Metabolic Control in Mammalian Fed-Batch Cell Cultures for Reduced Lactic Acid Accumulation and Improved Process Robustness

Abstract

Biomass and cell-specific metabolic rates usually change dynamically over time, making the “feed according to need” strategy difficult to realize in a commercial fed-batch process. We here demonstrate a novel feeding strategy which is designed to hold a particular metabolic state in a fed-batch process by adaptive feeding in real time. The feed rate is calculated with a transferable biomass model based on capacitance, which changes the nutrient flow stoichiometrically in real time. A limited glucose environment was used to confine the cell in a particular metabolic state. In order to cope with uncertainty, two strategies were tested to change the adaptive feed rate and prevent starvation while in limitation: (i) inline pH and online glucose concentration measurement or (ii) inline pH alone, which was shown to be sufficient for the problem statement. In this contribution, we achieved metabolic control within a defined target range. The direct benefit was two-fold: the lactic acid profile was improved and pH could be kept stable. Multivariate Data Analysis (MVDA) has shown that pH influenced lactic acid production or consumption in historical data sets. We demonstrate that a low pH (around 6.8) is not required for our strategy, as glucose availability is already limiting the flux. On the contrary, we boosted glycolytic flux in glucose limitation by setting the pH to 7.4. This new approach led to a yield of lactic acid/glucose (Y L/G) around zero for the whole process time and high titers in our labs. We hypothesize that a higher carbon flux, resulting from a higher pH, may lead to more cells which produce more product. The relevance of this work aims at feeding mammalian cell cultures safely in limitation with a desired metabolic flux range. This resulted in extremely stable, low glucose levels, very robust pH profiles without acid/base interventions and a metabolic state in which lactic acid was consumed instead of being produced from day 1. With this contribution, we wish to extend the basic repertoire of available process control strategies, which will open up new avenues in automation technology and radically improve process robustness in both process development and manufacturing.

Elevated pCO2 affects the lactate metabolic shift in CHO cell culture processes

“…However, reduced yields of lactate from glycolysis most probably derive from a reduced specific glucose consumption as described previously by Konakovsky et al. and Zalai et al. .…”

“…Low glucose feeding as applied from Konakovsky et al. can induce lactate consumption as shown in their study. Furthermore, pH‐shifts to lower values can strongly reduce glucose consumption rates and have been shown before to induce lactate consumption . (ii) Increasing the mitochondrial NAD⁺ regeneration would be the second approach to reduce R and initiate lactate consumption.…”

Effective bioreactor pH control using only sparging gases

“…By eliminating osmolality effects due to base addition, pH control loops with gas sparging could foster milieu for favorable lactate metabolism, thereby potentially improving overall process robustness. Indeed, controlling the lactate level or reducing lactate metabolism variations have been pursued for process improvement in multiple cases …”

A robust feeding strategy to maintain set‐point glucose in mammalian fed‐batch cultures when input parameters have a large error

“…For instance, GLC level may stay constant, while the cells consume less and less GLC. We know from historical runs that after a high qs phase of approximately 4 days, qs assumes a relatively constant value . This information is useful because it could be fed into the control strategy to improve control of the process using a priori experience.…”

“…In a simple scenario, a fixed amount of substrate can be assumed to be consumed by the cells per time. Knowing the cell count, this amount is then replenished and added in the form of feed . However, biomass estimation will come with an error, and the fixed amount that cells consume per time interval may not be constant during the time course of the fermentation, therefore bolus shot safeguards may still be required.…”