of anthrax protective antigen is 10°C. The melting temperature of tetanus toxoid is 2°C. The melting temperature of diphtheria toxoid is 5°C. To increase.

[0121] 4) Sugar: example Lactulose a) The buffer is prepared by mixing buffering agents (sodium dihydrogen phosphate, HEPES, histidine) and adjusting the pH to 7, 7.5, or 8. b) Lactulose is dissolved in a buffer solution at a concentration of 2M and the pH is readjusted to pH 7 or 7.5 or 8. c) Vaccine protein, anthrax protective antigen or CRM197 or tetanus toxoid, is added to the vehicle. Protein concentration is 0.1 mg / ml. Vehicle concentration is 1.7 M. d) The melting temperatures of the protein-excipient mixes are measured by differential scanning fluorimetry and compared to excipient-free protein solutions. e) Result: Lactulose is The melting temperature of anthrax protective antigen is 18°C. The melting temperature of tetanus toxoid is greater than 20° C. (The equipment does not allow accurate measurement of Tm at such high temperatures) The melting temperature of diphtheria toxoid is 24°C. To increase.

[0122] Additional Results : Melibios Prevention of aggregation by (Figures 28 and 29) Melibiose is dissolved in a buffer solution at a concentration of 2.75 M and the pH is adjusted to 7, 7.5 or 8. The vaccine protein suspension of anthrax protective antigen or CRM197 or tetanus toxoid is added to the excipient solution. The protein concentration is 0.1 mg / ml. The excipient concentration is 2M or 2.4M or 2.5M.

[0123] result: Diphtheria toxoid aggregation is inhibited at temperatures up to 80° C. when 2M or 2.4M excipients are used. No aggregation is observed when 2.5M excipients are used. Tetanus toxoid aggregation is inhibited when 2.4M or 2.5M excipients are used.

[0124] 5) Sugar acids and their salts: Example: Lactobionic acid a) The buffer is prepared by mixing buffering agents (HEPES, histidine) and adjusting the pH to 7 or 7.5. b) Lactobionic acid is dissolved in a buffer solution at a concentration of 0.6 M and the pH is readjusted to pH 7 or 7.5. c) Vaccine protein, anthrax protective antigen or tetanus toxoid is added to the vehicle. Protein concentration is 0.1 mg / ml. Vehicle concentration is 0.5M. d) The melting temperatures of the protein-excipient mixes are measured by differential scanning fluorimetry and compared to excipient-free protein solutions. e) Result: Lactobionic acid is The melting temperature of anthrax protective antigen is 7°C. The melting temperature of tetanus toxoid is 4°C. Increase.

[0125] 6) Amino sugars: example Meglumine a) Buffer solutions are prepared by mixing buffering agents (sodium phosphate or sodium citrate) and adjusting the pH to 6 or 7.6. b) Meglumine is dissolved in a buffer solution at a concentration of 3M and the pH is readjusted to pH 6 or 7.6 [if the pH is 7.6, 1 mM DTT (1,4-dithiothreitol) is added]. c) A protein, lactate dehydrogenase or lysozyme, is added to the excipient solution. The protein concentration is 0.1 mg / ml. The excipient concentration is 1.5 M. d) The melting temperatures of the protein-excipient mixes are measured by Differential Scanning Fluorimetry and compared to excipient-free protein solutions. e) Results: Meglumine The melting temperature of lactate dehydrogenase is increased by 3°C. The melting temperature of lysozyme is 6℃. To increase.

[0126] 7) Amino alcohols: e.g. mannitol a) A buffer solution is prepared consisting of a buffering agent (sodium phosphate) and adjusted to a pH of 6 or 7.6. b) Mannitol is dissolved in a buffer solution at a concentration of 0.5 M, the pH is readjusted to pH 7.6, and 1 mM DTT is added. c) The protein, lactate dehydrogenase, is added to the excipient solution in an amount that results in a protein concentration of 0.1 mg / ml. The excipient concentration is 0.45M. d) The melting temperatures of the protein-excipient mixes are measured by Differential Scanning Fluorimetry and compared to excipient-free protein solutions. e) Result: Mannitol is The melting temperature of lactate dehydrogenase is 2°C. To increase.

[0127] Figure 1 presents a summary of the results of the added excipients that show the best performance. This means that the results outline the added excipients that gave the largest change in the melting temperature of the tested proteins and that show the widest applicability. These excipients are suitable as stabilizers for all three vaccine proteins tested. Surprisingly, it has been found that these are the following excipients that themselves belong to very different substance groups: f) TMAO exhibits a thermostabilizing effect beginning at a concentration of 250 mM. Optimal stabilizing results are obtained at concentrations ranging from 1 to 4 M in solution. g) Choline dihydrogen phosphate exhibits a thermostabilizing effect beginning with a 1 molar solution. Optimal results are found for compositions where the concentration is in the range of 2-4M. h) Melibiose shows a thermostabilizing effect starting at 250 mM, with optimal results being found for compositions whose concentrations range between 1 and 2.5 M. i) Maltose shows a thermostabilizing effect starting at 500 mM. Optimal results are found for compositions whose concentrations range between 1 M and 1.8 M. j) Lactulose shows a heat stabilizing effect starting at 250 mM. Optimal results are found for compositions whose concentrations range between 0.5M and 1.7M.

[0128] Figure 2 presents a summary of the results on the change in melting temperature conferred by each excipient under optimal conditions:

[0129] Experimental Setup and Considerations To evaluate which additives or excipients are optimal for heat stabilization and in what amounts they must be added to produce the desired effect, test formulations with anthrax PA as the vaccine were prepared and tested over a 3-day period. The detailed formulation composition is outlined in Table 2 at the end of this chapter. The purpose of these experiments is to find the most suitable excipient or mixture of excipients for heat stabilization of protein compositions over several days.

[0130] To date, such compositions cannot be used to prepare pre-filled syringes because the stabilizer must be used at high concentrations, and patients will likely not tolerate the stabilizer. A dilution step is therefore required prior to administration to patients. Another focus of these experiments is also to determine the highest possible tolerated level of stabilizer for use in in vitro assays. Based on the results of the experiments, protein concentration can be adjusted and vaccine stock solutions can be produced, both of which can provide heat protection for solute proteins, but can still be effectively administered to patients after dilution.

[0131] For example, it is found that melibiose can be tolerated by patients up to a concentration of 250 mM with a very high probability. From previous screening, it is known that at least 2.25 M melibiose is essential to stabilize anthrax PA at high temperatures. Therefore, the protein concentration is increased by a factor of 2250 mM (stock) / 250 mM (final)=9. Thus, at least 9 times the amount of vaccine stock formulation is required.

[0132] Following these considerations, test formulations are prepared for the study of TMAO, choline dihydrogen phosphate, lactulose, maltose and melibiose as thermostabilizing excipients. Fluorescence spectroscopy is used to confirm whether all protein samples are natively folded and that the selected excipients are also capable of thermostabilizing elevated protein concentrations (up to 1.6 mg / ml). Incubations are then carried out at the following temperatures: 4° C.: Native (no excipients): These are unchanged control samples. 50°C: Native (no excipients), TMAO, choline dihydrogen phosphate, lactulose, maltose 55℃: TMAO, choline dihydrogen phosphate, lactulose, melibiose, maltose 60℃: TMAO, choline dihydrogen phosphate, lactulose, melibiose, maltose 65℃: Melibiose

[0133] Melibiose is tested at a higher temperature than all other excipients since it induces a higher thermal stability based on the transition midpoint.

[0134] After incubation periods of 6, 24, 32, 48 and 72 hours, small aliquots are removed and the structural state and stability of the contained proteins are assessed using fluorescence spectroscopy.

[0135] Table 2 : Overview of test preparations used herein. JPEG2024174888000004.jpg124163

[0136] Analysis parameters The results provided in the following sections are based on two parameters determined by fluorescence spectroscopy. The changes in these two parameters are monitored over time to determine if the anthracnose PA is still native or if it has been damaged. Both readouts described below are essential to determine if the protein involved remains stable throughout the duration of the experiment.

[0137] Anthracnose PA stabilized at temperatures above 50°C In its native formulation, anthrax PA has an unfolding transition temperature of 48° C., which would not allow storage of the protein at or above 50° C. Figure 3 shows the storage stability of anthrax PA at 50° C., assayed by fluorescence spectroscopy.

[0138] Figure 3 : Stability of anthracnose PA at 50°C. Figure 3a) (Left) Folded fraction. Natively formulated B. anthracis PA shows an immediate decay in the natively folded protein. All heat stabilizers tested prevent such decay. Figure 3b) (Right) Normalized transition temperatures. Data between the red lines indicate that the protein is stable.

[0139] Without the heat stabilizing excipients, the anthracnose PA would denature immediately. All selected excipients stabilized and allowed storage at 50° C. for 72 hours.

[0140] Figure 4 Figure 2 shows the storage stability of anthracnose PA at 55°C. Figure 4 : Stability of anthracnose PA at 55°C. Figure 4a) (Left) Folded fraction. Figure 4b) (Right) Normalized transition temperatures. Data between the red lines indicate that the protein is stable.

[0141] When the test formulations are incubated at 55° C., it is found that both melibiose and maltose are capable of inducing sufficient heat stabilization to allow storage at 55° C. for a period of 3 days.

[0142] In addition, the anthracnose PA was incubated at 60°C for 3 days. The results mentioned were: Figure 5 As shown in.

[0143] Figure 5: Stability of anthracnose PA at 60°C. Figure 5a) (Left) Folded fraction. Data below the red line indicate stable protein. Figure 5b) (right) Normalized transition temperatures. Data between the red lines indicate that the protein is stable.

[0144] At 60° C., the heat stabilizing capacity of the excipients used herein is terminated. Only melibiose can tolerate short exposure to 60° C. for more than 6 hours but less than 24 hours.

[0145] CRM197 stabilized at temperatures above 50°C In its native formulation, CRM197 shows a biphasic unfolding transition when assayed by fluorescence spectroscopy. The initial transition involves a localized loss of structure, while on a global scale the protein remains structurally intact. While the excipients used herein have an effect on the overall structural stability of CRM, they cannot prevent the more localized loss of structure. Therefore, the same criteria cannot be used for anthrax PA when studying the stability properties of CRM.

[0146] For data interpretation, two modifications are essential: first, the median unfolding temperature cannot be used as a readout. It has already been established that conformational recombination occurs due to local loss of structure, which cannot be stabilized by the excipients used here. Furthermore, changes in this initial transition may affect the determination of the second unfolding temperature, leading to a false assessment of the overall structural state based thereon. Secondly, the degree of damage inflicted in the thermolabile regions of the CRM affects the fluorescence emission ratio from 350 nm to 330 nm. Thus, the degree of structural damage may be an upper limit, rather than the actual degree of structure loss. To evaluate the effect of the excipients tested here, all results are compared to the protein in its native formulation, incubated and denatured at 50°C.

[0147] In its native formulation, CRM197 has a global unfolding temperature of 53° C., which does not allow for long-term storage at 50° C. Therefore, the negative control will be natively formulated CRM incubated at 50° C. In FIG. 6, the effect of melibiose and maltose is shown on the thermal stability of CRM.

[0148] Figure 6 Stability of CRM197 at 50°C. Melibiose and maltose clearly inhibit thermally induced unfolding for 3-4 days. The results show that maltose stabilizes CRM197 for 72 hours at 50° C. Melibiose stabilizes CRM for 96 hours at 50° C.

[0149] Figure 7 shows the storage stability of CRM197 at 55°C. Figure 7: Stability of CRM197 at 55°C. Melibiose and maltose clearly inhibit thermally induced unfolding for 3-4 days. The results show that maltose stabilizes CRM197 for 72 hours at 55° C. Melibiose stabilizes CRM for 96 hours at 55° C.

[0150] Figure 8 The figures show the storage stability of CRM197 at 60°C. Figure 8 : Stability of CRM197 at 60°C. Melibiose and maltose clearly inhibit thermally induced unfolding for 3-4 days.

[0151] While the 6 hour folded fraction is noticeable to be greater at 60° C. compared to 55° C. or 50° C., there are still clear differences for natively formulated and fully denatured CRM. The results of these studies show that maltose stabilizes CRM197 at 60° C. for 48 hours. Melibiose stabilizes CRM at 60° C. for 24 hours.

[0152] Further experiments for thermostabilizing enzymes as proteins As described for previous experiments and investigations with B. anthracis PA and CRM197, formulations with the enzymes lactate dehydrogenase and lysozyme as model proteins are also investigated here. The above mentioned excipients are added at suitable concentrations and examined for their possible heat stabilizing effect.

[0153] Preparation of ionic liquids based on choline chloride Additional potential thermal stabilizers from the group of four biodegradable ionic liquids are selected and prepared based on compounds that can be found regularly in the human body: choline chloride is found in bile, while glucose, sucrose, sorbitol, and glycerol are natural metabolites in the respective sugar or fat pathways.

[0154] The ionic liquids are prepared as hydrated deep eutectic mixtures in three steps. First, 0.5 moles of choline chloride are mixed with the respective amounts of the two sugar components sorbitol and glucose, used in an amount of 0.2 moles (ratio 5:2). Glycerol is used in an amount of 0.5 moles (ratio 1:1). Sucrose is used in an amount of 0.125 moles (ratio 4:1). The prepared mixture is then heated to 100° C. for 2 hours with stirring. These specific mixtures begin to melt, while no substances other than glycerol and sorbitol would take on a liquid form at this temperature. Subsequently, 20% w / w is added to the mixture, and when the solid substances are completely melted, the ionic liquids produced are cooled to room temperature where they maintain their liquid form.

[0155] To compare the amount of material used for heat stabilization with ionic liquids prepared in this manner to other excipients that are simply dissolved in water / buffer, the volume of the resulting liquid is determined and the concentration of choline chloride therein is calculated as if it were dissolved. Typically, about 100-150 ml of ionic liquid is prepared to represent a molar concentration of 3.3-4 M choline chloride.

[0156] b. Experimental approach As described previously, the unfolding transition midpoints of the proteins involved were determined by fluorescence spectroscopy, meaning that nano-differential scanning fluorimetry (nanoDSF) allows for high-throughput screening.

[0157] Here, proteins are used at concentrations of 0.05-0.1 mg / ml, while excipients are titrated to concentrations close to their solubility in the applied buffer solution. An appropriate amount of protein solution is then mixed with an appropriate amount of excipient solution, and the mixture is allowed to equilibrate at room temperature for 15 minutes.

[0158] The prepared protein solution is heated at a heating rate of 1 °C / min and the ratio of fluorescence emission at 350 / 330 nm is recorded and plotted against temperature. Typically, the resulting curve resembles a Boltzmann function, and the inflection point of this curve corresponds to the mean unfolding temperature, T m Represents.

[0159] From this curve, T m The change in m賦形剤 From T mネイティブ This is determined by subtracting dT m produces.

[0160] By definition, dT >5°C m Excipients that lead to dT are good stabilizers. m If dT is < 5°C and > 0°C, the excipient is defined as a poor stabilizer. m identifies the molecule as a protein destabilizing agent.

[0161] c. Results using lactate dehydrogenase as a model enzyme Lactate dehydrogenase (LDH) is formulated in 50 mM sodium phosphate, 1 mM DTT at pH 7.6. To explore the effect of molecules on LDH, the molecules are added at various concentrations to the protein formulated in exactly this buffer. The protein is used at a concentration of 0.05 mg / ml.

[0162] Figure 9: shows the effect of excipient candidates on the mean unfolding temperature of LDH. The black line indicates the dT m = 0℃. The red line indicates dT m= 5°C. Molecules that are able to stabilize LDH by more than 5°C are subjected to a second screen with a different model protein.

[0163] Figure 9 presents a first set of excipients from what is at this stage a very diverse group of compounds and their effect on the melting temperature of LDH. 4-Hydroxy-proline, ornithine, lactobionic acid and potassium dihydrogen phosphate are, by definition, good heat stabilizers, whereas others, such as ectoine, myo-inositol and citrulline, inhibit the T of LDH. m Due to its relatively small effect on , it is not suitable for the purposes examined.

[0164] Figure 10 is the change in melting temperature of LDH, dT m and this is determined with the addition of Na-Ser, OH-Lys, Choline Lactate, Choline Cl / Sorbitol, Choline Cl / Glycerol, Choline Cl / Glucose, Choline Cl / Sucrose.

[0165] Figure 10: Effect of excipient candidates on the mean unfolding temperature of LDH. The black line indicates the dT m = 0℃. The red line indicates dT m = 5°C. Molecules suitable for stabilizing LDH by more than 5°C are subjected to a second screen with different model proteins. In order to use the same plots for the hydrated deep eutectic liquids as for the other excipients, the molar concentration of choline chloride was estimated based on the weight of material used and the volume of liquid prepared.

[0166] In this set of excipients, hydrated deep eutectic mixtures of choline lactate and choline dihydrogen phosphate with sorbitol, glucose, and sucrose were identified as good thermal stabilizers and advanced to the second prescreening step.

[0167] In the next study, the effect of lactulose, magnesium gluconate, polysucrose 400, mannitol, maltose and ammonium acetate on the thermal stability of LDH was examined. These excipients were identified as suitable for thermally stabilizing the LDH herein. Figure 11: shows the effect of lactulose, magnesium gluconate, polysucrose 400, mannitol, maltose and ammonium acetate on the mean unfolding temperature of LDH.

[0168] The black line is dT m = 0℃. The red line indicates dT m = 5°C. Molecules that are able to stabilize LDH by more than 5°C are subjected to a second screen with a different model protein.

[0169] Lactulose and maltose in particular have been identified as good heat stabilizers. Ammonium acetate, magnesium gluconate, and mannitol have also been identified as protein-stabilizers.

[0170] d. Results using lysozyme as a model enzyme. Lysozyme is formulated in 20 mM Na-citrate, 55 mM NaCl, 1 mM DTT, pH 6.0. To explore the effect of molecules on lysozyme, the molecules are added at various concentrations to the protein formulated in exactly this buffer. Protein is used at a concentration of 0.1 mg / ml.

[0171] Figure 12: Effect of hydroxy-proline, L-citrulline, L-ornitine, potassium dihydrogen phosphate, lactobionic acid, meglumine, ectoine, myo-inositol, and methylsulfonate on the mean unfolding temperature of lysozyme. Black lines indicate dT m = 0℃. The red line indicates dT m =5℃.

[0172] From this set of molecules, hydroxyproline, L-ornitine, potassium dihydrogen phosphate, lactobionic acid, and meglumine show the required heat stabilizing effect. Ectoine, tris(2-hydroxyethyl)methylammonium methylsulfate (MeOSO3), and citrulline do not sufficiently stabilize lysozyme.

[0173] Figure 13 shows the results for a second group of substances tested in the lysozyme:ionic liquid system. Figure 13: The effect of excipient candidates on the mean unfolding temperature of LYZ. The black line indicates the dT m = 0℃. The red line indicates dT m = 5°C. Molecules that can stabilize LYZ by more than 5°C are identified as good thermal stabilizers according to the definitions mentioned previously. In order to use the same type of plot for the hydrated deep eutectic liquid as for the other excipients, the molar concentration of choline chloride is estimated based on the weight of material used and the volume of liquid prepared.

[0174] Of the excipient candidates tested here, choline lactate and a hydrated deep eutectic mixture of choline dihydrogen phosphate with sorbitol, glucose, and sucrose were identified as good heat stabilizers.

[0175] A summary of the concentration ranges used for each excipient that stabilized at least one model protein at or above 5° C. can be found in Table 3. Typically, molar excipient concentrations are given. For ionic liquids, the volume percent of excipient used is given, as the molar concentration of only one component would be misleading.

[0176] [Table 3]

[0177] Results showing the efficiency of excipient combinations with lactate dehydrogenase For initial feasibility assessment, the compatibility of various excipients combined with each other is examined. The goal is to ascertain whether the synergistic effect of the two excipients used in combination will improve the thermostabilizing effect of the two molecules. Salts and ionic liquids are combined with each other and with osmolytes and sugar acids. All experiments are carried out with lactate dehydrogenase in the corresponding native formulation (50 mM sodium phosphate, 1 mM DTT, pH 7.6). The protein concentration is adjusted to a concentration of 0.05 mg / ml. LDH is added to the solution containing the two excipients in various ratios and concentrations. The prepared compositions are incubated at room temperature for 15 minutes. The mean unfolding temperature of LDH is then measured at a heating rate of 1° C. / min using nano differential scanning fluorimetry as described above.

[0178] Figure 14 summarizes the results for the combination of potassium dihydrogen phosphate with lactobionic acid. Figure 14: Effect of various combinations of potassium dihydrogen phosphate and lactobionic acid on the model protein lactate dehydrogenase - blue text indicates T of the combined formulations m Stacked in black and red are the T determined separately for each excipient. m If the blue bar outweighs the stacked black and red bars, a synergistic effect would be detected.

[0179] In combination, potassium dihydrogen phosphate and lactobionic acid are found to induce a similar level of heat stabilization to LDH, as would be expected from the individual components. In relative terms, when using this combination, a heat stabilization efficacy of between 90% and 104% can be observed.

[0180] This leads to the conclusion that the corresponding salts and sugar acids are compatible in such protein solutions, leading to an additive effect.

[0181] Figure 15outlines the results for the combination of potassium dihydrogen phosphate and 4-OH-proline. Figure 15: The effect of various combinations of potassium dihydrogen phosphate and lactobionic acid on the model protein lactate dehydrogenase. The blue letters indicate the T of the combined formulations. m Stacked in black and red are the T determined separately for each excipient. m If the blue bar outweighs the stacked black and red bars, one would have to conclude a synergistic effect.

[0182] In combination, potassium dihydrogen phosphate and 4-OH-proline are found to induce a similar level of heat stabilizing effect on LDH as would be expected when the individual components are added. In relative terms, when these compounds are used in combination, a heat stabilizing efficacy in the range of between 95% and 100% can be observed.

[0183] It is therefore concluded that the thermostabilizing effects of salts and osmolytes are compatible and act in an additive manner.

[0184] Figure 16 summarizes the results for the combination of potassium dihydrogen phosphate and choline chloride / sorbitol. Figure 16: The effect of various combinations of potassium dihydrogen phosphate and a hydrated deep eutectic mixture of choline chloride and sorbitol (5:2 molar ratio) on the model protein lactate dehydrogenase. The blue text indicates the T of the combined formulations. m Stacked in black and red are the T determined separately for each excipient. m If the blue bar outweighs the stacked black and red bars, one would have to conclude a synergistic effect.

[0185] Here, it is observed that the combination of potassium dihydrogen phosphate with a hydrated deep eutectic mixture of choline chloride and sorbitol (molar ratio 5 / 2) is incompatible when the latter is used in excess compared to the former. With an excess of ionic liquid compared to the salt, an effectiveness of 58% to 81% is found. However, when the salt is added in excess of the ionic liquid, the thermal stabilization effect of both is additive, in the range of 99-102%.

[0186] As a result, the thermal stabilizing effect of salts and ionic liquids is: - When the salt is present in excess compared to the ionic liquid, it is compatible and additive. - When the ionic liquid is present in excess compared to the salt, it is incompatible but still thermostabilizing It behaves like this.

[0187] Figure 17 summarizes the results of the addition of a combination of choline Cl / sorbitol and lactobionic acid. These results are more complex than the data for previously examined combinations. Figure 17: The effect of various combinations of lactobionic acid with a hydrated deep eutectic mixture of choline chloride and sorbitol (molar ratio 5:2) in the formulation of the model protein lactate dehydrogenase. The blue text indicates the T of the combined formulations. m Stacked in black and red are the T determined separately for each excipient. m If the blue bar outweighs the stacked black and red bars, one would have to conclude a synergistic effect.

[0188] Different interactions are observed depending on the concentration of ionic liquid involved: when only low concentrations of ionic liquid are used (0.5 M) and lactobionic acid is added in a 2:1 ratio, a synergistic effect is obtained and the model protein is stabilized more than the individual components.

[0189] With 1.5 molar ionic liquid, a similar effect is observed, but under these conditions a larger excess of ionic liquid is necessary to produce a synergistic effect. When the excess of ionic liquid is increased 6-fold, synergistic stabilization is found. At very high concentrations (2.5M) of ionic liquid, only the effect of a small concentration of lactobionic acid can be explored. Using a 10-fold excess of ionic liquid over lactobionic acid leads to a synergistic effect of both. However, when only 0.15M lactobionic acid (16:1) is used, an additive effect is likely to be observed.

[0190] Regarding the interaction of ionic liquids with sugar acids in the model compositions, the following can be concluded: - The interaction between ionic liquids and sugar acids is concentration dependent - The interaction between ionic liquids and sugar acids also depends on the ratio of ionic liquid to sugar acid. - When a sufficiently large excess of ionic liquid is used, we observe a synergistic effect of both excipients on the thermal stability of the protein. - The ionic liquid:sugar acid ratio required for such synergistic effect increases with increasing ionic liquid fraction. - The sugar acid concentration must be a minimum of 0.25M in order to observe the synergistic effect of the two excipients.

[0191] Figure 18 outlines the results of the combination of choline chloride / sorbitol and 4-OH-proline. Figure 18: Effect of various combinations of hydrated deep eutectic mixtures of choline chloride and sorbitol (molar ratio 5:2) with 4-hydroxyl-proline in a model protein composition containing lactate dehydrogenase. Blue text indicates T of the combined formulations. m Stacked in black and red are the T determined separately for each excipient. m If the blue bar outweighs the stacked black and red bars, one would have to conclude a synergistic effect.

[0192] The measured results show that the combination of ionic liquids with amino acids over the full range of concentrations and ratios tested has a synergistic thermostabilizing effect on LDH, causing 5% to 36% higher thermostability than would be expected from the two single components.

[0193] A summary of the excipient combinations used, their concentration ranges and their effect on LDH can be found in Table 4 below. Table 4: JPEG2024174888000006.jpg242115

[0194] Screening for heat-stable excipients using vaccine proteins In the following sections, the results of screening excipient molecules with three very different vaccine proteins are discussed. Not all potential excipient molecules are tested for all vaccine proteins. The goal is to identify excipient molecules that can increase the average unfolding temperature of three very different proteins to above 60° C.

[0195] Experimental approach We employ fluorescence spectroscopy to determine the unfolding transition midpoint of our proteins. Specifically, we use a nano-differential scanning fluorimetric method that allows for high-throughput screening.

[0196] Proteins are used at a concentration of 0.1 mg / ml, which corresponds well to the protein concentration in commercial vaccine doses. Excipients are titrated to concentrations close to their solubility limits. All excipients are prepared in the vaccine protein's native formulation buffer, with the exception of choline dihydrogen phosphate. The latter is dissolved in water and the pH adjusted to match that of the native vaccine protein formulation (see below for details). In this case, dihydrogen phosphate ions will act as a buffering agent. The protein is mixed with the excipients and allowed to equilibrate for 15 min at room temperature.

[0197] Screening for heat-stable excipients using anthrax protective antigen. Anthrax protective antigen (ANTHRA PA) is the major protein component of the anthrax vaccine. Its native formulation is 5 mM HEPES with 50 mM NaCl at a pH of 7.5. The native mean unfolding temperature is 48°C. Three classes of excipients are used in the study to stabilize the anthrax PA: sugars, ionic liquids / salts, and osmolytes. The results obtained are compared with those using trehalose, an excipient widely used for various purposes.

[0198] Figure 19 shows results for heat stabilizing anthracnose PA with sugar. Figure 19: The effect of sugars as potential excipients on the mean unfolding temperature of B. anthracis PA. The black line indicates and represents dTm = 0 °C. The red line indicates and represents dT m = 5°C. Molecules that can stabilize anthrax PA by more than 5°C are subjected to a second screen with a different model protein.

[0199] In this first group, lactulose, melibiose, and maltose were identified as good thermostabilizers and will be used for further evaluation. Already at a relatively low concentration (250 mM), the requisite additional thermostability of 5°C is observed. At almost 30°C, melibiose reduces the T of P. anthracnose PA. mAll sugars tested in this group are more efficient heat stabilizers than trehalose.

[0200] Figure 20 shows results for heat stabilizing anthracnose PA using osmolytes Figure 20: The effect of osmolytes as potential excipients on the mean unfolding temperature of B. anthracis PA. The black line indicates the dT m = 0℃. The red line indicates dT m = 5°C. Molecules that can stabilize anthrax PA by more than 5°C are subjected to a second screen with a different model protein.

[0201] In this second group, there are some known protein stabilizers, such as taurine, which cannot stabilize the vaccine protein used herein. Among the osmolytes, TMAO, betaine, 4-hydroxyl-proline, hydroxyectoine, and lactobionic acid are found to be good heat stabilizers according to this definition, and are therefore selected for further examination. However, only TMAO outperforms trehalose as a heat stabilizer.

[0202] FIG. 21 shows the results for heat stabilizing anthracnose PA using salts and ionic liquids. Figure 21: The effect of excipient candidates on the mean unfolding temperature of B. anthracis PA. The black line indicates the dT m = 0℃. The red line indicates dT m = 5°C. Molecules that are able to stabilize B. anthracis PA by more than 5°C are subjected to a second screening with a different model protein. In order to use the same type of plot for the hydrated deep eutectic liquid as for the other excipients, the molar concentration of choline chloride was estimated based on the weight of material used and the volume of liquid prepared.

[0203] In this third group of molecules, according to the above definitions, it is found that hydrated deep eutectic mixtures of potassium dihydrogen phosphate, choline dihydrogen phosphate, choline lactate, and choline chloride with sorbitol, glucose, and sucrose, respectively, can be classified as good heat stabilizers under some conditions.

[0204] Screening for heat-stabilizing excipients using tetanus toxoid. Tetanus toxoid (T d ) is an inactive form of Clostridium tetanii toxin and is prepared by formaldehyde cross-linking of the toxin monomer. The formaldehyde cross-linked product is a large protein complex that is no longer toxic but still provides an antigenic structure for recognition by the immune system. Its native formulation is 100 mM histidine with 100 mM NaCl at pH 7.0. The native mean unfolding temperature is 67°C.

[0205] The three classes of excipients are d used in an effort to stabilize the glycerol: sugars, ionic liquids / salts, and osmolytes are compared to the results obtained with trehalose, a widely used excipient for various purposes.

[0206] Fig. 22 is a heat-stabilized T d The results for are shown below. Figure 22: T d The effect of sugars as potential excipients on the mean unfolding temperature of dT m = 0℃. The red line indicates dT m = 5℃. T d Molecules that are able to stabilize the α-terminal end of the β-terminal tail by more than 5° C. are subjected to a second screen with a different model protein.

[0207] In this first group, lactulose, melibiose, and maltose are identified as good heat stabilizers. The highest temperature line indicates that the complete unfolding transition could not be observed for the affected excipient concentrations because the instrument could not heat the sample any further and the unfolding transition was incomplete. As in the experiments with anthrax PA discussed previously, lactulose, melibiose, and maltose are more efficient heat stabilizers than trehalose.

[0208] Figure 23 The heat-stabilized T d The results for are shown below. Figure 23: T d The effect of osmolytes as potential excipients on the mean unfolding temperature of dTm = 0 °C. The black line indicates and represents dT m = 5℃. T d Molecules that are able to stabilize the α-terminal tail by more than 5° C. are subjected to a second screen with a different model protein.

[0209] In this second group of excipients we observe that only hydroxyectoine can be identified as a good heat stabilizer, it is also the only substance tested in this group that outperforms trehalose.

[0210] Fig. 24 The thermally stabilized T d The results for are shown below. Figure 24: T d The effect of excipient candidates on the mean unfolding temperature of dT m = 0℃. The red line indicates dT m = 5℃. T d Molecules that can stabilize >5° C. are subjected to a second screen with a different model protein. In order to use the same type of plot for the hydrated deep eutectic liquid as for the other excipients, the molar concentration of choline chloride was estimated based on the weight of material used and the volume of liquid prepared.

[0211] In this third group of excipients, choline dihydrogen phosphate is observed to be the most efficient heat stabilizer in this group and also more efficient than trehalose. The hydrated deep eutectic mixtures of choline chloride with sorbitol, glucose, and sucrose, respectively, can be classified as good heat stabilizers.

[0212] Screening for heat-stable excipients using diphtheria toxoid CRM197. Diphtheria toxoid (CRM197) is an inactive form of the corynebacterial diphtheria toxin. The introduction of point mutations renders the toxin harmless but still provides an antigenic structure for recognition by the immune system. Its native formulation is 20 mM HEPES with 150 mM NaCl at pH 8.0. The tertiary structure unfolding of CRM197, the type of unfolding monitored using fluorescence spectroscopy, is rather complex: it unfolds in a two-phase transition with the first transition representing local conformational changes, while the second transition represents a global unfolding of the protein. As no temperature can be specifically related to the ability of CRM197 to induce immunity, both transitions are analyzed with regard to the effectiveness of our excipients. Also here, three classes of excipients are used: sugars, ionic liquids / salts, and osmolytes, and are compared with the results obtained with trehalose, an excipient widely used for various purposes.

[0213] Fig. 25 shows results for the heat stabilization of CRM with sugars. Figure 25: The effect of sugars as potential excipients on the mean unfolding temperature of CRMs. The red line indicates the dT m = 5°C. Molecules that can stabilize the CRM locally (closed symbols) and globally (open symbols) by more than 5°C will be considered as excipient candidates and, if required, screened with various vaccine proteins.

[0214] In this first group, lactulose, melibiose, and maltose are identified as being good heat stabilizers that outperform trehalose.

[0215] Fig. 26 shows the results for the thermostabilization of CRM using osmolytes. Figure 26: Effect of osmolytes as potential excipients on the mean unfolding temperature of CRMs. The black line indicates the dT m = 0℃. The red line indicates dT m = 5°C. Molecules that can stabilize CRM locally (closed symbol) and globally (open symbol) by more than 5°C are considered as potential excipients.

[0216] It is observed that in this second group of excipients, only TMAO can be identified as a good thermal stabilizer.

[0217] Fig. 27 presents results on the thermal stabilization of CRM using ionic liquids. Figure 27: Effect of excipient candidates on the mean unfolding temperature of CRMs. The black line indicates the dT m = 0℃. The red line indicates dT m = 5°C. Molecules that can stabilize CRM by more than 5°C are subjected to a second screen with a different model protein. In order to use the same type of plot for the hydrated deep eutectic liquid as for the other excipients, the molar concentration of choline chloride was estimated based on the weight of material used and the volume of liquid prepared.

[0218] It is observed that in this third group of excipients, the local unfolding transition (Tm1) is not stabilized very much, but choline dihydrogen phosphate can be classified as a good thermostabilizer. Besides choline dihydrogen phosphate, all other choline-based ionic liquids thermostabilize CRM more efficiently than trehalose. However, choline chloride-based ionic liquids have a substantial destabilizing effect on the first unfolding transition of CRM. This is probably caused by the high amount of charge present in these ionic liquids.

[0219] Assessment of excipient effects on colloidal stability of vaccine proteins After the five molecules have been identified as potential excipients for inducing stability in proteins at high temperatures, these molecules are subjected to a more detailed evaluation. The key aspect here is to determine whether the selected excipient has any effect on the colloidal stability of the protein. In other words, it is necessary to determine whether the protein shows a change in the tendency to aggregate in the presence of the excipient. If aggregation is prevented, the respective excipient will show an additional benefit.

[0220] Dynamic light scattering is used to evaluate the aggregation behavior of proteins. The hydrodynamic radius of the protein is measured, and this value can be used to evaluate whether the protein has a monomeric or aggregated state. The five selected excipients are added to a solution containing the vaccine protein at a concentration of 0.1 mg / ml. As in the previous section, each vaccine protein is formulated in its native formulation, and each excipient is prepared in the corresponding native formulation of the vaccine protein. Also, in this set of experiments, choline dihydrogen phosphate is prepared without any buffer components, but the pH of the excipient stock solution is adjusted to match the pH of the vaccine protein formulation.

[0221] The vaccine protein and excipients are mixed together to produce a test formulation with 0.1 mg / ml vaccine protein content and the desired concentrations of excipients (details given below).

[0222] To assess the colloidal stability of vaccine proteins at elevated temperatures, samples are loaded into 316-well plates that are placed into a Dynapro Plate Reader III (Wyatt Technology). The hydrodynamic radius R of the vaccine protein is h R is determined at room temperature. The plate is then heated to 40° C. for 5 minutes and then cooled to room temperature, where R h is again determined. Thus, various temperatures ranging from 40 to 80° C. are assayed in 10° C. increments.

[0223] Fig. 28 shows the results for anthrax PA as an example. Figure 28: Hydrodynamic radius of anthracnose PA after 5 min of incubation at elevated temperatures. For natively formulated anthracnose PA, a low hydrodynamic radius is observed at room temperature. This low R h does not appear to be affected by a 5 min incubation at 40°C. However, after a subsequent 5 min incubation at 50°C, R h A dramatic increase in T was observed, indicating that anthracnose PA aggregates at 50°C. agg This allows the aggregation temperature T agg It can be concluded that the T is 50°C. The addition of various excipients agg The temperature is increased to a maximum of 80°C.

[0224] T agg To analyze whether the changes observed in T are, in fact, inhibition of aggregation or simply the result of higher conformational stability of the protein, agg T m , which is shown in FIG.

[0225] Figure 29: T agg vs. T m T using 10°C increments agg Since the question is being asked, m A 10° C. interval around the region where aggregation is thought to be strictly associated with unfolding (green area) is defined. T agg If T is elevated above that regime, the excipient is considered to inhibit aggregation (yellow area). agg If is reduced below that regime, the excipient would be considered to promote aggregation (red area).

[0226] Melibiose is observed to inhibit aggregation of CRM (2M and above) and tetanus toxoid (2.4M), the latter of which does not aggregate and therefore has no corresponding data point in Figure 29. Furthermore, maltose inhibits aggregation of CRM (1.5M and above), which also causes a complete lack of a data point in Figure 21.

[0227] Assessing the effect of excipients on the secondary structure stability of vaccine proteins In addition to probing the tertiary structure of the vaccine protein, the five excipients are subjected to analytical methods that report their effect on the secondary structure content of the vaccine protein.

[0228] One concentration of each excipient is analyzed for all three vaccine proteins. The protein concentration we use is 0.5mg / ml. Furthermore, in order not to interfere with the optical detection in this setup, it is necessary to use a different buffer system than in the previous experiment. However, it can be shown that this change in buffer does not affect the stability of the proteins used in this study.

[0229] Vaccine proteins are formulated in a buffer containing 5 mM sodium dihydrogen phosphate and sodium chloride (50-150 mM), maintaining the same pH level as in their respective native formulations. Spectra are recorded from 280-200 nm (in some cases 210 nm) with a data pitch of 1 nm, a slit width of 100 μm, a response of 2 s, a speed of 100 nm / s, and a 1 mm path length. Three spectra are averaged for each temperature.

[0230] Fig. 30 indicates the determined transition temperature. Fig. 30 Transition temperatures of anthrax PA, tetanus, and diphtheria toxoids in formulations containing newly identified heat stabilizers

[0231] Before outlining the results, there are two noteworthy observations that are important to put the data into the proper context:

[0232] First, two heat-induced transitions are observed for the anthrax PA. The lower transition temperature is used for analysis without prior information on how each transition affects the ability of the anthrax PA to affect the immunogenicity of the protein. In the formulation with lactulose, the two unfolding transitions occur in a rather narrow temperature regime, neither of which can be determined with the required precision. Therefore, the maximum temperature is recorded where no structural changes occurred. In general, the anthrax unfolding temperature should be considered as the lower limit, not the transition midpoint.

[0233] Second, there are conditions where no changes in secondary structure are observed, even at very high temperatures. However, information from DLS and fluorescence measurements is essential to properly assess the overall stability and mode of action of the excipients.

[0234] Table 5 summarizes the results of the experiment: JPEG2024174888000007.jpg64163

[0235] Effect of five selected excipients on vaccine protein stability The effect of excipients selected from three different substance groups on the conformational and colloidal stability of vaccine proteins is determined. With regard to conformational stability, it is possible to differentiate between effects on secondary and tertiary structure.

[0236] Fig. 31 gives an overview of all results for each protein and excipient combination. Figure 31: Summary of fluorescence, CD and DLS data obtained for anthrax PA, CRM197 and tetanus toxoid. The black line at 60 °C represents the desired stability target. With the information at hand, the effect of each excipient on each protein can be explained.

[0237] i) TMAO: Of the set of excipients used, TMAO appears to be the poorest of the very good heat stabilizers. ii) Choline dihydrogen phosphate: This ionic liquid is a stronger thermal stabilizer than TMAO, making CRM more stable than TMAO.

[0238] i) Melibiose: The sugars tested here generally show the best heat stabilization. Melibiose is a more efficient heat stabilizer than trehalose for all three vaccine proteins tested. Melibiose inhibits aggregation for tetanus and CRM. ii) Maltose: its heat stabilizing effect is similar to that of melibiose. Maltose stabilizes all vaccine proteins well above 60°C, but melibiose appears to have a stronger stabilizing effect. Maltose is a more efficient heat stabilizer than trehalose for all three vaccine proteins tested. On the other hand, maltose is used at a lower concentration, making this excipient easier to use. Maltose inhibits the aggregation of CRM and the secondary structure unfolding of CRM and Td. iii) Lactulose: Like the sugars mentioned previously, lactulose stabilizes all vaccine proteins up to temperatures above 60°C. However, in contrast to the other sugars, secondary structure unfolding of the CRM is observed and there is no aggregation prevention. However, lactulose shows similar effectiveness as a heat stabilizer as maltose and is more efficient than trehalose for all three vaccine proteins. Both sugars are used at similar concentrations (1.7 M lactulose and 1.8 M maltose), while melibiose is effective when more concentrated solutions are applied (2.4 M).

Claims

1. 1. A method for increasing the thermal stability of a liquid protein or peptide formulation, comprising: a) an osmolyte selected from the group consisting of trimethylamine-N-oxide; betaine, 4-hydroxyproline; ornitine; citrulline; N-acetyl-serine; hydroxylectoine; myo-inositol, allo-inositol, L-chiro-inositol, and D-chiro-inositol; and / or b) hydrated deep eutectic mixtures of choline dihydrogen phosphate, choline chloride with sugars, sugar alcohols or polyols selected from the group of glucose, sucrose, glycerol, sorbitol, and ionic liquids selected from the group of 2-hydroxyethyl-trimethylammonium L-(+)-lactate; and / or c) a salt selected from the group consisting of dihydrogen phosphate compounds such as potassium dihydrogen phosphate, calcium dihydrogen phosphate, sodium dihydrogen phosphate, and ammonium acetate; and / or d) sugars selected from the group consisting of maltose, melibiose, lactose, lactulose, cellobiose, maltulose, palatinose, turanose, sophorose, and nigerose; and / or e) Sugar acids and their salts selected from the group consisting of lactobionic acid, calcium lactobionate, and magnesium or calcium gluconate. and / or f) Amino sugars such as meglumine and / or g) sugar alcohols such as mannitol, maltitol, sorbitol, or mixtures thereof At least one excipient selected from the group consisting of: The method comprises combining at a concentration suitable to measurably increase the thermal stability of the protein or peptide formulation.

2. 2. The method of claim 1, wherein the single excipient is selected from the group specified in claim 1 consisting of an osmolyte or an ionic liquid or a salt or a sugar or a sugar acid and its salt or an amino sugar or a sugar alcohol.

3. The T from the measured melting curve indicates that the formulation is stable up to a temperature of at least 60°C. m 3. The method of claim 1 or 2, wherein the improved stabilization has been estimated by determining a value.

4. 4. The method of claim 1, 2 or 3, wherein a protein or peptide formulation containing the protein or peptide at a concentration of 0.05 to 2 mg / ml is stabilized.

5. 5. The method of any one of claims 1 to 4, wherein the protein or peptide formulation is stabilized and the protein or peptide is selected from the group consisting of viruses, attenuated viruses, virus-like particles, proteins derived from viruses, viral protein subunits, vaccines comprising viral protein subunits, enzymes, hormones, proteins used therapeutically or analytically, antibodies, antibody derivatives, nanobodies and monobodies.

6. 5. The method of any one of claims 1 to 4, wherein the protein or peptide formulation is stabilized and the protein or peptide is selected from the group consisting of a virus, an attenuated virus, a virus-like particle, a protein derived from a virus, a viral protein subunit, and a vaccine comprising a viral protein subunit.

7. The method according to any one of claims 1 to 4, wherein a protein or peptide formulation of a vaccine comprising a viral protein subunit is stabilized.

8. The method according to any one of claims 1 to 4, wherein the formulation comprising a vaccine selected from the group of anthrax PA (anthrax protective antigen), tetanus toxoid, and diphtheria toxoid (CRM197) is stabilized.

9. The method according to any one of claims 1 to 4, wherein the enzyme-containing protein or peptide formulation is stabilized.

10. The method according to any one of claims 1 to 4, wherein the formulation comprising an enzyme selected from the group of lactose dehydrogenase and lysozyme is stabilized.

11. 11. The method according to any one of claims 1 to 10, wherein the formulation is stabilized by the addition of at least one excipient selected from the group of trimethylamine-N-oxide, choline dihydrogen phosphate, melibiose, maltose and lactulose or mixtures thereof.

12. The method according to any one of claims 1 to 11, wherein the excipient(s) is / are added to the protein formulation at a concentration of >100 mM, preferably at a concentration in the range of 0.25 to 4 M / l.

13. The method according to any one of claims 1 to 12, wherein the pH value of the formulation is adjusted to pH 6 to 8, preferably 7 to 7.

5.

14. 14. The method according to any one of claims 1 to 13, wherein the pH value of the formulation is controlled by addition of a buffer selected from the group: potassium phosphate, sodium phosphate, sodium acetate, histidine, imidazole, sodium citrate, sodium succinate, HEPES, Tris, Bis-Tris, ammonium bicarbonate, and other carbonates.

15. 14. The method according to any one of claims 1 to 13, wherein the formulation comprises a polymer for stabilising the protein in an amount of at least 0.1% w / v and / or a surfactant in an amount of at least 0.005% w / v.

16. 14. The method of any one of claims 1 to 13, wherein the formulation comprises divalent cations at a concentration ranging from 0.1 to 100 mM and amino acids at a concentration ranging from about 0.1 to 1% w / v to stabilize the contained protein and to adjust the pH and osmolality of the solution.

17. A stabilized formulation prepared by the method of any one of claims 1 to 16.

18. 17. A stabilized formulation prepared according to the method of claim 16, characterized in that it contains lactate dehydrogenase and at least one reducing agent in a buffer at pH 7.

6.

19. 19. The stabilized formulation according to claim 18, characterized in that it contains lactate dehydrogenase and 1 mM DTT (1,4-dithiothreitol) as reducing agent in a buffer at pH 7.

6.

20. a) containing anthrax PA and maltose or anthrax PA and lactulose or anthrax PA and TMAO or anthrax PA and choline dihydrogen phosphate and is stable at a temperature of 50° C. for at least 72 hours; or b) contains anthrax PA and maltose or anthrax PA and melibiose and is stable at a temperature of 55° C. for at least 72 hours; or c) contains anthracnose PA and melibiose, and is stable at a temperature of 60° C. for at least 6 hours; 17. A stabilized formulation prepared according to the method of claim 16.

21. a) contains CRM197 and maltose and is stable at a temperature of 50° C. for at least 72 hours; or CRM197 and melibiose, and is stable at a temperature of 50° C. for at least 96 hours; or b) contains CRM197 and melibiose and is stable for at least 96 hours at a temperature of 55°C or CRM197 and maltose, and is stable at a temperature of 55° C. for at least 72 hours. or c) contains CRM197 and melibiose and is stable at a temperature of 60° C. for at least 72 hours or CRM197 and maltose, and is stable at a temperature of 60° C. for at least 72 hours.

17. A stabilized formulation prepared according to the method of claim 16.

22. 17. A stabilized protein formulation prepared according to the method of claim 16, which is intended to be formulated as a vaccination solution, and optionally finished as a solution for use in pharmaceutical injection vials.

23. 17. A stabilized protein formulation prepared according to the method of claim 16, filled into a container, such as a glass, plastic or foil vial, bottle, pouch, tube or bag, for further dosing, dilution or processing.

24. 20. A stabilized protein formulation prepared according to the method of claim 16 as part of an immunoassay test kit or in preparation and bottling for analysis.

25. 20. A kit comprising a stabilized protein formulation prepared according to the method of claim 16 and other container means containing necessary or convenient solutions for carrying out the method of use of said protein formulation.

26. 25. The kit of claim 24, comprising a stabilized protein formulation prepared according to the method of claim 16, and written or analytical information for carrying out a method of use of the protein solution.

27. 26. The kit of claim 24 or 25, comprising a stabilized protein formulation prepared according to the method of claim 16, and written information for carrying out a method of use of the protein solution by prefabrication and introduction into a small implantable pump or other device for therapeutic use.

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