Methods and Compositions Comprising Surfactant Protein D (SP-D)

Abstract

Some embodiments provided herein relate to methods and pharmaceutical compositions comprising recombinant human surfactant protein D or an active fragment thereof. Some such embodiments include solutions or suspensions of recombinant human surfactant protein D or an active fragment thereof, and lyophilized solid forms.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 650,142, filed March 29, 2018, entitled "METHODS AND COMPOSITIONS COMPRISING SURFACTANT PROTEIN D (SP-D)," which is incorporated herein by reference in its entirety.

[0002] Reference to sequence listing This application will be filed with a sequence listing in electronic format. The sequence listing will be approximately 14 Kb, 20 23 year 2 month 2 3 Created on 000280.000721 ​​Replacement Sequence Listing (16231966) The information in the electronic format of the Sequence Listing is provided as a file entitled "Sequence Listing 1.0.1 ...

[0003] Some embodiments provided herein relate to methods and pharmaceutical compositions comprising recombinant human surfactant protein D (SP-D) or an active fragment thereof. Some such embodiments include solutions and lyophilized solid forms of recombinant human surfactant protein D or an active fragment thereof. [Background technology]

[0004] Mammalian pulmonary surfactant is a mixture of protein (10%) and lipids (90%), with dipamyltoylphosphatidylcholine being the major lipid component (Zuo YY et al., Biochim Biophys Acta (2008) 1778:1947-77). The primary function of pulmonary surfactant is to ensure minimal surface tension within the lungs to avoid collapse during breathing. Furthermore, by interacting with inhaled pathogens, pulmonary surfactant also participates in host defense (Clements JA. Am Rev Respir Dis (1977) 115:67-71). Pulmonary surfactant deficiency is associated with pulmonary diseases such as asthma, bronchiolitis, respiratory distress syndrome (RDS), cystic fibrosis, and pneumonia (Griese M. Eur Respir J (1999) 13:1455-76). Surfactant preparations are indicated for the treatment of RDS, which affects approximately 1.5 million premature infants worldwide each year. Respiratory distress syndrome is a primary pulmonary surfactant deficiency disease caused by structural lung immaturity in premature infants, which makes breathing difficult, inhibits gas exchange, and promotes alveolar collapse (Notter RH. 2000 Lung Surfactants. Basic Science and Clinical Applications. New York, NY: Marcel Dekker Inc.). Successful treatment of complex lung diseases typically requires the creation of surfactant preparations whose composition matches as closely as possible with natural pulmonary surfactant (Robertson B et al., Biochim Biophys Acta (1998) 1408:346-61).

[0005] Pulmonary surfactant contains four distinct surfactant proteins. Two hydrophobic proteins, surfactant proteins B and C, are involved in reducing surface tension at the air-liquid interface, while two hydrophilic proteins, surfactant proteins A and D, are members of the collectin family and are involved in regulating host immune responses and surfactant pool recycling (Awasthi S. (2010) Recent Patents on Anti-Infective Drug Discovery, 5:115-123). Surfactant protein D (SP-D) is a C-type surfactant protein (Ca-type surfactant protein) containing four domains: a cysteine-linked N-terminal region required for intermolecular disulfide bond formation, a triple-helical collagen region, an α-helical coiled-coil trimerization neck peptide, and a C-terminal calcium-dependent carbohydrate recognition domain (CRD). 2+ SP-D is a lectin (protein-dependent) (Crouch E. et al. (1994) J Biol Chem 269:17311-9). Monomers form trimers by folding the collagen-like region into a triple helix and assembling a coiled-coil bundle of α-helices in the neck region. These trimers are stabilized by two disulfide bonds in the cysteine-rich N-terminal domain. SP-D trimers have a total molecular weight of 129 kDa, containing three identical 43 kDa polypeptide chains. SP-D trimers can form higher-order oligomerization states that differ in size and conformation. Higher oligomerization states may be important for SP-D function (Hakansson K et al., Protein Sci (2000) 9:1607-17; Crouch E. Respir Res (2000) 1:93-108; Crouch E. et al., (2006) J Biol Chem 281:18008-14).

[0006] In contrast to other surfactant proteins, SP-D is not involved in reducing surface tension at the air-liquid interface and does not alter the biophysical properties of pulmonary surfactants (Awasthi S. (2010) Recent Patents on Anti-Infective Drug Discovery, 5:115-123). SP-D plays a role in the innate immune system of the lung by providing anti-inflammatory and antibacterial activities to combat chronic lung diseases such as asthma, cystic fibrosis, and smoking-induced emphysema (Clark H et al., Immunobiology (2002) 205:619-31). SP-D also plays an important role in regulating surfactant pool homeostasis, and deficiency of SP-D during development results in an emphysematous phenotype (Wert, S. et al. (2000) Chest 117:248S; Korfhagen, TR et al. (1998) J Biol Chem 273: 28438-28443). Data based on preterm neonatal lambs suggest that administration of approximately 2-3 mg / kg of recombinant human SP-D (rhSP-D) in combination with 100 mg / kg Survanta® (a natural surfactant available in the USA) is more effective than Survanta® alone in preventing endotoxic shock and reducing lung inflammation caused by mechanical ventilation (Ikegami M et al., Am J Respir Crit Care Med (2006) 173:1342-7; Sato A et al., Am J Respir Crit Care Med (2010) 181:1098-105).

[0007] Traditionally, SP-D has been isolated from the supernatant of bronchoalveolar lavage fluid or amniotic fluid; however, most, if not all, of the SP-D is lost during the purification of commercial surfactants, in part due to the hydrophilic nature of SP-D (Dodagatta-Marri E et al., Methods Mol Biol (2014) 100:273-90). The use of rhSP-D in supplemental pulmonary surfactant formulations can ensure therapeutic efficacy, as current pulmonary surfactant formulations lack the ability to effectively modulate host immune responses in the absence of hydrophilic surfactant proteins. An important property of native SP-D that must be maintained in any pharmaceutical composition is the proper oligomerization state, because higher order multimerization in endogenous surfactant proteins increases the number of SP-D binding sites for carbohydrate ligands on the surface of pathogens, achieving potent bacterial and viral aggregation effects (White M et al., J Immunol (2008) 181:7936-43). The proper oligomerization state is also required for receptor recognition and receptor-mediated signaling for modulation of host immune responses (Yamazoe M. et al., J Biol Chem (2008) 283:35878-35888), as well as for maintaining surfactant homeostasis (Zhang L et al., J Biol Chem (2001) 276:19214-19219). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 8,865,643 [Patent Document 2] U.S. Patent Application Publication No. 2019 / 0071693 [Patent Document 3] U.S. Patent Application Publication No. 2019 / 0071694 [Patent Document 4] U.S. Provisional Patent Application No. 62 / 650138 [Non-patent literature]

[0009] [Non-licensed Document 1] Zuo YYら, Biochim Biophys Acta (2008) 1778:1947~77 pages [Non-licensed Document 2] Clements JA. Am Rev Respir Dis (1977) 115:67~71 pages [Non-licensed Document 3] Griese M. Eur Respir J (1999) 13: 1455~76 [Non-licensed Document 4] Notter RH. 2000 Lung Surfactants. Basic Science and Clinical Applications. New York, NY: Marcel Dekker Inc. [Non-licensed Document 5] Robertson B, Biochim Biophys Acta (1998) 1408: 346~61 pages [Non-licensed Document 6] Awasthi S. (2010) Recent Patents on Anti-Infective Drug Discovery, 5:115~123 pages [Non-licensed Document 7] Crouch E.ら(1994) J Biol Chem 269:17311~page 9 [Non-licensed Document 8] Hakansson Kら, Protein Sci (2000) 9:1607~17 pages [Non-licensed Document 9] Crouch E. Respir Res (2000) 1: 93-108 [Non-licensed Document 10] Crouch E.ら, (2006) J Biol Chem 281:18008~14 pages [Non-licensed Document 11] Awasthi S. (2010) Recent Patents on Anti-Infective Drug Discovery, 5:115~123 pages [Non-licensed Document 12] Clark Hら, Immunobiology (2002) 205:619~31 pages [Non-licensed Document 13] Wert, S. (2000) Chest 117:248S [Non-licensed Document 14] Korfhagen, TRら(1998) J Biol Chem 273: 28438~28443 pages [Non-licensed Document 15] Ikegami Mら、Am J Respir Crit Care Med (2006) 173:1342~7 pages [Non-licensed Document 16] Sato Aら、Am J Respir Crit Care Med (2010) 181:1098~105 pages [Non-licensed Document 17] Dodagatta-Marri Eら, Methods Mol Biol (2014) 100:273~page 90 [Non-licensed Document 18] White Mら, J Immunol (2008) 181:7936~43 pages [Non-licensed Document 19] Yamazoe M.ら, J Biol Chem (2008) 283:35878~35888 pages [Non-licensed Document 20] Zhang Lら, J Biol Chem (2001) 276:19214~19219 pages [Non-licensed Document 21] Knudsen L.ら, (2009) The Anatomical Record 292:183~189 pages [Non-licensed Document 22] Knudsen L.ら, (2013) J. Anat 223: pages 581~592 [Non-licensed Document 23] Winkler C.ら、(2014) Exp Lung Res 40:154~163 pages [Non-licensed Document 24] Ogasawara Y.ら, (1995) J Biol Chem 270:19052~19058 pages [Non-licensed Document 25] Kingma PSら, (2006) J Biol Chem 281:24496~24505 pages [Non-licensed Document 26] "Remington: The Science and Practice of Pharmacy", Lippincott Williams & Wilkins; 20th Edition (June 1, 2003) [Non-licensed Document 27] "Remington's Pharmaceutical Sciences", Mack Pub. Co.; 18th and 19th editions (それぞれ, December 1985, and June 1990) [Non-licensed Document 28] Arroyo Rら, (2017) Biophys J 112 (3): 503a [Non-licensed Document 29] Haagsman HPら、(2008) Neonatology 93:288~294 pages [Non-licensed Document 30] Vieira F. (2017) Ann Anat 211: 184-201 [Non-licensed Document 31] Malash AHら, (2016) Gene 592:23~28 pages [Non-licensed Document 32] Katz, MH, "Multivariate Analysis: A Practice Guide for Clinicians." Cambridge University Press, New York, pp. 158~162 (1999) [Non-licensed Document 33] Stahle, L., (1988) "Multivariate data analysis and experimental design in biomedical research." Prog. Med. Chem. 25: 291~338 pages [Non-Patent Document 34] Wold S. (2001) “PLS-regression: a basic tool of chemometrics.” Chemom. Intel. Lab. Syst. 58: pp. 109-130 [Non-Patent Document 35] Martens, H. et al. (2001) “Multivariate Analysis of Quality: An Introduction” Wiley and Sons, Chichester, UK Summary of the Invention [Means for solving the problem]

[0010] Some embodiments include a pharmaceutical composition comprising recombinant human surfactant protein D (rhSP-D) or an active fragment thereof. Some embodiments also include a buffer, a sugar, and a calcium salt.

[0011] In some embodiments, the buffer is selected from the group consisting of acetate, citrate, glutamate, histidine, succinate, and phosphate. In some embodiments, the buffer is histidine. In some embodiments, the concentration of histidine is about 0.1 mM to about 100 mM. In some embodiments, the concentration of histidine is about 5 mM.

[0012] In some embodiments, the sugar is selected from the group consisting of sucrose, maltose, lactose, glucose, fructose, galactose, mannose, arabinose, xylose, ribose, rhamnose, trehalose, sorbose, melezitose, raffinose, thioglucose, thiomannose, thiofructose, octa-O-acetyl-thiotrehalose, thiosucrose, and thiomaltose. In some embodiments, the sugar is lactose. In some embodiments, the concentration of lactose is 1 mM to 500 mM. In some embodiments, the concentration of lactose is about 265 mM.

[0013] In some embodiments, the calcium salt is selected from the group consisting of calcium chloride, calcium bromide, calcium acetate, calcium sulfate, and calcium citrate. In some embodiments, the calcium salt is calcium chloride. In some embodiments, the concentration of calcium chloride is about 0.1 mM to about 10 mM. In some embodiments, the concentration of calcium chloride is about 5 mM.

[0014] In some embodiments, the pharmaceutical composition has a pH of about 5.0 to about 9.0. In some embodiments, the pharmaceutical composition has a pH of about 5.5 to about 6.5. In some embodiments, the pharmaceutical composition has a pH of about 6.0.

[0015] In some embodiments, the concentration of rhSP-D is about 0.1 mg / ml to about 10 mg / ml. In some embodiments, the concentration of rhSP-D is about 1 mg / ml to about 4 mg / ml. In some embodiments, the concentration of rhSP-D is about 2 mg / ml. In some embodiments, the concentration of rhSP-D is about 4 mg / ml.

[0016] Some embodiments include pharmaceutical compositions comprising a population of rhSP-D polypeptides having oligomeric forms, wherein greater than 30% of the oligomeric forms comprise rhSP-D dodecamers. In some embodiments, greater than 40% of the oligomeric forms comprise rhSP-D dodecamers. In some embodiments, less than 15% of the oligomeric forms comprise aggregates of rhSP-D, the aggregates having a radius greater than 70 nm. In some embodiments, less than 10% of the oligomeric forms comprise aggregates of rhSP-D, the aggregates having a radius greater than 70 nm. In some embodiments, less than 5% of the oligomeric forms comprise aggregates of rhSP-D, the aggregates having a radius greater than 70 nm.

[0017] Some embodiments also include a bulking agent, hi some embodiments, the bulking agent is selected from the group consisting of mannitol, xylitol, sorbitol, maltitol, lactitol, glycerol, erythritol, arabitol, glycine, alanine, threonine, valine, and phenylalanine.

[0018] In some embodiments, the composition lacks a chelating agent selected from EDTA and EGTA.

[0019] In some embodiments, the rhSP-D polypeptide comprises the amino acid sequence of SEQ ID NO: 02, or an active fragment thereof. In some embodiments, the rhSP-D or active fragment thereof comprises a residue at a polymorphic position corresponding to a residue selected from the group consisting of Met 11, Thr 160, Ser 270, and Ala 286.

[0020] Some embodiments include a pharmaceutical composition comprising 2 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, wherein the solution has a pH of 6.0. Some embodiments include a pharmaceutical composition comprising 2 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 1 mM calcium chloride, wherein the solution has a pH of 6.0. Some embodiments include a pharmaceutical composition comprising 1 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, wherein the solution has a pH of 6.0. Some embodiments include a pharmaceutical composition comprising 4 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, wherein the solution has a pH of 6.0.

[0021] Some embodiments include a lyophilizate of any one of the preceding pharmaceutical compositions.

[0022] Some embodiments include an article comprising a container containing a lyophilized form of any one of the foregoing pharmaceutical compositions.

[0023] Some embodiments include a method of preparing a lyophilizate comprising lyophilizing any one of the preceding pharmaceutical compositions.

[0024] Some embodiments include a method of preparing a pharmaceutical composition comprising contacting a lyophilized form of the pharmaceutical composition of any one of claims 1 to 33 with a pharmaceutically acceptable diluent to form a solution or suspension of rhSP-D or an active fragment thereof.

[0025] In some embodiments, the diluent is suitable for pulmonary administration, hi some embodiments, the diluent is sterile water.

[0026] In some embodiments, the solution or suspension has a concentration of rhSP-D or an active fragment thereof of about 0.1 mg / ml to about 10 mg / ml. In some embodiments, the solution or suspension has a concentration of rhSP-D or an active fragment thereof of about 1 mg / ml to about 4 mg / ml. In some embodiments, the solution or suspension has a concentration of rhSP-D or an active fragment thereof of about 2 mg / ml. In some embodiments, the solution or suspension has a concentration of rhSP-D or an active fragment thereof of about 4 mg / ml.

[0027] In some embodiments, less than about 3% by weight of the rhSP-D oligomeric species in the solution or suspension are high molecular weight oligomeric forms having a radius greater than 70 nm.

[0028] In some embodiments, the solution or suspension comprises a population of rhSP-D polypeptides having oligomeric forms, wherein greater than 30% of the oligomeric forms comprise a rhSP-D dodecamer, in some embodiments, greater than 40% of the oligomeric forms comprise a rhSP-D dodecamer, and in some embodiments, greater than 45% of the oligomeric forms comprise a rhSP-D dodecamer.

[0029] In some embodiments, less than 10% of the oligomeric forms comprise aggregates of rhSP-D, the aggregates having a radius greater than 70 nm. In some embodiments, less than 5% of the oligomeric forms comprise aggregates of rhSP-D, the aggregates having a radius greater than 70 nm. In some embodiments, less than 3% of the oligomeric forms comprise aggregates of rhSP-D, the aggregates having a radius greater than 70 nm. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 is a schematic diagram depicting the formation of SP-D trimers and the structural features of SP-D trimers. [Figure 2]1 is a bar graph depicting a 6-month lyophilizate stability study showing the distribution of different SP-D oligomer species represented by either peak 1, peak 2, peak 3, or peak 4 of the AF4-MALLS analysis for a 2 mg / ml reconstituted lyophilizate of rhSP-D stored at either 5° C. or 25° C. for periods of 1 month, 3 months, or 6 months. [Figure 3] FIG. 1 is a set of bar graph panels depicting a 3-month lyophilizate stability study, showing the distribution of different SP-D oligomer species represented by either peak 1, peak 2, peak 3, or peak 4 of the AF4-MALLS analysis of rhSP-D lyophilizates reconstituted at either 2 mg / ml (upper panel) or 4 mg / ml (lower panel) and stored at either 5° C. or 25° C. for periods of 1 month or 3 months. [Figure 4] FIG. 1 is a set of bar graph panels depicting freeze / thaw stability studies, showing the distribution of different SP-D oligomer species, represented by either peak 1, peak 2, peak 3, or peak 4 of AF4-MALLS analysis of lyophilized rhSP-D that had been reconstituted at either 2 mg / ml (upper panel) or 4 mg / ml (lower panel), frozen either by rapid liquid nitrogen freezing or in a −80° C. freezer, and thawed for one, three, or five freeze / thaw cycles. [Figure 5] FIG. 1 is a set of bar graph panels depicting stability studies on reconstituted lyophilisates, showing the distribution of different SP-D oligomer species represented by either peak 1, peak 2, peak 3, or peak 4 of the AF4-MALLS analysis of rhSP-D lyophilisates reconstituted at either 2 mg / ml (upper panel) or 4 mg / ml (lower panel) and stored at either 5° C. or 25° C. for periods of 1 day or 3 days. DETAILED DESCRIPTION OF THE INVENTION

[0031] Surfactant protein D (SP-D) is a C-type (Ca) surfactant protein containing four domains: a cysteine-linked N-terminal region required for intermolecular disulfide bond formation, a triple-helical collagen region, an α-helical coiled-coil trimerization neck peptide, and a C-terminal calcium-dependent carbohydrate recognition domain (CRD). 2+ SP-D is a lectin (dependent on the ATP-dependent domain) (Crouch E. et al. (1994) J Biol Chem 269:17311-9). Monomers form trimers by folding the collagen-like region into a triple helix and assembling a coiled-coil bundle of α-helices in the neck region (Figure 1). These trimers are stabilized by two disulfide bonds in the cysteine-rich N-terminal domain. SP-D trimers have a total molecular weight of 129 kDa, containing three identical 43 kDa polypeptide chains. SP-D trimers can form higher-order oligomerization states that differ in size and conformation. Higher oligomerization states may be important for SP-D function (Hakansson K et al., Protein Sci (2000) 9:1607-17; Crouch E. Respir Res (2000) 1:93-108; Crouch E. et al., (2006) J Biol Chem 281:18008-14). The assembly of SP-D trimers into higher oligomerization states is sensitive to environmental factors and conditions during purification and storage. The pathways and types of interactions involved in the formation of large SP-D oligomers have not previously been elucidated.

[0032] Recombinant human SP-D (rhSP-D) has been produced in various mammalian systems, including certain human myeloid leukemia cell lines and Chinese hamster ovary (CHO) cells. Samples of rhSP-D may contain a diverse population of different rhSP-D oligomeric forms, including trimers, hexamers, dodecamers, hexamer and dodecamer multimers with a star-like appearance, and larger oligomeric species identified as aggregates. rhSP-D from various mammalian systems may differ substantially. For example, post-translational modifications of rhSP-D may affect the distribution of rhSP-D oligomeric forms within a given sample of rhSP-D. In addition, rhSP-D produced in certain human myeloid leukemia cell lines may have a higher amount of rhSP-D dodecamers and a lower amount of larger oligomeric species, such as rhSP-D aggregates, compared to rhSP-D produced from CHO cells.

[0033] Applicants have discovered certain formulations of rhSP-D and its active fragments that provide improved shelf life, improved stability of the active rhSP-D protein, and improved stability of certain rhSP-D oligomeric forms. Some embodiments of such formulations include rhSP-D for administration, rhSP-D for lyophilization, lyophilized rhSP-D, reconstituted lyophilized rhSP-D, and compositions comprising reconstituted lyophilized rhSP-D for administration. In some embodiments, the compositions are suitable for pulmonary administration, such as administration to the lung, e.g., neonatal lung.

[0034] Pharmaceutical Compositions Some embodiments of the compositions and methods provided herein include pharmaceutical compositions of recombinant human surfactant protein D (rhSP-D) or an active fragment thereof. In some embodiments, the rhSP-D or an active fragment thereof has activity in a bacterial agglutination assay or a TLR4 inhibition assay. In some embodiments, the pharmaceutical composition can be an aqueous solution, a suspension, or a solid. In some embodiments, the pharmaceutical composition of rhSP-D or an active fragment thereof is suitable for lyophilization into a solid form. In some embodiments, the solid form, such as a lyophilizate or powder, can be administered to the lungs and / or reconstituted to form a specific solution suitable for pulmonary administration. In some embodiments, the pharmaceutical composition comprising an aqueous solution or suspension of rhSP-D or an active fragment thereof is suitable for pulmonary administration.

[0035] A specific activity of rhSP-D or a fragment thereof can be readily determined using a bacterial agglutination assay, a Toll-like receptor 4 (TLR4) inhibition assay, and / or asymmetric flow field-flow fractionation combined with multi-angle laser light scattering (AF4-MALLS) analysis. In some embodiments, the activity of rhSP-D or an active fragment thereof can include a biological activity, such as activity measured in a bacterial agglutination assay or a TLR4 inhibition assay. In some embodiments, the activity of rhSP-D or an active fragment thereof can include the activity of a population of rhSP-D or an active fragment thereof to form a specific oligomeric form of rhSP-D and / or to form a specific distribution of oligomeric forms of rhSP-D. Exemplary methods for identifying the distribution of oligomeric forms of rhSP-D in a sample are provided herein.

[0036] SP-D contains four domains: a cysteine-linked N-terminal region required for intermolecular disulfide bond formation, a triple-helical collagen region, an α-helical coiled-coil trimerization neck peptide, and a C-terminal calcium-dependent carbohydrate recognition domain (CRD). An example of a rhSP-D polypeptide sequence is provided in TABLE 1. Certain portions of SP-D may have specific roles in SP-D function in vivo. For example, in SP-D knockout mice, a recombinant fragment of human SP-D consisting of a shorter collagen region (rather than the entire collagen region or the amino terminus of wild-type SP-D), the neck domain, and the CRD inhibits certain aspects of the development of emphysema-like pathology (Knudsen L. et al. (2009) The Anatomical Record 292:183-189, incorporated herein by reference in its entirety). The cysteine-linked N-terminal region may be active in the formation of oligomeric forms of SP-D. For example, an A to C nucleotide change resulting in a Met to Thr substitution at position 11 in the protein (Met(11)Thr) can affect the formation of oligomeric forms of SP-D. See, e.g., Knudsen L. et al., (2013) J. Anat 223:581-592; and Winkler C. et al., (2014) Exp Lung Res 40:154-163 (each incorporated herein by reference in its entirety). Deletion studies on mutant rat SP-D proteins lacking the rat triple-helical collagen domain demonstrated that the mutant rat SP-D was still able to form trimers but was unable to form higher-order oligomers, such as dodecamers, and lacked activity in pulmonary macrophage activation, airspace remodeling, and regulating surfactant lipid homeostasis. See Ogasawara Y. et al., (1995) J Biol Chem 270:19052-19058; and Kingma PS et al., (2006) J Biol Chem 281:24496-24505, each of which is incorporated herein by reference in its entirety.Further deletion studies on the rat SP-D protein demonstrated that the rat cysteine-linked N-terminal region plays a role in efficient virus neutralization and opsonization. See White M. et al. (2008) J. Immunol 181:7937-7942, incorporated herein by reference in its entirety. Examples of human SP-D lacking certain domains are disclosed in U.S. Patent No. 8,865,643, incorporated herein by reference in its entirety.

[0037] In some embodiments, the pharmaceutical composition may contain a buffer. Examples of buffers include acetate, citrate, glutamate, histidine, succinate, and phosphate. In some embodiments, the buffer is histidine. In some embodiments, the concentration of the buffer, such as histidine, is 0.1 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or a range between any two of the foregoing concentrations. In some embodiments, the concentration of the buffer, such as histidine, is about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, or a concentration in a range between any two of the foregoing concentrations.

[0038] In some embodiments, the pharmaceutical composition may contain a sugar. Examples of sugars include trehalose, sucrose, maltose, lactose, glucose, fructose, galactose, mannose, arabinose, xylose, ribose, rhamnose, trehalose, sorbose, melezitose, raffinose, thioglucose, thiomannose, thiofructose, octa-O-acetyl-thiotrehalose, thiosucrose, and thiomaltose. In some embodiments, the sugar is lactose. In some embodiments, the concentration of the sugar, such as lactose, is 0.1 mM, 1 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 265 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, or a concentration in a range between any two of the foregoing concentrations. In some embodiments, the concentration of the sugar, such as lactose, is about 0.1 mM, about 1 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 265 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, or a concentration in a range between any two of the foregoing concentrations.

[0039] In some embodiments, the pharmaceutical composition may contain a calcium salt. Examples of calcium salts include calcium chloride, calcium bromide, calcium acetate, calcium sulfate, and calcium citrate. In some embodiments, the calcium salt is calcium chloride. In some embodiments, the concentration of the calcium salt, such as calcium chloride, is 0.1 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or a range between any two of the foregoing concentrations. In some embodiments, the concentration of the calcium salt, such as calcium chloride, is about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, or a concentration in a range between any two of the foregoing concentrations.

[0040] In some embodiments, the pharmaceutical composition may contain an inorganic salt or an organic salt. Examples of inorganic salts include sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate. Examples of organic salts include sodium citrate, potassium citrate, and sodium acetate. In some embodiments, the inorganic salt is sodium chloride. In some embodiments, the concentration of the inorganic salt or organic salt, such as sodium chloride, is 0.1 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or a range between any two of the foregoing concentrations. In some embodiments, the concentration of an inorganic or organic salt, such as sodium chloride, is about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, or a concentration in a range between any two of the foregoing concentrations. In some embodiments, the pharmaceutical composition may lack an inorganic or organic salt, such as sodium chloride.

[0041] In some embodiments, the pharmaceutical composition may contain a surfactant. Examples of surfactants include hexadecanol, tyloxapol, dipalmitoylphosphatidylcholine (DPPC), PG, palmitoyl-oleoylphosphatidylglycerol, palmitic acid, tripalmitin, and polysorbates such as polysorbate-20, polysorbate-80, polysorbate-21, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-81, and polysorbate-85. Further examples of surfactants include poloxamers such as poloxamer 188, Triton Examples of surfactants include Triton X-100, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium octyl glycoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, lauroamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium cocoyl methyl taurate, disodium oleoyl methyl taurate, polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol. In some embodiments, the surfactant is tyloxapol. In some embodiments, the concentration of the surfactant, such as tyloxapol, is 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1% (v / v), or a range between any two of the foregoing concentrations. In some embodiments, the concentration of the surfactant, such as tyloxapol, is about 0.0001%, about 0.0005%, about 0.001%, about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1% (v / v), or a range between any two of the foregoing concentrations.In some embodiments, the pharmaceutical composition may lack a surfactant, such as tyloxapol.

[0042] In some embodiments, the pharmaceutical composition may have a pH of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, or a range between any two of the aforementioned values. In some embodiments, the pharmaceutical composition may have a pH of about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or a range between any two of the aforementioned values.

[0043] In some embodiments, the concentration of a protein, such as rhSP-D or an active fragment thereof, in a pharmaceutical composition can be 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, or a concentration in a range between any two of the foregoing concentrations. In some embodiments, the concentration of a protein, such as rhSP-D or an active fragment thereof, in a pharmaceutical composition can be about 0.01 mg / ml, about 0.05 mg / ml, about 0.1 mg / ml, about 0.5 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 20 mg / ml, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, or a concentration in a range between any two of the foregoing concentrations.

[0044] In some embodiments, the pharmaceutical composition may contain a bulking agent. Examples of bulking agents include the sugars disclosed herein. Further examples of bulking agents include mannitol, xylitol, sorbitol, maltitol, lactitol, glycerol, erythritol, arabitol, glycerin, glycine, alanine, threonine, valine, and phenylalanine. In some embodiments, the bulking agent is present at a concentration of 0.1 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, or a range between any two of the foregoing concentrations. In some embodiments, the concentration of the bulking agent is about 0.1 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, or a concentration in a range between any two of the foregoing concentrations.

[0045] In some embodiments, the pharmaceutical composition may include a chelating agent. In some embodiments, the pharmaceutical composition may lack a chelating agent. Examples of chelating agents include EDTA and EGTA.

[0046] In some embodiments, rhSP-D comprises a wild-type human SP-D polypeptide. In some embodiments, rhSP-D comprises a polymorphism of a human SP-D polypeptide. Exemplary SP-D polypeptide sequences are provided in TABLE 1. Polymorphisms in human SP-D polypeptides include residue 11, ATG (Met) → ACG (Thr); residue 25, AGT (Ser) → AGC (Ser); residue 160, ACA (Thr) → GCA (Ala); residue 270, TCT (Ser) → ACT (Thr); and residue 286, GCT (Ala) → GCC (Ala), where the positions are relative to the positions in a mature SP-D polypeptide, e.g., the exemplary polypeptide of SEQ ID NO: 02. In some embodiments, rhSP-D comprises certain residues at polymorphic positions selected from Met 11 / 31, Thr 160 / 180, Ser 270 / 290, and Ala 286 / 306, which residue positions are relative to the positions in a mature SP-D polypeptide, e.g., as exemplified by SEQ ID NO: 02, and the positions in an SP-D polypeptide with its leader polypeptide, e.g., as exemplified by SEQ ID NO: 01. In some embodiments, rhSP-D comprises Met 11 / 31. In some embodiments, rhSP-D comprises Met 11 / 31, Thr 160 / 180, Ser 270 / 290, and Ala 286 / 306. In some embodiments, the rhSP-D polypeptide has at least 80%, 90%, 95%, 99%, and 100% identity over the entire length of the polypeptide to the polypeptide of SEQ ID NO: 02, or any percentage in between.

[0047] [Table 1]

[0048] In some embodiments, the rhSP-D is derived from a human myeloid leukemia cell line that expresses rhSP-D from an integrated transgene. Examples of expression vectors, rhSP-D polypeptides, cell lines, and methods for purifying rhSP-D from such cells are provided in U.S. Patent Application Publication Nos. 2019 / 0071693 and 2019 / 0071694, each of which is expressly incorporated herein by reference in its entirety.

[0049] In some embodiments, a pharmaceutical composition, such as a solution or suspension, containing a population of rhSP-D polypeptides may have a particular distribution of oligomeric forms of rhSP-D. The composition of rhSP-D may contain different rhSP-D oligomeric forms, including: trimers, which contain three monomers and collectively have a mass of approximately 130-150 kDa on SDS-PAGE, which may have a rod-like appearance when visualized by atomic force microscopy (AFM); hexamers, which contain six monomers and have a mass of approximately 250 kDa on SDS-PAGE, which contain 12 monomers and may have an X-shaped appearance when visualized by AFM, as measured by AF4-MALLS. dodecamers with an estimated mass of about 520 kDa when determined by AFM; larger heterogeneous oligomeric species that contain trimers in multiples greater than four and may have a star-like or star-shaped appearance with a radius of about 70 nm when visualized and identified by AFM (such oligomers are known as star-shaped oligomers); and even larger oligomeric species that have a radius greater than 70 nm when visualized by AFM and measured by AF4-MALLS, known as aggregates.

[0050] In some embodiments, greater than about 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or a percentage within a range between any two of the foregoing percentages, of the oligomeric forms of rhSP-D may be the dodecameric oligomeric form of rhSP-D, as measured as relative peak area (RPA) in an AF4-MALLS analysis. In some embodiments, greater than about 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or a percentage within a range between any two of the foregoing percentages, of the mass of the oligomeric forms of rhSP-D, e.g., in a solution or suspension, may be the dodecameric oligomeric form of rhSP-D. In some embodiments, more than about 10%, more than 20%, more than 30%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than a percentage within a range between any two of the aforementioned percentages, of the number of oligomeric molecules of rhSP-D in a solution or suspension, etc., may be the dodecamer oligomeric form of rhSP-D.

[0051] In some embodiments, less than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 50%, or a percentage within a range between any two of the foregoing percentages, of the oligomeric form of rhSP-D may be aggregate oligomeric form of rhSP-D, as measured as RPA or adjusted RPA in an AF4-MALLS analysis. In some embodiments, less than about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 50%, or a percentage within a range between any two of the foregoing percentages, of the mass of oligomeric form of rhSP-D, e.g., in a solution or suspension, may be aggregate oligomeric form of rhSP-D. In some embodiments, less than about 0.5%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 10%, less than 20%, less than 30%, less than 50%, or less than a percentage within a range between any two of the aforementioned percentages, of the number of molecules of oligomeric form of rhSP-D in a solution or suspension, etc., may be aggregate oligomeric form of rhSP-D.

[0052] In some embodiments, the pharmaceutical composition consists of, consists essentially of, or comprises 1 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, having a pH of 6.0. In some embodiments, the pharmaceutical composition consists of, consists essentially of, or comprises 1 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 1 mM calcium chloride, having a pH of 6.0. In some embodiments, the pharmaceutical composition consists of, consists essentially of, or comprises 2 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 1 mM CaCl2, pH 6.0. In some embodiments, the pharmaceutical composition consists of, consists essentially of, or comprises 2 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, having a pH of 6.0. In some embodiments, the pharmaceutical composition consists of, consists essentially of, or comprises 4 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, having a pH of 6.0.

[0053] In some embodiments, the pharmaceutical compositions provided herein may comprise a mixture with a suitable carrier, diluent, or excipient, such as sterile water, saline, glucose, or the like, depending on the desired route of administration and preparation, and may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring agents, coloring agents, and the like. See, e.g., "Remington: The Science and Practice of Pharmacy," Lippincott Williams & Wilkins; 20th Edition (June 1, 2003); and "Remington's Pharmaceutical Sciences," Mack Pub. Co.; 18th and 19th Editions (December 1985 and June 1990, respectively). In some embodiments, such preparations may contain complexing agents, metal ions, polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, dextran, liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Suitable lipids for liposome formulation include monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponins, bile acids, etc. The presence of such additional components can affect the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and therefore the properties of the carrier can be selected according to the intended application, such that they are tailored to the selected route of administration, e.g., pulmonary delivery, such as delivery to the neonatal lung.

[0054] In some embodiments, the pharmaceutical composition is suitable for intratracheal, intrabronchial, or bronchoalveolar administration to the lungs. In some embodiments, intratracheal, intrabronchial, or bronchoalveolar administration can include nebulization, irrigation, inhalation, flushing, or placement using a physiologically acceptable composition in which the pharmaceutical composition is dissolved as a fluid. Methods of administration can include the use of continuous positive airway pressure (CPAP). Methods of administration can include direct intubation. In some embodiments, the pharmaceutical compositions provided herein can be delivered to the lungs via inhalation. Examples of forms that can be delivered include dry powders and aerosols. A wide variety of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including, but not limited to, nebulizers, metered-dose inhalers, and powder inhalers, all of which are well known to those skilled in the art. These devices use formulations suitable for dispensing the pharmaceutical composition. Typically, each formulation is specific to the type of device used and may include the use of an appropriate propellant in addition to diluents, adjuvants, and / or carriers useful in therapy.

[0055] Identification of oligomeric species of rhSP-D The distribution of rhSP-D oligomeric forms, such as the distribution of rhSP-D oligomeric forms in a solution or suspension, can be determined by various techniques. In some embodiments, a method for identifying rhSP-D oligomeric species can include performing atomic force microscopy (AFM) on a sample of rhSP-D and identifying and quantifying the rhSP-D oligomeric species in the AFM images. See, e.g., Arroyo R et al. (2017) Biophys J 112 (3): 503a, incorporated herein by reference in its entirety. In an example embodiment, a sample of rhSP-D can be diluted in a buffer, such as dilution buffer (200 mM NaCl, 20 mM Tris (pH 7.4), 1 mM EDTA), and placed on a freshly cleaved mica substrate. Samples can be imaged with an atomic force microscope, such as a microscope from Nanotec (Nanotec Electronica, Madrid, Spain) and a PointProbePlus tip, model PPP-NCH (Nanosensors, Neuchâtel, Switzerland). Raw images can be subjected to general plane subtraction, flattening with background subtraction, and artifact line removal. Oligomer species that can be identified in the resulting images can include trimers, hexamers, dodecamers, and star-shaped oligomers. Trimers can have a rod-like appearance, with some trimers having an average length of approximately 65 nm (±8.6 nm). Dodecamers can have an X-shaped appearance, with some dodecamers having a tip-to-opposite tip length of approximately 136 nm (±8.1 nm). Star-shaped oligomer species may contain members with a star-like or star-shaped appearance, consisting of 6 to 20 trimers, each linked through its center to a specific member of the species. Some star-shaped oligomers may have diameters of approximately 140 nm, similar to the diameter of a measured dodecamer.

[0056] In some embodiments, a method for identifying oligomeric species of rhSP-D can include performing asymmetric flow field-flow fractionation combined with multi-angle laser light scattering (AF4-MALLS) analysis on a sample of rhSP-D. AF4-MALLS can provide a fractogram containing a first peak (Peak 1), which may represent rhSP-D trimers and hexamers; a second peak (Peak 2), which may represent rhSP-D dodecamers; a third peak (Peak 3), which may represent an intermediate species between the rhSP-D dodecamers and rhSP-D star-shaped oligomer species; and a fourth peak (Peak 4), which may represent a heterogeneous mass of rhSP-D oligomers with a radius of approximately 70 nm. Other species of rhSP-D with a radius greater than 70 nm, which may include aggregate species, can be identified by AF4-MALLS.

[0057] In some embodiments, methods for identifying oligomeric species of rhSP-D can include performing size-exclusion chromatographic HPLC (SEC HPLC). In some embodiments, methods for identifying oligomeric species of rhSP-D can include performing polyacrylamide gel electrophoresis (PAGE). In some such methods, a sample of rhSP-D can be contacted with an anionic detergent such as SDS, and the sample can be contacted with a cross-linking reagent such as 1% glutaraldehyde to separate the rhSP-D species by size, such as by PAGE. Some methods can also include identifying the rhSP-D species, such as by performing a Western blot.

[0058] kit Some embodiments provided herein include kits. In some embodiments, the kits may include a pharmaceutical composition provided herein. Some embodiments include a sterile container containing a pharmaceutical composition provided herein. Some embodiments include a pharmaceutical composition provided herein in lyophilized form and a sterile reconstitution solution. In some embodiments, the kits may include a device for administering a pharmaceutical composition provided herein, such as an inhaler or nebulizer. [Example]

[0059] Example 1 Lyophilization of certain formulations Lyophilization of certain formulations without rhSP-D was investigated. The primary drying temperatures for lyophilization of various buffer formulations without rhSP-D were determined using subatmospheric differential scanning calorimetry (DSC). The formulations are listed in Table 2. The initial glass transition temperature (Tg'), midpoint Tg', and melting temperature for various formulations without rhSP-D were determined and are summarized in Table 3.

[0060] [Table 2]

[0061] [Table 3]

[0062] Formulations without rhSP-D and containing NaCl had detectable first glass transition temperatures (Tg'). The melting temperatures for formulations F01-01, F02-01, F06-01, and F07-01 were within the eutectic temperature range of NaCl. Lactose and lactose / NaCl have Tg' > -37°C. Therefore, a primary drying temperature of -35°C was feasible. Formulations without rhSP-D were lyophilized under the conditions shown in TABLE 4.

[0063] [Table 4]

[0064] A comparison of capacitance and Pirani manometers was used to determine completion of primary drying. The water content of the rhSP-D-free lyophilized formulations was determined by thermogravimetric analysis (TGA). The results are summarized in Table 5.

[0065] [Table 5]

[0066] Example 2 Preparation and analysis of formulations containing rhSP-D Formulations containing rhSP-D were prepared by dialysis of a 1 mg / ml SP-D solution with the specific buffers listed in Table 2. rhSP-D was obtained from H9D8WT Clone P1315-2A5 (Glycotope), a human myeloid leukemia cell line that expresses rhSP-D from an integrated transgene. Examples of expression vectors, rhSP-D polypeptides, cell lines, and methods for purifying rhSP-D from such cells are provided in U.S. Patent Application Publication Nos. 2019 / 0071693 and 2019 / 0071694, each of which is expressly incorporated herein by reference in its entirety. The pH and osmolality of the rhSP-D-containing formulations were determined to be substantially the same as those of the initial buffer, confirming that dialysis was substantially complete. The rhSP-D-containing formulations were lyophilized and stored at various temperatures, including 5°C, 25°C, and 40°C, for various periods of time. The protein concentrations of the reconstituted formulations were determined and the results are summarized in TABLE 6.

[0067] [Table 6]

[0068] The turbidity of the reconstituted formulations containing rhSP-D was determined by measuring the absorbance at 320 nm, and the results are summarized in TABLE 7.

[0069] [Table 7]

[0070] The turbidity of the protein solution was an indicator of protein precipitation. The low turbidity of F04-01 was likely due to its particularly low protein concentration. Formulations F03-01 and F05-01, each containing lactose and calcium, each had relatively low turbidity. In contrast, formulations F01-01, F02-01, F06-01, and F07-01, each containing NaCl and lacking lactose, were more than five times more turbid than F03-01 and F05-01 after 2 weeks of lyophilizate storage at 25°C. Thus, the presence of lactose and calcium, and the absence of NaCl, may promote the ability of rhSP-D to form a solution after reconstitution of the lyophilizate.

[0071] The distribution of different oligomeric species of rhSP-D in various reconstituted formulations containing rhSP-D was determined by asymmetric flow field-flow fractionation (AF4) analysis combined with multi-angle laser light scattering (AF4-MALLS). Exemplary methods are also provided in U.S. Provisional Patent Application No. 62 / 650,138, filed March 29, 2018, entitled "METHODS FOR CHARACTERIZING SURFACTANT PROTEIN D (SP-D) OLIGOMERS," which is incorporated herein by reference in its entirety. For AF4-MALLS analysis, rhSP-D samples were separated using an AF4 system (Eclipse Dual Tec, Wyatt Technology Corp., Santa Barbara, CA), followed by UV (Ultimate 3000 variable wavelength detector, Dionex Corporation, Sunnyvale, CA) and MALS analysis (Dawn Heleos II detector, Wyatt Technology Corp., Santa Barbara, CA). A Dionex Ultimate 3000 HPLC system (Dionex Corporation, Sunnyvale, CA) was used to inject samples into the AF4 system and deliver the mobile phase. The AF4 configuration used a short channel with a 350 μm thick spacer (Wyatt Technology Corp., Santa Barbara, CA). Data analysis and calculations were performed using Chromeleon (Dionex Corporation, Sunnyvale, CA) and Astra (Wyatt Technology Corp., Santa Barbara, CA) software. Results from the AF4-MALLS analysis included a fractogram with several peaks. TABLE 8 lists the parameters used for AF4.

[0072] [Table 8]

[0073] Data using AF4-MALLS were analyzed to determine the absolute molar mass and size of rhSP-D at specific times during elution. The size:mass ratio indicates the shape of rhSP-D. From the size-to-mass ratio, it was determined that rhSP-D molecules have a linear or rod-like structure during the early stages of elution (0–34 min). For the rod model calculations, the software assumed that the thickness of the rod-like particles was insignificant (0.0 nm) compared to their length. When the thickness was significant, the thickness or approximate thickness in nm was used. The rod thickness was estimated from the AFM data, and the rod length was determined to be consistent with the AFM measurement of 136 ± 8.1 nm. (Arroyo R et al. (2017) Biophysical Journal 112 (3): 503a, incorporated herein by reference in its entirety.) The later stages of elution for rhSP-D (34–45 min) indicated that a more compact structure was beginning to be observed. For the analysis of these stages of dissolution, a second-order Debye model was used. The second-order Debye model allows for very large (approximately 10 6 It provided better results over a wider range of molar masses, including those greater than 1000 Daltons or RMS radii greater than about 50 nm. For the dodecamer of rhSP-D, the molecular weight was determined to be 520.09 ± 4.61 kDa (N = 72 determinations).

[0074] The first peak in the fractogram (Peak 1) contained rhSP-D trimers and hexamers based on mass calculations using a stick model. The second peak in the fractogram (Peak 2) contained rhSP-D dodecamers. The third peak in the fractogram (Peak 3) contained an intermediate species between rhSP-D dodecamers and rhSP-D star oligomers based on the intermediate molecular weight as determined by a stick model. The fourth peak in the fractogram (Peak 4) contained a heterogeneous mass of rhSP-D oligomers with a constant RMS radius of approximately 70 nm, consistent with that observed by AFM measurements for star oligomer species, and larger species with radii greater than 70 nm. Beyond 36 min in the fractogram, the RMS radius further increased, indicating additional aggregate species. The relative peak area (RPA) for each peak was determined as a percentage of the total peak area of ​​the four peaks.

[0075] To determine the distribution of oligomeric species in solution using AF4-MALLS analysis at higher resolution, the species in Peak 4 were further analyzed. Adjusted RPAs were determined for each of Peak 3 and Peak 4. Specifically, aspects of the RPA of Peak 4 corresponding to star-shaped oligomeric species with a constant RMS radius of approximately 70 nm were determined, and these aspects were subtracted from the RPA of Peak 4 to provide the adjusted RPAs for Peak 4 and Peak 3, in addition to the RPA of Peak 3. Thus, the RPA of Peak 1, the RPA of Peak 2, the adjusted RPA of Peak 3, and the adjusted RPA of Peak 4 corresponded to the relative distributions of rhSP-D oligomeric species in the AF4-MALLS analysis for (1) trimers and hexamers, (2) dodecamers, (3) star-shaped oligomeric species with a radius of approximately 70 nm, and (4) aggregates with a radius greater than 70 nm, respectively.

[0076] Lyophilizates of formulations containing rhSP-D were stored under various conditions, reconstituted, and analyzed by AF4-MALLS. The results of the adjusted AF4-MALLS analysis are summarized in Table 9. The molecular weights of oligomeric species of rhSP-D were also determined for formulations stored at 5°C for 5 weeks and are summarized in Table 10.

[0077] [Table 9A]

[0078] [Table 9B]

[0079] [Table 9C]

[0080] [Table 9D]

[0081] [Table 10]

[0082] At week 0 and after 2 weeks of lyophilizate storage at 25°C, each formulation had a substantially similar distribution of RPA, indicating a substantially similar distribution of rhSP-D oligomeric species.

[0083] For adjusted peak 4, representing aggregate oligomeric species of rhSP-D with a radius greater than 70 nm, formulation F06-01 was observed to produce the largest increase, with an adjusted RPA of 5.96% at week 0 to 10.23% after 2 weeks of lyophilizate storage at 40° C. For peak 2, representing the rhSP-D dodecamer, formulations F01-01, F03-01, and F05-01, respectively, showed either an increase or no substantial change in RPA for peak 2 between week 0 and 2 weeks of lyophilizate storage at 40° C. In contrast, formulations F02-01, F04-01, F06-01, and F07-01, respectively, showed a decrease in RPA for peak 2 between week 0 and 2 weeks of lyophilizate storage at 40° C. The stability of rhSP-D dodecamer in lyophilized cakes was greater over time under all conditions tested in formulations containing lactose and calcium, including F03-01 and F05-01, which were also formulations that exhibited relatively low turbidity upon reconstitution, as shown herein.

[0084] Formulation F04-01 had the lowest adjusted RPA for peak 4, indicating the lowest protein concentration and the lowest relative proportion of aggregated rhSP-D species, suggesting that protein concentration may play a role in aggregate formation.

[0085] Example 3 Activity of formulations containing rhSP-D in bacterial agglutination assays The activity of reconstituted formulations containing rhSP-D was tested in a bacterial aggregation assay. In the bacterial aggregation assay, active rhSP-D aggregates bacterial cells, decreasing absorbance / increasing transmission through the bacterial suspension. The bacterial aggregation assay was performed in a manner substantially similar to the following: E. coli (ATCC: Y1088) was streaked onto a bacterial agar plate and incubated overnight at 37°C. A single colony was selected and used to inoculate an overnight culture, which was shaken overnight at 37°C. 1 mL of bacterial culture was pipetted into four 1.5 mL centrifuge tubes and centrifuged at 4,000 rpm for 5 minutes. The supernatant was discarded, and the pellet was resuspended in 1 mL buffer (150 mM HEPES, 20 mM NaCl pH 7.4). The tube was centrifuged at 4,000 rpm for 5 minutes, and the pellet was resuspended in 7 mL of buffer. The absorbance of the bacterial suspension was measured at 700 nm in a spectrophotometer. The bacterial suspension was adjusted to obtain an absorbance range of 1.0000–1.1000. 1 M CaCl2 was added to the suspension to obtain a final concentration of 5 mM CaCl2. Dilutions of rhSP-D in placebo buffer (total volume of 15 μl for each dilution) were made at concentrations of 5 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.1 μg / ml, and 0 μg / ml, and each was added to a cuvette containing 20 μL of HEPES-NaCl buffer. 600 μL of the bacterial suspension was then added to the cuvette, and the absorbance was measured at 700 nm for each cuvette every 2.5 minutes for a total of 120 minutes.

[0086] Test concentrations of reconstituted rhSP-D included 0 μg / ml, 0.1 μg / ml, 0.25 μg / ml, 0.5 μg / ml, 1.0 μg / ml, and 5.0 μg / ml. The percentage mean aggregation at 60 minutes was calculated from the absorbance values ​​at 60 minutes for each test concentration according to the following formula: (1-abs)*100=% agglomeration where 1 = measured absorbance of E. coli suspension without rhSP-D. abs = absorbance value of E. coli suspension + rhSP-D at 60 minutes.

[0087] Percentage (%) aggregation values ​​were averaged from three replicates and imported into GraphPad Prism v7.0c (GraphPad, La Jolla, CA 92037) along with the standard deviation. Averaged values ​​were fitted using a four-parameter logistic curve. EC 50 The resulting values ​​for pEC and span were determined for each reconstituted rhSP-D sample. 50 is an EC 50 -Log 10 is.

[0088] Example results are presented in TABLE 11. Formulations containing rhSP-D had substantially similar potency and efficacy (curve span) in the bacterial agglutination assay before and after lyophilization, indicating that lyophilization did not substantially affect the activity of the formulations tested in this assay. In addition, the formulations had substantially similar potency and efficacy under all conditions tested, indicating that although the stability of the dodecamer was affected under different conditions in some formulations, this did not appear to impair activity in the bacterial agglutination assay. See TABLE 11.

[0089] Example 4 Activity of formulations containing rhSP-D in the TLR4 assay Toll-like receptors (TLRs) play a role in both the innate and adaptive immune systems, and SP-D has the activity of regulating signaling through TLRs, such as Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4). See, e.g., Haagsman HP et al. (2008) Neonatology 93:288-294; Yamazoe M. et al. (2008) J. Biol Chem 283:35878-35888; and Vieira F. et al. (2017) Ann Anat 211:184-201 (each of which is incorporated herein by reference in its entirety). TLR4 activity may also regulate the severity of conditions such as bronchopulmonary dysplasia (BPD) (Malash AH et al. (2016) Gene 592:23-28, incorporated herein by reference in its entirety). Therefore, the activity of rhSP-D to modulate TLR4 activity was measured as an indicator of the effect of rhSP-D on the host immune response. The activity of reconstituted preparations containing rhSP-D was tested in an LPS-TLR4 assay. In the LPS-TLR4 assay, reconstituted rhSP-D can inhibit lipopolysaccharide (LPS)-induced inflammatory cell responses by preventing LPS from binding to / activating Toll-like receptor 4 (TLR4). See, for example, Yamazoe M. et al. (2008) J. Biol Chem 283:35878-35888, which is incorporated herein by reference in its entirety.

[0090] The activity of reconstituted rhSP-D in the TLR4 assay was tested in a manner substantially similar to the following: HEK-Blue™ hTLR4 cells (InvivoGen, San Diego, CA, USA) were plated in 384-well plates at a density of approximately 20,000 cells / well and incubated with various concentrations of rhSP-D for 2 hours at 37°C, 5% CO2. 80LPS (Escherichia coli O26:B6, L5543 Sigma-Aldrich) at a concentration of 1000 mg / mL was added to each well, and the cells were incubated for an additional 22 hours at 37°C and 5% CO2. TLR4 activity was measured by detaching the cells from the wells, washing the suspended cells, resuspending the cells in PBS, and gently pipetting to remove any clumps. 20 e 10 LPS of the washed cells were transferred to a 384-well plate containing HEK Blue Detection Medium (InvivoGen, San Diego, CA, USA) made up in endotoxin-free water containing 5 mM CaCl2 and 1% (v / v) BSA. 3 Cells were plated at a density of 100 cells / well. Cells were incubated at 37°C in 5% CO2 for 24 hours, and TLR4 activity was determined by measuring the activity of the secreted embryonic alkaline phosphatase (SEAP) receptor gene using a spectrophotometer at 655 nm. IC for rhSP-D 50 IC values ​​were determined by fitting the data to a four-parameter logistic equation using nonlinear regression analysis. 50 Only the logarithm of the values ​​is normally distributed, so with the goal of averaging the numbers from a series of experiments, we use -Log 10 (I C 50 ) defined as pIC 50 The curve span was calculated from the fitted maximum response (E max ) and the fitted minimum response (E min ) and corresponds to the amplitude of the dose-response curve or the potency of the response.

[0091] The results of the TLR4 assay for reconstituted rhSP-D, along with the results of the bacterial agglutination assay described in Example 3, are summarized in TABLE 11. The data presented in TABLE 11 for the bacterial agglutination assay include fitted curve parameters from a single dose-response curve composed of triplicate determinations obtained from the 60-minute time point in three independent experiments. The data presented for the TLR4 assay include the mean ± SD of fitted curve parameters obtained from the indicated number of independent experiments, with each dose-response curve composed of multiple concentrations performed in triplicate.

[0092] [Table 11A]

[0093] [Table 11B]

[0094] [Table 11C]

[0095] All tested formulations were active in the bacterial agglutination assay. In contrast, only a subset of formulations processed under various conditions were active in the TLR4 assay. For F01-01, F02-01, and F06-01, results in the TLR4 assay could not be determined for reconstituted lyophilizates after 2 weeks of storage at 40°C. For F01-01 and F02-1, no response was obtained in the TLR4 assay after 2 weeks of storage at either 25°C or 40°C. For F06-01, no response was obtained in the TLR4 assay under either condition. In contrast, formulations F03-01, F05-01, and F07-01 were active in the TLR4 assay for reconstituted lyophilizates after 2 weeks of storage at 40°C. Furthermore, unlike formulation F07-01, formulations F03-01 and F05-01, each containing lactose and calcium, were active in the TLR4 assay for reconstituted lyophilizates after 5 weeks of storage at 5°C. Notably, as shown in Example 2, F03-01 and F05-01 were also the only formulations that maintained dodecamer and aggregate stability under all conditions tested and exhibited relatively low turbidity upon reconstitution. Taken together, the data indicated that formulations containing lactose and CaCl2 exhibited a better ability to stabilize the SP-D oligomeric state and maintain activity than formulations containing NaCl. Therefore, formulations containing lactose and CaCl2 were selected for further study.

[0096] Example 5 Multivariate analysis of rhSP-D oligomer types Multivariate statistical analysis was performed to determine any correlation between the peaks observed in the fractograms in the AF4-MALLS analysis of various formulations of rhSP-D and the activity of the rhSP-D samples in either the bacterial agglutination assay or the TLR4 assay. Digitized fractograms from more than 40 different samples were used as a data matrix to compare the measured pIC from the TLR4 assay. 50 values ​​or pEC from bacterial agglutination activity assays50 Correlation with the results was determined using PLS.

[0097] Full cross-validation was performed on all calibration models using standard techniques. See, for example, Katz, MH, "Multivariate Analysis: A Practice Guide for Clinicians." Cambridge University Press, New York, pp. 158-162 (1999); Stahle, L. et al. (1988) "Multivariate data analysis and experimental design in biomedical research." Prog. Med. Chem. 25: pp. 291-338; Wold S. (2001) "PLS-regression: a basic tool of chemometrics." Chem. Intell. Lab. Syst. 58: pp. 109-130 (each of which is incorporated herein by reference in its entirety). Briefly, one sample was removed at a time, the data set was recalibrated, and a new model was constructed. This process was repeated until all of the calibration samples had been removed once and quantified as a validation model. Therefore, the initial set containing all samples was called the calibration set, and the one after cross-validation was called the validation set. A jackknife algorithm was used to determine statistical significance for any factor used to build a partial least squares (PLS) model (Martens, H. et al., (2001) "Multivariate Analysis of Quality: An Introduction", Wiley and Sons, Chichester, UK).

[0098] For various formulations of rhSP-D, correlations between specific peaks observed in AF4-MALLS analysis and activity in bacterial agglutination assays revealed that the majority of activity was present in peaks 1 and 2, with some activity persisting through peak 3. A negative correlation was found between peak 4 and activity in bacterial agglutination assays. For various formulations of rhSP-D, correlations between specific peaks observed in AF4-MALLS analysis and activity in TLR4 assays revealed that activity was almost exclusively localized in peak 2. Thus, the activity of rhSP-D in the TLR4 assay was directly related to the dodecameric species of rhSP-D. Notably, aggregate species with a radius of >70 nm in adjusted peak 4 were not associated with activity in either the bacterial agglutination or TLR4 assays, confirming that this species represents an inactive form of rhSP-D.

[0099] Example 6 Formulation containing rhSP-D, lactose, and CaCl2 The second generation formulations listed in TABLE 12 were prepared from a single batch of rhSP-D by dialysis of the protein solution against formulation buffer. The formulations were lyophilized by a method substantially similar to that described in Example 1 above.

[0100] [Table 12]

[0101] The lyophilizates were subjected to various conditions, reconstituted, and the activity of the reconstituted lyophilizates was examined in a bacterial aggregation assay. Various formulations of rhSP-D were active in the bacterial aggregation assay under the conditions tested for the various conditions. Example results for the activity of F02-02 and F07-02 in the bacterial aggregation assay are summarized in TABLE 13.

[0102] [Table 13]

[0103] The activity of the formulations under various conditions in the TLR4 assay was determined by methods substantially similar to those described herein, and the results are summarized in Table 14.

[0104] The distribution of rhSP-D oligomeric species in the reconstituted lyophilizates was also examined by AF4-MALLS analysis, using a method substantially similar to that described herein. In addition to summarizing the results of the activity of the formulations in the TLR4 assay, TABLE 14 also summarizes the relative peak area (RPA) for peak 2, which corresponds to the relative distribution of rhSP-D dodecamers, and the adjusted RPA for peak 4, which corresponds to the relative distribution of rhSP-D with a radius greater than 70 nm.

[0105] [Table 14A]

[0106] [Table 14B]

[0107] [Table 14C]

[0108] Activity in the TLR4 assay could not be determined for formulations F03-02 and F06-02, each containing tyloxapol, because the formulation buffer itself exhibited an antagonist effect in the TLR4 assay, which is the readout for the TLR4 assay. Formulations F09-02, F10-02, and F11-02 each lost activity in the TLR4 assay after the lyophilizates were stored at 25°C for 10 weeks. Formulations F10-02, F11-02, and F12-02 contained the lowest observed RPA for peak 2. Formulation F07-02 consistently had the highest RPA for peak 2 across all the various conditions studied, along with the lowest RPA for aggregates in peak 4.

[0109] Lyophilizates of formulations F01-02, F02-02, and F07-02 reconstituted in water were consistently active in the TLR4 assay under various storage conditions. These formulations also maintained dodecamer stability upon reconstitution for 72 hours at 2-8°C (TABLE 15). However, the level of dodecamer (peak 2) in the lyophilizates was higher over time for formulations F02-02 and F07-02, and the level of inactive aggregates in the reconstituted solutions was lower for F02-02 and F07-02 than for F01-02 after 72 hours of storage at 2-8°C (TABLE 15).

[0110] Of all the formulations evaluated, only F02-02 and F07-02 demonstrated consistent activity in the TLR4 assay after reconstitution over a 10-week period, regardless of the storage temperature of the lyophilizate, with consistently higher values ​​for peak 2 RPA associated with rhSP-D dodecamers and lower values ​​for adjusted peak 4 RPA associated with rhSP-D aggregates with radii greater than 70 nm. Collectively, the data indicated that rhSP-D stability and activity were maintained in formulations containing at least 1 mM CaCl2 in the absence of added NaCl, in histidine buffer at pH 6 with higher levels of lactose, and when the lyophilizate was reconstituted in water.

[0111] A second AF4-MALLS analysis was performed to examine the stability of the reconstituted formulations. Lyophilizates were subjected to various conditions, reconstituted, and stored at 2-8°C for 72 hours, after which the distribution of rhSP-D oligomer species in the reconstituted lyophilizates was examined by AF4-MALLS analysis using a method substantially similar to that described above. Table 15 lists the various conditions to which the lyophilizates were subjected, and lists the RPA for peak 2 and the adjusted RPA for peak 4, corresponding to the relative distribution of rhSP-D dodecamers and rhSP-D aggregates with a radius greater than 70 nm, respectively.

[0112] Comparison of the RPA for peak 2 in reconstituted samples after 72 hours of storage at 2-8°C (TABLE 15) with the RPA for peak 2 immediately after reconstitution (TABLE 14) revealed that the lyophilizate formulation and reconstitution solution affected duodecamer stability. Lyophilizates of formulations F04-02, F05-02, F08-02, and F10-02 stored at 25°C for 5 weeks, reconstituted in saline, and stored at 2-8°C for 72 hours all showed a >5% increase in RPA for peak 2 compared to the RPA for peak 2 stored at 25°C for 5 weeks, reconstituted in saline, and analyzed immediately thereafter. For lyophilizates stored at 5°C for 7.5 weeks, these same formulations resulted in a decrease in RPA for peak 2 compared to the RPA for peak 2 obtained immediately after reconstitution. However, lyophilizates of formulations F02-02 and F07-02 stored at either 25°C for 5 weeks or 5°C for 7.5 weeks, reconstituted in water, and stored at 2-8°C for 72 hours had substantially stable RPA, with <4% change in RPA for peak 2. Thus, reconstitution in saline reduced the stability of rhSP-D, as demonstrated in the AF4-MALLS analysis.

[0113] [Table 15A]

[0114] [Table 15B]

[0115] [Table 15C]

[0116] Example 7 Stability of lyophilized formulations containing rhSP-D, lactose, and CaCl2 Formulations of rhSP-D containing either 2 mg / ml or 4 mg / ml rhSP-D in 5 mM histidine, 265 mM lactose, and 5 mM CaCl, pH 6.0, were prepared from a single batch of rhSP-D by dialysis of the protein solution against buffer. The formulations were lyophilized by a method substantially similar to that described in Example 1 above. The lyophilizates were subjected to various conditions, reconstituted in water, and analyzed. The activity of the reconstituted lyophilizates was measured using bacterial agglutination and TLR4 assays. The distribution of rhSP-D oligomeric species in the reconstituted lyophilizates was also examined by AF4-MALLS analysis, using a method substantially similar to that described herein.

[0117] 6-month study (2mg / ml rhSP-D) A 6-month study was conducted on 2 mg / ml rhSP-D in 5 mM histidine, 265 mM lactose, and 5 mM CaCl, pH 6.0. Example results for the activity of reconstituted formulations in bacterial aggregation assays are summarized in Table 14. Example results for the activity of reconstituted formulations in TLR4 assays are summarized in Table 15. Example results for the distribution of oligomeric species in reconstituted lyophilizates prepared from batches of rhSP-D different from those used in the bacterial aggregation and TLR4 assays in Tables 14 and 15 are depicted in Figure 2. Samples from 3 months at 25°C were kept at 45°C for 1 week. In this study, oligomeric species tended to interconvert between peaks 2 and 3, and between peaks 1 and 2, during the first 3 months. After 3 months, aggregation peak 4 significantly increased. Samples kept at 25°C or 5°C had substantially similar activity and distribution of oligomeric species. As shown in TABLE 15, the antagonist potency in the TLR4 assay at 3 and 6 months for this batch of rhSP-D was similar to that at t0 and within the error range of the assay (data not shown). Taken together, the data from both SP-D batches indicate that samples kept at 25°C for 6 months did not perform substantially worse in the activity assay than those kept at 5°C for 6 months, and potency in the activity assay was maintained.

[0118] [Table 16]

[0119] [Table 17]

[0120] 3-month study (2mg / ml and 4mg / ml rhSP-D) A 3-month study was conducted with 2 mg / ml or 4 mg / ml rhSP-D in 5 mM histidine, 265 mM lactose, and 5 mM CaCl, pH 6.0. Example results for the activity of reconstituted formulations in the bacterial clumping assay are summarized in Table 16. Example results for the activity of reconstituted formulations in the TLR4 assay are summarized in Table 17. Example results for the distribution of oligomeric species in the reconstituted lyophilizates are depicted in Figure 3 (upper panel: 2 mg / ml; lower panel: 4 mg / ml). In this study, samples held at 25°C or 5°C had substantially similar activity and distribution of oligomeric species. Interconversion to lower-order (Peak 1) species was not observed. At 3 months, Peak 2 tended to interconvert to Peak 3. Activity was maintained in both the bacterial agglutination and TLR4 assays, regardless of storage temperature, as evidenced by similar potency values ​​(within the error range of the assays) over the entire 3-month period.

[0121] [Table 18]

[0122] [Table 19]

[0123] Example 8 Stability of formulations during freeze / thaw cycles Formulations of rhSP-D were prepared containing either 2 mg / ml or 4 mg / ml rhSP-D in 5 mM histidine, 265 mM lactose, and 5 mM CaCl, pH 6.0. Samples were aliquoted and frozen either by quick freezing, in which the samples were immersed in liquid nitrogen, or by placing the samples in a -80°C freezer. Freeze / thaw cycles involved freezing an aliquot and thawing it at room temperature. Samples were analyzed over several cycles, including t0, 1x, 3x, and 5x.

[0124] Example results for the distribution of oligomeric species in the reconstituted lyophilizates are depicted in Figure 4 (upper panel: 2 mg / ml; lower panel: 4 mg / ml). Example results for the activity of samples in the bacterial agglutination assay or TLR4 assay are summarized in Table 18 and Table 19, respectively. In this study, there was no significant difference between the 2 mg / ml and 4 mg / ml samples. Freezing samples at -80°C for five cycles increased peak 4 aggregates for both the 2 mg / ml and 4 mg / ml samples compared to quick freezing for five cycles. Therefore, quick freezing was the best freezing condition.

[0125] [Table 20]

[0126] [Table 21]

[0127] Example 9 Stability of reconstituted formulations Formulations of rhSP-D containing rhSP-D, 5 mM histidine, 265 mM lactose, and 5 mM CaCl, pH 6.0, were prepared. The solutions were lyophilized and reconstituted at either 2 mg / ml or 4 mg / ml rhSP-D, then kept at t0, 2-8°C for 1 or 3 days, or at 25°C for 1 or 3 days.

[0128] Example results for the distribution of oligomeric species in reconstituted lyophilizates are depicted in Figure 5 (upper panel: 2 mg / ml; lower panel: 4 mg / ml). Example results for the activity of samples in the TLR4 assay are summarized in Table 20. In this study, the distribution of oligomeric species was substantially stable under various conditions for the 2 mg / ml and 4 mg / ml formulations. There was no substantial difference between the distribution of oligomeric species for formulations held at 5°C and 25°C for 3 days. Thus, reconstituted rhSP-D was stable at both 5°C and 25°C for 3 days. Table 20 shows that the potency of rhSP-D in reconstituted samples did not change when the reconstituted samples were held at either 4°C or 25°C for up to 3 days, providing further evidence that the physical stability of reconstituted formulations translates into retention of biological activity.

[0129] [Table 22]

[0130] Example 10 Chemical stability of freeze-dried formulations The stability of lyophilized SP-D samples was evaluated using a RP HPLC assay. Twelve different formulations were prepared. The RP HPLC assay involved the use of an Agilent PLRP column, 300 Å, 3 μm, 2.1 × 150 mm (PL1912-3301). The first gradient (Gradient 1) was fairly shallow (1.7% B per minute to 6% B per minute after 6 minutes). The result was the detection of a major peak for the reduced sample, as well as minor peaks eluting both before and after the major peak.

[0131] All of the samples showed a purity close to 95% at t when the reduced samples were analyzed (TABLE 23After 5 weeks of storage at 25°C, there was no substantial change in the RP HPLC profiles for the majority of the samples; F11-02 and F12-02, both of which contained NaCl, showed the greatest change (TABLE 23). 24 (Table 24)). TABLE 2 3 Table 23 lists the relative areas of the peaks at t0 in the RP HPLC chromatograms of the reduced SP-D samples as formulations. 4 Table 24 lists the relative areas of the peaks at t5 (5 weeks at 25°C) in the RP HPLC chromatograms of the reduced SP-D samples as formulated.

[0132] [Table 23]

[0133] [Table 24]

[0134] As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0135] The above description discloses some methods and materials of the present invention. This invention is susceptible to modifications of the methods and materials, as well as changes in fabrication techniques and equipment. Such modifications will become apparent to those skilled in the art from consideration of this disclosure or practice of the invention disclosed herein. It is therefore not intended that this invention be limited to the particular embodiments disclosed herein, but rather that this invention cover all modifications and alternatives falling within the true scope and spirit of the invention.

[0136] All references cited herein, including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent that the publications and patents or patent applications incorporated by reference conflict with the present disclosure contained herein, it is intended that the present specification supersede and / or supersede any such conflicting material.

Claims

1. A pharmaceutical composition comprising recombinant human surfactant protein D (rhSP-D) or an active fragment thereof, a histidine buffer, a sugar, and a calcium salt.

2. the concentration of histidine is from about 0.1 mM to about 100 mM, optionally from about 1 mM to about 10 mM, optionally from about 3 mM to about 7 mM, and optionally about 5 mM; 10. The pharmaceutical composition of claim 1.

3. 3. The pharmaceutical composition of claim 1, wherein the sugar is selected from the group consisting of sucrose, maltose, lactose, glucose, fructose, galactose, mannose, arabinose, xylose, ribose, rhamnose, trehalose, sorbose, melezitose, raffinose, thioglucose, thiomannose, thiofructose, octa-O-acetyl-thiotrehalose, thiosucrose, and thiomaltose.

4. 4. The pharmaceutical composition of claim 3, wherein the sugar is lactose.

5. 5. The pharmaceutical composition of claim 4, wherein the concentration of lactose is from 1 mM to 500 mM, optionally from 200 mM to 300 mM, and optionally about 265 mM.

6. 6. The pharmaceutical composition of claim 1, wherein the calcium salt is selected from the group consisting of calcium chloride, calcium bromide, calcium acetate, calcium sulfate, and calcium citrate.

7. 7. The pharmaceutical composition of claim 6, wherein the calcium salt is calcium chloride.

8. 8. The pharmaceutical composition of claim 7, wherein the concentration of calcium chloride is from about 0.1 mM to about 10 mM, optionally from about 1 mM to about 10 mM, optionally from about 3 mM to about 7 mM, and optionally about 5 mM.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, having a pH of about 4.0 to about 9.0, optionally having a pH of about 5.0 to about 7.0, optionally having a pH of about 5.5 to about 6.5, and optionally having a pH of about 6.

0.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein the concentration of rhSP-D is from about 0.1 mg / ml to about 10 mg / ml, optionally from about 1 mg / ml to about 6 mg / ml, optionally from about 3 mg / ml to about 5 mg / ml, optionally about 4 mg / ml, optionally from about 1 mg / ml to about 3 mg / ml, and optionally about 2 mg / ml.

11. 11. A pharmaceutical composition according to any one of claims 1 to 10, comprising a population of rhSP-D polypeptides having oligomeric forms, wherein more than 30% of the oligomeric forms comprise a dodecamer of rhSP-D, optionally more than 35% of the oligomeric forms comprise a dodecamer of rhSP-D, and optionally more than 40% of the oligomeric forms comprise a dodecamer of rhSP-D.

12. 12. A pharmaceutical composition according to any one of claims 1 to 11, comprising a population of rhSP-D polypeptides having oligomeric forms, wherein less than 15% of the oligomeric forms comprise aggregates of rhSP-D, the aggregates having a radius greater than 70 nm, optionally less than 10% of the oligomeric forms comprise aggregates of rhSP-D, the aggregates having a radius greater than 70 nm, and optionally less than 5% of the oligomeric forms comprise aggregates of rhSP-D, the aggregates having a radius greater than 70 nm.

13. 13. The pharmaceutical composition of any one of claims 1 to 12, further comprising a bulking agent.

14. 14. The pharmaceutical composition of claim 13, wherein the bulking agent is selected from the group consisting of mannitol, xylitol, sorbitol, maltitol, lactitol, glycerol, erythritol, arabitol, glycine, alanine, threonine, valine, and phenylalanine.

15. 15. The pharmaceutical composition of any one of claims 1 to 14, which lacks a chelating agent.

16. 16. The pharmaceutical composition of claim 15, wherein the chelating agent is selected from EDTA and EGTA.

17. A pharmaceutical composition comprising 1 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, and having a pH of 6.0; A pharmaceutical composition comprising 2 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, and having a pH of 6.0; A pharmaceutical composition comprising 2 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 1 mM calcium chloride, and having a pH of 6.0; and A pharmaceutical composition comprising 4 mg / ml rhSP-D, 5 mM histidine, 265 mM lactose, 5 mM calcium chloride, and having a pH of 6.0; 2. The pharmaceutical composition of claim 1, wherein the compound is selected from the group consisting of:

18. 18. The pharmaceutical composition of any one of claims 1 to 17, wherein the rhSP-D polypeptide comprises the amino acid sequence of SEQ ID NO: 02 or an active fragment thereof.

19. 19. The pharmaceutical composition of claim 18, wherein rhSP-D or an active fragment thereof comprises a residue at a polymorphic position corresponding to a residue selected from the group consisting of Met 11, Thr 160, Ser 270, and Ala 286.

20. 20. The pharmaceutical composition of any one of claims 1 to 19, wherein the rhSP-D polypeptide or an active fragment thereof has activity in a bacterial agglutination assay or a TLR4 inhibition assay.

21. A lyophilisate of the pharmaceutical composition according to any one of claims 1 to 20.

22. 22. An article comprising a container containing a lyophilisate of the pharmaceutical composition of any one of claims 1 to 21.

23. 22. A method for preparing a lyophilisate, comprising the step of lyophilising a pharmaceutical composition according to any one of claims 1 to 21.

24. A method for preparing a pharmaceutical composition, comprising the step of contacting a lyophilized product of the pharmaceutical composition of any one of claims 1 to 21 with a pharmaceutically acceptable diluent to form a solution or suspension of rhSP-D or an active fragment thereof.

25. 25. The method of claim 24, wherein the diluent is suitable for pulmonary administration.

26. 26. The method of claim 24 or 25, wherein the diluent is sterile water.

27. the solution or suspension has a concentration of rhSP-D or an active fragment thereof of about 0.1 mg / ml to about 10 mg / ml, optionally about 1 mg / ml to about 6 mg / ml, optionally about 3 mg / ml to about 5 mg / ml, optionally about 4 mg / ml, optionally about 1 mg / ml to about 3 mg / ml, and optionally about 2 mg / ml; 27. The method of any one of claims 24 to 26.

28. 28. The method of any one of claims 24 to 27, wherein the solution or suspension comprises a population of rhSP-D polypeptides in oligomeric form.

29. The method of claim 28, wherein more than 30% of the oligomeric forms comprise a dodecamer of rhSP-D, optionally more than 35% of the oligomeric forms comprise a dodecamer of rhSP-D, optionally more than 40% of the oligomeric forms comprise a dodecamer of rhSP-D, optionally more than 45% of the oligomeric forms comprise a dodecamer of rhSP-D.

30. 30. The method of claim 28 or 29, wherein less than 10% of the oligomeric forms comprise aggregates of rhSP-D, said aggregates having a radius greater than 70 nm, optionally less than 5% of the oligomeric forms comprise aggregates of rhSP-D, said aggregates having a radius greater than 70 nm, optionally less than 3% of the oligomeric forms comprise aggregates of rhSP-D, said aggregates having a radius greater than 70 nm, and optionally less than about 3% by weight of the rhSP-D oligomeric species in the solution or suspension are high molecular weight oligomeric forms having a radius greater than 70 nm.

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