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Sugarcane wax is a co‐product with strong potential. It contains several bioactive components that can be used for pharmaceutical and cosmetic purposes. It is also considered a substitute for leading vegetable waxes in the international market. The extraction and refining technologies that have been reported in Cuba do not always manage to deliver a wax that meets the quality demanded by the market. This problem can be solved with the development of innovative technologies that allow increasing yields, higher quality, and the reduction of costs. The main objective of this work was to achieve a validated model of the Cuban sugarcane wax‐refining process using the SuperPro Designer simulator, and to evaluate the alternatives in order to reduce consumption rates and increase stability in the final product quality, as customers demand. The model successfully replicated 39 out of 44 variables used for process validation, with values falling within the historical range of the process. Deviations from historical values were less than 4 percentage points. Among the proposed modifications, the second of the two alternatives proposed resulted in lower consumption rates and more consistent quality of refined wax. [ABSTRACT FROM AUTHOR]

Polysorbates are the predominant surfactants used to stabilize protein formulations. Unfortunately, polysorbates can undergo hydrolytic degradation, which releases fatty acids that can accumulate to form visible particles. The detection and quantitation of these fatty acid degradation products are critical for assessing the extent of polysorbate degradation and the associated risks of particle formation. We previously developed a user-friendly mass spectrometric method called Fatty Acids by Mass Spectrometry (FAMS) to quantify the free fatty acids. The FAMS method was validated according to ICH Q2 (R1) guidelines and is suitable for a wide range of products, buffers and protein concentrations. The end-to-end workflow can be automated from sample preparation to data analysis. To broaden method accessibility, the QDa detector selected for fatty acid measurement does not require specific mass spectrometry experience. We provide here a detailed procedure for both manual and automated sample preparation for high-throughput analysis. In addition, we highlight in this protocol the critical operational details, procedural watchouts and troubleshooting tips to support the successful execution of this method in another laboratory. [ABSTRACT FROM AUTHOR]

Therapeutic oligonucleotides (ONs) are typically manufactured via solid-phase synthesis, characterized by limited scalability and huge environmental footprint, limiting their availability. Biomanufactured ONs have the potential to reduce the immunogenic side-effects, and to improve the sustainability of their chemical counterparts. Rhodovulum sulfidophilum was demonstrated a valuable host for the extracellular production of recombinant ONs. However, low viable cell densities and product titer were reported so far. In this work, perfusion cell cultures were established for the intensification of ON biomanufacturing. First, the perfusion conditions were simulated in 50 mL spin tubes, selected as a scale-down model of the process, with the aim of optimizing the medium composition and process parameters. This optimization stage led to an increase in the cell density by 44 % compared to the reference medium formulation. In addition, tests at increasing perfusion rates were conducted until achieving the maximum viable cell density (VCD max), allowing the determination of the minimum cell-specific perfusion rate (CSPR min) required to sustain the cell culture. Intriguingly, we discovered in this system also a maximum CSPR, above which growth inhibition starts. By leveraging this process optimization, we show for the first time the conduction of perfusion cultures of R. sulfidophilum in bench-scale bioreactors. This process development pipeline allowed stable cultures for more than 20 days and the continuous biomanufacturing of ONs, testifying the great potential of perfusion processes. • Medium composition and design space defined for perfusion cultures of Rhodovulum sulfidophilum. • Process scaled up to a 2 L bioreactor with ATF and effect of perfusion rate investigated. • Stable perfusion cultures for >20 days, with cell densities lager than in fed-batch. • Oligonucleotides continuously harvested, with titers 2 orders of magnitudes larger than literature. • First report on continuous biomanufacturing of oligonucleotides. [ABSTRACT FROM AUTHOR]

Escherichia coli has shouldered a massive workload with the discovery of recombinant DNA technology. A new era began in the biopharmaceutical industry with the production of insulin, the first recombinant protein, in E. coli and its use in treating diabetes. After insulin, many biopharmaceuticals produced from E. coli have been approved by the US Food and Drug Administration and the European Medicines Agency to treat various human diseases. Although E. coli has some disadvantages, such as lack of post‐translational modifications and toxicity, it is an important host with advantages such as being a well‐known bacterium in recombinant protein production, cheap, simple production system, and high yield. This study examined biopharmaceuticals produced and approved in E. coli under the headings of peptides, hormones, enzymes, fusion proteins, antibody fragments, vaccines, and other pharmaceuticals. The topics on which these biopharmaceuticals were approved for treating human diseases, when and by which company they were produced, and their use and development in the field are included. [ABSTRACT FROM AUTHOR]

Characterizing the phenotypic diversity and metabolic capabilities of industrially relevant manufacturing cell lines is critical to bioprocess optimization and cell line development. Metabolic capabilities of production hosts limit nutrient and resource channeling into desired cellular processes and can have a profound impact on productivity. These limitations cannot be directly inferred from measured data such as spent media concentrations or transcriptomics. Here, we present an integrated multi-omic analysis pipeline combining exo-metabolomics, transcriptomics, and genome-scale metabolic network analysis and apply it to three antibody-producing Chinese Hamster Ovary cell lines to identify reprogramming features associated with high-producing clones and metabolic bottlenecks limiting product formation in an industrial bioprocess. Analysis of individual datatypes revealed a decreased nitrogenous byproduct secretion in high-producing clones and the topological changes in peripheral metabolic pathway expression associated with phase shifts. An integrated omics analysis in the context of the genome-scale metabolic model elucidated the differences in central metabolism and identified amino acid utilization bottlenecks limiting cell growth and antibody production that were not evident from exo-metabolomics or transcriptomics alone. Thus, we demonstrate the utility of a multi-omics characterization in providing an in-depth understanding of cellular metabolism, which is critical to efforts in cell engineering and bioprocess optimization. • Multi-omics analysis permits synergistic evaluation of culture performance. • Overall phenotypic changes were traced back to phase-specific metabolic reprogramming. • Reprogramming nitrogen usage and metabolism was associated with increased productivity. • Amino acid usage and channeling towards desired products limited productivity. [ABSTRACT FROM AUTHOR]

The formulation of biopharmaceutical drugs is designed to eliminate chemical instabilities, increase conformational and colloidal stability of proteins, and optimize interfacial stability. Among the various excipients involved, buffer composition plays a pivotal role. However, conventional buffers like histidine and phosphate buffers may not always be the optimal choice for all monoclonal antibodies (mAbs). In this study, we investigated the effects of several alternative buffer systems on seven different mAbs, exploring various combinations of ionic strengths, concentrations of the main buffer component, mAb concentrations, and stress conditions. Protein stability was assessed by analyzing soluble aggregate formation through size exclusion chromatography. At low protein concentrations, protein instability after temperature stress was exclusively observed in the bis-TRIS/ glucuronate buffer. Conversely, freeze-thaw stress led to a significant increase in aggregate formation in tested formulations, highlighting the efficacy of several alternative buffers, particularly arginine/ citrate, in preserving protein stability. Under temperature stress, the introduction of arginine to histidine buffer systems provided additional stabilization, while the addition of lysine resulted in protein destabilization. Similarly, the incorporation of arginine into histi-dine/HCl buffer further enhanced protein stability during freeze--thaw cycles. At high protein concentrations, the histidine/citrate buffer emerged as one of the most optimal choices for addressing temperature and light-induced stress. The efficacy of histidine buffers in combating light stress might be attributed to the light-absorbing properties of histidine molecules. Our findings demonstrate that the development of biopharmaceutical formulations should not be confined to conventional buffer systems, as numerous alternative options exhibit comparable or even superior performance.

Abstract Multivariate interactions between process parameters can heavily impact product quality and process performance in biopharmaceutical manufacturing processes. Thus, multivariate interactions should be identified and appropriately controlled. This article describes an in-silico approach to establish multivariate acceptable ranges; these ranges help to illustrate the combined impact of multiple input variables on product quality and process performance. Additionally, this article includes a case study for a monoclonal antibody polishing application. Proven acceptable ranges are set by changing only one input parameter at a time while keeping all others constant to understand the impact of process variability on product quality or process performance, but the impact of synergistic variables are not evaluated. Within multivariate acceptable ranges, any combination of input parameters of a unit operation yields the desired product quality and process performance. The layered approach applied in this article is based on risk assessment and statistical models to leverage prior knowledge and existing data. The risk assessment is specific for a manufacturing facility but is applicable to multiple products manufactured in the same facility. No additional wet-lab experiments are required for building the statistical models when development and process characterization are executed using a design of experiments approach, compared to a univariate evaluation of data. The established multivariate acceptable range justifies revised normal operating ranges to ensure process control. Further, the determination of multivariate acceptable ranges adds to overall process knowledge, ultimately supporting the implementation of a more effective control strategy.

AbstractRecombinant fabs dominate the pharmaceutical pipelines today with microbial host systems continuing to be a major contributor toward their production. Escherichia coli is a versatile host for recombinant protein expression due to its simplicity, affordability, and ability to be cultivated at high cell density. It is particularly suitable for non-glycosylated proteins and small proteins. Despite the aforementioned benefits, the use of E. coli as the host for the synthesis of recombinant antibody fragments often suffers from low yield and reduced activity. In most cases, proteins are expressed as inclusion bodies and need to undergo refolding to achieve their active forms and this refolding step is generally low-yielding. In this article, we review the various approaches that researchers have taken to enhance the production of recombinant antibody fragments in E. coli. Molecular biology-oriented approaches such as cloning, chaperone-mediated folding, and host cell screening as well as process optimization involving examination of process parameters, media, and feeding have been addressed. [ABSTRACT FROM AUTHOR]

Aims To investigate an in-line Raman method capable of detecting accidental microbial contamination in pharmaceutical vessels, such as bioreactors producing monoclonal antibodies via cell culture. Methods and results The Raman method consists of a multivariate model built from Raman spectra collected in-line during reduced-scale bioreactor batches producing a monoclonal antibody, as well as a reduced-scale process with intentional spiking of representative compendial method microorganisms (n  = 4). The orthogonal partial least squares regression discriminant analysis model (OPLS-DA) area under the curve (AUC), specificity and sensitivity were 0.96, 0.99, and 0.95, respectively. Furthermore, the model successfully detected contamination in an accidentally contaminated manufacturing-scale batch. In all cases, the time to detection (TTD) for Raman was superior compared to offline, traditional microbiological culturing. Conclusions The Raman OPLS-DA method met acceptance criteria for equivalent decision making to be considered a viable alternative to the compendial method for in-process bioburden testing. The in-line method is automated, non-destructive, and provides a continuous assessment of bioburden compared to an offline compendial method, which is manual, results in loss of product, and in practice is only collected once daily and requires 3–5 days for enumeration. [ABSTRACT FROM AUTHOR]

The optimization of existing natural antioxidants that are highly effective is crucial for advancements in medicine and the food industry. Due to growing concerns regarding the safety of synthetic antioxidants, researchers are increasingly focusing on natural sources, particularly essential oils (EOs). Combining EOs might enhance antioxidant activity due to increased chemical diversity. This study investigates, for the first time, the antioxidant properties of EOs from Lavandula dentata, Rosmarinus officinalis, and Myrtus communis, both individually and in combination, using the augmented-simplex design methodology. The in vitro evaluation of the antioxidant activity was performed using DPPH and ABTS radical scavenging assays. Chromatography gas-mass spectrometry (CG-MS) revealed that 1,8-cineol (37.27%) and pinocarveol (12.67%) are the primary components of L. dentata; verbenone (16.90%), camphor (15.00%), and camphene (11.03%) are predominant in R. officinalis; while cineol (43.32%) is the main component of M. communis. The EOs showed varying scavenging activities against ABTS and DPPH radicals, with DPPH assay values ranging from 194.10 ± 3.01 to 541.19 ± 3.72 µg/mL and ABTS assay values ranging from 134.07 ± 1.70 to 663.42 ± 2.99 µg/mL. These activities were enhanced when the EOs were combined. The optimal antioxidant blend for DPPHIC50 consisted of 20% L. dentata, 50% R. officinalis, and 30% M. communis. For the highest ABTS radical scavenging activity, the best combination was 18% L. dentata, 43% R. officinalis, and 40% M. communis. These results highlight the potential of EO combinations as new natural formulations for use in cosmeceutical, food, and pharmaceutical sectors. [ABSTRACT FROM AUTHOR]

Long-lasting space missions as well as space tourism are technically possible today and economically in reach. It is a matter of time until the use of biopharmaceutical drug products in space will be common practice. Until drug product manufacturing in space is possible, the products need to be brought to space with rockets, which means that stable and light-weight products are preferred. Lyophilization is a promising approach to reduce weight during transportation and achieve storage stability at room temperature without cold-chain demands. This implies that recycled water in space needs to be used for reconstitution which poses a microbiological challenge and should be considered during formulation development. Furthermore, administration of the injectable drugs in space has an impact on the chosen packaging material which needs to be considered during drug product development. [ABSTRACT FROM AUTHOR]

Multivariate interactions between process parameters can heavily impact product quality and process performance in biopharmaceutical manufacturing processes. Thus, multivariate interactions should be identified and appropriately controlled. This article describes an in-silico approach to establish multivariate acceptable ranges; these ranges help to illustrate the combined impact of multiple input variables on product quality and process performance. Additionally, this article includes a case study for a monoclonal antibody polishing application. Proven acceptable ranges are set by changing only one input parameter at a time while keeping all others constant to understand the impact of process variability on product quality or process performance, but the impact of synergistic variables are not evaluated. Within multivariate acceptable ranges, any combination of input parameters of a unit operation yields the desired product quality and process performance. The layered approach applied in this article is based on risk assessment and statistical models to leverage prior knowledge and existing data. The risk assessment is specific for a manufacturing facility but is applicable to multiple products manufactured in the same facility. No additional wet-lab experiments are required for building the statistical models when development and process characterization are executed using a design of experiments approach, compared to a univariate evaluation of data. The established multivariate acceptable range justifies revised normal operating ranges to ensure process control. Further, the determination of multivariate acceptable ranges adds to overall process knowledge, ultimately supporting the implementation of a more effective control strategy. [ABSTRACT FROM AUTHOR]

Cell engineering strategies typically rely on energy‐consuming overexpression of genes or radical gene‐knock out. Both strategies are not particularly convenient for the generation of slightly modulated phenotypes, as needed in biosimilar development of for example differentially fucosylated monoclonal antibodies (mAbs). Recently, transiently transfected small noncoding microRNAs (miRNAs), known to be regulators of entire gene networks, have emerged as potent fucosylation modulators in Chinese hamster ovary (CHO) production cells. Here, we demonstrate the applicability of stable miRNA overexpression in CHO production cells to adjust the fucosylation pattern of mAbs as a model phenotype. For this purpose, we applied a miRNA chaining strategy to achieve adjustability of fucosylation in stable cell pools. In addition, we were able to implement recently developed artificial miRNAs (amiRNAs) based on native miRNA sequences into a stable CHO expression system to even further fine‐tune fucosylation regulation. Our results demonstrate the potential of miRNAs as a versatile tool to control mAb fucosylation in CHO production cells without adverse side effects on important process parameters. [ABSTRACT FROM AUTHOR]

Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained much interest in the context of modern biotechnology. In this review, we present the utilization of K. phaffii as a platform to produce several products of economic interest such as biopharmaceuticals, renewable chemicals, fuels, biomaterials, and food/feed products. Finally, we present synthetic biology approaches currently used for strain engineering, aiming at the production of new bioproducts. [ABSTRACT FROM AUTHOR]

Biocatalysis has been widely used to prepare drug leads and intermediates. Enzymatic synthesis has advantages, mainly in terms of strict chirality and regional selectivity compared with chemical methods. However, the enzymatic properties of wild-type enzymes may or may not meet the requirements for biopharmaceutical applications. Therefore, protein engineering is required to improve their catalytic activities. Thanks to advances in algorithmic models and the accumulation of immense biological data, artificial intelligence can provide novel approaches for the functional evolution of enzymes. Deep learning has the advantage of learning functions that can predict the properties of previously unknown protein sequences. Deep learning-based computational algorithms can intelligently navigate the sequence space and reduce the screening burden during evolution. Thus, intelligent computational design combined with laboratory evolution is a powerful and potentially versatile strategy for developing enzymes with novel functions. Herein, we introduce and summarize deep-learning-assisted enzyme functional adaptive evolution strategies based on recent studies on the application of deep learning in enzyme design and evolution. Altogether, with the developments of technology and the accumulation of data for the characterization of enzyme functions, artificial intelligence may become a powerful tool for the design and evolution of intelligent enzymes in the future.

Subcutaneous (SC) administration is transforming the delivery of biopharmaceuticals, facilitating care in a variety of healthcare settings, including home self-treatment. Large-volume single SC doses have gained attention for their potential to expand therapeutic applications and improve long-term, patient-centric dosing regimens, often at a reduced SC injection frequency. However, a systematic understanding of dose volumes and frequencies for large-volume (>2.0 mL) SC biopharmaceuticals (LVSCs) is lacking. Accordingly, this study systematically reviewed clinical-stage and approved intravenous (IV) and SC biopharmaceuticals, identifying 182 LVSCs – predominantly monoclonal or bispecific antibodies – which correspond to approximately 15% of all IV and SC biopharmaceuticals. These LVSCs are designed to target cancer and a range of non-cancer chronic disease states, including autoimmune, neurological, and cardiovascular diseases. Results show that anti-cancer LVSCs (n = 75) typically require 5.0 to 20.0 mL doses every three weeks and are administered by healthcare professionals. In contrast, non-cancer LVSCs (n = 107), which are typically self-administered monthly, show more significant dosing variability, with

The use of basil leaves as a vegetable and biopharmaceutical plant requires products that are safe for consumption, thus encouraging environmentally friendly cultivation using natural fertilizers, especially as a nitrogen source. This research aims to determine the effect of various types of organic nitrogen source fertilizer on the growth and production of several basil accessions. The study was conducted in July-September 2021 at the Jambu Luwuk Ciawi, Bogor experimental field. The research design used was factorial CRD. The first factor was basil accessions (Kadudampit, Ciaruteun and Cidolog), and the second factor was the source of N (without N fertilizer, urea, kipahit (Tithonia diversifolia L.) compost, kirinyuh (Chromolaena odorata L.) compost, and chicken manure). The variables observed were the number of leaves, plant height, number of branches and total branch length, leaf area, stem diameter, fresh and dry weight of the first to third harvest, and fresh and dry weight of roots and shoots. The Kadudampit accession has advantages in plant height, leaf area, wet and dry weight of first and second harvest as well as dry weight of third harvest and total harvest compared to other basil accessions. The application of kirinyuh compost increased the height of the basil plants, while the use of chicken manure increased the number of leaves, branches, total branch length, and wet and dry weight of the shoot and roots of the basil. Kirinyuh compost and chicken manure can be alternative sources N as substitute for urea in basil plants. [ABSTRACT FROM AUTHOR]

Many beneficial proteins have limited natural availability, which often restricts their supply and thereby reduces their potential for therapeutic or industrial usage. The advent of recombinant DNA (rDNA) technology enables the utilization of different microbes as surrogate hosts to facilitate the production of these proteins. This microbial technology continues to evolve and integrate with modern innovations to develop more effective approaches for increasing the production of recombinant biopharmaceuticals. These strategies encompass fermentation technology, metabolic engineering, the deployment of strong promoters, novel vector elements such as inducers and enhancers, protein tags, secretion signals, synthetic biology, high-throughput devices for cloning, and process screening. This appraisal commences with a general overview regarding the manufacture of recombinant proteins by microbes and the production of biopharmaceuticals, their trends towards the development of biopharmaceuticals, and then discusses the approaches adopted for accomplishing this. The design of the upstream process, which also involves host selection, vector design, and promoter design, is a crucial component of production strategies. On the other hand, the downstream process focuses on extraction and purification techniques. Additionally, the review covers the most modern tools and resources, methods for overcoming low expression, the cost of producing biopharmaceuticals in microbes, and readily available recombinant protein products. [ABSTRACT FROM AUTHOR]

Precise control over mammalian cell growth dynamics poses a major challenge in biopharmaceutical manufacturing. Here, we present a multi-level cell engineering strategy for the tunable regulation of growth phases in mammalian cells. Initially, we engineered mammalian death phase by employing CRISPR/Cas9 to knockout pro-apoptotic proteins Bax and Bak, resulting in a substantial attenuation of apoptosis by improving cell viability and extending culture lifespan. The second phase introduced a growth acceleration system, akin to a "gas pedal", based on an abscidic acid inducible system regulating cMYC gene expression, enabling rapid cell density increase and cell cycle control. The third phase focused on a stationary phase inducing system, comparable to a "brake pedal". A tetracycline inducible genetic circuit based on BLIMP1 gene led to cell growth cessation and arrested cell cycle upon activation. Finally, we developed a dual controllable system, combining the "gas and brake pedals", enabling for dynamic and precise orchestration of mammalian cell growth dynamics. This work exemplifies the application of synthetic biology tools and combinatorial cell engineering, offering a sophisticated framework for manipulating mammalian cell growth and providing a unique paradigm for reprogramming cell behaviour for enhancing biopharmaceutical manufacturing and further biomedical applications. • Engineering mammalian cell growth dynamics for biomanufacturing precision. • Achieving dynamic gene control with orthogonal inducible circuit –"a "gas and brake" system for growth control. • Tailoring growth phases yields a versatile mammalian platform for enhanced, predictable and high-level production. [ABSTRACT FROM AUTHOR]

One of the characteristics of urban areas is the increasingly limited land that can be used for plant cultivation. Only now, a lot of yard land has yet to be utilized optimally, especially for cultivating vegetables. Klitren Village, in the center of Yogyakarta City, has community-owned yard land that can be used even though the area is limited. This service activity aims to maximize the use of yard land for cultivating vegetable plants, herbal gardens, and food storage. This service activity was carried out in Klitren Village at the Asoka Farming Women's Group (FWG) in July 2022. The service activity was attended by 81 people who were a combination of women members of Asoka's FWG, the Village Head of Klitren Village, Village Agricultural Extension Officers, Lecturers, Students, Research and Community Service Units, and the Klitren Village community from various. The service is carried out through several activities, including making planting media for cultivating vegetable plants, arranging herbal gardens, and providing food barns. 100% of the evaluation results stated that the activity participants knew how to make planting media and food storage. In comparison, 66.7% said they knew how to organize and maintain an herbal garden. 66.7% of the participant's responses to the activities indicated that they were helpful and hoped they could be sustainable and improved. [ABSTRACT FROM AUTHOR]

Summary: Mammalian hearts lose their regenerative potential shortly after birth. Stimulating the proliferation of preexisting cardiomyocytes is a potential therapeutic strategy for cardiac damage. In a previous study, we identified 30 compounds that induced the bona-fide proliferation of human iPSC-derived cardiomyocytes (hiPSC-CM). Here, we selected five active compounds with diverse targets, including ALK5 and CB1R, and performed multi-omic analyses to identify common mechanisms mediating the cell cycle progression of hiPSC-CM. Transcriptome profiling revealed the top enriched pathways for all compounds including cell cycle, DNA repair, and kinesin pathways. Functional proteomic arrays found that the compounds collectively activated multiple receptor tyrosine kinases including ErbB2, IGF1R, and VEGFR2. Network analysis integrating common transcriptomic and proteomic signatures predicted that MAPK/PI3K pathways mediated compound responses. Furthermore, VEGFR2 negatively regulated endoreplication, enabling the completion of cell division. Thus, in this study, we applied high-content imaging and molecular profiling to establish mechanisms linking pro-proliferative agents to mechanisms of cardiomyocyte cell cycling.

This review explores the non-random distribution of chemical novelty among marine invertebrate phyla, with a particular focus on Porifera (sponges), which have exhibited the highest average count of novel natural products per species since 1990. Sponges, considered the oldest multicellular animals, boast approximately 15,000 species, with 8553 officially recognized. They are integral to marine ecosystems and have ecological, economic, and biopharmaceutical significance. The review outlines the structure and habits of sponges, their historical use, and their prevalence in various marine and freshwater habitats. Notably, sponges are recognized as key sources of marine natural products, with their potential medicinal applications extensively studied. Lead compounds derived from sponges have yielded marine pharmaceuticals, including cytarabine, vidarabine, and eribulin mesylate, approved for anticancer and antiviral purposes. Ongoing clinical studies involve novel sponge derivatives such as gemcitabine, hemiasterlin, discodermolide, and PM060184. The review underscores the global interest among researchers in natural product chemistry and pharmaceutical sciences in investigating marine sponges for medication development. The authors conducted a comprehensive literature review from 2000 to 2023, summarizing the chemistry and pharmacological activities of porifera-derived alkaloids based on 212 research papers. The chemical distribution section emphasizes the oceans as a vast repository of unexplored chemical diversity, with sponges yielding approximately 413 alkaloids from 2000 to 2023. Overall, the review provides a comprehensive overview of the chemical and pharmacological diversity of porifera-derived alkaloids, highlighting the importance of sponges as valuable subjects for chemical investigations with potential implications for medication development.

Abstract Cell engineering strategies typically rely on energy‐consuming overexpression of genes or radical gene‐knock out. Both strategies are not particularly convenient for the generation of slightly modulated phenotypes, as needed in biosimilar development of for example differentially fucosylated monoclonal antibodies (mAbs). Recently, transiently transfected small noncoding microRNAs (miRNAs), known to be regulators of entire gene networks, have emerged as potent fucosylation modulators in Chinese hamster ovary (CHO) production cells. Here, we demonstrate the applicability of stable miRNA overexpression in CHO production cells to adjust the fucosylation pattern of mAbs as a model phenotype. For this purpose, we applied a miRNA chaining strategy to achieve adjustability of fucosylation in stable cell pools. In addition, we were able to implement recently developed artificial miRNAs (amiRNAs) based on native miRNA sequences into a stable CHO expression system to even further fine‐tune fucosylation regulation. Our results demonstrate the potential of miRNAs as a versatile tool to control mAb fucosylation in CHO production cells without adverse side effects on important process parameters.

Animal cell cultures have found applications in various fields, from basic to advanced research. This includes studying the fundamentals of cell biology, mechanisms of the cell cycle, specialized cell functions, cell-cell and cell-matrix interactions, toxicity testing for the study of new drugs, gene therapy for replacing non-functional genes with functional cells, characterization of cancer cells, understanding the role of various chemical substances, viruses, and radiation in cancer cells, vaccine production, monoclonal antibodies, and pharmaceuticals. Additionally, the cultivation of viruses for use in vaccine production, such as for diseases like rabies, hepatitis B, and measles, is another important application of animal cell cultures. Aim. The purpose of this study was to analyze the literature data on the use of animal cell lines in genetic engineering, therapy, xenotransplantation, biopharmaceuticals, the food industry, and research. Methods. An analytical review of literature data was conducted using the information analysis of Medline (PubMed), Web of Science and Scopus databases, Google Scholar, the Cochrane Central Register of Controlled Trials (CENTRAL), and other sources up to the inclusive year 2023 using the keywords: “animal cell lines”, “immunobiological preparations”, “xenotransplantation”, “biopharmaceuticals”, “genetic engineering”. Results. An analysis of research related to the use of animal cells in the biopharmaceutical industry was carried out, and considerations regarding the prospects for their use in various research and production technologies were outlined. Conclusion. The technology of cultivating animal cells has become a fundamental tool in the development of research in the field of biotechnological sciences. The ability to culture animal cells in vitro has allowed the development of innovative methods, such as iPSC and organ-on-a-chip models, which have provided valuable information about disease mechanisms and potential therapeutic targets. Although there are some challenges with the use of animal cells related to variability in differentiation efficiency and concerns about safety and efficacy, further studies are needed to optimize protocols and overcome these limitations. Overall, animal cell culture technology remains an important component of modern biomedical research and has the potential to revolutionize the field of regenerative medicine.

Herein, a step-by-step protocol for simultaneous detection of 20 amino acids commonly present in cell culture media is described. The protocol facilitates detection of both primary and secondary amino acids through a two-step precolumn derivatization strategy using ortho-phthalaldehyde and 9-fluorenylmethyl chloroformate as derivatizing agents. The separation of derivatized amino acids with varying hydrophobicity is achieved through reverse-phase chromatography. The amino acids are simultaneously detected in a single workflow through the use of Variable Wavelength Detector at 338 and 262 nm. The protocol is applicable for both mammalian and bacterial cell culture matrices with an option for automation of precolumn derivatization.

Size exclusion chromatography (SEC) has become a powerful tool for analysing size variants of biologic drugs in their native form. Modern SEC can be defined by the use of chromatographic columns packed with sub-3 µm particles, allowing an increase in method throughput compared to that of conventional SEC.

Polysorbates are the predominant surfactants used to stabilize protein formulations. Unfortunately, polysorbates can undergo hydrolytic degradation, which releases fatty acids that can accumulate to form visible particles. The detection and quantitation of these fatty acid degradation products are critical for assessing the extent of polysorbate degradation and the associated risks of particle formation. We previously developed a user-friendly mass spectrometric method called Fatty Acids by Mass Spectrometry (FAMS) to quantify the free fatty acids. The FAMS method was validated according to ICH Q2 (R1) guidelines and is suitable for a wide range of products, buffers and protein concentrations. The end-to-end workflow can be automated from sample preparation to data analysis. To broaden method accessibility, the QDa detector selected for fatty acid measurement does not require specific mass spectrometry experience. We provide here a detailed procedure for both manual and automated sample preparation for high-throughput analysis. In addition, we highlight in this protocol the critical operational details, procedural watchouts and troubleshooting tips to support the successful execution of this method in another laboratory.

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Virus clearance, a critical aspect of biopharmaceutical manufacturing processes, ensures the safety of biopharmaceutics. Virus filtration is widely used during biopharmaceutical downstream processes to remove viruses effectively by separating small viruses (e.g., parvovirus with a size of 18–26 nm) from biopharmaceutics (e.g., monoclonal antibodies with a size of ~12 nm) based on a size-exclusion mechanism (i.e., more than 4-log10 removal of viruses with high product recovery). This paper presents a review of key factors such as filter morphology, feed solution composition, and the performance of high-throughput virus filtration, mainly focusing on the log reduction value for virus removal efficiency. Flow interruption and protein fouling issues are also discussed. [ABSTRACT FROM AUTHOR]

Animal cell cultures have found applications in various fields, from basic to applied research. This includes studying the fundamentals of cell biology, mechanisms of the cell cycle, specialized cell functions, cell-cell and cell-matrix interactions, toxicity testing for the study of new drugs, gene therapy for replacing non-functional genes with functional cells, characterization of cancer cells, understanding the role of various chemical substances, viruses, and radiation in cancer cells, vaccine production, monoclonal antibodies, and pharmaceuticals. Additionally, the cultivation of viruses for using in vaccine production, such as for diseases like rabies, hepatitis B, and measles, is another important application of animal cell cultures. Aim. The purpose of this study was to analyze the literature data on the use of animal cell lines in genetic engineering, therapy, xenotransplantation, biopharmaceuticals, the food industry, and research. Methods. An analytical review of literature data was conducted using the information analysis of Medline (PubMed), Web of Science and Scopus databases, Google Scholar, the Cochrane Central Register of Controlled Trials (CENTRAL), and other sources up to the current year 2023 using the keywords: “animal cell lines”, “immunobiological preparations”, “xenotransplantation”, “biopharmaceuticals”, “genetic engineering”. Results. An analysis of research related to the use of animal cells in the biopharmaceutical industry was carried out, and considerations regarding the prospects for their use in various research and production technologies were outlined. Conclusion. The technology of cultivating animal cells has become a fundamental tool in the development of research in the field of biotechnological sciences. The ability to culture animal cells in vitro has allowed the development of innovative methods, such as induced pluripotent stem cell (iPSC) and organ-on-a-chip models, which have provided valuable information about disease mechanisms and potential therapeutic targets. Although there are some challenges with the use of animal cells related to variability in differentiation efficiency and concerns about safety and efficacy, further studies are needed to optimize protocols and overcome these limitations. Overall, animal cell culture technology remains an important component of modern biomedical research and has the potential to revolutionize the field of regenerative medicine. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are the de facto standard host cells for biopharmaceuticals, and there is great interest in developing methods for constructing stable production cell lines. In this study, clones with a wide chromosome number distribution were selected from isolated antibody-producing strains, and subclones obtained from these clones were evaluated. The transgene copy number varied between the subclones. Even among subclones with similar copy numbers of antibody genes and maintained insertion sites, clones with different productivity were generated. Although the chromosome number distribution differed between these subclones, there was no correlation between the variability in chromosome number after cloning (genome instability) and productivity. Most of the subclones obtained from a parental strain with a wide chromosome number had the same wide chromosome number distribution as the parental strain. Less frequently, cells with less variation (remaining in one distribution) in chromosome number were isolated from cells with a wide chromosome number distribution, from which subclones with less variation in chromosome number were obtained when subcloning was performed again. These results imply that the characteristics of clones with chromosomal instability are inherited by subclones, and thus provide a better understanding of cell line stability/instability. [ABSTRACT FROM AUTHOR]

Introduction: The participation of immune components in inflammatory skin pathologies has brought about the possibility of intervening in the processes that trigger or perpetuate them, hence the importance of knowing the various biological therapies that target specific proteins that They are the result of dysfunction of the immune response in several skin diseases. In the present review work, various aspects related to the selection criteria of biological products (or biopharmaceuticals) are presented with particular interest in monoclonal antibodies (mAb) as well as the main dermatological pathologies in which they have been applied. This is due to the fact that the use of mAbs has been used mainly in the therapy of neoplasias and the possibility of their use in other skin conditions in addition to cancer is being explored. Development of the topic: This bibliographic review addresses the main aspects related to: a) Safety and immunogenicity of monoclonal antibodies, b) Therapeutic applications of mAbs in dermatology: Psoriasis, Atopic dermatitis, Hidrandenigtis suppurativa, Urticaria, Pemphigus, Alopecia areata, Pyoderma gangrenosum Pityriasis nubra pilaris, Cutaneous sarcoidosis, Behcet's syndrome and other pathologies, c) Biosimilars in dermatology and d) Skin lesions associated with the use of monoclonal antibodies. Discussion: mAb-based therapeutics is an area of constant growth and recombinant technology has allowed the obtaining of increasingly stable and less immunogenic molecules that will allow us to offer therapeutic options for various dermatological pathologies with characteristics of hyperinflammation or immunosuppression. Conclusion: The increase in clinical studies for the potential use of monoclonal antibodies in dermatology will allow their authorization by regulatory agencies and increase the therapeutic arsenal for conditions that are difficult to control. [ABSTRACT FROM AUTHOR]

Introduction: Tobacco (Nicotiana tabacum) cv Bright Yellow-2 (BY-2) cell suspension cultures enable the rapid production of complex protein-based biopharmaceuticals but currently achieve low volumetric productivity due to slow biomass formation. The biomass yield can be improved with tailored media, which can be designed either by laborious trial-and-error experiments or systematic, rational design using mechanistic models, linking nutrient consumption and biomass formation. Methods: Here we developed an iterative experiment-modeling-optimization workflow to gradually refine such a model and its predictions, based on collected data concerning BY-2 cell macronutrient consumption (sucrose, ammonium, nitrate and phosphate) and biomass formation. Results and discussion: The biomass formation was well predicted by an unstructured segregated mechanistic Monod-type model as long as the nutrient concentrations did not approach zero (we omitted phosphate, which was completely depleted). Multi-criteria optimization for sucrose and biomass formation indicated the best tradeoff (in a Paretian sense) between maximum biomass yield and minimum process time by reducing the initial sucrose concentration, whereas the inoculation biomass could be increased to maximize the biomass yield or minimize the process time, which we confirmed in calibration experiments. The model became inaccurate at biomass densities > 8 g L-1 dry mass when sucrose was almost depleted. We compensated for this limitation by including glucose and fructose as sucrose hydrolysis products in the model. The remaining offset between the simulation and experimental data might be resolved by including intracellular pools of sucrose, ammonium, nitrate and phosphate. Overall, we demonstrated that iterative models can be used to systematically optimize conditions for bioreactor-based processes. [ABSTRACT FROM AUTHOR]

Natural products and analogues are a source of antibacterial drug discovery. Considering drug resistance levels emerging for antibiotics, identification of bacterial metalloenzymes and the synthesis of selective inhibitors are interesting for antibacterial agent development. Peptide nucleic acids are attractive antisense and antigene agents representing a novel strategy to target pathogens due to their unique mechanism of action. Antisense inhibition and development of antisense peptide nucleic acids is a new approach to antibacterial agents. Due to the increased resistance of biofilms to antibiotics, alternative therapeutic options are necessary. To develop antimicrobial strategies, optimised in vitro and in vivo models are needed. In vivo models to study biofilm-related respiratory infections, device-related infections: ventilator-associated pneumonia, tissue-related infections: chronic infection models based on alginate or agar beads, methods to battle biofilm-related infections are discussed. Drug delivery in case of antibacterials often is a serious issue therefore this review includes overview of drug delivery nanosystems. [ABSTRACT FROM AUTHOR]

The aim of the work is to conduct a review of medications approved by the FDA in 2022.Materials and methods. In searching for the materials to write this review article, bibliographic databases including PubMed, Google Scholar and e-library.ru were utilized. The search was conducted for the publications spanning the period from 2008 to 2023. Herewith, the following keywords and word combinations were used: new drug approval, NDA, drug authorization, approval package, breakthrough medicine.Results. The discovery and development of medications are among the most crucial scientific processes in healthcare. Developing a new drug is a highly intricate, expensive, and time-consuming process. Nowadays, the problem of costs reduction and the process of expedited discovering of new medications are particularly pertinent. To optimize the search for active compounds, virtual and high-throughput screenings, machine learning, artificial intelligence, cryo-electron microscopy, and drug repurposing are employed. Simultaneously, the search for original molecules to serve as the basis for innovative drugs continues. This article presents a review of medications approved by the FDA in 2022 for the treatment of various pathologies.Conclusion. A drug development is a complex and resource-intensive process, with only a small fraction of candidates advancing to clinical trials. A drug design evolves in tandem with societal needs, and this review highlights some of the medications approved by the FDA in 2022. Technological advancements are expected to expedite drug development, potentially reducing the time to the market. Biotechnology, including cell therapy, holds significant prospects, and achievements in genetic mapping and chip technologies will enhance the accessibility of personalized pharmacology.

Abstract Background Process intensification is a major hurdle in pharmaceutical process scale-up. Solvent removal strategies have limited the effectiveness of the overall stability of pharmaceutical formulations. The main aim of present review article is to focus on the use of the freeze-drying process in pharmaceuticals, biopharmaceuticals and nanoderived therapeutics and their translation into commercial viable products. Unwavering efforts of scientists in the process intensification of lyophilization promote unique features of products for commercialization. Regulatory agencies are promoting the utilization of a quality-by-design approach to improve product characteristics. Among 300 FDA-approved pharmaceutical industries, 50% of products are freeze-dried. The freeze-drying process is costlier and requires more time than other drying methodologies. Unstable pharmaceutical dispersions and solutions can be preferably stabilized by using the freeze-drying method. Main text This review highlights the utilization of critical quality attributes and process parameters for the freeze-drying process, which helps to improve the integrity and stability of the formulation. The quality-by-design approach possibly cuts the cost of the process and saves money, time, and laborious work. The present review focuses preliminarily on the applications of freeze-drying in the development of biopharmaceuticals, including vaccines, proteins and peptides, and injectable products. In addition, a separate section demonstrating the potential of freeze-drying in nanoderived therapeutics has been illustrated briefly. The present clinical scenario of freeze-dried pharmaceuticals and biopharmaceuticals has also been described in later sections of the review. Conclusions This review underscores the value of integrating Quality by Design into the development of lyophilization processes for pharmaceutical and biopharmaceutical products. By identifying critical process parameters, delineating a design space, and leveraging advanced monitoring techniques, manufacturers can effectively address the intricacies of lyophilization. This approach empowers them to produce stable, superior quality products with confidence and consistency. Graphical abstract

Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained much interest in the context of modern biotechnology. In this review, we present the utilization of K. phaffii as a platform to produce several products of economic interest such as biopharmaceuticals, renewable chemicals, fuels, biomaterials, and food/feed products. Finally, we present synthetic biology approaches currently used for strain engineering, aiming at the production of new bioproducts.