Christoph Herwig

Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase

Pichia pastoris is a prominent host for recombinant protein production, amongst other things due to its capability of glycosylation. However, N-linked glycans on recombinant proteins get hypermannosylated, causing problems in subsequent unit operations and medical applications. Hypermannosylation is triggered by an α-1,6-mannosyltransferase called OCH1. In a recent study, we knocked out OCH1 in a recombinant P. pastoris CBS7435 MutS strain (Δoch1) expressing the biopharmaceutically relevant enzyme horseradish peroxidase. We characterized the strain in the controlled environment of a bioreactor in dynamic batch cultivations and identified the strain to be physiologically impaired. We faced cell cluster formation, cell lysis and uncontrollable foam formation.In the present study, we investigated the effects of the 3 process parameters temperature, pH and dissolved oxygen concentration on 1) cell physiology, 2) cell morphology, 3) cell lysis, 4) productivity and 5) product purity of the recombinant Δoch1 strain in a multivariate manner. Cultivation at 30°C resulted in low specific methanol uptake during adaptation and the risk of methanol accumulation during cultivation. Cell cluster formation was a function of the C-source rather than process parameters and went along with cell lysis. In terms of productivity and product purity a temperature of 20°C was highly beneficial. In summary, we determined cultivation conditions for a recombinant P. pastoris Δoch1 strain allowing high productivity and product purity.

A comprehensive and quantitative review of dark fermentative biohydrogen production

Biohydrogen production (BHP) can be achieved by direct or indirect biophotolysis, photo-fermentation and dark fermentation, whereof only the latter does not require the input of light energy. Our motivation to compile this review was to quantify and comprehensively report strains and process performance of dark fermentative BHP. This review summarizes the work done on pure and defined co-culture dark fermentative BHP since the year 1901. Qualitative growth characteristics and quantitative normalized results of H2 production for more than 2000 conditions are presented in a normalized and therefore comparable format to the scientific community.Statistically based evidence shows that thermophilic strains comprise high substrate conversion efficiency, but mesophilic strains achieve high volumetric productivity. Moreover, microbes of Thermoanaerobacterales (Family III) have to be preferred when aiming to achieve high substrate conversion efficiency in comparison to the families Clostridiaceae and Enterobacteriaceae.The limited number of results available on dark fermentative BHP from fed-batch cultivations indicates the yet underestimated potential of this bioprocessing application. A Design of Experiments strategy should be preferred for efficient bioprocess development and optimization of BHP aiming at improving medium, cultivation conditions and revealing inhibitory effects. This will enable comparing and optimizing strains and processes independent of initial conditions and scale.

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.

Microbials for the production of monoclonal antibodies and antibody fragments

Highlights • Glycosylated full length antibodies are currently produced in mammalian cells.• Antibody fragments can be produced in microbial organisms.• Strain engineering allows production of full length antibodies in microbials.• Microbials provide several advantages over mammalian cells.

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.

Real-time estimation of biomass and specific growth rate in physiologically variable recombinant fed-batch processes

The real-time measurement of biomass has been addressed since many years. The quantification of biomass in the induction phase of a recombinant bioprocess is not straight forward, since biological burden, caused by protein expression, can have a significant impact on the cell morphology and physiology. This variability potentially leads to poor generalization of the biomass estimation, hence is a very important issue in the dynamic field of process development with frequently changing processes and producer lines. We want to present a method to quantify “biomass” in real-time which avoids off-line sampling and the need for representative training data sets. This generally applicable soft-sensor, based on first principles, was used for the quantification of biomass in induced recombinant fed-batch processes. Results were compared with “state of the art” methods to estimate the biomass concentration and the specific growth rate µ. Gross errors such as wrong stoichiometric assumptions or sensor failure were detected automatically. This method allows for variable model coefficients such as yields in contrast to other process models, hence does not require prior experiments. It can be easily adapted to a different growth stoichiometry; hence the method provides good generalization, also for induced culture mode. This approach estimates the biomass (or anabolic bioconversion) in induced fed-batch cultures in real-time and provides this key variable for process development for control purposes.

Science-based bioprocess design for filamentous fungi.

Industrial bioprocesses are commonly based on empiricism rather than scientific process understanding. In this review, we summarize current strategies for science-based bioprocess design and control for filamentous fungi aiming at reducing development times and increasing process economics. We discuss recent developments and trends regarding three crucial aspects throughout the bioprocess life cycle of filamentous fungi, namely (i) strain and inoculum characterization, (ii) morphology, and (iii) rheology, as well as their effects on process performance. Complex interconnections between strain, inoculum, morphology, rheology, and process design are outlined and discussed. Only combining different hard type sensors with soft sensor technology and the development of simplified mechanistic models can enable science-based bioprocess design for filamentous fungi.

Knockout of an endogenous mannosyltransferase increases the homogeneity of glycoproteins produced in Pichia pastoris

The yeast Pichia pastoris is a common host for the recombinant production of biopharmaceuticals, capable of performing posttranslational modifications like glycosylation of secreted proteins. However, the activity of the OCH1 encoded α-1,6-mannosyltransferase triggers hypermannosylation of secreted proteins at great heterogeneity, considerably hampering downstream processing and reproducibility. Horseradish peroxidases are versatile enzymes with applications in diagnostics, bioremediation and cancer treatment. Despite the importance of these enzymes, they are still isolated from plant at low yields with different biochemical properties. Here we show the production of homogeneous glycoprotein species of recombinant horseradish peroxidase by using a P. pastoris platform strain in which OCH1 was deleted. This och1 knockout strain showed a growth impaired phenotype and considerable rearrangements of cell wall components, but nevertheless secreted more homogeneously glycosylated protein carrying mainly Man8 instead of Man10 N-glycans as a dominant core glycan structure at a volumetric productivity of 70% of the wildtype strain.

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.