Heparosan is a naturally occurring non-sulfated glycosaminoglycan. Heparosan serves as the substrate for chemoenzymatic synthesis of biopharmaceutically important heparan sulfate and heparin. ...Heparosan is biologically inert molecule, non-toxic, and non-immunogenic and these qualities of heparosan make it an ideal drug delivery vehicle. The critical-to-quality (CTQ) attributes for heparosan applications include composition of heparosan, absence of any unnatural moieties, and heparosan molecular weight size and unimodal distribution. Probiotic bacteria
E. coli
Nissle 1917 (EcN) is a natural producer of heparosan. The current work explores production of EcN heparosan and process parameters that may impact the heparosan CTQ attributes. Results show that EcN could be grown to high cell densities (OD
600
160–180) in a chemically defined media. The fermentation process is successfully scaled from 5-L to 100-L bioreactor. The chemical composition of heparosan from EcN was confirmed using nuclear magnetic resonance. Results demonstrate that heparosan molecular weight distribution may be influenced by fermentation and purification conditions. Size exclusion chromatography analysis shows that the heparosan purified from fermentation broth results in bimodal distribution, and cell-free supernatant results in unimodal distribution (average molecular weight 68,000 Da). The yield of EcN-derived heparosan was 3 g/L of cell free supernatant. We further evaluated the application of Nissle 1917 heparosan for chemical modification to prepare
N
-sulfo heparosan (NSH), the first intermediate precursor for heparin and heparan sulfate.
Key points
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High cell density fermentation, using a chemically defined fermentation media for the growth of probiotic bacteria EcN (E. coli Nissle 1917, a natural producer of heparosan) is reported.
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Process parameters towards the production of monodispersed heparosan using probiotic bacteria EcN (Nissle 1917) has been explored and discussed.
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The media composition and the protocol (SOPs and batch records) have been successfully transferred to contract manufacturing facilities and industrial partners.
Considering glycerol as an inexpensive alternative carbon source, the optimal glycerol concentration for succinic acid production with Anaerobiospirillum succiniciproducens was identified in ...shake‐flask trials. The addition of a defined amount of glucose improved the growth and succinic acid productivity significantly. In fed‐batch processes with glycerol as sole substrate, a maximum succinic acid concentration and product substrate yield were obtained. The addition of glucose led to a 2.5‐fold increased succinic acid concentration whereas the product substrate yield remained nearly constant.
The optimization of the glycerol concentration and the addition of small amounts of glucose improves growth and succinic acid production of Anaerobiospirillum succiniciproducens. In fed‐batch experiments the final succinic acid concentration could be improved with glycerol‐glucose mixtures by factor 2.5 in comparison to glycerol as sole carbon source.
In vitro transcription (IVT) is the main manufacturing method to produce mRNA vaccines. In this study, a fed-batch strategy was systematically optimized for IVT process with dual goals of achieving a ...high reaction rate and maximizing the final mRNA yield simultaneously. Initially various experimental conditions were investigated including Mg2+, nucleotide triphosphates (NTP), dithiothreitol (DTT), spermidine, as well as the temperature and ionic strength. It was found that the concentrations of Mg2+ and NTP had a significant impact on IVT process. Subsequently, under the optimized conditions, dividing the IVT reaction into three distinct phases was proposed to enable more efficient transcription. By optimizing the concentrations of Mg2+ and NTP in two replenishment processes, our fed-batch strategy resulted in the production of 367.8 μg of mRNA with reduced dsRNA byproducts within 180 min. This was achieved under conditions of a final volume of 30 μL, 250 U T7 RNA polymerase (RNAP), and 2 μg DNA template. In conclusion, the IVT process using a fed-batch approach, rather than an excessive one-time NTP input, is an efficient method to improve productivity given a fixed amount of T7 RNAP and DNA template.
•A three-stage fed-batch strategy was optimized for efficient mRNA manufacturing.•Mg2+ and nucleotide triphosphates concentrations were critical in fed-batch strategy.•The fed-batch strategy obtained three-fold mRNA compared to one-step operation.
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•Vitis labrusca cultures were scaled up to 5 L bioreactor cultures.•A small volume (3 × 50 mL) inocula were used.•Resveratrol production by the elicited culture was modeled.•This ...model shows that the system dynamic can be broken down into four phases.
The purpose of this work was to scale-up the culture of grapevine cells (Vitis labrusca) from shake-flasks (100 mL) to a 5L stirred bioreactor in order to develop a model able to describe the bioproduction of resveratrol under the controlled conditions of a fed-batch culture. For this study, the biomass, resveratrol and sugar concentrations as well as pH and dissolved oxygen were monitored daily. The experiments were conducted twice over a three month period. The culture was elicited during the exponential growth phase with methyl jasmonate, leading the cells to exhibit a complex behaviour during the resveratrol production phase. A model of the system behaviour involving simple mechanisms is proposed and successfully confronted to the experimental results. This model demonstrates that the system dynamic can be decomposed into four phases: a lag phase (cell growth slowing down), a starting phase (beginning of resveratrol production), a surge phase (significant resveratrol production accompanied by significant cell death) and a stationary phase. Thus, we were able to successfully scale-up resveratrol production from 100 mL flasks to 5L bioreactors.
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•High-performing biofilm process allowed high cheese whey acidification yields.•High organic acids titer was obtained by applying ED sequential batch processes.•The ED concentrated ...effluent allowed excellent PHAs production yield.•The employment of biowaste did not lead to inhibiting effects on PHAs accumulation.
The present work aimed to verify the feasibility of producing polyhydroxyalkanoates (PHAs) at high concentrations from an alternative and cheap carbon source such as the carboxylic acids (VFAs) obtained by the anaerobic fermentation of cheese whey (CW). An electrodialysis (ED) step was proposed for the obtainment of a concentrated acidic stream, suitable for being employed as feeding solution in the consecutive fed-batch culture system for PHAs production.
Experiments conducted in duplicate shown that a packed bed anaerobic bioreactor resulted a robust and high repeatable culture system for a high performing continuous production of VFAs from CW. The acidogenic effluent contained 12.55 ± 1.10 g L−1 of carboxylic acids (ca. 90% of the effluent soluble COD), including the hexanoic (4.13 ± 0.56 g L−1) and octanoic (3.12 ± 0.94 g L−1) acids. The application of 7 sequential batch ED processes allowed to obtain a carboxylic acids concentrated stream (ca. 63 g L−1); by the achievement of high molar flow. Finally, this stream was utilized as the feeding solution in a fed-batch fermentation aimed to produce PHAs. The attained PHAs yield (ca. 0.60 gPHAs gVFAs−1) was comparable to that obtained in a parallel test accomplished with a VFAs-water simulating solution and also to those previously reported from pure VFAs; i.e. no inhibition effects due to the employment of an actual biowaste as the feedstock were detected.
As a whole, the results allow to conclude that the proposed integrated valorisation scheme fed with CW for the production of PHAs by an ED-concentrated carboxylic acids solution is technically feasible and robust.
Bioprocess development today is slow and expensive compared to chemical process development. A drastic paradigm shift is necessary and possible by the consistent application of engineering strategies ...that are typically used in the process development phase already in the early product development. Aside from providing a consistent pathway, strategies such as statistical‐based design of experiments, fed‐batch, minibioreactors, new on‐line sensors, process modeling, and control tools in combination with automation of manual steps offer a higher success rate and the opportunity to find the optimum parameters and operation point. This also directly benefits the early phases of biomolecular screening and initial production of small amounts of the target molecule. The paper reviews the bioprocess developmental phases from a business perspective and the available systems and technologies.
The development of generic biopharmaceuticals is increasing the pressures for enhanced bioprocess productivity and yields. Autophagy (“self‐eating”) is a cellular process that allows cells to ...mitigate stresses such as nutrient deprivation. Reputed autophagy inhibitors have also been shown to increase autophagic flux under certain conditions, and enhance recombinant protein productivity in Chinese Hamster Ovary (CHO) cultures. Since peptides are commonly added to bioprocess culture media in hydrolysates, we evaluated the impact on productivity of an autophagy‐inducing peptide (AIP), derived from the cellular autophagy protein Beclin 1. This was analyzed in CHO cell batch and fed‐batch serum‐free cultures producing a human Immunoglobulin G1 (IgG1). Interestingly, the addition of 1–4 µM AIP enhanced productivity in a concentration‐dependent manner. Cell‐specific productivity increased up to 1.8‐fold in batch cultures, while in fed‐batch cultures a maximum twofold increase in IgG concentration was observed. An initial drop in cell viability also occurred before cultures recovered normal growth. Overall, these findings strongly support the value of investigating the effects of autophagy pathway modulation, and in particular, the use of this AIP medium additive to increase CHO cell biotherapeutic protein production and yields.
Autophagy (‘self‐eating’) is a biological process that allows cells to mitigate the impact of stresses such as nutrient deprivation. Braasch and colleagues have investigated CHO cell autophagy induction by an autophagy‐inducing peptide (AIP) derived from the autophagy protein Beclin 1. They have established that AIP can substantially increase antibody production in CHO cell batch and fed‐batch cultures. These findings provide compelling evidence supporting the further development of AIP and related peptides as defined medium additives to increase therapeutic protein manufacturing.
Bacillus coagulans, a thermophilic facultative anaerobe, is a favorable chassis strain for the biosynthesis of desired products. In this study, B. coagulans was converted into an efficient malic acid ...producer by metabolic engineering and promoter engineering. Promoter mapping revealed that the endogenous promoter Pldh was a tandem promoter. Accordingly, a promoter library was developed, covering a wide range of relative transcription efficiencies with small increments. A reductive tricarboxylic acid pathway was established in B. coagulans by introducing the genes encoding pyruvate carboxylase (pyc), malate dehydrogenase (mdh), and phosphoenolpyruvate carboxykinase (pckA). Five promoters of various strengths within the library were screened to fine‐tune the expression of pyc to improve the biosynthesis of malic acid. In addition, genes involved in the competitive metabolic pathways were deleted to focus the substrate and energy flux toward malic acid. Dual‐phase fed‐batch fermentation was performed to increase the biomass of the strain, further improving the titer of malic acid to 25.5 g/L, with a conversion rate of 0.3 g/g glucose. Our study is a pioneer research using promoter engineering and genetically modified B. coagulans for the biosynthesis of malic acid, providing an effective approach for the industrialized production of desired products using B. coagulans.
An integrated strategy using metabolic engineering and promoter engineering was designed to convert the thermophilic Bacillus coagulans into a malic‐acid producer. A reductive TCA pathway was established, and a promoter library based on promoter Pldh was developed to fine‐tune the expression of the gene encoding pyruvate carboxylase. The production and yield of malic acid were further improved via dual‐phase fermentation. An effective approach was developed for the industrialized production of desired products using B. coagulans.
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