PMID- 34154738
OWN - NLM
STAT- MEDLINE
DCOM- 20210924
LR  - 20210924
IS  - 1873-4863 (Electronic)
IS  - 0168-1656 (Linking)
VI  - 324S
DP  - 2020
TI  - Comparison of two glycoengineering strategies to control the fucosylation of a 
      monoclonal antibody.
PG  - 100015
LID - S2590-1559(20)30002-0 [pii]
LID - 10.1016/j.btecx.2020.100015 [doi]
AB  - Core fucosylation of an Fc N-linked glycan affects antibody effector functions, 
      as the absence of fucose increases the antibody dependent cell cytotoxicity 
      (ADCC) response with increased binding to the Fcgamma receptors. The work presented 
      here compares two different approaches to incrementally reduce core fucosylation 
      of a camelid heavy chain antibody, EG2-hFc expressed in CHO cells which targets 
      the EGFR receptor. The first method uses a fucosyltransferase (FUT) inhibitor, 2- 
      fluoro peracetylated fucose (2FF), which was added to cell cultures expressing 
      the EG2-hFc antibody in increasing concentrations up to 50muM. At this 
      concentration there was no observed effect on cell growth. Glycan analysis was 
      performed on antibodies collected from culture samples using HILIC-HPLC. The 
      inhibitor reduced total fucosylation from 80% to 17.5% at 20muM 2FF. The second 
      method involved transfecting the EG2-hFc producing cells with a prokaryotic GDP- 
      6-deoxy-D-lyxo-4-hexulose reductase (RMD) gene in order to deflect the fucose de 
      novo pathway into producing rhamnose which is not incorporated into a glycan. 
      Stable clones from transfected pools were isolated following flow cytometry using 
      the green fluorescent protein (GFP) marker which was co-expressed with the RMD 
      gene. High expressing RMD clones reduced the fucosylation of the antibody glycan 
      to as low as 16%. The addition of 2FF to cultures of these RMD clones reduced the 
      fucosylation level even further to 3% of the antibody glycan. An incremental 
      increase in fucosylation was obtained by step-wise addition of fucose (up to 1 
      mM) to the RMD cells, in which the fucosylation level increased to a maximum of 
      87%. We also used ESI-MS to analyze the fucosylation pattern of EG2-hFc with 
      addition of increasing concentrations of 2FF. This showed that 2FF inhibits the 
      addition of fucose in a concentration- dependent and specific manner with the 
      inhibition of fucose occurring one fucose at a time. Control cultures showed the 
      presence of a predominant peak indicating two fucose moieties per antibody. As 
      the 2FF inhibitor concentration was increased peaks corresponding to one fucose 
      per antibody and non-fucosylated antibody predominated with a gradual decrease of 
      the 2 fucose peak to insignificance at 15 muM 2FF.
CI  - Copyright (c) 2020. Published by Elsevier B.V.
FAU - Mishra, Neha
AU  - Mishra N
AD  - Department of Microbiology, University of Manitoba, Winnipeg, R3T 2N2, Canada.
FAU - Spearman, Maureen
AU  - Spearman M
AD  - Department of Microbiology, University of Manitoba, Winnipeg, R3T 2N2, Canada.
FAU - Donald, Lynda
AU  - Donald L
AD  - Department of Microbiology, University of Manitoba, Winnipeg, R3T 2N2, Canada.
FAU - Perreault, Helene
AU  - Perreault H
AD  - Department of Chemistry, University of Manitoba, Winnipeg, R3T 2N2, Canada.
FAU - Butler, Michael
AU  - Butler M
AD  - Department of Microbiology, University of Manitoba, Winnipeg, R3T 2N2, Canada; 
      National Institute for Bioprocessing Research & Training (NIBRT), Fosters Avenue, 
      Dublin4, Ireland. Electronic address: michael.butler@nibrt.ie.
LA  - eng
PT  - Journal Article
DEP - 20200220
PL  - Netherlands
TA  - J Biotechnol
JT  - Journal of biotechnology
JID - 8411927
RN  - 0 (Antibodies, Monoclonal)
RN  - 28RYY2IV3F (Fucose)
SB  - IM
MH  - Animals
MH  - *Antibodies, Monoclonal
MH  - CHO Cells
MH  - Cricetinae
MH  - Cricetulus
MH  - *Fucose
MH  - Glycosylation
OTO - NOTNLM
OT  - ADCC
OT  - Camelid heavy chain antibody
OT  - Fucosylation
OT  - Glycosylation
OT  - Monoclonal antibody therapeutics
COIS- Declaration of Competing Interest The authors declare that they have no known 
      competing financial interests or personal relationships that could have appeared 
      to influence the work reported in this paper
EDAT- 2020/01/01 00:00
MHDA- 2021/09/25 06:00
CRDT- 2021/06/22 05:42
PHST- 2019/08/16 00:00 [received]
PHST- 2020/01/16 00:00 [revised]
PHST- 2020/02/14 00:00 [accepted]
PHST- 2021/06/22 05:42 [entrez]
PHST- 2020/01/01 00:00 [pubmed]
PHST- 2021/09/25 06:00 [medline]
AID - S2590-1559(20)30002-0 [pii]
AID - 10.1016/j.btecx.2020.100015 [doi]
PST - ppublish
SO  - J Biotechnol. 2020;324S:100015. doi: 10.1016/j.btecx.2020.100015. Epub 2020 Feb 
      20.