Driving Consumption of Amino Acids
Although amino acid supplementation for the high nutrient condition had
minimal impact, the larger increases in cell growth and productivity
within Criterion 2 demonstrated an approach to identify limiting
nutrients in the moderate and low nutrient controls. However, to
determine if the limiting nutrients were indeed the model derived amino
acids, consumption rates were compared between model conditions and the
respective control within each nutrient feed level from Criterion 2
conditions (Fig. 8). The time dependent differences in amino acid
consumption rates between the process controls for each nutrient feed
level were represented as either a positive (green) difference
reflecting an increase in consumption or a negative (red) difference
reflecting a decrease in consumption (Fig. 7). For classification
purposes, the amino acids supplemented to the model-driven conditions
were highlighted and were grouped as either glucogenic or ketogenic.
Glucogenic amino acids can provide a carbon source to the cells to
biosynthesize glucose for metabolism whereas ketogenic amino acids
provide a carbon source to generate ketone bodies which can serve as a
secondary energy source in oxidative metabolism (Brosnan, 2003; Noguchi
et al., 2010).
Previously, it has been postulated that amino acid consumption is
largely dependent on the concentration of the corresponding amino acid
(Fan et al., 2015). Indeed, this was true for the high nutrient feed
conditions in which not only the model-supplemented amino acids, but
almost all amino acids showed an increase in consumption up to and
including day 11 for both models. The few with reduced consumption
included glutamic acid, which showed a marked decrease in consumption
across all time points after day 3. Although glutamic acid is a
nonessential amino acid for CHO cells, we posit that this decrease in
glutamic acid consumption is driven by a shift to increase consumption
of other supplemented nonessential amino acids, such as glycine and
alanine. However, due to the limited increase in cell growth and mAb
productivity from these conditions, overall increased consumption did
not shift the cells to a higher metabolic state, suggesting a saturation
state for cells and a point of maximum contribution by amino acids.
In contrast, moderate and low nutrient conditions showed a more drastic
difference in the consumption patterns when compared to the high
nutrient conditions. Most noticeably, there was a marked decrease in the
consumption of ketogenic amino acids and increased consumption of
glucogenic amino acids suggesting a greater need for energy and
biosynthesis due to the limited nutrient conditions. Furthermore, for
moderate nutrient conditions the growth model-derived amino acids had
increased consumption earlier in the culture and production
model-derived amino acids such as methionine were consumed more in the
latter half of the culture, supporting the distinct difference in
metabolic requirements between the cell growth phase and the high
mAb-producing stationary phase. In the low nutrient condition, there was
a decrease in majority of the amino acids within the growth model but
increase in glutamic acid and alanine in the latter half of the culture.
In the low nutrient production model, methionine and eventually lysine
showed increased consumption resulting in increased mAb productivity. In
all cases, model-derived amino acids were generally consumed more when
supplemented according to Criterion 2 than process control cultures for
each respective nutrient condition. Accordingly, these amino acids were
potential limiting nutrients that pushed cells into a higher metabolic
state when supplemented.