PMID- 28832690
OWN - NLM
STAT- MEDLINE
DCOM- 20171107
LR  - 20181113
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 12
IP  - 8
DP  - 2017
TI  - Development of a fluorescent reporter system for monitoring ER stress in Chinese 
      hamster ovary cells and its application for therapeutic protein production.
PG  - e0183694
LID - 10.1371/journal.pone.0183694 [doi]
LID - e0183694
AB  - Mammalian cell expression systems have become a workhorse for the production of 
      biotherapeutic proteins. As such, there is an ever increasing demand for higher 
      productivity from these expression platforms to reduce manufacturing costs. While 
      great advances have been made in the optimization of culture conditions and cell 
      line selection to improve productivity, protein mis-folding remains a common 
      limitation to high levels of production of therapeutic proteins. Accumulation of 
      mis- and unfolded protein in the endoplasmic reticulum (ER) causes ER stress and 
      initiates the unfolded protein response (UPR) that results in an activation of 
      protein folding machinery, translation attenuation in an effort to proper folding 
      of the newly synthesized peptides or may even lead to apoptosis if the correct 
      folding is not restored. As a result, UPR associated apoptosis often results in 
      lower protein expression. To better understand the molecular mechanisms in these 
      pathways, we developed a reporter construct that detects Inositol-requiring 
      enzyme 1 (IRE1)-alpha mediated splicing of X-box binding protein 1 (XBP1) to 
      monitor the course of UPR activation in cell lines expressing monoclonal 
      antibodies. Using this reporter we observed a clear activation of UPR in cells 
      treated with known ER stress causing pharmacological agents, such as Tunicamycin 
      (Tm) and Thapsigargin (Tg), as well as in stable IgG expressing cells during 
      fed-batch cultures. Furthermore, we developed a stress metric that we term as ER 
      stress index (ERSI) to gauge basal ER stress in cells which we used as a 
      predictive tool for isolation of high IgG expressing cell lines. This reporter 
      system, with its ability to monitor the stress involved in recombinant protein 
      expression, has utility to assist in devising engineering strategies for improved 
      production of biotherapeutic drugs.
FAU - Roy, Gargi
AU  - Roy G
AUID- ORCID: 0000-0002-8391-5067
AD  - Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, 
      Maryland, United States of America.
FAU - Zhang, Shu
AU  - Zhang S
AD  - Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, 
      Maryland, United States of America.
FAU - Li, Lina
AU  - Li L
AD  - Cell Culture and Fermentation Sciences, MedImmune LLC, Gaithersburg, Maryland, 
      United States of America.
FAU - Higham, Eileen
AU  - Higham E
AD  - Cell Culture and Fermentation Sciences, MedImmune LLC, Gaithersburg, Maryland, 
      United States of America.
FAU - Wu, Herren
AU  - Wu H
AD  - Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, 
      Maryland, United States of America.
FAU - Marelli, Marcello
AU  - Marelli M
AD  - Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, 
      Maryland, United States of America.
FAU - Bowen, Michael A
AU  - Bowen MA
AD  - Antibody Discovery and Protein Engineering, MedImmune LLC, Gaithersburg, 
      Maryland, United States of America.
LA  - eng
PT  - Journal Article
DEP - 20170823
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Recombinant Proteins)
SB  - IM
MH  - Animals
MH  - CHO Cells
MH  - Cricetinae
MH  - Cricetulus
MH  - *Endoplasmic Reticulum Stress
MH  - Fluorescent Dyes
MH  - *Genes, Reporter
MH  - Recombinant Proteins/biosynthesis
MH  - Unfolded Protein Response
PMC - PMC5568292
COIS- Competing Interests: This study was funded by MedImmune LLC. Gargi Roy, Shu 
      Zhang, Lina Li, Eileen Higham, Herren Wu, Marcello Marelli and Michael A Bowen 
      were employed by MedImmune during study design, data collection and analysis and 
      decision to publish. The cell lines used in the study express proprietary 
      products that are currently in clinical development and so cannot be made 
      available. This alters the authors' adherence to all the PLOS ONE policies on 
      sharing data and materials, as detailed online in the guide for authors. However, 
      the described experimental strategy has applicability for other recombinant 
      proteins produced from CHO cells and the relevant methods are shared in detail in 
      the article for others to use.
EDAT- 2017/08/24 06:00
MHDA- 2017/11/08 06:00
PMCR- 2017/08/23
CRDT- 2017/08/24 06:00
PHST- 2017/05/08 00:00 [received]
PHST- 2017/08/09 00:00 [accepted]
PHST- 2017/08/24 06:00 [entrez]
PHST- 2017/08/24 06:00 [pubmed]
PHST- 2017/11/08 06:00 [medline]
PHST- 2017/08/23 00:00 [pmc-release]
AID - PONE-D-17-17712 [pii]
AID - 10.1371/journal.pone.0183694 [doi]
PST - epublish
SO  - PLoS One. 2017 Aug 23;12(8):e0183694. doi: 10.1371/journal.pone.0183694. 
      eCollection 2017.