IP Library › Granted Patent US 12,385,894
Granted Patent B2
US 12,385,894 · App. 17/041,682 · Granted Aug 12, 2025

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

Inventors: Jan Susan Rosenbaum (Cincinnati, OH); Mark Cornell Manning (Johnstown, CO); Ryan R. Manning (Charlotte, MI)
Assignee: Airway Therapeutics, Inc.
G01N33/15C12Q1/025G01N33/5041G01N33/94G01N2500/10
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,385,894
App. No.
17/041,682
Granted
Aug 12, 2025
Kind
B2
Abstract

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

Claims (22)

1. A method to determine the activity of a pharmaceutical composition comprising surfactant protein-D (SP-D), the method 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,

wherein

the activity comprises activity in a toll-like receptor 4 (TLR4) assay,

the calculating comprises providing a model of the oligomeric species of the SP-D comprising rod-like geometries and spherical-like geometries,

the model comprises a Zimm model and a second order Debye model of the oligomeric species of the SP-D, and

the pharmaceutical composition having a relative proportion of SP-D dodecamers greater than or equal to 20% of the SP-D oligomeric species is indicative of the pharmaceutical composition having activity in the TLR4 assay.

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

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

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

5. The method of claim 1 , further comprising measuring the relative proportion of at least one SP-D oligomeric species in the pharmaceutical composition, wherein the SP-D oligomeric species is selected from the group consisting of a SP-D trimer, a SP-D hexamer, a SPD star-like oligomer having an average radius of 70 nm or RMS radius of 70 nm, and SP-D aggregate having an average radius greater than 70 nm or RMS radius greater than 70 nm.

6. A method to determine the relative proportion of oligomeric species of surfactant protein-D (SP-D) in a sample, comprising:

performing an asymmetric flow field-flow fractionation with multi-angle laser light scattering (AF4-MALLS) analysis on the SP-D sample;

determining the relative proportion of an oligomeric species of SP-D from the results of the AF4-MALLS analysis, wherein the SP-D oligomeric species is selected from the group consisting of a SP-D trimer, a SP-D hexamer, a SP-D dodecamer, a SP-D starlike oligomer having an average radius of 70 nm or RMS radius of 70 nm, and SP-D aggregate having an average radius greater than 70 nm or RMS radius greater than 70 nm, and differentiating the species using a Zimm model for rod-like geometries and a Debye model for spherical-like geometries, and

measuring the relative proportion of SP-D dodecamers in the sample,

wherein the sample having a relative proportion of SP-D dodecamers greater than or equal to 20% of the SP-D oligomeric species is indicative of the pharmaceutical composition having activity, and wherein the activity comprises activity in a toll-like receptor 4 (TLR4) assay.

7. The method of claim 6 , further comprising integrating a fractogram from the AF4-MALLS analysis into peaks.

8. The method of claim 6 , wherein the SP-D star-like oligomer has a molar mass less than or equal to 6 MDa.

9. The method of claim 6 , wherein the AF4-MALLS is performed in the absence of a chelating agent selected from the group consisting of EDTA and EGTA.

10. The method of claim 6 , wherein the relative proportion of the oligomeric species is with respect to mass.

11. The method of claim 6 , wherein the relative proportion of the oligomeric species is with respect to a relative peak area (RPA) or an adjusted RPA in an AF4-MALLS analysis.

Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 5, 2022
From: ROSENBAUM, JAN SUSAN; MANNING, MARK CORNELL; MANNING, RYAN R.
To: AIRWAY THERAPEUTICS, LLC
Reel/Frame 061318/0047 →
CHANGE OF NAME Recorded Oct 5, 2022
From: AIRWAY THERAPEUTICS, LLC
To: AIRWAY THERAPEUTICS, INC.
Reel/Frame 061607/0943 →
Continuity (2)
Provisional Application 62650138 · Mar 29, 2018
Related Publication 20210010988A1 · Jan 14, 2021
References Cited (94)
US 8865643B2 · Clark et al. · 2014 [cited by applicant]
US 9370555B2 · Whitsett et al. · 2016 [cited by applicant]
US 20110189104A1 · Whitsett et al. · 2011 [cited by applicant]
US 20110256133A1 · Dixit et al. · 2011 [cited by applicant]
US 20130316980A1 · Tchirikov · 2013 [cited by applicant]
US 20140302158A1 · Cabrera et al. · 2014 [cited by applicant]
US 20150335578A1 · Spira et al. · 2015 [cited by applicant]
US 20170189527A1 · Rinaldi et al. · 2017 [cited by applicant]
US 20190071693A1 · Rosenbaum et al. · 2019 [cited by applicant]
US 20190071694A1 · Rosenbaum et al. · 2019 [cited by applicant]
US 20210121536A1 · Rosenbaum et al. · 2021 [cited by applicant]
EP 1132402 · 2001 [cited by applicant]
JP 2003514864A · 2003 [cited by applicant]
JP 2006522751A · 2006 [cited by applicant]
JP 2009514958A · 2009 [cited by applicant]
JP 4744533B2 · 2011 [cited by applicant]
RU 2603093 · 2014 [cited by applicant]
RU 2643762 · 2016 [cited by applicant]
WO WO2001080633 · 2001 [cited by applicant]
WO WO2012093087 · 2012 [cited by applicant]
WO WO2017143024 · 2017 [cited by applicant]
WO WO2019191247 · 2019 [cited by applicant]
Yohannes, G., et al. 2011 Journal of Chromatography A 1218: 4104-4116. (Year: 2011). [cited by examiner]
Wyatt Technology Corporation Astra V User's Guide 2008: 372 pages. (Year: 2008). [cited by examiner]
Crouch, E.C. 1998 Am. J. Respir. Cell Mol. Biol. 19: 177-201. (Year: 1998). [cited by examiner]
Hartshorn, K. et al. “Interactions of recombinant human pulmonary surfactant protein D and SP-D multimers with influenza A.” Am. J. Physiol, vol. 271 No. 5, pt. 1, 1996, pp. L753-L762. [cited by applicant]
Kuroki, Y. “Innate immune function of pulmonary collectins.” Biochemistry, vol. 81 No. 3, 2009, pp. 182-188. [cited by applicant]
Kuroki, Y. “Pulmonary Surfactant Proteins: SP-A and SP-D.” Clinical Chemistry, vol. 32, 2003, pp. 216-226. [cited by applicant]
Kuroki, Y. “Collectin (SP-A, SP-D, MBL) and Infection Prevention.” Modern Medical Laboratory, vol. 42 No. 5, May 1, 2014, pp. 488-489. [cited by applicant]
Zherdev V.P et al. “Preclinical study the pharmacokinetics new anxiolytic drug of dipeptide structure GB-115.” Pharmacokinetics and Pharmacodynamics, vol. 1, 2015, pp. 52-59. (abstract only). [cited by applicant]
Kalmatov, R.K. et al. “Pathogenetic Role of Surfactant Protein Sp-D at Diseases Lungs and Airways.” Fundamental Research, No. 1, 2015, pp. 1591-1595. [cited by applicant]
“Respiratory Medicine.” A.G. Chuchalin, Ed., 2017, available at https://cpkmed.ru/materials/El_Biblio/AktualDoc/ftiziatrija/4.pdf. [cited by applicant]
Pozza G.M.-E. et al., Molecular and structural characterization of hybrid poly(ethylene oxide)-polyhedral oligomeric silesquioxanes star-shaped macromolecules, Phys Chem B, 2015, V.119, N. 4, p. 1669-1680. (Abstract onl… [cited by applicant]
Sluchanko N.N. et al., Monomeric 14-3-3ζ Has a Chaperone-Like Activity and Is Stabilized by Phosphorylated HspB6, Biochemistry, 2012, V. 51, N. 31, p. 6127-6138. (Abstract only). [cited by applicant]
Koeffler P. et al., Human Myeloid Leukemia Cell Lines: A Review, Blood, 1980, V. 56, I. 3, p. 344-350. (Abstract only). [cited by applicant]
Sorensen, G. “Surfactant Protein D in Respiratory and Non-Respiratory Disesases,” Sec. Pulmonary Medicine. (2018) 5: 1-37. [cited by applicant]
Wong, W. et al., “FungalmelaninstimulatessurfactantproteinD-mediated opsonization of and host immune response to Aspergillus fumigatus spores,” J. Biol. Chem. (2018) 293(13): 4901-4912. [cited by applicant]
Nousheen, L., et. al., “Molecular Docking and Mutational Studies of Human Surfactant Protein-D,” World J. Pharm. Res. (2014) 3(7): 1140-1147. [cited by applicant]
Sim, R. “SP-D,” The Complete FactsBook. (2000): 46-50. [cited by applicant]
Uniprot, Commercially available sequence for recombinant human SP-D. retrieved from https://www.uniprot.org/uniprotkb/P35247/entry#sequences on Oct. 10, 2023. [cited by applicant]
Watson, A. S. “Recombinant Expression of Functional Trimeric Fragments of Human SP-A and SP-D,” University of Southampton, Faculty of Medicine, PhD Thesis (2016). [cited by applicant]
International Search Report and Written Opinion mailed Jul. 12, 2019 in PCT/US2019/024320 (16 pgs.). [cited by applicant]
Mehn, D. “FFF-MALS method development and measurements of size and molecular weight”, EUNCL, Doc. ID EUNCL-PCC-22, Jan. 30, 2016, 27 pgs. [cited by applicant]
Arroyo et al. Structural Characterization of Human Pulmonary Surfactant Protein SP-D by Atomic Force Microscopy. Biophysical J. (2017) 112(3):503a Abstract 2478-Pos. [cited by applicant]
Arroyo et al., Supramolecular Assembly of Human Pulmonary Surfactant Protein SP-D. J Mol Biol. (2018) 430(10):1495-1509. [cited by applicant]
Awasthi S., Surfactant protein (SP)-A and SP-D as antimicrobial and immunotherapeutic agents. Recent Pat Antiinfect Drug Discov. (2010) 5(2):115-123. [cited by applicant]
Clark et al., Structural requirements for SP-D function in vitro and in vivo: therapeutic potential of recombinant SP-D. Immunobiology (2002) 205(4-5):619-631. [cited by applicant]
Clements J.A., Functions of the alveolar lining, Am Rev Respir Dis (1977) 115(6 Pt 2):67-71. [cited by applicant]
Crouch et al., Molecular structure of pulmonary surfactant protein D (SP-D). J Biol Chem. (1994) 269(25):17311-17319. [cited by applicant]
Crouch E.C., Surfactant protein-D and pulmonary host defense. Respir Res. (2000) 1(2):93-108. [cited by applicant]
Crouch et al., Contributions of phenylalanine 335 to ligand recognition by human surfactant protein D: ring interactions with SP-D ligands. J Biol Chem., (2006) 281(26):18008-18014. [cited by applicant]
Debye P., Molecular-weight determination by light scattering. J Phys Chem., (1947) 51:18-32. [cited by applicant]
Dodagatta-Marri et al., Purification of surfactant protein D (SP-D) from pooled amniotic fluid and bronchoalveolar lavage. Methods Mol Biol. (2014) 100:273-290. [cited by applicant]
Griese M., Pulmonary surfactant in health and human lung diseases: State of the art, Eur Respir J (1999) 13:1455-1476. [cited by applicant]
Haagsman et al., Surfactant collectins and innate immunity. Neonatology (2008) 93:288-294. [cited by applicant]
Håkansson et al., Collectin structure: a review. Protein Sci. (2000) 9:1607-1617. [cited by applicant]
Hartshorn et al., Role of viral hemagglutinin glycosylation in anti-influenza activities of recombinant surfactant protein D. Resp Res. (2008) 9(1):65 in 12 pgs. [cited by applicant]
Ikegami et al., Intratracheal Recombinant Surfactant Protein D Prevents Endotoxin Shock in the Newborn Preterm Lamb. Am J Respir Crit Care Med. (2006) 173(12):1342-1347. [cited by applicant]
Kingma et al., Correction of pulmonary abnormalities in Sftpd-/-mice requires the collagenous domain of surfactant protein D. J Biol Chem. (2006) 281:24496-24505. [cited by applicant]
Knudsen et al. A recombinant fragment of human surfactant protein D lacking the short collagen-like stalk fails to correct morphological alterations in lungs of SP-D deficient mice. The Anatomical Record (2009) 292(2):1… [cited by applicant]
Knudsen et al., Surfactant protein D (SP-D) deficiency is attenuated in humanised mice expressing the Met(11)Thr short nucleotide polymorphism of SP-D: implications for surfactant metabolism in the lung. J Anat. (2013) … [cited by applicant]
Korfhagen et al., Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo. J Biol Chem. (1998) 273(43):28438-28443. [cited by applicant]
Malash et al., Association of TLR polymorphisms with bronchopulmonary dysplasia. Gene (2016) 592(1):23-28. [cited by applicant]
Ogasawara et al., The role of the amino-terminal domain and the collagenous region in the structure and the function of rat surfactant protein D. J Biol Chem. (1995) 270(32):19052-19058. [cited by applicant]
Robertson et al., Principles of surfactant replacement, Biochim Biophys Acta (1998) 1408:346-361. [cited by applicant]
Sato et al., Surfactant Protein-D Inhibits Lung Inflammation Caused by Ventilation in Premature Newborn Lambs. Am J Respir Crit Care Med. (2010) 181(10):1098-1105. [cited by applicant]
Strong et al., A novel method of purifying lung surfactant proteins A and D from the lunglavage of alveolar proteinosis patients and from pooled amniotic fluid. J Immunol Methods. (1998) 220(1-2):139-149. [cited by applicant]
Vieira et al., Structure, Genetics and Function of the Pulmonary Associated SurfactantProteins A and D: The Extra-Pulmonary Role of These C Type Lectins.Ann Anat. (2017) 211:184-201. [cited by applicant]
Wagner et al., Asymmetric flow field-flow fractionation in the field of nanomedicine. Anal Chem (2014) 86(11):5201-5210. [cited by applicant]
Wert et al., Increased metalloproteinase activity, oxidant production, and emphysema in surfactant protein D gene-inactivated mice. PNAS (2000) 97(11): 5972-5977. [cited by applicant]
White et al., Multimerization of surfactant protein D, but not its collagen domain, is required for antiviral and opsonic activities related to influenza virus. J Immunol. (2008) 181(11):7936-7943. [cited by applicant]
Winkler et al., Impact of a Met(11)Thr single nucleotide polymorphism of surfactant protein D on allergic airway inflammation in a murine asthma model. Exp Lung Res. (2014) 40(4):154-163. [cited by applicant]
Wold et al., PLS-regression: a basic tool of chemometrics. Chemom. Intell. Lab. Syst. (2001) 58: 109-130. [cited by applicant]
Yamazoe et al., Pulmonary surfactant protein D inhibits lipopolysaccharide (LPS)-inducedinflammatory cell responses by altering LPS binding to its receptors. J Biol Chem. (2008) 283(51):35878-35888. [cited by applicant]
Zhang et al., Activity of pulmonary surfactant protein-D (SP-D) in vivo is dependent on oligomeric structure. J Biol Chem (2001) 276(22):19214-19219. [cited by applicant]
Zimm B.H., The scattering of light and the radial distribution function of high polymer solutions. J Chem Physics (1948) 16(12):1093-1099. [cited by applicant]
Zuo et al., Current perspective in pulmonary surfactant—Inhibition, enhancement and evaluation, Biochim Biophys Acta (2008) 1778:1947-1977. [cited by applicant]
Bharate et al., Interactions and incompatibilities of pharmaceutical excipients with active pharaceutical ingredients: A comprehensive review. J Excipients and Food Chem. (Nov. 2016) 1(3): 1131 in 24 pages. [cited by applicant]
Brooke et al., Universal buffers for use in biochemistry and biophysical experiments. AIMS Biophysics. (2015) 2(3): 336-342. [cited by applicant]
Carpenter et al., Rational design of stable lyophilized protein formulations: Theory and practice, rational design of stable protein formulations. Pharma Biotech. (2002) 109-127. [cited by applicant]
Muller et al., Spliceosomal peptide P140 for immunotherapy of systemic lupus erythematosus: Results of an early phase II clinical trial. Arth Rheuma. Official J Am College of Rheuma. (Dec. 2008) 58(12): 3873-3883. [cited by applicant]
Patel et al., Drug-excipient compatibility studies: First step for dosage form development. TPI Inter'I J. (2015) 4(5): 14-20. [cited by applicant]
Sako et al., Characterization of the ligand binding functionality of the extracellular domain of activin receptor type IIB. J Biol Chem. (Jul. 2010) 285(27): 21037-21048. [cited by applicant]
Schaefers, M., Reconstructing reconstitution. Packaging Digest (2015) downloaded from https://www.packagingdigest.com/sterilization/reconstructing-reconsitution; 3 pages. [cited by applicant]
Tang et al., Design of freeze-drying processes for pharmaceuticals: Practical advise. Pharm Res. (2004) 21(2): 191-200. [cited by applicant]
Tonnis et al., Size and molecular flexibility of sugars determine the storage stability of freeze-dried proteins. Mol Pharm. (2015) 12: 684-694. [cited by applicant]
Vuk-Pavlovic et al., Carbohydrate recognition domain of surfactant protein D mediates interactions with Pneumocystic carinii glycoprotein A. Am J Respir Cell Mol Biol. (2001) 24(4): 475-484. [cited by applicant]
Stahle, L. et al. “Multivariate Data Analysis and Experimental Design in Biomedical Research.” Progress in Medicinal Chemistry, vol. 25, 1988, pp. 291-338. (Abstract only). [cited by applicant]
Manning, R. et al. “Characterization of Oligomer Formation of Surfactant Protein-D (SP-D) Using AF4-MALLS.” Current Protein and Peptide Science, vol. 23, 2022, pp. 862-873. [cited by applicant]
Sano, H. et al. “The lung collectins, SP-A and SP-D, modulate pulmonary innate immunity”, Molecular Immunology, vol. 42, 2005, pp. 279-287. [cited by applicant]
Sarker et al., Lung Surfactant Protein A (SP-A) Interactions with Model Lung Surfactant Lipids and an SP-B Fragment. Biochem. (Jun. 2011), 50(22): 4867-4876. [cited by applicant]
White et al., Increased antiviral and opsonic activity of a highly multimerized collectin chimera. Biochem Biophys Res Commun. (2001) 286: 206-213. [cited by applicant]
Some, D. “The Chromatography Detector You Shouldn't Miss”, Nov. 20, 2015, at https://www.wyatt.com/blogs/the-chromatography-detector-you-shouldn-t-miss.html. [cited by applicant]
Rynkiewicz, M. et al. “Differential Ligand Binding Specificities of the Pulmonary Collectins Are Determined by the Conformational Freedom of a Surface Loop” Biochemistry, vol. 56 No. 31, 2017, pp. 4095-4105. (Abstract O… [cited by applicant]