TY  - THES
AB  - The increasing number of approved biopharmaceuticals produced in mammalian  cell lines 
emphasizes the importance of posttranslational modifications. A consistent product quality is 
a major goal in the production of biotherapeutics, especially recombinant glycoproteins. In 
contrast  to  the  biosynthesis  of  the  amino  acid  chain,  posttranslational  modifications  like 
glycosylation and protein folding may be influenced by changes within the production process. 
Glycosylation of recombinant proteins is important for their function, half-life and stability in 
vivo. In this study, glycosylation during a batch cultivation is evaluated. Cells and secreted 
protein  are  exposed  to  continuous  change  during  a  cultivation  batch  process.  In  early 
cultivation the supply situation is excellent and a complete glycosylation can be expected. 
However,  the  product´s long  dwell  time  may  influence  product quality.  Later  in  cultivation, 
substrate supply decreases while metabolites increase, which result in changes in cultivation. 
As the dwell time of the product produced at later cultivation stages is short, an extracellular 
degradation  has  a  less  significant  effect.  A  time-dependent  sampling  was  established  to 
examine the glycosylation at different cultivation stages. This sampling allows a comparison 
between product from early cultivation and product resulting from later cultivation stages. In 
this study, the glycosylation of five recombinant proteins (erythropoietin, an IgG1 antibody, 
C1-inhibitor, α-1-antitrypsin and antithrombin III) was analysed. The proteins were produced 
in  different  cell  lines  in batch  cultivations.  Products from three  different  cultivation  periods 
could be evaluated with the time-dependent sampling. Additional glycoprotein stability was 
considered after exposure to different cultivation-like scenarios to estimate the influence of 
extracellular processes.  
Results of the time-dependent analysis of the glycosylation showed that in early cultivation 
stages (excellent supply situation) the degree of galactosylation and sialylation (for sialylated 
products) was higher than in later cultivation stages. Galactosylation and sialylation decreased 
during  cultivation,  antennarity  however  stayed  consistent.  The  antibody  showed  less  core 
fucosylation but an increase in high mannose structures in later cultivation stages. Antennary 
fucoses  were  found  in  C1-inhibitor  and  α-1-antitrypsin.  The  antennary  fucosylation  of 
C1-inhibitor decreased with the cultivation time. Glycosylation was dependent on glycosidase 
activity in the supernatant, but not on chemical processes. This was confirmed via the stability 
experiments.  It  is  likely  that  the  secreted  product  is  degraded  by  glycosidases  with  a 
decreasing viability of the cell culture due to cell lysis. With the evidence of sialidase activity 
this assumption was confirmed. Furthermore, it was shown that sialidase activity is dependent 
on the producing cell line. Changes  of  intracellular  processes  due  to  varying  substrate  supply  and  accumulating 
metabolites were found to be a further reason for changes in glycosylation during a batch 
cultivation.  Mainly  glucose  supply  and  metabolism  of glutamine  appeared  to  influence  the 
glycosylation process.  
With the examination of five glycoproteins produced in different cell lines it could be shown 
that  product  heterogeneity  is  cell  line  dependent  as  well  as  product  dependent.  Such 
knowledge combined with the awareness of the requirements of the biotherapeutic is essential 
for optimising a production process.
DA  - 2019
DO  - 10.4119/unibi/2935606
LA  - ger
PY  - 2019
TI  - Glykosylierung rekombinant erzeugter Pharmazeutika in Abhängigkeit der Kultivierungsbedingungen
UR  - https://nbn-resolving.org/urn:nbn:de:0070-pub-29356065
Y2  - 2024-11-24T16:15:14
ER  -