Western equine encephalitis virus (WEEV) can cause lethal encephalitis in humans and equines and represents a serious public health threat in many countries. Therefore, development of efficient vaccines against WEEV remains an important challenge in the field of disease control. This study described for the first time successful production of WEEV virus-like particles (VLPs) in insect cells using recombinant baculoviruses. This well-established expression system is very suitable for production of WEEV VLPs. The immune experiment herein in mice showed that the VLPs formulated with 206-adjuvant were responsible for the stronger-VLP-specific cellular immune response, and were able to induce the secretion of IL-2, IL-4, IFN-γ and production of high titer antibodies that can effectively neutralize the WEEV pseudoviruses. The WEEV VLPs from insect cells could provide a new, safe, non-replicating and effective vaccine candidate against WEEV infections.
Bioprocess development and optimization is a challenging, costly, and time-consuming effort. In this multidisciplinary task, upstream processing (USP) and downstream processing (DSP) are conventionally considered distinct disciplines. This consideration fosters “one-way” optimization without considering interdependencies between unit operations; thus, the full potential of the process chain cannot be achieved. Therefore, it is necessary to fully integrate USP and DSP process development to provide balanced biotechnological production processes. The aim of the present study was to investigate how different host/leader/antigen binding fragment (Fab) combinations in E. coli expression systems influence USP and primary recovery performance and the final product quality. We ran identical fed-batch cultivations with 16 different expression clones to study growth and product formation kinetics, as well as centrifugation efficiency, viscosity, extracellular DNA, and endotoxin content, which are important parameters in DSP. We observed a severe influence on cell growth, product titer, extracellular product, and cell lysis, accompanied by a significant impact on the analyzed parameters of DSP performance. Our results provide the basis for establishing integrated process development considering interdependencies between USP and DSP. These interdependencies need to be understood for rational decision-making and efficient process development.
In recent years Next-Generation Sequencing (NGS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non-mutated antibody clone to generate spike-in mutations from ~60% down to ~0.3% frequencies. We subsequently tested the effect of 16 different sample and NGS library preparation protocols on the assay’s ability to quantify mutations and on the occurrence of false-positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ~0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low-level true mutations.
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.
We report a novel Cultured Neuronal Network on a chip as a viable alternative to current culture methods for the analysis of neuronal network formation and evolution of the structural properties of the network's graph . This innovative microfluidic chip fabricated from Polydimethylsiloxane, vinyl, and glass offers worthy features such as the posibility to develop and produce custom designs effortlessly, easy handling and monitoring, culture medium renewal, and reducing the exposure to contamination. Due to these benefits, longer survival of the neuronal networks are observed in comparison to conventional methods. In addition to the fast and cost-effective manufacturing of the chip, this technology provides a significant step forward in the studies of neuronal network development and many challening applications such as drug testing of in vitro cell culture models .
Biomolecules are increasingly attractive templates for the synthesis of functional nanomaterials. Chief among them are the plant Tobacco mosaic virus (TMV) and Barley stripe mosaic virus (BSMV) due to their high aspect ratio, narrow size distribution, diverse biochemical functionalities presented on the surface, and compatibility with a number of chemical conjugations. These properties are also easily manipulated by genetic modification to enable the synthesis of a range of metallic and non-metallic nanomaterials for diverse applications. This article reviews the characteristics of TMV, BSMV, and their virus-like particle (VLP) derivatives and how these may be manipulated to extend their use and function. A focus of recent efforts has been on greater understanding and control of the self-assembly processes that drive biotemplate formation. We briefly outline how these features have been exploited in engineering applications such as sensing, catalysis, and energy storage, and discuss emerging advances that promise to accelerate the development of these biotemplates for widescale industrial use.
Microbial conversion of plant biomass into fuels and chemicals offers a practical solution to global concerns on limited natural resources, environmental pollution, and climate change. Pursuant to these goals, researchers have put tremendous efforts and resources towards engineering the yeast Saccharomyces cerevisiae to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into various fuels and chemicals. Although many challenges remain, these research investments have facilitated efficient and rapid fermentation of xylose, simultaneous co-consumption of xylose with carbon sources in lignocellulosic hydrolysates, and enhanced production of a wide range of valuable chemicals from xylose. In particular, understanding of xylose-induced metabolic rewiring in engineered yeast has stimulated the use of xylose as a preferred carbon source for the production of various non-ethanol bioproducts. Here, we summarize recent advances in metabolic engineering in yeast to address bottlenecks of xylose assimilation, and to enable simultaneous co-utilization of xylose and other cellulosic carbon sources. We also highlight distinct characteristics of xylose metabolism which can be harnessed for the production of advanced biofuels and chemicals.
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.
Bioprocess optimization for cell-based therapies is a resource heavy activity. To reduce the associated cost and time, it is advantageous to carry out process development in small volume systems, with the caveat that such systems be predictive for process scaleup. The transport of oxygen from the gas phase into the culture medium, characterized using the volumetric mass transfer coefficient, kLa, has been identified as a critical parameter for predictive process scaleup. In both large- and small-scale bioreactors, kLa is controlled via mixing, with the method employed dependent upon the size of the reactor. However, existing microplate bioreactor platforms, beneficial for their low working volumes and throughput and automation capabilities, struggle to achieve desired kLa for mammalian cell cultures. Here, we describe the development and testing of a 96-well microplate with integrated Redbud Posts to provide mixing and thus enhanced kLa. Mixing characteristics were investigated, with actuating Redbud Posts shown (visually) to increase convective transport while producing enhanced kLa, providing means to mimic macroscale mammalian cell growth conditions at the microscale. Improved cell growth rates with mixing was demonstrated for two cell types, indicating the potential for this technology to play a valuable role in early stage bioprocess development and optimization.
Transient gene expression (TGE) using mammalian cells is an extensively used technology for production of antibodies and recombinant proteins and has been widely adapted by both academic and industrial labs. Chinese Hamster Ovary (CHO) cells have become one of the major work horses for TGE of recombinant antibodies due to their attractive features: post-translational modifications, adaptation to high cell densities, and use of serum-free media. In this study, we describe the optimization of parameters for TGE for antibodies from CHO cells. Through a matrix evaluation of multiple factors including inoculum, transfection conditions, amount and type of DNA used and post-transfection culture conditions, we arrived at an optimized process with higher titer and reduced costs and time, thus increasing the overall efficiency of early antibody material supply. We investigated the amount of coding DNA and the influence of size of the transfection complex on the in vitro efficiency of the transfection. Generation of the transfection complex in serum-free medium leads to the prompt formation of an optimal-sized polyplex, and is independent of the relative amount of coding DNA used for a successful transfection outcome.
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.
Interfaces between biomaterials and living system are critical in regulating their interactions. Poor biocontact properties always limited the performance of biomaterials in biological environment. Surface engineering aims to control the interface interaction to further enhance the desired behavior of biomaterials. Upon implantation of biomaterials into the biological environment, a series of host responses are initiated. Non-specific protein adsorption on biomaterials is the essential stage of all biological reactions that associated with implants failure, device-related infections and blood-coagulation. In this review, we first focused on surface modification techniques to eliminate protein adsorption by emphasizing PEGylation of both macroscopic surface and nanoparticle system. Next, recent developments in surface engineering of biomaterials to optimize interactions between biomaterials and specific host tissue and organs are discussed. Optimizing the biocontact property of blood-contact devices can improve their hemocompatibility and maintain vascular homeostasis. Surface modifications of orthopedic and dental implants confer improved osteointegration and tribology performance. Controlling the surface chemistry and topography, and immobilizing biomolecules can aid the expansion and direct the differentiation of stem cells.
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.
Today’s available therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication and transmission but fail to eliminate the virus. Although transplantation of an allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for three patients, this approach is not considered a general therapeutic strategy because of potential severe side effects. Conversely, genome editing to disrupt the CCR5 locus that encodes the major HIV coreceptor was shown to confer resistance to R5-tropic HIV strains on the cellular level. Here, we present a clinically relevant and highly efficient approach to produce HIV-1 resistant CD4+ T cells. After transferring mRNA coding for CCR5-targeting TALEN into CD4+ T cells by electroporation, up to 89% of CCR5 alleles were disrupted. Genotyping confirmed genetic stability of the edited cell product and off-target analyses established absence of relevant mutagenic events. When challenging these edited T cells with R5-tropic HIV, we observed protection in a dose-dependent manner. Functional assessments revealed no significant differences between edited and control CD4+ T cells in terms of proliferation capacity and their ability to secrete cytokines upon exogenous stimuli. Overall, we successfully engineered HIV-resistant CD4+ T cells under clinically relevant conditions, paving the way for clinical translation.
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.