Aerobic glycolysis and its by-product lactate accumulation are usually associated with adverse culture phenotypes such as poor cell viability and productivity. Due to the lack of knowledge on underlying mechanisms and accompanying biological processes, the regulation of aerobic glycolysis has been an ongoing challenge in culture process development for therapeutic protein productivity. NAD+ , a coenzyme and co-substrate in energy metabolism, promotes the conversion of inefficient glycolysis into an efficient oxidative phosphorylation pathway. However, the effect of NAD+ on CHO cells for biopharmaceutical production has not been reported yet. In this work, we aimed to elucidate the influence of NAD+ on cell culture performance by examining metabolic shifts and mAb productivity. The supplementation of NAD+ increased the intracellular concentration of NAD+ and promoted SIRT3 expression. Antibody titer and the specific productivity in the growth phase were improved by up to 1.82 and 1.88-folds, respectively, with marginal restrictions on cell growth. NAD+ significantly reduced the accumulation of ROS and the degree of glycolysis, determined by lactate accumulation versus glucose consumption. In contrast, oxidative phosphorylation capacity and amino acid consumption rate increased substantially. Collectively, these results suggest that NAD+ contributes to improving therapeutic protein productivity in bioprocessing via inducing an energy metabolic shift. This article is protected by copyright. All rights reserved.

Human Adipose-Derived Mesenchymal Stem/Stromal Cells (hAD-MSCs) have great potential for tissue regeneration. Since transplanted hAD-MSCs are likely to be placed in a hypoxic environment, culturing the cells under hypoxic conditions might improve their post-transplantation survival and regenerative performance. The combination of hAD-MSCs and PCL-nHA nanofibers synergically improves the contribution of both components for osteoblast differentiation. In this work, we hypothesized that this biomaterial constitutes a hypoxic environment for hAD-MSCs. We studied the cellular re-arrangement and the subcellular ultrastructure by Transmission Electron Microscopy (TEM) of hAD-MSCs grown into PCL-nHA nanofibers, and we compared them with the same cells grown in two-dimensional cultures, over tissue culture-treated plastic, or glass coverslips. Among the most evident changes, PCL-nHA grown cells showed enlarged mitochondria, and accumulation of glycogen granules, consistent with a hypoxic environment. We observed a 3.5 upregulation (p = 0.0379) of Hypoxia Inducible Factor (HIF)-1A gene expression in PCL-nHA grown cells. This work evidences for the first time intra-cellular changes in three-dimensional compared to two-dimensional cultures, which are adaptive responses of the cells to an environment more closely resembling that of the in vivo niche after transplantation, thus PCL-nHA nanofibers are adequate for hAD-MSCs pre-conditioning.

N6 -methyladenosine (m6A) is the most abundant internal modification on eukaryotic mRNA and has been implicated in a wide range of fundamental cellular processes. This modification is regulated and interpreted by a set of writer, eraser, and reader proteins. To date, there have been no reports on the potential of mRNA epigenetic regulators to influence recombinant protein expression in mammalian cells. In this study, the potential of manipulating the expression of the m6A YTH domain-containing readers, YTHDF1, 2 and 3 to improve recombinant protein yield based on their role in regulating mRNA stability and promoting translation were evaluated. Using siRNA-mediated gene depletion, cDNA over-expression, and methylation-specific RNA immunoprecipitation, it is demonstrated that (i) knock-down of YTHDF2 enhances (~2-fold) the levels of recombinant protein derived from GFP and EPO transgenes in CHO cells; (ii) the effects of YTHDF2 depletion on transgene expression is m6A-mediated; and (iii) YTHDF2 depletion, or over-expression of YTHDF1 increases viral protein expression and yield of infectious lentiviral (LV) particles (~2-3-fold) in HEK293 cells. We conclude that various transgenes can be subjected to regulation by m6A regulators in mammalian cell lines and that these findings demonstrate the utility of epitranscriptomic-based approaches to host cell line engineering for improved recombinant protein and viral vector production.

Auxotrophic marker genes have been widely used for genetic engineering in yeast. However, the effects of amino acids or nucleotides deficiency in auxotrophic strains on cell growth and product synthesis were rarely reported. In this study, a total of eight auxotrophic strains of Saccharomyces cerevisiae with single knockout of selection markers were obtained. Cell growth and free fatty acid (FFA) production of these auxotrophic strains were evaluated with supplementation of different concentrations of amino acids or nucleotides. Generally, except ade2Δ mutants, most auxotrophic strains showed decreased cell growth and FFA production, which could be recovered by adding higher concentrations of supplements. LEU2 deletion (leu2Δ) damaged both cell growth and FFA production even with supplementation of 1000 mg L-1 leucine. This study shows that growth and product biosynthesis of auxotrophs could be limited by insufficient supplementation of amino acids or nucleotides, and provides guidance on supplementation of these nutrients during fermentation to maximize cell growth and product biosynthesis.

Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) reportedly induce the osteogenic and tenogenic differentiation of anterior cruciate ligament (ACL)-derived stem cells (LDSCs), respectively. However, few studies have investigated the effect of BMP-2/TGF-β1 on the differentiation of LDSC. We developed a BMP-2/TGF-β1 gene insertion into an LDSC cell sheet that promotes tendon-bone healing in a mouse ACL reconstruction (ACLR) model. CD34+ LDSCs were isolated from human ACL stump tissues, virally transduced to express BMP-2 or TGF-β1, and then embedded within cell sheets. All mice underwent ACLR using an autograft wrapped with a cell sheet and were randomly divided into three groups: BMP-2-, TGF-β1-, and BMP-2/TGF-β1-transduced. At 4 and 8 weeks, tendon-bone healing was evaluated by micro-CT, biomechanical test, and histological analysis. BMP-2 and TGF-β1 promoted the osteogenic and tenogenic differentiation of LDSC in vitro. BMP-2/TGF-β1-transduced LDSC sheet application contributed to early improvement in mean failure load and graft stiffness, accelerated maturation of the tendon-bone junction, and inhibited bone tunnel widening. Furthermore, reduced M1 macrophage infiltration and a higher M2 macrophage percentage were observed in the BMP-2/TGF-β1-transduced LDSC group. This work demonstrated that BMP-2 and TGF-β1 promoted CD34+ LDSCs osteogenic and tenogenic differentiation in vitro and in vivo, which accelerated the tendon-bone healing after ACLR using autografts wrapped with cell sheets in a mouse model.

Enzymatic asymmetric synthesis of chiral amino acids has great industrial potential. However, the low catalytic efficiency of high-concentration substrates limits their industrial application. Herein, using a combination of substrate catalytic efficiency prediction based on "open to closed" conformational change and substrate specificity prediction, a novel leucine dehydrogenase (TsLeuDH), with high substrate catalytic efficiency toward benzoylformic acid for producing L-phenylglycine, was directly identified from 4,695 putative leucine dehydrogenases in a public database. The specific activity of TsLeuDH was determined to be as high as 4,253.8 U·mg-1 . Through reaction process optimization, a high-concentration substrate (0.7 M) was efficiently and completely converted within 90 min in a single batch, without any external coenzyme addition. Moreover, a continuous flow-feeding approach was designed using gradient control of the feed rate to reduce substrate accumulation. Finally, the highest overall substrate concentration of up to 1.2 M benzoylformic acid could be aminated to L-phenylglycine with conversion of > 99% in 3 h, demonstrating that this new combination of enzyme process development is promising for large-scale application of L-phenylglycine. This article is protected by copyright. All rights reserved.

Aptamers are a class of single-stranded (ss) nucleic acid molecules generated through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) that involves iterations of time-consuming and tedious selection, amplification, and enrichment steps. To compensate for the drawbacks of conventional SELEX, we have devised an in-silico methodology that facilitates a cost-effective and facile manner of aptamer selection. Here, we report the isolation of DNA aptamers against androgen receptors (ARs) using androgen response elements (ARE) that possess natural affinity toward AR. A virtual library of ARE sequences was prepared and subjected to a stringent selection criterion to generate a sequence pool having stable hairpin conformations and high GC content. The 3D-structures of the selected ss AREs were modeled and screened through rigid docking and molecular dynamic (MD) simulation to examine their potency as potential AR binders. The predicted sequences were further validated using direct enzyme-linked aptasorbent assay (ELASA), which includes the measurement of their binding affinity, specificity, and target discrimination properties under complex biological enviroments. A short, 15 nucleotides (nts), ssDNA aptamer, termed ARapt1 with the estimated Kd value of 5.5 ± 3 nm, was chosen as the most prominent aptamer against AR based on the coherence of both the in-silico and in-vitro evaluation results. The high target-binding affinity and selectivity of ARapt1 signify its potential use as a versatile tool in diagnostic applications relevant to prostate cancer and related diseases.

Engineered Saccharomyces cerevisiae expressing a lactic acid dehydrogenase can metabolize pyruvate into lactic acid. However, three pyruvate decarboxylase (PDC) isozymes drive most carbon flux toward ethanol rather than lactic acid. Deletion of endogenous PDCs will eliminate ethanol production, but the resulting strain suffers from C2 auxotrophy and struggles to complete a fermentation. Engineered yeast assimilating xylose or cellobiose produce lactic acid rather than ethanol as a major product without the deletion of any PDC genes. We report here that sugar flux, but not sensing, contributes to the partition of flux at the pyruvate branch point in S. cerevisiae expressing the Rhizopus oryzae lactic acid dehydrogenase (LdhA). While the membrane glucose sensors Snf3 and Rgt2 did not play any direct role in the option of predominant product, the sugar assimilation rate was strongly correlated to the partition of flux at pyruvate: fast sugar assimilation favors ethanol production while slow sugar assimilation favors lactic acid. Applying this knowledge, we created an engineered yeast capable of simultaneously converting glucose and xylose into lactic acid, increasing lactic acid production to approximately 17 g L-1 from the 12 g L-1 observed during sequential consumption of sugars. This work elucidates the carbon source-dependent effects on product selection in engineered yeast.

The high demand for monoclonal antibody (mAb) therapeutics in recent years has resulted in significant efforts to improve their costly manufacturing process. The high cost of manufacturing mAbs derives mainly from the purification process, which contributes to 50%-80% of the total manufacturing cost. One of the main challenges facing industry at the purification stage is the clearance of host cell proteins (HCPs) that are produced and often co-purified with the desired mAb product. One of the issues HCPs can cause is the degradation of the final mAb protein product. In this review, techniques are considered that can be used at different stages (upstream and downstream) of mAb manufacture to improve HCP clearance. In addition to established techniques, many new approaches for HCP removal are discussed that have the potential to replace current methods for improving HCP reduction and thereby the quality and stability of the final mAb product.

RNA aptamers bind specifically and selectively to various macromolecules, cell surfaces, and viruses and find broad applications as biosensors, diagnostics, and in therapeutic treatments and drug delivery. Currently, RNA aptamer production is via in vitro methods. Herein, a new E. coli-based approach has been demonstrated for the rapid production of multimeric RNA aptamer transcripts that are protected from degradation by burying the 5' and 3' ends of the transcript in a designed double-stranded spacer. Multimeric and fluorescent RNA aptamers were produced stably in vivo and readily isolated from RNase III-deficient cells, and their full functionalities were shown by binding assays and fluorescence measurements. This approach shows promise as a rapid and scalable bioprocess for the production of RNA aptamers at low cost.

Leptospirosis is a potentially life-threatening zoonosis caused by pathogenic Leptospira and for rapid diagnostics, direct detection is desirable. LipL32 protein is the most suitable biomarker for direct detection. DNA aptamers are sought to be generated against LipL32 by Systemic Evolution of Ligands via Exponential Enrichment (SELEX). LepDapt-5a is the most potent aptamer candidate among all the candidates, as determined by direct Enzyme-linked Aptasorbent Assay (ELASA). LepDapt-5a was predicted to form a G-quadruplex structure as predicted by QGRS Mapper and validated experimentally by direct ELASA. The diagnostic potential of the aptamer was further tested on a direct and sandwich ELASA platform. A LOD of 106 mL-1 and 105 mL-1 were estimated by direct and sandwich ELASA platforms, respectively, which are within the range associated with leptospiremia levels. The dot blot assay developed was able to attain a LOD of 104 CFU mL-1 against pathogenic Leptospira, which is also within the leptospiremia level. This is the first-ever DNA aptamer and hybrid-heterodimeric aptamer constructed against LipL32. The diagnostic potentiality of the LepDapt-5a DNA aptamer was proven on three major diagnostic platforms, which are direct ELASA, sandwich ELASA, and aptamer-based dot assay.

BACKGROUND: The identification of protein-protein interactions is a great challenge. In this study, we fabricated a gold surface-modified biochip with activated sophorolipids (SLs) in combination with 16-amino-1-hexadecanethiol hydrochloride to detect serum proteins. MAIN METHODS AND MAJOR RESULTS: The on-chip immunoassay reported here included a forward assay, in which a ligand is immobilized on the biochip surface and allowed to interact with its free specific receptor in liquid phase, and a reverse assay, in which a receptor is loaded on the biochip surface and combined with its free specific ligand in solution. The specificity of the molecular interactions on the biochip was evaluated using immunological blocking assays and chemiluminescent immunoassays (CLIA). Hemophagocytic lymphohistiocytosis (HLH) serum was used to test the potential utilization of the biochip. Reverse receptor CD25-based interleukin (IL)-2 and forward ligand IL-2-based CD25 assays revealed that the limit of detection of the target proteins was as low as 156 and 78 pg/ml, respectively. Using receptor- or ligand-based platforms, we found that the positive rates of free IL-2 and soluble CD25 (sCD25) monomers in the sera of HLH patients were 14.3% and 71.4%, respectively. In addition, the biochip showed good compatibility with CLIA for the measurement of sCD25 (r = 0.77, p < 0.01). CONCLUSIONS AND IMPLICATIONS: Biochip platforms, such as on-chip immunoprecipitation (IP), can be used to evaluate the interactions between proteins, ligands, and receptors, or enzymes and substrates in serum.

Recombinant adeno-associated virus (rAAV) has established itself as a highly efficacious gene delivery vector with a well characterised safety profile allowing broad clinical application. Recent successes in rAAV-mediated gene therapy clinical trials will continue to drive demand for improved rAAV production processes to reduce costs. Here, we demonstrate that small molecule bioactive chemical additives can significantly increase recombinant AAV vector production by human embryonic kidney (HEK) cells up to three-fold. Nocodazole (an anti-mitotic agent) and M344 (a selective histone deacetylase inhibitor) were identified as positive regulators of rAAV8 genome titre in a microplate screening assay. Addition of nocodazole to triple-transfected HEK293 suspension cells producing rAAV arrested cells in G2/M phase, increased average cell volume and reduced viable cell density relative to untreated rAAV producing cells at harvest. Final crude genome vector titre from nocodazole treated cultures was >2-fold higher compared to non-treated cultures. Further investigation showed nocodazole addition to cultures to be time critical. Genome titre improvement was found to be scalable and serotype independent across two distinct rAAV serotypes, rAAV8 and rAAV9. Furthermore, a combination of M344 and nocodazole produced a positive additive effect on rAAV8 genome titre, resulting in a three-fold increase in genome titre compared to untreated cells.

BACKGROUND AND AIMS: High-capacity adenoviral vectors (HC-AdV) show extended DNA payload and stability of gene expression in vivo due to the absence of viral coding sequences. However, production requires methods to trans-complement viral proteins, usually through Helper Viruses (HV). The Cre/loxP system is frequently employed to remove the packaging signal in HV genomes, in order to avoid their encapsidation. However, chronic exposure to the Cre recombinase in packaging cells is detrimental. We have applied the dimerizable Cre system to overcome this limitation. METHODS AND RESULTS: Cre was split in two fragments devoid of recombinase function (N-terminal 244 and C-terminal 99 amino-acids). In one version of the system, interaction with both moieties was favored by rapamycin-dependent heterodimerization domains (DiCre). Other version contained only Cre sequences (oCre). We generated packaging cells and HVs expressing the complementary fragments and studied their performance for HC-AdV production. We found that both conformations avoided interference with the growth of packaging cells, and the oCre system was particularly suitable for HC-AdV amplification. CONCLUSIONS: The split-Cre system improves the performance of packaging cells and can reduce the time and cost of HC-AdV amplification up to 30% and 15%, respectively. This may contribute to the standardization of HC-AdV production.

BACKGROUND/AIMS: Previous work developed a quantitative model using capacitance spectroscopy in an at-line setup to predict the dying cell percentage measured from a flow cytometer. This work aimed to transfer the at-line model to monitor lab-scale bioreactors in real-time, waiving the need for frequent sampling and enabling precise controls. METHODS AND RESULTS: Due to the difference between the at-line and in-line capacitance probes, direct application of the at-line model resulted in poor accuracy and high prediction bias. A new model with a variable range and offering similar spectral shape across all probes was first constructed, improving prediction accuracy. Moreover, the global calibration method included the variance of different probes and scales in the model, reducing prediction bias. External parameter orthogonalization, a preprocessing method, also mitigated the interference from feeding, which further improved model performance. The root-mean-square error of prediction of the final model was 6.56% (8.42% of the prediction range) with an R2 of 92.4%. CONCLUSION: The culture evolution trajectory predicted by the in-line model captured the cell death and alarmed cell death onset earlier than the trypan blue exclusion test. Additionally, the incorporation of at-line spectra following orthogonal design into the calibration set was shown to generate calibration models that are more robust than the calibration models constructed using the in-line spectra only. This is advantageous, as at-line spectral collection is easier, faster, and more material-sparing than in-line spectra collection.

BACKGROUND: Classical two-dimensional (2D) cell culture as a drug or nanoparticle test system only poorly recapitulates in vivo conditions. Animal studies are costly, ethically controversial, and preclude large-scale testing. METHODS AND RESULTS: We established a three-dimensional (3D) tissue slice air-liquid interface (ALI) culture model for nanoparticle testing. We developed an optimized procedure for the reproducible generation of large sets of tissue slices from tumor xenografts that retain their tissue architecture. When used for the analysis of nanoparticles based on chemically modified polyethylenimines (PEIs) to deliver siRNA or DNA, differences in transfection efficacy and cytotoxicity between nanoparticles were observed more clearly than in 2D cell culture. While nanoparticle efficacies between cell culture and the tissue slice model overall correlated, the tissue slice model also identified particularly suitable candidates whose efficacy was underestimated in 2D cell culture and had already been shown in previous in vivo studies. CONCLUSION: The ex vivo 3D tissue slice ALI culture model is a powerful system that allows the effective evaluation of biological nanoparticle efficacy and biocompatibility in an intact tissue environment. It is comparably inexpensive, time-saving, and follows the 3R principle, while allowing the identification of critical nanoparticle properties and optimal candidates for in vivo applications.

BACKGROUND: L-Asparaginase is an essential enzyme for the food and biopharmaceutical industry. The stability, however, of L-asparaginase is widely known to be an issue. Commercial manufacturing of any biopharmaceutical involves hold-ups during processing, and can result in product loss if stability is an issue, as is the case with L-asparaginase. This interplay of product intermediate stability and process design is the focus of this investigation. METHODS AND RESULTS: In this study, we propose a strategy to simultaneously increase the refolding yield and stability of refolded L-asparaginase so as to improve overall process yield. Using one variable at a time (OVAT) experiments, urea (6 M), solubilized inclusion bodies (15 mg/ml), refolding method (step dilution), and pH (8.6) were identified as significant process parameters. A design of experiment (DOE)-based optimization was then performed for the refolding step. The net outcome was more than a three-fold increase in enzyme recovery (i.e., 4.90 IU/ml) compared to unoptimized conditions (i.e., 1.26 IU/ml). Further, the L-asparaginase process intermediate was found to be stable for more than a week at room temperature and 2-8°C, while the unoptimized sample was stable at 2-8°C but did not show any activity at room temperature after 72 h. CONCLUSIONS: The current study elucidates how process intermediate stability needs to be given due consideration during process optimization, particularly for products such as L-asparaginase which are labile.

BACKGROUND: Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called serine integrase recombinational assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterized in vitro. METHODS AND MAJOR RESULTS: In this report, we compared the activities in vitro of 10 orthogonal LSIs to explore their suitability for multiplex SIRA reactions. We found that Bxb1, ϕR4, and TG1 integrases were the most active among the set we studied, but several others were also usable. As proof of principle, we demonstrated high-efficiency one-pot assembly of six DNA fragments (made by PCR) into a 7.5 kb plasmid that expresses the enzymes of the β-carotenoid pathway in Escherichia coli, using six different LSIs. We further showed that a combined approach using a few highly active LSIs, each acting on multiple pairs of att sites with distinct central dinucleotides, can be used to scale up "poly-part" gene assembly and editing. CONCLUSIONS AND IMPLICATIONS: We conclude that use of multiple orthogonal integrases may be the most predictable, efficient, and programmable approach for SIRA and other in vitro applications.