In the production of biopharmaceuticals depth filters followed by sterile filters are often employed to remove residual cell debris present in the feed stream. In the back drop of a global pandemic, supply chains associated with the production of biopharmaceuticals have been constrained. These constraints have limited the available amount of depth filters for the manufacture of biologics. This has placed manufacturing facilities in a difficult position having to choose between running processes with reduced number of depth filters and risking a failed batch or the prospect of plants going into temporary shutdown until the depth filter resources are replenished. This communication describes a modeling based method that leverages manufacturing scale filtration data to predict the depth filter performance with a reduced number of filters and an increased operational flux. This method can be used to quantify the acceptable level of area reduction before which the filtration process performance is affected. This enables facilities to manage their filter inventory avoiding potential plant shutdowns and reduces the risks of negative depth filter performance.
The production of high-quality recombinant proteins is critical to maintaining a continuous supply of biopharmaceuticals, such as therapeutic antibodies. Engineering mammalian cell factories presents a number of limitations typically associated with proteotoxic stress induced upon aberrant accumulation of off-pathway protein folding intermediates, which eventually culminate with the induction of apoptosis. Recent progress in mammalian synthetic biology provides unique opportunities to endow cells with programmable, user-defined behaviors, thereby addressing some of the challenges of current methods. In this review, we will discuss advances in synthetic biology to design efficient strategies for biomanufacturing.
The insect cell-baculovirus expression vector system (IC-BEVS) has shown to be a powerful platform to produce complex biopharmaceutical products, such as recombinant proteins and VLPs. More recently IC-BEVS has been also used as an alternative to produce adeno-associated virus (AAV). However, little is known about the variability of insect cell populations and the potential effect of heterogeneity on product titer and/or quality. In this study, transcriptomics analysis of Sf9 insect cells during the production of recombinant AAV using a low multiplicity of infection, dual-baculovirus system was performed via single-cell RNA-seq (scRNA-seq). Before infection, the principal source of variability in Sf9 insect cells was associated to cell cycle. Over the course of infection, an increase in transcriptional heterogeneity was detected, this being linked to the expression of baculovirus genes as well as to differences in AAV transgenes ( rep, cap and gfp) expression. Noteworthy, at 24 hours post-infection (hpi) only 29 % of cells showed to enclose all three necessary AAV transgenes to produce packed AAV particles, indicating limitations of the dual baculovirus system. In addition, the trajectory analysis herein performed highlighted biological processes such as protein folding, metabolic processes, translation and stress response has been significantly altered upon infection. Overall, this work reports the first application of scRNA-seq to the IC-BEVS and highlights significant variations in individual cells within the population, providing insight for rational cell and process engineering towards improved AAV production in IC-BEVS.
Reducing drug development timelines is an industry-wide goal to bring medicines to patients in need more quickly. This was exemplified in the COVID-19 pandemic where reducing development timelines had a direct impact on the number of lives lost to the disease. The use of drug substance produced using cell pools, as opposed to clones, has the potential to shorten development timelines. Toward this goal, we have developed a novel technology, GPEx® Lightning, that allows for rapid, reproducible, targeted recombination of transgenes into more than 200 Dock sites in the CHO genome. This allows for rapid production of high expressing stable cell pools and clones that reach titers of 4 to 12 g/L in generic fed-batch production. These pools and clones are highly stable in both titer and glycosylation, showing strong similarity in glycosylation profiles.
The integration of a transgene expression construct into the host genome is the initial step for the generation of recombinant cell lines used for biopharmaceutical production. The stability and level of recombinant gene expression in Chinese hamster ovary (CHO) can be correlated to the copy number, its integration site as well as the epigenetic context of the transgene vector. Also, undesired integration events, such as concatemers, truncated and inverted vector repeats, are impacting the stability of recombinant cell lines. Thus, to characterize cell clones and to isolate the most promising candidates it is crucial to obtain information on the site of integration, the structure of integrated sequence and the epigenetic status. Current sequencing techniques allow to gather this information separately but do not offer a comprehensive and simultaneous resolution. In this study, we present a fast and robust nanopore Cas9-targeted sequencing (nCats) pipeline to identify integration sites, the composition of the integrated sequence as well as its DNA methylation status in CHO cells that can be obtained simultaneously from the same sequencing run. A Cas9-enrichment step during library preparation enables targeted and directional nanopore sequencing with up to 724x median on-target coverage and up to 153 Kb long reads. The data generated by nCats provides sensitive, detailed and correct information on the transgene integration sites and the expression vector structure, which could only be partly produced by traditional Targeted Locus Amplification-Seq data. Moreover, with nCats the DNA methylation status can be analyzed from the same raw data without prior DNA amplification.
Next generation manufacturing (NGM) has evolved over the past decade to a point where large biopharmaceutical organizations are making large investments in the technology and considering implementation in clinical and commercial processes. There are many well-considered reasons to implement NGM. For the most part, organizations will not fund NGM unless the implementation benefits the funding organization by providing reduced costs, reduced time or additional needed capabilities. Productivity improvements gained from continuous purification are shown in this work, which used a new system that fully integrates and automates several downstream unit operations of a biopharmaceutical process to provide flexibility and easy implementation of NGM. The equipment and automation supporting NGM can be complicated and expensive. Biopharmaceutical Process Development considered two options: (1) design its own NGM system or (2) buy a pre-built system. PAK BioSolutions (Virginia, US), provides a turn-key automated and integrated system that can operate up to four continuous purification stages simultaneously, while maintaining a small footprint in the manufacturing plant. The PAK system provides significant cost benefits (~10x lower) compared to the alternative – integration of many different pieces of equipment through a Distributed Control System (DCS) that would require significant engineering time for design, automation and integration. Integrated and Continuous Biomanufacturing can lead to significant reductions in facility size, reduced manufacturing costs, and enhanced product quality when compared to the traditional batch mode of operation. The PAK system uses new automation strategies that robustly link unit operations. We present the optimized process fit, sterility and bioburden control strategy, and automation features (such as pH feedback control and in-line detergent addition) that enabled continuous operation of a 14 day end-to-end monoclonal antibody purification process at the clinical manufacturing scale.
The liver is one of the vital organs in the body, and the gold standard of treatment for liver function impairment is liver transplantation, which poses many challenges. The specific 3D structure of liver, which significantly impacts the growth and function of its cells, has made biofabrication with the 3D printing of scaffolds suitable for this approach. In this study, to investigate the effect of scaffold geometry on the performance of HepG2 cells, Poly-Lactic acid (PLA) polymer was used as the input of the Fused Deposition Modeling (FDM) 3D-printing machine. Samples with simple square and bioinspired hexagonal cross-section designs were printed. 1% and 2% of gelatin-coating were applied to the 3D printed PLA to improve the wettability and surface properties of the scaffold. SEM pictures were used to analyze the structural properties of PLA-Gel hybrid scaffolds, EDS to investigate the presence of gelatin, water contact angle measurement for wettability, and weight loss for degradation. In vitro tests were performed by culturing HepG2 cells on the scaffold to evaluate the cell adhesion, viability, cytotoxicity, and specific liver functions. Then, high-precision scaffolds were printed and the presence of gelatin was detected. Also, the effect of geometry on cell function was confirmed in viability, adhesion, and functional tests. The albumin and urea production of the Hexagonal PLA scaffold was about 1.22 ±0.02 fold higher than the square design in 3 days. This study will hopefully advance our understanding of liver tissue engineering toward a promising perspective for liver regeneration.
Herein we report the use of Pseudomonas putida F1 biofilms grown on carbonized cellulosic fibers to achieve biodegradation of airborne VOCs in the absence of any bulk aqueous phase media. It is believed that direct exposure of gaseous VOC substrates to biomass may eliminate aqueous phase mass transfer resistance and facilitate VOC capture and degradation. When tested with toluene vapor as a model VOC, the supported biofilm could grow optimally at 300 ppm toluene and 80% relative humidity, with a specific growth rate of 0.425 day -1. During long-term VOC biodegradation tests in a tubular packed bed reactor, biofilms achieved a toluene degradation rate of 2.5 mg g DCW -1 h -1 during the initial exponential growth phase. Interestingly, the P. putida F1 film kept biodegrading activity even at the subsequent stationary non-growth phase. The supported biofilms with a biomass loading of 20% (wt) could degrade toluene at a rate of 1.9 mg g DCW -1 h -1 during the stationary phase, releasing CO 2 at a rate of 6.4 mg g DCW -1 h -1 at the same time (indicating 100% conversion of substrate carbon to CO 2). All the specific degradation rates are much higher than what can be gleaned from previously reported work. It also demonstrates the feasibility of biofilm growth and direct gas phase degradation of VOCs without requiring any bulk aqueous phase.
Viral systems such as wild-type viruses, viral vectors, and virus-like particles are essential components of modern biotechnology and medicine. Despite their importance, the commercial-scale production of viral systems remains highly inefficient for multiple reasons. Computational strategies are a promising avenue for improving process development, optimization, and control, but require a mathematical description of the system. This article reviews mechanistic modeling strategies for the production of viral particles, both at the cellular and bioreactor scales. In many cases, techniques and models from adjacent fields such as epidemiology and wild-type viral infection kinetics can be adapted to construct a suitable process model. These process models can then be employed for various purposes such as in-silico testing of novel process operating strategies and/or advanced process control.
Extracellular production of target proteins simplifies downstream processing due to obsolete cell disruption. However, optimal combinations of a heterologous protein, suitable signal peptide and secretion host can currently not be predicted, resulting in large strain libraries that need to be tested. On the experimental side, this challenge can be tackled by miniaturization, parallelization and automation, which provide high-throughput screening data. These data need to be condensed into a candidate ranking for decision making to focus bioprocess development on the most promising candidates. We screened for Bacillus subtilis signal peptides mediating Sec secretion of two polyethylene terephthalate degrading enzymes (PETases), leaf-branch compost cutinase (LCC) and polyester hydrolase (PE-H) mutants, by Corynebacterium glutamicum. We developed a fully automated screening process and constructed an accompanying Bayesian statistical modeling framework, which we applied in screenings for highest activity in 4-nitrophenyl palmitate degradation. In contrast to classical evaluation methods, batch effects and biological errors are taken into account and their uncertainty is quantified. Within only two rounds of screening, the most suitable signal peptide was identified for each PETase. Results from LCC secretion in microliter-scale cultivation were shown to be scalable to laboratory-scale bioreactors. This work demonstrates an experiment-modeling loop that can accelerate early-stage screening in a way that experimental capacities are focused to the most promising strain candidates. Combined with high-throughput cloning, this paves the way for using large strain libraries of several hundreds of strains in a Design-Build-Test-Learn approach.
Thermobifida fusca cutinase ( TfC ) is a carboxylesterase (CE) that degrades the environmental pollutant, polyethylene terephthalate (PET). TfC also acts upon PET’s degradation intermediates (DIs), such as oligoethylene terephthalate (OET), and bis-/mono-hydroxyethyl terephthalate (BHET/MHET), to convert these into terephthalic acid (TPA), the terminal product of PET degradation. We examined TfC’s surface, compared it to that of other CEs, and performed molecular docking and MD simulations with an OET, 2HE-(MHET) 3, to understand interactions between TfC’s surface and the OET, at TfC’s active site as well as vicinal regions. We mutated 17 residues on TfC’s surface, mostly individually, but sometimes using pairs of mutations, to see how these modulate TfC’s activity. Most mutants/variants showed a decrease in activity against solid PET. Some killed activity completely. However, three mutations (L90F, F209I and F249R), made using a background mutation (G62A) already reported to improve activity by almost ~2.0-fold, yielded increases in activity that were between ~1.3- and ~2.0-fold higher than that of G62A TfC (which we found to display a ~1.7-fold increase in activity over TfC, in our own experiments). TfC variants, G62A/F249R, and G62A/F209I, exhibit the highest activities yet observed in any TfC mutants/variants, against PET, and BHET, respectively.
Autologous cell therapy has proven to be an effective treatment for hematological malignancies. Cell therapies for solid tumors are on the horizon, however the high cost and complexity of manufacturing these therapies remain a challenge. Routinely used open steps to transfer cells and reagents through unit operations further burden the workflow reducing efficiency and increasing the chance for human error. Here we describe a fully closed, autologous bioprocess generating MAGE-B2 TCR-T cells. This bioprocess yielded 5 – 12e9 MAGE-B2-specific TCR-expressing T cells, transduced at low MOIs, within 7 to 10 days, and cells exhibited an enriched memory T cell phenotype and enhanced metabolic fitness. It was demonstrated that activating, transducing, and expanding leuko-apheresed cells in a single bioreactor without a T cell enrichment step promoted lentivirus transduction efficiency while resulting in comparable level of T cell purity (~97%) as that of leukopak cells that went through CD8+ and CD4+ positive selection. The critical process parameters of the bioreactor, including culturing at a high cell density (7e6 cells/mL), adjusting rocking agitations during phases of scale up, lowering glycolysis through addition of 2-Deoxy-D-glucose (2-DG), and modulating IL-2 levels, were shown to positively regulate TCR expression and cell doubling time, and promote resistance to effector-associated apoptosis of TCR-T cells. The bioprocess described herein supports scale-out feasibility by enabling processing of multiple patients’ batches in parallel within a Grade C cleanroom.
Biobutanol produced in acetone-butanol-ethanol fermentation at batch mode cannot compete with chemically derived butanol because of the low reactor productivity. Continuous fermentation can dramatically enhance productivity and lower capital and operating costs but are rarely used in industrial fermentation because of increased risks in culture degeneration, cell washout, and contamination. In this study, cells of the asporogenous Clostridium acetobutylicum ATCC55025 were immobilized in a single-pass fibrous-bed bioreactor (FBB) for continuous production of butanol from glucose and butyrate at various dilution rates. Butyric acid in the feed medium helped maintaining cells in the solventogenic phase for stable continuous butanol production. At the dilution rate of 1.88 h -1, butanol was produced at 9.55 g/L with a yield of 0.24 g/g and productivity of 16.8 g/L∙h, which was the highest ever achieved for biobutanol fermentation and an 80-fold improvement over the conventional ABE fermentation. The extremely high productivity was attributed to the high density of viable cells (~100 g/L at >70% viability) immobilized in the fibrous matrix, which also enabled the cells to better tolerate butanol and butyric acid. The FBB was stable for continuous operation for an extended period of over one month.
Isoprenoids are a large family of natural products with diverse structures, which allow them to play diverse and important roles in the physiology of plants and animals. They also have important commercial uses as pharmaceuticals, flavouring agents, fragrances, and nutritional supplements. Recently, metabolic engineering has been intensively investigated and emerged as the technology of choice for the production of isoprenoids through microbial fermentation. Isoprenoid biosynthesis typically originates in plants from acetyl-coA in central carbon metabolism, however, a recent study reported an alternative pathway, the Isopentenol Utilization pathway (IUP), that can provide the building blocks of isoprenoid biosynthesis from affordable C5 substrates. In this work, we expressed the IUP in Escherichia coli to efficiently convert isopentenols into geranate, a valuable isoprenoid compound. We first established a geraniol-producing strain in E. coli that uses the IUP. Then, we extended the geraniol synthesis pathway to produce geranate through two oxidation reactions catalysed by two alcohol/aldehyde dehydrogenases from Castellaniella defragrans. The geranate titer was further increased by optimizing the expression of the two dehydrogenases and also parameters of the fermentation process. The best strain produced 764 mg/L geranate in 24 h from 2 g/L isopentenols (a mixture of isoprenol and prenol). We also investigated if the dehydrogenases could accept other isoprenoid alcohols as substrates.
The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate (MTX) or methionine sulfoximine (MSX), respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine–the selection medium for Gs-based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co-transfection of Gs-Enbrel and Aspg-Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in selection medium, the cells improved viability and growth while still achieving ~5-fold higher specific productivity and ~3-fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.
Microorganisms build fatty acids with biocatalytic assembly lines, or fatty acid synthases (FASs), that can be repurposed to produce a broad set of fuels and chemicals. Despite their versatility, the product profiles of FAS-based pathways are challenging to adjust without experimental iteration, and off-target products are common. This study uses a detailed kinetic model of the E. coli FAS as a foundation to model nine oleochemical pathways. These models provide good fits to experimental data and help explain unexpected results from in vivo studies. An analysis of pathways for alkanes and fatty acid ethyl esters, for example, suggests that reductions in titer caused by enzyme overexpression can result from shifts in pools of metabolic intermediates that are incompatible with the substrate specificities of downstream enzymes. In general, different engineering objectives (i.e., production, unsaturated fraction, and average chain length) show experimentally consistent sensitivities to pathway enzymes, and model-based compositional analyses indicate simple shifts in enzyme concentrations can alter the product profiles of pathways with promiscuous components. The study concludes by integrating all models into a graphical user interface. The models supplied by this work provide a versatile kinetic framework for studying oleochemical pathways in different biochemical contexts.
7-Methylxanthine, a derivative of caffeine (1,3,7-trimethylxanthine), is a high-value compound that has multiple medical applications, particularly with respect to eye health. Here, we demonstrate the biocatalytic production of 7-methylxanthine from caffeine using Escherichia coli strain MBM019, which was constructed for production of paraxanthine (1,7-dimethylxanthine). The mutant N-demethylase NdmA4, which was previously shown to catalyze N 3-demethylation of caffeine to produce paraxanthine, also retains N 1-demethylation activity toward paraxanthine. This work demonstrates that whole cell biocatalysts containing NdmA4 are more active toward paraxanthine than caffeine. We used four serial resting cell assays, with spent cells exchanged for fresh cells between each round, to produce 2,120 μM 7-methylxanthine and 552 μM paraxanthine from 4,331 μM caffeine. The purified 7-methylxanthine and paraxanthine were then isolated via preparatory-scale HPLC, resulting in 177.3 mg 7-methylxanthine and 48.1 mg paraxanthine at high purity. This is the first reported strain genetically optimized for the biosynthetic production of 7-methylxanthine from caffeine.