Method for in vitro glycoengineering of an erythropoiesis stimulating protein

Abstract

The present invention relates to a method for the production of in vitro glycoengineered erythropoiesis stimulating protein, comprising the steps of providing sialic acid free erythropoiesis stimulating protein, treating the erythropoiesis stimulating protein with N-Acetyl-Glucosamin-transferase B3GNT2, treating the erythropoiesis stimulating protein with galactosyltransferase, and treating the erythropoiesis stimulating protein with sialyltransferase.

1 . A method for the production of an in vitro glycoengineered erythropoiesis stimulating protein, comprising the steps of
 a) providing sialic acid free erythropoiesis stimulating protein,   b) treating the erythropoiesis stimulating protein with N-Acetyl-Glucosamin-transferase B3GNT2,   c) treating the erythropoiesis stimulating protein with galactosyltransferase,   d) treating the erythropoiesis stimulating protein with sialyltransferase.   
     
     
         2 . The method of  claim 1 , characterized in that step b) is performed in presence of manganese ions and calcium ions. 
     
     
         3 . The method of  claim 1 , the weight ratio between erythropoiesis stimulating protein and N-Acetyl-Glucosamin-transferase B3GNT2 in step b) is >1:1. 
     
     
         4 . The method of  claim 1 , characterized in that the method comprises a step of purifying the obtained erythropoiesis stimulating protein after step b) and prior to the treatment with galactosyltransferase. 
     
     
         5 . The method of  claim 1 , characterized in that step c) is performed using galactosyltransferase GalT1 in presence of UDP-galactose. 
     
     
         6 . The method of  claim 1 , characterized in that the method comprises a step of purifying the obtained erythropoiesis stimulating protein after step c) and prior to the treatment with sialyltransferase. 
     
     
         7 . The method of  claim 1 , characterized in that step d) is performed using sialyltransferase ST3. 
     
     
         8 . The method of  claim 1 , wherein the sialic acid free erythropoiesis stimulating protein comprises a relative frequency of N-linked glycans that include a sialic acid of 2%-0%. 
     
     
         9 . Use of the method of  claim 1  for increasing the number of poly-N-acetyl lactosamine repeats on the N-glycans of the erythropoiesis stimulating protein. 
     
     
         10 . Use of the method of  claim 1  for providing erythropoiesis stimulating protein with 2 or more poly-N-acetyl lactosamine repeats per N-glycosylation site. 
     
     
         11 . In vitro glycoengineered erythropoiesis stimulating protein, wherein the average number of poly-N-acetyl lactosamine repeats per erythropoiesis stimulating protein is more than 10, produced by a method of any one of  claims 1  to  8 . 
     
     
         12 . A population of erythropoiesis stimulating proteins, characterized in that the relative frequency of N-linked glycans comprising more than two poly-N-acetyl lactosamine repeats on each N-glycosylation site is more than 90%, and characterized in that the average number of poly-N-acetyl lactosamine repeats per erythropoiesis stimulating protein is more than 10. 
     
     
         13 . The population of erythropoiesis stimulating proteins of  claim 12 , wherein the population of erythropoiesis stimulating proteins is free of erythropoiesis stimulating proteins with zero or one poly-N-acetyl lactosamine repeats. 
     
     
         14 . The method of  claim 1 , the use of  claim 9  or  10 , the in vitro glycoengineered erythropoiesis stimulation protein of  claim 11 , or the population of erythropoiesis stimulating proteins of one of  claim 12  or  13 , wherein the erythropoiesis stimulating protein is erythropoietin. 
     
     
         15 . Use of the method of  claim 1  for improving the specific activity of the erythropoiesis stimulating protein.