Methods and products related to the improved analysis of carbohydrates

Abstract

The invention relates, in part, to the improved analysis of carbohydrates. In particular, the invention relates to the analysis of carbohydrates, such as N-glycans and O-glycans found on proteins and saccharides attached to lipids. Improved methods, therefore, for the study of glycosylation patterns on cells, tissue and body fluids are also provided. Information from the analysis of glycans, such as the glycosylation patterns on cells, tissues and in body fluids, can be used in diagnostic and treatment methods as well as for facilitating the study of the effects of glycosylation/altered glycosylation. Such methods are also provided. Methods are further provided to assess production processes, to assess the purity of samples containing glycoconjugates, and to select glycoconjugates with the desired glycosylation.

1 - 328 . (canceled) 
     
     
         329 . A method comprising steps of:
 a) providing a sample comprising a plurality of different glycans, such that the sample has a glycome which is all of the carbohydrates of the sample, which sample has a characteristic selected from the group consisting of function, cellular state, pathological condition, membership in a population, and combinations thereof;   b) analyzing the sample such that its glycome pattern is determined that is associated with the certain function, which analyzing comprises both:
 i) performing one or more analytical analyses of the sample, wherein the analytical comprise one or more of mass spectrometry, nuclear magnetic resonance, electrophoresis, chromatography, and combinations thereof; and 
 ii) performing one or more computational analyses of the sample, wherein the computational analyses include one or more of establishing and solving a mathematical relationship. 
   
     
     
         330 . The method of  claim 329 , wherein the step of providing comprises providing a sample obtained through one or more separating steps performed on a culture of a mammalian cell that expresses a glycoprotein of interest. 
     
     
         331 . The method of  claim 329 , wherein the at least some glycans of the plurality of glycans are part of a glycoconjugate. 
     
     
         332 . The method of  claim 329 , wherein at least some glycans of the plurality of glycans are in free form. 
     
     
         333 . The method of  claim 329 , further comprising steps of:
 comparing the determined glycome pattern with a reference glycome pattern;   based on the comparing, concluding that the sample shares a characteristic with the reference glycome pattern; and   in light of the conclusion, selecting a specific therapy, selecting a specific production method, or selecting production conditions that control generation of a specific glycoform.   
     
     
         334 . The method of  claim 329 , wherein the glycans in the sample are quantified. 
     
     
         335 . The method of  claim 329 , wherein the sample comprising a plurality of different glycans is a sample of a cell, group of cells, tissue or body fluid. 
     
     
         336 . The method of  claim 335 , wherein the body fluid is selected from the group consisting of serum, plasma, blood, urine, saliva, sputum, tears, CSF, seminal fluid, and feces. 
     
     
         337 . The method of  claim 329 , wherein the sample comprising a plurality of different glycans is a sample of a glycoprotein therapeutic. 
     
     
         338 . The method of  claim 329 , wherein performing one or more analytical analyses of the sample includes obtaining more than one glycoprofile spectra. 
     
     
         339 . The method of  claim 338 , wherein at least one of the spectra is of acidic glycans. 
     
     
         340 . The method of  claim 338 , wherein at least one of the spectra is of neutral glycans. 
     
     
         341 . The method of  claim 338 , wherein at least one of the spectra is of acidic glycans and at least one of the spectra is from neutral glycans. 
     
     
         342 . The method of  claim 329 , wherein the analyzing the sample includes determining one or more features selected from the group consisting of the presence or absence of a specific glycan, an amount of a specific glycan; a ratio between two or more glycans; a range of amounts of one or more glycans; and a range of ratios between two or more glycans. 
     
     
         343 . A method comprising steps of:
 providing a sample comprising a plurality of glycoconjugates;   determining one or more glycosylation sites of the glycoconjugates and identifying glycans that may be present at the one or more glycosylation sites; and   characterizing the glycans at each glycosylation site, thereby characterizing the glycosylation site occupancy of the one or more glycoconjugates.   
     
     
         344 . The method of  claim 343 , wherein the identifying glycans that may be present at the one or more glycosylation sites includes comparing unlabeled glycoconjugates to labeled glycoconjugates. 
     
     
         345 . The method of  claim 343 , wherein the identifying glycans that may be present at the one or more glycosylation sites includes comparing unlabeled deglycosylated fragments of the glycoconjugates to labeled deglycosylated fragments of the glycoconjugates. 
     
     
         346 . A method comprising steps of:
 a) providing a sample comprising a plurality of different glycans, such that the sample has a glycome which is all of the carbohydrates of the sample;   b) analyzing the sample such that the glycome is determined, which analyzing comprises both:
 i) performing one or more analytical analyses of the sample, wherein the analytical comprise one or more of mass spectrometry, nuclear magnetic resonance, electrophoresis, chromatography, and combinations thereof; and 
 ii) performing one or more computational analyses of the sample, wherein the computational analyses include one or more of establishing and solving a mathematical relationship. 
   
     
     
         347 . The method of  claim 346 , further comprising steps of:
 comparing the determined glycome with a glycome profile that has been associated with a certain function, cellular state, pathological condition, sample identity or population membership;   based on the comparing, concluding that the sample is obtained from a source characterized by the certain function, cellular state, pathological condition, sample identity or population membership; and   in light of the conclusion, selecting a specific therapy, selecting a specific production method, or selecting production conditions that control generation of a specific glycoform.