Christian Dietzsch

Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

AbstractΒackgroundThe methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains.ResultsA fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production.ConclusionsCo-overexpressing enzymes of the methanol utilization pathway significantly affected the specific growth rate, the methanol uptake and the specific productivity of recombinant P. pastoris MutS strains. A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development.

A dynamic method based on the specific substrate uptake rate to set up a feeding strategy for Pichia pastoris

BackgroundPichia pastoris is one of the most important host organisms for the recombinant production of proteins in industrial biotechnology. To date, strain specific parameters, which are needed to set up feeding profiles for fed batch cultivations, are determined by time-consuming continuous cultures or consecutive fed batch cultivations, operated at different parameter sets.ResultsHere, we developed a novel approach based on fast and easy to do batch cultivations with methanol pulses enabling a more rapid determination of the strain specific parameters specific substrate uptake rate qs, specific productivity qp and the adaption time (Δtimeadapt) of the culture to methanol. Based on qs, an innovative feeding strategy to increase the productivity of a recombinant Pichia pastoris strain was developed. Higher specific substrate uptake rates resulted in increased specific productivity, which also showed a time dependent trajectory. A dynamic feeding strategy, where the setpoints for qs were increased stepwise until a qsmax of 2.0 mmol·g-1·h-1 resulted in the highest specific productivity of 11 U·g-1·h-1.ConclusionsOur strategy describes a novel and fast approach to determine strain specific parameters of a recombinant Pichia pastoris strain to set up feeding profiles solely based on the specific substrate uptake rate. This approach is generic and will allow application to other products and other hosts.

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

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.

A dynamic fed batch strategy for a Pichia pastoris mixed feed system to increase process understanding.

Mixed substrate feeding strategies are frequently investigated to enhance the productivity of recombinant Pichia pastoris processes. For this purpose, numerous fed batch experiments or time‐consuming continuous cultivations are required to optimize control parameters such as the substrate mixing ratio and the applied methanol concentration. In this study, we decoupled the feeding of methanol and glycerol in a mixed substrate fed batch environment to gain process understanding for a recombinant P. pastoris Muts strain producing the model enzyme horseradish peroxidase. Specific substrate uptake rates (qs) were controlled separately, and a stepwise increased qGly‐control scheme was applied to investigate the effect of various substrate fluxes on the culture. The qs‐controlled strategy allowed a parallel characterization of the metabolism and the recombinant protein expression in a fed batch environment. A critical‐specific glycerol uptake rate was determined, where a decline of the specific productivity occurred, and a time‐dependent acceleration of protein expression was characterized with the dynamic fed batch approach. Based on the observations on recombinant protein expression, propositions for an optimal feeding design to target maximal productivities were stated. Thus, the dynamic fed batch strategy was found to be a valuable tool for both process understanding and optimization of product formation for P. pastoris in a mixed substrate environment.

Dynamic process conditions in bioprocess development

In this review, we summarise recent studies that purposefully employed dynamic conditions, such as shifts, pulses, ramps and oscillations, for fast physiological strain characterisation and bioprocess development. We show the broad applicability of dynamic conditions and the various objectives that can thereby be investigated in a short time. Dynamic processes reveal information about the analysed system faster than traditional strategies, like continuous cultivations, as process parameters can directly be linked to platform and product parameters. Furthermore, we demonstrate that dynamic operations can result in increased productivity and high product quality, making this strategy a valuable tool for bioprocess development. With this review, we would like to encourage bioprocess engineers to an increased use of dynamic conditions in bioprocess development.

On-line multiple component analysis for efficient quantitative bioprocess development

On-line monitoring devices for the precise determination of a multitude of components are a prerequisite for fast bioprocess quantification. On-line measured values have to be checked for quality and consistency, in order to extract quantitative information from these data. In the present study we characterized a novel on-line sampling and analysis device comprising an automatic photometric robot. We connected this on-line device to a bioreactor and concomitantly measured six components (i.e. glucose, glycerol, ethanol, acetate, phosphate and ammonium) during different batch cultivations of Pichia pastoris. The on-line measured data did not show significant deviations from off-line taken samples and were consequently used for incremental rate and yield calculations. In this respect we highlighted the importance of data quality and discussed the phenomenon of error propagation. On-line calculated rates and yields depicted the physiological responses of the P. pastoris cells in unlimited and limited cultures. A more detailed analysis of the physiological state was possible by considering the off-line determined biomass dry weight and the calculation of specific rates. Here we present a novel device for on-line monitoring of bioprocesses, which ensures high data quality in real-time and therefore refers to a valuable tool for Process Analytical Technology (PAT).

Purification of a recombinant plant peroxidase produced in Pichia pastoris by a simple 2-step strategy.

The enzyme horseradish peroxidase (HRP), which is frequently applied in industry and medicine, is primarily isolated from plant. This purification procedure is costly and the obtainable amount of HRP from the horseradish root is low. However, recombinant HRP (rHRP) produced in yeast is hyperglycosylated rendering the subsequent purification cumbersome and the recombinant production of HRP in yeast not competitive. In this study, we screened different common techniques to develop a fast and efficient purification strategy for hyperglycosylated rHRP expressed in Pichia pastoris. We demonstrated that the extensive glycosylation pattern on the surface of rHRP masked its physico-chemical properties, which is why standard purification strategies were rather unsuccessful. Only switching the strategies to a flowthrough mode gave satisfactory results. After determining the optimal operation conditions in a multivariate Design of Experiments approach, we present a simple 2-step strategy for the purification of hyperglycosylated rHRP. Combining a hydrophobic charge induction chromatography operated in flowthrough mode and a size-exclusion chromatography, we were able to purify rHRP more than 12-fold from a specific activity of 80U/mg to more than 1000U/mg.

Spore germination of Trichoderma atroviride is inhibited by its LysM protein TAL6

LysM motifs are carbohydrate‐binding modules found in prokaryotes and eukaryotes. They have general N‐acetylglucosamine binding properties and therefore bind to chitin and related carbohydrates. In plants, plasma‐membrane‐bound proteins containing LysM motifs are involved in plant defence responses, but also in symbiotic interactions between plants and microorganisms. Filamentous fungi secrete LysM proteins that contain several LysM motifs but no enzymatic modules. In plant pathogenic fungi, for LysM proteins roles in dampening of plant defence responses and protection from plant chitinases were shown. In this study, the carbohydrate‐binding specificities and biological function of the LysM protein TAL6 from the plant‐beneficial fungus Trichoderma atroviride were investigated. TAL6 contains seven LysM motifs and the sequences of its LysM motifs are very different from other fungal LysM proteins investigated so far. The results showed that TAL6 bound to some forms of polymeric chitin, but not to chito‐oligosaccharides. Further, no binding to fungal cell wall preparations was detected. Despite these rather weak carbohydrate‐binding properties, a strong inhibitory effect of TAL6 on spore germination was found. TAL6 was shown to specifically inhibit germination of Trichoderma spp., but interestingly not of other fungi. Thus, this protein is involved in self‐signalling processes during fungal growth rather than fungal–plant interactions. These data expand the functional repertoire of fungal LysM proteins beyond effectors in plant defence responses and show that fungal LysM proteins are also involved in the self‐regulation of fungal growth and development.

Determination of a dynamic feeding strategy for recombinant Pichia pastoris strains.

The knowledge of certain strain specific parameters of recombinant P. pastoris strains is required to be able to set up a feeding regime for fed-batch cultivations. To date, these parameters are commonly determined either by time-consuming and labor-intensive continuous cultivations or by several, consecutive fed-batch cultivations. Here, we describe a fast method based on batch experiments with methanol pulses to extract certain strain characteristic parameters, which are required to set up a dynamic feeding strategy for P. pastoris strains based on specific substrate uptake rate (q(s)). We further describe in detail the course of actions which have to be taken to obtain the desired dynamics during feeding.

Risk-based Process Development of Biosimilars as Part of the Quality by Design Paradigm

In the last few years, several quality by design (QbD) studies demonstrated the benefit of systematic approaches for biopharmaceutical development. However, only very few studies identified biosimilars as a special case of product development. The targeted quality profile of biosimilars is strictly defined by the originators product characteristic. Moreover, the major source of prior knowledge is the experience with the originator product itself. Processing this information in biosimilar development has a major effect on risk management and process development strategies. The main objective of this contribution is to demonstrate how risk management can facilitate the implementation of QbD in early-stage product development with special emphasis on fitting the reported approaches to biosimilars. Risk assessments were highlighted as important tools to integrate prior knowledge in biosimilar development. The risk assessment process as suggested by the International Conference on Harmonization (ICH Q9) was reviewed and three elements were identified to play a key role in targeted risk assessment approaches: proper understanding of target linkage, risk assessment tool compliance, and criticality threshold value. Adjusting these steps to biosimilar applications helped to address some unique challenges of these products such as a strictly defined quality profile or a lack of process knowledge. This contribution demonstrates the need for tailored risk management approaches for the risk-based development of biosimilars and provides novel tools for the integration of additional knowledge available for these products. LAY ABSTRACT: The pharmaceutical industry is facing challenges such as profit loss and price competition. Companies are forced to rationalize business models and to cut costs in development as well as manufacturing. These trends recently hinder the implementation of any concepts that do not offer certain financial benefit or promise a long return of investment. Quality by design (QbD) is a concept that is currently struggling for more acceptance from the side of the pharmaceutical industry. To achieve this, the major goals of QbD have to be revisited and QbD tools have to be subsequently developed. This contribution offers an example as to how implement risk management in early-stage biosimilar development as part of the QbD concept. The main goal was to go beyond the conventional QbD workflow and to adjust risk management to the challenges of biosimilar products. Accordingly, instead of using methods like failure mode and effects analysis, recommendations of the ICH Q9 guideline were reviewed and put into practice by creating tailored risk assessment tools. The novelty of this contribution is to report those tailored tools ready-to-use for early bioprocess development of biosimilars along QbD principles.