Means and Methods for Generating Complex Glycans Derived From Fungal Engineered Hosts

Abstract

The present application relates to the field of glyco-engineering, more specifically to glycosylation-engineered fungal cells, more specifically glycosylation-engineered yeast cells, optimized to produce highly homogenous forms of complex N-glycans on recombinant proteins. The invention specifically relates to methods to obtain pharmaceutical compositions comprising recombinant glycoproteins which have homogenous forms of complex N-glycans. In addition, the invention relates to novel pharmaceutical compositions which result from the methods of the invention.

1 . A method of producing a composition comprising different glycoforms of a glycoprotein, the method comprising:
 contacting complex N-glycan glycoforms of the glycoprotein secreted from a fungal organism with an endoglucosaminidase enzyme;   wherein the endoglucosaminidase enzyme acts on the secreted N-glycan glycoforms of the glycoprotein to produce the composition comprising different glycoforms of a glycoprotein, the composition comprising:
 a glycoform comprising a complex N-glycan and 
 a glycoform comprising an N-glycan structure consisting of a single GlcNAc; and 
   wherein the glycoprotein is exogenous to the fungal organism.   
     
     
         2 . The method according to  claim 1 , wherein the contacting occurs during purification of the glycoprotein. 
     
     
         3 . The method according to  claim 1 , wherein the contacting occurs after purification of the glycoprotein. 
     
     
         4 . The method according to  claim 1 , wherein contacting with the endoglucosaminidase takes place at a high salt concentration. 
     
     
         5 . The method according to  claim 2 , wherein contacting with the endoglucosaminidase takes place at a high salt concentration. 
     
     
         6 . The method according to  claim 3 , wherein contacting with the endoglucosaminidase takes place at a high salt concentration. 
     
     
         7 . The method according to  claim 1 , wherein the fungal organism expresses at least one exogenous nucleic acid sequence encoding an enzyme needed for complex N-glycosylation and/or wherein the fungal organism does not express an enzyme involved in the production of high-mannose type structures, which enzyme is normally expressed in a wild type from of the fungal organism. 
     
     
         8 . The method according to  claim 1 , wherein the endoglucosaminidase enzyme is an endo-beta-N-acetylglucosaminidase of EC3.2.1.96. 
     
     
         9 . The method according to  claim 1 , wherein the composition further comprises a glycoform comprising a complex N-glycan that is different from the glycoform comprising a complex N-glycan.