Fermentation remains a cornerstone of the modern biopharmaceutical industry due to its versatility and efficiency in producing a wide range of therapeutic products. By its very nature, fermentation facilitates the large-scale production of complex biomolecules. Its adaptability and cost-effectiveness make it an indispensable tool in this field. Scale-up and scale-down studies are critical components of fermentation process development. These studies ensure that processes developed on a small scale can be successfully transitioned to larger production scales. They help identify and address potential challenges that may arise due to changes in scale, thereby ensuring the efficiency, safety, and consistency of the final product.
Scaling Up
The purpose of scaling up the fermentation process is to translate the optimal operating conditions established in laboratory or pilot-scale fermenters to production-scale fermenters, thereby maximizing the productivity of the full-scale process. However, several essential tasks must be completed before scaling up the process: screening and improving host strains, adjusting the composition of the culture medium, and optimizing various process parameters. These tasks are typically conducted initially through parallel micro-scale culture setups for screening. Once the number of potential production strain candidates has been sufficiently narrowed, experiments are performed using benchtop culture equipment, such as 0.5 to 20 L stirred fermenters, for further strain characterization and the initial round of process parameter optimization. Finally, the process parameters are further refined in a pilot-scale facility.
The primary advantage of laboratory-scale cultures is their adaptability to various feeding schemes, and most standard sensor equipment can be integrated into the fermenters. However, a significant challenge associated with laboratory-scale fermenters, as well as larger pilot-scale fermenters, is the difficulty in comparing experiments across different scales with larger industrial fermenters. In real production environments, scale-up is often not based on mathematical process models or experimental design; instead, it typically relies on an empirical approach. In practice, process scale-up is usually conducted iteratively, by comparing the experimental results from the pilot plant with key data obtained from the industrial-scale process. If the results from the pilot plant can be replicated on the industrial scale, the scale-up is deemed successful. If not, additional rounds of pilot experiments may be conducted to investigate the impact of various process parameters on performance.
Fluid flow patterns in fermenters differ due to physical variations at different scales. In laboratory-scale fermenters, the fermentation broth can be considered perfectly mixed, while in large-scale fermenters, achieving 95% homogeneity may take only a few minutes. Insufficient mixing can lead to anaerobic conditions. Furthermore, gradients in glucose concentration, pH, and temperature may develop during production-scale fermentations. These heterogeneities in the biological environment can significantly impact biomass growth, which is a primary concern when scaling up a process.
The Reynolds number is a valuable metric for illustrating the differences in fluid flow regimes, as it varies with scale. When the same fermentation broth is used in fermenters of equivalent dimensions and with the same specific power input, a production-scale fermenter will yield a Reynolds number indicative of the turbulent regime, while a laboratory-scale fermenter will produce a Reynolds number characteristic of the transitional or laminar regime. These variations in mixing characteristics lead to differences in mass and heat transfer during the process. The transfer of oxygen into the fermentation broth and the removal of carbon dioxide are critical for the cultivation process. The volumetric mass transfer coefficient of oxygen (KLa) is often regarded as the key parameter for scale-up, indicating that successful scale-up depends on maintaining a constant KLa across different scales. Other significant scaling parameters include the oxygen transfer rate (OTR), specific power input (P/V), and superficial gas velocity (Vg). It is important to note that it is not feasible to keep all parameters consistent across scales; therefore, a suitable compromise must be reached. To achieve a high specific power input in small-scale fermenters, the agitation speed is typically very high, resulting in increased shear rates within the smaller vessel.
Scaling Down
Ideally, all the challenges associated with scaling up a process would be mitigated by conducting fermentation experiments at the required scale. However, this is often impractical due to prohibitive costs. These costs arise not only from the high consumption of substrates and energy but also, and particularly, from the loss of production time.
It is essential to recognize that scaling down presents a significant challenge for industrial fermentation technology. The primary objective of experiments conducted with small-scale equipment is to examine how variations in process parameters affect performance under conditions that are relevant to production-scale fermenters. Most biotechnological processes are designed for existing equipment, where a full-scale fermenter is installed and utilized for many years. Consequently, the range of available process equipment is established before a new process is designed, allowing bench-scale development to concentrate on maximizing specific productivity. This is contingent upon the availability of a suitable scaled-down version of the full-scale fermenter, ensuring that the results obtained in the smaller fermenter are applicable to the production scale.
Due to the need for cost-effective and rapid process development and optimization, a thorough understanding of critical process parameters, successful technology transfer, and a swift time to market, small-scale work is essential for acquiring initial knowledge. However, it can also pose a potential bottleneck for further development. Consequently, small-scale systems are receiving increasing attention. The objective of detailed studies on small-scale fermentation systems is twofold: first, to enhance our understanding of the engineering fundamentals of existing small-scale stirred systems, with a particular emphasis on identifying opportunities for improvement; and second, to design entirely new equipment, specifically scaled-down versions of large-scale fermenters. Experience has demonstrated that each small-scale system offers distinct benefits as well as unique challenges during cross-scale process development.
Shake flasks remain a widely used system for analyzing various fermentation conditions, such as growth rate, and for assessing the impact of these conditions on the initial recovery of exogenous proteins. The advancement of non-invasive optical sensing, combined with a solid understanding of engineering principles, enhances process comprehension and monitoring in these systems. The transfer of processes from shaking systems to stirred systems has been demonstrated to be feasible when appropriate scale-up criteria are applied. The development of milliliter-scale stirred fermenters has largely preserved geometrical similarities with benchtop reactors, positioning them as leaders in the application of existing scale-up methods based on specific power input, impeller tip speed, constant dissolved oxygen tension (DOT), oxygen mass transfer capacity (KLa), and mixing time.
Mass Transfer
Mass transfer is a critical factor in high biomass systems, as it is essential for the distribution of oxygen and nutrients, as well as the removal of potentially toxic compounds. The term and varies significantly depending on the specific organisms being cultured. Mass transfer encompasses various subprocesses that are greatly influenced by the viscosity of the fermentation broth. Consequently, the rheological properties of the fermentation broth are affected by both the biomass concentration and the morphology of the organism utilized as the expression system. The intricate interplay among these variables complicates the process of scaling up or down.
Agitation and aeration are essential prerequisites for nearly all types of fermentation and cell culture to ensure an adequate oxygen supply. However, agitation can also induce morphological changes that may impact product formation. Consequently, in an aerated stirred tank setup, the shear sensitivity of the organisms used becomes a critical consideration. Bacteria and yeast are generally regarded as more resilient to mechanical stress, and it has been proposed that their small size contributes to their resistance to shear stress, as they are smaller than the Kolmogorov turbulent microscale. As a result, their morphology is typically considered unaffected by mechanical mixing. Nevertheless, during scale-up, biomass concentration may decrease while viability can increase, which is attributed to the variations in homogeneity found in industrial-scale fermenters. Therefore, the challenge lies in disentangling the various effects to clearly associate the increase in productivity with the appropriate environmental conditions: specifically, the balance between the volume gradient in the fermentation medium and the internal gradient of the biomass, as well as the trade-off between higher OTR at lower viscosity and enhanced growth or secretion of proteins at higher biomass concentrations.
Monitoring and Control
The sensors commonly found in industrial fermenters are typically limited to pH, dissolved oxygen (DO), and temperature sensors, which are usually positioned at a single location within a large-volume vessel that often exhibits a concentration gradient. These sensors provide an average value for the entire process, which can be correlated to the conditions within the vessel; however, the information available regarding spatial heterogeneity is quite limited. The Proportional-Integral-Derivative (PID) controller is a standard method used to maintain the controlled variable at or near a specified set point or trajectory. Nevertheless, a significant challenge with the PID controller is its inability to ensure optimal fermentation performance consistently. The set point that the controlled variable must adhere to allows for optimal operation only under specific nominal conditions. When disturbances occur—such as changes in substrate quality or composition, moisture content, or ambient temperature—the set point must be adjusted accordingly, a process often referred to as monitoring or optimization.
A method to enhance the quality of online sensor data while adhering to Good Manufacturing Practice (GMP) regulations is the implementation of soft sensing techniques. This approach involves the simultaneous computational analysis of existing data to improve sensor accuracy and, consequently, the reliability of predictions. Through soft sensing, robust online measurements that are unaffected by time delays can be utilized to calculate the expected value of a new parameter that requires control. Additionally, soft sensing can be integrated into industrial applications within the framework of Quality by Design (QbD) and Process Analytical Technology (PAT) principles, as it offers a means to monitor processes in real time.
Computational fluid dynamics (CFD) methods possess high predictive power, and the reliability of these predictions is continually improving for laboratory, pilot, and full-scale fermentations. Such fluid dynamics models are increasingly utilized as powerful tools to assess mixing times, mass transfer, shear stress levels, and dead volumes. This type of numerical analysis can significantly facilitate experimental design and help reduce the number of experiments required. By evaluating certain configurations through CFD simulations, researchers can exclude them from experimental planning in advance based on the simulation results. This encompasses not only the geometric design of the fermenter but also operating modes, such as fed-batch operation, and the analysis of heterogeneity in the concentration of critical variables, including biomass concentration, substrate concentration, and product concentration.
DuoBioX® Explore Benchtop Multi-Parallel Glass Tank Fermenter
DuoBioX® Explore system from Duoning is a state-of-the-art microbial culture reactor designed for superior performance, comprehensive functionality, and user-friendly operation. Users can control one to eight glass tanks independently or in parallel using just a computer, significantly accelerating the process development timeline. The DuoBioX® Explore system is ideal for the development and research of fermentation processes within the biopharmaceutical industry.
500 mL, 3 L, 5 L, 7 L, 15 L and other tank volume specifications are available.
The satellite chassis design offers significant flexibility for system expansion, enabling rapid implementation of multi-link or parallel control for one to eight tanks. The Explore system facilitates easy parameter setting, process program execution, and data backup across different tanks, allowing for the comparison and analysis of trends from various batches on a single interface.
The operating system, developed on the WinCC industrial-grade architecture, complies with Good Manufacturing Practice (GMP) and 21 CFR Part 11 requirements. It is suitable for use in industries such as pharmaceuticals and healthcare.
Powerful software functions can enable comprehensive control based on feedback, time, conditions, and scripted recipes, effectively meeting the user's stringent process requirements. The software features a robust network communication interface that integrates with SCADA systems to facilitate data acquisition and remote monitoring.
The controller design includes additional analog input interfaces that can accommodate sensors such as CO2, weighing, and biomass. This allows for the integration of external sensors into the control feedback system. Furthermore, the capabilities for exhaust gas analysis and online biochemical analysis can be configurated.
A variety of Design of Experiments (DoE) methods are available, encompassing both screening and optimization functions. The system software is embedded, allowing for the experimental design to be assigned to the satellite tank with a single click for seamless connectivity. The results are analyzed visually and presented graphically to identify the optimal range.
Stainless Steel Fermentation Tanks And Supporting Systems
Duoning leverages its extensive engineering and manufacturing experience to offer customers modular and compact fermentation systems in capacities of 30 L, 50 L, and 100 L. Additionally, we provide customized fermentation systems for production use, starting at 200 L and above. Laboratory and pilot-scale fermentation systems are versatile tools for cultivating bacilli, yeast, and other microorganisms, enabling the optimization of process conditions for large-scale production or the preparation of samples for clinical trials. Production-scale systems can be tailored to align with anticipated production capacity plans based on market demand for commercial products. Furthermore, the level of automation can be customized according to the specific conditions of on-site facilities, ensuring enhanced cost-effectiveness for the final product.
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