The use of benchtop bioreactors (BRs) for the development of mammalian cell perfusion cultures is expensive and time consuming, given its complexity in equipment and operation. Scale-down models, going from liter to milliliter scale, are needed to support the rapid determination of suitable operating conditions in terms of viable cell density (VCD), perfusion rate, and medium composition. In this study, we compare the performance of steady-state perfusion cultures in orbitally shaken tube and BR systems for a given Chinese hamster ovary cell line. The developed scale-down model relied on a daily workflow designed to keep the VCD constant at specific target values. This includes: cell count, removal of excessive cells (bleeding), spin down of remaining cells, harvest of cell-free supernatant, and resuspension in fresh medium. Steady-state cultures at different VCD values, medium exchange rates and working volumes were evaluated. Shake-tube perfusion cultures allowed the prediction of cell-specific growth, glucose consumption, ammonia, and monoclonal antibody production rates for much larger BRs, but not lactate (LAC) production rates. Although charge variant profiles remained comparable, different glycosylation patterns were obtained. The differences in LAC production and glycosylation probably resulted from the discontinuous medium exchange, the poor carbon dioxide removal, and the deficient pH control. Therefore, if requested by the specific process to be developed, product quality has to be fine-tuned directly in the BR system. Altogether, the developed strategy provides a useful scale-down model for the design and optimization of perfusion cultures with strong savings in time and media consumption.

Lesch-Nyhan disease is caused by a deficiency of the purine salvage enzyme, hypoxanthine phosphoribosyl transferase (HPRT). The link between HPRT deficiency and the neuropsychiatric symptoms is unknown. In rat B103 neuroblastoma cell membranes and mouse Neuro2a neuroblastoma cell membranes, nucleoside 5 -triphosphatase (NTPase) activity is substantially reduced, whereas in fibroblast membranes from HPRT knock-out mice, NTPase activity is increased. Candidate genes for these NTPase activity changes are ecto-nucleoside 5 -triphosphate diphosphohydrolases (NTPDases). Therefore, we studied expression of NTPDases in B103 cells, Neuro2a cells and skin fibroblasts by reverse transcriptase polymerase chain reaction and restriction enzyme digestion of amplified cDNA fragments. In B103 cells, expression of NTPDases 1, 3 and 6 decreased, whereas expression of NTPDases 4 and 5 increased in HPRT deficiency. In Neuro2a cells, expression of NTPDases 3-6 increased in HPRT deficiency. In fibroblasts, NTPDase 3 expression decreased, and expression of NTPDases 4-6 increased in HPRT deficiency. Collectively, there are complex decreases and increases in NTPDase isoform expression in HPRT deficiency that depend on the specific cell type and species studied. These changes in NTPDase expression may reflect an (insufficient) attempt of cells to compensate for the changes in nucleotide metabolism caused by HPRT deficiency. © 2007 Elsevier Ireland Ltd. All rights reserved.Keywords: Lesch-Nyhan disease; Hypoxanthine phosphoribosyl transferase; Ecto-nucleoside 5 -triphosphate diphosphohydrolase; Fibroblasts; Neuroblastoma cells Lesch-Nyhan disease (LND) is caused by a defect of hypoxanthine phosphoribosyl transferase (HPRT) [10,6]. Clinical symptoms of LND are hyperuricemia, gouty arthritis, mental retardation, dystonia, spasticity, delayed motor development and a compulsive form of self-injurious behavior. The link between HPRT deficiency and the neuropsychiatric symptoms is unknown. To study the biochemical basis for the neuropsychiatric symptoms in LND, a HPRT knock-out mouse was generated [15]. Similar to LND patients, there are abnormalities in dopaminergic neurotransmission in the HPRT knock-out mouse [7,16]. In addition, several HPRT-deficient cell lines including primary human skin fibroblasts, immortalized mouse skin fibroblasts, mouse Neuro2a neuroblastoma cells and rat B103 neuroblastoma are used as biochemical models for LND [18,2,4,12,11]. In a recent study [11] we showed that nucleoside 5 -triphosphatase (NTPase) activity in B103-and Neuro2a cell membranes is reduced in HPRT-deficiency. In contrast, in mouse skin fibroblast membranes, NTPase activity is increased in HPRT deficiency. Thus, altered NTPase activity is a hallmark of HPRT deficiency and these changes are cell type-and species-specific. However, it is very difficult to reveal the molecular identity of the NTPase(s) in complex systems by measuring enzyme activity. Specifically, analysis of enzyme activity with various substrates and inhibitors revealed monoph...

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