Systems and methods for characterizing surfactant protein D (SP-D) oligomers

Abstract

Some embodiments of the methods and compositions provided herein include identifying and / or quantifying oligomeric species of surfactant protein-D (SP-D), and some embodiments include performing asymmetric flow field-flow fractionation with multi-angle laser light scattering (AF4-MALLS) analysis of SP-D.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 650,138, filed March 29, 2018, entitled "METHODS FOR CHARACTERIZING SURFACTANT PROTEIN D (SP-D) OLIGOMERS," the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing Reference This application is filed with an electronic Sequence Listing. The Sequence Listing is provided as a file named AIRWY013WOSEQLIST, created on March 20, 2019, and is approximately 7 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] FIELD OF THE INVENTION Some embodiments of the methods and compositions provided herein include identifying and / or quantifying oligomeric species of surfactant protein-D (SP-D), and some embodiments include performing asymmetric flow field-flow fractionation with multi-angle laser light scattering (AF4-MALLS) analysis of SP-D. [Background technology]

[0004] Mammalian pulmonary surfactant is a mixture of protein (10%) and lipids (90%), with dipalmitoylphosphatidylcholine as the major lipid component (Zuo YY et al., Biochim Biophys Acta (2008) 1778:1947-77). The primary function of pulmonary surfactant is to ensure a minimum surface tension in 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). Therefore, 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 caused by structural lung immaturity in premature infants, making breathing difficult, impairing gas exchange, and promoting alveolar collapse (Notter RH. 2000 Lung Surfactants. Basic Science and Clinical Applications. New York, NY: Marcel Dekker). However, current surfactant preparations lack surfactant protein D (SP-D), making treatment more difficult when the lung is infected or there are inflammatory or oxidative complications. Therefore, successful treatment of complex pulmonary diseases requires the production 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] SP-D plays a role in the pulmonary innate immune system by providing anti-inflammatory and antibacterial activities that address chronic lung diseases such as asthma, cystic fibrosis, and smoking-induced emphysema (Clark H et al., Immunobiology (2002) 205:619-31). Data based on premature lambs suggests that administration of approximately 2-3 mg / kg of recombinant human SP-D in combination with 100 mg / kg of Survanta® (a natural surfactant available in the United States) is more effective than Survanta® alone for preventing endotoxic shock and reducing ventilation-induced lung inflammation (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).

[0006] Traditionally, SP-D has been isolated from the supernatant of bronchoalveolar lavage fluid or amniotic fluid; however, most SP-D is lost during purification, due in part to its hydrophilic nature (Dodagatta-Marri E et al., Methods Mol Biol (2014) 100:273-90). Because current pulmonary surfactant preparations lack the ability to effectively modulate host immune responses in the absence of hydrophilic surfactant proteins, the use of recombinant human surfactant protein D (rhSP-D) to supplement pulmonary surfactant preparations can ensure therapeutic efficacy. Because higher-order multimerization of 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, a characteristic of native SP-D that must be maintained in any pharmaceutical composition is its proper oligomerization state (White M et al., J Immunol (2008) 181:7936-43). A proper oligomerization state is also required for receptor recognition and receptor-mediated signal transduction for modulation of host immune responses (Yamoze M et al., J Biol Chem (2008) 283:35878-35888) and for maintaining surfactant homeostasis (Zhang L et al., J Biol Chem (2001) 276:19214-19219). Low SP-D yields and variable oligomerization states make it difficult to use natural sources for the production of pharmaceutical SP-D (Strong P et al., J Immunol Methods (1998) 220:139-49). To overcome some of these limitations, recombinant SP-D can be produced in microorganisms or mammalian cell lines, potentially providing a large-scale platform for the production of homogeneous recombinant SP-D formulations. However, recombinant SP-D may exhibit variable oligomerization states and / or inactive aggregate forms, thus reducing the potential efficacy of such preparations. [Prior art documents] [Patent documents]

[0007] [License 1] U.S. Patent and Trademark Publication No. 2019 / 0071693 [License 2] U.S. Patent and Trademark Publication No. 2019 / 0071694 [Non-licensed literature]

[0008] [Non-licensed Document 1] Zuo YY et al., 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 [Non-licensed Document 5] Robertson B et al., Biochim Biophys Acta (1998) 1408: 346-61 [Non-licensed Document 6] Clark H et al., Immunobiology (2002) 205: 619-31 [Non-licensed Document 7] Ikegami M et al., Am J Respir Crit Care Med (2006) 173:1342~7 pages [Non-licensed Document 8] Sato A et al., Am J Respir Crit Care Med (2010) 181:1098~105 pages [Non-licensed Document 9] Dodagatta-Marri E et al., Methods Mol Biol (2014) 100:273~90 pages [Non-licensed Document 10] White M et al., J Immunol (2008) 181:7936~43 pages [Non-licensed Document 11] Yamoze M et al., J Biol Chem (2008) 283:35878~35888 pages [Non-licensed Document 12] Zhang L et al., J Biol Chem (2001) 276:19214~19219 pp. [Non-licensed Document 13] Strong P et al., J Immunol Methods (1998) 220: 139-49 [Non-licensed Document 14] Crouch E. et al., (1994) J Biol Chem, 269:17311-9 pages [Non-licensed Document 15] Hakansson K et al., Protein Sci (2000) 9:1607~17 [Non-licensed Document 16] Crouch E. Respir Res (2000) 1: 93-108 [Non-licensed Document 17] Crouch E. et al., (2006) J Biol Chem, 281:18008~14 pages [Non-licensed Document 18] Giddings, JC et al., Science 193: 1244-1245 (1976) [Non-licensed Document 19] Giddings, JC et al., Anal. Chem. 48: 1126-1132 (1976) [Non-licensed Document 20] Wagner et al., (2014) Anal Chem 86:5201-5210 [Non-licensed Document 21] Arroyo R et al., (2017) Biophysical Journal 112 (3):503a [Non-licensed Document 22] P. Debye, "Molecular-weight determination by light scattering", J. Phys. Coll. Chem., vol. 51, pages 18~32 (1947) [Non-licensed Document 23] BH Zimm, J. Chem. Phys., vol. 16, pp. 1093-1099 (1948) [Non-licensed Document 24] HC van de Hulst, Light Scattering by Small Particles, Wiley, New York (1957) [Non-licensed Document 25] Haagsman HP et al., (2008) Neonatology 93:288-294 [Non-licensed Document 26] Yamazoe M. et al., (2008) J. Biol Chem. 283:35878~35888 pages [Non-licensed Document 27] Vieira F. et al., (2017) Ann Anat 211: 184-201 [Non-licensed Document 28] Malash AH et al., (2016) Gene 592: 23-28 [Non-licensed Document 29] Katz, MH "Multivariate Analysis: A Practice Guide for Clinicians." Cambridge University Press, New York, pp. 158~162 (1999) [Non-licensed Document 30] Stahle, L. et al. (1988) "Multivariate data analysis and experimental design in biomedical research. Prog. Med. Chem. 25: 291~338 pages [Non-licensed Document 31] Wold S. (2001) "PLS-regression: a basic tool of chemometrics." Chemom. Intel. Lab. Syst. 58: pp. 109-130 [Non-Patent Document 32] Martens, H. et al. (2001) “Multivariate Analysis of Quality: An Introduction” Wiley and Sons, Chichester, UK Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, there is a need for a method that can be used to distinguish between active oligomeric and inactive aggregated forms and to quantify the oligomeric forms to ensure reproducible quality from the manufacturing process. [Means for solving the problem]

[0010] Some embodiments of the methods and compositions provided herein include methods for determining the activity of a pharmaceutical composition comprising surfactant protein-D (SP-D). Some such embodiments include methods comprising measuring the relative proportion of oligomeric species of SP-D in the pharmaceutical composition and calculating the relative proportion of SP-D dodecamers in the pharmaceutical composition, thereby determining the activity of the pharmaceutical composition. Some embodiments also include calculating the relative proportion of SP-D aggregates in the pharmaceutical composition having a mean radius greater than 70 nm or a root-mean-square (RMS) radius greater than 70 nm. In some embodiments, the measuring step comprises performing asymmetric flow field-flow fractionation using multi-angle laser light scattering (AF4-MALLS) analysis. In some embodiments, the calculating step comprises providing a model of the oligomeric species of SP-D, including rod-like and spherical shapes. In some embodiments, the model comprises a Zimm model and a second-order Debye model of the oligomeric species of SP-D. Some embodiments also include measuring the relative proportion of at least one SP-D oligomer species in the SP-D sample, wherein the SP-D oligomer species is selected from the group consisting of SP-D trimers, SP-D hexamers, SP-D dodecamers, SP-D star-shaped oligomers having a mean radius of 70 nm or an RMS radius of 70 nm, and SP-D aggregates having a mean radius greater than 70 nm or an RMS radius greater than 70 nm.

[0011] Some embodiments of the methods and compositions provided herein include determining the relative proportion of oligomeric species of surfactant protein-D (SP-D) in a sample, including performing asymmetric field-flow fractionation on an SP-D sample using multi-angle laser light scattering (AF4-MALLS) analysis; and determining the relative proportion of oligomeric species of SP-D from the results of the AF4-MALLS analysis, wherein the SP-D oligomeric species are selected from the group consisting of SP-D trimers, SP-D hexamers, SP-D dodecamers, SP-D star-shaped oligomers having a mean radius of 70 nm or an RMS radius of 70 nm, and SP-D aggregates having a mean radius of greater than 70 nm or an RMS radius of greater than 70 nm. Some embodiments also include measuring the relative proportion of SP-D dodecamers in the sample. Some embodiments also include measuring the relative proportion of SP-D aggregates having a mean radius of greater than 70 nm or an RMS radius of greater than 70 nm in the sample.

[0012] Some embodiments of the methods and compositions provided herein include methods for determining the relative proportions of active oligomeric species of surfactant protein-D (SP-D) in a sample, the method comprising: performing asymmetric field-flow fractionation on the SP-D sample using multi-angle laser light scattering (AF4-MALLS) analysis; measuring the relative proportion of SP-D dodecamers in the SP-D sample; measuring the relative proportion of at least one SP-D oligomeric species in the SP-D sample selected from the group consisting of SP-D trimers, SP-D hexamers, SP-D star-shaped oligomers having a mean radius of 70 nm or an RMS radius of 70 nm, and SP-D aggregates having a mean radius of greater than 70 nm or an RMS radius of greater than 70 nm; and calculating the ratio between each of the measured relative proportions. In some embodiments, the at least one SP-D oligomeric species comprises an SP-D aggregate having a mean radius of greater than 70 nm or an RMS radius of greater than 70 nm. Some embodiments also include integrating the fractogram from the AF4-MALLS analysis for each peak. Some embodiments also include measuring the relative peak area (RPA) of the fractogram for at least one peak representing an SP-D oligomer species selected from the group consisting of an SP-D trimer, an SP-D hexamer, an SP-D dodecamer, an SP-D star-shaped oligomer having a mean radius of 70 nm or an RMS radius of 70 nm, and an SP-D aggregate having a mean radius of more than 70 nm or an RMS radius of more than 70 nm. In some embodiments, the SP-D star-shaped oligomer has a molar mass of about 6 MDa or less. In some embodiments, the SP-D aggregate has a molar mass of more than 6 MDa.

[0013] Some embodiments also include measuring the relative proportion of SP-D star-shaped oligomeric species in the sample that have a mean radius of 70 nm or an RMS radius of 70 nm, and some embodiments also include measuring the relative proportion of SP-D aggregates in the sample that have a mean radius greater than 70 nm or an RMS radius greater than 70 nm.

[0014] In some embodiments, an SP-D sample is predicted to have activity in a lipopolysaccharide-Toll-like receptor 4 (LPS-TLR4) assay or a bacterial agglutination assay. In some embodiments, an SP-D sample having a relative proportion of SP-D dodecamers of 35% or more of the SP-D oligomeric species in the sample indicates an active SP-D sample. In some embodiments, an SP-D sample having a relative proportion of SP-D aggregates with a mean radius greater than 70 nm or an RMS radius greater than 70 nm of less than 5% of the total SP-D oligomeric species in the sample indicates an active SP-D sample.

[0015] In some embodiments, AF4-MALLS is performed in the absence of a chelating agent selected from the group consisting of EDTA and EGTA.

[0016] In some embodiments, the SP-D is recombinant human SP-D (rhSP-D). In some embodiments, the rhSP-D is derived from a human myeloid leukemia cell line that expresses rhSP-D from a transgene. In some embodiments, the rhSP-D comprises the amino acid sequence of SEQ ID NO: 02. In some embodiments, the rhSP-D comprises a residue at a polymorphic position corresponding to a residue selected from the group consisting of Met11, Thr160, Ser270, and Ala286.

[0017] In some embodiments, the relative proportions of oligomeric species are in terms of mass. In some embodiments, the relative proportions of oligomeric species of SP-D are in terms of number of molecules. In some embodiments, the relative proportions of oligomeric species are in terms of relative peak area (RPA) or adjusted RPA in AF4-MALLS analysis. In some embodiments, the SP-D dodecamer has a molecular weight of about 520 kDa.

[0018] Some embodiments of the methods and compositions provided herein include an electronic system for determining the activity of a pharmaceutical composition containing surfactant protein-D (SP-D). Some such embodiments include a system comprising a processor having instructions configured to perform the following steps: measuring the relative proportion of oligomeric species of SP-D in the pharmaceutical composition; calculating the relative proportion of SP-D dodecamer in the pharmaceutical composition; and determining the activity of the pharmaceutical composition based on the relative proportion of SP-D dodecamer, wherein a higher relative proportion of SP-D dodecamer indicates a composition having a higher level of activity compared to the activity of a composition having a lower relative proportion of SP-D dodecamer. In some embodiments, the activity includes activity in a toll-like receptor 4 (TLR4) assay or a bacterial agglutination assay. Some embodiments also include calculating the relative proportion of SP-D aggregates in the pharmaceutical composition having a mean radius greater than 70 nm or an RMS radius greater than 70 nm. In some embodiments, the measuring step includes performing asymmetric field-flow fractionation using multi-angle laser light scattering (AF4-MALLS) analysis. In some embodiments, the calculating step includes providing models of SP-D oligomeric species that include rod-like and spherical shapes. In some embodiments, the models include a Zimm model and a second-order Debye model of SP-D oligomeric species. Some embodiments also include measuring the relative proportion of at least one SP-D oligomeric species in the SP-D sample, wherein the SP-D oligomeric species is selected from the group consisting of SP-D trimers, SP-D hexamers, SP-D dodecamers, SP-D star-shaped oligomers with a mean radius of 70 nm or an RMS radius of 70 nm, and SP-D aggregates with a mean radius of greater than 70 nm or an RMS radius of greater than 70 nm.

[0019] Some embodiments of the methods and compositions provided herein include an electronic system for determining the relative proportions of surfactant protein-D (SP-D) oligomeric species in a sample. Some such embodiments include a system including a processor having instructions configured to perform the following steps: performing asymmetric flow field-flow fractionation using multi-angle laser light scattering (AF4-MALLS) analysis on an SP-D sample; and determining the relative proportions of SP-D oligomeric species from the results of the AF4-MALLS analysis, wherein the SP-D oligomeric species are selected from the group consisting of SP-D trimers, SP-D hexamers, SP-D dodecamers, SP-D star-shaped oligomers with a mean radius of 70 nm or an RMS radius of 70 nm, and SP-D aggregates with a mean radius of greater than 70 nm or an RMS radius of greater than 70 nm. Some embodiments also include measuring the relative proportions of SP-D dodecamers in the sample. Some embodiments also include determining the relative proportion of SP-D aggregates in the sample that have a mean radius greater than 70 nm or an RMS radius greater than 70 nm.

[0020] Some embodiments of the methods and compositions provided herein include an electronic system for determining the relative proportions of active oligomeric species of surfactant protein-D (SP-D) in a sample. Some such embodiments include a system comprising a processor having instructions configured to perform the following steps: performing asymmetric flow field-flow fractionation using multi-angle laser light scattering (AF4-MALLS) analysis on the SP-D sample; measuring the relative proportion of SP-D dodecamers in the SP-D sample; measuring the relative proportion of at least one SP-D oligomeric species in the SP-D sample selected from the group consisting of SP-D trimers, SP-D hexamers, SP-D star-shaped oligomers having a mean radius or RMS radius of 70 nm, and SP-D aggregates having a mean radius or RMS radius greater than 70 nm; and calculating the ratio between each of the measured relative proportions. In some embodiments, the at least one SP-D oligomeric species comprises an SP-D aggregate having a mean radius or RMS radius greater than 70 nm. Some embodiments also include integrating the fractogram from the AF4-MALLS analysis for each peak. Some embodiments also include measuring the relative peak area (RPA) of the fractogram for at least one peak representing an SP-D oligomer species selected from the group consisting of an SP-D trimer, an SP-D hexamer, an SP-D dodecamer, an SP-D star oligomer having a mean radius of 70 nm or an RMS radius of 70 nm, and an SP-D aggregate having a mean radius of greater than 70 nm or an RMS radius of greater than 70 nm. In some embodiments, the SP-D star oligomer has a molar mass of about 6 MDa or less. In some embodiments, the SP-D aggregate has a molar mass greater than 6 MDa. Some embodiments also include measuring the relative proportion of SP-D star oligomer species in the sample having a mean radius of 70 nm or an RMS radius of 70 nm. Some embodiments also include measuring the relative proportion of SP-D aggregates in the sample having a mean radius of greater than 70 nm or an RMS radius of greater than 70 nm.

[0021] In some embodiments, the SP-D sample is predicted to have activity in a lipopolysaccharide-Toll-like receptor 4 (LPS-TLR4) assay or a bacterial agglutination assay.

[0022] In some embodiments, an SP-D sample having a relative proportion of SP-D dodecamer of 35% or greater of the SP-D oligomeric species in the sample indicates an active SP-D sample.

[0023] In some embodiments, an SP-D sample having a relative proportion of SP-D aggregates with an average radius greater than 70 nm or an RMS radius greater than 70 nm of less than 5% of all SP-D oligomeric species in the sample indicates an active SP-D sample.

[0024] In some embodiments, AF4-MALLS is performed in the absence of a chelating agent selected from the group consisting of EDTA and EGTA.

[0025] In some embodiments, the SP-D is recombinant human SP-D (rhSP-D). In some embodiments, the rhSP-D is derived from a human myeloid leukemia cell line that expresses rhSP-D from a transgene. In some embodiments, the rhSP-D comprises the amino acid sequence of SEQ ID NO: 02. In some embodiments, the rhSP-D comprises a residue at a polymorphic position corresponding to a residue selected from the group consisting of Met11, Thr160, Ser270, and Ala286.

[0026] In some embodiments, the relative proportions of oligomeric species are in terms of mass. In some embodiments, the relative proportions of oligomeric species are in terms of relative peak area (RPA) or adjusted RPA in AF4-MALLS analysis. In some embodiments, the SP-D dodecamer has a molecular weight of about 520 kDa. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a schematic diagram showing the formation of SP-D trimers and the structural features of SP-D trimers. DETAILED DESCRIPTION OF THE INVENTION

[0028] Surfactant protein D (SP-D) is a C-type surfactant protein containing four domains: a cysteine-binding 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 through the folding of the collagenous region into a triple helix and the assembly of an α-helical coiled-coil bundle 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 vary 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.

[0029] Some embodiments include systems and methods for resolving SP-D oligomeric species using a variation of the asymmetric flow field-flow fractionation (AF4) technique. Some such embodiments were developed to resolve higher-order SP-D species (dodecamers and larger) into lower-order SP-D species (hexamers and smaller). These embodiments enable the use of multi-angle laser light scattering (MALLS) to clearly delineate the dodecameric form of SP-D within a complex fractogram envelope. Using this variation of the AF4 technique incorporating MALLS, the molar mass of the dodecameric species could be consistently determined to be 521.8 ± 3.6 kDa across the analysis of a large number (n = 102) of samples. As used herein, the term "AF4-MALLS" refers to a method that performs asymmetric flow field-flow fractionation followed by multi-angle laser light scattering to identify oligomeric species in an analyzed mixture. The accuracy of the AF4-MALLS method was calculated to be approximately 5%. Fractograms of SP-D also showed peaks associated with species larger than dodecamers, which appeared by atomic force microscopy (AFM) as star-like species arising from dodecamer and / or hexameric assemblies, as well as aggregates larger than 70 nm in radius. Thus, embodiments using the AF4-MALLS technique were found to provide detailed profiles of the various higher-order oligomeric association states of SP-D, which were found to be accurately and precisely determined.

[0030] Identification of oligomeric species of SP-D A sample of SP-D can contain several oligomeric species of SP-D protein, including trimers, hexamers, dodecamers, and higher-order oligomers, e.g., star-shaped oligomeric species with a root-mean-square (RMS) radius of approximately 70 nm, and aggregates that may have radii greater than 70 nm. As described herein, specific oligomeric species of SP-D are associated with specific useful activities. For example, SP-D dodecamers are associated with antagonist activity in Toll-like receptor 4 (TLR4) assays. The TLR4 assay can measure the activity of rhSP-D to inhibit lipopolysaccharide (LPS)-induced inflammatory cell responses by preventing LPS from binding to and activating the TLR4 complex. The activity of SP-D in assays such as the TLR4 assay can be a useful indicator for predicting the efficacy of SP-D in a particular treatment method. Furthermore, different samples of SP-D can contain different relative distributions of SP-D oligomeric species.

[0031] The distribution of oligomeric forms of recombinant human SP-D (rhSP-D) in a sample can be determined by various techniques. Some embodiments can include identifying the oligomeric species of rhSP-D by performing AFM. Some embodiments can include identifying the oligomeric species of rhSP-D by performing size-exclusion chromatography, such as high-performance liquid chromatography (HPLC). Some embodiments can include identifying the oligomeric species of rhSP-D by performing polyacrylamide gel electrophoresis. In some such methods, a sample of rhSP-D can be contacted with an anionic detergent, such as SDS, followed by contacting the sample with a cross-linking reagent, such as 1% glutardialdehyde. The cross-linked proteins can then be separated by size using techniques such as polyacrylamide gel electrophoresis. Some methods can also include identifying different oligomeric species of rhSP-D, such as by performing a Western blot.

[0032] Some embodiments include performing AF4-MALLS analysis on SP-D samples. AF4 is a type of asymmetric field-flow fractionation that allows for rapid fractionation and high-resolution characterization of various particles, including biomolecules. See, for example, Giddings, JC et al., Science 193:1244-1245 (1976); Giddings, JC et al., Anal. Chem. 48:1126-1132 (1976); and Wagner et al. (2014) Anal Chem 86:5201-5210, each of which is incorporated by reference in its entirety. AF4 can separate particles ranging from a few nanometers to a few micrometers in size. Field-flow fractionation separation occurs in a thin flow channel comparable to a chromatographic separation column. An aqueous or organic solvent carries the sample through this channel. Flow through the channel is laminar due to the low channel height, which is the primary force exerted on the sample. The second force is generated perpendicular to the flow of the channel. In AF4, one side of the flow channel is a membrane, and the second force is the flow of fluid across the channel through the membrane. These two forces result in particle separation in this system. First, the velocity gradient caused by laminar flow within the channel causes particles in the center of the channel to move more quickly along the channel, separating them from particles closer to the sides of the channel. Second, the second force propels the sample toward the membrane. Size separation occurs because smaller molecules diffuse back toward the center of the channel faster than larger particles, thus separating them from larger particles due to the faster solvent flow toward the center of the channel. In some embodiments, AF4-MALLS is performed in the absence of chelating agents such as EDTA and EGTA.

[0033] Some embodiments include determining the relative proportions of active oligomeric species in an SP-D sample, wherein the SP-D oligomeric species are selected from the group consisting of SP-D trimers, SP-D dodecamers, SP-D star oligomers having a mean radius or RMS radius of 70 nm, and SP-D aggregates having a mean radius or RMS radius of greater than 70 nm. In some embodiments, the SP-D star oligomers can have a molar mass of about 6 MDa or less. In some embodiments, the SP-D aggregates have a molar mass greater than 6 MDa.

[0034] Some embodiments also include integrating the fractogram from the AF4-MALLS analysis into peaks. The first peak in the fractogram (Peak 1) can include SP-D trimers and hexamers. The second peak in the fractogram (Peak 2) can include SP-D dodecamers. The third peak in the fractogram (Peak 3) can include intermediate species between SP-D dodecamers and SP-D star-shaped oligomers. The fourth peak in the fractogram (Peak 4) can include heterogeneous mass rhSP-D oligomers with a constant RMS radius of approximately 70 nm, consistent with that observed by AFM measurements for star-shaped oligomer species, and larger SP-D aggregate species with radii greater than 70 nm.

[0035] Some embodiments also include determining or measuring the relative peak area (RPA) for the peaks as a percentage of the total peak area of ​​the measured peaks, such as the four peaks identified above. Some such embodiments include measuring the relative peak area (RPA) of the fractogram for at least one peak indicative of an SP-D oligomer species, such as an SP-D trimer, an SP-D dodecamer, an SP-D star-shaped oligomer with a mean radius or an RMS radius of 70 nm, and an SP-D aggregate with a mean radius greater than 70 nm or an RMS radius greater than 70 nm.

[0036] In some embodiments, an SP-D sample having a relative ratio of SP-D dodecamers of a particular percentage or greater of the SP-D oligomer species in the sample indicates an active SP-D sample, which percentage can be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and any percentage between any two of the foregoing percentages.

[0037] In some embodiments, an SP-D sample having a relative proportion of SP-D aggregates of less than a certain percentage of the SP-D oligomeric species in the sample indicates an active SP-D sample, which percentage can be 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1%, and any percentage between any two of the aforementioned percentages.

[0038] In some embodiments, the relative proportions of SP-D oligomeric species can be in terms of mass, number of molecules / aggregates in a population of different oligomeric species of SP-D, or relative peak area (RPA) in AF4-MALLS analysis.

[0039] In some embodiments, SP-D comprises a wild-type human SP-D polypeptide. In some embodiments, SP-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 can include residue 11, ATG (Met) to ACG (Thr); residue 25, AGT (Ser) to AGC (Ser); residue 160, ACA (Thr) to GCA (Ala); residue 270, TCT (Ser) to ACT (Thr); and residue 286, GCT (Ala) to GCC (Ala), where the positions are relative to the positions in a mature SP-D polypeptide, such as the exemplary polypeptide of SEQ ID NO: 02. In some embodiments, rhSP-D comprises a particular residue at a polymorphic position, the residue selected from Met11 / 31, Thr160 / 180, Ser270 / 290, and Ala286 / 306, and the residue position is relative to the position in a mature SP-D polypeptide, such as exemplary SEQ ID NO: 02, and the position in an SP-D polypeptide with its leader polypeptide, such as exemplary SEQ ID NO: 01. In some embodiments, SP-D comprises Met11 / 31. In some embodiments, rhSP-D comprises Met11 / 31, Thr160 / 180, Ser270 / 290, and Ala286 / 306. In some embodiments, the SP-D polypeptide has at least 80%, 90%, 95%, 99%, and 100%, or any percentage in a range between any of the foregoing percentages, over the entire length of the polynucleotide to the polypeptide of SEQ ID NO: 02.

[0040] [Table 1]

[0041] In some embodiments, the SP-D is recombinant human SP-D (rhSP-D). In some embodiments, the rhSP-D is derived from a human myeloid leukemia cell line that expresses rhSP-D from an integrated transgene. Exemplary 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 by reference in its entirety.

[0042] Specific System Some embodiments of the methods and compositions provided herein include an electronic system for determining the activity of a pharmaceutical composition containing surfactant protein-D (SP-D). Some such embodiments include a system comprising a processor having instructions configured to perform the following steps: measuring the relative proportion of oligomeric species of SP-D in the pharmaceutical composition; calculating the relative proportion of SP-D dodecamer in the pharmaceutical composition; and determining the activity of the pharmaceutical composition based on the relative proportion of SP-D dodecamer, wherein a higher relative proportion of SP-D dodecamer indicates a composition having a higher level of activity compared to the activity of a composition having a lower relative proportion of SP-D dodecamer. In some embodiments, the activity includes antagonist activity in a toll-like receptor 4 (TLR4) assay. Some embodiments also include calculating the relative proportion of SP-D aggregates in the pharmaceutical composition having a mean radius greater than 70 nm or an RMS radius greater than 70 nm. In some embodiments, the measuring step includes performing asymmetric field-flow fractionation using multi-angle laser light scattering (AF4-MALLS) analysis. In some embodiments, the calculating step includes providing models of SP-D oligomeric species that include rod-like and spherical shapes. In some embodiments, the models include a Zimm model and a second-order Debye model of SP-D oligomeric species. Some embodiments also include measuring the relative proportion of at least one SP-D oligomeric species in the SP-D sample, wherein the SP-D oligomeric species is selected from the group consisting of SP-D trimers, SP-D hexamers, SP-D dodecamers, SP-D star-shaped oligomers with a mean radius of 70 nm or an RMS radius of 70 nm, and SP-D aggregates with a mean radius of greater than 70 nm or an RMS radius of greater than 70 nm.

[0043] Some embodiments of the methods and compositions provided herein include an electronic system for determining the relative proportions of surfactant protein-D (SP-D) oligomeric species in a sample. Some such embodiments include a system including a processor having instructions configured to perform the following steps: performing asymmetric flow field-flow fractionation using multi-angle laser light scattering (AF4-MALLS) analysis on an SP-D sample; and determining the relative proportions of SP-D oligomeric species from the results of the AF4-MALLS analysis, wherein the SP-D oligomeric species are selected from the group consisting of SP-D trimers, SP-D hexamers, SP-D dodecamers, SP-D star-shaped oligomers with a mean radius of 70 nm or an RMS radius of 70 nm, and SP-D aggregates with a mean radius of greater than 70 nm or an RMS radius of greater than 70 nm. Some embodiments also include measuring the relative proportions of SP-D dodecamers in the sample. Some embodiments also include determining the relative proportion of SP-D aggregates in the sample that have a mean radius greater than 70 nm or an RMS radius greater than 70 nm.

[0044] Some embodiments of the methods and compositions provided herein include an electronic system for determining the relative proportions of active oligomeric species of surfactant protein-D (SP-D) in a sample. Some such embodiments include a system comprising a processor having instructions configured to perform the following steps: performing asymmetric flow field-flow fractionation using multi-angle laser light scattering (AF4-MALLS) analysis on the SP-D sample; measuring the relative proportion of SP-D dodecamers in the SP-D sample; measuring the relative proportion of at least one SP-D oligomeric species in the SP-D sample selected from the group consisting of SP-D trimers, SP-D hexamers, SP-D star-shaped oligomers having a mean radius or RMS radius of 70 nm, and SP-D aggregates having a mean radius or RMS radius greater than 70 nm; and calculating the ratio between each of the measured relative proportions. In some embodiments, the at least one SP-D oligomeric species comprises an SP-D aggregate having a mean radius or RMS radius greater than 70 nm. Some embodiments also include integrating the fractogram from the AF4-MALLS analysis for each peak. Some embodiments also include measuring the relative peak area (RPA) of the fractogram for at least one peak representing an SP-D oligomer species selected from the group consisting of an SP-D trimer, an SP-D hexamer, an SP-D dodecamer, an SP-D star oligomer having a mean radius of 70 nm or an RMS radius of 70 nm, and an SP-D aggregate having a mean radius of greater than 70 nm or an RMS radius of greater than 70 nm. In some embodiments, the SP-D star oligomer has a molar mass of about 6 MDa or less. In some embodiments, the SP-D aggregate has a molar mass greater than 6 MDa. Some embodiments also include measuring the relative proportion of SP-D star oligomer species in the sample having a mean radius of 70 nm or an RMS radius of 70 nm. Some embodiments also include measuring the relative proportion of SP-D aggregates in the sample having a mean radius of greater than 70 nm or an RMS radius of greater than 70 nm.

[0045] In some embodiments, the SP-D sample is predicted to have antagonist activity in a lipopolysaccharide-Toll-like receptor 4 (LPS-TLR4) assay.

[0046] In some embodiments, an SP-D sample having a relative proportion of SP-D dodecamers of 35% or greater of the SP-D oligomer species in the sample indicates an active SP-D sample. In some embodiments, an SP-D sample having a relative proportion of SP-D dodecamers of a particular percentage or greater of the SP-D oligomer species in the sample indicates an active SP-D sample, which percentage can be 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and any percentage between any two of the foregoing percentages.

[0047] In some embodiments, an SP-D sample having a relative proportion of SP-D aggregates with an average radius greater than 70 nm or an RMS radius greater than 70 nm of less than 5% of the total SP-D oligomeric species in the sample indicates an active SP-D sample. In some embodiments, an SP-D sample having a relative proportion of SP-D aggregates of less than a certain percentage of the SP-D oligomeric species in the sample indicates an active SP-D sample, which percentage can be 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 3%, 2%, 1%, and any percentage between any two of the foregoing percentages.

[0048] In some embodiments, AF4-MALLS is performed in the absence of a chelating agent selected from the group consisting of EDTA and EGTA.

[0049] In some embodiments, the SP-D is recombinant human SP-D (rhSP-D). In some embodiments, the rhSP-D is derived from a human myeloid leukemia cell line that expresses rhSP-D from a transgene. In some embodiments, the rhSP-D comprises the amino acid sequence of SEQ ID NO: 02. In some embodiments, the rhSP-D comprises a residue at a polymorphic position corresponding to a residue selected from the group consisting of Met11, Thr160, Ser270, and Ala286.

[0050] In some embodiments, the relative proportions of oligomeric species are in terms of mass, hi some embodiments, the relative proportions of oligomeric species are in terms of relative peak area (RPA) or adjusted RPA in AF4-MALLS analysis. [Example]

[0051] Example 1 - Atomic Force Microscopy Analysis of rhSP-D Oligomeric species of recombinant human SP-D (rhSP-D) were characterized by atomic force microscopy (AFM). See, for example, Arroyo R et al. (2017) Biophysical Journal 112(3):503a, which is incorporated by reference in its entirety. A solution containing 0.85 ng / μL of rhSP-D in dilution buffer (200 mM NaCl, 20 mM Tris (pH 7.4), 1 mM EDTA) was placed on a freshly cleaved mica substrate. The sample was imaged using an atomic force microscope manufactured by Nanotec (Nanotec Electronica, Madrid, Spain) and a PointProbePlus tip, model PPP-NCH (Nanosensors, Neuchâtel, Switzerland). For quantitative studies, the image size was 1 μm × 1 μm with 512 pixels, acquired at a speed of 1 line / second. Raw images were subjected to general surface subtraction, flattening with background subtraction, and artifact line removal.

[0052] The oligomeric species identified in the resulting images included trimers, hexamers, dodecamers, and star-shaped oligomers. Trimers had a rod-like appearance, with some trimers measured to have an average length of approximately 65 nm (±8.6 nm). Dodecamers had an X-shaped appearance, with some dodecamers measured to have a tip-to-tip length of approximately 136 nm (±8.1 nm). Star-shaped species contained members with a star-shaped appearance composed of 6 to 20 trimers, with each trimer connected to a specific member of this species via a central hub. Some star-shaped species were measured to have a diameter of approximately 140 nm, similar to the diameter of the dodecamers measured. Table 2 summarizes the distribution and frequency of the observed oligomeric species.

[0053] [Table 2]

[0054] Example 2 - AF4-MALLS Analysis of rhSP-D The distribution of different oligomeric species of rhSP-D was determined by AF4-MALLS analysis. For AF4-MALLS analysis, rhSP-D samples were separated using an AF4 system (Eclipse Dual Tec, Wyatt Technology, Santa Barbara, CA), followed by UV (Ultimate 3000 variable wavelength detector, Dionex, Sunnyvale, CA) and MALS analysis (Dawn Heleos II detector, Wyatt Technology, Santa Barbara, CA). Samples were injected using a Dionex Ultimate 3000 HPLC system (Dionex, Sunnyvale, CA), and the mobile phase was delivered to the AF4 system. The AF4 configuration used a short channel with a 350 μm thick spacer (Wyatt Technology, Santa Barbara, CA). Data analysis and calculations were performed using Chromeleon (Dionex, Sunnyvale, CA) and ASTRA (Wyatt Technology, Santa Barbara, CA) software. Results from the AF4-MALLS analysis included a fractogram with several peaks.

[0055] ASTRA software (version 6.1.1.17) calculated the following: molar mass and root mean square (RMS) radial moment for each selected peak. The moments were based on an average over the entire sample, which can include many peaks. Equation (1) relates the number-average molar mass:

[0056]

number

[0057] ASTRA measurements typically required an independent concentration determination. The relationship between concentration (mg / mL) and number density (number / mL) was nM = c, so results could be determined from equation (2), which relates the mass-average molar mass:

[0058]

number

[0059] The polydispersity index value is ρ=M w / M n Typically, values ​​greater than 1.2 were considered polydisperse, while values ​​less than 1.1 were designated as having low polydispersity.

[0060] To determine molar mass, a mixed mathematical model was selected to calculate molecular size and MW in the analysis of rhSP-D using light scattering data. As shown herein, AFM images demonstrated that rhSP-D oligomeric species include monomers, dimers, trimers, hexamers, dodecamers, and higher-order oligomeric structures. AFM images demonstrated rod-, X-, and star-shaped structures. The star-shaped structures were measured by AFM and calculated to have a radius of approximately 70 nm and were best characterized by the second-order Debye model (P. Debye, "Molecular-weight determination by light scattering," J. Phys. Coll. Chem., vol. 51, pp. 18-32 (1947)), which is incorporated by reference in its entirety. The Debye model is expressed as Rθ / K * Using the c format, a very large mass (approximately 10 6 The Debye model provided good results over a wide range of molar masses, including 100 nm or greater than 100 daltons (RMS radius). The Debye model was applied to the analysis of peak 4 described herein. The rod-shaped structure was measured by AFM and calculated to have a length of approximately 65 nm. The X-shaped structure was measured by AFM and calculated to have a length of approximately 135 nm. Both the rod-shaped and X-shaped structures were best characterized by the rod model. The Zimm equation was applied to R θ / K *The theoretical shape factor P(θ) for the desired model was derived for spheres, coated spheres, and rods and is included in the work by van de Hulst (HC van de Hulst, Light Scattering by Small Particles, Wiley, New York (1957) which is incorporated by reference in its entirety). The spherical and coated sphere models yielded geometric radii, while the rod model yielded lengths. The rod model was applied to the analysis of peaks 1, 2, and 3 described herein. The resulting rod model is described by the following equation:

[0061]

number

[0062] In the formula, u=[(2πn o / λ o )L sin(θ / 2)], where L is the length of the rod, assumed to be much larger than its negligible diameter.

[0063] The diversity of SP-D structures could not be accommodated by a single model. Typically, the Zimm model can be flexible enough to accommodate multiple shapes, but in the case of SP-D, a mixed-mode analysis was used, combining the use of a second-order Debye model with a rod model. The second-order Debye model was applied to SP-D oligomeric species (peak 4), including aggregates with radii greater than 70 nm. The rod model was applied to rod-like SP-D structures (peaks 1 and 2), including monomers, dimers, trimers, hexamers, and dodecamers. There is a transition between the formation of dodecamers and star-like structures (peak 3), for which the rod model was applied to optimally characterize the MW. The mixed model provided a good fit to the AF4-MALLS data, consistent with the geometric measurements obtained by AFM.

[0064] Using a combination of UV signal analysis and analysis of molar mass determined by light scattering, the sample was integrated into four peaks, Peaks 1–4. All relative areas were calculated using drop-down integration at selected points. Peak 2 corresponds to the rhSP-D dodecamer peak, and its limits were determined by analyzing the molar mass and polydispersity index. A center point, usually the highest point in the UV trace, and boundaries were set equidistant from that point. The polydispersity of the region is measured by Mw / Mn, as described herein. Typically, values ​​greater than 1.2 are considered polydisperse, while values ​​less than 1.1 are designated as having low polydispersity. The boundaries were shifted until the polydispersity index was approximately 1.05. The index varied between 1.008 and 1.090. It was observed that slight shifts in the peak boundaries can cause changes in the index. The boundary of Peak 3 was set from the boundary after Peak 2 until a drop in the UV trace was observed at 36.5 min. The outer boundary of Peak 4 was set at 45 min. At 45 min, the crossflow was stopped. Any material remaining in the channel began to elute at that point, and virtually no separation occurred. Peak 1 contained all species lower than the rhSP-D dodecamer. In some cases, lower molecular weight species were resolved and separate integrations were performed. It was possible to overlay the MW trace on the UV trace, and integration points were found for the hexamer (258 kDa), trimer (129 kDa), dimer (86 kDa), and monomer (42 kDa). The peaks were very narrow and could be reliably determined.

[0065] In the first series of studies, cross-flow parameters were investigated using the original mobile phase composition (20 mM Tris buffer, pH 7.4, 200 mM NaCl, 1 mM EDTA). After establishing the cross-flow program, a series of experiments investigated the effect of mobile phase composition, resulting in the removal of EDTA from a mobile phase composed of 20 mM Tris, 200 mM NaCl, pH 7.4. Final adjustments were made to the focusing step, resulting in a final method with a runtime of 59.2 minutes. TABLE 3 lists the parameters used for AF4.

[0066] [Table 3]

[0067] In another series of studies, 102 lyophilized preparations of rhSP-D obtained from 17 different cell lines expressing rhSP-D from an integrated transgene were analyzed. These included cell lines S888, S990, S991, S1010, S2099, and 12 cell lines numbered 2428–2439. The reproducibility of the AF4 method was assessed using four different metrics: (i) the total area under the precision fractogram, (ii) the relative area of ​​the dodecamer, (iii) the calculated molar mass of the dodecamer from the MALLS data, and (iv) the polydispersity of the molar mass within the dodecamer peak envelope. Relative standard deviations (RSDs) were calculated for each metric across all samples. On average, the total area under the precision fractogram showed RSD values ​​ranging from 4.3% to 5.3%. In other words, the total area for these composite fractograms was reproducible within about 5% from run to run for a given sample. However, the relative areas of the rhSP-D dodecamer in these samples varied widely, from about 25% to 65%, and these relative amounts were reproducible, with rsd values ​​ranging from about 5% to 6.5%. The average rsd for the molar masses calculated from the MALLS data was very low at 0.86%. Finally, the polydispersity index (PDI) values ​​calculated from the MALLS data showed an average rsd value of about 1.5%.

[0068] The data using AF4-MALLS was analyzed to determine the absolute molar mass and size of rhSP-D at specific times during dissolution. The size-to-mass ratio indicated the shape of rhSP-D. From the size-to-mass ratio, it was determined that in the early stages of dissolution (0-34 min), rhSP-D molecules had a linear or rod-like shape. For the rod model calculation, the software assumed that the thickness of the rod-shaped particles was insignificant compared to their length (0.0 nm). The length of the rod was determined to be 136 ± 8.1 nm (N = 50 individual molecules), consistent with AFM measurements. Later stages of dissolution (34-45 min) for rhSP-D indicated that more compact structures were observed. A second-order Debye model was used to analyze these stages of dissolution. The second-order Debye model predicted very large (approximately 10 6 It provided good results over a wide range of molar masses, including >1000 daltons or an RMS radius of approximately 50 nm. For the dodecamer of rhSP-D, the molecular weight was determined to be 520.09 + / - 4.61 kDa (N=72 determinations).

[0069] The first peak in the fractogram (Peak 1) contained rhSP-D trimers and hexamers based on mass calculations according to the stick model. The second peak in the fractogram (Peak 2) contained rhSP-D dodecamers. The third peak in the fractogram (Peak 3) contained intermediate species between rhSP-D dodecamers and rhSP-D star oligomers based on intermediate molecular weights determined by the stick model. The fourth peak in the fractogram (Peak 4) contained heterogeneous mass 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.

[0070] The species in peak 4 were further analyzed to determine the distribution of oligomeric species in solution using AF4-MALLS analysis at higher resolution. The maximum molecular weight for star-shaped rhSP-D oligomers was assumed to be approximately 6 MDa based on the images from the AFM analysis. The maximum molecular weight was used as a nominal cutoff value for distinguishing star-shaped rhSP-D oligomers from larger aggregates. However, when 6 MDa was used as the cutoff value in the analysis, rhSP-D oligomeric species related to peak 3 were included in the aggregate determination. This did not coincide with the clear onset of elution of a new peak around 34 minutes. A size-based cutoff value of 70 nm was used in the analysis. The 70 nm cutoff value almost exactly coincided with the onset of peak 4, as seen by either the UV trace or the entire LS trace. Therefore, using a 70 nm cutoff value was a more accurate measure for identifying the presence of rhSP-D oligomeric aggregates.

[0071] Adjusted RPAs were determined for each of Peaks 3 and 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 removed from the RPA of Peak 4 and added to the RPA of Peak 3 to provide Peak 4 and the adjusted RPA for 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 an RMS radius of approximately 70 nm; and (4) aggregates with an RMS radius greater than 70 nm, respectively.

[0072] AF4-MALLS analysis was performed on three rhSP-D samples obtained from different human myeloid leukemia cell lines expressing rhSP-D from an integrated transgene. The results are summarized in TABLE 4.

[0073] [Table 4]

[0074] As shown in TABLE 4, rhSP-D sample 8B11 had an RPA for peak 2 of 44.37%, indicating that this sample had the highest relative amount of dodecamer among the three tested samples. rhSP-D sample 8B11 had an adjusted RPA for peak 4 of 1.04%, indicating that this sample had the lowest relative amount of aggregates with an RMS radius greater than 70 nm among the three tested samples.

[0075] Example 3 - Activity of rhSP-D in the TLR4 assay Toll-like receptors (TLRs) play roles in both the innate and adaptive immune systems, and SP-D has the activity of modulating signal transduction 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 by reference in its entirety. TLR4 activity can also modulate the severity of conditions such as bronchopulmonary dysplasia (BPD) (Malash AH et al. (2016) Gene 592:23-28, each of which is incorporated 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 formulations 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, e.g., Yamazoe M. et al. (2008) J. Biol Chem. 283:35878-35888.

[0076] The activity of reconstituted rhSP-D in the LPS-TLR4 assay was tested in a manner substantially similar to the following: HEK-Blue™ hTLR4 cells (InvivoGen, San Diego, CA, USA) were seeded in 384-well plates at a density of approximately 20,000 cells / well and incubated with various concentrations of SP-D for 2 hours at 37°C and 5% CO2. 80 LPS (E. coli O26:B6, L5543 Sigma-Aldrich) was added to each well at a concentration of 0.01 mg / mL, 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. The washed cells were then transferred to 20-well plates containing 20 e10 ng of 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 The cells were transferred to a 384-well plate at a density of 100 cells / well. The 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) reporter gene using a spectrophotometer at 655 nm. The IC for SP-D was calculated using nonlinear regression analysis by fitting the data to a four-parameter logistic equation. 50 The IC value was determined. 50 For the purposes of averaging values ​​from a series of experiments, pIC is used because only the logarithm of the values ​​is normally distributed. 50 Use the value -Log 10 (I C 50 ) was defined as the maximum fit response (E max ) and the fitted minimum response (E min ) to determine the curve span, which corresponds to the amplitude of the dose-response curve, or the potency of the response. The results of the LPS-TLR4 assay using rhSP-D samples are summarized in TABLE 5.

[0077] [Table 5]

[0078] As shown in TABLE 5, rhSP-D sample 8B11 was the only cell clone sample tested that had activity in the TLR4 assay. Notably, rhSP-D sample 8B11 was the only sample tested with an RPA for peak 2 greater than 35%.

[0079] Example 4 - Activity of rhSP-D in bacterial agglutination assay The activity of rhSP-D samples was tested in a bacterial agglutination assay. In the bacterial agglutination assay, active rhSP-D aggregates bacterial cells, decreasing absorbance and increasing transmission through the bacterial suspension. The bacterial agglutination assay was performed in a manner substantially similar to the following. Briefly, an exponential E. coli (ATCC: Y1088) culture was prepared, and an aliquot was resuspended in 1 mL of buffer (150 mM HEPES, 20 mM NaCl pH 7.4). The absorbance of the bacterial suspension was measured spectrophotometrically at 700 nm, and the bacterial suspension was adjusted to obtain an absorbance in the range of 1.0000 to 1.1000. 1 M CaCl2 was added to the suspension to obtain a final concentration of 5 mM CaCl2. rhSP-D samples in placebo buffer (15 μl total volume for each dilution) were prepared at the following concentrations: 5, 1, 0.5, 0.25, 0.1, and 0 μg / ml, and each was added to a cuvette containing 20 μL of Hepes-NaCl buffer. 600 μL of bacterial suspension was then added to the cuvette, and absorbance was measured at 700 nm for each cuvette every 2.5 minutes for a total of 120 minutes. Test concentrations of 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 average percentage of 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 min.

[0080] The percentage (%) of aggregation values ​​were averaged from three replicates and imported into GraphPad Prism v7.0c (GraphPad, Inc., La Jolla, CA 92037) along with the standard deviation. Mean values ​​were fitted using a four-parameter logistic curve. The resulting values ​​for EC50 and span were determined for each reconstituted rhSP-D sample. pEC50 is the -Log10 of EC50.

[0081] Each of the rhSP-D samples 7H8, 1C4 and 8B11 was active in the bacterial agglutination assay.

[0082] Example 5 - Multivariate analysis of rhSP-D oligomer species 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 determine the measured pIC from the TLR4 assay. 50 Correlation with values ​​or pEC from bacterial agglutination activity assay 50 The results were determined using PLS.

[0083] Full cross-validation was performed for all calibration models using standard techniques. See, for example, Katz, M. H., "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." Chemom. Intell. Lab. Syst. 58: pp. 109-130, each of which is incorporated 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 calibration samples were removed at once and quantified as a validation model. Thus, the first set containing all samples was referred to as the calibration set, and the set after cross-validation was referred to as the validation set. The jack-knife algorithm was used to determine statistical significance for any factors used in constructing the partial least squares (PLS) model (Martens, H. et al., (2001) "Multivariate Analysis of Quality: An Introduction" Wiley and Sons, Chichester, UK).

[0084] Regarding the correlation between the specific peaks observed in the AF4-MALLS analysis and the activity in the bacterial agglutination assay for various formulations of rhSP-D, the majority of the activity was found to be present in peaks 1 and 2, with some activity persisting throughout peak 3. A negative correlation was found between peak 4 and the activity in the bacterial agglutination assay. Regarding the correlation between the specific peaks observed in the AF4-MALLS analysis and the activity in the TLR4 assay for various formulations of rhSP-D, the activity was found to be almost exclusively localized in peak 2. Thus, the activity of rhSP-D in the TLR4 assay was directly related to the dodecamer species of rhSP-D. Thus, AF4-MALLS provides a method for determining the proportion of a sample containing the dodecamer that is active in the TLR4 assay. Notably, aggregate species with a radius greater than 70 nm found in the corrected peak 4 were not associated with activity in either the bacterial agglutination assay or the TLR4 assay, confirming that these species represent an inactive form of rhSP-D.

[0085] 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.

[0086] The above description discloses several methods and materials of the present invention. The invention is susceptible to modifications of the methods and materials, and to changes in the manufacturing methods and devices. Such modifications will become apparent to those skilled in the art from a consideration of the disclosure or practice of the invention disclosed herein. Therefore, it is not intended that the present invention be limited to the particular embodiments disclosed herein, but rather, it is intended to cover all modifications and alternatives falling within the true scope and spirit of the invention.

[0087] All references cited herein, including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated by reference herein in their entirety and made a part of this specification. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting material.

Claims

1. 1. A method for determining the activity of a pharmaceutical composition comprising surfactant protein-D (SP-D), comprising: determining the relative proportions of oligomeric species of SP-D in the pharmaceutical composition; calculating the relative proportion of SP-D dodecamer in the pharmaceutical composition, thereby determining the activity of the pharmaceutical composition; Including, the activity comprises activity in a toll-like receptor 4 (TLR4) assay; the calculating step includes providing a model of oligomeric species of SP-D that includes rod-like and spherical shapes; The model includes the Zimm model and the second-order Debye model of oligomeric species of SP-D; method.

2. The method of claim 1, wherein a composition having a higher relative proportion of SP-D dodecamers has higher activity compared to a composition having a lower relative proportion of SP-D dodecamers.

3. 3. The method of claim 1 or 2, further comprising the step of calculating the relative proportion of SP-D aggregates in the pharmaceutical composition having a mean radius greater than 70 nm or an RMS radius greater than 70 nm.

4. 4. The method of claim 1, wherein the measuring step comprises performing asymmetric flow field-flow fractionation using multi-angle laser light scattering (AF4-MALLS) analysis.

5. 5. The method of claim 1, further comprising measuring the relative proportion of at least one SP-D oligomer species in the SP-D sample, wherein the SP-D oligomer species is selected from the group consisting of SP-D trimers, SP-D hexamers, SP-D star-shaped oligomers having a mean radius of 70 nm or an RMS radius of 70 nm, and SP-D aggregates having a mean radius of more than 70 nm or an RMS radius of more than 70 nm.

6. 6. The method of claim 4 or 5, further comprising integrating the fractogram from the AF4-MALLS analysis into each peak.

7. 7. The method of claim 6, further comprising measuring the relative peak area (RPA) of the fractogram for at least one peak representing an SP-D oligomer species selected from the group consisting of SP-D trimers, SP-D hexamers, SP-D star oligomers having an average radius of 70 nm or an RMS radius of 70 nm, and SP-D aggregates having an average radius greater than 70 nm or an RMS radius greater than 70 nm.

8. 8. The method of any one of claims 5 to 7, wherein the SP-D star oligomer has a molar mass of less than or equal to 6 MDa.

9. 9. The method of claim 5, wherein the SP-D aggregates have a molar mass of more than 6 MDa.

10. 10. The method of any one of claims 5 to 9, further comprising the step of determining the relative proportion of SP-D star-oligomeric species in the sample having a mean radius of 70 nm or an RMS radius of 70 nm in the sample.

11. 11. The method of claim 5, further comprising measuring the relative proportion of SP-D aggregates in the sample that have a mean radius greater than 70 nm or an RMS radius greater than 70 nm.

12. 12. The method of any one of claims 4 to 11, wherein AF4-MALLS is carried out in the absence of a chelating agent selected from the group consisting of EDTA and EGTA.

13. 13. The method of any one of claims 1 to 12, wherein the SP-D is recombinant human SP-D (rhSP-D).

14. 14. The method of claim 13, wherein the rhSP-D is derived from a human myeloid leukemia cell line that expresses rhSP-D derived from a transgene.

15. 15. The method of any one of claims 1 to 14, wherein the relative proportions of oligomeric species relate to relative peak areas (RPA) or adjusted RPA in AF4-MALLS analysis.

16. 1. An electronic system for determining the activity of a pharmaceutical composition comprising surfactant protein-D (SP-D), comprising the steps of: determining the relative proportions of oligomeric species of SP-D in the pharmaceutical composition; calculating the relative proportion of SP-D dodecamer in the pharmaceutical composition; determining the activity of the pharmaceutical composition based on the relative proportion of SP-D dodecamers, wherein a higher relative proportion of SP-D dodecamers indicates a composition having a higher level of activity compared to the activity of a composition having a lower relative proportion of SP-D dodecamers; a processor having instructions configured to execute the activity comprises activity in a toll-like receptor 4 (TLR4) assay; the calculating step includes providing a model of oligomeric species of SP-D that includes rod-like and spherical shapes; The model includes the Zimm model and the second-order Debye model of oligomeric species of SP-D; system.

17. 17. The system of claim 16, further comprising the step of calculating the relative proportion of SP-D aggregates in the pharmaceutical composition having a mean radius greater than 70 nm or an RMS radius greater than 70 nm.

18. 18. The system of claim 16 or 17, wherein the measuring step comprises performing asymmetric flow field-flow fractionation using multi-angle laser light scattering (AF4-MALLS) analysis.

19. 20. The system of claim 18, wherein AF4-MALLS is performed in the absence of a chelating agent selected from the group consisting of EDTA and EGTA.

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