Logistic PCA explains differences between genome-scale metabolic models in terms of metabolic pathways.

Genome-scale metabolic models (GSMMs) offer a holistic view of biochemical reaction networks, enabling in-depth analyses of metabolism across species and tissues in multiple conditions. However, comparing GSMMs Against each other poses challenges as current dimensionality reduction algorithms or clustering methods lack mechanistic interpretability, and often rely on subjective assumptions. Here, we propose a new approach utilizing logisitic principal component analysis (LPCA) that efficiently clusters GSMMs while singling out mechanistic differences in terms of reactions and pathways that drive the categorization. We applied LPCA to multiple diverse datasets, including GSMMs of 222 Escherichia-strains, 343 budding yeasts (Saccharomycotina), 80 human tissues, and 2943 Firmicutes strains. Our findings demonstrate LPCA's effectiveness in preserving microbial phylogenetic relationships and discerning human tissue-specific metabolic profiles, exhibiting comparable performance to traditional methods like t-distributed stochastic neighborhood embedding (t-SNE) and Jaccard coefficients. Moreover, the subsystems and associated reactions identified by LPCA align with existing knowledge, underscoring its reliability in dissecting GSMMs and uncovering the underlying drivers of separation.

Proton-transfer-reaction mass spectrometry (PTR-MS) for online monitoring of glucose depletion and cell concentrations in HEK 293 gene therapy processes.

OBJECTIVES: The applicability of proton-transfer-reaction mass spectrometry (PTR-MS) as a versatile online monitoring tool to increase consistency and robustness for recombinant adeno-associated virus (rAAV) producing HEK 293 bioprocesses was evaluated. We present a structured workflow to extract process relevant information from PTR-MS data. RESULTS: Reproducibility of volatile organic compound (VOC) measurements was demonstrated with spiking experiments and the process data sets used for applicability evaluation consisted of HEK 293 cell culture triplicates with and without transfection. The developed data workflow enabled the identification of six VOCs, of which two were used to develop a soft sensor providing better real-time estimates than the conventional capacitance sensor. Acetaldehyde, another VOC, provides online process information about glucose depletion that can directly be used for process control purposes. CONCLUSIONS: The potential of PTR-MS for HEK 293 cell culture monitoring has been shown. VOC data derived information can be used to develop soft sensors and to directly set up new process control strategies.

Enhancement of rAAV titers via inhibition of the interferon signaling cascade in transfected HEK293 suspension cultures.

The production of recombinant adeno-associated virus (rAAV) for gene therapy applications relies on the use of various host cell lines, with suspension-grown HEK293 cells being the preferred expression system due to their satisfactory rAAV yields in transient transfections. As the field of gene therapy continues to expand, there is a growing demand for efficient rAAV production, which has prompted efforts to optimize HEK293 cell line productivity through engineering. In contrast to other cell lines like CHO cells, the transcriptome of HEK293 cells during rAAV production has remained largely unexplored in terms of identifying molecular components that can enhance yields. In our previous research, we analyzed global regulatory pathways and mRNA expression patterns associated with increased rAAV production in HEK293 cells. Our data revealed substantial variations in the expression patterns between cell lines with low (LP) and high-production (HP) rates. Moving to a deeper layer for a more detailed analysis of inflammation-related transcriptome data, we detected an increased expression of interferon-related genes in low-producing cell lines. Following upon these results, we investigated the use of Ruxolitinib, an interferon pathway inhibitor, during the transient production of rAAV in HEK293 cells as potential media additive to boost rAAV titers. Indeed, we find a two-fold increase in rAAV titers compared to the control when the interferon pathways were inhibited. In essence, this work offers a rational design approach for optimization of HEK293 cell line productivity and potential engineering targets, ultimately paving the way for more cost-efficient and readily available gene therapies for patients.

Scaling strategy for cell and gene therapy bioreactors based on turbulent parameters.

So far, power input has been used as the main parameter for bioreactor scale-up/-down in upstream process development and manufacturing. The rationale is that maintaining a consistent power input per unit volume should result in comparable mixing times at different scales. However, shear generated from turbulent flow may compromise the integrity of non-robust cells such as those used during the production of cell and gene therapies, which may lead to low product quality and yield. Of particular interest is the Kolmogorov length parameter that characterizes the smallest turbulent eddies in a mixture. To understand its impact on scale-up/-down decisions, the distribution of Kolmogorov length along the trajectory flow of individual particles in bioreactors was estimated in silico with the help of computational fluid dynamics simulations. Specifically, in this study the scalability of iPSC-derived lymphocyte production and the impact of shear stress across various differentiation stages were investigated. The study used bioreactors of volumes from 0.1 to 10 L, which correspond to the scales most used for parameter optimization. Our findings, which align with in vitro runs, help determine optimal agitation speed and shear stress adjustments for process transfer between scales and bioreactor types, using vertically-oriented wheel and pitched-blade impellers. In addition, empirical models specific to the bioreactors used in this study were developed. The provided computational analysis in combination with experimental data supports selection of appropriate bioreactors and operating conditions for various cell and gene therapy process steps.

Orthogonal characterization of rAAV9 reveals unexpected transgene heterogeneity.

Recombinant adeno-associated virus (rAAV) is the most widely used viral vector for in vivo human gene therapy. To ensure safety and efficacy of gene therapy products, a comprehensive analytical profile of the rAAVs is needed, which provides crucial information for therapeutic development and manufacturing. Besides information on rAAV quantities and possible contaminating DNA and protein species, assessing rAAV quality is of utmost importance. In vitro biopotency and methods to determine the full/empty ratio of rAAV capsids are commonly applied, but methods to assess the integrity of the viral genome are still rarely used. Here we describe an orthogonal approach to characterize rAAV quality. Two biologically different rAAV9s from different stages of the bioprocess, generated each with two different transfection reagents, were investigated. In vitro biopotency tests in all cases demonstrated that rAAV9s generated with transfection reagent FectoVIR® possessed a higher biological activity. Mass-based analytical methods, such as sedimentation velocity analytical ultracentrifugation (AUC) and mass photometry, showed a high share of full capsids (>80 %) at late process stages but did not detect any differences in the rAAV9s from the different transfection reagents. Multiplex dPCR and Nanopore long-read sequencing both demonstrated that, also in late-stage process samples, sample heterogeneity was relatively high with a rather small share of full-length transgenes of ∼10-40 %. Intriguingly, both methods detected a higher share of complete transgenes in rAAV9 generated with transfection reagent FectoVIR® instead of Polyethylenimine (PEI), and thereby explain the differences already observed in the biopotency assays. This study therefore emphasizes the necessity to utilize multiple, orthogonal methods to gain a better understanding of recombinantly manufactured AAVs.

Optimizing VLP production in gene therapy: Opportunities and challenges for in silico modeling.

Over the past decades, virus-like particle (VLP)-based gene therapy (GT) evolved as a promising approach to cure inherited diseases or cancer. Tremendous costs due to inefficient production processes remain one of the key challenges despite considerable efforts to improve titers. This review aims to link genome-scale metabolic models (GSMMs) to cell lines used for VLP synthesis for the first time. We summarize recent advances and challenges of GSMMs for Chinese hamster ovary (CHO) cells and provide an overview of potential cell lines used in GT. Although GSMMs in CHO cells led to significant improvements in growth rates and recombinant protein (RP)-production, no GSMM has been established for VLP production so far. To facilitate the generation of GSMM for these cell lines we further provide an overview of existing omics data and the highest production titers so far reported.

Comprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for gene therapy applications.

Human embryonal kidney cells (HEK-293) are the most common host cells used for transient recombinant adeno-associated virus (rAAV) production in pharmaceutical industry. To better cover the expected gene therapy product demands in the future, different traditional strategies such as cell line sub-cloning and/or addition of chemical substances to the fermentation media have been used to maximize titers and improve product quality. A more effective and advanced approach to boost yield can be envisaged by characterizing the transcriptome of different HEK-293 cell line pedigrees with distinct rAAV productivity patterns to subsequently identify potential gene targets for cell engineering. In this work, the mRNA expression profile of three HEK-293 cell lines, resulting in various yields during a fermentation batch process for rAAV production, was investigated to gain basic insight into cell variability and eventually to identify genes that correlate with productivity. Mock runs using only transfection reagents were performed in parallel as a control. It finds significant differences in gene regulatory behaviors between the three cell lines at different growth and production stages. The evaluation of these transcriptomics profiles combined with collected in-process control parameters and titers shed some light on potential cell engineering targets to maximize transient production of rAAV in HEK-293 cells.

Assessment of the percentage of full recombinant adeno-associated virus particles in a gene therapy drug using CryoTEM.

In spite of continuous development of gene therapy vectors with thousands of drug candidates in clinical drug trials there are only a small number approved on the market today stressing the need to have characterization methods to assist in the validation of the drug development process. The level of packaging of the vector capsids appears to play a critical role in immunogenicity, hence an objective quantitative method assessing the content of particles containing a genome is an essential quality measurement. As transmission electron microscopy (TEM) allows direct visualization of the particles present in a specimen, it naturally seems as the most intuitive method of choice for characterizing recombinant adeno-associated virus (rAAV) particle packaging. Negative stain TEM (nsTEM) is an established characterization method for analysing the packaging of viral vectors. It has however shown limitations in terms of reliability. To overcome this drawback, we propose an analytical method based on CryoTEM that unambiguously and robustly determines the percentage of filled particles in an rAAV sample. In addition, we show that at a fixed number of vector particles the portion of filled particles correlates well with the potency of the drug. The method has been validated according to the ICH Q2 (R1) guidelines and the components investigated during the validation are presented in this study. The reliability of nsTEM as a method for the assessment of filled particles is also investigated along with a discussion about the origin of the observed variability of this method.

Stable overexpression of miR-17 enhances recombinant protein production of CHO cells.

miRNAs negatively regulate gene expression at post-transcriptional level, and consequently play an important role in the control of many cellular pathways. The use of miRNAs to engineer Chinese hamster ovary (CHO) cells is an emerging strategy to improve recombinant protein production. Here, we describe the effect of transient and stable miRNA overexpression on CHO cell phenotype. Using an established transient miRNA screening protocol, the effects of miR-17, miR-92a and cluster miR17-92a on CHO growth and protein productivity were studied and followed by analysis of cell pools with stable overexpression of these miRNAs. CHO cells stably engineered with miR-17 exhibited both enhanced growth performance and a 2-fold increase in specific productivity, which resulted in a 3-fold overall increase in EpoFc titer. While further studies of miRNA-mRNA interactions will be necessary to understand the molecular basis of this effect, these data provide valuable evidence for miR-17 as a cell engineering target to enhance CHO cell productivity.

Reduced quenching and extraction time for mammalian cells using filtration and syringe extraction.

In order to preserve the in vivo metabolite levels of cells, a quenching protocol must be quickly executed to avoid degradation of labile metabolites either chemically or biologically. In the case of mammalian cell cultures cultivated in complex media, a wash step previous to quenching is necessary to avoid contamination of the cell pellet with extracellular metabolites, which could distort the real intracellular concentration of metabolites. This is typically achieved either by one or multiple centrifugation/wash steps which delay the time until quenching (even harsh centrifugation requires several minutes for processing until the cells are quenched) or filtration. In this article, we describe and evaluate a two-step optimized protocol based on fast filtration by use of a vacuum pump for quenching and subsequent extraction of intracellular metabolites from CHO (Chinese hamster ovary) suspension cells, which uses commercially available components. The method allows transfer of washed cells into liquid nitrogen within 10-15s of sampling and recovers the entire extraction solution volume. It also has the advantage to remove residual filter filaments in the final sample, thus preventing damage to separation columns during subsequent MS analysis. Relative to other methods currently used in the literature, the resulting energy charge of intracellular adenosine nucleotides was increased to 0.94 compared to 0.90 with cold PBS quenching or 0.82 with cold methanol/AMBIC quenching.

Dynamic mRNA and miRNA profiling of CHO-K1 suspension cell cultures.

In spite of the importance of Chinese hamster ovary (CHO) cells for recombinant protein production, very little is known about the molecular and gene regulatory mechanisms that control cellular phenotypes such as enhanced growth under serum-free conditions or high productivity. Most microarray analyses to this purpose are performed with samples taken during the exponential growth phase. However, the cellular transcriptome is dynamic, changing in response to external and internal stimuli and thus reflecting the current functional capacity of cells as well as their ability to adapt to a changing environment. Therefore, during batch or fed-batch cultivations it can be expected that the transcription pattern of genes will change and that such changes may give indications on the cellular state in terms of viability, growth, and productivity. In the current study we monitored the change in expression patterns of mRNAs and microRNAs (miRNA) during lag, exponential, and stationary phases in CHO-K1 suspension cell cultures. In total, over 1400 mRNAs and more than 100 miRNAs were differentially regulated (p<0.05) relative to the batch culture at the starting point. Functional clustering revealed groups of genes with similar expression patterns, which were subjected to functional pathway analysis. In addition, as miRNAs generally act as negative post-transcriptional regulators of mRNAs, we looked for changes in their expression that were inverse to those of their predicted target mRNAs.

Growth, productivity and protein glycosylation in a CHO EpoFc producer cell line adapted to glutamine-free growth.

A primary objective of cell line development and process optimisation in animal cell culture is the improvement of culture performance as indicated by desirable properties such as high cell concentration, viability, productivity and product quality. The inefficient energy metabolism of mammalian cells in culture is still a major limiting factor for improvements in process performance. It results in high uptake rates of glucose and glutamine and the concomitant accumulation of waste products which in turn limits final cell concentrations and growth. To avoid these negative side effects, a CHO host cell line was established recently which is able to grow in completely glutamine free medium (Hernandez Bort et al., 2010). To determine the influence of this adaptation on productivity and product quality, the same procedure was repeated with a recombinant CHO cell line producing an erythropoietin-Fc fusion protein (CHO-EpoFc) for this publication. After adaptation to higher cell densities and glutamine free medium, culture performance was monitored in batch bioprocesses and revealed comparable growth properties and EpoFc product formation in both cell lines. The level of reactive oxygen species was elevated in the adapted cells, reflecting a higher level of oxidative stress, however, at the same time the level of the oxido-protective glutathione was also higher, so that cells seem adequately protected against cellular damage. Analysis of nucleotides and nucleotide sugars revealed elevated UDP-sugars in cells grown in the absence of glutamine. Furthermore, the antennarity of N-glycans was moderately higher on the Epo part of the protein produced by the adapted cell line compared to the parental cell line. Except for this, the glycosylation, with respect to site occupancy, degree of sialylation and glycoform structure, was highly comparable, both for the Epo and the Fc part of the protein.