The production of recombinant proteins usually reduces cell fitness and the growth rate of producing cells. The growth disadvantage favors faster-growing non-producer mutants. Therefore, continuous bioprocessing is hardly feasible in Escherichia coli due to the high escape rate. We investigated the stability of E. coli expression systems under long-term production conditions and how metabolic load triggered by recombinant gene expression influences the characteristics of mutations. We conducted iterated fed-batch-like microbioreactor cultivations under production conditions. We used the easy-to-produce green fluorescent protein (GFP) and a challenging antigen-binding fragment (Fab) as model proteins, and BL21(DE3) and BL21Q strains as expression hosts. In comparative whole genome sequencing analyses, we identified mutations that allowed cells to grow unhindered despite recombinant protein production. A T7 RNA polymerase expression system is only conditionally suitable for long-term cultivation under production conditions. Mutations leading to non-producers occur in either the T7 RNA polymerase gene or the T7 promoter. The host RNA polymerase-based BL21Q expression system remained stable in the production of GFP in long-term cultivations. For the production of Fab, mutations in lacI of the BL21Q derivatives had positive effects on long-term stability. Our results indicate that adaptive evolution carried out with genome-integrated E. coli expression systems in microtiter cultivations under industrial relevant production conditions is an efficient strain development tool for production hosts.
Nascent advanced therapies, including regenerative medicine and cell and gene therapies, rely on the production of cells in bioreactors that are highly heterogeneous in both space and time. Unfortunately, these promising therapies have failed to reach a wide patient population due to unreliable manufacturing processes that result in batch variability and cost prohibitive production. This can be attributed largely to a void in existing process analytical technologies (PATs) capable of characterizing the secreted critical quality attributes (CQAs) biomolecules that correlate with the final product quality. The Dynamic Sampling Platform (DSP) is a PAT for cell bioreactor monitoring that can be coupled to a suite of sensor techniques to provide real-time feedback on spatial and temporal CQA content in situ. In this study, DSP is coupled with electrospray ionization mass spectrometry (ESI-MS) and direct-from-culture sampling to obtain measures of CQA content in bulk media and the cell microenvironment throughout the entire cell culture process (~3 weeks). Post hoc analysis of this real-time data reveals that DSP output is heavily dependent on spatial context. Importantly, these results demonstrate that an effective PAT must incorporate both spatial and temporal resolution to serve as an effective input f or feedback control in advanced therapy production.
As a milestone in soft and hard tissue engineering, a precise control over the micropatterns of scaffolds have lightened new opportunities for the recapitulation of native body organs through 3D bioprinting approaches. Well-printable bioinks are pre-requisites for the bioprinting of tissues/organs where hydrogels play a critical role. Despite the outstanding developments in 3D engineered microstructures, current printer devices, suffer from the risk of exposing loaded living agents to mechanical (nozzle-based) and thermal (nozzle-free) stresses. Thus, tuning the rheological, physical, and mechanical properties of hydrogels are promising solutions to address these issues. The relationship between the mechanical characteristics of hydrogels and their printability is important to control printing quality and fidelity. Recent developments in defining this relationship have highlighted the decisive role of main additive manufacturing strategies. These strategies are applied to enhance the printing quality of scaffolds and determine the nurture of cellular morphology. In this regard, it is beneficial to use external and internal stabilization, photo curable biopolymers, and cooling substrates containing the printed scaffolds. The objective of this study was to review cutting-edge developments in hydrogel-type bioinks and discuss the optimum simulation of the zonal stratification in osteochondral and cartilage units.
The current industrial production of polymer building blocks such as ε-caprolactone (ε-CL) and 6-hydroxyhexanoic acid (6HA) is a multi-step process associated with critical environmental issues such as the generation of toxic waste and high energy consumption. Consequently, there is a demand for more eco-efficient and sustainable production routes. This study deals with the generation of a platform organism that converts cyclohexane to such polymer building blocks without the formation of byproducts and under environmentally benign conditions. Based on kinetic and thermodynamic analyses of the individual enzymatic steps, we rationally engineered a 4-step enzymatic cascade in Pseudomonas taiwanensis VLB120 via stepwise biocatalyst improvement on the genetic level. We found that the intermediate product cyclohexanol severely inhibits the cascade and optimized the cascade by enhancing the expression level of downstream enzymes. The integration of a lactonase enabled exclusive 6HA formation without side products. The resulting biocatalyst showed a high activity of 44.8 ± 0.2 U gCDW-1 and fully converted 5 mM cyclohexane to 6HA within 3 h. This platform organism can now serve as a basis for the development of greener production processes for polycaprolactone and related polymers.
Viral vectors have a great potential for gene delivery, but manufacturing at pharmaceutical scale is a big challenge for the industry. The baculovirus-insect cells system is one of the most scalable platforms to produce clinical grade recombinant Adeno-Associated Virus (rAAV) vectors, however, the standard procedure to generate recombinant baculovirus based on Tn7 transposition is time consuming and still suffers technical constraints. Indeed, we recently shown that baculoviral sequences adjacent to the AAV ITRs are preferentially encapsidated into the rAAV vector particles. This observation raised concern about safety for clinical applications due to the presence of bacterial and antibiotic resistance coding sequences with Tn7-mediated system for the construction of recombinant baculoviruses. Here, we investigated a faster and safer method to generate baculovirus reagents based on homologous recombination (HR) for its use in rAAV manufacturing compared to the Tn7-based system. First, we confirmed the functionality of inserted cassette and the absence of undesirable genes into HR-derived baculoviral genomes. Strikingly, we found that the exogenous cassette shown an increased stability over passages when using HR system. Finally, we tested these materials to produce rAAV vectors. The baculoviruses originated from either system lead to high rAAV vector genome yields, with the advantage for the HR method being that the rAAV lots are exempted of undesirable gentamycin and kanamycin genes derived sequences which provides an additional level of safety for the manufacturing of rAAV vectors. Overall, this study highlights the importance of the upstream process and starting biologic materials to generate safer rAAV biotherapeutic products.
The accelerating development of gene therapy from research towards clinical trials and beyond has elevated the demand for practical viral vector manufacturing solutions. The use of disposable upstream technology is gaining traction in clinical manufacturing. The world's first disposable, fully integrated, high-cell density fixed-bed bioreactor was launched approximately one decade ago. By now, the iCELLis fixed-bed technology has obtained the broadest customer base. This system is available in small scale but also provides the largest GMP compliant commercial system. However, there are several alternative technologies, which have been widely used for the manufacturing of different viral vectors, allowing for complementation within the market. This article will review virus production using the latest disposable fixed-bed bioreactors, present highlights of an interview with the inventor of these bioreactors, and share some user experience. It is predicted that single-use fixed-bed bioreactors will receive even more attention in the field of viral vector manufacturing and commercialization, especially with high virus yields.
Benzoic acid is one of the most commonly used food preservatives, but currently exclusively produced in petrochemical processes. In this study, we describe a bio-based production pathway using an engineered strain of Pseudomonas taiwanensis. In a phenylalanine-overproducing strain, we heterologously expressed bacterial, yeast, and plant genes to achieve production of benzoate via a β-oxidation pathway. Strategic disruption of the native Pseudomonas benzoate degradation pathway further allowed the production of catechol and cis,cis-muconate. Taken together, this work demonstrates new routes for the microbial production of these industrially relevant chemicals from renewable resources.
Adeno-associated viral vectors (AAV) are one of the most efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs goes hand in hand with the need to increase manufacturing capacities and to develop appropriate quality controls. In particular, accurate methods to assess the level of residual DNA in AAV vector stocks are needed, considering the potential risk of co-transferring oncogenic or immunogenic sequences with the therapeutic vectors. Our laboratory has developed an assay based on high-throughput sequencing (HTS) to exhaustively identify and quantify DNA species in recombinant AAV batches. Compiled with a computational analysis of the single nucleotide variants (SNV), Single-Stranded Virus Sequencing (SSV-Seq) also provides information regarding rAAV genome identity. In this study, we show that the PCR amplification of regions with high GC content and including mononucleotide stretches can be biased during the DNA library preparation, leading to drops in the sequencing coverage along the AAV vector genome. To circumvent this problem, we have developed a PCR-free protocol, named SSV-Seq 2.0, that is optimized for the sequencing of rAAV genomes that contain sequences with a high percentage of GC and homopolymers, such as the CAG promoter. HTS-based assays are indispensable to provide accurate data to the regulatory agencies regarding nucleic acids content in AAV vector batches and to improve the safety and efficacy of these viral vectors.
Graphene quantum dots (GQDs), the latest member of graphene family, have attracted outstanding interest in the last few years, due to their outstanding physical, chemical, electrical, optical and biological properties. Their strong size-dependent photoluminescence (PL) and the presence of many reactive groups on the graphene surface allow their multimodal conjugation with therapeutic agents, targeting ligands, polymers, light responsive agents, fluorescent dyes, and functional nanoparticles, making them valuable agents for cancer diagnosis and treatment. In this review, the very recent advances covering the last three years on the applications of GQDs as drug delivery systems (DDS) and theranostic tools for anticancer therapy are discussed, highlighting the relevant factors which regulate their biocompatibility. Among these factors, the size, kind and degree of surface functionalization have shown to greatly affect their use in biological systems. Toxicity issues, which still represent an open challenge for the clinical development of GQDs based therapeutic agents, are also discussed at cellular and animal levels.
Synthetic biology has promoted the development of biosensors as tools for detecting trace substances. In the past, biosensors based on synthetic biology have been designed on living cells, but the development of cell biosensors has been greatly limited by defects such as the obstruction of cell membrane. However, the advent of cell-free synthetic biology addresses these limitations. Biosensors based on the cell-free protein synthesis system have the advantages of higher safety, higher sensitivity, and faster response time over cell biosensors, which makes cell-free biosensors have a broader application prospect. This review summarizes the workflow of various cell-free biosensors, including the identification of analytes and signal output. The detection range of cell-free biosensors is greatly enlarged by different recognition mechanisms and output methods. In addition, the review also discusses the applications of cell-free biosensors in environmental monitoring and health diagnosis, as well as existing deficiencies and aspects that should be improved. In the future, through continuous improvement and optimization, the potential of cell-free biosensors will be stimulated, and their application fields will be expanded.
The pandemic outbreaks of coronavirus disease 2019 (COVID-19) was first discovered in Wuhan, Hubei, China in December 2019. The COVID-19 was caused by the novel coronavirus, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It took 30 days to spread to all provinces of China . Recently, the confirmed cases of COVID-19 have been reported from about 200 countries or regions on March 30, 2020, and killed almost 30 thousand people . Efficient identification of the infection by SARS-CoV-2 has been one of the most important tasks to facilitate all the following counter measurements in dealing with infectious disease. In Taiwan, a COVID-19 Open Science Platform adhering to the spirit of open science: sharing sources, data, and methods to promote progress in academic research while corroborating findings from various disciplines has established in mid-February 2020, for collaborative research in support of the development of detection methods, therapeutics, and a vaccine for COVID-19. Research priorities include infection control, epidemiology, clinical characterization and management, detection methods (including viral RNA detection, viral antigen detection, and serum antibody detection), therapeutics (neutralizing antibody and small molecule drugs), vaccines, and SARS-CoV-2 pathogenesis. In addition, research on social ethics and the law are included to take full account of the impact of the COVID-19 virus.
There is a vast number of biomaterials ranging from drug-eluting stents, coated implants, drug delivery devices and artificial organs, among others, that have been developed in recent years. However, translation of many of these biomaterials to clinic is often plagued by biocompatibility challenges. This review focuses on strategies implemented in some of the recently developed biomaterials -- particularly for soft and hard tissue regeneration, organ manufacturing and disease remediation -- to overcome potential foreign body response to the incorporation of the biomaterials in the host.