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.
Highlights
The microorganism Pichia pastoris is a commonly used microbial host for the expression of recombinant proteins in biotechnology and biopharmaceutical industry
In our previous study we developed a fast approach based on batch experiments with methanol pulses to extract a minimal set of strain characteristic parameters (i.e. Δtimeadapt - time for adaptation, qs adapt - specific substrate uptake rate during adaptation, qs max - maximum specific substrate uptake rate), which are required to set up a subsequent feeding regime based on qs
A fast approach to derive a minimal set of strain characteristic parameters relevant for bioprocess development Each P. pastoris strain was cultivated in an easy-to-do batch system with methanol pulses to obtain certain strain characteristic parameters during the adaptation period (Δtimeadapt - time for adaptation of the culture to methanol, qs adapt - specific substrate uptake rate during the adaptation pulse) and the maximum specific substrate uptake rate
Summary
The microorganism Pichia pastoris is a commonly used microbial host for the expression of recombinant proteins in biotechnology and biopharmaceutical industry. A fast and easy-to-do characterization of recombinant Pichia strains to extract bioprocess-relevant strain characteristic parameters for the applied to the culture which is increased to a predefined maximum and constantly maintained throughout the whole cultivation time [5,6], and 2) the glycerol flow in the reactor is decreased following a linear function during a concomitant addition of methanol, a period which is called transition phase, to slowly adapt the culture to methanol [7,8,9,10] These methods are often based on specific experiences with a certain strain, but are often used as a general approach for different Pichia strains in following studies, without taking into account the specific requirements of the single strains during adaptation. Based on those findings and motivated by problems which occur, when more traditional feeding strategies are applied (e.g. possible accumulation of methanol caused by changing cell capacities during cultivation or the need of expensive monitoring equipment to allow μ-controlled feeding [9,14]), we have focused our research on the specific substrate uptake rate (qs) and have recently shown optimization potential using dynamic feeding profiles based on this parameter [18]
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