A cell culture process is typically developed in bench scale bioreactors and then scaled up for commercial production. However, although the recent advances in cell technologies have enabled high titer processes, these high cell density cultures begin to become a challenge from the perspective of process scale-up. Especially factors such as mixing time, oxygen transfer, and carbon dioxide removal need to be carefully considered when scaling up high cell density processes. For example, poor mixing can result in local nutrient gradients within the bioreactor, leading to reduced cell growth and productivity. However, at the same time, low-power input mixing is recommended because of high sensitivity of mammalian cells to shear stress. The mixing will, in turn, affect both oxygen transfer and dissolved CO2 removal. The former is of critical importance, as mammalian cultures are aerobic processes and often oxygen is the limiting nutrient, while the latter has been linked with lower cell productivities and even to product quality due to a different glycosylation. In other words, optimizing oxygen supply and carbon dioxide removal, while avoiding cell damage, is the key to the mammalian cell culture scale-up.
In terms of manufacturability and scalability, mammalian cells have historically been considered difficult to work with due to factors such as low yield, medium complexity, serum requirement, and shear sensitivity, although the latter has generally been incorrectly overemphasized. After two decades of intensive development work in cell line, media, and bioreactor condition optimization, cell densities of 15–25 million viable cells/ml can be routinely achieved for monoclonal antibody fed-batch processes, giving production titers of 3–5 g/L; high titers up to ~10 g/L, and cell densities of more than 50 million viable cells/mL in fed-batch processes, which have been recently reported by a few companies at major conferences [24,25]. The enhancement of specific productivity per cell is achieved not only by selection of highly productive clones, but also by optimization of medium composition and bioreactor operation conditions.
Cell line stability is another factor that should be considered because volumetric and specific productivity decline as cell age increases for some cell lines. Such unstable clones are not suitable for large-scale production because cell age increases with scale as the cell culture process is scaled up through serial culture passages of the seed train and inoculum train. In addition to cell line stability, growth and metabolite characteristics that can affect process robustness and scalability also need to be assessed. Robust cell growth with high viability and low lactate synthesis is usually desirable. High-lactate producing clones are not preferred to avoid the osmolality increase that accompanies the addition of base needed to maintain pH.
Therapeutic antibodies are produced in mammalian host cell lines, including NS0 murine myeloma cells, and Chinese hamster ovary (CHO) cells. The selection of expression system is determined by its ability to deliver high productivity with acceptable product quality attributes and the preferences of individual companies, which is often influenced by their historical experiences.
A typical cell culture manufacturing process begins with thawing of a cryopreserved cell-bank vial, followed by successive expansions into larger culture vessels such as shake flasks, spinners, rocking bags, and stirred bioreactors. When culture volume and cell density meet predetermined criteria, the culture is transferred to the production bioreactor in which cells continue to grow and eventually express product. Getting the required cell density and volume of culture to inoculate the production bioreactor is achieved via a cell culture seed train. The cells are usually run through many cultivation systems that become larger with each passage. The seed train steps have a significant impact on the product titer and cell growth in production scale, as well as the success and the reproducibility of the seed train as a whole. Batch-to-batch transfers from small to subsequently larger bioreactors commonly have split ratios from 1:5 to 1: 10.
Generally, the scale of the production bioreactor is determined by the capacity needs of the facility to meet the market requirements. However, the duration of cell culturing and the length and composition of the seed culture train is determined by the cell clone and culturing conditions. The seed train expansion will typically be determined during the process development phase, based on the expansion capabilities of the chosen cell line. Typically, durations are assumed for the culture phases of each bioreactor. What can be noted is that the production bioreactor is usually the longest step in a batch or fed-batch cell culture train and process. As such, it becomes the rate-determining step within the overall production process. The correct assumptions around the cell culture durations is important, as it will determine if the production demand of the annual number of batches required each year will be fulfilled.
At the commercial scale, facility utilization is key, therefore to increase the productivity of a batch or fed-batch cell culture train, the upstream process design may consider the use of multiple production bioreactors to increase the batch throughput of the process. This allows either parallel processing (i.e., multiple batches to be harvested at the same time), or staggered processing, whereby line 2 is started a few days after line 1 and line 3 is started a few days after line 2.
This approach allows for a high degree of flexibility of operation; however, it is expensive, given that multiple seed and production bioreactors are needed with large space requirements within the facility. An alternative option is that of inoculating multiple production bioreactors with a single seed train. The rationale behind this approach is that the seed bioreactors are typically culturing cells for less time than the production bioreactor. Therefore, when batches are run sequentially with the minimum downtime between operations, the production bioreactor is usually still culturing a batch while the seed train may already be completed for the next successive batch. Increasing the number of production bioreactors facilitates the ability of the seed train to run in the most productive manner. Further advantages of such an approach are that the total number of bioreactors is minimized to reduce the impact on cost and facility space.
Within this approach, however, the flexibility of operation is slightly diminished compared with having multiple parallel lines supporting each production bioreactor. With the use of dedicated seed trains supporting each production bioreactor, batch frequency is limited only by the number of production bioreactors chosen. In the case where the number of seed lines are minimised to support multiple production bioreactors; batch frequency is a function of the number of production bioreactors chosen, as well as the number of seed lines. It can be noted that as the number of production bioreactors increases, the minimum number of seed trains required to support their inoculation also increases.
The availability of production bioreactors for inoculation from the seed line increases as their number increases. Should one production bioreactor be used, its availability for inoculation is only once every 14 days. Should 6 bioreactors be utilized, then a production bioreactor would be available for inoculation once every ~2 days. As the number of production bioreactors is increased, the rate-determining step of the cell culture line falls more on the seed bioreactors. We see that the longest seed culture step is 4 days. As such, maintaining this as a single seed bioreactor would limit the operation of the six production bioreactors to a batch once every 4 days as opposed to the potential of every ~2days. To make full use of the maximum productivity of, for instance, six bioreactors, the seed bioreactor with the longest occupancy duration would need to be replicated. Should the seed bioreactors all have the same occupancy duration, then the whole line would need to be replicated to ensure maximum productivity can be achieved.
It should be noted, the determination of the number of seed trains may not solely be down to productivity-related aspects. Running multiple bioreactors intensely with a single seed train does have risks associated with equipment breakdown. If a single or multiple bioreactors break down, the whole cell culture cycle may need to be started again, resulting in significant loss in production time. As such, multiple seed bioreactors may be designed as redundancy to allow mitigation against any equipment failure.
Given that productivity considerations are a concern in large-scale process design, another option open to the process designer is changing the cell culturing technology from batch/fed-batch to a more continuous operation, such as a perfusion process. This could be implemented within the seed train or the production bioreactor. In comparison with batch operation, a seed bioreactor in perfusion mode is extremely flexible in allowing an extended window of inoculation. Because higher cell densities can be achieved in perfusion mode relative to batch mode, a perfusion seed reactor can inoculate larger reactor volumes or multiple bioreactors simultaneously. Alternatively, use of a production bioreactor in perfusion mode would allow a continuous daily harvest from one bioreactor; hence, overall productivity per bioreactor volume would increase as compared with the fed-batch/batch technologies. The use of perfusion technology will require significant process development work at the bench scale and its choice of use should be driven by results at that stage of activity. It would not be advisable to develop a fed-batch process and try to switch to a perfusion mode to increase productivity at the large scale. In addition to the cell line adaptation required for such technology, the facility requirements of a perfusion process differ significantly compared with that of a fed-batch process. Most notably on the upstream side, the cell culture media requirement will be much greater in the perfusion case, and tasks such as media preparation, and requirements such as hold vessels will need to be designed and sized appropriately in the facility.
Regardless of the upstream technology used, the batch frequency emanating from the cell culture will significantly impact the sizing of the downstream purification (DSP) train. The DSP will therefore need to be sized to match the frequency of batch being passed on from the upstream. The solutions available to the designer may be sizing the equipment appropriately to meet the productivity need, or in the worst case, designing for additional DSP lines.
