Enhanced Feed Medium and Process Development
Although high titers are typically not required for early-stage development because of relatively low material requirements
in early clinical stages, they often are desirable for some late-stage processes because of market demand or manufacturing
capacity constraints. There are two reasons to develop enriched media. First, to gain a better understanding of the determinants
for high volumetric productivity in cell culture fed-batch processes. Second, to develop a general cell-line–specific medium
optimization methodology to achieve higher titers.
Although a platform process emphasizes simplicity of feeding strategy and ease of implementation during manufacturing, it
might not lead to the maximum possible volumetric productivity that might be achieved with any specific cell line. The aim
of developing the enhanced process was to achieve higher titers with a specific cell line through feed media optimization
and the development of feeding strategies.
Iterative process: Enhanced feed medium optimization was achieved through an iterative process to customize the formulation for specific cell
lines. In this approach, metabolism and other key performance indicators were monitored to determine levels of lactate, ammonia,
and osmolality. Nutrient and metabolite consumption rates from fed-batch bioreactor experiments were analyzed and used to
adjust the feed formulation and feeding strategy in accordance with the nutrient requirements for a specific cell line. To
achieve this, daily supernatant spent medium samples were collected and used to obtain amino acid and vitamin consumption
profiles. With accumulating components reduced or omitted, more essential components could be added. The feed medium formulation
was adjusted when components were either increased or reduced. In this case, amino acids and vitamins were further enriched.
Because of variability associated with cell growth processes and the measurement of nutrient consumption rates, this had to
be conducted iteratively to adequately optimize the culture medium. Thus, the feed medium was reformulated for each subsequent
study, with the goal of maintaining a high specific productivity by supplementing key nutrients at desired levels. In our
study this was achieved by 2–3 rounds of optimization studies (Figure 7).
Figure 7. Development of enhanced feed medium and feeding strategy in an iterative optimization process
The enhanced fed-batch process was operationally more complicated than the platform fed-batch process. In both cases, however,
the timing of specific volume additions needed to be optimized for each cell line, but for the enhanced process, the feed
volume was calculated daily based on a combination of viable cell counts, cell size, and viability. This iterative process
for reformulating feed and feeding strategy was tested on two model cell lines with different growth and productivity characteristics.
MAb A exhibited high growth but average specific productivity, whereas MAb B exhibited low growth but high specific productivity.
The customized enhanced feed resulted in improved cell growth and significant titer increase. Titers as high as 9 g/L (MAb
A) and 6.7 g/L (MAb B) were achieved compared in the platform medium to 4.2 and 4.6 respectively (Figure 8). Our results indicate
that compared with the platform feeding strategy, the enhanced feeding strategy prevented excessive osmolality increases,
nutrient depletion, and enhanced volumetric productivity.
Figure 8. Feed optimization by the iterative process (enhanced feed) resulted in significant titer increases for two model
cell lines compared to the platform feed
Using a CD medium in cell development workflows could potentially lead to more comparable results between cell line development
and early-stage process development by minimizing changes or eliminating cell adaptation when using a different production
medium. Some cell lines also can adapt slowly to CD media during the later stages of development for inoculum expansion and
protein production.8 To transition from early-stage to late-stage processes, cell-line–specific process optimization is more straightforward
in CD media compared to media containing undefined raw materials, as exact concentrations of all components are known.
It was essential to balance solubility and osmolality of the platform basal medium for ease of use, manufacturability, and
long-term storage. The enriched feed medium, on the other hand, was formulated to minimize the dilution of culture volume
during feeding without resulting in an excessive increase in osmolality. Excessive concentration of feed media should be avoided
because this could lead to the precipitation of specific media components if their concentration exceeds their solubility.
Developing a CD feed platform and enhanced feed formulations for fed-batch processes resulted in overall benefits with more
favorable metabolic profiles and culture growth. Interestingly, tested cell lines that were lactogenic in the peptone-containing
medium exhibited a shift from lactate production to consumption during the later stage of cultures in the CD medium. At the
end of these processes, most cell lines had almost undetectable lactate concentrations that resulted in higher peak cell densities
and extended viability during production. The importance of the newly adjusted amino acid and vitamin concentrations were
clearly identified to improve lactate metabolism, enhance growth, extend viability, and increase volumetric production rates.9,10