High concentration phosphatase compositions

Abstract

The invention relates to an aqueous composition comprising at least 20 mg / mL of an alkaline phosphatase, a non-ionic surfactant, an additive, and a multivalent metal, having a pH of 6-8. The invention further relates to said aqueous composition for use as a medicament.

Description

[0001] P136822PC00 Title: High concentration phosphatase compositions FIELD The invention relates to compositions comprising concentrations of more than 20 mg / mL of a phosphatase, and to their use as a medicament, in particular for subcutaneous use. The invention also relates to compositions comprising between 2 – 20 mg / ml of a phosphatase that are devoid of sorbitol, and to their use as a medicament, in particular for intravenous use in pediatric patients. BACKGROUND OF THE INVENTION Alkaline phosphatase (AP) is a common endogenous enzyme present in many cells and organs, such as intestines, placenta, liver, bone, kidney and granulocytes. AP (EC 3.1.3.1 according to IUBMB Enzyme nomenclature) is an enzyme that catalyzes the reaction of a phosphatase monoester and H2O to an alcohol and phosphate. AP is a broad specificity enzyme, it also catalyses transphosphorylations. In humans and other mammals at least four distinct, but related alkaline phosphatases are known. These are intestinal (ALPI), placental (ALPP; only in human and primates), placental-like (GCAP), and liver / bone / kidney (or tissue non- specific) alkaline phosphatase (TNAP). In human, genes encoding the first three Aps are located on chromosome 2, while a gene encoding the tissue non-specific form is located on chromosome 1. The amino acid sequence of alkaline phosphatases and the relative positions of the catalytic and crown domains are known by the skilled person. As an example, reference is made to the textbook on mammalian alkaline phosphatases by Millán (Mammalian Alkaline Phosphatases, Wiley-VCH (2006), ISBN-13: 978-3- 527-31079-1) which shows, amongst others, the amino acid sequence of the four human alkaline phosphatases. It has been shown that AP may be beneficial as a medicament to treat a wide range of diseases, including Acute Kidney Injury (AKI), Sepsis, Inflammatory Bowel Disease (IBD), and hypophosphatasia (HPP). These studies have used naturally occurring APs, such as isolated bovine as well as isolated and recombinant human AP. In some animal models, a recombinant chimeric alkaline phosphatase has been used that comprises the catalytic domain of a human intestinal AP and a crown domain of a human placental AP (described in WO2008133511). A pharmaceutical composition comprising a novel and improved recombinant chimeric alkaline phosphatase having a sequence as depicted in SEQ ID NO:1 is being developed for use as a medicament. For most medicinal uses, AP is to be applied intravenously (i.v.). In some instances, a subcutaneous (s.c.) administration is preferred. To allow for an effective amount of an AP in an acceptable volume, in particular when administered s.c., an aqueous composition comprising at least 20 mg / mL of an alkaline phosphatase, would be advantageous. However, such formulations comprising increased levels of an AP were found to be prone to particle formation upon prolonged storage. There is thus a need to develop formulations comprising more than 20 mg / mL AP that can be used for therapeutic administration, in particular s.c. administration of AP to a person in need thereof. There is also a need to develop formulations comprising between 2 - 20 mg / mL AP for intravenous administration, that are devoid of sorbitol, having similar stability characteristics as those comprising sorbitol. BRIEF DESCRIPTION OF THE INVENTION A pharmaceutical composition comprising a protein of interest does not show visible particle formation (vpf) during stability testing at 2- 8° C for 2 months, whereby vpf generally are defined as particles greater than 50 µm. In addition, the number of subvisible particles that are greater than 10 µm must also remain within limits. The international patent application WO 2015 / 112015 describes specific downstream processing and final drug formulation of recombinantly produced AP in order to reduce particle formation due to remaining host cell proteins. The resulting pharmaceutical composition of up to 10 mg / mL of AP, further comprising histidine, sorbitol, MgCl2, ZnCl2, in a citrate buffer at pH between 6.5 and 7.5, showed no visible particle formation during stability testing. However, it was found that particle formation, especially of subvisible particles comprising AP, did occur during stability testing when using AP concentrations of more than 10 mg / mL. The invention therefore provides an aqueous composition comprising at least 20 mg / mL of an alkaline phosphatase, 0.05-5 mg / mL of a non-ionic surfactant, 1- 1000 mM of an additive, and 0.1-5 mM of a multivalent metal, having a pH of 6-8. Said composition was found to be stable and did not incite visual particle formation during rigorous stress testing, involving freeze-thaw cycles and mechanical stress. Said aqueous composition may comprise 20-200 mg / mL of an alkaline phosphatase. Said multivalent metal in an aqueous composition according to the invention is one or more of Mg, Zn, Ca, and Al, such as Mg and Zn. Said additive in an aqueous composition according to the invention is a non- metal salt, a sugar, and / or an amino acid, preferably a sugar, such as trehalose. Said non-ionic surfactant in an aqueous composition according to the invention may be an ethylene oxide / propylene oxide block copolymer, such as an ethylene oxide / propylene oxide triblock copolymer. Said non-ionic surfactant in an aqueous composition according to the invention may comprise a central propylene oxide chain which is flanked at both sides by an ethylene oxide chain, such as a poloxamer. Said non-ionic surfactant comprises preferably comprises a central chain of about 29 propylene oxide moieties, which is flanked by two chains of about 38 ethylene oxide moieties. An aqueous composition according to the invention may comprise 0.1-1 mg / mL of a non-ionic surfactant. An aqueous composition according to the invention may comprises a recombinant alkaline phosphatase, preferably a recombinant human alkaline phosphatase. Said recombinant alkaline phosphatase may be obtained from a cell- based expression system. An aqueous composition according to the invention comprising a recombinant alkaline phosphatase preferably comprises less than 100 ppm of a host cell protein (HCP). In embodiments, the invention provides an aqueous composition according to the invention, for use as a medicament. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. A: Pictures of vials during visual inspection after stirring (150 rpm, 24h, RT). From left to right, duplicate samples of 8 mg / mL AP in histidine; 8 mg / mL AP in citrate; 40 mg / mL AP in histidine; 40 mg / mL AP in citrate. B: Pictures of vials during visual inspection after pumping. From left to right, duplicate samples of 8 mg / mL AP in histidine; 8 mg / mL AP in citrate; 40 mg / mL AP in histidine; 40 mg / mL AP in citrate. C: Pictures of vials during visual inspection after syringe stress. From left to right, duplicate samples of 8 mg / mL AP in histidine; 8 mg / mL AP in citrate; 40 mg / mL AP in histidine; 40 mg / mL AP in citrate. Figure 2. Subvisible particle counts after inducing mechanical stress. Particle counts below 5 µm (A), 5-10 µm (B), above 10 µm (C), and above 25 µm (D). The induces stress conditions were, from left to right, starting formulations at T=0; after overhead rotation; after orbital shaking; after stirring, After PSPump; after syringe. Figure 3. Subvisible particle counts after oxidation and repeated freeze- thawing at -20 °C and -80 °C as determined by Flowcam measurements. Particle counts below 5 µm (A), 5-10 µm (B), above 10 µm (C), and above 25 µm (D). The number of freeze-thaw cycles and temperatures were, from left to right, starting formulations at T=0; after 2 freeze / thaw cycles at -20 °C; after 2 freeze / thaw cycles at -80 °C; after 5 freeze / thaw cycles at -20 °C; after 5 freeze / thaw cycles at -80 °C; after oxidation. Figure 4. (A-F) Relative peak areas of RP-HPLC measurements before (left) and after oxidation (right). Panels show the relative peak areas of Isodimer A and degradation species. Bars and error bars indicate the mean of 3 replicates and the standard deviated, respectively. Figure 5. Comparison total subvisible particle counts as measured by Flowcam at t0. Panels show subvisible particle counts dependent on presence of Kleptose (left), and dependent on presence of Poloxamer 188 (right). Figure 6. Subvisible particle counts stirring (light grey) as determined by Flowcam measurements. Particle counts below 5 µm (top left), 5-10 µm (top right), above 10 µm (bottom left), and above 25 µm (bottom right). Bar show pharmacopeia limits adjusted to fill volume of 4.5 mL. Figure 7. Delta in relative main peak area as determined by SE-HPLC from t = 0 to 1 month storage at 30°C, dependent on tonicity agent. Figure 8. Osmolality [mOsmol / kg] of formulations after TFF over DS concentration [mg / mL]. Figure 9. A: Dynamic Viscosity [mPa*s] of formulations over concentration [mg / mL]. B: Turbidity [NTU] of formulations over concentration [mg / mL]. Figure 10. Subvisible particle counts of formulations. Particle counts [counts / mL] over concentration [mg / mL] below 5 μm (top left), above 5 μm (top right), above 10 μm (bottom left), and above 25 μm (bottom right). Figure 11. Circulating PPi (A) and PLP (B) plasma levels in untreated wildtype (WT) mice and Prrx1Cre-Alplfl / - (PAKO) mice after 12 weeks of treatment with either ilofotase alfa (PAKO_IF) or placebo (PAKO_VEH). Figure 12. Various parameters measured during graded maximal exercise testing (GTX) on a metabolic treadmill in wildtype (WT) mice and Prrx1Cre-Alplfl / - (PAKO) after 12 weeks of treatment with either ilofotase alfa (PAKO_IF) or placebo (PAKO_VEH). A: VO2max; B: run time until exhaustion; C: total meters run; and D: hang time. DETAILED DESCRIPTION OF THE INVENTION The invention is directed to an aqueous composition comprising at least 20 mg / mL of an alkaline phosphatase, 0.05-5 mg / mL of a non-ionic surfactant, 1-1000 mM of an additive, and 0.1-5 mM of a multivalent metal, having a pH of 6-8. Said aqueous composition has acceptable viscosity, turbidity, visible and subvisible particle counts. With visible particles is meant that particle(s) can be observed with the naked eye by a person skilled in the art, optionally using a means for magnification, such as a magnifying glass. In the regulatory world, there is a distinction between visible and non-visible particulate matter. A visible particulate is loosely defined as any particulate that can be detected with the unaided eye. Typically, visible objects are defined as objects that are 0.05 mm or larger. With the term “visible particles”, as used in the present invention, is meant particles that are 0.05 mm or larger, preferably 0.1 mm or larger, more preferably 0.2 mm or larger, more preferably 0.5 mm or larger, most preferably 1 mm or larger. Micron-sized protein aggregates and particles (subvisible particles, SVP) are important quality attributes of therapeutic protein formulations due to their risk of enhancing an immunogenic response. Hence pharmacopoeias require quantification of SVP larger than 10 μm and 25 μm. Currently, the pharmacopoeial acceptance criteria for SVP are: NMT 6000 particles ≥10 µm per container and NMT 600 particles ≥25 µm per container. SVP < 10 µm have to be monitored, however specific acceptance criteria are not defined. It has to be mentioned that the Compendial methods Ph. Eur. 2.9.19 and the USP 787 / 788 specify Light Obscuration, as method for subvisible particle counts. Flow imaging, as performed here, produces significantly higher particle counts than Light Obscuration. Thus, if limits are met using this methods, it is to be expected that the pharmacopeia limits will be met. Said alkaline phosphatase (AP; EC 3.1.3.1 according to IUBMB Enzyme nomenclature), is an enzyme that catalyzes the reaction of a phosphatase monoester and H2O to an alcohol and phosphate. Other name(s) for AP are alkaline phosphomonoesterase; phosphomonoesterase; glycerophosphatase; alkaline phosphohydrolase; alkaline phenyl phosphatase; orthophosphoric- monoester phosphohydrolase (alkaline optimum). The systemic name of AP is phosphate-monoester phosphohydrolase (alkaline optimum). AP is a broad specificity enzyme, it also catalyses transphosphorylations. In humans and other mammals at least four distinct, but related alkaline phosphatases are known. These are intestinal (ALPI), placental (ALPP; only in human and primates), placental-like (GCAP), and liver / bone / kidney (or tissue non- specific) alkaline phosphatase (TNAP). The first three are located together on chromosome 2 while the tissue non-specific form is located on chromosome 1. The amino acid sequence of alkaline phosphatases and the relative positions of the catalytic and crown domains are known by the skilled person. As an example, reference is made to the textbook on mammalian alkaline phosphatases by Millán (Mammalian Alkaline Phosphatases, Wiley-VCH (2006), ISBN-13: 978-3- 527-31079-1) which shows, amongst others, the amino acid sequence of the four human alkaline phosphatases. Previously it has been shown that AP is beneficial as a medicament in a wide range of diseases (Acute Kidney Injury (AKI), Sepsis, Inflammatory Bowel Disease (IBD), etc.). These studies have used naturally occurring APs, such as isolated bovine as well as isolated and recombinant human AP. In some animal models, a recombinant chimeric alkaline phosphatase has been used that comprises the catalytic domain of a human intestinal AP and a crown domain of a human placental AP (described in the international patent application WO2008 / 133511). At present, a pharmaceutical composition comprising a recombinant chimeric human alkaline phosphatase having a sequence as depicted in SEQ ID NO: 1, termed, herein ‘recAP’ (International Nonproprietary Name: ilofotase alfa), is being developed for use as a medicament. An AP as identified in the claims preferably is 70-100% identical to the amino acid sequence of SEQ ID NO: 1, such as at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical to the amino acid sequence of SEQ ID NO: 1. An aqueous composition according to the invention may comprise 20-250 mg / mL of an AP, such as at least 50 mg / mL, at least 60 mg / mL, at least 70 mg / mL, at least 80 mg / mL, at least 90 mg / mL, at least 100 mg / mL, at least 110 mg / mL, at least 120 mg / mL, at least 130 mg / mL, at least 140 mg / mL, at least 150 mg / mL, at least 160 mg / mL, at least 170 mg / mL, at least 180 mg / mL. An aqueous composition of the invention may comprise 50-250 mg / mL, such as 100-200 mg / mL, such as about 170 mg / mL, about 180 mg / mL, about 190 mg / mL AP. In embodiments, the AP is a recombinant AP that is obtained from a cell- based expression system. Suitable cell-based expression systems are known in the art and include mammalian expression systems such as HeLa cells, Human Embryonic Kidney (HEK) 293 cells, Hep G2 cells, Chinese Hamster Ovary (CHO) cells and Baby hamster kidney (BHK) cells. In embodiments, a recombinant AP such as recAP is produced in a CHO cell line, such as a CHO cell line lacking dihydrofolate reductase (DHFR) activity. CHO cell lines and derivatives thereof can be obtained from a number of biological resource centers such as the European Collection of Authenticated Cell Cultures (ECACC). As has been described previously, expression of AP in a cell line such as a CHO cell line, may result in the co-purification of a host cell protein (HCP) using standard downstream processing techniques such as affinity purification, anion exchange and mixed mode chromatography, resulting in particle formation in a purified fraction (see WO 2015 / 112015). The co-purification of the HCP could be minimized by employment of specific columns and washing and elution steps, as described in WO 2015 / 112015. An aqueous composition of the invention may have been purified as described in WO 2015 / 112015 and comprises less than 100 ppm of a host cell protein (HCP). An aqueous composition according to the invention has a pH between 6 and 8, including endpoints, such as 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9, preferably at a pH of about 7.0. Said composition is maintained at the indicated pH by the presence of a buffer. In embodiments, said buffer may be selected, for example, from citric acid, acetic acid, aspartic acid, glutamic acid, tartaric acid, succinic acid, malic acid, fumaric acid, alpha- ketoglutaric acid, a combination of histidine and lactid acid, or a combination of tromethamine (2-amino-2-(hydroxymethyl)propane-1,3-diol) and gluconic acid. Phosphoric acid is preferably not included as a buffer. As is indicated herein below, the anion of some of the indicated buffers, such as tartrate, aspartate, and citrate, may also function as counterion for a multivalent metal that is included in an aqueous composition according to the invention. In embodiments, citrate is used as a buffer for keeping the pH at about 7.0. A multivalent metal in an aqueous composition according to the invention may be one or more of Mg, Zn, Ca, and Al, such as Mg and Zn; Mg and Ca; Mg and Al; Zn and Ca; Zn and Al; Ca and Al; Mg, Zn, and Ca; Mg, Zn, and Al; Mg, Ca and Al, Zn, or Ca and Al. Said multivalent metal may be present in an aqueous composition according to the invention in an amount of 0.1-5 mM, such as 0.2-4 mM, 0.3-3 mM, 0.4-2 mM, or 0.5-1 mM. In embodiments an aqueous composition according to the invention comprises two or more multivalent metals. One or more of said metals may be present in an amount of 0.01-1 mM, such as 0.02-0.5 mM, 0.03-0.4 mM, 0.05-0.2 mM, or 0.08-0.1 mM. One other of said two or more multivalent metals may be present in an amount of 0.1-5 mM, such as 0.2-4 mM, 0.3-3 mM, 0.4-2 mM, or 0.5-1 mM. Said multivalent metal may be present in an aqueous composition according to the invention with a counterion selected from gluconate, lactate, acetate, formate, pyruvate, galacturonate, chloride, nitrite, bromide, nitrate, glutarate, succinate, carbonate, tartrate, benzoate, aspartate, benzenesulfonate, besylate, bicarbonate, bitartrate, camsylate, citrate, decanoate, edetate, esylate, fumarate, gluceptate, glutamate, glycolate, hexanoate, hydroxynaphthoate, iodide, isethionate, lactobionate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, octanoate, oleate, pamoate, pantothenate, phosphate, polygalacturonate, propionate, salicylate, stearate, sulfate, teoclate, and tosylate. In embodiments, said anionic counterion is an inorganic counterion such as bromide, chloride nitrite, nitrate, phosphate, and sulphate. In embodiments, said anionic counterion is chloride. In embodiments, said aqueous composition of the invention comprises Mg and Zn. Said aqueous composition may comprise 0.1-5 mM of Mg, and 0.01-1 mM of Zn. Said aqueous composition may comprise an inorganic counterion such as chloride as a counterion for both the Zn and Mg multivalent metals. An aqueous composition of the invention comprises 1-1000 mM of an additive. In embodiments, said additive is a non-metal salt such as sodium chloride, calcium citrate, calcium lactate, sodium lactate, calcium fumarate, calcium sulphate dehydrate, calcium silicate, phosphosilicide, calcium carbonate, calcium phosphate, calcium phosphate, sodium phosphate, potassium phosphate, potassium sulphate, potassium chloride, and / / or sodium carbonate. Said non-metal salt may mask surface charges of proteins, leading to weakened intermolecular repulsions and a prevalence of hydrophobic interactions. Hence, the addition of a non-metal salt may decrease melting and aggregation temperature of a protein such as AP. In embodiments, said additive is a sugar including, for example, a disaccharide sugar such as sucrose, trehalose, maltose, and lactose, and a polyol such as mannitol and glycerol. Said sugar may act as a stabilizing agent, for example by increasing the free energy of unfolding of a protein, thereby thermodynamically favoring the native folded state. In embodiments, said sugar is trehalose at a concentration of 200-300 mM, including 250 mM. As is shown in the examples, 250 mM trehalose showed the highest main peak retention of recAP in a buffer comprising 20 mM citrate, 2 mM MgCl2, 0.05 mM ZnCl2, and 0.05 mg / mL of poloxamer 188, in the absence of arginine and methionine, and surprisingly outperformed a comparable composition comprising sorbitol or sodium chloride, instead of trehalose. In embodiments, said additive is an amino acid such as DL-methionine, glycine, L-alanine, L-arginine, L-aspartate, L-glutamic acid, L-leucine, L-lysine, L- methionine, L-phenylalanine. L-proline, L-serine, L-threonine and / or L-valine. The addition of one or more amino acids may help in preservation of shelf life of a protein such as AP, and / or prevent crystallization of a protein such as AP. In addition, L-methionine may prevent oxidation of a protein such as AP. An aqueous composition of the invention may comprise 1-1000 mM of the additive or additives, such as 10-750 mM, 50-500 mM, 100-400 mM, including 200 mM, 250 mM, 300 mM or 350 mM. An aqueous composition of the invention comprises 0.05-5 mg / mL of a non- ionic surfactant. A non-ionic detergent comprises molecules with uncharged head groups. A non-ionic detergent is considered to be non-denaturing and useful in formulations to preserve the structure of protein such as AP. In addition, a non- ionic detergent may prevent protein adsorption to hydrophobic surfaces and unfolding at interfaces, and prevent the formation of aggregates and particles. Examples of a non-ionic detergent are 1-oleoyl-rac-glycerol, Brij® L23, Igepal® CA- 630, Kolliphor® P407, N-octanoyl-N-methylglucamine, Poloxamer 407, saponin, and Span® 80. A preferred non-ionic detergent is based on polyoxyethylene and related compounds, such as an ethylene oxide / propylene oxide block copolymer. Said ethylene oxide / propylene oxide block copolymer may comprise a core of polyoxypropylene that is flanked by two chains of polyoxyethylene. Because the lengths of the polymer blocks can be customized, many different ethylene oxide / propylene oxide block copolymers exist that have slightly different properties. These copolymers are commonly named with the letter "P" (for poloxamer) followed by three digits: the first two digits x 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 gives the percentage polyoxyethylene content. For example, P407 refers to a poloxamer with a polyoxypropylene molecular mass of 4,000 g / mol and a 70% polyoxyethylene content. In embodiments, said poloxamer comprises a central chain of about 29 propylene oxide moieties, which is flanked by two chains of about 38 ethylene oxide moieties, such as P188. An aqueous composition of the invention may comprise 0.1-2 mg / mL of a non- ionic detergent, such as comprise 0.2-1 mg / mL, 0.3-0.8 mg / mL, 0.4-0.6 mg / mL, including 0.5 mg / mL. In embodiments, the invention provides a composition comprising about 2-20 mg / mL of an alkaline phosphatase such as recAP, 0.5 mg / mL of a non-ionic surfactant such as P188, 250 mM of an additive such as trehalose, 0.05 mM of a zinc salt such as zinc chloride, and 2 mM of a magnesium salt such as magnesium chloride, and which is buffered to a pH of 7, for example with 20 mM of a citric acid-based buffer. Surprisingly, trehalose outperformed also sorbitol and sodium chloride in a composition of the invention comprising 2-20 mg / mL of an alkaline phosphatase such as recAP. Such composition comprising trehalose instead of sorbitol is, for instance, useful for i.v. administration to pediatric patients as in many countries, sorbitol is not allowable in intravenous compositions for children. A composition comprising about 2-20 mg / mL of an alkaline phosphatase such as recAP, 0.5 mg / mL of a non-ionic surfactant such as P188, 250 mM of an additive such as trehalose, 0.05 mM of a zinc salt such as zinc chloride, and 2 mM of a magnesium salt such as magnesium chloride, and which is buffered to a pH of 7, for example with 20 mM of a citric acid-based buffer, may be used for intravenous (i.v.) administration of an alkaline phosphatase such as recAP, including i.v. injection and i.v. infusion, in particular in pediatric patients. Said composition comprising about 2-20 mg / mL of an alkaline phosphatase such as recAP is for use in a method of treating hypophosphatasia (HPP), or renal damage, such as cardiac surgery-associated renal damage, sepsis-associated kidney injury, especially sepsis- associated acute kidney injury, or ischemia-induced kidney injury. AP displays activity towards dephosphorylating and detoxifying damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), ATP, ADP and other extracellular substrates that drive acute inflammation, coagulation and microvascular ischemia found in kidney following sepsis or ischemia-induced damage (Tang et al., 2020. Medicine 99: e18788). Said aqueous composition comprising 2-20 mg / mL of an alkaline phosphatase such as recAP, 0.5 mg / mL of a non-ionic surfactant such as P188, 250 mM of an additive such as trehalose, 0.05 mM of a zinc salt such as zinc chloride, and 2 mM of a magnesium salt such as magnesium chloride, and which is buffered to a pH of 7, for example with 20 mM of a citric acid-based buffer, may be injected, either as a single injection or multiple injections, or infused at a dose between 0.5 mg / kg and 5 mg / kg, such as between 1 mg / kg and 3 mg / kg, such as between 1,5 mg / kg and 1,7 mg / kg, such as about 1.6 mg / kg, or as a flat dose of between 35 – 500 mg, preferably between 70 – 300 mg, more preferably between 100 – 170 mg, most preferably of about 128 mg. Said aqueous composition comprising 2-20 mg / mL of an alkaline phosphatase such as recAP, may be injected or infused at a dose between 0.5 mg / kg and 20 mg / kg once daily, every other day, twice weekly, once weekly, bimonthly or once monthly. A composition comprising about 2-20 mg / mL of an alkaline phosphatase such as recAP, 0.5 mg / mL of a non-ionic surfactant such as P188, 250 mM of an additive such as trehalose, 0.05 mM of a zinc salt such as zinc chloride, and 2 mM of a magnesium salt such as magnesium chloride, and which is buffered to a pH of 7, for example with 20 mM of a citric acid-based buffer is essentially free of sorbitol. With essentially free is meant that sorbitol is undetectable in the composition employing, for example, isocratic HPLC (Simonzadeh and Ronsen, 2012. J Chrom Science 50: 644–647). In embodiments, an aqueous composition of the invention comprises 20-250 mg / mL of an alkaline phosphatase such as recAP, 0.5 mg / mL of a non-ionic surfactant such as P188, 250 mM of an additive such as trehalose, 0.05 mM of a zinc salt such as zinc chloride, and 2 mM of a magnesium salt such as magnesium chloride, and which is buffered to a pH of 7, for example with 20 mM of a citric acid-based buffer, may be used for parenteral administration of an alkaline phosphatase such as recAP, in particular for subcutaneous injection. An aqueous composition of the invention comprising 20-250 mg / mL of an alkaline phosphatase such as recAP, 0.5 mg / mL of a non-ionic surfactant such as P188, 250 mM of an additive such as trehalose, 0.05 mM of a zinc salt such as zinc chloride, and 2 mM of a magnesium salt such as magnesium chloride, and which is buffered to a pH of 7, for example with 20 mM of a citric acid-based buffer, may be for use in a method of treating renal damage, such as cardiac surgery-associated renal damage, sepsis-associated kidney injury, especially sepsis-associated acute kidney injury, or ischemia-induced kidney injury, or HPP or other acute or chronic diseases or metabolic diseases. Said aqueous composition of the invention comprising 20-250 mg / mL of an alkaline phosphatase such as recAP, 0.5 mg / mL of a non-ionic surfactant such as P188, 250 mM of an additive such as trehalose, 0.05 mM of a zinc salt such as zinc chloride, and 2 mM of a magnesium salt such as magnesium chloride, and which is buffered to a pH of 7, for example with 20 mM of a citric acid-based buffer, in particular is for treating HPP or other chronic disease by parenteral administration, especially by subcutaneous injection, and is provided for instance in a pre-filed syringe or a smart pen. A patient, such as a patient suffering from HPP, that is treated with an aqueous composition of the invention comprising 20-250 mg / mL of an alkaline phosphatase such as recAP, preferably is at least 6 months of age, including at least 2 years of age, including at least 6 years of age, such as at least 12 years of age. In embodiments, said patient suffering from HPP that is treated with an aqueous composition of the invention comprising 20-250 mg / mL of an alkaline phosphatase such as recAP, is an adult being at least 18 years of age. A composition comprising about 20-250 mg / mL of an alkaline phosphatase such as recAP, 0.5 mg / mL of a non-ionic surfactant such as P188, 250 mM of an additive such as trehalose, 0.05 mM of a zinc salt such as zinc chloride, and 2 mM of a magnesium salt such as magnesium chloride, and which is buffered to a pH of 7, for example with 20 mM of a citric acid-based buffer is preferably essentially free of sorbitol. With essentially free is meant that sorbitol is undetectable in the composition employing, for example, isocratic HPLC (Simonzadeh and Ronsen, 2012. J Chrom Science 50: 644–647). Said aqueous composition of the invention, comprising 20-250 mg / mL of an alkaline phosphatase such as recAP, may be injected or infused at a dose between 0.5 mg / kg and 5 mg / kg, such as between 1 mg / kg and 3 mg / kg, such as between 1.5 mg / kg and 1.7 mg / kg, such as about 1.6 mg / kg. For practical considerations, the dose may be fixed, for example at 180 mg or 270 mg. In embodiments, said aqueous composition comprising 180 mg or 270 mg of recAP may be formulated as a 1 ml, or 1.5 ml, respectively, formulation in a pre- filled syringe or smart pen for s.c. administration. These doses are envisaged for adults weighing at least 70 kg, preferably at least 100 kg, more preferably at least 120 kg. A fixed dose can be adjusted for patients weighing less, such as younger children. For a child of about 30 kg, for instance, the fixed dose may be 60 or 90 mg. The specific activity of the AP may be about 625 units / mg. The specific activity of an alkaline phosphatase such as recAP may be determined by decomposition of benzene disodium phosphate into free phenol and phosphoric acid in the presence of AP, after which phenol may be reacted with 4-aminopyrline in alkaline solution in the presence of potassium ferricyanide into a red quinone derivative. The enzyme activity can be calculated indirectly by measuring the OD value at 520 nm. Such commercial tests for determining AP activity are available, for example from Invitrogen. As an alternative, the specific activity of an alkaline phosphatase such as recAP may be determined by using p-nitrophenyl phosphate (pNPP) as a substrate, of which the dephosphorylated product may be determined by measuring the OD value at 405 nm. Such commercial tests for determining AP activity are available, for example from Abcam. An aqueous composition of the invention comprises no visible particles and less than 6000 particles of ≥10 µm per container and less than 600 particles ≥25 µm per container, even after prolonged storage at elevated temperatures, after repeated cycles of freeze-thawing or after mechanical stress such as shear stress. Methods for determining particle formation are known to a person skilled in the art and include light obscuration, by which the size of a particle in a product is determined by the amount of light that it blocks; electrical sensing zone (Coulter), by which the size of a particle is measured in terms of the change in resistance as the particle passes through a microchannel, flow imaging microscopy, by which a high-speed camera records images as a sample flows through a flow cell, thereby capturing images of particles in real-time, or laser diffraction, by which the size of a particle is determined by measuring the angle of the scattered light. In addition, an aqueous composition of the invention should have acceptable viscosity, turbidity, osmolality and injectability. The viscosity of a composition may be a limiting factor when administering a pharmaceutical composition comprising AP such as recAP. Especially when administration is by subcutaneous injection, which may require a fast injection rate for injecting a volume of at most 2 mL. Viscosity (mPa*s) may be determined by a rotational viscometer such as a HAAKE MARS II rheometer (Thermo Fisher Scientific) at a shear rate of 200 / s at 20 °C, or by Cone-Plate Rheometry, for example by using a RSX Cone Plate Rheometer (AMETEK Brookfield) at 1000 s-1. A viscosity of less than 20 mPa*s is considered feasible, while a viscosity of less than 15 mPa*s may be desired for an injectable composition. As is shown in Figure 9A, an aqueous composition of the invention comprising up to 260 mg / mL of an AP such as recAP was found to have a viscosity of 15 mPa*s or less. An aqueous composition of the invention comprising up to 281 mg / mL of an AP such as recAP was found to have a viscosity of at most 20 mPa*s. Turbidity or opalescence is a measure of the relative clarity of a composition. By determining the scattering effect that particles may have on light. Turbidity may be determined by any method known in the art, including visual evaluation and dynamic light scattering (DLS) / static light scattering (SLS) by determining the difference in intensity between the transmitted beam and the incident beam. Suitable instruments include DynaPro® NanoStar® (Wyatt Technology Corp, Santa Barbara, CA) and NEPHELOstar Plus (BMG Labtech). Turbidity is preferably determined according to standards such as USP <855>, Ph. Eur.2.2.19, USEPA Method 180.1, or ISO7027. There are two standard units for reporting turbidity: Formazin Nephelometric Units (FNU) from ISO 7027 and Nephelometric Turbidity Units (NTU) from USEPA Method 180.1. A turbidity of less than 20 NTU is desired for an injectable composition. As is shown in Figure 9B, all compositions of the invention comprising up to 200 mg / mL of an AP such as recAP was found to have a turbidity of less than 20 NTU. High-concentration biological products, such as an aqueous composition of the invention comprising 80-160 mg / mL of an alkaline phosphatase such as recAP, may cause tonicity-related discomfort / pain upon injection such as subcutaneous injection, which may be associated with the osmolality of the composition. Osmotic pressure of an injectable composition can be expressed as either osmolality or osmolarity. Osmolality is defined as the number of milliosmoles of solute per kilogram of solvent and can be calculated experimentally using sodium chloride equivalents or determined with an osmometer, as is known to a person skilled in the art. Methods for determining the osmolality of a composition are known, including the use of a freezing point depression osmometer, a vapor pressure osmometer or a membrane osmometer. Suitable osmometers include a freezing point depression osmometer such as an Osmomat 3000 D-M (Gonotec GmbH) and Precision Systems Osmette II™ and Osmette III™ Osmometers (Thermo Fisher Scientific). The upper osmolality limit of a composition for intramuscular or subcutaneous injection in general may be below 1000 mOsm / kg for small-volume injections (≤100 mL) and below 500 mOsm / kg for large-volume injections (>100 mL). Several options are available for minimization of hypertonicity-induced pain upon product administration. As is shown in Figure 6, all AP concentrations tested up to 182 mg / mL of an AP such as recAP in an aqueous composition of the invention, osmolality was found to be at most 447 mOsm / kg. Injectability of a composition according to the invention, refers to, for example, pressure or force required for injection, evenness of flow, and freedom from clogging. Injectability may be measured by determining a break-loose force and a glide force, as is known to a person skilled in the art. It was found that different stress conditions, including incubation for one month at -20 °C, 5 °C and 40 °C, and repeated freeze-thaw cycles, did not significantly amend the determined break loose force of about 9 N at 204 mm / min, or the highest force during glide phase between 10 and 12 N. Examples Example 1 Starting formulations (SF) Formulations were prepared by weighing the solid components and by dissolving with 80 % of the final volume of water for injection (WFI; Aqua B Braun). Afterwards, the solution was stirred at ambient temperature until all components were dissolved. The pH value was carefully adjusted to target pH with NaOH or HCl (or a dilution thereof), whereby over-titration was avoided. The solution was adjusted with WFI to the final volume. Solutions were sterile filtered (Stericup®- GC 0.22 µm Durapore (PVDF)-membrane, propelled by a vacuum pump) and subsequently filled in vials. Small volumes for analytical purposes (e.g. dilution) were filtered using a Steriflip® 0.22 µm Durapore (PVDF)-membrane syringe filter prior to use. Poloxamer 188 was added after dialysis from sterile stock solutions, since this macro molecule does not pass a 10kDa membrane. Table 1. Alkaline phosphatase solutions Material name Buffer Concentration(mg / mL) recAP (PF-06853082) 20 mM histidine, 250 mM41.5 sorbitol, 2 mM MgCl2, 50 µM ZnCl2, pH 7.0 recAP (130986-20-RS) 20 mM histidine, 250 mM10 sorbitol, 2 mM MgCl2, 50 µM ZnCl2, pH 7.0 recAP (VLT_Demo_BDS 20 mM citrate, 250 mM sorbitol,8 2 mM MgCl2, 50 µM ZnCl2, pH 7.0 Dialysis for buffer exchange The buffer exchange for re-formulation was performed by dialysis in Slide-A- Lyzer™ Dialysis Cassettes (Thermo Fisher) with a nominal cut-off of 10 kDa. Per formulation, 25 mL of SF was dialyzed in three steps, each lasting for at least 2 hours and slowly stirred at 2 –8 °C (volume ratio per dialysis step 1:43). The last step was performed overnight (14 –16 h, 2 –8 °C). After dialysis, Minisart syringe top filter with 0.22 µm PES-membrane (Sartorius) was pre-equilibrated with placebo formulation for sterile filtration and subsequently samples were sterile filtered. The required amount of detergents and macromolecules (where applicable) was added as defined by adding appropriate volumes of the respective stock-solution (sterile filtered, 0.22 µm PVDF-filter) after dialysis to ensure the defined concentration of those excipients as they do not pass the dialysis membrane. Diafiltration for buffer exchange The buffer exchange for the experiments was obtained by performing diafiltration (DF) on an Ambr Crossflow system equipped with 4-channel TFF module (Sartorius). To this end, ~300 mL of SF was thawed for ~20 hours (h) at 2-8°C. The DS was aliquotted for the 4 formulation preparations and connected to the system at product ports alongside the placebo buffers at diafiltration buffer port. Buffer exchange was performed with a target exchange of >96%, using Ambr CF filters (Sartorius) with Polyethersulfone (PES)-membrane and 30 kDa molecular weight cut-off (MWCO). Ultrafiltration for buffer exchange The buffer exchange for the buffer screening was conducted using ultrafiltration in an Unchained Labs Big Tuna device. The Unchained Labs Big Tuna is an ultrafiltration device used for rebuffering of up to 96 samples in parallel using high pressure. Target buffers were transferred into two 96-well deep-well plates (1.7 mL per well) and placed onto the Big Tuna. The drug substance is transferred into a 96-well unfilter plate (Unchained Labs ) (250 µL per well, 30 kDa MWCO) at 40 mg / mL and placed into the pressure chamber. The instrument was set to 875 rpm mixing speed, 60 psi pressure, and an exchange per cycle of 50 Vol% until a minimum exchange of 96 % was reached. The exchange was performed at controlled room temperature of 22 °C. To this end, 6 cycles were need to reach 96 % exchange in all wells with a total run duration of 4 h 30 min. pH adjustment The final pH of all formulations and buffers was adjusted and the pH of formulated AP (f_AP) was determined using a calibrated pH-electrode connected to a SevenExcellence pH-Meter with a micro-electrode with temperature probe at room temperature between 21 °C and 27 °C). The pH values were always adjusted to the desired target value ± 0.03 pH units. Conductivity Measurement of conductivity of placebo formulations was performed with a calibrated conductivity-electrode InLab 731-ISM (VWR) connected to a SevenExcellence pH-Meter with a multimode module (Mettler Toledo) at a temperature between 23 °C and 25 °C. Osmolality Determination of osmolality was performed by freezing point depression in an osmometer (Osmomat 3000 D-M, Gonotec). Each measurement was performed in 15 µL aliquots after calibration with sodium chloride standard (600 and 500 mOsmol / kg) and purified water. The used calibration range is bracketing the actual range of formulation osmolalities. If a deviation of > 10 % of both replicates is observed, a third measurement is done. UV spectroscopy for concentration measurements The concentration of the formulated AP (f-AP) was determined by absorption spectroscopy at 280 nm with correction for background absorption (aggregates) at 320 nm. Analysis was performed with a NanoPhotometer N120 (Implen).2 µL samples were either measured diluted with placebo (dilution factor 1:10) samples or non-diluted for WP4 samples against the respective placebo as blank. Protein concentration was calculated by the device based on the customer-provided extinction coefficient of 1.01 mL*mg-1*cm-1using the Lambert-Beer law. Concentrations were reported as the mean of three replicates. Vial filling For accelerated aging, a volume of 1.8 mL for the three months’ study and 1.8 mL of each formulation for mechanical and freeze / thaw stress were inserted into sterile, particle-free 2R-vials (Adelphi) under a laminar air flow. The vials were closed with sterile 13 mm injection stoppers (Adelphi) and crimped with suitable aluminum caps (Adelphi). Sample storage During basic characterization, accelerated aging vials were stored at the indicated temperature and protected from light. The samples stored at 5 ± 3 °C were kept in fridges with external monitoring of the temperature. Samples subjected to an accelerated aging were stored (International Council for Harmonisation (ICH) Q1- compliant in cabinets (Memmert) with controlled humidity at 25 ± 2 °C / 60 ± 5 % relative humidity (rH), 30 °C ± 2 °C / 65 ± 5 % rH and 40 ± 2 °C / 75 ± 5 % rH, respectively. The temperature was additionally monitored independently during the complete storage period. Freeze / thaw stress was performed in alternating storage at -80 °C and +20 °C (uncontrolled room temperature), whereby the thawed state lasted 2 h in each freeze / thaw cycle. Shear stress was applied by rotating overhead at 30 rpm for 24 h and orbital shaking at 400 rpm for 24 h, both at room temperature. Differential scanning fluorimetry (nanoDSF) The thermal unfolding profile of the AP in various buffer systems (pH conditions and salt levels) was assessed using nanoDSF. Buffer substances were citrate (pH 6.0, 6.5, 7.0), histidine (pH 6.0, 6.5, 7.0), succinate (pH 6.0) and TRIS (pH 7.5), each with molar concentrations of 0 –400 mM NaCl. The method determines the changes in intrinsic fluorescence of the protein sample, while the sample is subjected to a thermal ramp inducing denaturation of the protein. The fluorescence is detected at wavelengths λ=330 nm and λ=350 nm while the sample is ramped from 25-95°C at 1K / min. The Tm and Tonset are determined from the ratio of 330nm / 350nm readout over temperature ramp by fitting either a two-state or three-state thermodynamic model to the data. Size exclusion (SE) chromatography Samples (50 µg recAP) were separated on YMC-Pack Diol-200, 20 nm, (8 mm ID x 300 mm) YMC on a Thermo Ultimate 3000 or Thermo Vanquish Flex HPLC system, both with UV detection (ThermoFisher Scientific). The samples were diluted in the mobile phase. The measurements were performed using a mobile phase buffer at an isocratic flow of 0.75 mL / min (Table 2). Data were recorded at 280 nm by UV detector. For column performance check, a molecular weight marker (1511901; Bio-Rad) comprising thyroglobulin, ɣ-globulin, ovalbumin, myoglobin, and vitamin B12 was used prior to and after every set of reference standard and 10 samples. Table 2. Parameters of SE-HPLC Mobile phase 0.02 mol / L (20 mM)TRIS(hydroxymethyl)aminomethane (TRIS), 0.1 mol / L (100 mM) NaCl, pH 7.5 Solvents A: Mobile Phase B: Water C: Water D: WaterFlow-rate 0.75 mL / min Flow up 0.1 mL / min Flow down 0.1 mL / min Flow-cell Semi-Micro Flow cell VWD-3x00(RS) Stainless Steel 2.5 µL; 7 mm Column YMC-Pack Diol-200, 20nm, 8 mm ID x 300 mm Pre-column Frit PEEK 0.5 µm, blue (0.094 in x 0.065 in x 0.250 in) Oven temperature 30 °C ± 2 °C Autosampler 5 °C ± 3 °C temperature Detection 280 nm Injection volume 25 µLAmount of Injection 50 µg AP Running time 20 min Reverse Phase HPLC (RP-HPLC) Samples (15 µg recAP) were separated on Waters Xbridge BEH300 C4, 3.5 µm, 250 x 2.1 mm column on Ultimate 3000 HPLC system (Thermo Fisher). The samples were diluted in solvent (see Table 3). The measurements were performed using two mobile phase buff-ers (see Table 3) in gradient mode at an isocratic flow of 0.3 mL / min. Data were recorded at 280 nm by UV detector. For column performance check, RP in original customer buffer (reference RP) was injected every 10 sample injections after blanking the column with a solvent injection (water). Table 3: Parameters of RP-HPLC Mobile Phase A: 0.1% (v / v) TFA in water B: 0.1% TFA (v / v) in 90% acetonitrileC: Water D: Water Solvent Water HPLC Grade Flow rate 0.3 mL / min Flow up 0.1 mL / min Flow down 0.1 mL / min Flow cell Semi-micro flow cell VWD-3x00 (RS) Stainless steel 11 µL Column Waters Xbridge BEH300 C4, 3.5 µm, 250 x 2.1 mm Pre-column Frit PEEK 0.5 µm, blue (0.094 in x 0.065 in x 0.250 in) Column temperature 40 °C ± 2 °CAutosampler temperature 5 °C ± 3 °CDetector 220 nm Injection volume 30 µL (β(DS) = 0.5 mg / mL) Injected amount AP 15 µg AP substanceAnalysis time 80 min Maximum pressure 415 barStorage solution Mobile Phase A / Mobile Phase B (50:50)Cleaning buffer 1 Water / 2-Propanol / Methanol / Acetonitrile(25:25:25:25) Cleaning buffer 2 Water / Acetonitrile (50:50) Dynamic light scattering (DLS) Dynamic light scattering was measured with a DynaPro Plate Reader (Wyatt) at 25 °C. In DLS, particles with hydrodynamic radii in the nanometer scale are monitored. Degradation products can be either fragments with smaller radii than the radius of the monomer DS or aggregates with higher radii that each appears as additional populations if the difference in hydrodynamic radius exceeds at least 5- fold. If the difference in hydrodynamic radius is smaller, the change can be only monitored, if at all, by an increase in polydispersity. Samples were not diluted prior to analysis but directly transferred to the analysis plate. For analysis (Dynamics, version 7.10.1.21) the default settings for refractive index and viscosity of water were used. Three replicates á 30 µL of each sample were transferred to a 384-Well-Plate. The whole plate was measured twice, which results in 6 measurements per sample. Within the implementation of the method, 35 acquisitions of 1 second each were chosen as measurement parameters. DLS data are presented as D50-values over radius scatter plots of all measured peaks with intensity weighted symbol size in order to allow for easier comparison of the different formulations during storage time. The radii for each population (x- axis) and the D50 value of all particles measured (y-axis) are plotted in a log-log scatter plot. The depicted symbol size of each peak is proportional to the relative intensity of the respective peak (%Intensity weighted). The overlay of replicate measurements in one graph enables the evaluation of the reproducibility of each measurement by increasing color intensity. Subvisible particles (SVP) Micron-sized protein aggregates and particles (subvisible particles, SVP) are important quality attributes of therapeutic protein formulations due to their risk of enhancing an immunogenic response. Hence pharmacopoeias require quantification of SVP larger than 10 μm and 25 μm. Currently, the pharmacopoeial acceptance criteria for SVP are: NMT 6000 particles ≥10 µm per container and NMT 600 particles ≥25 µm per container. SVP < 10 µm have to be monitored, however specific acceptance criteria are not defined. It has to be mentioned that the Ph. Eur. and the USP compendial methods specify Light Obscuration, as method for subvisible particle counts. Flow imaging, as performed here, produces significantly higher particle counts than Light Obscuration. Thus, if limits are met using this methods, it is to be expected that the pharmacopeia limits will be met. Formulations were analyzed by flow imaging microscopy using a FlowCam® 8100 system (ANASYSTA). In order to achieve an optimal flow imaging efficiency in the range of 60 - 70%, the following analysis settings were used: 0.300 mL / min flow rate with imaging rate of 60 frames per second. Prior to performing sample analysis a system suitability test (SST) was run in order to verify operational system status (e.g. system free from foreign particle contamination or other defects). For the SST water (B. Braun / freshly opened) was analyzed with the acceptance criteria for the SST defined beforehand: Total particle count <50 particles. For the analysis of formulations 350 µL of sample were manually injected, of which ~180 µL were imaged by the system. The SVP data was size-grouped according to EP / USP utilizing the following filters: Total particle count, below 5 µm, 5 - 10 µm, above 10 µm, and above 25 µm. The SVP of all formulations at all storage time points were determined. For each formulation 2 technical replicates were measured non-diluted. Appearance in vial according Ph. Eur. method 2.9.20 Assessment of visible particles was performed using a visual inspection box equipped with non-flickering fluorescent lamps and a black and white background plate. Light intensity in the visual box was tested first with a lux meter at inspection height and was in accordance within the limit stated in pharmacopoeia (2000-3750 lux). The samples were equilibrated to room temperature and the lights were switched on 30 minutes before evaluation in order to stabilize the light. Window blinds were closed to avoid incidence of light from outside the black and white box. The outside of the sample vials were cleaned with a lint-free tissue followed by an inspection of each sample for approximately 5 seconds in front of black and white background, respectively, without magnification in order to determine the number (if possible) and material of visual particles and visual detectable clarity. If possible, particles were further characterized. Particles of different color can be detected easier in front of the two different backgrounds. In order to minimize the particles sticking to the vial walls, the vials were softly swayed. The following scoring system was used: no, few, many or high number of particles / uncountable. Table 4: Letter code for Visual Appearance Letter code Description AAir bubbleC Colour, Ph. Eur. 2.2.2F Fibre F* Multiple fibre (more than one) HHurricane, tornado, e.g. because of sedimenting orfloating particles LParticles that are on the limit of being visible asdistinct particles SSchlieren (optical inhomogeneities or phaseseparation) TTurbidity, opalescence, cloudiness, hazinessV Viscosity X Non-inherent particles: metal, glitter, rubber parts, glass sheds Table 5. Score for visible particles Score for visible particles Description0 No particles visible within first 5 s1 Few particles visible within first 5 s (for e.g. belowor equal to 5 particles per vial) 10 High number / uncountable of particles visibleimmediately Turbidity according Ph. Eur. method 2.2.1 Turbidity (degree of opalescence) of samples was measured in specific glass cuvettes (HACH-Lange) with an inner diameter of 11 mm in a daily calibrated turbidimeter LAB 2100AN (HACH-Lange). The degree of opalescence was determined by comparing the measured turbidity of samples to standards with known turbidity (<0.1, 3, 6, 18, 20, 30 and if necessary 200 nephelometric turbidity units (NTU), according to European Pharmacopeia 10 as described in the StablCal®-Kit user manual; HACH-Lange). For each measurement the whole 1.8 mL present in 2R vial were used to respect the minimum volume of at least 1.5 mL for the analysis method. Tangential Flow-Filtration, sample preparation The four formulations (Table 6) were prepared by tangential flow-filtration (TFF), sterile filtered, and then stability stored for up to 3 weeks in 2R vials filled with 1.8 mL AP, each. The formulations for the stability and mechanical stress study were prepared by tangential flow-filtration (TFF) using an Ambr®crossflow –4 channel system at a flow-rate 20 mL / min and a set transmembrane pressure of 1000 mbar. The filters used were Ambr®CF Filter Hydrosart®with a 30 kD cutoff and 10 cm2membrane area. The samples were dialyzed with 5 times against their original volume. The permeate flow rate of buffer exchange indicated that the samples rebuffered to citrate allowed a faster trans-membrane flow than the samples rebuffered to histidine. The impaired flow in presence of histidine might already hint at presence of aggregates clogging the membrane. Stability study A stability study was performed on the four formulations described in Table 6. Each of the formulations was placed for 3 weeks on 5 °C ± 3 °C, 25 °C ± 2 °C RH = 60 % ± 5 %, 30 ± 2 °C RH = 65 ± 5 % and 40 °C ± 2 °C RH = 75 % ± 5 %. Samples were analyzed on T = 0 W, T = 1 W, T = 2 Ws, and T = 3 Ws. Mechanical stress study A mechanical stress study was performed by subjecting the samples to pumping stress (24 h, RT, 0.89 mm inner diameter, 100 rpm, Imatic peristaltic pump), oxidation stress (by addition of 0.01 % H2O2 + 0.1 mM Fe(II), incubation for 4 h) , repeated syringe deployment (3x, RT, 150 mL / h, 21 g needle), repeated freeze / thaw cycles at -20 °C / RT (>18 h per freeze cycle > 7 h per thaw cycle), and repeated freeze / thaw cycles at -80 °C / RT (>18 h per freeze cycle > 7 h per thaw cycle). Visual Appearance Visual appearance of samples was assessed using an inspection light box equipped with non-flickering fluorescent lamps and a black and a white background plate (Portable Inspection Hood MIH-PORT, BOSCH). The samples were evaluated by eye in their storage container without magnification to assess sample appearance in respect to visible particles, colour, and clarity. LC-MS 100 μg of each sample (12.5 μL for 8 mg / mL or 2.5 for 40 mg / mL) were mixed with lyse buffer (Preomics) to reach 100 μL. Samples were processed by resuspension in 100 μl of LC-LOAD Buffer (the iST Preomics Kit, Preomics).1 μL of each sample (1 μg) was sepa-rated in an Ultimate 3000nRSLC (ThermoFisher Scientific) coupled to a in a 25-cm ana-lytical column (75μm ID, 1.6 Qexactive HF C18, IonOpticks) with a 50-min gradient from 2 to 37 % acetonitrile in 0.1% formic acid. The effluent from the HPLC was directly elec-trosprayed into a Qexactive HF (ThermoFisher Scientific) operated in data dependent mode to automatically switch between full scan MS and MS / MS acquisition (survey full scan MS spectra (m / z 375–1600) were acquired with resolution R=60,000 at m / z 400 (AGC target of 3x106), the 10 most intense peptide ions with charge states between 2 and 5 were sequentially isolated to a target value of 1x105, and fragmented at 27 % normalized collision energy. Mass spectrometric conditions were: •spray voltage, 1.5 kV •no sheath and auxiliary gas flow •heated capillary temperature, 250°C •ion selection threshold, 33.000 counts Raw data was analyzed using Mascot 2.6.0 against a DB containing the recAP sequence as provided by the customer including oxidation in C, D, M, F, H, W, K, N, P, Y. Results were parsed in Scaffold 4.9.0. LC-MS measurements were performed at the protein analysis unit of the LMU Munich. Kinetic Activity This method is to confirm the presence and determine the recAP enzymatic activity using para-Nitrophenyl Phosphate (pNPP) substrate for different formulations and stress conditions. Reference material and samples were pre-diluted in three steps to a concentration of 700 ng / mL. Between dilution steps dilutions were mixed by pipetting up and down. A pipetting robot performed the dilution steps for the following concentration: 466 ng / mL, 311 ng / mL and 207 ng / mL. The dilution plate was equilibrated at 25 °C in a water bath. Kinetic measurements were performed row wise. After addition of pNPP substrate to row A, the measurement was started. Then, pNPP substrate was added to row B and measurement was started again, continuing in the same way, until all rows were measured. Particle Characterization (Unchained Labs Hound) The Unchained Labs Hound is a device that combines microscopy with directed Raman and Laser-induced breakdown spectroscopy. 200 µL of each sample was filtered onto gold filter-plates with 10 µm pores using a vacuum pump. The filter plates were measured before and after adding the sample to confirm cleanliness of the plates. Particles were focused manually using 20x magnification to take pictures and measure sizes. The microscope was then further used to focus the Raman and LIBS lasers onto individual particles to characterize their composition. Results Study design The basic characterization comprised of two studies. 1. Forced degradation / accelerated aging study 2. Buffer screening study The forced degradation / accelerated aging (mechanical stress) and stability study examined four formulations of recAP at 8 mg / mL and 40 mg / mL in two buffers each (see Table 6). The samples were stored up to 3 weeks at 5 °C, 25 °C, 30 °C and 40 °C. Additionally, samples were exposed to various stress conditions (Table 7). This was performed to identify degradation pathways and conditions that might lead to particle formation. The buffer screening study using melting Temperature (Tm) and kD measurements were performed to find suitable conditions that might reduce particle formation and render the AP more stable (Table 7). The findings of this Buffer Screening were used as a starting point for a formulation round. The buffers chosen for this study are shown in Table 7. The original AP was formulated as depicted in Table 1. Results of the stability and mechanical stress study AM-Pharma observed a strong increase in particle formation after shifting from citrate buffer to histidine buffer for their drug product. Two formulations of recAP were prepared in histidine buffer and two in citrate buffer to infer on the impact of histidine on recAP tendency to aggregate. In both systems, the AP was diluted to 8 mg / mL to reflect the target concentration of the final product or to 40 mg / mL Table 6. Formulations used in the Stability and mechanical stress study. Formulations prepared from PF-06853082. Concentration Sample recAP) Buffer Other Excipients [mg / mL] WP3_01 8 20 mM histidine 250 mM Sorbitol 2 mM MgCl2 0.05 mM ZnCl52WP3_02 8 20 mM citrate 250 mM Sorbitol 2 mM MgCl2 0.05 mM ZnCl2WP3_03 40 20 mM histidine 250 mM Sorbitol 2 mM MgCl2 0.05 mM ZnCl2WP3_04 40 20 mM citrate 250 mM Sorbitol 2 mM MgCl2 0.05 mM ZnCl2

[0002] Table 7: Samples for Buffer, Osmolality screening study. Sample Target Buffer pH 01 40 mM citrate, 0 mM NaCl 6 02 40 mM citrate, 50 mM NaCl 6 03 40 mM citrate, 100 mM NaCl 6 04 40 mM citrate, 200 mM NaCl 6 05 40 mM citrate, 400 mM NaCl 6 06 40 mM citrate, 0 mM NaCl 6.5 07 40 mM citrate, 50 mM NaCl 6.5 08 40 mM citrate, 100 mM NaCl 6.5 09 40 mM citrate, 200 mM NaCl 6.5 10 40 mM citrate, 400 mM NaCl 6.5 11 40 mM citrate, 0 mM NaCl 7.0 12 40 mM citrate, 50 mM NaCl 7.0 13 40 mM citrate, 100 mM NaCl 7.0 14 40 mM citrate, 200 mM NaCl 7.0 15 40 mM citrate, 400 mM NaCl 7.0 16 40 mM histidine, 0 mM NaCl 6.0 17 40 mM histidine, 50 mM NaCl 6.0 18 40 mM histidine, 100 mM NaCl 6.0 19 40 mM histidine, 200 mM NaCl 6.0 20 40 mM histidine, 400 mM NaCl 6.0 21 40 mM histidine, 0 mM NaCl 6.5 22 40 mM histidine, 50 mM NaCl 6.5 23 40 mM histidine, 100 mM NaCl 6.5 24 40 mM histidine, 200 mM NaCl 6.5 25 40 mM histidine, 400 mM NaCl 6.5 26 40 mM histidine, 0 mM NaCl 7.0 27 40 mM histidine, 50 mM NaCl 7.0 28 40 mM histidine, 100 mM NaCl 7.0 29 40 mM histidine, 200 mM NaCl 7.0 30 40 mM histidine, 400 mM NaCl 7.0 31 40 mM Succinate, 0 mM NaCl 6.0 32 40 mM Succinate, 50 mM NaCl 6.0 33 40 mM Succinate, 100 mM NaCl 6.0 34 40 mM Succinate, 200 mM NaCl 6.0 35 40 mM Succinate, 400 mM NaCl 6.0 36 40 mM TRIS, 0 mM NaCl 7.5 37 40 mM TRIS, 50 mM NaCl 7.5 38 40 mM TRIS, 100 mM NaCl 7.5 39 40 mM TRIS, 200 mM NaCl 7.5 40 40 mM TRIS, 400 mM NaCl 7.5 respectively, reflecting the concentration that is present throughout downstream processing during production. Stability Study Visual Appearance of the samples directly after rebuffering showed no particles in the samples with a concentration of 8 mg / mL. The samples at 40 mg / mL showed a slightly higher turbidity than those at 8 mg / mL and contained less than 5 visible particles, each. All samples were colorless. RecAP at 40 mg / mL in histidine showed a higher turbidity than the sample buffered in citrate (data not shown). Already after one week of storage, a high number of visible particles formed in all four formulations. Turbidity increased for all samples, but predominantly in the sample stored at 40 mg / mL in histidine. Turbidity also increased with increasing storage temperature (data not shown). This trend continued throughout the stability study. Mechanical Stress Study Mechanical stress also led to an increase in turbidity predominantly in the samples with histidine Buffer. Also samples at 40 mg / mL always showed higher turbidity than samples at 8 mg / mL. The stress condition which led to the highest increase in turbidity was stirring at 150 rpm for 24 h, see Figure 1A, followed by peristaltic pumping (100 rpm, 60 min), repeated syringe deployment (Figures 1B, C). Overhead rotation and orbital shaking showed lesser but still significant increase (data not shown). The relative unspecificity of the occurrence of turbidity and hence bigger particles points to causes that are inherent to all of the used mechanical stressing forms: shear stress and oxidation. Similarly to mechanical stressing, also freeze-thawing resulted in an increase in visual particles and turbidity, with larger, fiber-like particles being visible predominantly in the samples buffered in histidine. Overall, for both the mechanical stress and stability testing of the various formulations, recAP in citrate is less turbid and contains less particles compared to histidine buffer, especially when recAP is dissolved in higher concentration of 40 mg / mL. Subvisible Particles (SvP) by Flowcam Particle sizes in the subvisible range were determined by flow imaging analysis (as also cited in USP <787>) using a FlowCam 8100 device. Currently, subvisible particles < 10^µm have to be monitored but acceptance criteria are not defined. For larger subvisible particles, the acceptance criteria are 6000^particles > 10^µm per container and 600^particles > 25^µm per container. It has to be noted, however, that these limits refer to subvisible particle counts determined by light obscuration (LO). As flow imaging is known to produce consistently higher particle counts than LO due to higher sensitivity for translucent (e.g. proteinaceous) particles, applying the same thresholds does not pose any risks. Overall, recAP showed high but still acceptable numbers of subvisible particles at t0. During storage, no systematic trends over time or temperature could be detected. RecAP showed an increase in subvisible particles after stirring, peristaltic pumping and repeated syringe deployment, as well as repeated freeze- thawing. Throughout the study, particle counts were higher in the higher concentrated samples, and citrate samples showed slightly lower counts than samples in histidine. While samples after orbital shaking and overhead rotation only showed a modest increase in subvisible particle counts, stirring, peristaltic pumping, and repeated syringe deployment led to a drastic increase in subvisible particles (Figure 2). Stirring caused an increase in more than one order of magnitude in all size categories monitored, with the higher concentrated samples showing higher particle counts above 10 µm and above 25 µm. Peristaltic pumping also led to higher particle counts predominantly in the samples at 40 mg / mL drug concentration, where particle counts increased more than 10-fold. Deploying the sample three times through a 21-needle gauge at 150 mL / h caused an increase in subvisible particle counts that was more pronounced in the histidine samples than in the samples buffered in citrate. Again, the increase in particle counts was more severe in the higher concentrated samples. Subjecting the samples to oxidation stress via addition of 0.01 % H2O2 and 0.1 mM of Fe(II) did not lead to a significant increase in particle counts, indicating that the oxidation might not play a major role in particle formation of recAP, and the role of shear stress is more important for particle formation (Figure 3). Repeated freeze-thawing also generally increased the number of particles especially below 5 µm as can be seen in Figure 3. Here the increase was most severe for the lower concentrated samples independent of buffer. Freeze-thawing at –20 °C showed a more distinct increase in particle counts already after cycles, while for freeze-thawing at -80°C, 5 cycles were necessary to reach similar levels of subvisible particles. Size Exclusion Chromatography (SEC) Size exclusion chromatography was performed as described. It was performed for all samples at t0 and after 3 weeks at all temperatures. Peaks were integrated as higher molecular weight species (HMWS), Main Peak, and lower molecular weight species (LMWS). SEC data showed a decrease in relative main peak area after three weeks at 30 °C and 40 °C. The decrease was slightly higher in the samples stored at 40 mg / mL than in those stored at 8 mg / mL (data not shown). After storage for three weeks the amount of higher molecular weight species (HMWS) increased for the higher temperatures (30 °C, 40 °C). Here, the samples concentrated to 40 mg / mL showed a more pronounced increase to a relative peak area of 1.2% and 1.5% for histidine and citrate, respectively (not shown). The only other condition that led to a detectable increase in HMWS was oxidation. Here, the samples buffered in histidine showed an increase to 0.7 % HMWS relative peak area at 8 mg / mL and 1.0 % at 40 mg / mL concentration. Samples in histidine generally showed a lower amount of LMWS compared to samples stored in citrate. The only stress condition affecting the amount of LMWS was Oxidation (data not shown). Here, all formulations increased in LMWS except 40 mg / mL recAP in histidine. However, storage at high temperatures, oxidation, and stirring resulted om a decrease in total area. Specifically the samples in histidine stored for three weeks at 40°C showed a loss of area of more than 20 %. This indicates the abundance of aggregates that are too large to actually pass the column and that therefore will not contribute to total area. Considering the high amount of visible particles that were seen in these samples, this appears very likely. Besides relative peak areas, an important measure of quality for SEC measurements is recovery, i.e. the total area under the curve, which should stay similar if the same amount of protein is used. Although this implies the Table 8. Recovery of Recap in SE-HPLC as concentration-adjusted absolute area of the chromatogram. Total Average recovery (%) area t=0 3w 3w 3w 3w OH OS 2xFT 5xFT 2xFT 5xFT PSSyringe Oxi Stir(%) 5°C 25°C 40°C 5°C 20°C 20°C 80°C 80°C pump 8 102.8 93.6 94.3 82.8 74.1 103.7 104.8 104.1 105.4 103.6 105.7 106.4 105.4 95.8 94.5 mg / mL in His 8 101.2 100.4 96.1 97.5 98.3 101.8 102.9 100.5 102.2 100.1 102.6 101.8 105.9 102.1 106.5 mg / mL in Cit 40 100.6 91.2 85.5 83.1 78.9 99.9 99.0 98.6 98.1 102.4 99.3 98.6 101.1 91.8 89.8 mg / mL in His 40 100.3 99.9 98.7 100.7 101.9 97.3 97.2 98.5 95.9 103.6 102.0 98.8 105.4 92.3 100.8 mg / mL in Cit

[0003] approximation that all degraded and aggregated species visible in SEC have the same extinction coefficient at 280 nm, it is expected that the total area under the curve should stay within ± 10 % of its original value at t0 (see Table 8). Hound Hound measurements were performed to further characterize the particles present in recAP samples by Raman microscopy. Reasonable amounts of particles to conduct optical and Raman characterization were only obtained in 40 mg / mL samples. Generally, the amount of found particles was low (3-15 particles per filter plate), except after stirring stress (>20 particles per filter plate). The particles found had two sets of shapes. One type of particle was more globular with sharp detectable edges. The other type was more fibrillar and also often larger. Both particles types could later be identified as proteinaceous. Although the limited number of particles did not allow a precise quantitative assessment, some trends were still apparent. Samples buffered in histidine showed higher numbers of fibrillar particles compared to samples buffered in citrate. In citrate, there were generally less particles. The particles found looked more globular but few fibrillar particles were still present. The only exception is after stirring, where also in the histidine buffered samples more globular particles seem to be present (data not shown). Raman spectra of particles were recorded using a laser with 532 nm wavelength. By comparison with a spectral database, all particles could be identified as either protein or sorbitol or a combination thereof. As the spectral database did not contain any phosphatase spectra, Bovine IGG was used which showed the highest correlation with the signal (protein spectra are generally very similar). Using Raman spectra at 785 nm laser wavelength did not give any additional insight and showed a lower signal to noise ratio. Laser-induced breakdown spectroscopy (LIBS) did not reveal any higher local metal concentrations. However, that does not exclude metals from being present in the particles, as LIBS would require high concentrations and therefore a strong local enrichment of Mg or Zn to allow detection. Dynamic Light Scattering (DSL) DLS measurements were performed as described. Most formulations yielded predominantly a single peak between 5 and 8 nm diameter following all conditions. Stability Study The recAP protein had an apparent hydrodynamic radius of 7 nm. However, particle populations of higher sizes were also present in the formulation from t0. In the samples buffered in citrate, these higher sized aggregates were only present in individual technical replicates with intensity percentages of < 5 %, whereas in the histidine buffered samples, these aggregates caused ~50 % of the scattering intensity. Significant differences over time were only detectable for the samples stored at 40 °C / 75 % rH where the intensity of higher-size particle population increased over time for the samples buffered in histidine. This indicates that the amount of aggregates increases over time at 40°C for the histidine-buffered samples (data not shown). Mechanical Stress Study Overhead rotation, syringe deployment, and orbital shaking of the samples did not affect the particle size distribution systematically. Repeated freeze-thawing and oxidation, similarly, had no effect on the size distribution . Peristaltic pumping and stirring led to an increase of larger submicron particles in the samples buffered in citrate. Since these slight increases in the amount of aggregates do not seem to agree with the strong increase seen in subvisible particle measurements and visual appearance, one must treat these findings with caution, as large particles might not be detected by DLS. Very large particles (> 2 µm, depending on particle density) tend to sink down in the well and do therefore not diffuse through the focus of the laser of the DLS, rendering these very big aggregates undectable by DLS measurements. RP-HPLC Degradation of recAP after Oxidation was monitored using RP-HPLC. Oxidation was performed by spiking 0.01 % H2O2 and 0.1 mM Fe(II) to the formulations and incubating for 4 h. See Figure 4. The relative peak area of the intact recAP protein (Isodimer A) decreased after oxidation for all four formulations. However, this decrease was more pronounced in the histidine buffered samples. In favor of Isodimer A, most degradation species increased in relative area. The severe increase in the peak termed “related species” was similar in all four samples from 7 % to approx. 25 %. In Isodimer B1, the relative areas also increased but only by a small margin and seemingly unspecific. The most pronounced increases were detected in both peaks of Isodimer B2. Here, also clear differences between the buffers and the concentrations were visible. The histidine buffered samples increased significantly more in area than their citrate-buffered counterparts. This indicates that the related species is indeed an oxidation product, where the presence of citrate (or the lack of histidine) cannot prevent the degradation process. Conversely, degradation to both Isodimer B2 species is heavily influenced by the presence of citrate. LC-MS LC-MS measurements were performed to determine the prevalently oxidized amino acid in recAP. This, in turn, allowed to choose a suitable antioxidant (in this case methionine) to be used in the subsequent formulation round. The results indicate that about ~20 % of Methionine and a ~10 % of Tryptophan were already oxidized in the original material. The amount of oxidized methionine was considerably increased after inducing oxidation with 0.1 mM Fe(II) and 0.01 % H2O2. As the variance of the amount of oxidized methionine was high, the difference between samples in histidine and samples in citrate were non-significant. Based on these results, it was chosen to use methionine as an antioxidant in the formulation round. Kinetic Activity Kinetic Activity measurements were performed as described. Goal of the method was to identify differences in enzymatic activity in different formulations after storage for 1 month at 30°C and after subjecting the samples to various mechanical stress conditions. No significant differences could be detected in kinetic activity over storage or mechanical stresses. Nephelometry At t0, turbidity is lowest for recAP at 8 mg / mL buffered in citrate with 4 nephelometric turbidity units (NTU). The sample at 8 mg / mL in histidine shows slightly higher turbidity at 6 NTU. The higher concentrated samples show a significantly higher turbidity, with recAP at 40 mg / mL in citrate starting at 10 NTU and in histidine as high as 28 NTU. Table 9: Turbidity over 3 weeks storage at various temperatures. Turbidity (nephelometric turbidity units) Formulation t=0 1w 5°C 1w1w 1w 2w 2w 2w 2w 3w 3w 3w 3w 25°C 30°C 40°C 5°C 25°C 30°C 40°C 5°C 25°C 30°C 40°C 8mg / mL in histidine 6 8 7 7 8 7 6 7 8 11 9 11 98 mg / mL in citrate 4 3 3 3 3 3 3 3 3 6 4 5 440 mg / mL in histidine 28 27 27 26 43 25 27 26 50 27 29 30 6140 mg / mL in citrate 10 10 10 10 13 9 12 11 12 10 15 11 14Table 10: Turbidity after various mechanical stresses. Turbidity (nephelometric turbidity units) Formulation t=0 OverheadOrbitalStirring Syringe Oxydation 2 F / T5 F / T 2 F / T 5 F / T Rotation Shaking -20°C -20°C -80°C -80°C 8mg / mL in histidine 6 6 6 32 7 7 8 8 7 88 mg / mL in citrate 4 3 3 9 3 3 3 3 3 440 mg / mL in histidine 28 26 25 132 26 26 26 26 26 2740 mg / mL in citrate 10 10 10 24 10 10 10 10 10 11

[0004] Table 11: Conductivity over 3 weeks storage at various temperatures. Turbidity (nephelometric turbidity units) Formulation t=0 1w 5°C 1w1w 1w 2w 2w 2w 2w 3w 3w 3w 3w 25°C 30°C 40°C 5°C 25°C 30°C 40°C 5°C 25°C 30°C 40°C 8mg / mL in histidine 668 640 644 684 649 673 657 656 658 637 470 638 6438 mg / mL in citrate 4067 4173 4074 4323 4127 4173 4140 4055 4074 3749 3908 3823 383040 mg / mL in histidine 624 635 622 656 631 625 641 634 622 626 541 557 42140 mg / mL in citrate 3848 3791 3827 4009 3933 3955 3871 3915 3949 3884 3753 3734 3623Table 12: Conductivity after various mechanical stresses. Turbidity (nephelometric turbidity units) Formulation t=0 OverheadOrbitalStirring Syringe Oxydation 2 F / T5 F / T 2 F / T 5 F / T Rotation Shaking -20°C -20°C -80°C -80°C 8mg / mL in histidine 668 668 679 704 660 591 681 644 598 6488 mg / mL in citrate 4067 4316 4307 4455 4029 3078 4030 3946 3071 395440 mg / mL in histidine 624 656 680 675 627 421 608 605 604 54840 mg / mL in citrate 3848 4053 4283 4283 3860 2354 3870 3890 3837 3841

[0005] At 5 °C, 25 °C, and 30 °C the turbidity did not change over the course of three weeks. At 40°C, an increase in turbidity for the higher concentrated sample in histidine to 61 NTU was detected (see Table 9). Mechanical Stress Study The only mechanical stress condition that caused a detectable increase in turbidity was stirring, where again the samples buffered in histidine showed a much higher increase than the samples buffered in citrate, reaching 132 NTU. See Table 10. Conductivity, Osmolality, pH Over the period of three weeks, the conductivity of the solutions is stable (See Table 11). Conductivity is mostly caused by the total dissolved solids, with strongest effect being caused by metal ions. A decrease in conductivity can therefore only be caused by complexation, covalent binding of these ions, or precipitating out of solution. The conductivity of citrate is much higher than the conductivity of histidine. This is caused by citrate having three carboxyl groups, being able to provide highly conductive OH-anions. A decrease in conductivity is detectable after oxidation (Table 12), implying that the metal ions present in the formulation might play a role in oxidative degradation of recAP. In osmolality and pH no changes were detected the values are within the variance of the assays. The sample pH, osmolality and conductivity stay mostly constant for both the storage and the mechanical stress study. Only oxidation led to a decrease in conductivity implying that either the metal ions take part in a reaction, resulting in a less conductive compound, or hydrolysis is taking place in the sample, as both ions resulting from water (H3O+ and OH-) have the highest conductivity. Results of the Buffer, Osmolality screening study For the buffer / osmolality screening study, recAP (PF-06853082) was rebuffered at 41.5 mg / mL using pressure filtration (Unchained Labs, Big Tuna, 96-well 30 kDa MWCO filter plates). As target buffers, citrate buffer at pH 6.0, 6.5, and 7.0, histidine buffer at pH 6.0, 6.5, and 7.0, succinic acid buffer at pH 6.0, and TRIS buffer at pH 7.5 were used. A salt concentration of 0, 50, 100, 200, and 400 mM NaCl is used for each pH-buffer combination, respectively (Table 7). The nano-DSF measurements were performed at 10 mg / mL recAP concentration. Using nanoDSF, the melting temperature as well as the aggregation temperature of recAP were measured in the target buffers mentioned in Table 7. Therefore, recAP samples were further diluted in the target buffer to a concentration of 10 mg / mL. With 82.1°C, the average melting temperature of recAP is high in comparison with other proteins indicating a high conformational stability of the enzyme towards increased temperatures. The melting temperature (Tm) of recAP declined with increasing NaCl concentration in all buffer systems at each pH. At low osmolality, histidine at pH 7 and TRIS at pH 7.5 showed the highest Tm, whereas the Tm of recAP in citrate was lowest. At higher salt concentrations (reflecting realistic osmolality ranges of the actual drug product), the samples in Succinate buffer at pH 6 and in TRIS buffer at pH 7.5 had the highest Tm -values of > 82 °C. Both Succinate and TRIS were therefore also used later in the formulation round. As this study is focused on aggregation, nanoDSF was also used to determine the aggregation temperature (Tagg) of recAP in the above mentioned buffers. The aggregation temperature is measured as the inflection temperature of the scattering signal. The average aggregation temperature (Tagg) of recAP across all samples was 79.7 °C (data not shown). The highest Tagg was measured in citrate buffer at pH 7 and 0 mM NaCl. However, aggregation temperature decreased with increasing salt concentration, particularly in citrate buffer. At higher osmolality recAP in Succinate pH 6 and TRIS pH 7.5 showed a higher Tagg, indicating that these buffers might be suitable to reduce aggregation at the osmolality levels of the final drug product. The aggregation temperature was not directly correlated to the melting temperature, indicating that aggregation is not a simple by-product of unfolding alone. To conclude, recAP shows a high melting and a high aggregation temperature compared to other therapeutic proteins, indicating a high conformational stability. Both melting and aggregation temperature decrease with sodium chloride concentration. As a salt, NaCl masks surface charges of proteins, leading to weakened intermolecular repulsions and a prevalence of hydrophobic interactions. The effect of NaCl on the transition temperatures could also be related to the increased osmolalities in the samples with NaCl. At osmolalities targeted for the actual drug product (330 mOsm / kg, corresponds to 165 mM NaCl), aggregation temperature and melting temperature are highest in Succinate buffer at pH 6.5 and TRIS buffer at pH 7.5. Summary The results of the basic characterization show high numbers of particles across all size ranges monitored in both original formulations in both concentrations. Characterization by microscopy combined with Raman spectroscopy identified these particles as proteinaceous, which was in line with images derived from Flowcam measurements. Throughout all analytics, the prevalence of particles is higher at 40 mg / mL than at 8 mg / mL recAP concentration. This is expected as aggregation is ultimately linked to the frequency of collisions of the protein in solution, and hence, its concentration. It was further shown that citrate seemed to reduce the amount of particles created throughout storage and after stress. However, in the citrate-buffered samples the particle numbers of visible particles and subvisible particles were still high. This might be caused by the pre-exposure of the drug substance to histidine in the initial drug substance sent and throughout tangential flow filtration. It was therefore decided to compare this drug substance to another batch that did not have any pre-exposure to histidine. The discrepancy material pre-exposed to histidine vs. citrate buffer can have multiple causes. Besides being a buffer, citrate acts as a chelator and anti-oxidant. This is especially seen in RP-HPLC, where samples buffered in citrate show a drastically higher main peak retention after oxidation. However, LC-MS data does not reveal great differences in oxidation patterns between citrate- and histidine- buffered samples, indicating that oxidation plays a role in particle formation, but cannot be the sole cause of aggregation. Example 2 Materials and Methods As in Example1. Results Study design The aim of this work was to find suitable buffers, excipient combinations, and pH that reduce particle counts and contribute to the storage and stress stability of recAP. Based on the results described in Example 1, seven formulations with minor tweaks, one reference formulation (recAP in original buffer) and eight formulations with additional excipients and further adaptations based on current formulation were prepared. Additionally, two samples using recAP DS from another production batch were included in the study. This material was not pre-exposed to Histidine. As depicted in Table 30, formulation WP4_01 to 15 and WP4_Ref were prepared from AM-Pharma drug substance PF-06853082 sent in October 2021. This material originally contained 41.5 mg / mL recAP in 20 mM Histidine, 250 mM Sorbitol, 2 mM MgCl2, 50 µM ZnCl2, at pH 7.0. Formulations 16 and PC were prepared from the AM-Pharma Drug substance “VLT_Demo_BDS”. This material originally contained: 8 mg / mL recAP in 20 mM Citrate, 250 mM Sorbitol, 2 mM MgCl2, 50 µM ZnCl2, at pH 7.0. Formulation WP4_PC was prepared by sterile filtering and filling in 2R vials. Formulation WP4_16 was prepared by re-concentrating to 40 mg / mL and was subsequently dialyzed against the respective formulation buffer, diluted to 8mg / mL, sterile filtered, and filled into sterile 2R vials. This was done, to analyze which effect the handling and dialysis during formulation preparation has on the material. Visual Appearance, according Ph. Eur. method 2.9.20 All formulations were colorless and contained visual fiber-like particles already at t0. Particularly low amounts were seen in formulations WP4_16 and WP4_PC, which originate from different DS Material, which has never been in contact with Histidine. Partially, the amount of visible particles decreased over the first two weeks of storage at 30°C, but generally the amount of visible particles after 1 month is high. Mechanical Stress Study After mechanical stressing and freeze-thaw stressing, all samples showed high numbers of visual particles, except samples prepared from material that was not pre-exposed to histidine (WP4_16 & WP4_PC). Table 13 Formulations

[0006] Stirring caused a visible increase in turbidity compared to the other stressing methods. The results of the visual inspection indicate, that abundance of particles is mostly found in material “PF-06853082”. The two samples prepared from ”VLT_Demo_BDS” contained low amounts of particles in comparison. Here, stirring, repeated syringe deployment and peristaltic pumping were the only conditions that led to an increased presence of particles. Subvisible Particles (SvP) by Flowcam Particle sizes and quantities in the subvisible range were determined by flow imaging analysis (as also cited in USP <787>) using a FlowCam 8100 device. Currently, subvisible particles < 10^µm have to be monitored but acceptance criteria are not defined. For larger subvisible particles, the acceptance criteria are 6000^particles ≥10^µm per container and 600^particles ≥25^µm per container. Assuming a container filling of 4.5 mL for recAP, these numbers correspond to: 1333 Particles per mL >10 µm 133 Particles per mL > 25 µm It has to be noted, however, that these limits refer to subvisible particle counts determined by light obscuration. As SvP is known to produce consistently higher particle counts than LO, applying the same thresholds does not pose any risks. Stability Study At the start of the storage, generally lower particle counts were observed, when compared to earlier experiments. The highest particle counts at t0 were found in Formulations 04, 07, and 15. Comparing Formulations 16, Ref, and PC (all 20 mM Citrate, 250 mM Sorbitol, 2 mM MgCl2, 50 µM ZnCl2, at pH 7.0), the samples prepared from ”VLT_Demo_BDS” (without pre-exposure to Histidine, formulations 16 and PC) showed considerably less particles compared to formulation Ref (and compared to all other formulations prepared from “PF-06853082”, see Figure 5). Across all timepoints and temperatures, samples 3 & 10 (containing Kleptose HPB) and samples 6 & 11 (containing Poloxamer 188) show generally lower subvisible particle counts. During storage at 5 °C, the number of particles below 10 µm size remained on similar levels, while the particle counts at higher sizes (>10 µm and > 25 µm) decreased. Similar trends are seen at 25 °C and 30 °C, with the exception of formulation 07, were an increase in larger particles was seen after 1 month. Formulations 1,2,4,5,7,8,9,11,12, and 14, as well as reference have more than 133 Particles per mL > 25 µm at t0, but decrease in number over time at 5°C, 25°C, and 30°C. Generally, no relevant increase in particle counts after storage at 30°C except in formulation WP4_Ref. Formulations 03, 06, 15, as well as 16 and PC (prepared from ”VLT_Demo_BDS”) material was always below the relevant Ph. Eur. limits at all timepoints. Formulations 05, 07, and 08 were also below, except at t0. Mechanical Stress Study After Orbital shaking, only minor increases in particle counts were detected, and all formulations were below the Ph. Eur. / USP limit for particles ≥10 µm and only one replicate of formulation 01 and one replicate of formulation Ref was above the pharmacopeia limits for particles ≥25 µm. Repeated syringe deployment led to a higher increase in particle counts. Here, formulations 05 and 08 contained more particles ≥10 µm than specified by Ph. Eur. / USP. For subvisible particles ≥25 µm, formulations 03, 06, 10, 11, 13, 14, 16, PC and Ref met the pharmacopeia criteria. The strongest increase at all particle sizes was measured for the arginine-containing formulations. Peristaltic Pumping was performed for Formulations 16, PC, and Ref. Here, an increase in subvisible particle counts by one order of magnitude was detected for formulations 16 and PC, while a 100 fold increase occurred for formulation Ref. This again indicates that the samples prepared form material ”VLT_Demo_BDS” were much less prone to particle formation, than material “PF-06853082”. Particle formation was strongest after stirring (Figure 6). This is to be expected, as this is by experience the harshest mechanical stress condition. Here, formulations 03 and 10 (containing Kleptose), as well as 06 and 11 (containing Poloxamer 188) showed drastically lower particle counts after stress, even in comparison with formulation 16 (prepared from material “VLT Demo BDS”). This indicates an involvement of hydrophobic interactions in particle formation, as both Kleptose and Poloxamer typically reduce the exposed hydrophobic surface area of a protein. Kleptose by masking aromatic residues, Poloxamer by acting as a detergent. Repeated freeze-thaw stress resulted in an increase of particle counts. In general, freeze-thaw cycles from - 20 °C to RT resulted in a stronger increase in particle counts compared to freeze-thaw cycles from - 80 °C to RT (data not shown). The lowest increase in particle counts upon freeze-thaw cycling was found in the Formulations with Kleptose (3 & 10), Poloxamer 188 (6 & 11), and the samples prepared from material “VLT Demo BDS”. To conclude, presence of Kleptose and Poloxamer 188 reduce the amount subvisible particles, especially formulations 03 and 06 showed reduced counts to other formulations throughout all conditions. Moreover, formulations 16 and PC (prepared from material “VLT Demo BDS”) showed reduced counts. SE-HPLC Size exclusion chromatography was performed as described. It was performed for all samples at t0 and after 3w at all temperatures. Peaks were integrated as higher molecular weight species (HMWS), Main Peak, and lower molecular weight species (LMWS). Storage Study At t0, Formulations containing Citrate or Kleptose showed an additional post-peak in the size exclusion chromatograms. SE-HPLC measurements of the formulation buffers without recAP confirmed, that these were caused by the presence of citrate and Kleptose themselves. Therefore, it was mutually decided to not integrate these peaks. Generally, over the course of storage, the retention of main peak area is on a high level. Here, relative main peak areas range from 98.9 % through 99.8 % even after one month of storage at 30 °C (data not shown). The decrease of main peak area is accompanied by a corresponding increase in HMWS in all formulations. The lowest increase was seen in samples 02 (Citrate pH 7, Sorbitol, Methionine), 06 (Citrate pH 7, Sorbitol, Poloxamer 188), 13 (Succinate pH 6.5, Trehalose), and 15 (Succinate pH 6.5, Sorbitol). LMWS are present in negligible amounts and do only show unsystematic change within the variance of the measurement. There are correlations between the decrease of main peak area and presence of certain excipients and original material. Samples with succinate at pH 6.5 show the highest main peak retention, indicating that either lower pH or succinate buffer itself leads to reduced aggregation. Samples in citrate at pH 7 shows slightly lower main peak retention, while samples buffered in TRIS at pH 7.5 show the highest decrease in main peak area. This suggests that the tendency to form aggregates is pH dependent, increasing with increasing pH. However, it has to be noted that the differences between buffers are relatively small. There were also differences seen in main peak retention between different tonicifiers used. Trehalose and Sorbitol improve main peak retention compared to NaCl. Presence of NaCl masks charges and thereby prevents electrostatic protein- protein interactions, which, in turn, can cause aggregation. As NaCl does not have a positive effect on main peak retention, electrostatic interactions seem to be of less importance for the aggregation of recAP. Sugars and polyols like Trehalose and Sorbitol, stabilize proteins by preferential exclusion. This seems to have a beneficial effect on the colloidal stability of recAP. For recAP, samples containing Trehalose showed the highest main peak retention (Figure 7). Samples prepared form material ”VLT_Demo_BDS” show a lower amount of HMWS at t0, but increase with storage at 30°C similarly fast as the samples prepared from PF-06853082. With regards to recovery, there were no relevant discrepancies between t0 and 1 month of storage at 30 °C. Recovery was assessed as total absolute area of protein peaks (UV signal) in the chromatograms compared to concentration of the sample prior to injection. Mechanical Stress Study With regards to relative peak areas, only little differences between t0 and the stress conditions were found between mechanical stress conditions data not shown). After each condition, the relative main peak area for all formulations is still beyond 99 %. While LMWS peaks increased by approx. 0.05 % after mechanical stresses, relative area of HMWS peaks decreased in the range of 0.05 %, compared to t0. After freeze-thaw cycles from - 80 °C and - 20 °C to RT, relative main peak areas are still above 99 %. Freeze-thaw cycles from -20 °C lead to a higher increase in LMWS, whereas freeze-thawing from –80°C to RT increased HMWS slightly. However retention of relative main peak are is very high throughout the work package. While relative main peak areas showed good retention upon mechanical stress, the absolute areas of the SE chromatograms decreased upon stirring, syringe deployment and peristaltic pumping (Table 14). As concentrations stayed the same (as measured by UV 280), this means that part of the sample does not pass the column. This is indicative of very large aggregates, which cannot pass the frit (0.5 µm). Freeze-thawing or storage, as well as orbital shaking showed no decrease in absolute areas. Table 14. Recovery in SE-HPLC chromatograms, calculated from the sum of absolute peak areas, corrected by injection volume and actually measured concentration of the samples. Average Recovery (%) Formulation t0 OS Stir PSPump Syringe1 95.9 96.3 61.1 86.12 101.1 98.0 62.4 89.73 102.6 95.2 67.9 87.74 99.9 99.0 62.9 81.95 101.7 96.1 69.5 85.16 101.8 95.0 60.9 85.77 101.4 96.4 63.5 81.28 102.5 95.2 65.2 83.59 98.8 96.0 61.4 81.810 102.0 98.0 69.2 84.411 99.0 96.4 67.4 84.412 102.7 95.0 62.7 82.113 96.7 97.1 61.8 81.814 101.5 98.6 61.9 82.515 103.4 96.5 62.1 84.616 102.9 78.8 90.5 89.1Ref 102.7 96.0 60.7 83.3 81.3PC 82.1 89.5 91.2To conclude, recAP shows a high main peak retention after storage, freeze-thaw and orbital shaking stress. More severe mechanical stressing like stirring, peristaltic pumping, and repeated syringe deployment lead to a decrease in total area, presumably by forming larger aggregates that cannot pass the column. Although variance between formulations is comparably small, samples 16 and PC (prepared from ”VLT_Demo_BDS”) showed higher main peak areas compared to samples prepared from PF-06853082. The reduction of main peak over 1m at 40 °C was similar for both materials. DLS DLS measurements were performed as described. Most formulations yielded predominantly a single peak between 5 and 8 nm diameter following all conditions (data not shown). Stability Study There were no significant changes in DLS data with storage (data not shown). Small peaks in the order of 1 nm were present in samples 03 and 10. Buffer measurements confirmed that these are caused by the presence of Kleptose itself. Mechanical Stress Study Stirring and peristaltic pumping were the only mechanical stress conditions that led to higher sized particle populations. Here, a second particle population with a size of 100 –200 nm appears after the stressing. This indicates that harsh shear stress conditions (as they are present in both stirring and peristaltic pumping) contribute to the formation of aggregates. To conclude, recAP is stable with regards to colloidal stability over storage and most mechanical stress types, with only harsh mechanical stress conditions, as stirring and peristaltic pumping, leading to presence of larger aggregates. Kinetic Activity Kinetic Activity measurements were performed as described. Goal of the method was to identify differences in enzymatic activity in different formulations after storage for 1 month at 30°C and after subjecting the samples to various mechanical stress conditions. Stability Study At t0, no significant differences in kinetic activity could be detected between formulations. Upon storage for 1 month at 30°C, only slight differences in kinetic activities were detected. The only excipient effect was is that presence of methionine seems to lead to a slightly higher kinetic activity after 1 month. Generally, the kinetic activity of recAP is not affected by storage for 1 month at 30°C. Mechanical Stress Study Mechanical stress, as well as repeated freeze-thaw stress did not have a significant effect on kinetic activity of recAP. To conclude, none of the imposed storage or stress conditions had a significant effect on kinetic activity. This indicates that presence of aggregates (as for example after stirring) does not affect the enzymatic activity of recAP. Moreover, there is no difference in activity between the samples prepared from materials PF-06853082 and VLT Demo BDS Nephelometry Nephelometry was performed as described. In brief, turbidity of samples was measured in specific glass cuvettes (HACH-Lange) with an inner diameter of 11 mm in a daily calibrated turbidimeter LAB 2100AN (HACH-Lange). The degree of opalescence was determined by comparing the measured turbidity of samples to standards with known turbidity (<0.1, 3, 6, 18, 20, 30 and if necessary 200 nephelometric turbidity units, NTU). Stability Study There were no significant changes in turbidity with storage. Turbidity was low with the measured values being in the range of 1.9 –3.4 NTU. Differences between formulations are within the variance of the assay. Mechanical Stress Study Of the various stressing conditions, only stirring and peristaltic pumping caused an increase in turbidity values. Here, especially formulation 04 (the only formulation with neither Sorbitol nor Trehalose) increased in turbidity. Other than that, there were no differences detectable between formulations. To conclude, turbidity is on a low level for recAP after storage and for most of the stress conditions. Only harsh stress conditions like peristaltic pumping or stirring caused an increase in turbidity (which is in good agreement with subvisible particle data). Osmolality and pH Conductivity, Osmolality, and pH of the samples were monitored at each storage timepoint and stress condition as described. Stability Study Measured osmolality was in good agreement with theoretically calculated values for the formulation buffers. Here, no significant changes occurred during storage. The pH values of the formulations did also not show changes upon storage for up to 1 month at any of the subjected temperatures. Mechanical Stress Study The osmolality of the formulations stayed constant after all mechanical stress conditions, except formulations 03 and 10 (both Kleptose HPB-containing formulations) were the osmolality dropped upon induction of mechanical stress (Table 15). This is especially drastic after repeated syringe deployment, where the osmolality of these formulations drops by more than half. The special structure of Kleptose (with its hydrophobic interior) allows it to complex hydrophobic substances on the inside, which could theoretically alter the osmolality of the solution. However, it is not clear by what mechanism this extreme drop in osmolality is caused. Additinally, cyclodextrins like Kleptose are reported self-assemble over time (Couto et al., 2018. Materials 11: 1971). The other analytics do suggest that this has no impact on kinetic activity or formation of aggregates. The pH values did not change after any of the mechanical stress conditions Summary All formulations contained few visual particles at the start of the experiments. Over the course of storage and after stress conditions, the two samples prepared from ”VLT_Demo_BDS” (16 and PC) contained lower amounts of particles in comparison to the other samples (prepared from material PF-06853082). Kleptose HPB and Poloxamer 188 reduce the amount of subvisible particles. Similar to what was seen by visual inspection, also samples prepared from ”VLT_Demo_BDS” contained lower amounts of subvisible particles in comparison to the other samples (prepared from material PF-06853082. RecAP shows good stability during storage in SE-HPLC measurements. Trehalose and Sorbitol have a small positive impact on main peak retention upon storage. Samples prepared from material ”VLT_Demo_BDS” show a higher main peak area at t=0, but the main peak area decreases by similar amounts as for the other samples. Only harsh mechanical stressing conditions like peristaltic pumping or stirring cause a loss in total peak area of the chromatograms, suggesting the presence of larger aggregates. This finding is consistent with the results of DLS, where peristaltic pumping and stirring were the only stress conditions that led to presence of aggregates. Nephelometry data suggests that only stirring and peristaltic pumping lead to a significant increase in sample turbidity. Upon stirring, formulation 04 (the only formulation with neither Trehalose nor Sorbitol) shows the highest turbidity increase, suggesting a positive effect of sugars or sugar alcohols. All samples were stable with regards to osmolality and pH, except samples containing Kleptose HPB (03 and 10), where mechanical stressing causes a decrease in osmolality. Although it is not clear what causes this decrease, it had no negative effect on any of the other analytics. Methionine has a positive effect on the kinetic activity after 1 month of storage at 30°C. Generally, the kinetic activity of recAP seems to be unaffected by mechanical stress conditions. Example 3 The aim was to detect the effect of L-Methionine on the chemical stability of recAP determined by RP-HPLC. To this end, Formulations WP4_02 and WP4_09 (the methionine-containing formulations), as well as WP4_Ref and WP4_PC (as control and to identify differences in the original DS material) were used. Partially, storage was re-performed (for 2 week samples at 5°C and 25 °C). For the time points > 1 month, the material for further storage was pulled after 2 and 3 months. For all other time points, frozen retains of the stored samples were used. Formulation WP4_02, WP4_09, and WP4_Ref were prepared from drug substance 17L151O002 (W07394). This material originally contained: 41.5 mg / mL recAP in 20 mM Histidine, 250 mM Sorbitol, 2 mM MgCl2, 50 µM ZnCl2, at pH 7.0. Formulations 16 and PC (referred to as “citrate_only” material) were prepared from the AM-Pharma Drug substance “VLT_Demo_BDS”. This material originally contained: 8 mg / mL recAP in 20 mM Citrate, 250 mM Sorbitol, 2 mM MgCl2, 50 µM ZnCl2, at pH 7.0. Formulation WP4_PC was prepared by sterile filtering and filling in 2R vials. Materials and Methods As in Example1. Results RP-HPLC RP-HPLC measurements resulted in reproducible chromatograms and were generally well in agreement with the reference chromatogram, regarding peak positions. Peak areas were analyzed specifically for peaks Isodimer A, Related Species, Isodimer B1, Isodimer B2 Peak 1, and Isodimer B2 Peak 2. Relative peak areas at t0 were at 84 % for formulations WP4_02, WP4_09, and WP4_Ref (Formulations produced from material initially buffered in Histidine, PF- 06853082). These formulations showed a reduced relative peak area of Isodimer A1, compared to Formulation WP4_PC (which was prepared from material initially buffered in citrate) at t0 (89 %). As WP4_Ref and WP4_PC have the same formulation buffer, this difference must be present already in the original material. The relative area of Isodimer A1 decreases upon storage at 5 °C, 25 °C, and 30 °C, with the higher temperatures leading to a higher decrease over time (data not shown). The decrease was slowest for formulations 02 and 09 (both containing methionine) suggesting a positive effect of methionine on chemical stability, which also suggest oxidation as the oxidation pathway. Although formulation PC had a higher Isodimer A area at t0, the decrease over time is similar to that of formulation Ref, suggesting that degradation over time is similar in samples prepared from both original materials. The decrease in Isodimer A peak area is mirrored by an increase in Related Species and Isodimer B2 peaks. The relative peak area of Isodimer B1 also decreases over time. The increase in related species peak area over time is much stronger in the formulations without methionine, suggesting the “related species” to be an oxidation product. Formulations 02 and 09 show a much slower increase in related species, with no difference between both formulations. Isodimer B1 decreased only very slow at 5 °C. The decrease is fastest for Formulation 09 (the only sample in TRIS at pH 7.5), followed by 02, Ref, and PC in that order (all in citrate at pH 7). Here, no correlation between degradation and presence of methionine is detectable. In turn, increase in Isodimer B2 peak 1 and 2 is strongest in formulation 09, suggesting an effect of higher pH (or TRIS as buffer substance) on this degradation pathway. As the decrease of Isodimer A is very linear at all temperature, kinetic modelling and a linear extrapolation was performed to predict long-term stability after 12 months and 24 months. For this, a 0thOrder kinetic model (linear over time) was used: ^^= ^^^^−^^⋅^^^^^^^^ Conclusion Formulation development for recombinant human Alkaline Phosphatase (recAP) was performed with the main objectives to understand the reasons for particle formation and to optimize the original formulation with minor tweaks in order to minimize particle formation. In the course of a basic characterization, it recAP’s sensitivity to temperature, mechanical, and freeze / thaw stress by forced degradation studies, was investigated. For these experiments the AP was tested at two concentrations in two different buffers. These buffers (histidine vs. citrate) had previously been tested. Citrate seemed to reduce the amount of particles created throughout storage and after stress. However, in the citrate-buffered samples the particle numbers of visible particles and subvisible particles were still high. Later findings suggested that a major contributor here, is the original material. The material used (Material PF- 06853082) had previously been buffered in histidine and showed less Isodimer A peak in RP-HPLC from the beginning compared to material provided later (Material “VLT_Demo_BDS”). It can therefore be concluded that most of this high particle counts are attributable to some sort of “pre-damage”of the original material. Although the pre-exposure to histidine seems a plausible cause, as histidine samples had higher particle counts, it cannot be excluded that other forms of stress during the production or DSP of this material contributed to the high particle counts. The AP used in had been delivered in histidine buffer (Material PF-06853082). To test the possible influence of storage in histidine, it was decided to add a comparison of this drug substance with another batch that did not have any pre- exposure to histidine. That batch was to be tested as reference material.

[0007] Table 15. Osmolality values of formulations at t0 and after mechanical stress. Osmolality determination for mechanical stress study (mOsmol / kg) Formulation t=0 Orbital Stirring Syringe Peristaltic2 F / T 5 F / T 2 F / T 5 F / T Shaking pumping -20°C / RT -20°C / RT -80°C / RT -80°C / RT WP4_01 452 414 422 439 n.a. 415 418 419 420WP4_02 360 342 342 343 n.a. 334 325 340 334WP4_03 593 470 470 240 n.a. 487 461 465 450WP4_04 305 294 300 301 n.a. 280 292 288 288WP4_05 476 450 450 464 n.a. 451 466 459 448WP4_06 379 355 361 363 n.a. 350 363 368 355WP4_07 382 358 358 368 n.a. 370 370 362 373WP4_08 434 419 407 430 n.a. 414 427 412 428WP4_09 322 348 314 324 n.a. 313 312 318 318WP4_10 552 471 481 230 n.a. 429 458 457 457WP4_11 316 322 307 311 n.a. 304 302 303 299WP4_12 462 484 445 460 n.a. 438 433 439 446WP4_13 358 376 356 361 n.a. 340 344 344 346WP4_14 343 349 336 337 n.a. 328 323 336 335WP4_15 341 324 317 332 n.a. 316 326 319 318WP4_16 330 n.a. 327 346 334 321 324 327 333WP4_Ref 340 330 330 345 316 332 322 328 334WP4_PC 354 n.a. 335 342 334 331 328 325 333

[0008] The aim was to find suitable buffers, excipient combinations, and pH that reduce particle counts and contribute to the storage and stress stability of recAP. Based on previous re-sults, seven formulations with minor tweaks, one reference formulation (recAP in original citrate based buffer) and eight formulations with additional excipients and further adaptations based on current formulation were prepared. Additionally, two samples using recAP from another production batch were included in the study. This material was not pre-exposed to histidine, it was pre- exposed to citrate (Material “VLT_Demo_BDS”). It was shown that the samples prepared from material without pre-exposure to histidine performed better than most samples re-buffered from histidine, especially in visual appearance. There was no clear indication to which buffer substance performs best, therefore Citrate pH 7 would be recommended. Neither was there a significant difference between formulations with Trehalose and formulations with Sorbitol. Presence of Kleptose HPB or Poloxamer 188 reduced the amount subvisible particles drastically, with the formulations 03 and 06 (Table 2) performing well in all analytics except visual appearance. This could, however, also be attributable to the pre-exposure of the originator material to histidine. The results from Example 3 confirmed, that material PF-06853082 contained considerably less Isodimer A1 species in RP-HPLC, compared to material “VLT_Demo_BDS”. Also it was demonstrated that the presence of methionine reduces chemical degradation (presumably oxidation) of recAP during storage, pointing at oxidation being a degradation pathway for recAP. Example 4 In this project, the goal was to find a suitable liquid formulation for subcutaneous injection, that allows a high recAP concentration (> 100 mg / mL) with acceptable stability. Additionally, the formulation should be acceptable for pediatric purposes. Here, a formulation based on the outcomes of Examples 1-3 was used: Citrate 20.00 mM pH 7.0 Trehalose 250.00 mM Poloxamer 188 0.50 g / L Zinc Chloride 0.05 mM Magnesium Chloride 2.00 mM Materials and methods As in Example 1, except for the following. Tangential Flow-Filtration (TFF) for buffer exchange and up-concentration The buffer exchange and concentration processes were conducted using tangential flow filtration on a Sartorius Ambr Crossflow device, using the buffers described below. Sartorius Sartocon Slice 50 filter cassettes (30 kDa) were used. The buffer exchange and concentration process was performed sequentially as initial concentration-diafiltration-final concentration process: At first, the drug substance was concentrated to reduce the process volume; followed by buffer exchange with 5 diafiltration volumes for continuous buffer exchange, resulting in a theoretical buffer exchange of > 99%. Finally, the drug substance was concentrated to its target concentration. The process was performed at room temperature. (Sterile-) Filtration of formulations, spiking of detergents and adjustment of concentration Following TFF, the buffer exchanged and concentrated drug substance was sterile- filtered as a bulk (Sartorius Sartolab P20 pressure filter, 0.22 µm, PES). Here, the first 15 droplets of filtered drug substance were discarded to avoid dilution effects and leaching of filter components. The filtered volume was split into 16 equal parts. Formulations were produced by spiking variable volumes of methionine and poloxamer 188 stock-solutions respectively as well as placebo formulations to adjust the concentration. For the reference formulation, thawed and filtered (Sartorius Minisart syringe top filter, 0.22 µm, PES) customer material was used without any further processing. Nitrogen Overlay For the nitrogen overlay investigation, two vials each of formulation 09, 10, 11 and 12 were removed from the 1m_40C storage samples prior to storage. The two vials were pooled in one. 150 µL were used for pH measurement. Visual appearance was performed within the vial and the vial was subsequently again split into two vials. One vial directly went to storage for one month at 40 °C, the other vial was overlaid with nitrogen and also stored for one month at 40 °C. The nitrogen overlay step was performed in a freeze-dryer: Vacuum was applied to the vials. Then, nitrogen was applied to the chamber, aerating the vials with nitrogen. This was repeated five times. Finally, the vials were closed under nitrogen atmosphere and put to storage. After storage, visual appearance, SE-HPLC, RP-HPLC, UV280, pH and measurement of osmolality were performed. Turbidity according Ph. Eur. method 2.2.1 Turbidity of formulations was analyzed using the NEPHELOstar Plus (BMG Labtech). 200 μL of turbidity standards and samples respectively were pipetted into wells of a 96 well transparent microplate. The device’s standard method was used for analysis. The measurement was per-formed in duplicates. Data analysis was performed by MARS data analysis software. Kinetic Activity This method is to confirm the presence and determine the recAP enzymatic activity using para-Nitrophenyl Phosphate (pNPP) substrate for different formulations and stress conditions. Reference material and samples were pre-diluted in three steps to a concentration of 700 ng / mL. Between dilution steps dilutions were mixed by pipetting up and down. A pipet robot performed the dilution steps for the following concentration: 466 ng / mL, 311 ng / mL and 207 ng / mL. The dilution plate was equilibrated at 25 °C in a water bath. Kinetic measurements were performed row wise. After addition of pNPP substrate to row A, the measurement was started. Then, pNPP substrate was added to row B and measurement was started again, continuing in the same way, until all rows were measured. Results Study Design The objective was to find highest concentration of recAP with acceptable viscosity, turbidity and subvisible particle counts. The formulation used was: Citrate 20.00 mM pH 7.0, Trehalose 250.00 mM Poloxamer 188 0.50 g / L Zinc Chloride 0.05 mM Magnesium Chloride 2.00 mM The formulation was prepared without Poloxamer 188. First, the original material at 8 mg / mL was concentrated to 40 mg / mL using tangential flow filtration (TFF). Then, the original buffer was exchanged against the new formulation, using Diafiltration into 5 Diavolumes of Citrate 20.00 mM, Trehalose 100.00 mM, Zinc Chloride 0.05 mM, Magnesium Chloride 2.00 mM , pH 7.0. Within the same process, the material was up-concentrated to 197 mg / mL. Subsequently, the residual 150 mM of Trehalose (this was done to keep viscosity during the process low) and Poloxamer 188 were spiked to the formulation and sterile filtration was performed. This resulted in fully formulated recAP at 182 mg / ml. From this, dilutions from 80 to 180 mg / mL (in the full formulation buffer) were prepared and assessed for subvisible particles (SvP), visual appearance, osmolality, turbidity, viscosity (Cone-Plate Rheometer), and determination of concentration by UV280. Analytics were performed under non-GMP conditions. Diafiltration / Up-Concentration In the formulation development process using the AMBR crossflow TFF device with two channels (to assess cassette variation) and Sartoslice 50 cassettes (30 kDa MWCO, PES, 50 cm^2 membrane area), the following program was executed: TFF Concentration Steps: Initial concentration from 8 mg / mL to 40 mg / mL. Diafiltration with 5 Diavolumes of Citrate (20.00 mM), Trehalose (100.00 mM), Zinc Chlo-ride (0.05 mM), Magnesium Chloride (2.00 mM), at pH 7.0. Final concentration to 197 mg / mL with no visual observations of concern. Post-TFF Formulation Adjustments: Poloxamer 188 and residual Trehalose were spiked post-TFF to achieve the target formulation, to avoid up-concentration of detergent and ensuring low viscosity during the process. This resulted in a fully formulated Drug Substance (DS) at 183 mg / mL. After sterile-filtration, a final concentration of 182 mg / mL was achieved. Quality Checks: Only minimal differences in flux were observed between the two cassettes in flow and achieved final concentration, indicating consistency of the process. Additional Concentration Evaluation: To assess the upper concentration limit, 182 mg / mL was included in a dilution series: 182 mg / mL, 160 mg / mL, 141 mg / mL, 123 mg / mL, 101 mg / mL, and 78 mg / Ml. Originally, only concentrations up to 160 mg / mL were planned. This TFF process demonstrates the successful up-concentration of the formulation, including con-centration steps, diafiltration, and post-TFF adjustments. The final concentration of 182 mg / mL was achieved after sterile-filtration, exceeding the specified target. Concentration and Osmolality Osmolality data showed that recAP itself makes a significant contribution to the formulation osmolality, with the additional osmolality almost being equal to the AP concentration in mg / mL. See Figure 8. But even at the highest concentration of 182 mg / mL, the osmolality was at a level acceptable for subcutaneous injection (< 500 mOsmol / kg). Visual Inspection Vials show no visible particles. Formulations were clear, transparent solutions with a slight yellow coloration (Y6 / Y7) at elevated concentration. Viscosity Viscosity was measured by Cone-Plate Rheometry at 1000 s-1 at 20°C with a 0.5° cone angle. All concentrations tested (SEE Figure 9A) are well within acceptable viscosity limits for subcutaneous injections: desired: < 15 mPa*s feasible: < 20 mPa*s As expected, rheometry showed an exponential increase of viscosity with concentration, which was best described with the following fit formula: η=η0+η+⋅ exp (k1*CrecAP), wherein η0 = 0.974 mPa⋅s η+ = 0.372 mPa⋅s k1 = 0.014 ml / mg. Extrapolation of this fit curve suggest the following concentrations for the given viscosity limits: 15 mPa*s at 260 mg / ml 20 mPa*s at 281 mg / ml A shear rate scan (from 10-1to 10-4s-1up- and downscan) showed no hysteresis at 182 mg / ml, indicating that the protein is stable at relevant shear rates for pumping, filtration, and during the viscosity measurements (not shown). Turbidity Formulation turbidity was measured by a Nephelostar plate reader device. All concentrations tested (see Figure 9B) are well within typical turbidity limits: desired: < 20 NTU The measurement showed, that the turbidity was higher for the reference formulation at 8 mg / ml compared to the reformulated recAP at higher concentrations. This is most likely the influence of Poloxamer 188, as the reference formulation did not contain any detergent. Subvisible Particles Subvisible particles (SvPs) were measured using a Flowcam device (Figure 10). The formulations showed generally low levels of SvPs. Higher particle counts were seen in the original formulation at lowest concentration (8 mg / mL). This is in line with the nephelometry results and most likely the effect of Poloxamer 188. Subvisible particles are compendially assessed by Light Obscuration with the following limits: < 6000 particles > 10 µm < 600 particles > 25 µm per container. Here measurements were performed by Flowcam, which generally gives higher particle counts, especially for particles with low difference in refractive index compared to the solvent. Despite this, all formulations measured were within compendial limits. Conclusion The formulated solution exhibits a satisfactory profile with no visual particles and a slight yellow stain observed at higher concentrations. Key parameters such as viscosity, turbidity, and subvisible particles are well within acceptable ranges, even at the highest concentration of 182 mg / mL. Overall, the formulation's robust performance allows for flexibility in concentration levels for different stages of the development process while providing avenues for optimization if specific at-tributes, such as osmolality, need adjustment. Example 5 The objective was to investigate the sensitivity to temperature during storage, mechanical and freeze / thaw stress of recAP in trehalose formulation and at increasing concentrations for 3 months. Selected formulations were stored and analysed for up to 12 months.Three different concentrations of recAP were planned (8, 120, 150 and 180 mg / mL). They were tested in four different formulations each. All contained 20 mmol / L citric acid, 250 mmol / L Trehalose, 0.05 mmol / L Zinc chloride, 2 mmol / L Magnesium chloride, at pH 7.0. Previous work on recAP demonstrated that Citrate provides enough buffer capacity in combination with AP at pH 7.0. Once this has been used without any extra excipients, once with Methionine, once with Poloxamer and once with both. The reference formulation contained sorbitol and no detergent and was only used at the original concentration of 8 mg / mL. The composition of the reference solution is consistent with the original formulation. Table 16 shows a list of the formulations. Analytics consisted of measurements of subvisible particles (SvP), visual appearance, osmolality, turbidity, viscosity (Cone-Plate Rheometer), determination of concentration by UV280 and pH, analysis by SE-HPLC and RP-HPLC, and determination of kinetic activity. Analytics were per-formed under non-GMP conditions. Results Diafiltration / Up-Concentration In the up-concentration process utilizing a 4-channel AMBR crossflow TFF device with Sartoslice 50 cassettes (30 kDa MWCO, PES, 50 cm^2 membrane area), the program involved the following steps: TFF Concentration Steps: (1) Initial concentration from 8 mg / mL to 140 mg / mL. (2) Diafiltration with 5 Diavolumes of Citrate (20.00 mM), Trehalose (250.00 mM), Zinc Chlo-ride (0.05 mM), Magnesium Chloride (2.00 mM), at pH 7.0. (3) Final concentration achieved was 222 mg / mL with no observed issues. Post-TFF Formulation Adjustments: Table 16. Composition of formulations. Formulation Citric acidTrehalose L- PoloxamerSorbitol ZinkMagnesiumpH APmono- dihydrate Methionine 188 chloride chloride concentration hydrate hexahydrate mmol / L mmol / L mmol / L g / L mmol / L mmol / L mmol / L mg / mLWP6b_01 20 250 0.05 2 7 8WP6b_02 20 250 10 0.05 2 7 8WP6b_03 20 250 0.5 0.05 2 7 8WP6b_04 20 250 10 0.5 0.05 2 7 8WP6b_05 20 250 0.05 2 7 120WP6b_06 20 250 10 0.05 2 7 120WP6b_07 20 250 0.5 0.05 2 7 120WP6b_08 20 250 10 0.5 0.05 2 7 120WP6b_09 20 250 0.05 2 7 150WP6b_10 20 250 10 0.05 2 7 150WP6b_11 20 250 0.5 0.05 2 7 150WP6b_12 20 250 10 0.5 0.05 2 7 150WP6b_13 20 250 0.05 2 7 180WP6b_14 20 250 10 0.05 2 7 180WP6b_15 20 250 0.5 0.05 2 7 180WP6b_16 20 250 10 0.5 0.05 2 7 180WP6b_Ref 20 250 0.05 2 7 8

[0009] Following TFF, Poloxamer 188 and Methionine were spiked into the formulations to reach the target specifications. This process demonstrated a successful up-concentration strategy utilizing the specified TFF device and cassettes. The final formulation, after post-TFF adjustments, meets the target specifications. Process Parameters In more detail, the process data allowed for the following conclusions: Loaded volumes for each channel: Channel 1: 2258.4 ml Channel 2: 2253.3 ml Channel 3: 2244.6 ml Channel 4: 2250.1 ml Each channel was harvested at a target volume of 80 ml, resulting in an average up-concentration factor of 28.15. Starting with a concentration of 8 mg / ml, the expected concentration after up-concentration would be 225.16 mg / ml. The actual concentration of the pooled material from the four channels was measured at 222 mg / ml, indicating a recovery of approximately 98.5%. These calculations are based on the weight of the retentate vessel and permeate flow. The high recovery indicated, that only minor losses across the 30 kDa membrane occurred. Given the molecular weight of recAP at 105 kDa, significant losses on a 30 kDa membrane were not to be expected. No analysis on the permeate was conducted during or after this process. Concentration by UV 280 nm Concentration measurements after filling revealed a slight overconcentration of ~15 %. The concentrations refer to the actual achieved concentrations and not to the planned concentration. Concentrations were not affected by mechanical, freeze / thaw stress, or storage, including long term storage. Visual Inspection Visual Inspection was performed as described. The initial visual assessment at t0 revealed the absence of visible particles. However, after the addition of spiking agents and filling, a slight opalescence was noted in the samples, which vanished after a few hours. Formulation 13, which excluded Methionine and Poloxamer, displayed a fiber-like particle in one of the inspected vials, likely an extrinsic contamination. Across all samples surpassing 8 mg / ml, a slight yellow stain (Y6 / Y7) was consistently observed. Mechanical and Freeze / Thaw Stress Subsequent to five freeze-thaw cycles (-80°C / RT), schlieren (slight optical inhomogeneities) were observed in samples exceeding 8 mg / ml. Similar schlieren patterns were also identified in samples above 140 mg / ml following 24 hours of orbital shaking at 400 rpm. Short-Term Storage After one month of storage, the samples exhibited slight turbidity at elevated storage temperatures. Furthermore, particles were detected in some formulations lacking Poloxamer 188 after one month at 5°C and 30°C. Most formulations exhibited particles after storage at 40°C. At the intended storage temperatures of 5°C and -20°C, no relevant change occurred during the 3 months storage for the formulation containing Poloxamer 188. For the formulations without detergent, individual vials showed low numbers of visible particles during storage, indicating the necessity of Poloxamer 188 to prevent the particle formation. Visual inspection of samples under N2-overlay revealed no remarkable findings. However, after three months at 30°C, particles were observed in the vials of Formulations 03, 05, and 07. Long-Term Storage A slight color change was noted after six months in formulations containing Poloxamer. Additionally, one particle was identified in one vial of Formulations 08 and 11. Extended storage periods of nine months at 5°C and -20°C did not result in visible particles. Also after 12 months at -20°C, no particles were observed, however, one fiber was present in one vial after 12 months at 5°C. These observations provide comprehensive insights into the visual characteristics and stability of the formulations across various conditions and timeframes. Viscosity The viscosity profile over concentrations remained consistent independent of the addition of Methionine, Poloxamer 188, or both. Notably, the viscosity levels were well below the acceptable limit (typically around 20 mPa*s). Formulations at 8 mg show a viscosity of 1.5 – 1.7 mPa*s. Formulations at 140 mg / mL showed viscosities of 3.5 – 3.8 mPa*s. Formulations at 175 mg showed a viscosity of 5.2 – 6.4 mPa*s. Formulations at 210 mg / mL showed viscosities of 7.2 – 8.8 mPa*. y There was no change in viscosity after any of the performed mechanical or freeze- thaw stresses, nor after any of the analyzed short-term storage periods. There was no change in viscosity after any of the analyzed long-term storage periods. A minor decline of viscosity for the formulation at 210 mg / mL is small in comparison to the general variance of the data. pH The pH of the formulations was determined as described. Target pH was 7, which was achieved for all formulations, independent of concentration. There was no change of pH upon any mechanical or freeze-thawing stress. Storage at –20°C and 5°C did not cause any changes in pH. Storage at 40°C led to a pH drop to pH 4.3 to 5.7. Stored material at 30°C was pH stable for 3 months, except the formulations at 150 mg / ml, were pH seemed to show a high variance. Here, material was only sufficient for the minimum required volume to perform the measurement. This is therefore likely a measurement artifact. 1 month storage at 30°C did not show any pH changes. Nitrogen overlay samples showed a stable pH at 40°C storage, hinting at some involvement of oxygen in the cause of the pH drop. In the long-term storage performed at 5°C and -20°C, no pH changes were detected even after full storage time of 12 months. Osmolality Osmolality was determined as described. At t0, the recAP contribution to the formulation osmolality was observed. There was no change of osmolality upon any mechanical or freeze-thawing stress. Similar to the observed pH behavior, the osmolality was stable upon storage at 5°C and -20°C for up to 3 months for all formulations, but at 30°C and 40°C, the higher concentrated formulations showed an increase in osmolality (after 3m at 30°C and 1m at 40°C). This change was not detected in the N2 overlay samples. An increase in osmolality implies that more solute is present in the formulation, indicating a degradation mechanism, where macromolecules are separated into multiple degradants. The elevated temperature at which this is observed and the concentration dependence suggest an auto-effect of recAP itself, splitting ester bonds of other recAP proteins in the solution. However, this mechanism would not explain why oxygen would be required. In the long-term storage performed at 5°C and -20°C, no osmolality changes were detected even after full storage time of 12 months. SE-HPLC SE-HPLC was performed. At t0, 99.5 % relative main peak area were found, with 0.2 % higher molecular weight species (HMWS) and 0.3 % lower molecular weight species (LMWS). This in good agreement with data of the previous study and independent of concentration. There was no change in chromatogram detected upon freeze-thawing stress with all formulations. Total chromatogram area suggests a close-to 100 % recovery for all measurements. Orbital shaking lead to a minimal increase in HMWS of < 0.1 %. All formulations exhibit stability when stored at 5°C and -20°C for up to 3 months. After one month at 30°C, the highest concentrated samples show a modest ~1.5% decrease in the relative main peak area. At three months and 40°C (the most severe condition within the short time study), there is an approximate ~7% decrease in the relative main peak areas for the highest concentrated samples. The emergence of HMWS at elevated temperatures was observed to rise proportionally with concentration and in the presence of Methionine. The inclusion of Poloxamer shows a slight reduction in the emergence of HMWS, indicating a mitigating effect. The emergence of HMWS is also positively correlated with the recAP concentration, as expected for any high concentration biologic. Notably, any decrease in the monomeric form is consistently accompanied by a corresponding increase in HMWS, suggesting an interconnected relationship between these two species in the formulations. These observations contribute to a nuanced understanding of the impact of concentration, additives, and formulation components on the high molecular weight species. Extrapolating the data (linear function) suggests that after 24 months, samples stored at 5°C with a concentration of 210 mg / ml would result in a 2.2% presence of high molecular weight species (HMWS), for the formulation without methionine and without Poloxamer 188 (worst case). Also, a slight decrease in LMWS (approx. 0.05 %) was detected over 3m at all temperature, suggesting that these degradation products might become part of larger aggregates over time. In perspective of other highly concentrated protein therapeutics, the aggregation tendency of recAP seems low, even at higher concentration and storage at higher temperatures. The formulation used in the long-term study (- Methionine, + Poloxamer 188) showed little change in SE-HPLC profile over up to 12 months of storage at 5°C and -20°C. The decrease in relative main peak area was ~0.7 % after 12m for the formulation without Methionine, but with Poloxamer 188 at 5°C at the highest concentration of 210 mg / mL, demonstrating good stability with regards to aggregation. This is also lower than the worst-case extrapolated data from the short term study. All monomer decrease is accompanied by a similar increase in HMWS. Also, a minor decrease of LMWS was detected over time (< 0.1 %). RP-HPLC RP-HPLC was measured as described. At t0, Isodimer A (Main Peak) relative peak area was at 88 %, which is similar to the data from the previous study. Similarly, the other peaks show relative areas in agreement with previous data on recAP, with related species at ~ 6.4 %, Isodimer B1 at 3 %, and Isodimer B2 at 1.9 % (data not shown). There was no change in chromatogram detected upon freeze-thawing stress or orbital shaking. In the short-term study, there was only little change in chromatograms upon storage at -20°C and 5°C for the storage of up to 3 months. Behavior of recAP chromatograms at these temperatures over time was slightly concentration dependent, with the formulation at 210 mg / mL showing a slightly higher Isodimer A reduction after 3 months (7 % at 5°C, worst case), with a corresponding increase in related species and Isodimer B2. An increase in related species was detected after 1m 40°C for samples at 8 mg / mL. Other than that, the relative Isodimer A content was comparably stable even at 40°C, which is surprising, giving the detected changes in pH and osmolality. The recovery was good, indicating no loss of absolute peak area upon storage. Storage at 40°C resulted in a decrease of Isodimer B1 area after 1m at 40°C, with a corresponding increase of Isodimer B2 area after at 40°C, especially at low concentrations. The 30°C samples showed a higher heterogeneity amongst the formulations and across time points. Also, a stronger decrease in Isodimer A at 30°C than at 40°C was detected. This might indicate that the changes that recAP seemingly underwent at 40°C might stabilize the Isodimer A form. Similarly, there was a stronger increase in related species at 30°C than at 40°C. Systematic differences between formulations with and without Poloxamer and / or Methionine were not detectable. At long term storage, RP-chromatograms demonstrated a high Isodimer A retention at -20°C and 5°C. No relevant changes in chromatogram patterns were seen for up to 12m at 5°C and -20°C. As the relative peak areas were consistent over all time points, the 3m data can confidently be treated as outlier, showing a higher deviation than later pull points and also a high variance between replicates. Kinetic Activity Kinetic activity measurements were performed as described. At t0, recAP showed specific activities between 479 and 693 U / mg. Within the variance of the assay, there were no relevant differences in activity between formulations. During the entire study, all quality criteria of the assay were passed. There were only minor changes in specific activity after any of the performed mechanical or freeze-thaw stresses. The strongest decrease was found after orbital shaking with the lowest recAP concentration affecting the formulation without methionine with poloxamer. Specific activity was independent of concentration, indicating no competition effects at the used concentrations and amounts of substrate. All formulations exhibited consistent specific activities when stored at 5°C, 40°C, and -20°C for up to 3 months, while there were few individual outliers. For example, the sample at 1 month 40°C for the formulation with methionine and Poloxamer 188 showed a low activity, but the same formulation stored for 2 months showed normal activity again. As biological assays generally show higher variance than biophysical methods, such behavior is to be expected and analyzing the entire data collectively, it can be concluded that the activity is consistent for all formulations detected across temperatures and storage points. Longer-term storage for up to 12 months of the formulation with Poloxamer 188 and without Methionine demonstrated consistent specific activity, independent of concentration. Turbidity Turbidity was measured, as described. Turbidity data at t0 already indicated differences between formulations. Generally, formulations with Poloxamer 188 show significantly lower turbidity than formulations without it. While the formulations with Poloxamer 188 showed relatively consistent turbidity values of 1 – 3 NTU, samples without the detergent showed values form 4.7 – 12.7 NTU and also higher variances within each sample set. While turbidity did not change upon freeze-thawing, orbital shaking for 24 h at 400 rpm led to a strong increase in turbidity for the formulations without detergent, while formulations containing Poloxamer 188 showed no change in turbidity. Upon storage for up to 3 months, strong changes in turbidity were observed. At the elevated temperature of 40°C, there was a substantial increase in turbidity, signifying a considerably heightened sensitivity to stress under these conditions, accompanying the change in pH and osmolality at this temperature. Upon storage for up to 3 months, strong changes in turbidity were observed. At the elevated temperature of 40°C, there was a substantial increase in turbidity, signifying a considerably heightened sensitivity to stress under these conditions, accompanying the change in pH and osmolality at this temperature. Especially, the samples containing methionine show a higher increase in turbidity, than the samples without it. The increase in turbidity is most pronounced at the concentration of 140 mg / ml, but seems to decline for higher concentrations. Conversely, when subjected to a milder stress condition of 30°C, the increase in turbidity is comparatively minor, suggesting a temperature-dependent effect on the visual clarity of the samples. Notably, formulations containing Poloxamer 188 exhibit lower turbidity levels, indicating a mitigating effect of this component on the stress-induced increase in turbidity. At 5°C and -20°C the turbidity stayed low throughout the 3 months for the formulations with Poloxamer 188. Upon storage for up to 12 months, the formulation without methionine and with Poloxamer 188 showed no changes in turbidity. Again, the 3 month data points seem to be an outlier, as later pull points consistently show significantly lower NTU values. At 5°C, there seems to be a slight concentration effect with turbidities at 210 mg / ml being generally slightly higher, than those at all other concentrations. The reference formulation showed a slight increase in turbidity after about 6 months at 5°C. Subvisible Particles (FlowCam) FlowCam measurements were performed as described. At t0, subvisible particle counts were generally low, especially at the higher sizes of >10 µm and > 25 µm. While subvisible particle counts did not change significantly upon orbital shaking (24 h, 400 rpm), repeated freeze-thawing from -80°C to room temperature resulted in an increase in subvisible particles for the formulations without Poloxamer 188. This is mostly the case for small particles < 5 µm. Formulations with Poloxamer 188 showed consistently low subvisible particle counts after all stress conditions in all particle sizes. For samples stored at -20°C or 5°C, particle counts stayed consistently low throughout the entire storage period. This is the case for all formulations and concentrations. Upon storage for up to 3 months, the pattern observed at t0 is confirmed also for samples stored at 40°C, as the increase in small particles (< 5 µm) is higher for samples with methionine, than for samples without it. Generally, during storage at 40°C, there was a notable increase in smaller subvisible particles (SvPs) after one month, indicating a temperature-dependent impact on particle size distribution. Upon storage for 2 months at 40°C, particle counts for particles < 5 µm further increase for the higher concentrated samples. After 2 months at 40°C, there is also an increase in larger particles (> 10 µm and > 25 µm) for the highly concentrated formulations. Sub-visible particle counts remained generally low during 12 months of storage, both at 5°C and 20°C. While there is an increase in subvisible particles after 12 months at 5°C, the numbers are low in comparison to other highly concentrated protein products and are well within acceptable limits. Conclusion During the complete storage study, short- and long-term up to 12 months, the formulation consisting of 20 mM Citrate, 250 mM Trehalose, 0.5 g / L Poloxamer 188, 0.05 mM ZnCl2, 2 mM MgCl2, at pH 7 provided stability across all applied analytical methods and at all concentrations. Example 6. Short-term stability study at higher recAP concentrations The goal of these experiments was to investigate the short-term stability study at higher recAP concentrations. Materials and methods As described in the previous examples. Upconcentration using AMBR crossflow TFF device using Sartoslice 50 casettes (30 kDA MWCO, PES, 50 cm2membrane area). Program TFF included a first concentration step from 8 mg / mL to 140 mg / mL, Diafiltration into 5 Diavolumes of Citrate 20.00 mM, Trehalose 250.00 mM, Zinc Chloride 0.05 mM, Magnesium Chloride 2.00 mM , pH 7.0, concentration to 222 mg / mL without any visual observations. After TFF: Spiking Poloxamer 188 and Methionine to reach the target formulation. Resulting formulations are provided in Table 17. Results Visual Inspection No visible particles were observed at t0. Samples showed slight opalescence after spiking and filling. All samples having more than 8 mg / ml recAP showed slight yellow staining (Y6) Schlieren were observed in samples having more than 8 mg / ml recAP after 5x freeze-thawing at -80°C / RT, and after orbital shaking (24 h / 400 rpm). Formulation 13 (without Methionine and Poloxamer) showed a fiber-like particle in one of the inspected vials. Samples were slightly turbid after storage for 1 month.

[0010] Table 17. Formulations. Formulation Citric acidTrehalose L- Poloxamer Sorbitol Zink MagnesiumpH APmonohydrate dihydrate Methionine 188 chloride chloride concentration hexahydrate PEO100_0_WP6b_ mmol / L mmol / L mmol / L g / L mmol / L mmol / L mmol / L mg / mL01 20 250 0.05 2 7 802 20 250 10 0.05 2 7 803 20 250 0.5 0.05 2 7 804 20 250 10 0.5 0.05 2 7 805 20 250 0.05 2 7 14006 20 250 10 0.05 2 7 14007 20 250 0.5 0.05 2 7 14008 20 250 10 0.5 0.05 2 7 14009 20 250 0.05 2 7 17510 20 250 10 0.05 2 7 17511 20 250 0.5 0.05 2 7 17512 20 250 10 0.5 0.05 2 7 17513 20 250 0.05 2 7 21014 20 250 10 0.05 2 7 21015 20 250 0.5 0.05 2 7 21016 20 250 10 0.5 0.05 2 7 210Ref 20 250 0.05 2 7 8

[0011] Some formulations showed particles without Poloxamer 188 after 1 month at 5°C and 30°C. Particles were observed in most formulations after storage at 40°C. Particles were observed in the vials of Formulation 03, 05, and 07 after 3m at 30°C. No special findings in N2-overlay samples. pH pH drop at 40°C for the high concentrations of recAP, and for low concentrations in sam-ples containing methionine. A pH drop was also observed for formulation 16. No pH change for samples at 30°C for 1 month A pH increase was observed in formulation 10 after 1m 40°C with nitrogen overlay.A pH increase was observed in Formulation 11 after 1m 40°C without nitrogen overlay. Osmolality No increase after 1 month at 30 °C. An increase in osmolality was observed in higher concentrated samples at 40°C. A slight increase in osmolality was observed after 3m 30°C for samples at 175 mg / ml and 210 mg / ml. No osmolality change was observed after 1m 40°Cwith nitrogen overlay. SE-HPLC All formulations were stable at 5°C and 20°C. A ~1.5 % decrease was observed in the relative main peak after 1m 30°C for highest concentrated samples. A ~ 7 % decrease was observed in relative main peak areas after 2m 40°C for highest concentrated samples. Extrapolation would result in 2.2 % HMWS after 24m for samples at 210 mg / ml at 5°C. A HMWS increase of approx. 7 % after 3 month at 40°C was observed for highest concentrated samples. The HMWS emergence at higher temperatures increased with concentration and presence of methionine. Poloxamer slightly reduced the emergence of HMWS. No relevant difference was observed from original 40C data to N2 overlay and N2 overlay control. RP-HPLC - Storage Isodimer A areas were at ~ 88 % area for all formulations at t0. A good stability was observed at -20°C and 5°C. An increase was observed in related species after 1m 40°C for samples at 8 mg / mL. A stronger decrease in Isodimer A was observed at 30°C than at 40°C, coinciding with a stronger increase in Related Species at 30°C than at 40°C. Also higher decrease in Isodimer A after 3 m 5°C at 210 mg / ml. A decrease of Isodimer B1 area was observed after 1m at 40°C. An increase of Isodimer B2 area was observed after 40°C for low recAP concentrations. After mechanical stress, Isodimer A areas were at ~ 88 % area for all formulations. Related Species at ~ 6.4 %, Isodimer B1 at 3 % and Isodimer B2 at 1.9 %. Kinetic Activity No significant difference was observed between Formulations or concentrations. All quality criteria were passed. No relevant decrease after storage. A low activity was observed for 8 mg / ml recAP sample with methionine and poloxamer 188 after 1m 40°C. No significant difference was observed between Formulations or concentrations after mechanical stress. All quality criteria were passed. Turbidity High increase in Turbidity was observed at 40°C. Increase was highest at 140 mg / ml. Only minor increase at 30°C. A lower turbidity was observed in samples with Poloxamer 188. After storage, a higher turbidity increase was observed in samples with Methionine. Higher concentration of recAP seemed to reduce turbidity. A high turbidity increase was observed at 40°C. After mechanical stress, the presence of Poloxamer 188 decreased turbidity, especially after orbital shaking. Turbidity between 1.1 –27 NTU at t0. Subvisible Particles (SvP) After storage, higher amounts of subvisible particles were observed in samples without Poloxamer. An increase in smaller SvPs was observed after 1m 40°C. Very high counts of small SvPs were observed after 2m 40°C, The number of subvisible particles >10 µm and >25 µm still acceptable except Ref after 1m at 5°C and Sample 11 after 1m 40°C (potential outliers). The increase in smaller SvPs was more pronounced in samples with methionine after storage at 40°C. Lower amounts of small SvPs were observed in samples with Poloxamer 188 after freeze-thaw stress. In addition, an increase in Particle counts was observed after repeated freeze-thawing -80°C / RT in samples without Poloxamer 188. All samples were within acceptable range, except WP6b_14 (210 mg / ml (+Methionine –Poloxamer188) after Freeze-Thaw stress. Example 7 Continued Long-term storage for up to 12 months was performed with best performing formulations from Example 6. Formulations PEO100_0_WP6c_, 03, 07, 11, 15 and Ref (see Table 17) were included. Results Visual inspection A slight change of color was observed after 6m in formulations with Poloxamer. Additionally, one particle was identified in one vial of Formulations 08 and 11. Extended storage periods of nine months at 5°C and -20°C did not result in visible particles. Also after 12 months at -20°C, no particles were observed, however, one fiber was present in one vial after 12 months at 5°C. These observations provide comprehensive insights into the visual characteristics and stability of the formulations across various conditions and timeframes. Viscosity The viscosity profile over concentrations remained consistent independent of the addition of methionine, Poloxamer 188, or both. Notably, the viscosity levels were well below the acceptable limit (typically around 20 mPa*s). This aligns with the findings from Example 6, indicating good agreement in viscosity values. Formulations at 8 mg show a viscosity of 1.5 – 1.7 mPa*s. Formulations at 140 mg / mL show viscosities of 3.5 – 3.8 mPa*s. Formulations at 175 mg show a viscosity of 5.2 – 6.4 mPa*s. Formulations at 210 mg / mL show viscosities of 7.2 – 8.8 mPa*s. There was no change in viscosity after any of the analyzed long-term storage periods. The seeming minor decline of viscosity for the formulation at 210 mg / mL was small in comparison to the general variance of the data. pH In the long-term storage performed at 5°C and -20°C, no pH changes were detected even after full storage time of 12 months. Osmolality In the long-term storage performed at 5°C and -20°C, no osmolality changes were detected even after full storage time of 12 months. SE-HPLC The formulation used in the long-term study (- Methionine, + Poloxamer 188) showed little change in SE-HPLC profile over up to 12 months of storage at 5°C and -20°C. The decrease in relative main peak area is ~0.7 % after 12m for the formulation without Methionine, but with Poloxamer 188 at 5°C at the highest concentration of 210 mg / mL, demonstrating good stability with regards to aggregation. This is also lower than the worst-case extrapolated data from the short term study. All monomer decrease is accompanied by a similar increase in HMWS. Also, a minor decrease of LMWS was detected over time (< 0.1 %). RP-HPLC No relevant change in was observed in Related Species after 12m at 5°C and -20°C. A slight decrease of Isodimer B1 area was observed at 5°C and -20°C. No relevant change in Isodimer B2 after 12m at 5°C and -20°C. Kinetic activity Longer-term storage for up to 12 months of the formulation with Poloxamer 188 and without Methionine demonstrated consistent specific activity, independent of concentration. Turbidity Upon storage for up to 12 months, the formulation without methionine and with Poloxamer 188 showed no changes in turbidity. At 5°C, there seems to be a slight concentration effect with turbidities at 210 mg / ml being generally slightly higher, than those at all other concentrations. The reference formulation showed a slight increase in turbidity after about 6 months at 5°C. Subvisible particles (SvP) No increase in SvPs was observed after 12 months compared to t0. SvP counts remained generally low during 12 months of storage, both at 5°C and 20°C. While there was an increase in subvisible particles after 12 months at 5°C, the numbers were low in comparison to other highly concentrated protein products and are well within acceptable limits. Conclusion Formulations show no visual particles and slight yellow stain at higher concentrations of recAP. Viscosity, Turbidity, and Subvisible particles were well within acceptable range at highest concentration up to 182 mg / ml of recAP. Example 8 Effect of subcutaneous administration of ilofotase alfa in a mouse model of adult hypophosphatasia (HPP). Materials and methods Mice The Adult HPP mouse model, Prrx1Cre-Alplfl / - (PAKO) was created by mating Prrx1Cre-Alplfl / fl males x Alpl + / - females. All PAKO (Crefl / -) and littermate + / f controls were bred, tested and maintained in an ALAAC accredited animal vivarium at the MaineHealth Institute for Research. All studies were approved under IACUC number #2209. Ilofotase alfa treatment PAKO and control mice were treated with either ilofotase alfa (16 mg / kg) or vehicle (n=8 / geno / treatment) daily by subcutaneous injection from 16 to 28 weeks of age. Plasma PPi and PLP levels At sacrifice, blood was collected via cardiac puncture into lithium heparin treated tubes and immediately spun down at 7000 RPM x 10 min. Plasma was then collected and spun down in Spin-X columns (0.22 μM, Coster 8160) at 7000 RPM x 2 min and eluted plasma was snap frozen and stored at -80℃. Prior to assay, plasma samples were thawed on ice and de-proteinated with 10 kD cutoff column (Amicon UFC501024) (10000 RPM x 10 min at 4℃). Plasma PPi and PLP levels were then measured utilizing the PPiLight® Inorganic Pyrophosphate Assay (Lonza) or the Pyridoxal 5’-phosphate (vit B6) Assay Kit (Abcam), per the manufacturer’s instructions. Graded Maximal Exercise Tests (GXT) Animals were acclimated to the Promethion Core Metabolic Treadmill (Sable Systems) and allowed to rest for one week prior to performing the GXT. Acclimation consisted of 3 training sessions with 48 hours recovery between sessions. During acclimation, mice were placed on a motionless treadmill for 3 min, with the shock grid activated (1 Hz and 0.75 mA). Then treadmill was engaged to a walking speed of 6 m / min for 5 min and progressively increased up to 12 m / min for a total duration of 12 min of exercise at 0° incline. A Graded Maximal Exercise Testing Protocol (GXT) (adapted from: Petrosino at al, 2016) was then performed at baseline, 4, 8, and 12 weeks of treatment. GXT protocol consisted of the following steps (speed, time, incline): 0 m / min, 3 min, 0°; 6 m / min, 2 min, 0°; 9 m / min, 2 min, 5°; 12 m / min, 2 min, 10°; 15 m / min, 2 min, 15°; 18 m / min, 1 min, 15°; 21 m / min, 1 min, 15°; 23 m / min, 1 min, 15°; 24 m / min, 1 min, 15°; 25 m / min, 0.5 min, 15°; 26 m / min, 0.5 min, 15°; 27 m / min, 0.5 min, 15°; 28 m / min, 0.5 min, 15°; 29 m / min, 0.5 min, 15°; 30 m / min, 0.5 min, 15°; 31 m / min, 0.5 min, 15°; 32 m / min, 0.5 min, 15°; 33 m / min, 0.5 min, 15°; 34 m / min, 0.5 min, 15°. Exhaustion (endpoint for treadmill cessation) was defined as the point at which mice maintained continuous contact with the shock grid for 5 seconds. VO2max was determined by the peak oxygen consumption reached during the maximum running speed attained during the test. Wire Hang Testing Mice were inverted on a wire lid for up to 60 seconds. If the mice were able to hang on for 60 seconds, no further trials were performed. If the mice did not hang on for 60 seconds, they were immediately tested again after falling. If they failed to hang on for 60 seconds the second time, they were given a break for a few minutes prior to the third test. The best time out of the 3 trials were recorded. Results Ilofotase alfa treatment reduced circulating PPi and PLP to control levels in PAKO mice after 12 weeks of treatment (Fig.11). Graded maximal exercise testing (GTX) on a metabolic treadmill revealed a significant improvement in VO2max in PAKO_IF treated mice vs. baseline (Fig. 12A), while no such improvement was noted in the PAKO_VEH treated cohort. Furthermore, PAKO_IF mice had significantly improved run time until exhaustion (Fig.12B) and total meters run (Fig.12C) vs. PAKO_VEH treated mice. In addition, PAKO_IF treated mice exhibited significant improvements in hang time vs. PAKO_VEH mice after 12 weeks of treatment (Fig. 12D). Taken together these results show that subcutaneous ilofotase alfa treatment results in improvements in metabolic rate, muscle strength, and endurance in an Adult HPP mouse model. Example 9 The objectives of this study were to determine the local tolerance and bioavailability of ilofotase alfa, when given via the subcutaneous route on Days 1 and 22, or via intravenous (slow bolus) injection on Day 1 to the Göttingen minipig. Göttingen minipigs received a subcutaneous injection with ilofotase alfa dose solution (185 mg / mL) and with Placebo dose solution at distinct injection sites on Days 1 and 22 at two different dose volumes (Group 1: 1 mL, Group 2: 1.5 mL) to assess the local tolerance and bioavailability. In addition, a third group of minipigs was treated with an intravenous (slow bolus) injection of ilofotase alfa (8 mg / mL) on Day 1 at a dose level of 9 mg / kg body weight. Table 18: Schematic overview of study design SC = Subcutaneous; IV = Intravenous; M = Males; F = FemalesaBased on the most recent body weight measurement. The following parameters and endpoints were evaluated in this study: mortality, clinical signs, injection site evaluation (Groups 1 and 2), body weights, food consumption, blood collection for toxicokinetic evaluation and anti-drug antibody (ADA), macroscopic examination and microscopic examination of selected tissues. No mortality occurred and no ilofotase alfa-related changes were noted in clinical signs, body weight, and food consumption in all groups, and no erythema and edema were present at the subcutaneous administration sites in Group 1 and 2 animals. Also, there were no macroscopic findings suggestive of systemic toxicity at the tested doses via subcutaneous or intravenous administration. On Day 24, i.e. two days after subcutaneous administration on Day 22 of Placebo (site 4) or ilofotase alfa (site 3), microscopic findings mild of nature were noted in the subcutaneous tissue. These findings consisted of necrosis / inflammation of myofibers (up to mild), mononuclear cell infiltrates (minimal), and / or hemorrhages (up to mild) with a macroscopic correlate red discoloration. The mild severity of necrosis / inflammation of myofibers (up to mild) was only noted in ilofotase alfa-treated sites (site 3) of one animal out of six in Group 1 (1 mL) and one animal out of six in Group 2 (1.5 mL) However, the limited number of animals (3 / sex / group) requires caution in drawing conclusions. On Day 24, i.e. 23 days after subcutaneous administration on Day 1 of Placebo (site 1) or ilofotase alfa (site 2), degeneration / regeneration of myofibers, mononuclear cell infiltrates, and / or pigmented macrophages were noted at minimal severity and low incidences. This indicates almost complete recovery for both Placebo-injection sites and ilofotase alfa injection sites. Microscopic findings of the draining lymph nodes consisted of intrasinusoidal erythrocytes (minimal) in the left ventral superficial lymph node of 3 / 3 males and 3 / 3 females of Group 1 (1 mL) and 1 / 3 females of Group 2 (1.5 mL). These were most likely related to the hemorrhages noted, 2 days post administration, in Administration Site 3 (with ilofotase alfa). There were no microscopic correlates for the small size of the ventral superficial lymph node in a single Group 1 male (1 mL) and a single Group 2 female (1.5 mL), and for the brown discoloration noted in 1 / 3 males and 2 / 3 females of Group 2 (1.5 mL). Toxicokinetic evaluation revealed that systemic exposure to ilofotase alfa was independent of sex for all groups, and the test material was quantifiable in minipigs up to 168 or 336 hours post-dose for all groups. For subcutaneous administration, the average absolute bioavailability, calculated with AUC0-inf / Dose, was 45.2%. Systemic exposure increased with increasing subcutaneous dose volume in an approximately dose proportional manner. Average t1 / 2 values were 67.3 hours after subcutaneous administration and 78.4 hours after intravenous administration. Anti-drug antibody (ADA) evaluation on Day 24 showed that no (specific) antibodies were detected in any animal after subcutaneous dosing with ilofotase alfa on Days 1 and 22, while after a single intravenous dose on Day 1, one out of six animals had a titer of 1800 for anti-ilofotase alfa antibodies. In conclusion, subcutaneous administrations of an ilofotase alfa dosing solution of 185 mg / mL at dose volumes of 1 or 1.5 ml per injection on Days 1 and 22 to Göttingen minipigs were locally well tolerated during in-life, with minimal to mild histopathological changes in the subcutaneous tissue two days after administration for Placebo and ilofotase alfa. Both Placebo-injection sites and ilofotase alfa- injection sites had near complete recovery 23 days after injection. The average absolute bioavailability of ilofotase alfa after subcutaneous administration was 45.2%. SEQ ID NO:1 SEQUENCE OF RECAP VIPAEEENPAFWNRQAAEALDAAKKLQPIQKVAKNLILFLGDGLGVPTVTATRILKGQK NGKLGPETPLAMDRFPYLALSKTYNVDRQVPDSAATATAYLCGVKANFQTIGLSAAARF NQCNTTRGNEVISVMNRAKQAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADMPAS ARQEGCQDIATQLISNMDIDVILGGGRKYMFPMGTPDPEYPADASQNGIRLDGKNLVQE WLAKHQGAWYVWNRTELMQASLDQSVTHLMGLFEPGDTKYEILRDPTLDPSLMEMTEAA LRLLSRNPRGFYLFVEGGRIDHGHHEGVAYQAVTEAVMFDDAIERAGQLTSEEDTLTLV TADHSHVFSFGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESE SGSPEYRQQSAVPLDEETHGGEDVAVFARGPQAHLVHGVQEQSFVAHVMAFAACLEPYT ACDLALPACTTD

Claims

Claims 1. An aqueous composition comprising at least 20 mg / mL of an alkaline phosphatase, 0.05-5 mg / mL of a non-ionic surfactant, 1-1000 mM of an additive, and 0.1-5 mM of a multivalent metal, having a pH of 6-8.

2. The aqueous composition of claim 1, comprising 20-200 mg / mL of an alkaline phosphatase.

3. The aqueous composition of claim 1 or 2, wherein the multivalent metal is one or more of Mg, Zn, Ca, and Al.

4. The aqueous composition of any one of claims 1-3, wherein multivalent metal is Mg and Zn.

5. The aqueous composition of any one of claims 1-4, wherein the additive is a non-metal salt, a sugar, and / or an amino acid.

6. The aqueous composition of any one of claims 1-5, wherein the additive is a sugar, preferably trehalose.

7. The aqueous composition of any one of claims 1-6, wherein the non-ionic surfactant comprises an ethylene oxide / propylene oxide block copolymer.

8. The aqueous composition of any one of claims 1-7, wherein the non-ionic surfactant comprises an ethylene oxide / propylene oxide triblock copolymer.

9. The aqueous composition of any one of claims 1-8, wherein the non-ionic surfactant comprises a central propylene oxide chain which is flanked at both sides by an ethylene oxide chain, such as a poloxamer.

10. The aqueous composition of any one of claims 1-9, wherein the non-ionic surfactant comprises a central chain of about 29 propylene oxide moieties, which is flanked by two chains of about 38 ethylene oxide moieties.

11. The aqueous composition of any one of claims 1-10, comprising 0.5 mg / mL of a non-ionic surfactant.

12. The aqueous composition of any one of claims 1-10, comprising a recombinant alkaline phosphatase, preferably a recombinant human alkaline phosphatase.

13. The aqueous composition of any one of claims 1-12, wherein the alkaline phosphatase is obtained from a cell-based expression system.

14. The aqueous composition of claim 13, wherein the composition comprises less than 100 ppm of a host cell protein (HCP).

15. The aqueous composition of any one of claims 1-14, for use as a medicament.

Images