Knowledge of concentrations of intracellular metabolites is important for quantitative analysis of metabolic networks. As far as the very fast response of intracellular metabolites in the millisecond range is concerned, the frequently used pulse technique shows an inherent limitation. The time span between the disturbance and the first sample is constrained by the time necessary to obtain a homogeneous distribution of the pertubation within the bioreactor. For determination of rapid changes, a novel sampling technique based on the stopped-flow method has been developed. A continuous stream of biosuspension leaving the bioreactor is being mixed with a glucose solution in a turbulent mixing chamber. Through computer-aided activation of sequentially positioned three-way valves, different residence times and thus reaction times can be verified. The application of this new sampling method is illustrated with examples including measurements of adenine nucleotides and glucose-6-phosphate in Saccharomyces cerevisiae as well as measurements related to the PTS system in Escherichia coli.

Near-infrared (NIR) spectroscopy as a process monitoring and process supervision technique is reviewed in the context of biomanufacturing.An industrial pilot-plant mammalian cell cultivation process has been chosen to illustrate the use of on-line in-situ NIR monitoring by means of an immersion transflectance NIR probe.NIR calibration development must be performed carefully and should incorporate a number of steps to obtain a properly validated model which exhibits long-term robustness and is independent of process scale. A description of such good modelling practises is given. In general, NIR can be as accurate as the reference methods employed and at least as precise provided that sufficient spectral selectivity and sensitivity exists.NIR can also be used as a direct technique for very fast process monitoring and process supervision, thus enabling one to follow the trajectory of a process. This alternative to the indirect use of NIR through laborious calibration development with direct reference methods has been little explored. Since NIR is sensitive to both chemical and physical properties, the analysis of whole samples enables relevant process information to be captured and thus generates better process state estimates than by simply looking at defined process parameters one at a time.

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Process analytical technology (PAT), the regulatory initiative for incorporating quality in pharmaceutical manufacturing, is an area of intense research and interest. If PAT is effectively applied to bioprocesses, this can increase process understanding and control, and mitigate the risk from substandard drug products to both manufacturer and patient. To optimize the benefits of PAT, the entire PAT framework must be considered and each elements of PAT must be carefully selected, including sensor and analytical technology, data analysis techniques, control strategies and algorithms, and process optimization routines. This chapter discusses the current state of PAT in the biopharmaceutical industry, including several case studies demonstrating the degree of maturity of various PAT tools. Graphical Abstract Hierarchy of QbD components.