Modulation of nonspecific binding in ultrafiltration protein binding studies

Kyoung Jin Lee, Rachel Mower, Tom Hollenbeck, Jesus Castelo, Nikole Johnson, Perry Gordon, Patrick J. Sinko, Kevin Holme, Yong Hee Lee

Research output: Contribution to journalArticlepeer-review

86 Scopus citations


Purpose. The aim of this study was to reduce or prevent nonspecific binding (NSB) of compounds to ultrafiltration (UF) protein binding (PB) testing units. Methods. UF units (regenerated cellulose, MWCO 10K) were used for PB and NSB measurements with or without pretreatment with 5% tween 80 (TW 80) or 5% benzalkonium chloride (BAK) on the filter membrane. Dosing solutions (10 μM) in human serum and pH 7.4 phosphate-buffered saline were centrifuged at 3,000 g and room temperature after 1-h incubation in UF testing units. In parallel, a 96-well equilibrium dialyzer was used for PB and NSB measurements in equilibrium dialysis (ED) at 37°C for 4 h. Samples of UF and ED were analyzed by LC/MS or LSC. Results. Severe NSB was observed for etoposide, hydrocortisone, propranolol, and vinblastine in UF. In contrast, TW 80 or BAK pretreatment on the filter membrane decreased the NSB from 87-95% to 13-64% without causing a significant change in membrane integrity. When NSB was below 50% as a result of pretreating agents, PB data of marker compounds were comparable to those of ED. Conclusions. The pretreated membrane with TW 80 or BAK showed significantly less NSB for compounds that had a tendency toward high membrane binding. A modified UF method with pretreatment improved the performance of UF and was able to produce comparable PB results to ED.

Original languageEnglish (US)
Pages (from-to)1015-1021
Number of pages7
JournalPharmaceutical research
Issue number7
StatePublished - Jul 1 2003

ASJC Scopus subject areas

  • Biotechnology
  • Molecular Medicine
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry
  • Pharmacology (medical)


  • Equilibrium dialysis
  • Nonspecific binding
  • Protein binding
  • Ultrafiltration


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