rAAV viral vectors (VV) used for in vivo gene therapy are commonly produced using transient transfection of HEK293 cells. Although this method is well established, challenges remain in achieving high ...VV titer and a high ratio of full-to-empty capsids. Our aim is to build a mechanistic understanding and knowledge of amino acid (AA) consumption to guide the development of advanced manufacturing processes. The resulting model-driven approach is used to design, predict, test, and optimize VV production processes – scaling up and transferring to a continuous process at bioreactor scale.
Media optimization can be a laborous process involving multiple DoEs. Rapid and simple analytical tools at the point-of-use are required to quickly assess key-nutrient consumption and provide feedback to optimize feed and supplement addition strategies. In this study, we performed data-driven media screening and optimization experiments leveraging a fast, at-line amino acid analyser, REBEL.
Utilising mechanistic models for HEK293 rAAV production and at-line spent media analysis for data-driven process intensification and yield improvement by cell culture media and feed optimisation.
For initial experiments, rAAV was produced via transient transfection in a fed-batch 300 mL mini-bioreactor and in the shake flasks. Samples were collected to identify potential AA depletion that may impact capsid titre.
Asn is an important AA building block and constitutes ∼10% of capsid protein composition. Results show clear differences in fluxes of Asn and Asp, which were reported previously as candidate AAs for producing viral particles. Other essential AA also exhibited strong depletion, indicating opportunities to optimize cell culture conditions. With subsequent experiments, the impact of AA addition to the cell culture was assessed with the goal of improving vector titre.
This work is relevant to addressing key challenges of rAAV production platforms via process engineering and optimization of cell culture conditions to scale-up vector production and development of continuous bioprocessing of rAAVs. The technology used uniquely enables at-line AA measurements, leading to deeper insights and informed feeding strategy optimization.
As the use of CAR T-cell therapies advances, testing different CAR T-cells, and associating metabolic characteristics to well-performing, desired phenotypes of CAR T-cells enables optimization of CAR ...constructs and manufacturing processes. Vasoactive intestinal peptide (VIP) is an emerging checkpoint pathway for T-cell function. Antagonization of VIP/VIPR axis in T-cells enhances T-cell-mediated anti-tumor function. In this work, we elucidate the mechanisms by which CAR T-cells engineered to secrete potent VIPR antagonistic peptides (CAR/VIPRa) can overcome the immunosuppression of VIP-rich tumor microenvironments.
CAR T-cell manufacturing processes enhanced by characterisation of products through spent media amino acid analysis – correlating metabolism with desired, less-differentiated and less-exhaustive phenotype CAR T-cell populations.
To interrogate if VIPR antagonism impacts metabolic pathways, spent media analysis was performed using 908 Devices REBEL at-line amino acid analyzer to determine overall amino acid usage differences between CAR and CAR/VIPRa T-cells. Spent media analysis revealed differences in the consumption of amino acids such as alanine, glutamate, and serine. This prompted further investigation of the differences in metabolic pathway utilization and alterations in metabolites within these CAR T-cells. LC/MS metabolomics, cellular bioenergetics and RNA sequencing were used to elucidate differences between metabolic states of CAR and CAR/VIPRa T-cells.
We found that when the manufactured cells are antigen-stimulated, CAR/VIPRa T-cells become significantly more energetic. In contrast, CAR T-cells become less energetic and more glycolytic, which are known features of T-cell exhaustion. Further, metabolic assessment in terms of oxygen consumption and amino acid levels in spent media correlated with these findings. CAR T-cell activity, potency, and cytotoxicity assessments showed a correlation with anti-tumor activity. Together, this work demonstrates that distinct metabolic features allow CAR/VIPRa T-cells, when antigen-stimulated, to have enhanced effector functions against tumors.
At-line spent media analysis of amino acids, in combination with other assays and measurements, was used to understand, characterise and optimise CAR T-cell manufacturing process, correlate with cell performance, and develop bioprocess parameters.
There are several options available to clinics culturing IVF embryos, including group and individual droplet culture, sequential or single-step medium culture and the possibility to renew the medium ...during culture. The absence of standardised culturing conditions or molecular testing methodologies, including whole genome amplification (WGA) used for non-invasive preimplantation genetic testing for aneuploidy (NI-PGT-A) may explain the variable rates of concordance reported between the spent embryo culture media and embryo biopsy results to-date. Culture conditions contribute to the accumulation of embryonic and contaminating DNA in spent embryo culture media. Optimisation of either the culturing conditions, molecular testing methodologies, or both, should yield the highest accuracy results for NI-PGT-A. This study examined rates of concordance between spent embryo culture media and embryo biopsies with the aim of evaluating the impact of culturing conditions on NI-PGT-A results.
Spent embryo culture media was collected from single embryo culture droplets following biopsy of the embryo for PGT-A, then stored at -20°C, with ethics approval. Spent embryo culture media samples from 10ul-60ul culture droplets were whole genome amplified using DOPlify® kit reagents (PerkinElmer). WGA DNA yield was assessed following gel electrophoresis and high sensitivity Qubit instrument quantification (ThermoFisher). Next generation sequencing libraries and sequencing was performed according to the standard PG-SeqTM kit 48 sample protocol on a MiSeq® instrument sequencer (Illumina). Data was bioinformatically aligned to hg19 and analysed using PG-SeqTM kit software. WGA DNA yield, NGS metrics, and whole chromosome aneuploidy concordance with the PGT-A result for the embryo biopsy were determined.
Results were collated for each set of culturing conditions, including whether the media was renewed during culture. Whole genome amplification using DOPlify® kit reagents resulted in the amplification of 78-100% of spent embryo culture media samples (WGA failure rate 0-22%). Ploidy concordance with the embryo biopsy ranged from 33-55% for autosomal chromosomes and 47-53% for sex chromosomes using a single-step culturing system (n=3 protocols), compared with concordance rates of 60-95% and 50-97% respectively when media was changed during the 5-6 day culture (n=4 protocols).
Successful NI-PGT-A using spent embryo culture media will possibly require specific culturing conditions and/or specialised molecular methodologies for accurate and representative amplification and testing of the embryonic DNA. In a step toward this, we identified that renewing culture media during IVF improves overall concordance rates between the embryo biopsy and spent embryo culture media for NI-PGT-A.
The clinical use of non-invasive preimplantation genetic testing for aneuploidy (PGT-A) requires concordance of the spent embryo culture media result to embryo biopsy result and the ability to ...distinguish maternal contamination from the embryonic DNA, especially for a euploid female result.
Although concordance of spent embryo culture media and trophectoderm biopsy has been reported at as high as 95% following the collection of samples at day 5-7 using DOPlify™ (Lane et al, 2017), the ability to test media collected earlier in culture requires an increased level of sensitivity. Additionally, there are a number of known PCR inhibitors in culture media, including salts and proteins, which need to be overcome. Here we describe acustomised whole genome amplification (WGA) protocol designed specifically for the amplification of DNA contained in spent embryo culture media.
Spent embryo culture media was collected by clinics with ethics approval from single embryo culture droplets and pooled prior to storage at -20°C. Pooling was used to provide a consistent test sample to analyse across a range of protocol variants to overcome sample to sample variability. To accommodate the use of a larger sample input volume into the WGA and the composition of culture media post embryo culture, samples were amplified using a re-formulated single step version of DOPlify™. The single step protocol removes the DOPlify™ lysis step and requires the addition of only one reagent mastermix into the PCR tube containing the culture media, making this protocol ideal for automation. The same pooled media samples were amplified in parallel using the standard DOPlify™ protocol. WGA DNA yield was assessed following gel electrophoresis and HS Qubit quantification (ThermoFisher) and sequenced on a MiSeq sequencer (Illumina) according to the standard PG-Seq™ 48 sample protocol. The sequencing data for each sample was bioinformatically aligned to hg19 and the sequencing QC metrics for each protocol were collated and compared.
A total of 32 protocol variants were tested in triplicate. Outputs measured were yield, size distribution of PCR amplicons, %GC content and PG-Seq™ NGS software quality scores. With the optimal protocol, WGA DNA yield per sample increased on average 5-fold following the amplification of pooled media samples using a re-formulated version of DOPlify™ with output measures comparable to biopsy results. It is also possible to detect mitochondrial DNA from the amplified spent culture media, although with fewer reads per sample than biopsies provide. The same increased yield and acceptable QC results have since been obtained for individual culture droplets from embryos cultured in 10-60ul of media from a range of manufacturers.
Successful non-invasive PGT-A using spent embryo culture media requires a customised approach to amplify DNA found within this complex sample type. Accurate and representative amplification of the embryo DNA is essential for the success of this non-invasive approach. Further incorporation of RHS’ target sequence enrichment protocol would provide a mechanism to detect maternal DNA within a spent culture media sample.
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•Use of spent media reduces water footprint and nutrient demand in algae cultivation.•Spent media fostered enhanced algal PUFA and carbohydrate production.•Nitrogen uptake, TCA cycle, ...and phospholipid synthesis downregulated in spent media.•Extracellular matter accumulated inhibiting algal growth past the 2nd use cycle.•Humic acid and free fatty acids were the major extracellular inhibitors.
The recycling of unfiltered spent media during cultivation of Chlorella vulgaris was studied using metabolomics in an effort to enhance water and nutrient sustainability and reduce operating costs in algal biorefineries. Cultivation in spent media resulted in reduced biomass and lipid productivity by 14% and 19%, respectively, compared to fresh media. The decrease was related to a detected lower nutrient uptake. Nevertheless, carbohydrate content (28% of dry cell weight) and α-linolenic acid content (27 % of fatty acids) were higher in spent media cultures than in fresh media. Metabolomics analysis of intracellular metabolites revealed downregulation of nitrogen assimilation, tricarboxylic acid cycle, structural lipids, and energy metabolism, but upregulation of stress mitigation and carbohydrate synthesis. No growth was supported by spent media during a second cultivation cycle and was likely due to the identified extracellular accumulation of humic acid and free fatty acids that acted as growth auto-inhibitors.
•A fast, simple and sensitive CE-MS method for amino acid analysis is reported.•The method is a monitoring and screening tool for upstream media development.•Cell culture media is analysed directly ...by CE-MS, avoiding sample preparation.•The approach could be used for near real time at-line process monitoring.
The development and optimization of cell culture media for biotech applications is a fundamental step of process development. The composition of cell culture media requires an ideal blend of amino acids, vitamins, nucleosides, lipids, carbohydrates, trace elements and other components. The ability to monitor these constituents is required to ensure that cells receive sufficient nutrients to facilitate growth, viability and productivity. Analysis of cell culture media is challenging due to the range and diversity of compounds contained in this matrix and normally requires time consuming methods. A rapid, simple and sensitive microfluidic chip CE-MS method is described to monitor amino acids in chemically defined cell culture media from a Chinese hamster ovary cell line cultured over a period of 10 days. The described platform enabled the separation of 16 amino acids in less than 2 minutes and without the requirement for extensive sample preparation. The analytical parameters evaluated were precision, linearity, limit of detection and limit of quantification. The majority of essential amino acids were present in cell culture growth in high concentrations compared to non-essential amino acids. Over the course of the 10 days cell culture the concentration of certain amino acids declined by up to 100%. Microfluidic chip based CE-MS methods can be used effectively to obtain the consumption rates of amino acids in cell culture media during cell growth and to perform at-line monitoring and screening of cell culture status.
In this study, we investigated the thermal decomposition mechanisms of perfluoroalkyl ether carboxylic acids (PFECAs) and short-chain perfluoroalkyl carboxylic acids (PFCAs) that have been ...manufactured as replacements for phased-out per- and polyfluoroalkyl substances (PFAS). C–C, C–F, C–O, O–H, and CC bond dissociation energies were calculated at the M06-2X/Def2-TZVP level of theory. The α-C and carboxyl-C bond dissociation energy of PFECAs declines with increasing chain length and the attachment of an electron-withdrawing trifluoromethyl (−CF3) group to the α-C. Experimental and computational results show that the thermal transformation of hexafluoropropylene oxide dimer acid to trifluoroacetic acid (TFA) occurs due to the preferential cleavage of the C–O ether bond close to the carboxyl group. This pathway produces precursors of perfluoropropionic acid (PFPeA) and TFA and is supplemented by a minor pathway (CF3CF2CF2OCFCF3COOH → CF3CF2CF2· + ·OCFCF3COOH) through which perfluorobutanoic acid (PFBA) is formed. The weakest C–C bond in PFPeA and PFBA is the one connecting the α-C and the β-C. The results support (1) the C–C scission in the perfluorinated backbone as an effective PFCA thermal decomposition mechanism and (2) the thermal recombination of radicals through which intermediates are formed. Additionally, we detected a few novel thermal decomposition products of studied PFAS.
Optimization and monitoring of bioprocesses requires the measurement of several process parameters and quality attributes. Mass spectrometry (MS)-based techniques such as those coupled to gas ...chromatography (GCMS) and liquid Chromatography (LCMS) enable the simultaneous measurement of hundreds of metabolites with high sensitivity. When applied to spent media, such metabolome analysis can help determine the sequence of substrate uptake and metabolite secretion, consequently facilitating better design of initial media and feeding strategy. Furthermore, the analysis of metabolite diversity and abundance from spent media will aid the determination of metabolic phases of the culture and the identification of metabolites as surrogate markers for product titer and quality. This review covers the recent advances in metabolomics analysis applied to the development and monitoring of bioprocesses. In this regard, we recommend a stepwise workflow and guidelines that a bioprocesses engineer can adopt to develop and optimize a fermentation process using spent media analysis. Finally, we show examples of how the use of MS can revolutionize the design and monitoring of bioprocesses.
Dynamic flux balance analysis (FBA) allows estimation of intracellular reaction rates using organism‐specific genome‐scale metabolic models (GSMM) and by assuming instantaneous pseudo‐steady states ...for processes that are inherently dynamic. This technique is well‐suited for industrial bioprocesses employing complex media characterized by a hierarchy of substrate uptake and product secretion. However, knowledge of exchange rates of many components of the media would be required to obtain meaningful results. Here, we performed spent media analysis using mass spectrometry coupled with liquid and gas chromatography for a fed‐batch, high‐cell density cultivation of Escherichia coli BL21(DE3) expressing a recombinant protein. Time course measurements thus obtained for 246 metabolites were converted to instantaneous exchange rates. These were then used as constraints for dynamic FBA using a previously reported GSMM, thus providing insights into how the flux map evolves through the process. Changes in tri‐carboxylic acid cycle fluxes correlated with the increased demand for energy during recombinant protein production. The results show how amino acids act as hubs for the synthesis of other cellular metabolites. Our results provide a deeper understanding of an industrial bioprocess and will have implications in further optimizing the process.
The authors applied dynamic flux balance analysis (FBA) to metabolomics data obtained from spent culture media to decipher the metabolic variations associated with industrial fed‐batch process of E. coli on complex media. Exchange fluxes determined from time‐course measurements of spent media components provided the essential constraints for dynamic FBA. This approach revealed the importance of energy metabolism and amino acid pathways in response to the increased energy demand during recombinant protein expression, and can guide bioprocess design and optimization.
With the advances in tissue engineering and regenerative medicine, various approaches have been developed for peripheral nerve tissue repair and regeneration. In the current study, we have ...synthesized a cryogel matrix from chitosan and gelatin incorporated with polypyrrole for neural tissue regeneration. The three-dimensional (3-D) cryogel matrix was fabricated to mimic the in vivo microenvironment and analyzed for stem cell differentiation. Isolated bone marrow stem cells (BMSCs) cultured on a 3-D cryogel matrix differentiated into neural lineage on the basis of scaffold properties, in a co-culture system and by treatment with the spent media of Neuro 2a cells. To validate the cell–cell contact and BMSCs differentiation, scanning electron micrography and fluorescent imaging were done, which revealed the differentiation of the BMSCs. Immunostaining and gene expression analysis showed the BMSCs differentiation in all of the three cases studied. However, BMSCs in the co-culture system showed increased neurotransmitter levels of dopamine (34%) and acetylcholine (16%) with a respective concentration of 2.04 ± 0.03 ng/mL and 15.06 ± 0.19 pg/mL. Based on these properties, an in vivo study explored the potential of the synthesized cryogel in regeneration of a 1.5 cm nerve gap in the sciatic nerve of rats for a period of 12 weeks. The biocompatibility analysis showed that the scaffold did not induce any adverse immune response. Moreover, the walking track analysis and electrophysiological and immunostaining analyses revealed enhanced sciatic nerve regeneration in comparison to the negative control group. This study reveals the regenerative potential of the cryogel matrix for peripheral nerve regeneration.
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