Production et glycosylation d'EPO par cellules CHO : caractérisation par électrophorèse capillaire au cours de procédés discontinus

Abstract : The scope of this work is to improve the control of the glycosylation of recombinant proteins produced by animal cell culture by studying the influence of the culture process on the glycan structure of a model protein : CHO cell produced EPO. For building the system, several step were necessary : the generation of a high-EPO-producer CHO cell clone, the set-up of an immuno-affinity purification method and the adaptation of a technique for analysis of protein glycosylation to our system constraints. Then, two parameters of the culture process were studied more precisely : the culture time and the presence of serum. By monitoring EP9 glycosylation in the course of serum-containing batch cultures, we observed a progressive desialylation of the protein during the second part of the culture and an important cell lysis. Since a significative siaiidase activitv was also found in the same time, this phenomenon seemed to be induced by an enzymatic degradation following cell lysis in serum-containing cultures. On the other band, after serum-free adaptation, EPO sialylation was found constant during the whole serum-free cultures. Anaiysis of EPO glycosylation produced in serum-containing medium by serum-free adapted cells showed that the occurring desialylation was directly linked to the presence of serum and not to the serum-free adaptation procedure. To conclude, this work clearly shows the important influence of sorne process parameters on the quality of EPO when produced in our system.
Document type :
Theses
Complete list of metadatas

https://hal.univ-lorraine.fr/tel-01749708
Contributor : Thèses Ul <>
Submitted on : Thursday, March 29, 2018 - 12:24:19 PM
Last modification on : Thursday, January 31, 2019 - 1:27:49 AM
Long-term archiving on : Friday, September 14, 2018 - 9:53:53 AM

File

INPL_T_2003_LE_FLOCH_F.pdf
Files produced by the author(s)

Identifiers

  • HAL Id : tel-01749708, version 1

Collections

Citation

François Le Floch. Production et glycosylation d'EPO par cellules CHO : caractérisation par électrophorèse capillaire au cours de procédés discontinus. Alimentation et Nutrition. Institut National Polytechnique de Lorraine, 2003. Français. ⟨NNT : 2003INPL079N⟩. ⟨tel-01749708⟩

Share

Metrics

Record views

40

Files downloads

198