Improving speed and quality, and reducing the cost of technology transfers is becoming increasingly important in the biopharmaceutical
industry. Microbial fermentation processes have challenging equipment requirements, such as high heat and oxygen transfer
rates, and ensuring minimal and consistent medium concentration changes resulting from condensate gain during steam-in-place
and evaporative losses during media hold and fermentation. This article presents a platform approach to equipment characterization
for microbial fermenters. These tests are used to gain understanding of the equipment capabilities before starting process
runs. The platform presented here outlines a wet testing approach that has been successfully executed at pilot scale (300-L)
for a dual-purpose reactor (used for both mammalian cell culture and microbial fermentation) and in a large-scale (10,000-L)
fermenter. Comprehensive equipment characterization using a platform approach streamlines the technology transfer and maximizes
success rates during process runs.
Eden Biodesign Ltd.
An optimized and fast-growing microbial culture is a dynamic process, and ensuring that the equipment is suitable for meeting
these challenges is necessary for project success. These cultures tend to have a high oxygen demand that the fermenter must
be able to meet to sustain cell growth and desired productivity. The culture also generates significant amounts of heat that
the fermenter must remove to maintain temperature control. To ensure success during actual process runs, it is important to
understand oxygen supply and heat removal capabilities before operating the process in the fermenter. Water-based tests were
developed to characterize heat removal and oxygen supply capacity to understand if any equipment modifications would be required
to meet process requirements. This testing should be performed early in technology transfer facility fit activities to allow
time for equipment modifications, if needed.
Microbial media generally are batched into the fermenter and then steam sterilized-in-place (SIP). The SIP cycle is automated
to ensure control within the required temperature range. Temperatures outside the acceptable range could result in insufficiently
sterilized medium or overheated medium. Additionally, evaporation or condensation can occur during the SIP cycle. The change
in fermenter weight should be characterized or eliminated during SIP operations to ensure the target medium concentration
for optimal growth is achieved. After sterilization, the fermentation medium often is held at specified conditions until inoculation.
Determining the evaporation rate during the medium hold is essential for achieving the correct medium concentration at the
time of inoculation. To compensate for the evaporation rate, additional water can be added during media preparation, and adjustments
to the air flow rate can be made to minimize evaporative losses. Water-based testing was developed to understand parameters
around media SIP and hold conditions to ensure the target starting media concentration would be met.
Materials and Methods
Table 1 lists the fermenter dimensions and operational settings for both pilot and large scales.
Table 1. Pilot- and large-scale fermenter characteristics. The agitation setting was scaled between the pilot- and large-scale
fermenters by maintaining the ratio of agitator power draw (P) per unit volume (V).
Heat Removal Characterization
To maintain constant temperature control the fermenter must have sufficient heat removal capacity to remove metabolic heat
generated by the cells and mechanical heat generated by the agitator. A common heat transfer scale-up challenge stems from
the fact that the relative heat transfer area decreases with increasing fermenter scale. The heat transfer rate (HTR) can
be calculated using a simple heat transfer equation:
in which m is the mass of water in the fermenter (kg), C
p is the heat capacity of water at 37 °C (4.181 kJ/kg/°C),1
T is the temperature of water in the fermenter (°C), t is time (h), and dT/dt is the rate of temperature change as the slope of water temperature versus time curve.
Strategies for maintaining temperature control involve equipment modifications such as: lowering chilled water or glycol temperature
to increase the driving force for heat removal (dT/dt), ensuring jacket and coils have minimum resistance, and adding internal cooling coils to increase heat transfer surface
The fermenters were filled to different target weights and heated to approximately 75 °C. The temperature set point was changed
to 10 °C, which resulted in a 100% cooling output on the temperature control loop. The rate of temperature change was estimated
by the slope of the linear portion of the temperature-versus-time curve from 41 °C to 34 °C. This range encompasses the process
temperature set point of 37 °C. The HTR was then calculated using Equation 1.