Experimental Validation with Amino Acid Supplementation
To validate the OPLS Model for each Criterion, experimental conditions were designed similarly to the high nutrient process control with the addition of amino acid cocktail feeds. CHO cell cultures fed with amino acid cocktails based on Criterion 1 showed a nominal increase in total cell growth at day 9 for both models (Growth Model vs Control: no significant change; Production Model vs Control: no significant change) (Fig. 3a). Day 14 CVC was also analyzed to assess if the early additions of amino acid up to day 9 would have an impact on the overall cell growth of the culture. Similar to day 9, there was only a slight increase of less than 10% in totals cells from the growth model (p = 0.1712) suggesting that increased supplementation of amino acids that were already being consuming in excess based on the theoretical demands did not further drive cell growth. In some instances, overfeeding nutrients can have deleterious effects on the cell culture (Salazar, Keusgen, & von Hagen, 2016). For instance, the growth model showed a significant drop in titer near 20% (p < 0.05) and a slight drop in titer for the production model (p = 0.159) (Fig. 3b). For comparison, day 9 titer was also analyzed to determine if the growth model for Criterion 1 had any early impact on productivity. Unfortunately, no deviation from the control was observed (Growth Model vs Control: no significant change; Production Model vs Control: no significant change).
In contrast, Criterion 2 was designed to test those amino acids that were being consumed below their theoretical demand. However, since Criterion 2 was built on a basis of high nutrient feeds in the training dataset, we postulated that information gained could also support various phases of media development such as moderate nutrient or low nutrient feeds. Therefore, three feed media conditions for the growth and production models were included with biological replicates for the validation run. Similarly to Criterion 1, amino acid cocktail feeds were prepared and supplemented on model-informed days. For statistical comparisons of the model conditions to the control, CVC and relative titer values were only considered from day 8 to day 14 because a majority of the cultures showed similar CVC and mAb titer during the first half of the culture. Although day 9 CVC did not show a significant change for high nutrient conditions, day 14 CVC showed a modest increase of about 10% for the growth model and about 20% for production model (Fig. 4). A more significant increase was observed within the moderate nutrient and low nutrient conditions when supplemented with the same amino acid cocktail feeds. Increase in CVC was noticed as early as day 7 for both models in the moderate nutrient condition reaching about a 35% increase in total cells by day 14 for the growth model (p < 0.001) and about 30% increase for the production model (p < 0.05). The low nutrient condition also showed an increase in CVC as early as day 7 but relative cell growth propagated to near 55% increase in total cells for both models by day 14 (Growth Model vs Control: p < 0.01; Production Model vs Control: p < 0.05). Interestingly, the relative cell growth rates were only slightly impacted by the amino acid supplementations (Fig. S3). The growth rate for high nutrient conditions showed a minor increase at day 3 during the log-phase of growth but also maintained the slight increased up to day 7 (p < 0.05) which could have been the driver to the 20% increase in CVC. Similarly, the growth rates for moderate and low nutrient conditions also slightly increased, but unlike the high nutrient condition, the growth rate remained elevated for a longer duration up to day 9. Moderate nutrient conditions had a 5% increase in growth rate at day 3 in the growth model whereas the low nutrient condition had a 5% increase around day 5 reflecting a delayed log-phase but overall greater increase in CVC relative to the control.
The increase in cell growth for both models was further reflected in mAb productivity. Although the high nutrient growth model did not result in any significant increase in titer, the production model yielded about 25% increased productivity (p = 0.163) (Fig. 4). The moderate and low nutrient productivity in contrast reflected a significant increase in cell growth by producing about 80% more relative titer by day 9 and 60% more relative titer by day 14 when compared to control cultures (p < 0.001). Although low nutrient conditions had a higher boost in cell growth, it also resulted in a similar 60% increase in productivity by day 14 suggesting an upper threshold for mAb production by this cell line with those particular feeds. The large increases in mAb titer for moderate and low nutrient conditions were further reflected by the significant increases in relative specific productivity (qP) (Fig. S2). For example, the moderate nutrient condition showed a 20% increase in qP for the growth model (p < 0.05) and showed a near 30% increase for the production model during the stationary phase (p < 0.01). In contrast, the low nutrient condition showed about a 40% increase in qP starting from day 7 (p < 0.05) suggesting the increased mAb production was not just an artefact of increased cell growth.
To further understand the differences between the resulting cell growth and mAb productivity phenotypes observed, glucose consumption rates were analyzed to assess how the model-driven conditions and supplementation of presumably limiting amino acids could alter cellular metabolism. Posed differently, was differential glucose consumption artefact of the increased percentage of cells within each model condition or a result of altered metabolic activity at the cellular specific level? Total glucose consumption within high nutrient feeds did not show any change in overall consumption whereas under moderate and low nutrient conditions, there was about a 2 g/L decrease in peak glucose consumption (Fig. 5). In contrast, the specific glucose consumption rates highlighted a more significant difference between control and model conditions within moderate and low nutrient feeds. Growth and production model conditions resulted in almost a 1000 pmol/cell*day decrease in specific glucose consumption in moderate feed and about a 2000 pmol/cell*day decrease in low nutrient feeds (p < 0.01). Accordingly, the marked increase in cell growth and mAb production for moderate and low nutrient conditions when compared to the high nutrient condition was further reflected by the larger magnitude of specific glucose consumption. Taken together, the decreased specific glucose consumption rates in model-driven conditions suggests that cells within these conditions presumably consumed other nutrients such desired amino acids for bioproduction and energy generation that ultimately resulted the cells to be in a higher metabolic state to produce more mAb.
Since the amino acid cocktails were supplemented in addition to process feed media and prepared at 100x concentrations, modulations in feed volume, pH, and osmolality were warranted. Specific control conditions for each model at all three nutrient feed levels for Criterion 2 were included within the experiment to understand if the increase in CVC or productivity was in fact due to the amino acids and no other cell culture variations. The solution/pH control was designed for the high nutrient feed only and was supplemented with base at the same volume and pH as the amino acid cocktail with time points representative of either of the growth or production models. Similarly, osmolality control cultures were fed with a NaCl solution with the same osmolality and volume as the highest osmolality among the amino acid feed cocktails. High nutrient feed cultures supplemented with the solution/pH control feed showed about a 20% decrease in day 14 CVC and day 14 titer (Fig. S3). Due to the high pH levels, supplementation of the solution/pH control could have increased cell death and a decline in productivity. Comparably, cultures supplemented with the osmolality control feed for high nutrient conditions also showed about a 20% drop in total cell growth and productivity (p < 0.35). However, moderate and low nutrient conditions supplemented with the osmolality feed control did not show any significant differences from the control cultures suggesting that the accumulation of additional osmolality by the cultures in high nutrient conditions was an artefact of the already nutrient rich environment and not of the control feed. Therefore, the effects of osmolality and the pH change from the amino acid cocktail feeds did not contribute to the overall increase in cell growth or productivity, but it was in fact a result of the key amino acids supplementation.
The growth and production models for Criterion 3 represented those nutrients that were consumed less than their theoretical demand but were not deemed important by the model VIP statistic. Presumably, these nutrients were considered not to significantly impact cell growth or productivity within all three nutrient conditions. Although day 9 CVC was chosen as the response variable, relative cell growth rates from Criterion 2 showed that the peak cell growth occurred prior to day 7. This is further reflected in the experimental results for Criterion 3 growth model where there was insignificant change to total cells measured in response to day 8 amino acid supplementation for all three nutrient feed levels (Fig. 6a). Similarly, growth model conditions for all three nutrient levels resulted in about a 20% decrease in relative titer when compared to the control (Fig. 6b). Overall, the production model resulted in about a 5% increase in total cells for high and moderate nutrient conditions, and about a 15% decrease in total cells for the low nutrient conditions when compared to the control. Relative titer on the other hand showed a 10% increase for the high nutrient condition (p < 0.3) and about a 40% increase for both moderate and low nutrient conditions (p < 0.05). Although 40% increase was presumed to be significant, it was not as significant as the 90% increase in the analogous cultures within Criterion 2 (p < 0.001). We presume that the large increase in titer from moderate and low nutrient feed conditions in Criterion 3 was due to the low nutrient levels in the culture and that any additional nutrients could provide a positive effect.