RESUMEN

Ectoine, a biologically significant compound, was successfully produced by a strain of bacteria capable of utilizing sucrose. In a ground-breaking approach, we harnessed the potential of sugar beet molasses, a by-product rich in sucrose, amino acid, and vitamins, as a growth medium for this purpose. Through meticulous investigation, we identified the ideal conditions for maximizing ectoine synthesis. This remarkable milestone was reached by introducing only 1 g of (NH4)2SO4 and 5 mL of molasses per liter, maintaining a pH level of 8.0, upholding a 7.5% NaCl concentration, employing agitation at 120 rpm, and sustaining a temperature of 30 °C. This study marks a pioneering endeavour as it represents the first instance where molasses has been effectively employed to produce ectoine through the cultivation of Nesterenkonia sp. We showcased the production of 75.56 g of the valuable compound ectoine utilizing 1 L of waste molasses with this specific bacterial strain. These findings hold tremendous promise, not only in terms of resource utilization but also for the potential applications of ectoine in various biological contexts.

Asunto(s)

RESUMEN

Modern machine learning has the potential to fundamentally change the way bioprocesses are developed. In particular, horizontal knowledge transfer methods, which seek to exploit data from historical processes to facilitate process development for a new product, provide an opportunity to rethink current workflows. In this work, we first assess the potential of two knowledge transfer approaches, meta learning and one-hot encoding, in combination with Gaussian process (GP) models. We compare their performance with GPs trained only on data of the new process, that is, local models. Using simulated mammalian cell culture data, we observe that both knowledge transfer approaches exhibit test set errors that are approximately halved compared to those of the local models when two, four, or eight experiments of the new product are used for training. Subsequently, we address the question whether experiments for a new product could be designed more effectively by exploiting existing knowledge. In particular, we suggest to specifically design a few runs for the novel product to calibrate knowledge transfer models, a task that we coin calibration design. We propose a customized objective function to identify a set of calibration design runs, which exploits differences in the process evolution of historical products. In two simulated case studies, we observed that training with calibration designs yields similar test set errors compared to common design of experiments approaches. However, the former requires approximately four times fewer experiments. Overall, the results suggest that process development could be significantly streamlined when systematically carrying knowledge from one product to the next.

Asunto(s)

RESUMEN

Automation of metabolite control in fermenters is fundamental to develop vaccine manufacturing processes more quickly and robustly. We created an end-to-end process analytical technology and quality by design-focused process by replacing manual control of metabolites during the development of fed-batch bioprocesses with a system that is highly adaptable and automation-enabled. Mid-infrared spectroscopy with an attenuated total reflectance probe in-line, and simple linear regression using the Beer-Lambert Law, were developed to quantitate key metabolites (glucose and glutamate) from spectral data that measured complex media during fermentation. This data was digitally connected to a process information management system, to enable continuous control of feed pumps with proportional-integral-derivative controllers that maintained nutrient levels throughout fed-batch stirred-tank fermenter processes. Continuous metabolite data from mid-infrared spectra of cultures in stirred-tank reactors enabled feedback loops and control of the feed pumps in pharmaceutical development laboratories. This improved process control of nutrient levels by 20-fold and the drug substance yield by an order of magnitude. Furthermore, the method is adaptable to other systems and enables soft sensing, such as the consumption rate of metabolites. The ability to develop quantitative metabolite templates quickly and simply for changing bioprocesses was instrumental for project acceleration and heightened process control and automation. ONE-SENTENCE SUMMARY: Intelligent digital control systems using continuous in-line metabolite data enabled end-to-end automation of fed-batch processes in stirred-tank reactors.

Asunto(s)

RESUMEN

ß-carotene, a precursor to vitamin A, holds significant promise for health and nutrition applications. This study introduces an optimized approach for ß-carotene production in Saccharomyces cerevisiae, leveraging metabolic engineering and a novel use of agricultural waste. The GAL80 gene deletion facilitated efficient ß-carotene synthesis from sucrose, avoiding the costly galactose induction, and achieved titers up to 727.8 ±â€¯68.0 mg/L with content levels of 71.8 ±â€¯0.4 mg/g dry cell weight (DCW). Furthermore, the application of agricultural by-products, specifically molasses and fish meal as carbon and nitrogen sources, was investigated. This approach yielded a substantial ß-carotene titer of 354.9 ±â€¯8.2 mg/L and a content of 60.5 ±â€¯4.3 mg/g DCW, showcasing the potential of these sustainable substrates for industrial-scale production. This study sets a new benchmark for cost-effective, green manufacturing of vital nutrients, demonstrating a scalable, eco-friendly alternative for ß-carotene production.

RESUMEN

Host cell proteins (HCPs) are process-related impurities expressed by the host cells during biotherapeutics' manufacturing, such as monoclonal antibodies (mAbs). Some challenging HCPs evade clearance during the downstream processing and can be co-purified with the molecule of interest, which may impact product stability, efficacy, and safety. Therefore, HCP content is a critical quality attribute to monitor and quantify across the bioprocess. Here we explored a mass spectrometry (MS)-based proteomics tool, the sequential window acquisition of all theoretical fragment-ion spectra (SWATH) strategy, as an orthogonal method to traditional ELISA. The SWATH workflow was applied for high-throughput individual HCP identification and quantification, supporting characterization of a mAb purification platform. The design space of HCP clearance of two polishing resins was evaluated through a design of experiment study. Absolute quantification of high-risk HCPs was achieved (reaching 1.8 and 4.2 ppm limits of quantification, for HCP A and B respectively) using HCP-specific synthetic heavy labeled peptide calibration curves. Profiling of other HCPs was also possible using an average calibration curve (using labeled peptides from different HCPs). The SWATH approach is a powerful tool for HCP assessment during bioprocess development enabling simultaneous monitoring and quantification of different individual HCPs and improving process understanding of their clearance.

Asunto(s)

RESUMEN

As the need for higher volumetric productivity in biomanufacturing grows, biopharmaceutical companies are increasingly investing in a perfusion cell culture process, most commonly one that uses a hollow fiber filter as the cell retention device. A current challenge with using hollow fiber filters is fouling of the membrane, which reduces product sieving and can increase transmembrane pressure (TMP) past process limitations. In this work, the impact of hollow fiber filter geometries on product sieving and hydraulic membrane resistance profiles is evaluated in a tangential flow filtration (TFF) perfusion system. The hollow fibers tested had lengths ranging from 19.8 to 41.5 cm, inner diameters (IDs) ranging from 1.0 to 2.6 mm, and pore sizes of 0.2 or 0.65 µm. The results showed that the shortest hollow fibers experienced higher product sieving while larger IDs contributed to both higher product sieving and lower hydraulic membrane resistances, illustrating the impact of filter geometry on process performance. The results also showed 0.2 µm pore size filters maintain higher product sieving, but also higher membrane resistances compared to 0.65 µm pore size filters. This study highlights the need for optimized hollow fiber filter geometries to maximize use of the membrane area, which in turn can reduce production costs and increase scalability of the perfusion process.

Asunto(s)

RESUMEN

Miniaturized cultivation systems offer the potential to enhance experimental throughput in bioprocess development. However, they usually lack the miniaturized pumps necessary for fed-batch mode, which is commonly employed in industrial bioprocesses. An alternative are enzyme-mediated glucose release systems from starch-derived polymers, facilitating continuous glucose supply. Nevertheless, while the glucose release, and thus the feed rate, is controlled by the enzyme concentration, it also strongly depends on the type of starch derivative, and the culture conditions as well as pH and temperature. So far it was not possible to implement controlled feeding strategies (e.g., exponential feeding). In this context, we propose a model-based approach to achieve precise control over enzyme-mediated glucose release in cultivations. To this aim, an existing mathematical model was integrated into a computational framework to calculate setpoints for enzyme additions. We demonstrate the ability of the tool to maintain different pre-defined exponential growth rates during Escherichia coli cultivations in parallel mini-bioreactors integrated into a robotic facility. Although in this case study, the intermittent additions of enzyme and dextrin were performed by a liquid handler, the approach is adaptable to manual applications. Thus, we present a straightforward and robust approach for implementing defined continuous fed-batch processes in small-scale systems, where continuous feeding was only possible with low accuracy or high technical efforts until now.

RESUMEN

BACKGROUND: Diabetes is a disease affecting over 500 million people globally due to insulin insufficiency or insensitivity. For individuals with type 1 diabetes, pancreatic islet transplantation can help regulate their blood glucose levels. However, the scarcity of cadaveric donor islets limits the number of people that could receive this therapy. To address this issue, human pluripotent stem cells offer a potentially unlimited source for generating insulin-producing cells through directed differentiation. Several protocols have been developed to make stem cell-derived insulin-producing cells. However, there is a lack of knowledge regarding the bioprocess parameters associated with these differentiation protocols and how they can be utilized to increase the cell yield. METHODS: We investigated various bioprocess parameters and quality target product profiles that may influence the differentiation pipeline using a seven-stage protocol in a scalable manner with CellSTACKs and vertical wheel bioreactors (PBS-Minis). RESULTS: Cells maintained > 80% viability through all stages of differentiation and appropriately expressed stage-specific markers. During the initial four stages leading up to the development of pancreatic progenitors, there was an increase in cell numbers. Following pancreatic progenitor stage, there was a gradual decrease in the percentage of proliferative cells, as determined by Ki67 positivity, and a significant loss of cells during the period of endocrine differentiation. By minimizing the occurrence of aggregate fusion, we were able to enhance cell yield during the later stages of differentiation. We suggest that glucose utilization and lactate production are cell quality attributes that should be considered during the characterization of insulin-producing cells derived from stem cells. Our findings also revealed a gradual metabolic shift from glycolysis, during the initial four stages of pancreatic progenitor formation, to oxidative phosphorylation later on during endocrine differentiation. Furthermore, the resulting insulin-producing cells exhibited a response to several secretagogues, including high glucose. CONCLUSION: This study demonstrates process parameters such as glucose consumption and lactate production rates that may be used to facilitate the scalable manufacture of stem cell-derived insulin-producing cells.

Asunto(s)

RESUMEN

Chinese hamster ovary (CHO) cells are essential to biopharmaceutical manufacturing and production instability, the loss of productivity over time, is a long-standing challenge in the industry. Accurate prediction of cell line stability could enable efficient screening to identify clones suitable for manufacturing saving significant time and costs. DNA repair genes may offer biomarkers to address this need. In this study, over 40 cell lines representing various host lineages from three companies/organizations were evaluated for expression of five DNA repair genes (Fam35a, Lig4, Palb2, Pari, and Xrcc6). Expression measured in cells with less than 30 population doubling levels (PDLs) was correlated to stability profiles at 60+ PDL. Principal component analysis identified markers which separate stable and unstable CHO-DG44 cell lines. Notably, two genes, Lig4 and Xrcc6, showed higher expression in unstable CHO-DG44 cell lines with copy number loss identified as the mechanism of production instability. Expression levels across all cell ages showed lower DNA repair gene expression was associated with increased cell age. Collectively, DNA repair genes provide critical insight into long-term behavior of CHO cells and their expression levels have potential to predict cell line stability in certain cases.

Asunto(s)

RESUMEN

The biopharmaceutical industry is under increased pressure to maximize efficiency, enhance quality compliance, and reduce the cost of drug substance manufacturing. Ways to reduce costs associated with manufacturing of complex biological molecules include maximizing efficiency of chromatography purification steps. For example, process analytical technology (PAT) tools can be employed to improve column resin life, prevent column operating failures, and decrease the time it takes to solve investigations of process deviations. We developed a robust method to probe the shape of the chromatogram for indications of column failure or detrimental changes in the process. The approach herein utilizes raw data obtained from manufacturing followed by a pre-processing routine to align chromatograms and patch together the different chromatogram phases in preparation for multivariate analysis. A principal component analysis (PCA) was performed on the standardized chromatograms to compare different batches, and resulted in the identification specific process change that affected the profile. In addition, changes in the chromatogram peaks were used to create predictive models for impurity clearance. This approach has the potential for early detection of column processing issues, improving timely resolution in large-scale chromatographic operations.

Asunto(s)

RESUMEN

Raman spectroscopy is widely used in monitoring and controlling cell cultivations for biopharmaceutical drug manufacturing. However, its implementation for culture monitoring in the cell line development stage has received little attention. Therefore, the impact of clonal differences, such as productivity and growth, on the prediction accuracy and transferability of Raman calibration models is not yet well described. Raman OPLS models were developed for predicting titer, glucose and lactate using eleven CHO clones from a single cell line. These clones exhibited diverse productivity and growth rates. The calibration models were evaluated for clone-related biases using clone-wise linear regression analysis on cross validated predictions. The results revealed that clonal differences did not affect the prediction of glucose and lactate, but titer models showed a significant clone-related bias, which remained even after applying variable selection methods. The bias was associated with clonal productivity and lead to increased prediction errors when titer models were transferred to cultivations with productivity levels outside the range of their training data. The findings demonstrate the feasibility of Raman-based monitoring of glucose and lactate in cell line development with high accuracy. However, accurate titer prediction requires careful consideration of clonal characteristics during model development.

Asunto(s)

RESUMEN

Chemically defined mineral media are widely used in bioprocesses, as these show less batch to batch variation compared with complex media. Nonetheless, the recommended media formulations often lead to the formation of precipitants at elevated pH values. These precipitates are insoluble and reduce the availability of macronutrients to the cells, which can result in limiting growth rates and lower productivity. They can also damage equipment by clogging pipes, hoses, and spargers in stirred tank fermenters. In this study, the observed precipitate was analyzed via X-ray fluorescence spectroscopy and identified as the magnesium ammonium phosphate salt struvite (MgNH4 PO4 × 6H2 O). The solubility of struvite crystals is known to be extremely low, causing the macronutrients magnesium, phosphate, and ammonium to be bound in the struvite crystals. Here, it was shown that struvite precipitates can be redissolved under common fermentation conditions. Furthermore, it was found that the struvite particle size distribution has a significant effect on the dissolution kinetics, which directly affects macronutrient availability. At a certain particle size, struvite crystals rapidly dissolved and provided unlimiting growth conditions. Therefore, struvite formation should be considered during media and bioprocess development, to ensure that the dissolution kinetics of struvite are faster than the growth kinetics.

Asunto(s)

RESUMEN

Vaccines are integral to human life to protect them from life-threatening diseases. However, conventional vaccines often suffer limitations like inefficiency, safety concerns, unavailability for non-culturable microbes, and genetic variability among pathogens. Chimeric vaccines combine multiple antigen-encoding genes of similar or different microbial strains to protect against hyper-evolving drug-resistant pathogens. The outbreaks of dreadful diseases have led researchers to develop economical chimeric vaccines that can cater to a large population in a shorter time. The process development begins with computationally aided omics-based approaches to design chimeric vaccines. Furthermore, developing these vaccines requires optimizing upstream and downstream processes for mass production at an industrial scale. Owing to the complex structures and complicated bioprocessing of evolving pathogens, various high-throughput process technologies have come up with added advantages. Recent advancements in high-throughput tools, process analytical technology (PAT), quality-by-design (QbD), design of experiments (DoE), modeling and simulations, single-use technology, and integrated continuous bioprocessing have made scalable production more convenient and economical. The paradigm shift to innovative strategies requires significant attention to deal with major health threats at the global scale. This review outlines the challenges and emerging avenues in the bioprocess development of chimeric vaccines.

RESUMEN

The increased occurrence of antibiotic resistance and the harmful use of pesticides are a major problem of modern times. A ban on the use of antibiotics as growth promoters in animal breeding has put a focus on the probiotics market. Probiotic food supplements are versatile and show promising results in animal and human nutrition. Chemical pesticides can be substituted by biopesticides, which are very effective against various pests in plants due to increased research. What these fields have in common is the use of spore-forming bacteria. The endospore-forming Bacillus spp. belonging to this group offer an effective solution to the aforementioned problems. Therefore, the biotechnological production of sufficient qualities of such endospores has become an innovative and financially viable field of research. In this review, the production of different Bacillus spp. endospores will be reviewed. For this purpose, the media compositions, cultivation conditions and bioprocess optimization methods of the last 20 years are presented and reflected.

RESUMEN

OBJECTIVES: Research on hydrogenases from Cupriavidus necator has been ongoing for more than two decades and still today the common methods for culture inoculation are used. These methods were never adapted to the requirements of modified bacterial strains, resulting in different physiological states of the bacteria in the precultures, which in turn lead prolonged and different lag-phases. RESULTS: In order to obtain uniform and always equally fit precultures for inoculation, we have established in this study an optimized protocol for precultures of the derivative of C. necator HF210 (C. necator HP80) which is used for homologous overexpression of the genes for the NAD+-reducing soluble hydrogenase (SH). We compared different media for preculture growth and determined the optimal time point for harvest. The protocol obtained in this study is based on two subsequent precultures, the first one in complex nutrient broth medium (NB) and a second one in fructose -nitrogen mineral salt medium (FN). CONCLUSION: Despite having two subsequent precultures our protocol reduces the preculture time to less than 30 h and provides reproducible precultures for cultivation of C. necator HP80.

Asunto(s)

RESUMEN

Bacillus coagulans is a promising probiotic, because it combines probiotic properties of Lactobacillus and the ability of Bacillus to form endospores. Due to this hybrid relationship, cultivation of this organism is challenging. As the probiotics market continues to grow, there is a new focus on the production of these microorganisms. In this work, a strain-specific bioprocess for B. coagulans was developed to support growth on one hand and ensure sporulation on the other hand. This circumstance is not trivial, since these two metabolic states are contrary. The developed bioprocess uses a modified chemically defined medium which was further investigated in a one-factor-at-a-time assay after adaptation. A transfer from the shake flask to the bioreactor was successfully demonstrated in the scope of this work. The investigated process parameters included temperature, agitation and pH-control. Especially the pH-control improved the sporulation in the bioreactor when compared to shake flasks. The bioprocess resulted in a sporulation efficiency of 80%-90%. This corresponds to a sevenfold increase in sporulation efficiency due to a transfer to the bioreactor with pH-control. Additionally, a design of experiment (DoE) was conducted to test the robustness of the bioprocess. This experiment validated the beforementioned sporulation efficiency for the developed bioprocess. Afterwards the bioprocess was then scaled up from a 1 L scale to a 10 L bioreactor scale. A comparable sporulation efficiency of 80% as in the small scale was achieved. The developed bioprocess facilitates the upscaling and application to an industrial scale, and can thus help meet the increasing market for probiotics.

RESUMEN

Robotic facilities that can perform advanced cultivations (e.g., fed-batch or continuous) in high throughput have drastically increased the speed and reliability of the bioprocess development pipeline. Still, developing reliable analytical technologies, that can cope with the throughput of the cultivation system, has proven to be very challenging. On the one hand, the analytical accuracy suffers from the low sampling volumes, and on the other hand, the number of samples that must be treated rapidly is very large. These issues have been a major limitation for the implementation of feedback control methods in miniaturized bioreactor systems, where observations of the process states are typically obtained after the experiment has finished. In this work, we implement a Sigma-Point Kalman Filter in a high throughput platform with 24 parallel experiments at the mL-scale to demonstrate its viability and added value in high throughput experiments. The filter exploits the information generated by the ammonia-based pH control to enable the continuous estimation of the biomass concentration, a critical state to monitor the specific rates of production and consumption in the process. The objective in the selected case study is to ensure that the selected specific substrate consumption rate is tightly controlled throughout the complete Escherichia coli cultivations for recombinant production of an antibody fragment.

RESUMEN

BACKGROUND: Modern genome editing enables rapid construction of genetic variants, which are further developed in Design-Build-Test-Learn cycles. To operate such cycles in high throughput, fully automated screening, including cultivation and analytics, is crucial in the Test phase. Here, we present the required steps to meet these demands, resulting in an automated microbioreactor platform that facilitates autonomous phenotyping from cryo culture to product assay. RESULTS: First, an automated deep freezer was integrated into the robotic platform to provide working cell banks at all times. A mobile cart allows flexible docking of the freezer to multiple platforms. Next, precultures were integrated within the microtiter plate for cultivation, resulting in highly reproducible main cultures as demonstrated for Corynebacterium glutamicum. To avoid manual exchange of microtiter plates after cultivation, two clean-in-place strategies were established and validated, resulting in restored sterile conditions within two hours. Combined with the previous steps, these changes enable a flexible start of experiments and greatly increase the walk-away time. CONCLUSIONS: Overall, this work demonstrates the capability of our microbioreactor platform to perform autonomous, consecutive cultivation and phenotyping experiments. As highlighted in a case study of cutinase-secreting strains of C. glutamicum, the new procedure allows for flexible experimentation without human interaction while maintaining high reproducibility in early-stage screening processes.

Asunto(s)

RESUMEN

Optimization and monitoring of bioprocesses requires the measurement of several process parameters and quality attributes. Mass spectrometry (MS)-based techniques such as those coupled to gas chromatography (GCMS) and liquid Chromatography (LCMS) enable the simultaneous measurement of hundreds of metabolites with high sensitivity. When applied to spent media, such metabolome analysis can help determine the sequence of substrate uptake and metabolite secretion, consequently facilitating better design of initial media and feeding strategy. Furthermore, the analysis of metabolite diversity and abundance from spent media will aid the determination of metabolic phases of the culture and the identification of metabolites as surrogate markers for product titer and quality. This review covers the recent advances in metabolomics analysis applied to the development and monitoring of bioprocesses. In this regard, we recommend a stepwise workflow and guidelines that a bioprocesses engineer can adopt to develop and optimize a fermentation process using spent media analysis. Finally, we show examples of how the use of MS can revolutionize the design and monitoring of bioprocesses.

Asunto(s)

RESUMEN

Since natural resources for the bioproduction of commodity chemicals are scarce, waste animal fats (WAF) are an interesting alternative biogenic residual feedstock. They appear as by-product from meat production, but several challenges are related to their application: first, the high melting points (up to 60 °C); and second, the insolubility in the polar water phase of cultivations. This leads to film and clump formation in shake flasks and microwell plates, which inhibits microbial consumption. In this study, different flask and well designs were investigated to identify the most suitable experimental set-up and further to create an appropriate workflow to achieve the required reproducibility of growth and product synthesis. The dissolved oxygen concentration was measured in-line throughout experiments. It became obvious that the gas mass transfer differed strongly among the shake flask design variants in cultivations with the polyhydroxyalkanoate (PHA) accumulating organism Ralstonia eutropha. A high reproducibility was achieved for certain flask or well plate design variants together with tailored cultivation conditions. Best results were achieved with bottom baffled glass and bottom baffled single-use shake flasks with flat membranes, namely, >6 g L-1 of cell dry weight (CDW) with >80 wt% polyhydroxybutyrate (PHB) from 1 wt% WAF. Improved pre-emulsification conditions for round microwell plates resulted in a production of 14 g L-1 CDW with a PHA content of 70 wt% PHB from 3 wt% WAF. The proposed workflow allows the rapid examination of fat material as feedstock, in the microwell plate and shake flask scale, also beyond PHA production. KEY POINTS: • Evaluation of shake flask designs for cultivating with hydrophobic raw materials • Development of a workflow for microwell plate cultivations with hydrophobic raw materials • Production of polyhydroxyalkanoate in small scale experiments from waste animal fat.

Asunto(s)