Journal of Biotechnology 117 (2005) 325–336 A multiwavelength fluorescence probe: Is one probe capable for on-line monitoring of recombinant protein production and biomass activity? Steve Hisiger, Mario Jolicoeur ∗ Canada Research Chair on the Development of Metabolic Engineering Tools, Bio-P 2 Research Unit, Department of Chemical Engineering, ´ Ecole Polytechnique de Montr´ eal, P.O. Box 6079, Centre-ville Station, Montr´ eal, Que., Canada Received 18 July 2004; received in revised form 16 March 2005; accepted 18 March 2005 Abstract Monitoring cell culture performance requires maximizing the number and the quality of measured parameters and in situ 2D fluorescence spectroscopy could allow intensification of simultaneous data acquisition. The use of a multiwavelength fluorescence probe is proposed for monitoring GFP-producing cultures in bioreactor. The yeast Pichia pastoris and NSO mammalian cells were studied as model systems. Tryptophan, NAD(P)H and riboflavins (riboflavin, FMN, FAD) signals were effective for on-line yeast biomass estimation during the growth phase. During the GFP production phase, in situ measurements of the GFP concentration from the fluorescence probe were well correlated with off-line analyses. Tryptophan and NAD(P)H signals diverged from that of biomass during GFP production. With NSO mammalian cells, results showed that the culture parameters have to be optimized for the use of a fluorescence probe. The use of serum and phenol-red interfered with NAD(P)H and riboflavins fluorescence signals. Nevertheless, it appears that a multiwavelength probe could be useful for culture monitoring of biomass, cell activity and recombinant protein expression in an optimized culture medium. © 2005 Elsevier B.V. All rights reserved. Keywords: In situ fluorescence spectroscopy; GFP; NAD(P)H; Tryptophan; Pichia pastoris; NSO 1. Introduction Bioprocess development and industrial biotechnol- ogy require adequate monitoring of cell culture state ∗ Corresponding author. Tel.: +1 514 340 4711x4525; fax: +1 514 340 4159. E-mail address: mario.jolicoeur@polymtl.ca (M. Jolicoeur). parameters. In addition to commonly used O 2 , CO 2 , pH, redox potential and conductivity probes, attention is now being placed on developing probes capable to follow on-line and non-invasively cellular physiolog- ical state, cell mass and production level in bioprod- ucts. Many metabolites such as pyridine nucleotides (NADH and NADPH as NAD(P)H), tryptophan, ri- boflavins (riboflavin, FMN, FAD) and pyridoxine are 0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.03.004