title: A sensitive WST-8-based bioassay for PEGylated granulocyte colony stimulating factor using the NFS-60 cell line.
abstract: BACKGROUND
  Granulocyte colony stimulating factor (G-CSF) has been commonly used to treat neutropenia caused by chemotherapy, radiotherapy, and organ transplants. Improved in vitro efficacy of G-CSF has already been observed by conjugating it to polyethylene glycol (PEG).
  
  
  OBJECTIVE
  The in vivo bioassay using tetrazolium dye with the NFS-60 cell line has been recommended for G-CSF but no such monographs are available for PEGylated G-CSF in pharmacopeias. In the present study, the assay recommended for G-CSF was evaluated for its suitability to PEGylated G-CSF.
  
  
  METHODS
  The generally used MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based assay was compared with a bioassay employing a water-soluble tetrazolium dye, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium], using NFS-60 cells at a concentration of 7 × 10(5) cells/ml against 800 IU/ml of PEGylated G-CSF at 24, 48, 72, and 72 h time points to determine the efficacy of PEGylated G-CSF. Further, the optimized WST-8 dye-based assay was used to test the potency of various commercially available PEGylated G-CSF preparations.
  
  
  RESULTS
  The results demonstrated enhanced sensitivity of the WST-8-based assay over the conventional MTS-based assay for determining the potency of PEGylated G-CSF using the NFS-60 cell line.
  
  
  CONCLUSIONS
  Our study demonstrates the potential application of WST-8-based bioassays for other biotherapeutic proteins of human and veterinary interest.
authors.affiliation: Gennova Biopharmaceuticals Ltd., Rajeev Gandhi International Biotech Park , Phase-II, Pune , India .
authors.forename: Krishna
authors.initials: K
authors.lastname: Tiwari
authors.forename: Madan
authors.initials: M
authors.lastname: Wavdhane
authors.forename: Shafiul
authors.initials: S
authors.lastname: Haque
authors.forename: Thavendran
authors.initials: T
authors.lastname: Govender
authors.forename: Hendrik G
authors.initials: HG
authors.lastname: Kruger
authors.forename: Maheshwari K
authors.initials: MK
authors.lastname: Mishra
authors.forename: Ramesh
authors.initials: R
authors.lastname: Chandra
authors.forename: Dileep
authors.initials: D
authors.lastname: Tiwari
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D051379:Mice; D011092:Polyethylene Glycols / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D013778:Tetrazolium Salts / Q000494:pharmacology*
chemical_list: C476329:2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D016179:Granulocyte Colony-Stimulating Factor; D011092:Polyethylene Glycols; C423652:pegylated granulocyte colony-stimulating factor
keywords: Bioassay; G-CSF; PEGylation; neutropenia; tetrazolium dye