Protein formulation additives to improve thermal stability

Abstract

The present invention relates to excipients for specific protein formulations which are suitable for improving thermal stability against denaturation and inactivation. In particular, the present invention relates to additives for thermal stabilization of vaccine formulations.

Description

Technical Field

[0001] The present invention relates to protein formulations comprising excipients suitable for improving thermal stability against denaturation and inactivation. In particular, the present invention relates to additives for thermally stabilizing vaccine formulations and methods for preparing these formulations. Prior art

[0002] Proteins or peptides are used commercially in a variety of pharmaceutical forms (e.g., vaccines). Many of them contain proteins and protein-based structures / complexes. The proteins are usually produced by recombinant DNA technology or other means. The proteins used in vaccine preparations can be of bacterial or viral origin. The proteins can be divided into several categories, including but not limited to toxins, toxoids or mutated proteins. In addition, vaccines can be derived from the bacterial or viral proteins / peptides, for example, in the form of epitope vaccines, chemically inactivated toxins (toxoids), glycoproteins or conjugated proteins, in which the pharmaceutically 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. 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 (these amino acid residues are covalently linked together by peptide bonds to form linear unbranched polypeptide chains), and their relative molecular weights range from several thousand to several million Da. The useful properties of proteins as drugs depend on the polypeptide chains adopting a unique three-dimensional folded conformation, i.e., the tertiary structure of the protein. It is this unique folding that causes the protein to be highly selective for the molecules it will recognize. However, the ability to maintain a unique three-dimensional structure is precisely one of the obstacles that makes the use of polypeptides in human and animal diseases full of difficulties.

[0004] Therefore, the ability of vaccines or protein particles to maintain resistance to degradation is a major challenge in providing adequate immunization services, as biological materials (including those in solution) are well known to be susceptible to inactivation by heat, oxidants, salts, etc.

[0005] Viruses, bacteria and other infectious agents used in vaccines are easily inactivated after a short time at ambient temperature, but storage at low temperatures is costly. Even brief exposure to high temperatures can lead to loss of vaccine activity, even at low temperatures, limiting the use of vaccines and increasing costs, thus limiting their distribution, especially in underdeveloped third world countries.

[0006] It is known from the literature that up to 80% of the cost of vaccination programs is due to cold chain issues (used to keep the vaccine cold), which means that the vaccine must be kept cold and maintain its effectiveness.

[0007] Typically, this requires keeping vaccines refrigerated from the time they are produced to the time they are administered - a major undertaking, especially in remote areas of the developing world.

[0008] Known as the cold chain problem, the high costs and risks associated with protecting vaccines from degradation have been identified by the World Health Organization as one of the most important challenges to expanding global vaccination programs. Therefore, there are a variety of approaches to stabilize immune preparations, and attempts have been made to address this problem by adding low-cost biocompatible additives and slowing down the degradation of viral particles. For example, it has been proposed to stabilize immune preparations by adding anionic gold nanoparticles (10 -8 –10 -6 M) or polyethylene glycol (PEG, molecular weight ~8,000Da, 10 -7 –10 -4M) to increase the half-life of adenovirus expressing green fluorescent protein at 37°C from ~48h to 21 days (from 7 days to>30 days at room temperature) to improve the storage time of adenovirus type 5. The stabilizing effect of sucrose (which is added in low concentrations) is also known. It was found that the addition of PEG and sucrose can maintain the in vivo immunogenicity of viruses stored at 37°C for 10 days. However, these stabilizing effects are not enough to achieve storage stability at high temperatures exceeding 60°C. However, the latter is necessary for the safe use of vaccine formulations in tropical areas, and in this regard, it is surprising that liquid protein formulations (such as vaccine formulations) are currently produced without any specific thermal stabilizers or excipients that will lead to thermal stability at temperatures above 60°C. One of the reasons is that adding additives to existing vaccines usually means going through the expensive approval process of the health department again, which is commercially unattractive. Therefore, it is expected that the vaccine formulation will be stabilized by adding additives or excipients (preferably those that have been considered as compatible components of pharmaceutical compositions) to prevent inactivation at high temperatures. As mentioned in the review by Pellicca, M. et al., one approach is to add sucrose to the vaccine formulation (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 purpose, but the desired effect can only be found at high concentrations rather than at temperatures above 40°C. As reported by Pellicca, M. et al., the most effective thermal stabilizing additive PEG has been shown to be unsuitable for vaccine formulations because up to 50% of people show allergic reactions to it.

[0009] In WO 2012 / 125658 A1, a method for producing a stabilized vaccine composition of an immunogen, especially an unstable immunogen, is provided, wherein oral particles are nebulized with an antigenic virus culture at a temperature of about 25° C. to about 50° C., preferably at about 30° C., so that the immunogen coating is dried onto the particles. The vaccine product contains particles having a moisture content of about 0.1% w / w to 10% w / w to obtain 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 stabilized by a combination of an amorphous excipient selected from sucrose, lactose, trehalose, dextrose, xylose, cellobiose, raffinose, isomaltose and cyclodextrin and a buffer of a salt selected from citrate, histidine, phosphate, tris, succinate and acetate buffers.

[0011] In addition, there are a series of publications and patent documents that attempt to address the stability problem of corresponding protein formulations at higher temperatures in a similar manner, but at the expense of the need to re-establish the injectable solution before application. However, the latter will be a major problem in underdeveloped countries and crisis areas. Here, even a few hours of improvement in protein formulation stability will represent an improvement with respect to elevated ambient temperatures.

[0012] Purpose of the Invention

[0013] Thermal stability and cold chain disruptions are major obstacles to ensuring consistent vaccine doses are delivered to remote areas, especially in poor countries. Currently, vaccines are refrigerated during transport, but 25% of all liquid vaccine doses and 50% of all freeze-dried vaccine doses delivered to developing countries are lost because continuous refrigeration cannot be guaranteed.

[0014] The main object of the present invention is to provide a thermostabilized vaccine formulation which can preferably be obtained with the same quality as comparable products and which is stable for a long time when stored at temperatures up to 60°C and above.

[0015] Summary and Subject Matter of the Invention

[0016] An excipient from one of the molecular classes listed below is added at an optimal concentration to a protein or peptide formulated in an aqueous buffer. The melting temperature T of the protein or peptide m This significantly increases the temperature so that it can withstand temperatures up to 60°C and above.

[0017] Another subject of the present invention is an excipient as disclosed below which, at suitable concentrations, confers sufficient thermostability to vaccine formulations so that they can be stored at temperatures above 60° C. while retaining their folded protein structure for a sufficiently long time.

[0018] The present invention provides stabilized protein solutions, advantageously having a relatively high concentration of about 2.0 mg / ml, comprising at least one stabilizer which minimizes degradation and inactivation of the contained protein, which directly has a positive impact on maintaining the efficacy of the protein and allows these formulations of the present invention to be stored and even transported to underdeveloped markets in tropical regions.

[0019] More specifically, the present invention relates to a method for thermally stabilizing a liquid protein formulation as described in claim 1 and in specific embodiments according to claims 2 to 16. The present invention also comprises a formulation as described in claims 17 to 24 and prepared according to the method of claims 1 to 16. A further embodiment of the present invention resides in a kit as described in claims 25 to 27 for a stabilized protein formulation.

[0020] DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

[0021] The present invention relates to a method for obtaining a liquid protein or peptide preparation, which is heat-stabilized and, if desired, in a ready-to-use preparation, comprising proteins produced by recombinant DNA technology or otherwise, protein conjugates (i.e. protein-sugar, protein-nucleotide, protein-RNA / DNA or protein-peptide), cross-linked proteins (e.g. formaldehyde-treated proteins, e.g. tetanus toxoid) and other proteins used in the final larger structure or vaccine preparation, which may be of bacterial or viral origin, viral protein subunits, enzymes, hormones or others. Therefore, hereinafter, when referring to a protein preparation, this is always also meant to be a peptide preparation, unless a specific preparation containing one or the other is explicitly mentioned.

[0022] The invention also relates to stabilized formulations in which the protein retains its folded effective structure even when the temperature is raised to values ​​as high as 60°C. The invention also relates to such formulations, which retain their stabilization for up to three or even four days. Advantageously, stabilized protein formulations can be easily obtained by adding an effective amount of the selected excipients to the solution, which is optionally buffered, and their pH adjusted to the optimal range for the contained protein. The formulation may also contain a reducing agent.

[0023] Liquid preparations of the present invention are prepared, comprising proteins at a concentration of 0.05 to 1.8 mg / ml. The heat stabilizing effect of the added excipients is demonstrated by an increase in the melting temperature of the contained protein; and at the same time, the increase in the melting temperature of the protein is an indicator of increased thermal stability of the protein in the preparation. It was found through specific experiments that, on the one hand, depending on the type of protein and the added excipients, different amounts of excipients must be added to achieve the desired stabilization of the contained protein, and it is also advantageous to adjust the pH value in the stabilizing solution according to the contained protein. Depending on the excipient, a concentration of 0.25 to 4 M is used in the protein solution for the desired protein stabilization to minimize degradation and inactivation during storage and exposure to temperatures above room temperature (especially temperatures above 40° C.). As previously mentioned, reliable stabilization of proteins also depends in particular on the pH of the solution. Depending on the protein, the optimal pH can be in the range of pH 6 to 8, and is advantageously set with a suitable buffer.

[0024] In this way, protein solutions can be stabilized against the inactivating effects of high temperatures. Depending on the protein and excipients added, stabilization up to 20°C can be determined by the following experiments. For improving thermal stability, only those experiments were considered in which it could be determined that the protein melting temperature was increased by at least 5°C by the addition of a specific excipient.

[0025] For further use, such stabilized preparations can be filled into bottles for further administration, dilution or processing. However, they can also be formulated into immune solutions, which can optionally be made into solutions for use in pharmaceutical injection bottles, unless further processing or dilution is required. In addition, in another application form, such stabilized protein solutions can be presented in immunoassay test kits or in preparations and bottles for analysis. In addition, stock preparations can be stabilized in this way, which are shipped globally for subsequent final filling.

[0026] The kit comprising the heat-stabilized protein formulation may also comprise other container means comprising solutions necessary or convenient for carrying out the method using the protein formulation. The container means may be made of glass, plastic or foil and may be a vial, bottle, bag, tube, bag, etc. The kit may comprise written information, such as procedures or analytical information for carrying out the method using, such as procedures or analytical information for carrying out the method using the protein solution, such as the amount of reagents contained in the first container means. The container means may be another container means together with the written information, such as a box or bag.

[0027] Since it is liquid, in another embodiment of the present invention, the pharmaceutical composition of the present invention can be pre-formulated to be made later for injection into a living body. In an alternative embodiment, the pharmaceutical composition can be pre-formulated to be introduced into a small implantable pump or introduced into other devices for therapeutic application.

[0028] Excipients suitable for use as heat stabilizers for use in the present invention can be selected from different types of organic molecules. A 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 in both animal and human organisms without causing side effects or allergic reactions if used in appropriate concentrations.

[0029] Liquid formulations containing proteins may contain, in addition to the excipients added according to the invention, one or more other compounds, such as amino acids, proteins, chelating agents, buffers, salts, preservatives, stabilizers, antioxidants, emulsifiers, plasticizers or lubricants.

[0030] A peptide is an organic chemical compound containing peptide bonds between amino acids. Oligopeptides are distinguished from polypeptides with a few amino acids by their number. Long polypeptide chains are also called proteins, especially those formed by protein biosynthesis. Due to this chemical relationship of peptides and proteins, if proteins are mentioned below, both types of substances are meant.

[0031] In the literature, the expression "thermal stability" is often used, but often in a misleading way. In the literature, this usually only means that protein solutions can be stored at slightly elevated temperatures below 40°C. By increasing the storage temperature to 40°C, this temperature is still well below the unfolding transition midpoint (T m )(For example, AnthraxPA T m =48°C), the spontaneous unfolding rate of the protein increases. In terms of equilibrium thermodynamics, there is a certain probability that the protein may spontaneously unfold at any point in time, but the probability of such an unfolding event is temperature dependent. By increasing the storage temperature, the frequency of potential unfolding events is increased, and thus the long-term storage of the protein formulation can be evaluated in a reduced period of time.

[0032] Here, according to the invention, the expression "thermostability" is used in the context that the protein or peptide remains predominantly in its native, rather than unfolded, form during storage at temperatures above 40°C, preferably above 50°C, in particular up to 60°C.

[0033] The current analytical work involves determining the thermostability by determining the midpoint of the thermodynamic transition of heat-stressed proteins. To this end, the effect of model molecules or selected excipients is examined to determine their stabilizing effect on the structure of the model protein at elevated temperatures, which means at temperatures above 40°C, preferably up to 60°C. At the same time, the concentration range in which the thermostable excipients are effective is analyzed experimentally.

[0034] By definition, causing a dT > 5°C m The excipient is a good stabilizer. If dT m <5°C and >0°C, the excipient is defined as a poor stabilizer. Negative dT m Identification of molecules as protein destabilizers.

[0035] Transition midpoint T m Determined by fluorescence spectroscopy, this means using nano differential scanning fluorimetry (nanoDSF) of protein solutions containing protein at a concentration of 0.05-1.8 mg / ml.

[0036] The prepared protein solution was heated at a heating rate of 1°C / min, and the fluorescence emission ratio at 350 / 330 nm was recorded and plotted against temperature. Typically, the resulting curve resembles a Boltzmann function, and the inflection point of the curve represents the average unfolding temperature T m From this curve, by m赋形剂 Subtract T from the preparation m天然 To determine T m The change of dT m .

[0037] The expression "folding fraction" is used below and indicates the amount of native protein in the liquid formulation. Secondly, the expression "thermal denaturation midpoint" is also used in conjunction with the stability of the protein and it defines that a deviation of 10% in either direction is acceptable. It is important to note that any change in the transition midpoint (also including higher temperatures) is the result of changes in protein conformation or data quality. The latter can be affected, for example, by protein aggregation or many other effects. Therefore, it is required that the transition temperature deviates from the initial measured value by less than 10%.

[0038] As used herein, the term "sugar" refers to any of a group of water-soluble carbohydrates with relatively low molecular weight. The term sugar includes reducing sugars (e.g., maltose), non-reducing sugars (e.g., sucrose), sugar alcohols (e.g., sorbitol), and sugar acids (e.g., lactobionic acid).

[0039] All ranges shown herein are intended to include the lower and upper limits of the range. The term "about" is intended to slightly encompass a given range. Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the method used to determine the value.

[0040] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the words "a" or "an" may mean "one / an", but is also consistent with the meaning of "one or more / one or more", "at least one / at least one" and "one or more than one / one or more than one".

[0041] As used in this specification and claims, the words "comprising" (and any forms of "comprising," such as "including" and "containing"), "having" (and any forms of "having," such as "containing" and "having"), "including" (and any forms of "including," such as "including" and "containing"), or "containing" (and any forms of "containing," such as "including" and "including") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0042] It should also be specifically understood that any numerical value listed herein includes all values ​​from the lower limit to the upper limit, that is, all possible combinations of numerical values ​​between the listed minimum and maximum values ​​are considered to be explicitly indicated in this application. For example, if the range is stated as 1% to 20%, it is intended to explicitly list values ​​such as 2% to 5%, 10% to 15% or 3% to 4.5% in this specification.

[0043] "Contacting" refers to the process of bringing at least two different species into contact so that they can interact or react.

[0044] "Excipient" generally refers to a compound or material added to ensure or increase the stability of a therapeutic agent (ie, for long-term stability).

[0045] A "stable" formulation or composition is one in which the biologically active material or protein therein substantially retains its physical stability and / or chemical stability and / or biological activity upon storage. Stability can be measured at a selected temperature and over 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).

[0046] "Pharmaceutically acceptable" refers to those active agents, salts, and excipients which 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, etc., commensurate with a reasonable benefit / risk ratio, and effective for their intended use.

[0047] As described above, the protein solution of the present invention is adjusted to a specific pH. In order to keep the pH value stable, an appropriate buffer is added to the protein solution.

[0048] The buffer can be added in the form of a carrier liquid suitable for dissolving and / or dispersing the protein to be dissolved. The buffer is usually selected from a pharmaceutically acceptable buffer system. A preferred buffer is a pharmaceutically acceptable buffer system that has the ability to resist pH changes after the addition of an acid, base, inorganic compound, organic compound, or solvent or diluent. Buffer components such as phosphate or citrate are included to control the pH of the solution containing the vaccine, and to adjust the permeability of the solution. The buffer concentration can be in the range of 5mM to 2M, wherein the pH of the solution is adjusted to a range of about pH 4 to about pH 10, preferably adjusted to a range of about pH 6 to about pH 8.

[0049] Pharmaceutically acceptable buffers can be selected from potassium phosphate, sodium phosphate, sodium acetate, histidine, imidazole, sodium citrate, sodium succinate, HEPES, Tris, Bis-Tris, ammonium bicarbonate and other carbonates. The buffer can comprise a pH of about pH 4 to about pH 10, preferably about pH 6 to pH 8, and about pH 6 to about pH 7.

[0050] If one or more types of sugar are added to the protein solution, the sugar is preferably selected from monomeric and / or dimer molecules, and in particular may be selected from glucose, galactose, maltose, maltulose, melibiose, sucrose, trehalose, lactose, lactulose, fructose, lactose, mannitol, sorbitol, xylitol, cellobiose, Palatinose, turanose, sophorose, nigerose and combinations thereof. The most preferred sugar is selected from 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 concentrations to very high concentrations. Therefore, for stabilization, the selected sugar may be added in a relatively small amount, but if the concentration of the sugar contained is relatively high, i.e. when added to the solution in a few molar amounts, there is generally a particularly effective heat stabilization of the protein. Thus, it has been found experimentally that particularly good stabilization results can be achieved if the selected excipient is added in an amount of 0.25 to 4 M / l protein solution.

[0051] The aqueous solution may further comprise pharmaceutically acceptable surfactants, polymers, amino acids and other pharmaceutically acceptable excipients. Polymers for stabilizing proteins may be included. These polymers may be added in amounts of 0.1% w / v and more. Surfactants may be added to reduce surface tension and displace protein molecules from the surface. Surfactants may also increase the solubility of other formulation components. Surfactants may be included in amounts of 0.005% and more by weight of the protein-containing formulation.

[0052] Divalent cations and amino acids may be included to stabilize proteins and adjust the pH and osmotic pressure 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 a concentration ranging from about 0.1% to about 1% (w / v).

[0053] Overall, this means that depending on the protein or peptide solution according to the invention to be used and on the nature of the protein or peptide contained in the solution, different components of the final formulation may be added.

[0054] Preferably, according to the present invention, the thermostable excipient or the combination of thermostable excipients is selected from the following classes of molecules:

[0055] 1) Osmolytes: trimethylamine-N-oxide; betaine, 4-hydroxyproline; ornithine; citrulline; N-acetylserine; hydroxylectoine; inositol, iso-inositol, L-chiro-inositol, D-chiro-inositol

[0056] 2) Ionic liquids: choline dihydrogen phosphate; hydrated deep eutectic mixtures of choline chloride and sugars, sugar alcohols or polyols such as glucose, sucrose, glycerol or sorbitol; L-(+)-2-hydroxyethyl-trimethylammonium lactate

[0057] 3) Salts: dihydrogen phosphate compounds, such as potassium dihydrogen phosphate, calcium dihydrogen phosphate or sodium dihydrogen phosphate, ammonium acetate,

[0058] 4) Sugar: maltose, melibiose, lactose, lactulose, cellobiose, maltulose, palatinose, turanose, sophorose, nigerose

[0059] 5) Sugar acids and their salts: lactobionic acid, calcium lactobionic acid, magnesium gluconate or calcium gluconate

[0060] 6) Amino sugars: meglumine

[0061] 7) Sugar alcohols: mannitol, maltitol, sorbitol,

[0062] Thermostability excipients were added to the protein formulations at various concentrations. Protein concentrations ranged from 0.05 to 1.8 mg / ml. Increased thermostability was detectable starting with 100 mM excipient, but best results were obtained in the range of 0.4-4 M. Concentrations were expressed in volume % for the hydrated depth eutectic mixture of choline chloride and the second component and for 2-hydroxyethyl-trimethylammonium L-(+)-lactate. Increased thermostability was detectable starting with 15%, and best results were obtained with 95% solutions.

[0063] The aqueous protein solution of the present invention can be provided in a primary container, for example in a vial made of glass or plastic / resin, or a foil bag device or any other suitable and compatible device. Depending on the desired subsequent use, the solution provided in this manner can be present in an amount ranging from a few microliters to several liters.

[0064] The present invention provides a method by which proteins or peptides used in pharmacy and medicine in solution can be protected against the destabilization and inactivation effects of high temperatures, which means that the protein can be thermostable. In particular, this stabilization effect involves protection against the occurrence of protein unfolding and aggregation, which usually leads to inactivation and loss of protein activity and / or drug efficacy. The method of the present invention is applicable to any protein or vaccine comprising viral protein subunits, which can be used in microgram amounts. The protein can be a protein as a vaccine component, but it can also be an enzyme, hormone or other therapeutic or analytical protein, for example, an enzyme, an antibody, a nanobody or a single antibody (monobody). Depending on the type of protein and its application, the solution is provided in different amounts, packaging or devices, but is also provided in combination with other substances required for the desired application composition. Therefore, they can be part of a bioassay, or can be already present as a solution in an application or analysis or diagnostic kit or in an enzyme-containing molecular biology kit. This means that the protein can be included in a vaccine and in a diagnostic assay for detecting pathogens in a sample of a subject suffering from a disease. The pathogen of the disease can be from an infection, a tumor or an allergen or other.

[0065] As mentioned above, the present invention relates to the thermal stabilization of the corresponding protein preparations. By means of a planned experimental procedure, the influence of the selected additives on the thermal stability was investigated using the sample proteins listed above.

[0066] In order to test the thermal stabilization of as different proteins as possible by adding suitable excipients as listed above, experiments were performed with the following sample proteins:

[0067] Enzymes, especially lactate dehydrogenase and lysozyme as model enzymes,

[0068] and

[0069] Vaccine proteins, especially AnthraxPA (anthrax protective antigen), tetanus toxoid and diphtheria toxoid (CRM197) were used as model vaccines.

[0070] In order to demonstrate effective thermal stabilization by adding one or more excipients, liquid formulations were prepared, which contained the protein in a concentration of 0.05 to 1.8 mg / ml. For this purpose, a stock solution of the protein was prepared. In addition, a stock solution of the excipient was prepared in a suitable buffer. The protein, excipient and (if necessary) buffer were combined to produce the formulations described in the Examples. As shown in the "Examples" chapter, the melting temperatures (T) of these proteins in the presence of the most effective and most suitable thermal stabilizers were tested. m ), since an increase in protein melting temperature is an indicator of increased thermal stability of the protein in a liquid formulation.

[0071] Denaturation temperature or melting temperature T m The determination of T can be performed in a high-throughput manner using differential scanning fluorimetry (NanoDSF: Prometheus.NT48, equipped with standard capillaries, NanoTemper Technologies GmbH) for small volumes. In addition, for some preparations, circular dichroism spectroscopy was used to determine T m To obtain additional structural information about the proteins in the preparation (spectropolarimeter J-815, Jasco Deutschland GmbH, using 0.1 cm quartz Suprasil cuvettes model 110-QS from Hellma).

[0072] Proteins and peptides are usually folded into stable three-dimensional structures, which are stabilized by a large number of weak interactions of residues within the polypeptide chain. By increasing the temperature of the preparation, increased thermal motion and other factors lead to protein denaturation. To quantify the thermal stability of a protein, a parameter called T is used. m temperature.

[0073] T m represents the temperature of the midpoint of the equilibrium transition obtained here for the heat-induced denaturation of the model protein. Assuming two-state transitions from native to non-native state (unfolded / partially unfolded), T m represents the temperature at which the two states are equivalent. The measurement of the midpoint of a transition is affected by the kinetic stability of the substance, and therefore only such values ​​obtained under identical conditions are comparable. All T values ​​reported in this paper are m The values ​​were recorded at a heating rate of 1°C / min. m Values ​​indicate stabilization of the protein.

[0074] Protein denaturation can be measured by various spectroscopic techniques that exploit the specific optical properties of proteins.

[0075] Differential scanning fluorimetry is a fluorescence-based method that exploits the specific emission properties of the natural proteinogenic amino acid tryptophan. Due to its two unique dipole axes, the fluorescence emission of the indole side chain is solvatochromic. When embedded inside a protein (which is a more lipophilic environment), tryptophan typically displays a fluorescence emission maximum of about 328-330nm. When the protein is denatured, the tryptophan residues become progressively less isolated from the surrounding water. Therefore, the emission maximum of tryptophan approaches 350nm. This spectral migration to higher wavelengths is called a red shift. NanoDSF records this migration by recording the emission of the protein at 330nm and 350nm and plotting its ratio against temperature. This typically results in an S-shaped curve with a positive amplitude.

[0076] Fluorescence spectroscopy is primarily used to probe tertiary structural transitions. It should be noted that the fluorescence spectrum of a protein represents the superposition of all emission spectra of the amino acids contained therein, and therefore the emission characteristics of a specific protein may differ from the values ​​given above.

[0077] CD spectroscopy exploits the differential absorption of circularly polarized light by chiral substances or macromolecules with asymmetric structures, such as proteins. Depending on the wavelength range used, CD spectroscopy can be used to probe the secondary or tertiary structure of proteins. Here, we only use CD spectroscopy to obtain information about the secondary structure of proteins. Natively folded proteins exhibit very unique CD spectra based on their structural composition. After thermally induced unfolding, the absorption difference of circularly polarized light decreases and the CD signal approaches zero. In order to determine T m , the CD signal at a specific wavelength (usually 222nm, 218nm, 208nm or 196nm) is plotted against temperature and then fitted with a sigmoid function.

[0078] Regardless of which method is used, there are specific properties of the protein that report on the structural features of the protein. If the midpoint of the transition increases after the addition of an excipient, then the added excipient stabilizes the protein. Additionally, the steepness of the S-shaped curve can be modified by the addition of an excipient. The steepness of the curve reflects the kinetic stability of the protein. Although T m describes the temperature at which the protein unfolds, but the steepness of the unfolding curve indicates how quickly the transition occurs.

[0079] In the case of fluorescence spectroscopy, additional features can change the behavior of the sigmoid curve. The curve can show a negative amplitude, which indicates a shift of fluorescence to lower wavelengths, rather than the previously described red shift. This can be explained by protein aggregation. If not all tryptophan residues are buried within the protein, the emission of the natively folded protein may be at wavelengths greater than 330nm. After unfolding, the tryptophan residues are more exposed to water, but after aggregation, these residues are buried in the lipophilic core of the aggregate. Despite the negative amplitude, the sigmoid fit can still be used to determine the temperature reflecting the thermal stability of the protein.

[0080] Now, in order to evaluate which additives or excipients are most suitable for the thermostabilization of proteins and in what amounts they must be added to produce the desired effect, in a first step test formulations of AnthraxPA as a model vaccine were prepared and examined over three days. The detailed formulation compositions are summarized in Table 1 of the "Examples" section. The purpose of these experiments was to find the most suitable excipient or excipient mixture for thermostabilizing protein compositions of different origins over several days.

[0081] Since stabilizers have to be used in high concentrations so far, such compositions cannot be used to prepare prefilled syringes, since patients may not tolerate the stabilizers. Therefore, a dilution step is required before administration to patients. Another aim of these experiments is also to determine the highest possible tolerance level of stabilizers for in vitro assays. Based on the experimental results, the protein concentration can be adjusted and a vaccine stock solution can be generated, which can both provide thermal protection for the solute protein and can still be effectively administered to patients after dilution.

[0082] For example, it has been found that patients are most likely to tolerate melibiose at concentrations up to 250 mM. However, it is known from previous screening that at least 2.25 M of melibiose is required to stabilize AnthraxPA at high temperatures. Therefore, the protein concentration is increased by 2250 mM (stock) / 250 mM (final value) = 9 times. However, for patient use, this stabilized vaccine stock preparation needs to be diluted at least 9 times.

[0083] Based on these considerations, test formulations were prepared to examine the model proteins listed above, whose excipients were most suitable as physiologically acceptable additives for the most likely thermal stabilization at temperatures above 40°C, preferably above 60°C. From the available potential excipients that are considered acceptable to patients in liquid pharmaceutical formulations, lactulose and maltose were first selected as promising temperature stabilizing additives, and the corresponding protein formulations were prepared and examined with these additives. TMAO, choline dihydrogen phosphate and melibiose were also tested under the same conditions, and the results were compared with those of lactulose and maltose.

[0084] First, the samples are those spiked with the selected excipients, which are appropriately subjected to fluorescence spectroscopy to verify that all protein samples are natively folded, and then to determine whether they are suitable to stabilize the protein at elevated temperatures, including at elevated protein levels (up to 1.8 mg / ml). Following these tests, incubations are carried out at the specified temperatures, especially at 4°C and in the range of 50°C to 60°C, and if appropriate at higher temperatures. Tests have shown that, in particular, when melibiose is added as a stabilizing additive to protein formulations, generally better thermal stabilization occurs, and the formulations are generally stable at higher temperatures and have higher melting points, as can be demonstrated by determination of melting curves and transition midpoints.

[0085] To verify the thermostabilizing effect of various excipients over longer periods of time, small samples were taken after 6, 24, 32, 48 and 72 h of incubation time and the structural state and stability of the contained proteins were assessed by fluorescence spectroscopy.

[0086] In order to determine whether the protein-containing sample being examined is still in a native or damaged state, two parameters are now repeatedly monitored during the experiment by fluorescence spectroscopy. The first parameter relates to the amount of damaged or unfolded protein in the liquid preparation. The second parameter relates to the thermal denaturation midpoint. (However, it is crucial for the quality and reliability of the determined data that the deviations of the determined transition temperatures are less than 10%. This deviation can be caused by various effects, such as aggregation or other influences.)

[0087] Based on the experiments conducted, it has been found that by adding the selected excipients according to the present invention, a significant improvement in temperature stability can be achieved for protein formulations. For example, in its natural formulation, AnthraxPA has an unfolding transition temperature of 48°C, which does not allow storage at or above 50°C. At this temperature, AnthraxPA denatures immediately. But experiments have shown that AnthraxPA is stable up to 50°C if the formulation contains appropriate amounts of TMAO or choline dihydrogen phosphate. In addition, melibiose, maltose, is suitable for stabilizing liquid AnthraxPA formulations at 55°C for at least three days. If the storage temperature is increased to 60°C during storage, the formulation remains stable for at least 6 hours when formulated with melibiose.

[0088] However, it is not always possible to obtain comparable results in all cases. For example, the evaluation of the thermal stability of CRM197 in a liquid formulation is slightly different from that of the AnthraxPA formulation. When measured by fluorescence spectroscopy, the natural preparation of CRM197 shows a biphasic unfolding transition. The initial transition is associated with a local loss of structure, while in the overall range, the protein remains structurally intact. This is why different criteria must be used to study the stability of CRM197. In this case, two modified conditions are required. First, the median unfolding temperature cannot be used as a readout because conformational disturbances occur due to local loss of structure, which cannot be stabilized by simply adding the selected excipient. In addition, changes in its initial transition may affect the determination of the second unfolding temperature, resulting in an erroneous assessment of its overall structural state based on it. Secondly, the extent of damage to the temperature-dependent region of CRM affects the fluorescence emission rate from 350nm to 330nm. In order to evaluate the role of the excipients tested herein, all results were compared with those measured in a preparation containing the protein in its natural preparation, incubated at about 50°C and denatured. Under the same conditions, CRM197 showed unfolding in its native preparation at a temperature of 53°C. By adding melibiose or maltose to the corresponding CRM197 preparation, the unfolding was significantly inhibited for at least 72 hours when the preparation was incubated at 50°C. At this temperature, but also at 55°C, the addition of melibiose had a stabilizing effect lasting even for 96 hours, while maltose only stabilized CRM197 for 72 hours. Incubation at 60°C reversed this ratio. The results of the measurements showed that maltose stabilized CRM197 for 48 hours at 60°C, while melibiose only stabilized CRM for an incubation time of 24 hours at 60°C. In addition, TMAO, choline dihydrogen phosphate, and lactulose have been shown to be suitable stabilizers for CRM197. As mentioned above, these excipients also stabilized for a period of at least 72 hours when incubated at 50°C, while melibiose and maltose were still stabilized at 55°C and 60°C.

[0089] The formulation can be heat stabilized for three days by adding TMAO, choline dihydrogen phosphate, lactulose, melibiose and maltose to the protein formulation, wherein the protein is included at a concentration of up to 1.8 mg / ml. The protein can be a vaccine protein. It can be a protein such as AnthraxPA (anthrax protective antigen) or, for example, CRM197 or similar proteins. The protein can be a heterologously expressed protein or an overexpressed protein.

[0090] The experiments identified the best stabilization of liquid protein formulations containing AnthraxPA or CRM as the protein. The AnthraxPA formulation contained 5 mM HEPES as a buffer and 50 mM NaCl. As a stabilizing excipient, melibiose was included at a concentration of 2.25 M, and the pH of the formulation was 7.5. The CRM formulation contained 20 mM HEPES as a buffer and 150 mM NaCl. As a stabilizing excipient, melibiose was included at a concentration of 2.4 M, and the pH of the formulation was 8.0.

[0091] As mentioned for previous experiments and studies using AnthraxPA and CRM197, the thermal stabilization of formulations with lactate dehydrogenase and lysozyme as model proteins was also tested. The excipients of Table 1 were added at appropriate concentrations and tested for their possible thermal stabilization effects. In this context, other potential thermal stabilizers were also tested and selected from the group of ionic liquids, which were four biodegradable compounds and they were selected and prepared based on compounds that can be commonly 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.

[0092] In this context, it has been found through the studies conducted herein on the thermal stabilization of the vaccine and enzyme proteins 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 protein type.

[0093] Surprisingly, based on the results found, one cannot easily assume from the fact that a selected excipient belongs to a certain class of substances that it is also a good thermostability additive for a specific protein in solution, because a related substance provides good results for another protein.

[0094] The summary results of the study showed that the added excipients that caused the greatest changes in the melting temperatures of the tested proteins and showed the broadest applicability and suitability as stabilizers for all three vaccine proteins tested belonged to very different groups of substances.

[0095] These excipients are TMAO, choline dihydrogen phosphate, melibiose, maltose and lactulose.

[0096] a) TMAO shows a thermal stabilization effect starting from a concentration of 250 mM. The best stabilization results are obtained when the concentration is in the range of 1 M to 4 M in solution.

[0097] b) Choline dihydrogen phosphate showed a thermal stabilization effect starting from a 1 molar solution. The best results were found for the composition when the concentration was in the range of 2M to 4M.

[0098] c) Melibiose showed a thermostabilizing effect starting from 250 mM. Herein, the best results were found for the composition when the concentration was in the range of 1 M to 2.5M.

[0099] d) Maltose showed a thermal stabilization effect starting from 500 mM. The best results were found for the composition when the concentration was in the range of 1 M to 1.8 M.

[0100] and

[0101] e) Lactulose showed a thermal stabilization effect starting from 250 mM. The best results were found for the composition when the concentration was in the range of 0.5M to 1.7M.

[0102] The corresponding results are shown and commented in the next chapter “Examples”.

[0103] In addition, the preparation of lactate dehydrogenase and lysozyme as model proteins was studied. The above excipients were added at appropriate concentrations and tested for their possible thermal stabilization effect. Experiments performed accordingly for stabilizing lactate dehydrogenase showed that 4-hydroxyproline, L-ornithine, lactobionic acid and potassium dihydrogen phosphate were good thermal stabilizers, which means that the excipients increased the midpoint of the thermal transition of LDH by 5°C or more.

[0104] In addition to this, other potential thermal stabilizers from the group of ionic liquids, four biodegradable ionic liquids, were selected and prepared based on compounds commonly 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 were prepared as hydrated deep eutectic mixtures in three steps. First, 0.5 moles of choline chloride were mixed with the corresponding amounts of the two sugar (sorbitol and glucose) components and used in an amount of 0.2 moles (ratio 5:2). Glycerol was used in an amount of 0.5 moles (ratio 1:1). Sucrose was used in an amount of 0.125 moles (ratio 4:1), and 20% w / w H2O was added.

[0105] Tests herein have shown that hydrated deep eutectic mixtures of choline lactate and choline chloride with sorbitol, glucose and sucrose are good thermal stabilizers for lactate dehydrogenase.

[0106] 4-Hydroxyproline, L-ornithine, potassium dihydrogen phosphate, lactobionic acid and meglumine showed the desired heat stabilization effect on lactate dehydrogenase (LDH). Excipients identified as suitable for heat stabilization of LDH were also used in experiments to heat stabilize vaccine proteins. These screening experiments have shown that lactulose and maltose are good heat stabilizers in formulations containing vaccine proteins.

[0107] In experiments with the enzyme lysozyme, hydrated deep eutectic mixtures of choline lactate and choline chloride with sorbitol, glucose, and sucrose, respectively, were identified as good thermal stabilizers.

[0108] However, since the thermostabilization of the added excipients was not satisfactory in each case, it was additionally tested whether the synergistic effect of the two excipients used in combination would improve the thermostabilization of the two molecules. Next, salts and ionic liquids were combined with each other and with osmotic pressure agents and sugar acids. All experiments were performed using lactate dehydrogenase in the corresponding natural preparation (50 mM sodium phosphate, 1 mM DTT (1,4-dithiothreitol), pH 7.6). The protein concentration was adjusted to a concentration of 0.05 mg / ml.

[0109] It was found here that the combination of potassium dihydrogen phosphate and lactobionic acid resulted in a similar level of thermal stability to LDH as would be expected from the individual components. This result suggests that if the corresponding salts and sugar acids are compatible with each other in this protein solution, their use will result in an additive effect. This result was confirmed by using a combination of potassium dihydrogen phosphate and 4-hydroxyproline, which resulted in a similar level of thermal stabilization to LDH.

[0110] The results using potassium dihydrogen phosphate in combination with a hydrated deep eutectic mixture of choline chloride and sorbitol (molar ratio 5:2) are less clear and can be summarized as follows:

[0111] Thermal stabilization of salts and ionic liquids

[0112] - Salts are compatible and additive when present in excess compared to ionic liquids

[0113] - Ionic liquids are incompatible when present in excess compared to the salt, but are still thermally stabilized.

[0114] However, the effects of different combinations of hydrated deep eutectic mixtures of choline chloride and sorbitol (molar ratio 5:2) with lactobionic acid in the composition of the model protein lactate dehydrogenase were different. In this context, it can be concluded that the interaction of ionic liquids and sugar acids in this model composition is:

[0115] -The interaction between ionic liquids and sugar acids is concentration-dependent.

[0116] -The interaction between ionic liquids and sugar acids also depends on the ratio of ionic liquid to sugar acid.

[0117] - When a sufficient excess of ionic liquids was used, a synergistic effect of the two excipients on the thermal stability of the protein was observed.

[0118] -The ratio of ionic liquid to sugar acid required for this synergistic effect increases with the increase of ionic liquid fraction.

[0119] - The concentration of sugar acid must be minimum 0.25 M to observe a synergistic effect of the two excipients.

[0120] In a model protein composition comprising lactate dehydrogenase, the effect of different combinations of a hydrated deep eutectic mixture of choline chloride and sorbitol (molar ratio 5:2) with 4-hydroxyproline (an amino acid) had a synergistic thermostabilizing effect on LDH over the entire concentration range and ratio range tested, resulting in 5% to 36% higher thermostability than expected for the two single components.

[0121] The effects of these combinations on the vaccine proteins were similar.

[0122] Among this third group of selected excipients, it was 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 thermal stabilizers by definition under certain conditions.

[0123] After evaluating all the tests, it was found that

[0124] 1. There are many excipients that can stabilize protein formulations against the deleterious effects of elevated temperatures.

[0125] 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; a hydrated deep eutectic mixture of choline chloride and a second component (sorbitol or glucose or sucrose or glycerol); lactose; lactulose; maltose; melibiose; meglumine; choline dihydrogen phosphate; TMAO; betaine; Hydroxylectoine; Ectoine); inositol; citrulline; ammonium acetate; magnesium gluconate; mannitol; tris(2-hydroxyethyl)methylammonium sulfate.

[0126] b. Particularly effective heat stabilizers are: 4-OH-proline; L-ornithine; lactobionic acid; potassium dihydrogen phosphate; 2-hydroxyethyl-trimethylammonium L-(+)-lactic acid; a hydrated deep eutectic mixture of choline chloride and a second component (sorbitol or glucose or sucrose); lactose; lactulose; maltose; melibiose; meglumine; choline dihydrogen phosphate; TMAO; betaine; Hydroxylectoine.

[0127] c. The most effective and widely used excipients are TMAO, choline dihydrogen phosphate, lactulose, maltose, and melibiose.

[0128] 2. The concentration range required for each excipient is different, and the range in which each individual excipient a) stabilizes a particular protein and b) stabilizes a particular protein at temperatures more than 5°C above the temperature at which the protein is still effective is also different.

[0129] 3. Experiments show that each of the excipients listed in Table 4 can stabilize the protein at temperatures above 60°C.

[0130] 4. The excipient is compatible with a wide variety of buffers used in biology / biochemistry (HEPES, phosphate buffer, histidine buffer, citrate buffer).

[0131] 5. The excipient can be used in the pH range of 6 to 8.

[0132] 6. The excipient is compatible with the reducing agent (ie DTT).

[0133] 7. The excipient is compatible with chloride salts.

[0134] 8. Experiments show that melibiose and maltose are suitable for inhibiting protein aggregation.

[0135] 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.

[0136] [The molten globule state is a compact, partially folded conformation of a protein with near-native compactness, significant secondary structure, little detectable tertiary structure, and increased solvent-exposed hydrophobic surface area relative to the native state. (Anthony L Fink, University of California, Santa Cruz, California, USA, published online in 2001)]

[0137] 10. The combined use of two excipients can have very different effects:

[0138] a. Salts in combination with sugar acids exhibit additive behavior: the resulting T m The change in is similar to the sum of the changes in the unfolding temperature caused by the individual components.

[0139] b. Salts in combination with osmotic agents exhibit additive behavior: the resulting change in Tm is similar to the sum of the changes in unfolding temperature caused by the individual components.

[0140] c. Salts in combination with osmotic agents exhibit additive behavior: the resulting change in Tm is similar to the sum of the changes in unfolding temperature caused by the individual components.

[0141] d. Salts in combination with the hydrated deep eutectic mixture of choline chloride and sorbitol exhibit complex behavior:

[0142] i. When the use of salt exceeds that of ionic liquid, it exhibits additive behavior

[0143] ii. When the ionic liquid is used in excess of the salt, it is still thermally stable, but less efficient than the sum of the two individual components

[0144] e. Hydrated deep eutectic mixture of choline chloride and sorbitol in combination with lactobionic acid:

[0145] i. The interaction between ionic liquids and sugar acids is concentration-dependent

[0146] ii. The interaction between ionic liquids and sugar acids also depends on the ratio of ionic liquids to sugar acids

[0147] iii. When a sufficient excess of ionic liquids was used, a synergistic effect of the two excipients on the thermal stability of the protein was observed.

[0148] iv. The ratio of ionic liquid to sugar acid required for this synergistic effect increases with the increase of ionic liquid fraction

[0149] v. The concentration of sugar acid must be a minimum of 0.25 M to observe a synergistic effect of the two excipients f. The hydrated deep eutectic mixture of choline chloride and sorbitol in combination with osmotic pressure agents / amino acids has a synergistic thermal stabilization effect on LDH, resulting in a thermal stability that is 5%-36% higher than expected from the effects of the two individual components

[0150] The above results were obtained by formulating a panel of different proteins in the test solutions to study the effects of the previously examined excipients as well as the aggregation inhibition by the addition of melibiose and maltose. The proteins selected were from bacterial and animal sources. Among these proteins were enzymes, pore formers and two inactivated toxins. One toxin was inactivated by mutation, while the second toxin was inactivated by formaldehyde cross-linking.

[0151] Since these results have been obtained with these different proteins, it can be inferred from these results that the addition of these selected excipients produces similar effects in formulations in which soluble proteins are contained as the main component.

[0152] Another conclusion drawn from these results is also acceptable, namely that these effects also occur when other components are covalently bound to the corresponding protein, for example, if 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.

[0153] Preparation of specific protein formulations as disclosed herein can be performed by the methods described above and as shown in the examples below.

[0154] This description combined with the given examples enables one of ordinary skill in the art to fully practice the invention.

[0155] The examples given below, which illustrate the invention, will be reviewed in detail in conjunction with the results in the following sections. These reviews are also intended to broadly describe the invention in the context of various protein formulations.

[0156] Therefore, even without further comments, it is assumed that one of ordinary skill in the art will be able to utilize the above description in the broadest scope.

[0157] If there is anything unclear, it is understood that reference should be made to publications and patent documents cited and known to the skilled person. Therefore, the cited documents are considered to be part of the disclosure content of this specification and are incorporated herein by reference.

[0158] In order to better understand and illustrate the present invention, the following examples are given, which are within the scope of the present invention. These examples are also used to illustrate possible variations.

[0159] Furthermore, it is self-evident to the person skilled in the art that in the given examples as well as in the rest of the description, the amounts of the components present in the compositions always only add up to 100% by weight or mol% (based on the entire composition) and that this percentage cannot be exceeded even if higher values ​​can be derived from the percentage range indicated. Therefore, unless otherwise indicated, the percentage data are weight percentages or molar percentages, except for the proportions indicated in the volume data. Example

[0160] Model proteins such as lactate dehydrogenase (rabbit muscle) and lysozyme (hen egg, white) are used herein, as well as vaccine proteins such as anthrax protective antigen (AnthraxPA), tetanus toxoid and diphtheria toxoid (CRM197), to demonstrate effective thermal stabilization by the addition of one or more of the excipients listed above. Liquid formulations are prepared containing proteins at a concentration of 0.05 to 1.8 mg / ml. Described below are the melting temperatures of these proteins in the presence of the most effective and most suitable thermal stabilizers tested. An increase in the melting temperature of a protein is an indicator of increased thermal stability of the protein in the formulation.

[0161] Materials and methods

[0162] Table 1:

[0163]

[0164]

[0165]

[0166] 1) Osmotic pressure agents:

[0167] Examples TMAO

[0168] a) A buffer solution composed of a buffering agent (sodium dihydrogen phosphate, HEPES, histidine) was prepared and the pH value was adjusted to 7, 7.5 or 8.

[0169] b) TMAO was dissolved in a buffer solution at a concentration of 4.2 M and the pH was readjusted to pH 7, 7.5 or 8.

[0170] c) Add vaccine protein anthrax protective antigen or CRM197 or tetanus toxoid to the excipient. Protein concentration is 0.1 mg / ml. Excipient concentration is 4M.

[0171] d) The melting temperature of the protein-excipient mixture was measured by differential scanning fluorimetry and compared with that of the protein solution without excipients

[0172] e) Results: TMAO increased the melting temperature of anthrax protective antigen by 20°C, the melting temperature of tetanus toxoid by 3°C, and the melting temperature of diphtheria toxoid by 7°C.

[0173] 2) Ionic liquids:

[0174] Examples Choline dihydrogen phosphate

[0175] a) Dissolve choline dihydrogen phosphate in water at a concentration of 4 M and adjust the pH to pH 7, 7.5 or 8.

[0176] b) Add vaccine protein anthrax protective antigen or CRM197 or tetanus toxoid to the excipient. Protein concentration is 0.1 mg / ml. Excipient concentration is 3M.

[0177] c) The melting temperatures of the protein-excipient mixtures were measured by differential scanning fluorimetry and compared with protein solutions without excipients

[0178] d) Results: Choline dihydrogen phosphate increased the melting temperature of anthrax protective antigen by 19°C, the melting temperature of tetanus toxoid by 5°C, and the melting temperature of diphtheria toxoid by 13°C

[0179] 3) Salt:

[0180] Examples Potassium dihydrogen phosphate

[0181] a) Prepare a buffer solution containing buffering agents (sodium dihydrogen phosphate, HEPES, histidine) and adjust the pH value to 7, 7.5 or 8.

[0182] b) Dissolve potassium dihydrogen phosphate at a concentration of 1.2 M in the buffer solution and readjust the pH to pH 7, 7.5 or 8.

[0183] c) Add vaccine protein anthrax protective antigen or CRM197 or tetanus toxoid to the excipient. Protein concentration is 0.1 mg / ml. Excipient concentration is 1M.

[0184] d) The melting temperatures of protein-excipient mixtures were measured by differential scanning fluorimetry and compared with protein solutions without excipients.

[0185] e) Results: Potassium dihydrogen phosphate increased the melting temperature of anthrax protective antigen by 10°C, the melting temperature of tetanus toxoid by 2°C, and the melting temperature of diphtheria toxoid by 5°C

[0186] 4) Sugar:

[0187] Examples Lactulose

[0188] a) Prepare a buffer solution composed of buffering agents (sodium dihydrogen phosphate, HEPES, histidine) and adjust the pH value to 7, 7.5 or 8

[0189] b) Dissolve lactulose at a concentration of 2 M in a buffer solution and readjust the pH to pH 7, 7.5 or 8

[0190] c) Add vaccine protein anthrax protective antigen or CRM197 or tetanus toxoid to the excipient. Protein concentration is 0.1 mg / ml. Excipient concentration is 1.7 M

[0191] d) The melting temperature of the protein-excipient mixture was measured by differential scanning fluorimetry and compared with that of the protein solution without excipients

[0192] e) Results: Lactulose increased the melting temperature of anthrax protective antigen by 18°C ​​and that of tetanus toxoid by more than 20°C (the equipment did not allow accurate measurement of T at such high temperatures). m ), which increases the melting temperature of diphtheria toxoid by 24°C

[0193] Other results :pass Melibiose Prevent gatherings

[0194] ( Fig.28 and 29 )

[0195] Melibiose was dissolved in a buffer solution at a concentration of 2.75 M and the pH was adjusted to 7, 7.5 or 8.

[0196] Vaccine protein suspension of anthrax protective antigen or CRM197 or tetanus toxoid was added to the excipient solution. Protein concentration was 0.1 mg / ml. Excipient concentration was 2 M or 2.4 M or 2.5 M.

[0197] result:

[0198] When 2M or 2.4M excipients were used, aggregation of diphtheria toxoid was inhibited up to temperatures of 80° C. When 2.5M excipient was used, no aggregation was observed.

[0199] Aggregation of tetanus toxoid was inhibited when 2.4M or 2.5M excipient was used.

[0200] 5) Sugar acids and their salts:

[0201] Examples Lactobionic acid

[0202] a) Prepare a buffer solution composed of a buffering agent (HEPES, histidine) and adjust the pH to 7 or 7.5

[0203] b) Lactobionic acid was dissolved in a buffer solution at a concentration of 0.6 M and the pH was readjusted to pH 7 or 7.5

[0204] c) Add vaccine protein anthrax protective antigen or tetanus toxoid to the excipient. Protein concentration is 0.1 mg / ml. Excipient concentration is 0.5 M

[0205] d) The melting temperature of the protein-excipient mixture was measured by differential scanning fluorimetry and compared with that of the protein solution without excipients

[0206] e) Results: Lactobionic acid increased the melting temperature of anthrax protective antigen by 7°C and that of tetanus toxoid by 4°C

[0207] 6) Amino sugars:

[0208] Examples Meglumine

[0209] a) Prepare a buffer solution consisting of a buffer (sodium phosphate or sodium citrate) and adjust the pH to 6 or 7.6

[0210] b) Dissolve meglumine at 3M in buffer solution and readjust the pH to pH 6 or 7.6 [if pH is 7.6, add 1 mM DTT (1,4-dithiothreitol)]

[0211] c) Add protein lactate dehydrogenase or lysozyme to the excipient solution. Protein concentration is 0.1 mg / ml. Excipient concentration is 1.5 M.

[0212] d) The melting temperatures of protein-excipient mixtures were measured by differential scanning fluorimetry and compared with protein solutions without excipients.

[0213] e) Results: Meglumine increased the melting temperature of lactate dehydrogenase by 3°C and that of lysozyme by 6°C

[0214] 7) Amino alcohols: Examples Mannitol

[0215] a) Prepare a buffer solution consisting of a buffer (sodium phosphate) and adjust the pH to 6 or 7.6.

[0216] b) Mannitol was dissolved in a buffer solution at a concentration of 0.5 M, the pH was readjusted to pH 7.6, and 1 mM DTT was added.

[0217] c) The protein lactate dehydrogenase was added to the excipient solution in an amount resulting in a protein concentration of 0.1 mg / ml. The excipient concentration was 0.45M.

[0218] d) The melting temperatures of protein-excipient mixtures were measured by differential scanning fluorimetry and compared with protein solutions without excipients.

[0219] e) Results: Mannitol increased the melting temperature of lactate dehydrogenase by 2°C.

[0220] Figure 1 The results for the added excipients that showed the best performance are summarized. This means that the results for the added excipients that caused the largest change in the melting temperature of the proteins tested and showed the broadest applicability are summarized. These excipients were suitable as stabilizers for all three vaccine proteins tested. Surprisingly, these were found to be the following excipients, which themselves belong to very different groups of substances:

[0221] f) TMAO starts to show thermal stabilization effect from a concentration of 250 mM. The best stabilization results are obtained when the concentration in the solution is in the range of 1 M to 4 M.

[0222] g) Choline dihydrogen phosphate showed a thermal stabilization effect starting from a 1 molar solution. The best results were found for the composition when the concentration was in the range of 2M to 4M.

[0223] h) Melibiose showed a thermostabilizing effect starting from 250 mM. In this context, the best results were found for the composition when the concentration was in the range of 1 M to 2.5M.

[0224] i) Maltose showed a thermal stabilization effect starting from 500 mM. The best results were found for the composition when the concentration was in the range of 1 M to 1.8 M,

[0225] and

[0226] j) Lactulose showed a thermal stabilizing effect starting from 250 mM. The best results were found for the composition when the concentration was in the range of 0.5M to 1.7M.

[0227] Figure 2 The results on the changes in melting temperature caused by various excipients under optimal conditions are summarized as follows:

[0228] Experimental setup and considerations

[0229] In order to evaluate the most suitable additive or excipient for thermal stabilization and the amount that needs to be added to produce the desired effect, test formulations of AnthraxPA as a vaccine were prepared and examined over three days. The detailed formulation compositions are summarized in Table 2 at the end of this chapter. The purpose of these experiments was to find the most suitable excipient or mixture of excipients for thermal stabilization of protein compositions over several days.

[0230] Since stabilizers have to be used in high concentrations so far, such compositions cannot be used to prepare prefilled syringes, since patients may not tolerate the stabilizers. Therefore, a dilution step is required before administration to patients. Another aim of these experiments is also to determine the highest possible tolerance level of stabilizers for in vitro assays. Based on the experimental results, the protein concentration can be adjusted and a vaccine stock solution can be generated, which can both provide thermal protection for the solute protein and can still be effectively administered to patients after dilution.

[0231] For example, it was found that patients are most likely to tolerate melibiose at concentrations up to 250 mM. From previous screening, it is known that at least 2.25 M of melibiose is required to stabilize AnthraxPA at high temperatures. Therefore, the protein concentration is increased by 2250 mM (stock) / 250 mM (final value) = 9 times. Therefore, at least a nine-fold amount of vaccine stock preparation is required.

[0232] Based on these considerations, test preparations of TMAO, choline dihydrogen phosphate, lactulose, maltose and melibiose were prepared for investigation as temperature-stable additives. Fluorescence spectroscopy was used to verify whether all protein samples were natively folded and whether the selected excipients could also thermally stabilize elevated protein concentrations (up to 1.6 mg / ml). Incubations were then performed at the following temperatures:

[0233] 4°C: Native (no excipients): These are unchanged control samples.

[0234] 50℃: Natural (no excipients), TMAO, choline dihydrogen phosphate, lactulose, maltose

[0235] 55℃: TMAO, choline dihydrogen phosphate, lactulose, melibiose, maltose

[0236] 60℃: TMAO, choline dihydrogen phosphate, lactulose, melibiose, maltose

[0237] 65℃: Melibiose

[0238] Melibiose was tested at higher temperatures compared to all other excipients because it induces higher thermal stability based on the transition midpoint.

[0239] After each incubation time of 6 h, 24 h, 32 h, 48 h and 72 h, a small sample was taken and the structural state and stability of the contained protein was assessed by fluorescence spectroscopy.

[0240] Table 2 : Summary of test formulations used herein.

[0241]

[0242] Analysis parameters

[0243] The results presented in the following sections are based on two parameters measured by fluorescence spectroscopy. The changes in these two parameters are monitored over time to determine whether AnthraxPA is still native or has been destroyed. The two readouts described below are needed to find out whether the included protein remains stable throughout the experiment.

[0244] AnthraxPA is stabilized at temperatures above 50°C

[0245] In its native preparation, AnthraxPA has an unfolding transition temperature of 48°C, which does not allow storage of the protein at temperatures of 50°C or higher. Figure 3 The storage stability of Anthrax PA at 50°C as determined by fluorescence spectroscopy is shown.

[0246] Figure 3 : Stability of AnthraxPA at 50℃.

[0247] Figure 3 a) (Left) Folded fraction. Natively formulated Anthrax PA shows an immediate drop in natively folded protein. All tested thermal stabilizers prevent such a drop.

[0248] Figure 3 b) (right) Normalized transition temperatures. Data between the red lines indicate that the protein is stable.

[0249] Anthrax PA without heat stabilizing excipients denatured immediately. All selected excipients were stabilized and allowed to be stored at 50°C for 72 hours.

[0250] Figure 4 The storage stability of Anthrax PA at 55°C is shown.

[0251] Figure 4 : Stability of AnthraxPA at 55°C.

[0252] Figure 4 a) (Left) Folded fraction.

[0253] Figure 4 b) (right) Normalized transition temperatures. Data between the red lines indicate that the protein is stable.

[0254] When the test formulations were incubated at 55°C, it was found that both melibiose and maltose were able to induce sufficient thermal stabilization to allow storage at 55°C for three days.

[0255] In addition, AnthraxPA was incubated at 60°C for three days. Figure 5 shown.

[0256] Figure 5 : Stability of AnthraxPA at 60℃.

[0257] Figure 5 a) (Left) Folded fraction. Data below the red line indicate stable protein.

[0258] Figure 5 b) (right) Normalized transition temperatures. Data between the red lines indicate that the protein is stable.

[0259] At 60° C., the heat stabilizing capacity of the excipients used herein ends. Only melibiose was able to tolerate a brief exposure to 60° C. for more than 6 hours, but less than 24 hours.

[0260] CRM197 is stabilized at temperatures above 50°C

[0261] CRM197 in its native formulation shows a biphasic unfolding transition when measured by fluorescence spectroscopy. The initial transition is associated with a local loss of structure, while on a global scale the protein remains structurally intact. Although the excipients used herein have an effect on the overall structural stability of the CRM, they cannot prevent more localized losses of structure. Therefore, the same criteria as AnthraxPA cannot be used when studying the stability of the CRM.

[0262] For data interpretation, the following two modifications are necessary: ​​First, the median unfolding temperature cannot be used as a readout. It has been determined that conformational perturbations occur due to localized loss of structure that cannot be stabilized by the excipients used herein. In addition, changes in this initial transition may affect the determination of the second unfolding temperature and lead to an erroneous assessment of the overall structural state based on this. Secondly, the extent of damage caused to the temperature-dependent region of the CRM affects the fluorescence emission rate from 350nm to 330nm. Therefore, the extension of structural damage may be an upper limit rather than the actual extent of structural loss. In order to evaluate the role of the excipients tested herein, all results were compared with the protein in its native preparation incubated at 50°C and denatured.

[0263] In its native formulation, CRM197 has an overall unfolding temperature of 53°C, which does not allow for prolonged storage at 50°C. Therefore, the negative control will be the native formulated CRM incubated at 50°C. Figure 6 In Figure 2, the effect of melibiose and maltose on the thermal stability of CRM is shown.

[0264] Figure 6 : Stability of CRM197 at 50°C. Melibiose and maltose significantly inhibited the heat-induced unfolding, which lasted for 3-4 days.

[0265] The results showed that maltose stabilized CRM197 for 72 hours at 50°C. Melibiose stabilized CRM for 96 hours at 50°C.

[0266] Figure 7 The storage stability of CRM197 at 55°C is shown.

[0267] Figure 7 : Stability of CRM197 at 55°C. Melibiose and maltose significantly inhibited the heat-induced unfolding, which lasted for 3-4 days.

[0268] The results showed that maltose stabilized CRM197 for 72 hours at 55°C. Melibiose stabilized CRM for 96 hours at 55°C.

[0269] Figure 8 The storage stability of CRM197 at 60°C is shown in turn.

[0270] Figure 8 : Stability of CRM197 at 60°C. Melibiose and maltose significantly inhibited the heat-induced unfolding, which lasted for 3-4 days.

[0271] It is noteworthy that the fraction folded was greater at 60°C for 6 hours compared to 55°C or 50°C, but there was still a clear difference from the native preparation and the fully denatured CRM. The results of these tests show that maltose stabilizes CRM197 for 48 hours at 60°C. Melibiose stabilizes CRM for 24 hours at 60°C.

[0272] Further experiments with thermostable enzymes as proteins

[0273] As described previously for experiments and studies using AnthraxPA and CRM197, formulations with lactate dehydrogenase and lysozyme as model proteins were also investigated. The excipients described above were added at appropriate concentrations and tested for their possible thermal stabilization effects.

[0274] a. Preparation of ionic liquids based on choline chloride

[0275] Other potential thermal stabilizers from a group of ionic liquids, four biodegradable ionic liquids, were selected and prepared based on compounds commonly 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.

[0276] The ionic liquids were prepared as hydrated deep eutectic mixtures in three steps. First, 0.5 moles of choline chloride were mixed with respective amounts of the two sugar (sorbitol and glucose) components and used in an amount of 0.2 moles (ratio 5:2). Glycerol was used in an amount of 0.5 moles (ratio 1:1). Sucrose was used in an amount of 0.125 moles (ratio 4:1). The prepared mixture was then heated to 100°C for two hours with stirring. Although none of the substances except glycerol and sorbitol would become liquid at this temperature, these particular mixtures began to melt. Subsequently, 20% w / w was added to the mixture, and when the solid substances were completely melted, the resulting ionic liquids were cooled to room temperature, where they remained in their liquid form.

[0277] In order to compare the amount of substance used for thermal stabilization using the ionic liquid prepared in this way with other excipients that are only soluble in water / buffer, the volume of the resulting liquid is measured and the concentration of choline chloride therein (as if it were dissolved) is calculated. Typically, about 100-150 ml of ionic liquid is prepared that exhibits a molar concentration of choline chloride of 3.3-4 M.

[0278] b. Experimental methods

[0279] As described previously, the unfolding transition midpoints of the included proteins were determined by fluorescence spectroscopy, which allowed high throughput screening by nano-differential scanning fluorimetry (nanoDSF).

[0280] Here, the protein was used at a concentration of 0.05-0.1 mg / ml, while the excipient was titrated to a concentration close to its solubility in the applied buffer. The appropriate amount of protein solution was then mixed with the appropriate amount of excipient solution, and the mixture was equilibrated at room temperature for 15 minutes.

[0281] The prepared protein solution was heated at a heating rate of 1 °C / min, and the fluorescence emission ratio at 350 / 330 nm was recorded and plotted against temperature. In general, the resulting curve resembles a Boltzmann function, and the inflection point of the curve represents the average unfolding temperature T m .

[0282] From this curve, by m赋形剂 Subtract T from the preparation m天然 To determine T m The change of dT m .

[0283] By definition, causing a dT > 5°C m The excipient is a good stabilizer. If dT m <5°C and >0°C, the excipient is defined as a poor stabilizer. Negative dTm Identification of molecules as protein destabilizers.

[0284] c. Results using lactate dehydrogenase as a model enzyme

[0285] Lactate dehydrogenase (LDH) was formulated in 50 mM sodium phosphate, 1 mM DTT, pH 7.6. To probe the effect of the molecules on LDH, the molecules were added at various concentrations to the protein formulated in exactly this buffer. The protein was used at a concentration of 0.05 mg / ml.

[0286] Fig. 9 : Shows the effect of excipient candidates on the mean unfolding temperature of LDH. The black line indicates dT m = 0°C. The red line indicates dT m = 5° C. Molecules capable of stabilizing LDH by more than 5° C. were subjected to a second screening using different model proteins.

[0287] Fig. 9 The first group of excipients from the group of various compounds at this stage and their effect on the melting temperature of LDH are shown. By definition, 4-hydroxyproline, ornithine, lactobionic acid and potassium dihydrogen phosphate are good thermal stabilizers, while other substances such as ectoine, inositol and citrulline have a negative effect on the T of LDH. m The relatively small effect makes it unsuitable for testing purposes.

[0288] Fig.10 The melting temperature dT of LDH is shown m The changes in , which were determined by the addition of Na-Ser, OH-Lys, choline lactate, choline Cl / sorbitol, choline Cl / glycerol, choline Cl / glucose, choline Cl / sucrose.

[0289] Fig.10 : Effect of excipient candidates on the mean unfolding temperature of LDH. The black line indicates dT m = 0°C. The red line indicates dT m = 5°C. Molecules suitable for stabilizing LDH by more than 5°C were subjected to a second screen using different model proteins. To use the same plot 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.

[0290] Among this group of excipients, hydrated deep eutectic mixtures of choline lactate and choline dihydrogen phosphate with sorbitol, glucose and sucrose, respectively, were identified as good thermal stabilizers and entered the second pre-screening step.

[0291] In the following tests, the effects of lactulose, magnesium gluconate, polysucrose 400, mannitol, maltose and ammonium acetate on the thermal stability of LDH were tested. These excipients are identified herein as being suitable for thermally stabilizing LDH. Fig.11 : Shows the effect of lactulose, magnesium gluconate, polysucrose 400, mannitol, maltose and ammonium acetate on the mean unfolding temperature of LDH. The black line indicates dT m = 0°C. The red line indicates dT m = 5° C. Molecules capable of stabilizing LDH by more than 5° C. were subjected to a second screening using different model proteins.

[0292] In particular, lactulose and maltose were identified as good heat stabilizers. Ammonium acetate, magnesium gluconate and mannitol were also identified as protein stabilizers.

[0293] d. Results using lysozyme as a model enzyme

[0294] Lysozyme was formulated as 20 mM sodium citrate, 55 mM NaCl and 1 mM DTT at pH 6.0. To probe the effect of the molecules on lysozyme, the molecules were added at various concentrations to the protein formulated in exactly this buffer. The protein was used at a concentration of 0.1 mg / ml.

[0295] Fig.12 : Effects of hydroxyproline, L-citrulline, L-ornithine, potassium dihydrogen phosphate, lactobionic acid, meglumine, ectoine, inositol and methanesulfonic acid on the average unfolding temperature of lysozyme. The black line indicates dT m = 0°C. The red line indicates dT m =5℃.

[0296] From this group of molecules, hydroxyproline, L-ornithine, potassium dihydrogen phosphate, lactobionic acid and meglumine showed the desired thermal stabilization effect. Ectoine, tris(2-hydroxyethyl)methylammonium sulfate (MeOSO3) and citrulline were not able to stabilize lysozyme sufficiently.

[0297] Fig.13 Results are shown for a second set of substances tested in lysozyme:ionic liquids.

[0298] Fig.13 : Effect of excipient candidates on the mean unfolding temperature of LYZ. The black line indicates dT m = 0°C. The red line indicates dT m = 5°C. Based on the above definition, molecules that can stabilize LYZ by more than 5°C are identified as good thermal stabilizers. To use the same type of plot for hydrated deep eutectic liquids as for other excipients, the molar concentration of choline chloride was estimated based on the weight of material used and the volume of liquid prepared.

[0299] Among the excipient candidates tested here, hydrated deep eutectic mixtures of choline lactate and choline dihydrogen phosphate with sorbitol, glucose, and sucrose, respectively, were identified as good thermal stabilizers.

[0300] A summary of the concentration ranges used for each excipient to stabilize at least one model protein by 5°C or more is shown in Table 3. In general, the excipient molar concentration is given. For ionic liquids, the volume percentage of the excipient used is given, as the molar concentration of only one component would be misleading.

[0301] Table 3:

[0302]

[0303] DNQ: Failed; None: None of the tested concentrations induced the required thermal stability

[0304] Results show the efficiency of excipient combinations using lactate dehydrogenase

[0305] In order to conduct a preliminary feasibility assessment, the compatibility of various excipients combined with each other was tested. The purpose is to clarify whether the synergistic effect of the two excipients used in combination will improve the thermal stabilization of the two molecules. Salts and ionic liquids are combined with each other and with osmotic pressure agents and sugar acids. All experiments are carried out using lactate dehydrogenase in the corresponding natural preparations (50mM sodium phosphate, 1mM DTT, pH 7.6). The protein concentration is adjusted to a concentration of 0.05mg / ml. LDH is added to a solution containing two excipients of different proportions and concentrations. The prepared composition was incubated at room temperature for 15 minutes. Then, as described above, the average unfolding temperature of LDH was measured using nano differential scanning fluorimetry at a heating rate of 1°C / min.

[0306] Fig.14 Summarizes the results of the combination of potassium dihydrogen phosphate and lactobionic acid

[0307] Fig.14 : Effects of different combinations of potassium dihydrogen phosphate and lactobionic acid on the model protein lactate dehydrogenase - blue indicates the T of the combined preparations m The stacked black and red are the T values ​​determined for the individual excipients, respectively. m If the blue bar exceeds the black and red stacked bars, a synergy will be detected.

[0308] The combination of potassium dihydrogen phosphate and lactobionic acid was found to induce a similar level of thermal stability for LDH as would be expected from the individual components. In contrast, when this combination was used, a thermal stabilization effect of 90% to 104% could be observed.

[0309] This leads to the conclusion that the corresponding salts and sugar acids are compatible in this protein solution and lead to an additive effect.

[0310] Fig.15 The results for the combination of potassium dihydrogen phosphate and 4-OH-proline are summarized.

[0311] Fig.15 : Effects of different combinations of potassium dihydrogen phosphate and lactobionic acid on the model protein lactate dehydrogenase. Blue indicates the T of the combined preparations m The stacked black and red are the T values ​​determined for the individual excipients, respectively. m If the blue bar exceeds the black and red stacked bars, a synergy conclusion will be drawn.

[0312] The combination of potassium dihydrogen phosphate and 4-OH-proline was found to induce a similar level of thermal stability for LDH as would be expected if the individual components were added. In contrast, when these components were used in combination, a thermal stabilization effect of 95% to 100% could be observed.

[0313] Therefore, it was concluded that the thermal stabilization effects of salt and osmotic agents are compatible and act in an additive manner.

[0314] Fig.16 Summarizes the results for potassium dihydrogen phosphate and the choline chloride / sorbitol combination

[0315] Fig.16 : Effects of different combinations of potassium dihydrogen phosphate and choline chloride with sorbitol (molar ratio 5:2) on the model protein lactate dehydrogenase. Blue indicates the T of the combined preparations m The stacked black and red are the T values ​​determined for the individual excipients, respectively. m If the blue bar exceeds the black and red stacked bars, a synergy conclusion will be drawn.

[0316] It was observed herein that the combination of potassium dihydrogen phosphate with the hydrated deep eutectic mixture of choline chloride and sorbitol (molar ratio 5:2) was incompatible when the hydrated deep eutectic mixture of choline chloride and sorbitol (molar ratio 5:2) was used in excess compared to potassium dihydrogen phosphate. In case of excess of ionic liquid compared to salt, 58% to 81% effectiveness was found. However, when salt was added in excess to ionic liquid, the thermal stabilization effect of both was additive in the range of 99-102%.

[0317] Therefore, the thermal stabilization effect of salts and ionic liquids is manifested as follows:

[0318] - Salts are compatible and additive when present in excess compared to ionic liquids

[0319] - Ionic liquids are incompatible when present in excess compared to salts, but are still thermally stable.

[0320] Fig.17 The results for the combination of added choline Cl / sorbitol and lactobionic acid are summarized. These results are more complex than the data for the previously tested combinations.

[0321] Fig.17 : Effects of different combinations of hydrated deep eutectic mixtures of choline chloride and sorbitol (molar ratio 5:2) with lactobionic acid in model protein lactate dehydrogenase compositions. Blue indicates T of the combined formulations m The stacked black and red are the T values ​​determined for the individual excipients, respectively. m If the blue bar exceeds the black and red stacked bars, a synergy conclusion will be drawn.

[0322] Depending on the concentration of the ionic liquid included, different interactions were observed: if only a low concentration of ionic liquid was used (0.5 M) and lactobionic acid was added in a 2:1 ratio, a synergistic effect was obtained and the model protein was more stabilized than would be caused by the individual components.

[0323] Using 1.5 molar ionic liquid, similar effects were observed, but under these conditions, a greater excess of ionic liquid was required to produce a synergistic effect. When the excess of ionic liquid was increased to 6 times, synergistic stabilization was found. At very high concentrations of ionic liquid (2.5 M), only a small concentration of lactobionic acid could be detected. Using a 10-fold excess of ionic liquid over lactobionic acid resulted in a synergistic effect of the two. However, when only 0.15 M lactobionic acid (16:1) was used, an additive effect was more likely to be observed.

[0324] The following conclusions were drawn regarding the interaction between ionic liquids and sugar acids in the model composition:

[0325] - The interaction between ionic liquids and sugar acids is concentration-dependent

[0326] -The interaction between ionic liquids and sugar acids also depends on the ratio of ionic liquid to sugar acid

[0327] - When a sufficient excess of ionic liquid is used, a synergistic effect of the two excipients on the thermal stability of the protein is observed

[0328] -The ratio of ionic liquid to sugar acid required for this synergistic effect increases with the increase of ionic liquid fraction

[0329] - The concentration of sugar acid must be at least 0.25 M to observe a synergistic effect of the two excipients Fig.18 Summarizes the results of the combination of choline chloride / sorbitol and 4-OH-proline

[0330] Fig.18 : Effects of different combinations of hydrated deep eutectic mixtures of choline chloride and sorbitol (molar ratio 5:2) with 4-hydroxyproline in a model protein composition containing lactate dehydrogenase. Blue indicates T of the combined formulations m The stacked black and red are the T values ​​determined for the individual excipients, respectively. m If the blue bar exceeds the black and red stacked bars, a synergy conclusion will be drawn.

[0331] The results of the measurements indicate that the combination of ionic liquids and amino acids has a synergistic thermostabilizing effect on LDH over the entire concentration range and ratios tested, resulting in 5% to 36% higher thermostability than would be expected from the two single components.

[0332] Table 4 below lists a summary of the excipient combinations used, their concentration ranges, and their effects on LDH.

[0333] Table 4:

[0334]

[0335] *Molar concentration is based on the amount of choline chloride in the volume produced.

[0336] Screening of thermostable excipients using vaccine proteins

[0337] In the next chapter, the results of a screen of excipient molecules using three rather different vaccine proteins are discussed. Not all potential excipient molecules were tested on all vaccine proteins. The aim was to identify excipient molecules that were able to increase the mean unfolding temperature of three very different proteins to temperatures above 60°C.

[0338] Experimental methods

[0339] We employ fluorescence spectroscopy to determine the unfolding transition midpoint of our proteins. Specifically, we use nanoscale differential scanning fluorimetry, which allows for high-throughput screening.

[0340] Protein is used with a concentration of 0.1 mg / ml, which corresponds well to the protein concentration of commercial vaccine dosage. Excipients are titrated to a concentration close to their solubility limit. All excipients are prepared in the natural preparation buffer of vaccine protein, except choline dihydrogen phosphate. The latter is dissolved in water, and pH is adjusted to match the pH (detailed information see below) of the natural vaccine protein preparation. In this case, dihydrogen phosphate ions will serve as buffers. Protein is mixed with excipients, and it is balanced at room temperature for 15 minutes.

[0341] Screening of thermostable excipients using anthrax protective antigen

[0342] Anthrax protective antigen (AnthraxPA) is the main protein component of anthrax vaccine. Its native formulation is 5 mM HEPES and 50 mM NaCl at pH 7.5. The native average unfolding temperature is 48°C. Three classes of excipients were used in the tests to stabilize AnthraxPA: sugars, ionic liquids / salts, and osmotic agents. The results obtained were compared with those using trehalose, a widely used excipient for various purposes.

[0343] Fig.19 Results for thermal stabilization of AnthraxPA using sugars are shown.

[0344] Fig.19 : Effect of sugars as excipient candidates on the mean unfolding temperature of AnthraxPA. The black line indicates dT m = 0°C. The red line indicates dT m = 5° C. Molecules capable of stabilizing AnthraxPA by more than 5° C. were subjected to a second screening using different model proteins.

[0345] In the first group, lactulose, melibiose, and maltose were identified as good thermal stabilizers and will be used for further evaluation. The required 5°C additional thermal stability was observed at relatively low concentrations (250 mM). Melibiose was clearly most effective in reducing the T of AnthraxPA to 1.5°C at approximately 30°C. m All sugars tested in this group were more effective thermal stabilizers than trehalose.

[0346] Fig. 20 Results showing the use of an osmotic agent to heat stabilize AnthraxPA

[0347] Fig. 20 : Effect of osmotic pressure agents as excipient candidates on the mean unfolding temperature of AnthraxPA. The black line indicates dT m = 0°C. The red line indicates dT m= 5° C. Molecules capable of stabilizing AnthraxPA by more than 5° C. were subjected to a second screening using different model proteins.

[0348] In this second group, there are several known protein stabilizers, such as taurine, which cannot stabilize the vaccine proteins used herein. Among the osmotic agents, TMAO, betaine, 4-hydroxyproline, Hydroxylectoine and lactobionic acid were found to be good thermal stabilizers according to the definition of the present invention and were therefore selected for further testing. However, only TMAO was superior to trehalose as a thermal stabilizer.

[0349] Fig.21 Results are shown for the thermal stabilization of AnthraxPA using salts and ionic liquids.

[0350] Fig.21 : Effect of excipient candidates on the mean unfolding temperature of AnthraxPA. The black line indicates dT m = 0°C. The red line indicates dT m = 5°C. Molecules capable of stabilizing AnthraxPA by more than 5°C were subjected to a second screen using a different model protein. To use the same type of plot 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.

[0351] In this third group of molecules, it was found that the 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 thermal stabilizers under certain conditions according to the above definition.

[0352] Screening of thermostable excipients using tetanus toxoid

[0353] Tetanus toxoid (T d ) is an inactivated form of Clostridium tetanii toxin and is prepared by formaldehyde cross-linking of toxin monomers. The use of formaldehyde cross-linking produces a large protein complex that is no longer toxic but still presents an antigenic structure recognized by the immune system. Its natural preparation is 100 mM histidine and 100 mM NaCl at pH 7.0. The natural average unfolding temperature is 67°C. In order to make T d For stabilization, three classes of excipients were used: sugars, ionic liquids / salts and osmolytes were compared with the results obtained for trehalose, a widely used excipient for various purposes.

[0354] Fig. 22 The use of sugars to thermally stabilize T d result.

[0355] Fig. 22 : Sugars as excipient candidates for T d The black line indicates dT m = 0°C. The red line indicates dT m = 5°C. This will enable T d Molecules stabilizing more than 5°C were subjected to a second screen using a different model protein.

[0356] In the first group, lactulose, melibiose and maltose are considered good thermal stabilizers. The highest temperature line indicates that for the affected excipient concentrations, a complete unfolding transition cannot be observed because the equipment cannot heat the sample further and the unfolding transition is incomplete. Like the previously discussed experiments with AnthraxPA, lactulose, melibiose and maltose are more effective thermal stabilizers compared to trehalose.

[0357] Fig.23 The use of osmotic pressure agents to thermally stabilize T d result.

[0358] Fig.23 : Osmotic pressure agents as excipient candidates for T d The black line indicates dT m = 0°C. The red line indicates dT m = 5°C. This will enable T d Molecules stabilizing more than 5°C were subjected to a second screen using a different model protein.

[0359] In this second group of excipients, we observed that only Hydroxylectoine could be identified as a good thermal stabilizer. It was also the only substance tested in this group that performed better than trehalose.

[0360] Fig.24 The use of ionic liquids to thermally stabilize T d result.

[0361] Fig.24 : Excipient candidates for T d The black line indicates dT m = 0°C. The red line indicates dT m = 5°C. This will enable T d Molecules that stabilized more than 5°C were subjected to a second screen using a different model protein. To use the same type of plots for the hydration 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.

[0362] In this third group of excipients, choline dihydrogen phosphate was observed to be the most effective thermal stabilizer in this group and more effective than trehalose. The hydrated deep eutectic mixtures of choline chloride with sorbitol, glucose and sucrose, respectively, can be classified as good thermal stabilizers.

[0363] Screening of thermostable excipients using diphtheria toxoid CRM197

[0364] Diphtheria toxoid (CRM197) is an inactivated form of the toxin of Corynebacterium diphteria. The introduction of point mutations renders the toxin harmless, but still presents an antigenic structure recognized by the immune system. Its natural preparation is 20mM HEPES and 150mM NaCl at pH 8.0. The tertiary structure unfolding of CRM197, which is the type of unfolding monitored using fluorescence spectroscopy, is very complex: it unfolds in a two-phase transition, the first transition representing a local structural change, and the second transition representing the overall unfolding of the protein. Since neither temperature can be specifically associated with the ability of CRM197 to induce immunity, both transitions are analyzed based on the effectiveness of our excipients. Again, three categories of excipients are used here: sugars, ionic liquids / salts, and osmotic agents, and compared with the results using trehalose, which is a widely used excipient for various purposes.

[0365] Fig.25 Results are shown for heat stabilization of CRMs using sugars.

[0366] Fig.25 : Effect of sugars as excipient candidates on the mean unfolding temperature of CRMs. The red line indicates dT m = 5° C. Molecules that are able to stabilize the CRM by more than 5° C. locally (closed symbols) and globally (open symbols) are considered excipient candidates and screened with different vaccine proteins if necessary.

[0367] In this first group, lactulose, melibiose and maltose were identified as good thermal stabilizers that performed better than trehalose.

[0368] Fig.26 Results are shown for heat stabilization of CRM using an osmotic agent.

[0369] Fig.26 : Effect of osmotic pressure agents as excipient candidates on the mean unfolding temperature of CRMs. The black line indicates dT m = 0°C. The red line indicates dT m = 5° C. Molecules that were able to stabilize the CRM by more than 5° C. locally (closed symbols) and globally (open symbols) were considered excipient candidates.

[0370] In this second group of excipients, it was observed that only TMAO could be identified as a good thermal stabilizer.

[0371] Fig. 27 Results for thermal stabilization of CRMs using ionic liquids are shown.

[0372] Fig. 27 : Effect of excipient candidates on the mean unfolding temperature of CRMs. The black line indicates dT m = 0°C. The red line indicates dT m = 5°C. Molecules capable of stabilizing the CRM by more than 5°C were subjected to a second screen using a different model protein. To use the same type of 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.

[0373] In this third group of excipients, it was observed that choline dihydrogen phosphate can be classified as a good thermostabilizer, although the local unfolding transition (Tm1) was not stabilized very much. Except for choline dihydrogen phosphate, all other choline-based ionic liquids thermostabilized the CRM more effectively than trehalose. However, choline chloride-based ionic liquids had a significant destabilizing effect on the first unfolding transition of the CRM. This may be due to the large amount of charge present in these ionic liquids.

[0374] Evaluation of the effects of excipients on the colloidal stability of vaccine proteins

[0375] After five molecules have been identified as potential excipients for initiating stability of proteins at elevated temperatures, these molecules were evaluated in more detail. A key aspect here is to determine whether the selected excipient has any effect on the colloidal stability of the protein. In other words: it needs to be determined whether the protein shows an altered aggregation tendency in the presence of the excipient. If aggregation is prevented, the respective excipient will show additional benefits.

[0376] In order to evaluate the aggregation behavior of protein, dynamic light scattering was used. The hydrodynamic radius of the protein is measured, and this value can be used to evaluate whether the protein is in monomeric form or has an aggregated state. Five selected excipients are added to a solution of vaccine protein containing a concentration of 0.1 mg / ml. As described in the previous section, each vaccine protein is prepared in its natural preparation, and each excipient is prepared in the corresponding natural preparation of the vaccine protein. Similarly, in this group of experiments, choline dihydrogen phosphate without any buffer component is prepared, but the pH of the excipient stock solution is adjusted to match the pH of the vaccine protein preparation.

[0377] Vaccine protein and excipients were mixed together to produce test formulations with 0.1 mg / ml vaccine protein content and desired concentrations of excipients (details below).

[0378] To evaluate the colloidal stability of vaccine proteins at elevated temperatures, samples were loaded into 316-well plates and placed in a Dynapro plate reader III (Wyatt Technology). The hydrodynamic radius R of the vaccine proteins was determined at room temperature. h The plate was then heated to 40°C for 5 minutes and then cooled back to room temperature, at which point R was measured again. h In this way, different temperatures in the range of 40-80°C were measured in 10°C increments.

[0379] Fig.28 Results for AnthraxPA are shown as an example.

[0380] Fig.28 : Hydrodynamic radius of AnthraxPA after 5 min incubation at elevated temperature.

[0381] For naturally formulated AnthraxPA, a lower hydrodynamic radius was observed at room temperature. This low R h It did not seem to be affected by the 5-minute incubation at 40°C. However, after a subsequent 5-minute incubation at 50°C, R h The significant increase in the concentration of AnthraxPA at 50°C indicates that AnthraxPA aggregates. This temperature is called T agg It can be concluded that for naturally formulated AnthraxPA, the aggregation temperature T agg is 50°C. The addition of various excipients will agg Raised to temperatures as high as 80°C.

[0382] In order to analyze the observed T agg Whether the change in T is actually an inhibition of aggregation, or whether it is simply a consequence of a higher conformational stability of the protein, will be agg Relative to T m Make a drawing, such as Fig.29 shown.

[0383] Fig.29 :T agg Relative T m Since 10°C increments were used to probe T agg , so in T m An interval of 10°C is defined nearby, where aggregation is considered to be strictly accompanied by unfolding (green area).

[0384] If T agg If the T increases beyond this range, the excipient is considered to inhibit aggregation (yellow area). agg Below this range, the excipient is considered to promote aggregation (red area).

[0385] Melibiose was observed to inhibit the aggregation of CRM (2M and above) and tetanus toxoid (2.4M). Fig.29 In addition, maltose inhibits the aggregation of CRM (1.5M and above), which also leads to Fig.21 The complete lack of data points.

[0386] Evaluation of the effect of excipients on the secondary structure stability of vaccine proteins

[0387] In addition to probing the tertiary structure of the vaccine proteins, analytical methods were performed on the five excipients to report their effects on the secondary structure content of the vaccine proteins.

[0388] One concentration of each excipient was analyzed with all three vaccine proteins. We used a protein concentration of 0.5 mg / ml. In addition, it was necessary to use a different buffer system than in previous experiments in order to not interfere with light detection in this setup. However, it could be demonstrated that this change in buffer did not affect the stability of the proteins used in this study.

[0389] The vaccine protein was formulated in a buffer containing 5 mM sodium dihydrogen phosphate and sodium chloride (50-150 mM) to maintain the same pH level as in the respective natural preparations. Spectra were recorded from 280-200 nm (210 nm in some cases) with a data spacing of 1 nm, a slit width of 100 μm, a response time of 2 s, a speed of 100 nm / s, and an optical path length of 1 mm. The average of three spectra was taken for each temperature.

[0390] Fig.30 The determined transition temperatures are shown.

[0391] Fig.30 Transition temperatures of AnthraxPA, tetanus, and diphtheria toxoids in formulations containing a newly identified thermal stabilizer

[0392] Before summarizing the results, two observations are important to note, which are important to put the data into proper context:

[0393] First, for AnthraxPA, two thermally induced transitions were observed. In the absence of prior information on how each transition affects the ability of AnthraxPA and influences the immunogenicity of the protein, the lower transition temperature was used for the analysis. In the formulation with lactulose, the two unfolding transitions occurred within a fairly narrow temperature range, and neither could be determined with the required precision. Therefore, the highest temperature, where no structural changes occurred, is reported. Overall, the unfolding temperature of anthrax should be considered a lower limit, rather than a transition midpoint.

[0394] Secondly, there are conditions where no changes in the secondary structure are observed even at very high temperatures. However, information from DLS and fluorescence measurements is needed to properly assess the overall stability and mode of action of an excipient.

[0395] Table 5 The experimental results are summarized as follows:

[0396]

[0397] Effects of the five selected excipients on vaccine protein stability

[0398] The effect of excipients selected from three different substance groups on the conformational and colloidal stability of the vaccine proteins was determined. For the conformational stability, effects on the secondary and tertiary structure can be distinguished.

[0399] Fig.31 All results are summarized for each protein and excipient combination.

[0400] Fig.31 : Summary of fluorescence, CD and DLS data obtained for AnthraxPA, CRM197 and tetanus toxoid. The black line at 60°C represents the desired stability target. With the available information, the effect of each excipient on each protein can be interpreted.

[0401] i) TMAO: TMAO appears to be the weakest of the very good thermal stabilizers among the group of excipients used.

[0402] ii) Choline dihydrogen phosphate: This ionic liquid is a stronger thermal stabilizer than TMAO and stabilizes CRM better than TMAO.

[0403] i) Melibiose: The sugars tested here generally showed the best thermal stability. Melibiose was a more effective thermal stabilizer than trehalose for all three vaccine proteins tested. Melibiose inhibited aggregation of tetanus and CRM.

[0404] ii) Maltose: Its thermostabilizing effect is similar to that of melibiose. Maltose significantly stabilizes all vaccine proteins above 60°C, but melibiose appears to have a stronger stabilizing effect. For all three vaccine proteins tested, maltose is a more effective thermostabilizer than trehalose. On the other hand, maltose is used in much lower concentrations, which makes this excipient easier to use. Maltose inhibits the aggregation of CRM and inhibits the secondary structure unfolding of CRM and Td.

[0405] iii) Lactulose: Similar to the previously mentioned sugars, lactulose stabilized all vaccine proteins to temperatures above 60°C. However, in contrast to the other sugars, secondary structure unfolding of the CRM was observed and aggregation was not prevented. However, lactulose showed similar thermal stabilizer efficacy to maltose and was more effective than trehalose for all three vaccine proteins. Both sugars were used at similar concentrations (1.7 M lactulose and 1.8 M maltose), while melibiose was effective if a more concentrated solution (2.4 M) was used.

Claims

1. A method for increasing the thermal stability of a liquid protein or peptide formulation, said method comprising the step of combining a protein or peptide solution with at least one excipient selected from the group consisting of: choline dihydrogen phosphate at a concentration of 1-4 M, melibiose at a concentration of 0.25-4 M, maltose at a concentration of 0.5-4 M and lactulose at a concentration of 0.25-4 M or a mixture thereof, wherein the protein or peptide concentration is 0.05 to 2 mg / ml and remains in its native rather than unfolded form during storage at temperatures above 50°C and up to 60°C.

2. The method according to claim 1, wherein T has been determined from a measured melting curve m values ​​to estimate the improved stabilization.

3. The method according to claim 1 or 2, wherein the concentration of choline dihydrogen phosphate is 2-4 M, the concentration of melibiose is 1-2.5 M, the concentration of maltose is 1-1.8 M, and the concentration of lactulose is 0.5-1.7 M.

4. The method according to any one of claims 1 to 2, wherein a protein or peptide preparation is stabilized, and wherein the protein or peptide is selected from the group consisting of proteins of viral origin, vaccines comprising viral protein subunits, enzymes, hormones and antibodies.

5. The method according to any one of claims 1 to 2, wherein a protein or peptide preparation is stabilized, and wherein the protein or peptide is selected from the group consisting of a viral protein subunit, a nanobody and a monoclonal antibody.

6. The method of claim 4, wherein the protein or peptide preparation of the vaccine comprising viral protein subunits is stabilized.

7. The method according to claim 4, wherein a protein or peptide preparation comprising an enzyme is stabilized.

8. The method according to any one of claims 1 to 2, wherein the pH value of the formulation is adjusted to pH 6-8.

9. The method according to any one of claims 1 to 2, wherein the pH value of the formulation is adjusted to 7-7.

5.

10. The method according to claim 8, wherein the pH of the formulation is controlled by adding a buffer selected from the group consisting of potassium phosphate, sodium phosphate, sodium acetate, histidine, imidazole, sodium citrate, sodium succinate, HEPES, Tris, Bis-Tris, ammonium bicarbonate.

11. The method according to any one of claims 1 to 2, wherein the formulation comprises a polymer for stabilizing 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.

12. The method according to any one of claims 1 to 2, wherein the formulation comprises divalent cations at a concentration of 0.1 to 100 mM and amino acids at a concentration of 0.1 to 1% w / v to stabilize the contained proteins and to adjust the pH and osmolarity of the solution.

13. A stabilized protein formulation prepared by the method according to any one of claims 1 to 12.

14. The stabilized protein formulation according to claim 13 as part of an immunoassay test kit, or in preparation and vial for analysis.

15. A kit comprising a stabilized protein formulation according to claim 13 and further container means comprising solutions necessary or convenient for carrying out a method using the protein formulation.

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