A fast approach to determine a fed batch feeding profile for recombinant Pichia pastoris strains

Abstract

BackgroundThe microorganism Pichia pastoris is a commonly used microbial host for the expression of recombinant proteins in biotechnology and biopharmaceutical industry. To speed up process development, a fast methodology to determine strain characteristic parameters, which are needed to subsequently set up fed batch feeding profiles, is required.ResultsHere, we show the general applicability of a novel approach to quantify a certain minimal set of bioprocess-relevant parameters, i.e. the adaptation time of the culture to methanol, the specific substrate uptake rate during the adaptation phase and the maximum specific substrate uptake rate, based on fast and easy-to-do batch cultivations with repeated methanol pulses in a batch culture. A detailed analysis of the adaptation of different P. pastoris strains to methanol was conducted and revealed that each strain showed very different characteristics during adaptation, illustrating the need of individual screenings for an optimal parameter definition during this phase. Based on the results obtained in batch cultivations, dynamic feeding profiles based on the specific substrate uptake rate were employed for different P. pastoris strains. In these experiments the maximum specific substrate uptake rate, which had been defined in batch experiments, also represented the upper limit of methanol uptake, underlining the validity of the determined process-relevant parameters and the overall experimental strategy.ConclusionIn this study, we show that a fast approach to determine a minimal set of strain characteristic parameters based on easy-to-do batch cultivations with methanol pulses is generally applicable for different P. pastoris strains and that dynamic fed batch strategies can be designed on the specific substrate uptake rate without running the risk of methanol accumulation.

Quantifying the Effects of Frequency and Amplitude of Periodic Oxygen-Related Stress on Recombinant Protein Production in Pichia pastoris

“…The medium, for the batch and fed batch cultivations as well as the detailed composition of the feeds are available elsewhere [4].…”

“…The feed rate in this phase was adjusted in such a way that specific substrate uptake rate of 1 mmol/L· h was set. The detailed description of each phase was fully described before [4].…”

“…In induction phase, the stirrer speed was automatically adjusted to achieve the desired dissolved oxygen level. pH was adjusted by means of a PID controller which added NH 4 OH to keep the pH at 5. The off gas passed through the gas analyzer (Servomex, M. Müller AG, Egg, Switzerland) to quantify CO 2 and O 2 content, using infrared and paramagnetic principles, respectively.…”

“…They were processed to measure the biomass concentration and the protein activity. The activity of HRP was determined using an enzymatic robot as described elsewhere [4].…”

Integrated Application of Bioprocess Engineering and Biotechniques for Quality & Bulk Drug Manufacturing

“…After a series of such reactions (which take place in large vessels called fermenters or fermentation tanks), the initial raw materials are chemically changed to form the desired end product. Although it sounds quite simple, this procedure presents two major challenges [5].…”