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April 01, 2009 (Vol. 29, No. 7)

Strategies for Measuring Viable Biomass

Novel Technique Aims to Extend Application of RF Impedance to Single-Use Bioreactors

  • Probes for Disposable Bioreactors

    Click Image To Enlarge +
    Figure 2. Flow-through viable biomass probe is used with silicone tubing.

    Many biopharma companies are now switching from conventional glass or stainless steel vessels to disposable, single-use bioreactors. On-line probes are provided in some disposable bioreactors but typically provide only limited options—just pH, temperature, and pO2. The majority of these bioreactors are used in cell culture for production of seed or for low cell densities using batch production processes where accurate measurement and control is not so critical and the systems often do not require validation. 

    New processes requiring cGMP production are now being developed using disposable bioreactors and consequently there will be the increasing demand for the same range of sensors (including viable biomass) used with conventional bioreactors.

    The Biomass Monitor with conventional reusable probes can be used with  most of the larger, single-use, disposable bioreactors. For example, with the Hyclone Single- Use Bioreactor (SUB) from Thermo Fisher Scientific, it is possible to use existing half-inch ports on the bags for insertion of a reusable 12 mm biomass probe designed to the correct insertion length. The Hyclone SUB uses a Pall Kleenpak connector to allow the presterilized probe to be inserted into the bioreactor, and there is a special tray to support the array of conventional stainless steel probes including pH, pO2, and biomass.  

    This approach has the advantage that the protocols and validation processes developed for using biomass probes with conventional bioreactors can be immediately transferred to the disposable bioreactor. The main disadvantage is that the insertion of any probe into the bioreactor can compromise the sterility of the system.   

    An alternative solution for measuring viable biomass in disposable (and conventional) bioreactors is to use a reusable, small diameter flow-through probe. The probe (Figure 2) has four annular electrodes on the inside making it ideal for insertion into a silicone tubing, recirculation loop from a disposable (or conventional) bioreactor.

    The flow-through probe can also be used for measuring the bleed off of biomass in a continuous or perfused bioreactor system or for confirming the transfer of the correct amount of seed to a production bioreactor. The latter application is similar to the process used in conventional brewery processes where the viable biomass probe is used to accurately dose the correct amount of live seed (in this case, yeast) into a production fermentor.

    A disposable biomass probe is also available from Aber Instruments. It has been designed to be welded into most single-use bioreactors and is suitable for bags with agitators (e.g., the Hyclone SUB) or those using the rocker type platform (e.g., the Appliflex single-use bioreactor from Applikon).   

    The disposable biomass probe has pure platinum electrodes with the same dimensions as the existing reusable production biomass probes with flush electrodes that are often used in cGMP manufacture. As both the Aber disposable and reusable biomass probes will produce the same radio-frequency field, this will allow users to directly compare viable cell density data between conventional and disposable bioreactors.

    The electrode support material is HDPE, which meets FDA and USP Class VI requirements, and the probe can withstand gamma sterilization and be stored for prolonged periods before use. The disposable probe is easily connected to a mini-lightweight preamplifier (Figure 3) so that the weight load or torque on the bag is minimal and the bulk of the electronics is then located well away from the bag. Trials of the single-use biomass probe are under way.

    The use of RF impedance to monitor and control cell culture processes is well established in conventional stainless steel or glass bioreactors, and it is crucial that RF impedance can also be applied to single-use bioreactors. Recent developments addressing the validation of the reusable probe and a complimentary range of disposable probes that can be used in bags will allow this technology to be used in confidence with most types of bioreactors from process development through to cGMP production.

  • Click Image To Enlarge +
    Figure 3. Disposable biomass probe is used with single-use bioreactors.