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We have developed a simple and accurate isocratic HPLC method, without any prederivatisation, for the determination of glucose, lactate, glutamine, glutamate, pyrrolidone carboxylic acid and alanine in samples from mammalian cell cultures. The method has been successfully validated with enzyme analysis for each of the compounds. Quantification of pyrrolidone carboxylic acid makes the correction for glutamine decrease due to chemical decomposition very simple and accurate, and avoids some possibly erroneous calculations.

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In the context of process transfer, test runs and validation runs done in the start-up phase of a GMP facility aimed at producing a monoclonal antibody (MAb) with CHO cells, scale-down experiments (satellite cultures) were performed at the 2 L bioreactor size in parallel with 13.5 KL production bioreactors.Time courses of growth parameters (packed cell volume, cell count, viability) as well as other physicochemical parameters (glucose, lactate, ammonia, pCO 2 ) gathered at the small scale over several runs were established and compared to data coming from GMP production lots. Monoclonal antibody titer was determined and specific productivity calculated. Kinetic data were shown to compare well between both scales allowing to establish a scale down baseline model for lab-scale experiments with defined cultivation parameters. A comparison between both scales is presented here.The scale-down model, representing the performance of the commercial process, can be useful for technical support/troubleshooting activities in relationship with large scale workshops to assess for performance of cells, media or equipment in case of discrepancies observed during GMP batches (investigation studies).Scale down studies were part of the set-up of a technical support lab nearby the production workshops. This lab might be essential in the case small scale validation, new raw materials testing (new lot, quality change, other issue), analytical equipment evaluation, etc., as well as training of production staff, are needed and may help addressing regulatory expectations.

A murine hybridoma line (Zac3), secreting an IgA monoclonal antibody, was cultivated in different systems: a BALB/c mouse, a T-flask, a stirred-tank bioreactor and a hollow fiber reactor. These systems were characterized in terms of cell metabolism and performances for IgA production. Cultures in T-flask and batch bioreactor were found to be glutamine-limited. Ammonia and lactate were produced in significant amounts. IgA productivity was found to be constant and growth associated. Final IgA concentration was similar in both systems. In fed-batch cultures, supplemented with glutamine and glucose, maximum viable cell concentration was increased by 60% and final IgA concentration by 155%. The hollow fiber reactor was able to produce very large amounts of IgA at very high concentrations, similar to the value found in ascites fluid. The productivity ofZac3 is similar to the values reported for IgG-producing cell lines.