Versatility of mesenchymal stem cell-derived extracellular vesicles in tissue repair and regenerative applications.

Mesenchymal stem/stromal cells (MSCs) are multipotent somatic cells that have been widely explored in the field of regenerative medicine. MSCs possess the ability to secrete soluble factors as well as lipid bound extracellular vesicles (EVs). MSCs have gained increased interest and attention as a result of their therapeutic properties, which are thought to be attributed to their secretome. However, while the use of MSCs as whole cells pose heterogeneity concerns and survival issues post-transplantation, such limitations are absent in cell-free EV-based treatments. EVs derived from MSCs are promising therapeutic agents for a range of clinical conditions and disorders owing to their immunomodulatory, pro-regenerative, anti-inflammatory, and antifibrotic activity. Recent successes with preclinical studies using EVs for repair and regeneration of damaged tissues such as cardiac tissue, lung, liver, pancreas, bone, skin, cornea, and blood diseases are discussed in this review. We also discuss delivery strategies of EVs using biomaterials as delivery vehicles through systemic or local administration. Despite its effectiveness in preclinical investigations, the application of MSC-EV in clinical settings will necessitate careful consideration surrounding issues such as: i) scalability and isolation, ii) biodistribution, iii) targeting specific tissues, iv) quantification and characterization, and v) safety and efficacy of dosage. The future of EVs in regenerative medicine is promising yet still needs further investigation on enhancing the efficacy, scalability, and potency for clinical applications.

Systematic assessment of process analytical technologies for biologics.

The application of process analytical technology (PAT) for biotherapeutic development and manufacturing has been employed owing to technological, economic, and regulatory advantages across the industry. Typically, chromatographic, spectroscopic, and/or mass spectrometric sensors are integrated into upstream and downstream unit operations in in-line, on-line, or at-line fashion to enable real-time monitoring and control of the process. Despite the widespread utility of PAT technologies at various unit operations of the bioprocess, a holistic business value assessment of PAT has not been well addressed in biologics. Thus, in this study, we evaluated PAT technologies based on predefined criteria for their technological attributes such as enablement of better process understanding, control, and high-throughput capabilities; as well as for business attributes such as simplicity of implementation, lead time, and cost reduction. The study involved an industry-wide survey, where input from subject matter industry experts on various PAT tools were collected, assessed, and ranked. The survey results demonstrated on-line liquid Chromatography (LC), in-line Raman, and gas analysis techniques are of high business value especially at the production bioreactor unit operation of upstream processing. In-line variable path-length UV/VIS measurements (VPE), on-line LC, multiangle light scattering (MALS), and automated sampling are of high business value in Protein A purification and polishing steps of the downstream process. We also provide insights, based on our experience in clinical and commercial manufacturing of biologics, into the development and implementation of some of the PAT tools. The results presented in this study are intended to be helpful for the current practitioners of PAT as well as those new to the field to gauge, prioritize and steer their projects for success.

Understanding Design Rules for Optimizing the Interface between Immobilized Enzymes and Random Copolymer Brushes.

A long-standing goal in the field of biotechnology is to develop and understand design rules for the stabilization of enzymes upon immobilization to materials. While immobilization has sometimes been successful as a strategy to stabilize enzymes, the design of synthetic materials that stabilize enzymes remains largely empirical. We sought to overcome this challenge by investigating the mechanistic basis for the stabilization of immobilized lipases on random copolymer brush surfaces comprised of poly(ethylene glycol) methacrylate (PEGMA) and sulfobetaine methacrylate (SBMA), which represent novel heterogeneous supports for immobilized enzymes. Using several related but structurally diverse lipases, including Bacillus subtilis lipase A (LipA), Rhizomucor miehei lipase, Candida rugosa lipase, and Candida antarctica lipase B (CALB), we showed that the stability of each lipase at elevated temperatures was strongly dependent on the fraction of PEGMA in the brush layer. This dependence was explained by developing and applying a new algorithm to quantify protein surface hydrophobicity, which involved using unsupervised cluster analysis to identify clusters of hydrophobic atoms. Characterization of the lipases showed that the optimal brush composition correlated with the free energy of solvation per enzyme surface area, which ranged from -17.1 kJ/mol·nm2 for LipA to -11.8 kJ/mol·nm2 for CALB. Additionally, using this algorithm, we found that hydrophobic patches consisting of aliphatic residues had a higher free energy than patches consisting of aromatic residues. By providing the basis for rationally tuning the interface between enzymes and materials, this understanding will transform the use of materials to reliably ruggedize enzymes under extreme conditions.

Carotenoids from Cyanobacteria: Biotechnological Potential and Optimization Strategies.

Carotenoids are tetraterpenoids molecules present in all photosynthetic organisms, responsible for better light-harvesting and energy dissipation in photosynthesis. In cyanobacteria, the biosynthetic pathway of carotenoids is well described, and apart from the more common compounds (e.g., ß-carotene, zeaxanthin, and echinenone), specific carotenoids can also be found, such as myxoxanthophyll. Moreover, cyanobacteria have a protein complex called orange carotenoid protein (OCP) as a mechanism of photoprotection. Although cyanobacteria are not the organism of choice for the industrial production of carotenoids, the optimisation of their production and the evaluation of their bioactive capacity demonstrate that these organisms may indeed be a potential candidate for future pigment production in a more environmentally friendly and sustainable approach of biorefinery. Carotenoids-rich extracts are described as antioxidant, anti-inflammatory, and anti-tumoral agents and are proposed for feed and cosmetical industries. Thus, several strategies for the optimisation of a cyanobacteria-based bioprocess for the obtention of pigments were described. This review aims to give an overview of carotenoids from cyanobacteria not only in terms of their chemistry but also in terms of their biotechnological applicability and the advances and the challenges in the production of such compounds.

Application of machine learning methods to pathogen safety evaluation in biological manufacturing processes.

The production of recombinant therapeutic proteins from animal or human cell lines entails the risk of endogenous viral contamination from cell substrates and adventitious agents from raw materials and environment. One of the approaches to control such potential viral contamination is to ensure the manufacturing process can adequately clear the potential viral contaminants. Viral clearance for production of human monoclonal antibodies is achieved by dedicated unit operations, such as low pH inactivation, viral filtration, and chromatographic separation. The process development of each viral clearance step for a new antibody production requires significant effort and resources invested in wet laboratory experiments for process characterization studies. Machine learning methods have the potential to help streamline the development and optimization of viral clearance unit operations for new therapeutic antibodies. The current work focuses on evaluating the usefulness of machine learning methods for process understanding and predictive modeling for viral clearance via a case study on low pH viral inactivation.

Development of a packed bed reactor for the removal of aromatic hydrocarbons from soil using laccase/mediator feeding system.

Polyaromatic hydrocarbons (PAH) are persistent pollutants of great concern due to their potential toxicity, mutagenicity and carcinogenicity. A biotechnological approach to remove PAH from soil was evaluated in this work using a laccase mediator system. Initially, laccase was produced by fungal co-cultivation, using kiwi peels as substrate. The produced laccase was applied to PAH contaminated soil to evaluate its efficiency on enzymatic bioremediation. Results showed that laccase mediator system was effective in the degradation of pyrene, fluorene, chrysene and a lower extension anthracene. Mediators improved the PAH degradation and natural mediators (ferulic acid and p-coumaric acid) were as effective as the synthetic mediator ABTS. However, the process was not effective in the benzo[a]pyrene degradation, one of the most recalcitrant and toxic PAH. This low degradation rate could be related to the low activity of the laccase mediator system in an environment lacking water. To overcome this issue, a PAH contaminated soil degradation system was developed in packed bed reactor (PBR) fed with laccase/mediator. Continuous flow of laccase/mediator improved the PAH degradation, achieving 74.8 %, 71.9 %, 72.2 %, 81.8 % and 100 % degradation for fluorene, anthracene, phenanthrene, chrysene and pyrene, respectively. This system was able to degrade 96 % benzo[a]pyrene, which was 90 % higher than the degradation in batch system. Results indicated that the produced laccase as well as the fed-batch degradation system developed in PBR could be successfully applied in the degradation of soil PAH pollutants, with the advantage of achieving higher degradation rates than in simple batch, as well as being a faster and simpler process than microorganism bioremediation.

Economic assessment of continuous processing for manufacturing of biotherapeutics.

Continuous processing offers a promising approach to revolutionize biotherapeutics manufacturing as reflected in recent years. The current study offers a comparative economic assessment of batch and continuous processing for the production of biotherapeutic products. Granulocyte-colony stimulating factor (GCSF), a protein expressed in E. coli, and an IgG1 monoclonal antibody, were chosen as representatives of microbial and mammalian derived products for this assessment. Economic indicators-cost of goods (COGs), net present value (NPV), and payback time have been estimated for the assessment. For the case of GCSF, conversion from batch to integrated continuous manufacturing induced a $COGs/g reduction of 83% and 73% at clinical and commercial scales, respectively. For the case of mAb therapeutic, a 68% and 35% reduction in $COGs/g on translation from batch to continuous process was projected for clinical and commercial scales, respectively. Upstream mAb titer was also found to have a significant impact on the process economics. With increasing mAb titer, the $COG/g decreases in both operating modes. With titer increasing from 2 to 8 g/L, the $COG/g of batch process was reduced by 53%, and that of the continuous process was reduced by 63%. Cost savings in both the cases were attributed to increased productivity, efficient equipment and facility utilization, smaller facility footprint, and reduction in utilization of consumables like resin media and buffers actualized by the continuous processing platform. The current study quantifies the economic benefits associated with continuous processing and highlights its potential in reducing the manufacturing cost of biotherapeutics.

Safety risk management for low molecular weight process-related impurities in monoclonal antibody therapeutics: Categorization, risk assessment, testing strategy, and process development with leveraging clearance potential.

Process-related impurities (PRIs) derived from manufacturing process should be minimized in final drug product. ICH Q3A provides a regulatory road map for PRIs but excludes biologic drugs like monoclonal antibodies (mAbs) that contain biological PRIs (e.g. host cell proteins and DNA) and low molecular weight (LMW) PRIs (e.g., fermentation media components and downstream chemical reagents). Risks from the former PRIs are typically addressed by routine tests to meet regulatory expectations, while a similar routine-testing strategy is unrealistic and unnecessary for LMW PRIs, and thus a risk-assessment-guided testing strategy is often utilized. In this report, we discuss a safety risk management strategy including categorization, risk assessment, testing strategy, and its integrations with other CMC development activities, as well as downstream clearance potentials. The clearance data from 28 mAbs successfully addressed safety concerns but did not fully reveal the process clearance potentials. Therefore, we carried out studies with 13 commonly seen LMW PRIs in a typical downstream process for mAbs. Generally, Protein A chromatography and cation exchange chromatography operating in bind-and-elute mode showed excellent clearances with greater than 1,000- and 100-fold clearance, respectively. The diafiltration step had better clearance (greater than 100-fold) for the positively and neutrally charged LMW PRIs than for the negatively charged or hydrophobic PRIs. We propose that a typical mAb downstream process provides an overall clearance of 5,000-fold. Additionally, the determined sieving coefficients will facilitate diafiltration process development. This report helps establish effective safety risk management and downstream process design with robust clearance for LMW PRIs.

Biodegradable Plastics: Standards, Policies, and Impacts.

Plastics are ubiquitous in our society. They are in our phones, clothes, bottles, and cars. Yet having improved our lives considerably, they now threaten our environment and our health. The associated carbon emissions and persistency of plastics challenge the fragile balance of many ecosystems. One solution is using biodegradable plastics. Ideally, such plastics are easily assimilated by microorganisms and disappear from our environment. This can help reduce the problems of climate change, microplastics, and littering. However, biodegradable plastics are still only a tiny portion of the global plastics market and require further efforts in research and commercialization. Here, a critical overview of the state of the art of biodegradable plastics is given. Using a material flow analysis, the challenges of the plastic market are highlighted, and with it the large market potential of biodegradable plastics. The environmental and socio-economic impact of plastics, government policies, standards and certifications, physico-chemical properties, and analytical techniques are covered. The Review concludes with a personal outlook on the future of bioplastics, based on our own experience with their development and commercialization.

Implementation of food safety management systems that meets ISO 22000:2018 and HACCP: A case study of capsule biotechnology products of chaga mushroom.

In our study, a food safety management system was developed for a chaga mushroom biotechnology product manufacturer, with the purpose of meeting the quality demands of customers and enhancing the manufacturer's reputation. The study focused on identifying the potentially significant hazards present at each stage of the production process for chaga mushroom capsule products, and on ensuring that the biotechnology company in question has fully implemented ISO22000:2018 and the HACCP methodology. The results indicate that, in the 1 year following the implementation of ISO 22000:2018, there was a statistically significant drop (P < 0.05) in the coliforms level of the tested biotechnology product samples that started in the ninth month following implementation. The rapid screening of mycotoxin, heavy metal, and pesticide residue levels also increased monitoring intensity and strengthened the periodic rotation plan, enabling control over potential problems relating to raw materials and ensuring product quality. This finding reveals the importance and necessity of rapid screening for small- and medium-sized food industry enterprises. Furthermore, 1 year after the system's implementation, the defect rate for chaga products was also observed to have declined, and the number of process flow anomalies requiring correction was also found to have decreased significantly (P < 0.05), indicating that safety and quality levels were improving and stabilizing. If implemented over a longer period of time, the food safety management system's benefits would stand out even more, and significant improvements would be observed for more monitored items. PRACTICAL APPLICATION: Few studies have touched on food safety management systems (FSMSs) developed for capsule health food products. The enterprise examined in this study had actively worked to improve its quality system and meet its customers' needs through the implementation of the FSMSs.

[Assessment and empirical analysis of the competitiveness of bioindustry in various regions of China].

The era of bioeconomy has ushered in a new wave of technological and industrial revolution for mankind. The strategic deployment for the bioindustry in China has achieved remarkable results. However, there are still problems such as unbalanced regional development in the process of bioindustry development. In order to comprehensively assess the current situation of the competitiveness of the bioindustry in various regions of China, the assessment indicator system of the overall competitiveness of the bioindustry is constructed from the perspective of the four sub-industries of biomedicine, bioenergy, bioagriculture and bio-based industry. The weight of each assessment indicator is determined by the analytic hierarchy process. According to geographical administrative division and regional economic relations, an empirical analysis of the comprehensive competitiveness score of the bioindustry in seven regions of China is carried out. The assessment results show that the competition of bioindustry in various regions of China presents a gradient distribution in space. In view of this, relevant policy recommendations are put forward from four aspects: (1) implementing the strategy of rural revitalization, (2) implementing the strategy of regional coordinated development, (3) deepening the supply-side structural reform for the bioindustry, and (4) establishing regional unified information collaboration network system.

The Formidable Challenge of Controlling High Mannose-Type N-Glycans in Therapeutic mAbs.

The clinical efficacy and safety of therapeutic monoclonal antibodies (mAbs) are significantly affected by their Fc-glycosylation profile. High mannose-type N-glycans (HM) affect efficacy (in terms of antibody-dependent cell cytotoxicity), pharmacokinetics, and stability. While in endogenous IgGs the HM levels are very low, they are significantly higher in marketed therapeutic mAbs. In order to meet the demands for late-phase clinical trial and market supply, process intensification is required. Since glycosylation profiles are sensitive to process variations and changes, controlling HM levels in robust manufacturing processes presents a formidable challenge and requires a thorough understanding of the cellular processes as well as the biotechnical aspects that govern the production of HM glycans.

Technologies and Standardization in Research on Extracellular Vesicles.

Extracellular vesicles (EVs) are phospholipid bilayer membrane-enclosed structures containing RNAs, proteins, lipids, metabolites, and other molecules, secreted by various cells into physiological fluids. EV-mediated transfer of biomolecules is a critical component of a variety of physiological and pathological processes. Potential applications of EVs in novel diagnostic and therapeutic strategies have brought increasing attention. However, EV research remains highly challenging due to the inherently complex biogenesis of EVs and their vast heterogeneity in size, composition, and origin. There is a need for the establishment of standardized methods that address EV heterogeneity and sources of pre-analytical and analytical variability in EV studies. Here, we review technologies developed for EV isolation and characterization and discuss paths toward standardization in EV research.

Good design practices for an integrated containment and production system for advanced therapies.

Advances in molecular biology and the possibility of differentiating stem cells have opened up new scenarios in therapies that use progenitor or variously differentiated cells. Regardless of the choice of the system, designing a plant for producing advanced therapies requires a clear understanding of the final objective (the product), taking into account all the regulatory, environment, process, risk assessment, asepsis, and validation aspects involved until its implementation. Good Manufacturing Practice (GMP) compliant procedures are a prerequisite for cell production in clinical application, and clean rooms are zones for producing cell therapies. Clean rooms for clinical application require high running and maintenance costs and need trained operators and strict procedures to prepare the rooms and the people involved in the processes. While today production mainly occurs in open systems (clean rooms), there is evidence of processes in closed systems (isolators). The isolator is a Grade A aseptic closed system that requires a controlled environment and at least a Grade D environment in the case of sterile productions (A in D closed system). The use of isolators can ensure a very high level of protection against the risk of product contamination and, at the same time, provide the operators with a very safe working environment. Furthermore, working with closed systems can optimize and facilitate the production of Advanced Therapy Medical Products in GMP environments, by providing an easily reproducible working tool even for large-scale production, with generally lower costs compared to a classical clean room approach. In conclusion, the isolator workstation as a possible alternative to the classic clean room, due to its small size and the simplification of the working and maintenance operational procedures, may represent an interesting solution in the perspective of the increasingly more stringent requests for cost reductions of GMP in clinical application.

Variations in novel cellular therapy products manufacturing.

BACKGROUND AIMS: At the frontier of transfusion medicine and transplantation, the field of cellular therapy is emerging. Most novel cellular therapy products are produced under investigational protocols with no clear standardization across cell processing centers. Thus, the purpose of this study was to uncover any variations in manufacturing practices for similar cellular therapy products across different cell processing laboratories worldwide. METHODS: An exploratory survey that was designed to identify variations in manufacturing practices in novel cellular therapy products was sent to cell processing laboratory directors worldwide. The questionnaire focused on the manufacturing life cycle of different cell therapies (i.e., collection, purification, in vitro expansion, freezing and storage, and thawing and washing), as well as the level of regulations followed to process each product type. RESULTS: The majority of the centers processed hematopoietic progenitor cells (HPCs) from peripheral blood (n = 18), bone marrow (n = 16) or cord blood (n = 19), making HPCs the most commonly processed cells. The next most commonly produced cellular therapies were lymphocytes (n = 19) followed by mesenchymal stromal cells (n = 14), dendritic cells (n = 9) and natural killer (NK) cells (n = 9). A minority of centers (<5) processed pancreatic islet cells (n = 4), neural cells (n = 3) and induced-pluripotent stem cells (n = 3). Thirty-two laboratories processed products under an investigational status, for either phase I/II (n = 27) or phase III (n = 17) clinical trials. If purification methods were used, these varied for the type of product processed and by institution. Environmental monitoring methods also varied by product type and institution. CONCLUSION: This exploratory survey shows a wide variation in cellular therapy manufacturing practices across different cell processing laboratories. A better understanding of the effect of these variations on the quality of these cell-based therapies will be important to assess for further process evaluation and development.

A systematic review protocol for crime trends facilitated by synthetic biology.

BACKGROUND: When new technologies are developed, it is common for their crime and security implications to be overlooked or given inadequate attention, which can lead to a 'crime harvest'. Potential methods for the criminal exploitation of biotechnology need to be understood to assess their impact, evaluate current policies and interventions and inform the allocation of limited resources efficiently. Recent studies have illustrated some of the security implications of biotechnology, with outcomes of misuse ranging from compromised computers using malware stored in synthesised DNA, infringement of intellectual property on biological matter, synthesis of new threatening viruses, 'genetic genocide,' and the exploitation of food markets with genetically modified crops. However, there exists no synthesis of this information, and no formal quality assessment of the current evidence. This review therefore aims to establish what current and/or predicted crimes have been reported as a result of biotechnology. METHODS: A systematic review will be conducted to identify relevant literature. ProQuest, Web of Science, MEDLINE and USENIX will be searched utilizing a predefined search string, and Backward and Forward searches. Grey literature will be identified by searching the official UK Government website (www.gov.uk) and the Global database of Dissertations and Theses. The review will be conducted by screening title/abstracts followed by full texts, utilising pre-defined inclusion and exclusion criteria. Papers will be managed using Eppi-center Reviewer 4 software, and data will be organised using a data extraction table using a descriptive coding tool. A predefined rating system (speculative, experimental or currently occurring) will be used to sort studies, and a thematic synthesis of the results will be presented. DISCUSSION: Despite the concerns raised about the misuse of biotechnology, no previous work has been conducted from a Crime Science perspective to collate and assess the literature. This systematic review aims to identify the types of offending activity facilitated by biotechnology, including synthetic biology and genetic engineering. The objective of the review is to examine whether this offending activity can be prevented by assessing the conditions necessary for the crime events to occur. It is anticipated that evidence generated from this review will guide future research in this area and aid relevant stakeholders to prioritise and allocate limited resources to biotechnology crime prevention. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42019131685.

Update and Revalidation of Ghose's Cellulase Assay Methodology.

New studies on cellulolytic enzymes aiming to improve biofuels production lead to a concern over the assaying methods commonly applied to measure their activity. One of the most used methods is Ghose's cellulase and endoglucanase assay, developed by the International Union of Pure and Applied Chemistry in 1987. Carrying out this method demands high volumes of reagents and generation of high amounts of chemical residues. This work aimed to adapt Ghose's methodology to reduce its application cost and residue generation and validate the adjustments. To do so, International and Brazilian laws were applied to validate methodologies. Method's modifications were successfully validated according to all institutions and were considered linear, accurate, precise, and reproducible. It was possible to reduce the volume of reagents and residues in 12 times. Considering the routine work of most laboratories, it is a great reduction on material costs and residue treatment, which reflects in sustainability and environmental impacts.

Developing synthetic biology for industrial biotechnology applications.

Since the beginning of the 21st Century, synthetic biology has established itself as an effective technological approach to design and engineer biological systems. Whilst research and investment continues to develop the understanding, control and engineering infrastructural platforms necessary to tackle ever more challenging systems - and to increase the precision, robustness, speed and affordability of existing solutions - hundreds of start-up companies, predominantly in the US and UK, are already translating learnings and potential applications into commercially viable tools, services and products. Start-ups and SMEs have been the predominant channel for synthetic biology commercialisation to date, facilitating rapid response to changing societal interests and market pull arising from increasing awareness of health and global sustainability issues. Private investment in start-ups across the US and UK is increasing rapidly and now totals over $12bn. Health-related biotechnology applications have dominated the commercialisation of products to date, but significant opportunities for the production of bio-derived materials and chemicals, including consumer products, are now being developed. Synthetic biology start-ups developing tools and services account for between 10% (in the UK) and ∼25% (in the US) of private investment activity. Around 20% of synthetic biology start-ups address industrial biotechnology targets, but currently, only attract ∼11% private investment. Adopting a more networked approach - linking specialists, infrastructure and ongoing research to de-risk the economic challenges of scale-up and supported by an effective long-term funding strategy - is set to transform the impact of synthetic biology and industrial biotechnology in the bioeconomy.