Methods and compositions for preparing surfactant protein d (sp-d)

Abstract

Some embodiments of the methods and compositions provided herein relate to the preparation surfactant protein-D (SP-D). Some embodiments include the expression of human SP-D in certain cell lines, and the purification of human SP-D from such cell lines. Some embodiments include the preparation of certain oligomeric forms of human SP-D.

1 .- 46 . (canceled) 
     
     
         47 . A composition comprising:
 a plurality of recombinant human SP-D (rhSP-D) polypeptides, wherein the rhSP-D polypeptides have a glycosylation pattern that is substantially similar to wild-type human SP-D polypeptides; and   a histidine buffer.   
     
     
         48 . The composition of  claim 47 , wherein the plurality of recombinant human SP-D (rhSP-D) polypeptides were produced in a cell selected from the group consisting of: NM-H9D8, NM-H9D8-E6Q12, and NM-F9 cells. 
     
     
         49 . The composition of  claim 47 , wherein the plurality of rhSP-D polypeptides have an amino acid sequencing comprising the polypeptide sequence of SEQ ID NO:05. 
     
     
         50 . The composition of  claim 47 , wherein the plurality of rhSP-D polypeptides have an amino acid sequence comprising the amino acid sequence of positions 22 to 376 of SEQ ID NO:04. 
     
     
         51 . The composition of  claim 47 , wherein the plurality of rhSP-D polypeptides comprise at least one residue at a polymorphic position, wherein the residue is selected from the group consisting of: Met11/31, Thr160/180, Ser 270/290, and Ala 286/306. 
     
     
         52 . The composition of  claim 51 , wherein the plurality of rhSP-D polypeptides comprises Met11/31. 
     
     
         53 . The composition of  claim 47 , wherein the plurality of rhSP-D polypeptides each comprise a complex-type carbohydrate attached at an N-glycosylation site, and wherein the plurality has a glycosylation pattern comprising the following characteristics:
 (i) at least 70% of the complex-type carbohydrates include a core fucose;   (ii) at least 10% of the complex-type carbohydrates include at least one sialic acid residue;   (iii) at least 50% of the complex-type carbohydrates include at least a biantennary carbohydrate structure;   (iv) at least 10% of the complex-type carbohydrates include a bisecting N-acetylglucosamine;   (v) less than 10% of the carbohydrates are high-mannose type structures; and   (vi) a detectable amount of α2,6-coupled sialic acid residues.   
     
     
         54 . The composition of  claim 53 , wherein the glycosylation pattern comprises at least 70% of the complex-type carbohydrates including a core fucose. 
     
     
         55 . The composition of  claim 53 , wherein the glycosylation pattern comprises at least 10% of the complex-type carbohydrates including at least one sialic acid residue. 
     
     
         56 . The composition of  claim 53 , wherein the glycosylation pattern comprises at least 50% of the complex-type carbohydrates including at least a biantennary carbohydrate structure. 
     
     
         57 . The composition of  claim 53 , wherein the glycosylation pattern comprises at least 10% of the complex-type carbohydrates including a bisecting N-acetylglucosamine. 
     
     
         58 . The composition of  claim 53 , wherein the glycosylation pattern comprises less than 10% of the carbohydrates having high-mannose type structures. 
     
     
         59 . The composition of  claim 53 , wherein the glycosylation pattern comprises a detectable amount of α2,6-coupled sialic acid residues 
     
     
         60 . The composition of  claim 53 , wherein the population has a glycosylation pattern comprising one or more of the following characteristics:
 (i) at least 20% of the complex-type carbohydrates include a bisecting N-acetylglucosamine; and   (ii) at least 85% of the complex-type carbohydrates include a core fucose.   
     
     
         61 . The composition of  claim 47 , wherein the plurality of SP-D polypeptides have an antennarity number within a range from 190 to 215. 
     
     
         62 . The composition of  claim 47 , wherein the plurality of rhSP-D polypeptides comprise a population of oligomeric forms of rhSP-D, wherein greater than about 40% of the oligomeric forms comprise dodecamers of the SP-D. 
     
     
         63 . The composition of  claim 62 , wherein greater than about 60% of the oligomeric forms comprise dodecamers of the SP-D. 
     
     
         64 . The composition of  claim 62 , wherein greater than about 62% of the oligomeric forms comprise dodecamers of the SP-D. 
     
     
         65 . The composition of  claim 62 , wherein greater than about 64% of the oligomeric forms comprise dodecamers of the SP-D. 
     
     
         66 . The composition of  claim 62 , wherein each SP-D polypeptide in the oligomeric forms comprises a complex-type carbohydrate attached at an N-glycosylation site, wherein the population has a glycosylation pattern comprising the following characteristics:
 (i) at least 70% of the complex-type carbohydrates include a core fucose;   (ii) at least 10% of the complex-type carbohydrates include at least one sialic acid residue;   (iii) at least 50% of the complex-type carbohydrates include at least a biantennary carbohydrate structure;   (iv) at least 10% of the complex-type carbohydrates include a bisecting N-acetylglucosamine;   (v) less than 10% of the carbohydrates are high-mannose type structures; and   (vi) a detectable amount of α2,6-coupled sialic acid residues.