Miniaturization of hiPSC-derived 3D neural cultures in stirred-tank bioreactors for parallelized preclinical assessment of rAAV.

Engineered 3D models employing human induced pluripotent stem cell (hiPSC) derivatives have the potential to recapitulate the cell diversity and structure found in the human central nervous system (CNS). Therefore, these complex cellular systems offer promising human models to address the safety and potency of advanced therapy medicinal products (ATMPs), such as gene therapies. Specifically, recombinant adeno-associated viruses (rAAVs) are currently considered highly attractive for CNS gene therapy due to their broad tropism, low toxicity, and moderate immunogenicity.

SWATH-MS as a strategy for CHO host cell protein identification and quantification supporting the characterization of mAb purification platforms.

Host cell proteins (HCPs) are process-related impurities expressed by the host cells during biotherapeutics' manufacturing, such as monoclonal antibodies (mAbs). Some challenging HCPs evade clearance during the downstream processing and can be co-purified with the molecule of interest, which may impact product stability, efficacy, and safety. Therefore, HCP content is a critical quality attribute to monitor and quantify across the bioprocess.

Identification of Mispairing Omic Signatures in Chinese Hamster Ovary (CHO) Cells Producing a Tri-Specific Antibody.

Monoclonal antibody-based therapy has shown efficacy against cancer, autoimmune, infectious, and inflammatory diseases. Multispecific antibodies (MsAbs), including trispecifics (tsAbs), offer enhanced therapeutic potential by targeting different epitopes. However, when co-expressed from three or more different polypeptide chains, MsAb production can lead to incorrect chain assembly and co-production of mispaired species with impaired biological activity.

Towards a scalable bioprocess for rAAV production using a HeLa stable cell line.

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems.

Unveiling Human Proteome Signatures of Heart Failure with Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent but still poorly understood clinical entity. Its current pathophysiological understanding supports a critical role of comorbidities and their chronic effect on cardiac function and structure. Importantly, despite the replication of some HFpEF phenotypic features, to this day, experimental models have failed to bring new effective therapies to the clinical setting.

Assessing Multi-Attribute Characterization of Enveloped and Non-Enveloped Viral Particles by Capillary Electrophoresis.

Virus-based biopharmaceutical products are used in clinical applications such as vaccines, gene therapy, and immunotherapy. However, their manufacturing remains a challenge, hampered by the lack of appropriate analytical tools for purification monitoring or characterization of the final product. This paper describes the implementation of a highly sensitive method, capillary electrophoresis (CE)-sodium dodecyl sulfate (SDS) combined with a laser-induced fluorescence (LIF) detector to monitor the impact of various bioprocess steps on the quality of different viral vectors.

Biolayer Interferometry Analysis for a Higher Throughput Quantification of In-Process Samples of a Rotavirus Vaccine.

Rotavirus A infection is a global leading cause of severe acute gastroenteritis associated with life-threatening diarrheal episodes in infants and young children. The disease burden is being reduced, namely due to a wider access to rotavirus vaccines. However, there is a demand to expand rotavirus vaccination programs, and to achieve this, it is critical to improve high-throughput in-process product quality control and vaccine manufacturing monitoring.

Exploring Metabolic Signatures of Ex Vivo Tumor Tissue Cultures for Prediction of Chemosensitivity in Ovarian Cancer.

Predicting patient response to treatment and the onset of chemoresistance are still major challenges in oncology. Chemoresistance is deeply influenced by the complex cellular interactions occurring within the tumor microenvironment (TME), including metabolic crosstalk. We have previously shown that ex vivo tumor tissue cultures derived from ovarian carcinoma (OvC) resections retain the TME components for at least four weeks of culture and implemented assays for assessment of drug response.

Expression of Extracellular Vesicle PIWI-Interacting RNAs Throughout hiPSC-Cardiomyocyte Differentiation.

Extracellular Vesicles (EV) play a critical role in the regulation of regenerative processes in wounded tissues by mediating cell-to-cell communication. Multiple RNA species have been identified in EV, although their function still lacks understanding. We previously characterized the miRNA content of EV secreted over hiPSC-cardiomyocyte differentiation and found a distinct miRNA expression in hiPSC-EV driving its bioactivity.

Glycation modulates glutamatergic signaling and exacerbates Parkinson's disease-like phenotypes.

Alpha-synuclein (aSyn) is a central player in the pathogenesis of synucleinopathies due to its accumulation in typical protein aggregates in the brain. However, it is still unclear how it contributes to neurodegeneration. Type-2 diabetes mellitus is a risk factor for Parkinson's disease (PD).

Insect Cells for High-Yield Production of SARS-CoV-2 Spike Protein: Building a Virosome-Based COVID-19 Vaccine Candidate.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) homotrimeric spike (S) protein is responsible for mediating host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, thus being a key viral antigen to target in a coronavirus disease 19 (COVID-19) vaccine. Despite the availability of COVID-19 vaccines, low vaccine coverage as well as unvaccinated and immune compromised subjects are contributing to the emergence of SARS-CoV-2 variants of concern. Therefore, continued development of novel and/or updated vaccines is essential for protecting against such new variants.

Bioactivity and miRNome Profiling of Native Extracellular Vesicles in Human Induced Pluripotent Stem Cell-Cardiomyocyte Differentiation.

Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.

Multi attribute method implementation using a High Resolution Mass Spectrometry platform: From sample preparation to batch analysis.

Quality control of biopharmaceuticals such as monoclonal antibodies (mAbs) has been evolving and becoming more challenging as the requirements of the regulatory agencies increase due to the demanding complexity of products under evaluation. Mass Spectrometry (MS)-based methods such as the multi-attribute method (MAM) are being explored to achieve a deeper understanding of the attributes critical for the safety, efficacy, and quality of these products. MAM uses high mass accuracy/high-resolution MS data that enables the direct and simultaneous monitoring of relevant product quality attributes (PQAs, in particular, chemical modifications) in a single workflow, replacing several orthogonal methods, reducing time and costs associated with these assays.

Leveraging rAAV bioprocess understanding and next generation bioanalytics development.

Recombinant adeno-associated (rAAV) vector-based gene therapy has been the focus of intense research driven by the safety profile and several recent clinical breakthroughs. As of April 2021, there are two rAAV-based gene therapies approved and more than two-hundred active clinical trials (approximately thirty in Phase III). However, the expected increase in demand for rAAV vectors still poses several challenges.

The role of muscle in the susceptibility and progression of axial Spondyloarthritis: The MyoSpA Study Protocol.

Background: Axial Spondyloarthritis (axSpA) is a chronic, inflammatory rheumatic disease that affects the axial skeleton, causing pain, stiffness, and fatigue. Genetics and environmental factors such as microbiota and microtrauma are known causes of disease susceptibility and progression. Murine models of axSpA found a decisive role for biomechanical stress as an inducer of enthesitis and new bone formation.

Muscle dysfunction in axial spondylarthritis: the MyoSpA study.

Objectives: We aimed to investigate muscle physical properties, strength, mass, physical performance, and the prevalence of sarcopenia in patients with axial spondylarthritis (axSpA) compared to the healthy controls (HC).

Methods: We performed a cross-sectional study on 54 participants: 27 patients with axSpA and 27 HC, matched by age, gender, and level of physical activity. Muscle physical properties (stiffness, tone and elasticity), muscle strength (five-times sit-to-stand [5STS] test), muscle mass, physical performance (measured through gait speed) and sarcopenia were compared between the groups.

Stem cells characterization: OMICS reinforcing analytics.

Stem cells hold outstanding potential to model and treat disease and are valuable tools in pharmacology and toxicology. Characterization of stem cells and derivatives still poses many challenges to ensure safe, efficacious, and reliable therapies. Regulatory agencies have defined key mandatory attributes related to identity, purity, sterility, and genomic integrity, however robust analytics to determine cell's potency are still a major challenge, in most cases assessed case-by-case.

Lumbar myofascial physical properties in healthy adults: myotonometry vs. shear wave elastography measurements.

Objective: The human resting myofascial tone maintains the body tone in a neutral posture, the assessment of this and other muscle physical properties (MPP) is relevant, since, it is altered in many pathological states.

Patients And Methods: Seventeen healthy subjects (8 males), between 18-50 years old, were assessed. The MPP of lower lumbar muscles was evaluated on right and left sides during prone resting position using two devices; myotonometry (stiffness, elasticity and tone) and ultrasound-based shear-wave elastography (SWE) (shear modulus).

Proteomic and Glyco(proteo)mic tools in the profiling of cardiac progenitors and pluripotent stem cell derived cardiomyocytes: Accelerating translation into therapy.

Research in stem cells paved the way to an enormous amount of knowledge, increasing expectations on cardio regenerative therapeutic approaches in clinic. While the first generation of clinical trials using cell-based therapies in the heart were performed with bone marrow and adipose tissue derived mesenchymal stem cells, second generation cell therapies moved towards the use of cardiac-committed cell populations, including cardiac progenitor cells and pluripotent stem cell derived cardiomyocytes. Despite all these progresses, translating the aptitudes of R&D and pre-clinical data into effective clinical treatments is still highly challenging, partially due to the demanding regulatory and safety concerns but also because of the lack of knowledge on the regenerative mechanisms of action of these therapeutic products.

Production of high-quality SARS-CoV-2 antigens: Impact of bioprocess and storage on glycosylation, biophysical attributes, and ELISA serologic tests performance.

Serological assays are valuable tools to study SARS-CoV-2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID-19 therapeutics.

Toward a Microencapsulated 3D hiPSC-Derived Cardiac Microtissue for Recapitulation of Human Heart Microenvironment Features.

The combination of cardiomyocytes (CM) and non-myocyte cardiac populations, such as endothelial cells (EC), and mesenchymal cells (MC), has been shown to be critical for recapitulation of the human heart tissue for cell-based modeling. However, most of the current engineered cardiac microtissues still rely on either (i) murine/human limited primary cell sources, (ii) animal-derived and undefined hydrogels/matrices with batch-to-batch variability, or (iii) culture systems with low compliance with pharmacological high-throughput screenings. In this work, we explored a culture platform based on alginate microencapsulation and suspension culture systems to develop three-dimensional (3D) human cardiac microtissues, which entails the co-culture of human induced pluripotent stem cell (hiPSC) cardiac derivatives including aggregates of hiPSC-CM and single cells of hiPSC-derived EC and MC (hiPSC-EC+MC).

Mining for Peaks in LC-HRMS Datasets Using Finnee - A Case Study with Exhaled Breath Condensates from Healthy, Asthmatic, and COPD Patients.

Separation techniques hyphenated to high-resolution mass spectrometry are essential in untargeted metabolomic analyses. Due to the complexity and size of the resulting data, analysts rely on computer-assisted tools to mine for features that may represent a chromatographic signal. However, this step remains problematic, and a high number of false positives are often obtained.

Bioreactor-based 3D human myocardial ischemia/reperfusion in vitro model: a novel tool to unveil key paracrine factors upon acute myocardial infarction.

During acute myocardial infarction (AMI), Ischemia/Reperfusion (I/R) injury causes cardiomyocyte (CM) death and loss of tissue function, making AMI one of the major causes of death worldwide. Cell-based in vitro models of I/R injury have been increasingly used as a complementary approach to preclinical research. However, most approaches use murine cells in 2D culture setups, which are not able to recapitulate human cellular physiology, as well as nutrient and gas gradients occurring in the myocardium.

Exploring the analytical power of the QTOF MS platform to assess monoclonal antibodies quality attributes.

The biopharmaceutical industry is growing at a fast pace, making nowadays 20% of the pharma market. Within this market, therapeutic monoclonal antibodies (mAbs) are the dominant product class. With the patent expirations, biosimilars and, perhaps more relevant, biobetters, are in fast development.

Definition of a cell surface signature for human cardiac progenitor cells after comprehensive comparative transcriptomic and proteomic characterization.

Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Assisted by complementary RNAseq analysis, we defined the fraction of the CPC proteome associable with specific functions by comparison with human bone marrow mesenchymal stem cells (MSC), the reference population for cell therapy, and human dermal fibroblasts (HDF), as a distant reference. Label-free proteomic analysis identified 526 proteins expressed differentially in CPC.