Fulvic acid foliar application: a novel approach enhancing antioxidant capacity and nutritional quality of pistachio (Pistacia vera L.).

BACKGROUND: The global growth of pistachio production has prompted exploration into sustainable agricultural practices, on the application of humic substances such as fulvic acid in enhancing the quality of horticultural crops. The present study was carried out in Qom province, Iran, on 20 years old pistachio (Pistacia vera L. cv. Kaleh-Ghoochi) trees and investigated the impact of foliar spraying of fulvic acid at varying concentrations (1.5, 3, and 4.5 g L- 1) on the antioxidant and quality properties of pistachio. The different concentrations of fulvic acid were applied at two key stages: at the initiation of pistachio kernel formation (late June) and the development stage of pistachio kernel (late August), as well as at both time points. Following harvest at the horticulturally mature phase, various parameters, including total phenols, flavonoids, soluble proteins, soluble carbohydrate content, antioxidant capacity, and antioxidant enzyme activity, were assessed. RESULTS: Results indicated that foliar application of fulvic acid, particularly at 1.5 g L- 1 during both late June and August, effectively increased phenolic compounds (31.8%) and flavonoid content (24.53%). Additionally, this treatment also augmented antioxidant capacity and heightened the activity of catalase (CAT) (37.56%), ascorbate peroxidase (APX) (63.86%), and superoxide dismutase (SOD) (76.45%). Conversely, peroxidase (POX) (41.54%) activity was reduced in fulvic acid-treated nuts compared with controls. Moreover, the content of chlorophyll (45%) and carotenoids (46.7%) was enhanced using this organic fertilizer. In terms of mineral elements, the increment was observed in zinc (Zn) (58.23%) and potassium (K) (28.12%) amounts in treated nuts. Additionally, foliar application of fulvic acid led to elevated levels of soluble carbohydrates and proteins in treated nuts. CONCLUSIONS: In the present study, application of fulvic acid resulted in enhancement of antioxidant activity and quality traits of pistachio nut through an increase in total phenol, flavonoids, chlorophyll, carotenoids, K, Zn, and also activity of antioxidant enzymes. Therefore, use of fulvic acid emerges as a promising strategy to enhance the quality and nutritional attributes of pistachios, contributing to sustainable agricultural practices and improved crop outcomes.

Isolation and characterization of rabies monoclonal antibody charge variants.

Current postexposure prophylaxis of rabies includes vaccines, human rabies immunoglobulin (RIG), equine RIG, and recombinant monoclonal antibodies (mAb). In the manufacturing of rabies recombinant mAb, charge variants are the most common source of heterogeneity. Charge variants of rabies mAb were isolated by salt gradient cation exchange chromatography (CEX) to separate acidic and basic and main charge variants. Separated variants were further extensively characterized using orthogonal analytical techniques, which include secondary and tertiary structure determination by far and near ultraviolet circular dichroism spectroscopy. Charge and size heterogeneity were evaluated using CEX, isoelectric focusing (IEF), capillary-IEF, size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and western blotting. Antigen binding affinity was assessed by enzyme linked immuno-sorbent assay and rapid florescence foci inhibition test. Results from structural and physicochemical characterizations concluded that charge variants are formed due to posttranslational modification demonstrating that the charge heterogeneity, these charge variants did neither show any considerable physicochemical change nor affect its biological function. This study shows that charge variants are effective components of mAb and there is no need of deliberate removal, until biological functions of rabies mAb will get affected.

Development of a Robust Consensus Modeling Approach for Identifying Cellular and Media Metabolites Predictive of Mesenchymal Stromal Cell Potency.

Mesenchymal stromal cells (MSCs) have shown promise in regenerative medicine applications due in part to their ability to modulate immune cells. However, MSCs demonstrate significant functional heterogeneity in terms of their immunomodulatory function because of differences in MSC donor/tissue source, as well as non-standardized manufacturing approaches. As MSC metabolism plays a critical role in their ability to expand to therapeutic numbers ex vivo, we comprehensively profiled intracellular and extracellular metabolites throughout the expansion process to identify predictors of immunomodulatory function (T-cell modulation and indoleamine-2,3-dehydrogenase (IDO) activity). Here, we profiled media metabolites in a non-destructive manner through daily sampling and nuclear magnetic resonance (NMR), as well as MSC intracellular metabolites at the end of expansion using mass spectrometry (MS). Using a robust consensus machine learning approach, we were able to identify panels of metabolites predictive of MSC immunomodulatory function for 10 independent MSC lines. This approach consisted of identifying metabolites in 2 or more machine learning models and then building consensus models based on these consensus metabolite panels. Consensus intracellular metabolites with high predictive value included multiple lipid classes (such as phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins) while consensus media metabolites included proline, phenylalanine, and pyruvate. Pathway enrichment identified metabolic pathways significantly associated with MSC function such as sphingolipid signaling and metabolism, arginine and proline metabolism, and autophagy. Overall, this work establishes a generalizable framework for identifying consensus predictive metabolites that predict MSC function, as well as guiding future MSC manufacturing efforts through identification of high-potency MSC lines and metabolic engineering.

Considerations for the development of iPSC-derived cell therapies: a review of key challenges by the JSRM-ISCT iPSC Committee.

Since their first production in 2007, human induced pluripotent stem cells (iPSCs) have provided a novel platform for the development of various cell therapies targeting a spectrum of diseases, ranging from rare genetic eye disorders to cancer treatment. However, several challenges must be tackled for iPSC-based cell therapy to enter the market and achieve broader global adoption. This white paper, authored by the Japanese Society for Regenerative Medicine (JSRM) - International Society for Cell Therapy (ISCT) iPSC Committee delves into the hurdles encountered in the pursuit of safe and economically viable iPSC-based therapies, particularly from the standpoint of the cell therapy industry. It discusses differences in global guidelines and regulatory frameworks, outlines a series of quality control tests required to ensure the safety of the cell therapy, and provides details and important considerations around cost of goods (COGs), including the impact of automated advanced manufacturing.

Simultaneous prediction of 16 quality attributes during protein A chromatography using machine learning based Raman spectroscopy models.

Several key technologies for advancing biopharmaceutical manufacturing depend on the successful implementation of process analytical technologies that can monitor multiple product quality attributes in a continuous in-line setting. Raman spectroscopy is an emerging technology in the biopharma industry that promises to fit this strategic need, yet its application is not widespread due to limited success for predicting a meaningful number of quality attributes. In this study, we addressed this very problem by demonstrating new capabilities for preprocessing Raman spectra using a series of Butterworth filters. The resulting increase in the number of spectral features is paired with a machine learning algorithm and laboratory automation hardware to drive the automated collection and training of a calibration model that allows for the prediction of 16 different product quality attributes in an in-line mode. The demonstrated ability to generate these Raman-based models for in-process product quality monitoring is the breakthrough to increase process understanding by delivering product quality data in a continuous manner. The implementation of this multiattribute in-line technology will create new workflows within process development, characterization, validation, and control.

A high-titer scalable Chinese hamster ovary transient expression platform for production of biotherapeutics.

Transient gene expression (TGE) in Chinese hamster ovary (CHO) cells offers a route to accelerate biologics development by delivering material weeks to months earlier than what is possible with conventional cell line development. However, low productivity, inconsistent product quality profiles, and scalability challenges have prevented its broader adoption. In this study, we develop a scalable CHO-based TGE system achieving 1.9 g/L of monoclonal antibody in an unmodified host. We integrated continuous flow-electroporation and alternate tangential flow (ATF) perfusion to enable an end-to-end closed system from N-1 perfusion to fed-batch 50-L bioreactor production. Optimization of both the ATF operation for three-in-one application-cell growth, buffer exchange, and cell mass concentration-and the flow-electroporation process, led to a platform for producing biotherapeutics using transiently transfected cells. We demonstrate scalability up to 50-L bioreactor, maintaining a titer over 1 g/L. We also show comparable quality between both transiently and stably produced material, and consistency across batches. The results confirm that purity, charge variants and N-glycan profiles are similar. Our study demonstrates the potential of CHO-based TGE platforms to accelerate biologics process development timelines and contributes evidence supporting its feasibility for manufacturing early clinical material, aiming to strengthen endorsement for TGE's wider implementation.

Antibody-Drug Conjugate Overview: a State-of-the-art Manufacturing Process and Control Strategy.

Antibody-drug conjugates (ADCs) comprise an antibody, linker, and drug, which direct their highly potent small molecule drugs to target tumor cells via specific binding between the antibody and surface antigens. The antibody, linker, and drug should be properly designed or selected to achieve the desired efficacy while minimizing off-target toxicity. With a unique and complex structure, there is inherent heterogeneity introduced by product-related variations and the manufacturing process. Here this review primarily covers recent key advances in ADC history, clinical development status, molecule design, manufacturing processes, and quality control. The manufacturing process, especially the conjugation process, should be carefully developed, characterized, validated, and controlled throughout its lifecycle. Quality control is another key element to ensure product quality and patient safety. A patient-centric strategy has been well recognized and adopted by the pharmaceutical industry for therapeutic proteins, and has been successfully implemented for ADCs as well, to ensure that ADC products maintain their quality until the end of their shelf life. Deep product understanding and process knowledge defines attribute testing strategies (ATS). Quality by design (QbD) is a powerful approach for process and product development, and for defining an overall control strategy. Finally, we summarize the current challenges on ADC development and provide some perspectives that may help to give related directions and trigger more cross-functional research to surmount those challenges.

A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency.

Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the cap gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.

High-humidity hot-air impingement blanching conditions for the inhibition of potato-browning enzymes and for quality retention.

BACKGROUND: Potato is an important non-cereal crop. It provides carbohydrates, a major source of energy in the human diet. Blanching during the processing of fresh fruits and vegetables is essential for their preservation. High-humidity hot-air impingement blanching (HHAIB) is a promising emerging technology for pretreating different food materials. This research aimed to identify the optimum HHAIB conditions for the inhibition of potato-browning enzymes, maintaining their nutritional and physical quality, and to compare this with conventional hot-water blanching (HWB). RESULTS: Polyphenol oxidase (PPO) inactivation, total phenol content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, color, textural attributes, thermal properties, microstructure, and particles crystallinity were evaluated. The relative humidity (RH), temperature, and duration of HHAIB required for PPO inactivation (2.59%) were 50%, 105 °C, and 4 min, respectively, which resulted in a complete gelatigination of potato starches, based on the thermal properties and the microstrcture of the blanched potatoes. These conditions led to improvements in TPC to 312.54 µg GAE.g-1 FP, DPPH scavenging to 1.99 µmol TE.g-1 FP, as well as enhancements in color and crystallinity. When HHAIB was conducted at lower temperatures (85 and 95 °C) there were negative effects on the blanched potatoes' color and crystallinity, along with a non-safe level of PPO activity. CONCLUSION: High-humidity hot-air impingement blanching was superior to HWB, inhibiting PPO, maintaining nutrients, and preserving physical properties, especially under the optimum conditions revealed by the principal component analysis. It provides an excellent technique for blanching and pretreating potatoes, preserving them, and maintaining their quality. © 2023 Society of Chemical Industry.

Quality attributes and functional properties of whole wheat bread baked from frozen dough with the addition of enzymes and hydrocolloids.

BACKGROUND: The increased demand for healthy and standardized bread has led to a demand for an efficient and promising dough improver, of natural origin, to reduce the deterioration of whole wheat bread baked from frozen dough caused by the high levels of dietary fiber and by freezing treatment. In this study, the combined effects of xylanase (XYL), lipase (LIP), and xanthan gum (XAN) on the quality attributes and functional properties of whole wheat bread baked from frozen dough were evaluated. RESULTS: The optimal combination, which contained XYL (0.12 g kg-1 ), LIP (0.25 g kg-1 ), and XAN (3.1 g kg-1 ), was obtained using response surface methodology (RSM). The addition of the optimal combination endowed frozen dough bread with a higher specific volume, softer texture, better brown crumb color, and greater overall acceptability. The optimal combination had no adverse impact on the volatile organic compounds (VOCs) of frozen dough bread. In terms of the functional properties of bread, the water-holding capacity (WHC), oil-holding capacity (OHC), and swelling capacity (SWC) of dietary fiber in frozen dough bread decreased in the presence of the optimal combination, whereas the glucose adsorption capacity (GAC) did not affect them. Correspondingly, the in vitro digestive glucose release was not significantly different between the control group and the optimal combination group after frozen storage. CONCLUSION: The optimal combination could improve the quality attributes and functional properties of whole wheat bread baked from frozen dough effectively, thereby increasing consumption. © 2023 Society of Chemical Industry.

Development and evaluation of earth air heat exchanger cum evaporative cool system as an energy-efficient method for storage of tomatoes.

BACKGROUND: The lack of adequate on-farm storage facilities is one of the leading causes of enormous postharvest losses of fresh commodities, negatively affecting farmers' livelihoods and the sector's economic contribution. The aim of the current study was to develop and evaluate a solar-powered earth air heat exchanger cum evaporative cooling (EAHE-EC) system as an energy-efficient method for the storage of fresh produce for smallholders. RESULTS: The postharvest quality parameters of tomatoes stored under ambient storage (AS) and in an EAHE-EC system were assessed at regular time intervals during storage. These include weight loss, total soluble solids, titratable acidity, fruit firmness, color, pH, ascorbic acid, lycopene content, total phenolic acid, and antioxidant activity. The average temperature and relative humidity inside the EAHE-EC system varied from 20.59 to 22.61 °C and 82.60% to 89.43%, respectively, in comparison with AS (26.39-39.21 °C and 22.09-43.58%). Storage methods and time had a significant (P < 0.05) effect on tomato quality. Based on the retention of the overall quality until the end of the storage period, the shelf life of tomatoes was recorded as 21 days in the EAHE-EC system, which was 1.5 times more than AS (14 days). CONCLUSIONS: The extra shelf-life gained by tomatoes in the EAHE-EC system may provide a sufficient buffer period for smallholders for retailing and marketing. This suggests that the developed system can maintain postharvest quality and increase the shelf life of tomatoes. It therefore has potential as an energy-efficient system for the storage of fruits and vegetables, including tomatoes. © 2023 Society of Chemical Industry.

Predicting Human Dermal Drug Concentrations Using PBPK Modeling and Simulation: Clobetasol Propionate Case Study.

Quantitative in silico tools may be leveraged to mechanistically predict the dermato-pharmacokinetics of compounds delivered from topical and transdermal formulations by integrating systems of rate equations that describe permeation through the formulation and layers of skin and pilo-sebaceous unit, and exchange with systemic circulation via local blood flow. Delivery of clobetasol-17 propionate (CP) from DermovateTM cream was simulated using the Transdermal Compartmental Absorption & Transit (TCATTM) Model in GastroPlus®. The cream was treated as an oil-in-water emulsion, with model input parameters estimated from publicly available information and quantitative structure-permeation relationships. From the ranges of values available for model input parameters, a set of parameters was selected by comparing model outputs to CP dermis concentration-time profiles measured by dermal open-flow microperfusion (Bodenlenz et al. Pharm Res. 33(9):2229-38, 2016). Predictions of unbound dermis CP concentrations were reasonably accurate with respect to time and skin depth. Parameter sensitivity analyses revealed considerable dependence of dermis CP concentration profiles on drug solubility in the emulsion, relatively less dependence on dispersed phase volume fraction and CP effective diffusivity in the continuous phase of the emulsion, and negligible dependence on dispersed phase droplet size. Effects of evaporative water loss from the cream and corticosteroid-induced vasoconstriction were also assessed. This work illustrates the applicability of computational modeling to predict sensitivity of dermato-pharmacokinetics to changes in thermodynamic and transport properties of a compound in a topical formulation, particularly in relation to rate-limiting steps in skin permeation. Where these properties can be related to formulation composition and processing, such a computational approach may support the design of topically applied formulations.

[Optimization of processing technology of bark of Ilicis Rotundae Cortex based on quality by design concept].

Based on the concept of quality by design(QbD), this study optimized the processing technology of Ilicis Rotundae Cortex. According to the processing method and ingredient requirements of Ilicis Rotundae Cortex in the Chinese Pharmacopoeia, the content of syringin and pedunculoside, alcohol extract, fragmentation rate, and moisture content were taken as the critical quality attributes(CQAs). The soaking time, moistening time, and drying time were taken as critical process parameters(CPPs) by single factor tests. The weight coefficients of CQAs were determined by the analytic hierarchy process(AHP)-entropy weighting method, and the comprehensive score was calculated. With the comprehensive score as the response value, Box-Behnken design was employed to establish a mathematical model between CPPs and CQAs, and the design space for the processing of Ilicis Rotundae Cortex was built and verified. The results of ANOVA showed that the mathematical model had the P value below 0.05, the lack of fit greater than 0.05, adjusted R~2=0.910 5, and predicted R~2=0.831 0, which indicated that the proposed model had statistical significance and good prediction performance. Considering the factors in production, the best processing conditions of Ilicis Rotundae Cortex were decoction pieces of about 1 cm soaking for 1 h, moistening for 4 h, and drying at 60-70 ℃ in a blast drier for 2 h. The optimized processing technology of Ilicis Rotundae Cortex was stable and feasible, which can provide a reference for the standardized preparation and stable quality of Ilicis Rotundae Cortex.

[Extraction process optimization and link evaluation of Reyanning Mixture based on quality by design idea].

Reyanning Mixture is one of the superior Chinese patent medicine varieties of "Qin medicine". Based on the idea of quality by design(QbD), the extraction process of the Reyanning Mixture was optimized. The caffeic acid, polydatin, resveratrol, and emodin were used as critical quality attributes(CQAs). The material-liquid ratio, extraction temperature, and extraction time were taken as critical process parameters(CPPs) by the Plackett-Burman test. The mathematical model was established by the star design-effect surface method, and the design space was constructed and verified. The optimal extraction process of the Reyanning Mixture was obtained as follows: material-liquid ratio of 11.84 g·mL~(-1), extraction temperature at 81 ℃, and two extractions. A partial least-square(PLS) quantitative model for CQAs was established by using near-infrared spectroscopy(NIRS) combined with high-performance liquid chromatography(HPLC) under the optimal extraction process. The results showed that the correlation coefficients of the correction set(R_c) and validation set(R_p) of the quantitative models of four CQAs were more than 0.9. The root mean square error of the correction set(RMSEC) were 0.744, 6.71, 3.95, and 1.53 µg·mL~(-1), respectively, and the root mean square error of the validation set(RMSEP) were 0.709, 5.88, 2.92, and 1.59 µg·mL~(-1), respectively. Therefore, the optimized extraction process of the Reyanning Mixture is reasonable, feasible, stable, and reliable. The NIRS quantitative model has a good prediction, which can be used for the rapid content determination of CQAs during extraction. They can provide an experimental basis for the process research and quality control of Reyanning Mixture.

[Application of process analysis technology in traditional Chinese medicine manufacturing industry].

In the traditional Chinese medicine(TCM) manufacturing industry, quality control determines the safety, effectiveness, and quality stability of the final product. The traditional quality control method generally carries out sampling off-line testing of drugs after the end of the batch production, which is incomprehensive, and it fails to find the problems in the production process in time. Process analysis technology(PAT) uses process testing, mathematical modeling, data analysis, and other technologies to collect, analyze, feedback, control, and continuously improve the critical quality attributes(CQA) in all aspects of the production of TCM preparations in real time. The application of PAT in the TCM manufacturing industry is one of the research hotspots in recent years, which has the advantages of real-time, systematic, non-destructive, green, and rapid detection for the production quality control of TCM preparations. It can effectively ensure the stability of the quality of TCM preparations, improve production efficiency, and play a key role in the study of the quantity and quality transfer law of TCM. Commonly used PAT includes near-infrared spectroscopy, Raman spectroscopy, online microwave, etc. In addition, the establishment of an online detection model by PAT is the key basic work to realize intelligent manufacturing in TCM production. Obtaining real-time online detection data through PAT and establishing a closed-loop control model on this basis are a key common technical difficulty in the industry. This paper adopted systematic literature analysis to summarize the relevant Chinese and foreign literature, policies and regulations, and production applications, and it introduced the development trend and practical application of PAT, so as to provide references for accelerating the application of PAT in the TCM manufacturing industry, the intelligent transformation and upgrading, and high-quality development of the TCM industry.

Development and optimization of a LC-MS based multi-attribute method (MAM) workflow for characterization of therapeutic Fc-fusion protein.

The growing complexity of novel biopharmaceutical formats, such as Fc-fusion proteins, in increasingly competitive environment has highlighted the need of high-throughput analytical platforms. Multi-attribute method (MAM) is an emerging analytical technology that utilizes liquid chromatography coupled with mass spectrometry to monitor critical quality attributes (CQAs) in biopharmaceuticals. MAM is intended to supplement or replace the conventional chromatographic and electrophoretic approaches used for quality control and drug release purpose. In this investigation, we have developed an agile sample preparation approach for deploying MAM workflow for a complex VEGFR-targeted therapeutic Fc-fusion protein. Initially, a systematic time course evaluation of tryptic digestion step was performed to achieve maximum amino acid sequence coverage of >96.5%, in a short duration of 2 h, with minimum assay artifacts. This approach facilitated precise identification of five sites of N-glycosylation with successful monitoring of other CQAs such as deamidation, oxidation, etc. Subsequently, the developed MAM workflow with suitable tryptic digestion time was qualified according to the International council for harmonisation (i.e. ICH) Q2R1 guidelines for method validation. Post-validation, the analytical workflow was also evaluated for its capability to identify unknown moieties, termed as 'New Peak Detection' (i.e. NPD), and assess fold change between the reference and non-reference samples, in a representative investigation of pH stress study. The study, thus, demonstrated the suitability of the MAM workflow for characterization of heavily glycosylated Fc-fusion proteins. Moreover, its NPD feature could offer an all-encompassing view if applied for forced degradation and stability studies.

A CHO stable pool production platform for rapid clinical development of trimeric SARS-CoV-2 spike subunit vaccine antigens.

Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time-consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non-clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP-toxicology and early-phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS-CoV-2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost-effective clinical evaluation of potentially life-saving vaccines.

Early dynamic changes in iPSC oxygen consumption rate predict future cardiomyocyte differentiation.

Human induced pluripotent stem cells (iPSCs) hold great promise for reducing the mortality of cardiovascular disease by cellular replacement of infarcted cardiomyocytes (CMs). CM differentiation via iPSCs is a lengthy multiweek process and is highly subject to batch-to-batch variability, presenting challenges in current cell manufacturing contexts. Real-time, label-free control quality attributes (CQAs) are required to ensure efficient iPSC-derived CM manufacturing. In this work, we report that live oxygen consumption rate measurements are highly predictive CQAs of CM differentiation outcome as early as the first 72 h of the differentiation protocol with an accuracy of 93%. Oxygen probes are already incorporated in commercial bioreactors, thus methods presented in this work are easily translatable to the manufacturing setting. Detecting deviations in the CM differentiation trajectory early in the protocol will save time and money for both manufacturers and patients, bringing iPSC-derived CM one step closer to clinical use.

Design space determination to optimize DNA complexation and full capsid formation in transient rAAV manufacturing.

Recombinant adeno-associated virus (rAAV) vectors are a promising platform for in vivo gene therapies. However, cost-effective, well-characterized processes necessary to manufacture rAAV therapeutics are challenging to develop without an understanding of how process parameters (PPs) affect rAAV product quality attributes (PQAs). In this work, a central composite orthogonal experimental design was employed to examine the influence of four PPs for transient transfection complex formation (polyethylenimine:DNA [PEI:DNA] ratio, total DNA/cell, cocktail volume, and incubation time) on three rAAV PQAs related to capsid content (vector genome titer, vector genome:capsid particle ratio, and two-dimensional vector genome titer ratio). A regression model was established for each PQA using partial least squares, and a design space (DS) was defined in which Monte Carlo simulations predicted < 1% probability of failure (POF) to meet predetermined PQA specifications. Of the three PQAs, viral genome titer was most strongly correlated with changes in complexation PPs. The DS and acceptable PP ranges were largest when incubation time and cocktail volume were kept at mid-high setpoints, and PEI:DNA ratio and total DNA/cell were at low-mid setpoints. Verification experiments confirmed model predictive capability, and this work establishes a framework for studying other rAAV PPs and their relationship to PQAs.

Preservation of critical quality attributes of mesenchymal stromal cells in 3D bioprinted structures by using natural hydrogel scaffolds.

Three dimensional (3D) bioprinting is an emerging technology that enables complex spatial modeling of cell-based tissue engineering products, whose therapeutic potential in regenerative medicine is enormous. However, its success largely depends on the definition of a bioprintable zone, which is specific for each combination of cell-loaded hydrogels (or bioinks) and scaffolds, matching the mechanical and biological characteristics of the target tissue to be repaired. Therefore proper adjustment of the bioink formulation requires a compromise between: (i) the maintenance of cellular critical quality attributes (CQA) within a defined range of specifications to cell component, and (ii) the mechanical characteristics of the printed tissue to biofabricate. Herein, we investigated the advantages of using natural hydrogel-based bioinks to preserve the most relevant CQA in bone tissue regeneration applications, particularly focusing on cell viability and osteogenic potential of multipotent mesenchymal stromal cells (MSCs) displaying tripotency in vitro, and a phenotypic profile of 99.9% CD105+ /CD45,- 10.3% HLA-DR,+ 100.0% CD90,+ and 99.2% CD73+ /CD31- expression. Remarkably, hyaluronic acid, fibrin, and gelatin allowed for optimal recovery of viable cells, while preserving MSC's proliferation capacity and osteogenic potency in vitro. This was achieved by providing a 3D structure with a compression module below 8.8 ± 0.5 kPa, given that higher values resulted in cell loss by mechanical stress. Beyond the biocompatibility of naturally occurring polymers, our results highlight the enhanced protection on CQA exerted by bioinks of natural origin (preferably HA, gelatin, and fibrin) on MSC, bone marrow during the 3D bioprinting process, reducing shear stress and offering structural support for proliferation and osteogenic differentiation.