Production Cross Research Articles

Intensified production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration

Intensified production of recombinant vesicular stomatitis virus-based vectors by tangential flow depth filtration

Discriminative label correlation based robust structure learning for multi-label feature selection

Feature selection is a key technique to tackle the curse of dimensionality in multi-label learning. Lots of embedded multi-label feature selection methods have been developed. However, they face challenges in identifying and excluding redundant features. To address these issues, this paper proposes a multi-label feature selection method that combines robust structural learning and discriminative label regularization. The proposed method starts from the feature space rather than data space, motivated by the principle that redundant features have high similarity or strong correlation. To exclude redundant features, a regularization on the feature selection matrix is designed by combining ℓ2,1-norm penalty with inner products of feature weight vectors. This regularization can help to learn a robust structure in the feature selection matrix. Meanwhile, both of the similarity and dissimilarity of labels of instances are involved in exploring discriminative label correlations. Extensive experiments verified the effectiveness of the proposed model for feature selection.

The relationship between imaging features, therapeutic response, and overall survival in pediatric diffuse intrinsic pontine glioma

We aimed to evaluate the relationship between imaging features, therapeutic responses (comparative cross-product and volumetric measurements), and overall survival (OS) in pediatric diffuse intrinsic pontine glioma (DIPG). A total of 134 patients (≤ 18 years) diagnosed with DIPG were included. Univariate and multivariate analyses were performed to evaluate correlations of clinical and imaging features and therapeutic responses with OS. The correlation between cross-product (CP) and volume thresholds in partial response (PR) was evaluated by linear regression. The log-rank test was used to compare OS patients with discordant therapeutic response classifications and those with concordant classifications. In univariate analysis, characteristics related to worse OS included lower Karnofsky, larger extrapontine extension, ring-enhancement, necrosis, non-PR, and increased ring enhancement post-radiotherapy. In the multivariate analysis, Karnofsky, necrosis, extrapontine extension, and therapeutic response can predict OS. A 25% CP reduction (PR) correlated with a 32% volume reduction (R2 = 0.888). Eight patients had discordant therapeutic response classifications according to CP (25%) and volume (32%). This eight patients’ median survival time was 13.0 months, significantly higher than that in the non-PR group (8.9 months), in which responses were consistently classified as non-PR based on CP (25%) and volume (32%). We identified correlations between imaging features, therapeutic responses, and OS; this information is crucial for future clinical trials. Tumor volume may represent the DIPG growth pattern more accurately than CP measurement and can be used to evaluate therapeutic response.

Optimization of Culture Media and Feeding Strategy for High Titer Production of an Adenoviral Vector in HEK 293 Fed-Batch Culture.

Adenoviruses are efficient and safe vectors for delivering target antigens and adenovirus-based vaccines have been used against a wide variety of pathogens, including tuberculosis and COVID-19. Cost-effective and scalable biomanufacturing processes are critical for the commercialization of adenovirus-vectored vaccines. Adenoviral vectors are commonly produced through the infection of batch cultures at low cell density cultures, mostly because infections at high cell densities result in reduced cell-specific virus productivity and does not improve volumetric productivity. In this study, we have investigated the feasibility of improving the volumetric productivity by infecting fed-batch cultures at high cell densities. Four commercial and one in-house developed serum-free media were first tested for supporting growth of HEK 293 cells and production of adenovirus type 5 (Ad5) in batch culture. Two best media were then selected for development of fed-batch culture to improve cell growth and virus productivity. A maximum viable cell density up to 16 × 106 cells/mL was achieved in shake flask fed-batch cultures using the selected media and commercial or in-house developed feeds. The volumetric virus productivity was improved by up to six folds, reaching 3.0 × 1010 total viral particles/mL in the fed-batch culture cultivated with the media and feeds developed in house and infected at a cell density of 5 × 106 cells/mL. Additional rounds of optimization of media and feed were required to maintain the improved titer when the fed-batch culture was scaled up in a bench scale (3 L) bioreactor. Overall, the results suggested that fed-batch culture is a simple and feasible process to significantly improve the volumetric productivity of Ad5 through optimization and balance of nutrients in culture media and feeds.

Quasi-perfusion studies for intensified lentiviral vector production using a continuous stable producer cell line

Quasi-perfusion culture was employed to intensify lentiviral vector (LV) manufacturing using a continuous stable producer cell line in an 8-day process. Initial studies aimed to identify a scalable seeding density, with 3, 4, and 5 x 104 cells cm-2 providing similar specific productivities of infectious LV. Seeding at 3 x 104 cells cm-2 was selected, and the quasi-perfusion was modulated to minimise inhibitory metabolite accumulation and vector exposure at 37 °C. Similar specific productivities of infectious and physical LV were achieved at 1, 2, and 3 vessel volumes per day (VVD), with 1 VVD selected to minimise downstream processing volumes. The optimised process was scaled 50-fold to 1,264 cm2 flasks, achieving similar LV titres. However, scaling up beyond this to a 6,320 cm2 multilayer flask reduced titres, possibly from suboptimal gas exchange. Across three independent processes in 25 cm2 to 6,320 cm2 flasks, reproducibility was high with a coefficient of variation of (7.7 ± 2.9)% and (11.9 ± 3.0)% for infectious and physical LV titres, respectively. The optimised flask process was successfully transferred to the iCELLis™ Nano (Cytiva) fixed-bed bioreactor, with quasi-perfusion at 1 VVD yielding 1.62 x 108 TU.

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

An Exact Bound for the Inner Product of Vectors in ℂn

It is shown that 1/2 is the exact upper bound on the difference ∑ k = 1 n | x k | 2 | y k | 2 − | ∑ k = 1 n x k y k | 2 for any unit vectors x = ( x 1 , … , x n ) and y = ( y 1 , … , y n ) in C n .

Symmetric doubly periodic gravity-capillary waves with small vorticity

We construct small amplitude gravity-capillary water waves with small nonzero vorticity, in three spatial dimensions, bifurcating from uniform flows. The waves are symmetric, and periodic in both horizontal coordinates. The proof is inspired by Lortz' construction of magnetohydrostatic equilibria in reflection-symmetric toroidal domains [23]. It relies on a global representation of the vorticity as the cross product of two gradients, and on prescribing a functional relationship between the Bernoulli function and the orbital periods of the water particles.The presence of the free surface introduces significant new challenges. In particular, the resulting free boundary problem is not elliptic, and the involved maps incur a loss of regularity under Fréchet differentiation. Nevertheless, we show that a version of the Crandall–Rabinowitz local bifurcation method still applies in this setting, by carefully tracking the loss of regularity.

Onto-semiotic analysis of Colombian engineering students’ mathematical connections to problems-solving on vectors: A contribution to the natural and exact sciences

The mathematical connections Colombian engineering students activated when they solved vector problems were explored. The study was based on the extended theory of connections and the onto-semiotic approach. We followed a qualitative methodology that consisted of three stages: (1) selection of engineering students as participants; (2) application of a questionnaire with 15 tasks on vectors to the participating students; and (3) analysis of these data based on the theoretical articulation. The results show that students perform arithmetic operations with vectors, find the scalar and vector product, the norm of a vector, the angle between vectors, and unit vector based on mathematical connections (<i>procedural, meaning, different representations, </i>and<i> implication</i>), detail from an onto-semiotic point of view. However, some students have difficulty finding the angle between vectors because they misuse the norm. Furthermore, the new metaphorical connection based on mnemonics activated by the “<i>law of the ear</i>” is reported. The connections activated by engineering students to solve problems about vectors may have been influenced by the explanations provided by their calculus teacher, who promotes connections for the teaching and learning mathematical concepts.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to4.64×107TU mL-1in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38×107TU mL-1in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of>1×107TUmL-1day-1for up to 29 consecutive days in a non-optimized 5L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

A compressed 2D-DOA and polarization estimation algorithm for mmWave polarized massive MIMO systems

Due to providing greater degrees-of-freedom (DOFs) and securer communication guarantees than traditional scalar array, polarized massive multiple-input multiple-output (MIMO) technique offers a prospective insight into millimeter-wave (mmWave) communication. In this paper, a compressed two-dimensional (2D) direction-of-arrival (DOA) and polarization estimation algorithm combining the compressive sampling propagator method with reduced-dimension multiple signal classification (CSPM-RDMUSIC) is developed for mmWave polarized massive MIMO systems. First, a synchronous compressive network is constructed to compress the high-dimensional output into a low-dimensional one. Thereafter, a propagator is structured to acquire the signal subspace. Subsequently, accurate 2D-DOA and polarization estimation are achieved using a coarse-refined strategy, where the vector cross-product technology is investigated for coarse 2D-DOA estimation and then RDMUSIC is exploited to perform 2D local search for accurate parameter estimation. Owing to integrating the CSPM framework with the RDMUSIC strategy, the developed approach provides high-precision parameter estimation with relative computational economy, supporting mmWave communication demands. What's more, it's suitable for arbitrary array geometry with high flexibility. Several experimental results validate the superiorities of the developed framework.

Multivariate data analysis on multisensor measurement for inline process monitoring of adenovirus production in HEK293 cells.

In the era of Biopharma 4.0, process digitalization fundamentally requires accurate and timely monitoring of critical process parameters (CPPs) and quality attributes. Bioreactor systems are equipped with a variety of sensors to ensure process robustness and product quality. However, during the biphasic production of viral vectors or replication-competent viruses for gene and cell therapies and vaccination, current monitoring techniques relying on a single working sensor can be affected by the physiological state change of the cells due to infection/transduction/transfection step required to initiate production. To address this limitation, a multisensor (MS) monitoring system, which includes dual-wavelength fluorescence spectroscopy, dielectric signals, and a set of CPPs, such as oxygen uptake rate and pH control outputs, was employed to monitor the upstream process of adenovirus production in HEK293 cells in bioreactor. This system successfully identified characteristic responses to infection by comparing variations in these signals, and the correlation between signals and target critical variables was analyzed mechanistically and statistically. The predictive performance of several target CPPs using different multivariate data analysis (MVDA) methods on data from a single sensor/source or fused from multiple sensors were compared. An MS regression model can accurately predict viable cell density with a relative root mean squared error (rRMSE) as low as 8.3% regardless of the changes occurring over the infection phase. This is a significant improvement over the 12% rRMSE achieved with models based on a single source. The MS models also provide the best predictions for glucose, glutamine, lactate, and ammonium. These results demonstrate the potential of using MVDA on MS systems as a real-time monitoring approach for biphasic bioproduction processes. Yet, models based solely on the multiplicity and timing of infection outperformed both single-sensor and MS models, emphasizing the need for a deeper mechanistic understanding in virus production prediction.

Volume and Surface Area of 3D Mesh Objects : A Matlab GUI

In many applications of science and engineering, the real physical related objects are usually modelled in terms of mesh objects, where, the mesh will be used during computation or simulation. For most cases, a good mesh is required in order to increase the accuracy of the results. As the mesh represents the original object, the mesh should have similar physical properties of the original object such as the surface area or the volume. However, finding the value of these parameters could require extra computational cost and increase overall time taken to solve the problem. Thus, it might be useful if these parameters can be determined separately to ensure good mesh is used before any simulation is performed. In this study, a Matlab GUI is developed to enable user for easily computing the volume and the surface area of three-dimensional mesh objects. In order to use the GUI, user will firstly need to import the Mesh that is generated by the software Netgen Mesh generator. Once any mesh is imported as an input to the GUI, the program will use the concepts in vectors, which includes dot and cross products, to compute both parameters. Apart from presenting briefly the steps in creating the GUI with Matlab, this paper will also provide some examples to demonstrate the use of the GUI.

On the expressivity of embedding quantum kernels

One of the most natural connections between quantum and classical machine learning has been established in the context of kernel methods. Kernel methods rely on kernels, which are inner products of feature vectors living in large feature spaces. Quantum kernels are typically evaluated by explicitly constructing quantum feature states and then taking their inner product, here called embedding quantum kernels. Since classical kernels are usually evaluated without using the feature vectors explicitly, we wonder how expressive embedding quantum kernels are. In this work, we raise the fundamental question: can all quantum kernels be expressed as the inner product of quantum feature states? Our first result is positive: Invoking computational universality, we find that for any kernel function there always exists a corresponding quantum feature map and an embedding quantum kernel. The more operational reading of the question is concerned with efficient constructions, however. In a second part, we formalize the question of universality of efficient embedding quantum kernels. For shift-invariant kernels, we use the technique of random Fourier features to show that they are universal within the broad class of all kernels which allow a variant of efficient Fourier sampling. We then extend this result to a new class of so-called composition kernels, which we show also contains projected quantum kernels introduced in recent works. After proving the universality of embedding quantum kernels for both shift-invariant and composition kernels, we identify the directions towards new, more exotic, and unexplored quantum kernel families, for which it still remains open whether they correspond to efficient embedding quantum kernels.

On split-octonionic curves

Abstract In this paper, we first define the vector product in Minkowski space $\mathbb{R}_{4}^{7}$, which is identified with the space of spatial split-octonions. Next, we derive the $G_{2}-$ frame formulae for a seven dimensional Minkowski curve by using the spatial split-octonions and the vector product. We show that Frenet–Serret formulas are satisfied for a spatial split octonionic curve. We obtain the congruence of two spatial split octonionic curves and give relationship between the $G_{2}-$ frame and Frenet–Serret frame. Furthermore, we present the Frenet–Serret frame with split octonions in $\mathbb{R}_{4}^{8}$. Finally, we give illustrative examples with Matlab codes.

Lighter and faster simulations on domains with symmetries

A strategy to improve the performance and reduce the memory footprint of simulations on meshes with spatial reflection symmetries is presented in this work. By using an appropriate mirrored ordering of the unknowns, discrete partial differential operators are represented by matrices with a regular block structure that allows replacing the standard sparse matrix–vector product with a specialised version of the sparse matrix-matrix product, which has a significantly higher arithmetic intensity. Consequently, matrix multiplications are accelerated, whereas their memory footprint is reduced, making massive simulations more affordable. As an example of practical application, we consider the numerical simulation of turbulent incompressible flows using a low-dissipation discretisation on unstructured collocated grids. All the required matrices are classified into three sparsity patterns that correspond to the discrete Laplacian, gradient, and divergence operators. Therefore, the above-mentioned benefits of exploiting spatial reflection symmetries are tested for these three matrices on both CPU and GPU, showing up to 5.0x speed-ups and 8.0x memory savings. Finally, a roofline performance analysis of the symmetry-aware sparse matrix–vector product is presented.

Development of a Potency Assay for Nous-209, a Multivalent Neoantigens-Based Genetic Cancer Vaccine.

Quality control testing of vaccines, including potency assessment, is critical to ensure equivalence of clinical lots. We developed a potency assay to support the clinical advancement of Nous-209, a cancer vaccine based on heterologous prime/boost administration of two multivalent viral vector products: GAd-209 and MVA-209. These consist of a mix of four Adeno (Great Ape Adenovirus; GAd) and four Modified Vaccinia Ankara (MVA) vectors respectively, each containing a different transgene encoding a synthetic polypeptide composed of antigenic peptide fragments joined one after the other. The potency assay employs quantitative Reverse Transcription PCR (RT-Q-PCR) to quantitatively measure the transcripts from the four transgenes encoded by each product in in vitro infected cells, enabling simultaneous detection. Results showcase the assay's robustness and biological relevance, as it effectively detects potency loss in one component of the mixture comparably to in vivo immunogenicity testing. This report details the assay's setup and validation, offering valuable insights for the clinical development of similar genetic vaccines, particularly those encoding synthetic polypeptides.

Elliptical magnetic trajectories of a point on the elliptical 2-sphere

Elliptical trajectories play numerous significant roles in mathematics and physics. The focus of this work is to analyze the trajectories of a point on the ellipsoid S a 1 , a 2 , a 3 2 while it is under the influence of a killing vector field K. For this purpose, we introduce the generalized Darboux frame and the variational vector fields of S a 1 , a 2 , a 3 2 . We were able to get the trajectory equations of the charged particle on the ellipsoid by utilizing the elliptical structure and elliptic inner and cross product. We adopted a clearer and more comprehensible strategy in this way. With the aid of a mathematical program, we were able to display the paths we had determined based on the particle's velocity vector's position in the magnetic field. These trajectories are spiral, helical, and elliptical.

A novel nucleic acid linker for multi-gene expression enhances plant and animal synthetic biology.

The production of compact vectors for gene stacking is hindered by a lack of effective linkers. Here, we report that a 26-nt nucleic acid linker, NAL1, from the fungus Glarea lozoyensis and its truncated derivatives could connect two genes as a bicistron, enabling independent translation in a maize protoplast transient expression system and human 293 Tcells. The optimized 9-nt NAL10 linker was then used to connect four genes driven by a bidirectional promoter; this combination was successfully used to reconstruct the astaxanthin biosynthesis pathway in transgenic maize. The short and efficient nucleic acid linker NAL10 can be widely used in multi-gene expression and synthetic biology in animals and plants.