Search Results (344)

The biopharmaceutical industry is undergoing a fundamental transformation from traditional batch manufacturing to continuous manufacturing (CM) for recombinant drugs and biosimilars, driven by regulatory support through the International Council for Harmonization (ICH) Q13 guidance and compelling economic advantages. This comprehensive review examines the technical, economic, and regulatory aspects of implementing continuous manufacturing specifically for recombinant protein production and biosimilar development, synthesizing validated data from peer-reviewed research, regulatory sources, and global implementation case studies. The analysis demonstrates that continuous manufacturing offers substantial benefits, including a reduced equipment footprint of up to 70%, a 3- to 5-fold increase in volumetric productivity, enhanced product quality consistency, and facility cost reductions of 30–50% compared to traditional batch processes. Leading biomanufacturers across North America, Europe, and the Asia–Pacific region are successfully integrating perfusion upstream processes with connected downstream bioprocesses, enabling the fully end-to-end continuous manufacture of biopharmaceuticals with demonstrated commercial viability. The regulatory framework has been comprehensively established through ICH Q13 guidance and region-specific implementations across the FDA, EMA, PMDA, and emerging market authorities. This review provides a critical analysis of advanced technologies, including single-use perfusion bioreactors, continuous chromatography systems, real-time process analytical technology, and Industry 4.0 integration strategies. The economic modeling presents favorable return-on-investment profiles, accompanied by a detailed analysis of global market dynamics, regional implementation patterns, and supply chain integration opportunities. Full article

Chinese hamster ovary (CHO) cells are the most common protein production platform for glycosylated biopharmaceuticals due to their relatively efficient secretion systems, post-translational modification (PTM) machinery, and quality control mechanisms. However, high productivity and titer demands can overburden these processes. In particular, the endoplasmic reticulum (ER) can become overwhelmed with misfolded proteins, triggering the unfolded protein response (UPR) as evidence of ER stress. The UPR increases the expression of multiple genes/proteins, which are beneficial to protein folding and secretion. However, if the stressed ER cannot return to a state of homeostasis, a prolonged UPR results in apoptosis. Because ER stress poses a substantial bottleneck for secreting protein therapeutics, CHO cells are both selected for and engineered to improve high-quality protein production through optimized UPR and ER stress management. This is vital for optimizing industrial CHO cell fermentation. This review begins with an overview of common ER-stress related markers. Next, the optimal UPR profile of high-producing CHO cells is discussed followed by the context-dependency of a UPR profile for any given recombinant CHO cell line. Recent efforts to control and engineer ER stress-related responses in CHO cell lines through the use of various bioprocess operations and activation/inhibition strategies are elucidated. Finally, this review concludes with a discussion on future directions for engineering the CHO cell UPR. Full article

Heterologous expression of caseins in non-mammalian systems offers a sustainable and scalable alternative for producing milk proteins, with potential applications in the food and biopharmaceutical industries. However, a significant challenge in these systems is achieving proper phosphorylation, a critical post-translational modification required for casein functionality and stability. This review explores the current state of research on heterologous casein production, with a particular focus on the biological and technical hurdles associated with phosphorylation. Specifically, we examine the absence of the mammalian-specific kinase Fam20C in plant and yeast systems and the broader lack of secretory kinase machinery in bacteria, which collectively contribute to impaired phosphorylation fidelity. While some endogenous kinases may partially compensate, they are typically insufficient to replicate the phosphorylation pattern required for functionality. We evaluate potential strategies to address these limitations, analyze the role of phosphorylation in casein functionality, provide insights into existing patents and experimental approaches, and highlight ongoing research efforts. By synthesizing current knowledge and proposing new avenues for innovation, this review aims to provide a roadmap for the successful production of functional heterologous caseins. Full article

CRISPR technology, which is derived from the bacterial adaptive immune system, has transformed traditional genetic engineering techniques, made strain engineering significantly easier, and become a very versatile genome editing system that allows for precise, programmable modifications to a wide range of microbial genomes. The economies of fermentation-based manufacturing are changing because of its quick acceptance in both academic and industry labs. CRISPR processes have been used to modify industrially significant bacteria, including the lactic acid producers, Clostridium spp., Escherichia coli, and Corynebacterium glutamicum, in order to increase the yields of bioethanol, butanol, succinic acid, acetone, and polyhydroxyalkanoate precursors. CRISPR-mediated promoter engineering and single-step multiplex editing have improved inhibitor tolerance, raised ethanol titers, and allowed for the de novo synthesis of terpenoids, flavonoids, and recombinant vaccines in yeasts, especially Saccharomyces cerevisiae and emerging non-conventional species. While enzyme and biopharmaceutical manufacturing use CRISPR for quick strain optimization and glyco-engineering, food and beverage fermentations benefit from starter-culture customization for aroma, texture, and probiotic functionality. Off-target effects, cytotoxicity linked to Cas9, inefficient delivery in specific microorganisms, and regulatory ambiguities in commercial fermentation settings are some of the main challenges. This review provides an industry-specific summary of CRISPR–Cas9 applications in microbial fermentation and highlights technical developments, persisting challenges, and industrial advancements. Full article

Proteins play pivotal roles in safeguarding plants against numerous biotic and abiotic stresses. Understanding their biological functions and mechanisms of action is essential for advancing plant biology, agriculture, and biotechnology. This review considers the diversity and potential applications of plant defense proteins including pathogenesis-related (PR) proteins, chitinases, glucanases, protease inhibitors, lectins, and antimicrobial peptides. Recent advances, such as the omics technologies, have enabled the discovery of new plant defense proteins and regulatory networks that govern plant defense responses and unveiled numerous roles of plant defense proteins in stress perception, signal transduction, and immune priming. The molecular affinities and enzymatic activities of plant defense proteins are essential for their defense functions. Applications of plant defense proteins span agriculture, biotechnology, and medicine, including the development of resistant crop varieties, bio-based products, biopharmaceuticals, and functional foods. Future research directions include elucidating the structural bases of defense protein functions, exploring protein interactions with ligands and other proteins, and engineering defense proteins for enhanced efficacy. Overall, this review illuminates the significance of plant defense proteins against biotic stresses in plant biology and biotechnology, emphasizing their potential for sustainable agriculture and environmental management. Full article

The recent advancement of 3D-printed drugs is an emerging technology that has the potential for effective and safe oral delivery of personalized treatment regimens to patients, replacing the current “one size fits all” philosophy. The objective of this literature review is to highlight the importance of 3D-printing technology in the development of personalized treatments, focusing on Levetiracetam, the first FDA-approved 3D-printed drug, for the treatment of epilepsy. Levetiracetam serves as an ideal paradigm for exploring how precision medicine and 3D printing can be applied to improve treatment outcomes for other complex diseases such as diabetes, cardiovascular diseases, and cancer. 3D printing enables precise dosage and time-release profiles by modifying factors such as shape and size, and the combination of active pharmaceutical ingredients (APIs) and excipients, ensuring consistent therapeutic levels over the treatment period. Design of oral tablets with multiple compartments allows for simultaneous treatment with multiple APIs, each one with a different release profile, minimizing drug–drug interactions and side effects. This technology also supports on-demand production, making it particularly beneficial in resource-limited or urgent situations, and offers the flexibility to customize dosage forms. Additive manufacturing could be an important tool for developing personalized treatments to address the diverse medical needs of patients with complex diseases. Therefore, there is a need for more 3D-printed FDA-approved drugs in the biopharmaceutical industry to enable personalized treatment, improved patient compliance, and precise drug release control. Full article

Background/Objectives: Increasing the solid-state landscape of an active pharmaceutical ingredient (API) by generating new crystalline forms (e.g., polymorphs, cyclodextrin (CD) inclusion complexes, co-crystals, and salts) can yield products with significantly enhanced biopharmaceutical properties (especially increased water solubility), thereby improving API delivery and extending its lifetime. The aim of this study was the isolation of new solid forms of the poorly water-soluble non-steroidal anti-inflammatory drug fenbufen (FBF), for which relatively few solid phases have been reported to date. Further motivation for the study is the recent finding that it has potential for repurposing to treat acute pancreatitis. Methods: Interventions for generating new solid forms of FBF included (a) polymorph screening with a variety of solvent media, (b) attempts to form solid inclusion complexes with the native cyclodextrins α-, β-, and γ-CD using various preparative methods, and (c) co-crystallization with a series of coformers to produce co-crystals and/or molecular salts. Results: No new polymorphic forms of FBF were identified, but screening with CDs resulted in isolation and characterization of a new solid inclusion complex with γ-CD. However, co-crystallization of FBF with the water-soluble coformer isonicotinamide yielded two new products, namely a 1:1 co-crystal and an unusual multi-component ionic co-crystal, whose aqueous solubility indicated significant enhancement of FBF solubility. Conclusions: Due to its extremely low water solubility, FBF presented challenges during the study aimed at modifying its crystalline form. However, two new supramolecular forms, a co-crystal and an ionic co-crystal, were isolated, the latter phase having potential for further formulation owing to its significantly enhanced solubility. Full article

Background: Recombinant monoclonal antibodies represent a vital category of biologics, constituting the largest class of molecules used to treat autoimmune disorders, cancers, rheumatoid arthritis, and other chronic conditions. The IgG1 subclass is the most potent among all the immunoglobulin gamma (IgG) antibodies, inducing Fc-related effector functions. N-linked glycan distribution of therapeutic IgG1s affects Fc-related effector functions such as CDC (complement-dependent cytotoxicity) and ADCC (antibody dependent cell-mediated cytotoxicity) biological activities and efficacy in vivo. Hence, as a critical quality attribute (CQA), the glycosylation profile of therapeutic IgG1s must be consistently preserved, which is primarily influenced by manufacturing process factors. In the era of biosimilars, it is challenging for biopharmaceutical manufacturers to not only obtain the desired glycan distribution consistently but also to meet the innovator molecule specifications as per the regulatory agencies. Methods: This study investigates the CHO fed-batch process parameters that affect the titer and terminal galactosylation of the TNF-α blocker-IgG1. It was hypothesized that galactose supplementation would enhance the galactosylation of TNF-α blocker-IgG1. Results: It was observed that such in-cultivation process shift does not affect cell culture parameters yet significantly enhances the galactosylation of TNF-α blocker-IgG1. Interestingly, the results indicate that supplementing D-galactose from the exponential phase of the CHO fed-batch process had the greatest effect on Fc galactosylation, increasing the amount of total galactosylated TNF-α blocker-IgG1 from 7.7% to 15.8%. Conclusions: Our results demonstrate a relatively easy and viable technique for cell culture engineering that is more appropriate for industrial production than costly in vitro glycoengineering. Full article

Plant Molecular Farming (PMF) capitalizes on the unique properties of plants as bioreactors to efficiently produce valuable proteins, pharmaceuticals, and enzymes. This review emphasizes the critical role of transient expression systems, particularly in Nicotiana benthamiana, due to its susceptibility to various pathogens. Viral vector-based transient expression has proven essential during health emergencies like COVID-19, enabling rapid recombinant protein production. The review also evaluates different transient expression platforms and highlights their applications in biopharmaceutical production, education, synthetic biology, and gene editing. Advances in viral vector modification, hydroponics, and Controlled Environment Agriculture (CEA) are presented as transformative innovations enhancing scalability and regulatory compliance. Furthermore, glycoengineering advancements broaden the range of producible biopharmaceuticals, improving global medication access. By exploring these advancements, this review underscores the vast potential of transient expression systems to meet dynamic scientific and market demands, positioning PMF as a vital component in modern biotechnology. Full article

The development of ion exchange chromatography to polish biopharmaceuticals requires extensive experimental benchmarking. As part of the Design of Experiments (DoE), statistical models increased efficiency somewhat and are still state of the art; however, the capability to predict process conditions is limited due to their nature as interpolating models. Applying the DoE still requires numerous experiments and is constrained to the design space, posing a risk of missing the potential optimum. To make a leap in model-based process development, applying extrapolating models can tremendously extend the design space and also allow for process understanding and knowledge transfer. While existing chromatography modeling software explains experimental data, it often lacks predictive power for new conditions. In academic–industrial cooperation, we demonstrate a new high-fidelity model based on biophysics for developing ion-exchange chromatography in biomanufacturing, making it a general tool in rationalizing process development for the present demand of recombinant proteins and monoclonal antibodies and the emerging demand of new modalities. Using the new computational tool, we achieved predictability and attained high accuracy; with minimal experimental effort to calibrate the system, the mathematical model predicted sensitive process conditions, and even described product-related impurities, antibody charge variants. Thus, the computational tool can be deployed for process-by-design and material-by-design approaches. Full article

To address the limitations of traditional endotoxin adsorbents, which exhibit poor endotoxin removal efficiency and low sample recovery when processing high-concentration samples, a novel cryogel, CG(HEMA-co-AM), based on acrylamide (AM) and hydroxyethyl methacrylate (HEMA) as the second monomer, was successfully designed and synthesized. After optimizing the epoxidation and polymyxin B (PMB) conjugation processes, leading to the successful preparation of the functionalized cryogel CG(HEMA-co-AM)@ECH@PMB, flow-through experiments showed that in Tris-HCl buffer at a flow rate of 6 mL/min, the endotoxin removal efficiency reached 99.82%, with a maximum adsorption capacity of 1408.38 EU/mg. In a complex protein system containing BSA, HSA, Hb, LYS, and OVA (each at 10 mg/mL), the maximum endotoxin removal efficiency was 99.62%. Further investigations revealed that pH and ionic strength critically influenced the regulation of hydrophobic and electrostatic interactions, thus significantly affecting the endotoxin adsorption efficiency. Additionally, weakly hydrophobic and basic lysozyme exhibited significantly higher recovery rates compared to strongly hydrophobic and acidic proteins such as BSA and OVA. This functionalized cryogel integrates a high adsorption capacity with excellent specificity and features a macroporous structure suitable for dynamic chromatographic separation. It offers a novel, reusable adsorbent material for endotoxin removal in protein preparation, biopharmaceutical production, and clinical blood purification applications. Full article

Background/Objectives: The Self-Nanoemulsifying Drug Delivery System (SNEDDS) has been widely applied in oral drug delivery, particularly for poorly water-soluble compounds. The successful development of SNEDDS largely depends on the precise composition of its components. This narrative review provides an in-depth analysis of Quality by Design (QbD), Design of Experiment (DoE), and in silico approach applications in SNEDDS development. Methods: The review is based on publications from 2020 to 2025, sourced from reputable scientific databases (Pubmed, Science direct, Taylor and francis, and Scopus). Results: Quality by Design (QbD) is a systematic and scientific approach that enhances product quality while ensuring the robustness and reproducibility of SNEDDS, as outlined in the Quality Target Product Profile (QTPP). DoE was integrated into the QbD framework to systematically evaluate the effects of predefined factors, particularly Critical Material Attributes (CMAs) and Critical Process Parameters (CPP_S), on the desired responses (Critical Quality Attributes/CQA), ultimately leading to the identification of the optimal SNEDDS formulation. Various DoEs, including the mixture design, response surface methodology, and factorial design, have been widely applied to SNEDDS formulations. The experimental design facilitates the analysis of the relationship between CQA and CMA/CPP, enabling the identification of optimized formulations with enhanced biopharmaceutical, pharmacokinetic, and pharmacodynamic profiles. As an essential addition to this review, in silico approach emerges as a valuable tool in the development of SNEDDS, offering deep insights into self-assembly dynamics, molecular interactions, and emulsification behaviour. By integrating molecular simulations with machine learning, this approach enables rational and efficient optimization. Conclusions: The integration of QbD, DoE, and in silico approaches holds significant potential in the development of SNEDDS. These strategies enable a more efficient, rational, and predictive formulation process. Full article

Insect cell lines are a cornerstone of recombinant protein production, providing a versatile platform for biopharmaceutical and research applications. In the early 20th century, scientists first attempted to culture insect cells in vitro, developing continuous cell lines to produce the first insect cell-derived recombinant protein, IFN-β. Initial successes, along with advancements in the use of insect cells for recombinant protein manufacturing, primarily relied on baculovirus expression vector systems (BEVSs), which enable heterologous gene expression in infected cells. Today, growing attention is focused on baculovirus-free systems based on the transfection of insect cells with plasmid DNA. This approach simplifies the final product purification process and facilitates the development of stable monoclonal cell lines that produce recombinant proteins or protein complexes, particularly virus-like particles (VLPs). Thanks to advancements in genetic engineering and the application of adaptive laboratory evolution (ALE) methods, significant strides have been made in overcoming many limitations associated with insect cell BEVSs, ultimately enhancing the reliability, yield, and quality of the biomanufacturing process. Our manuscript discusses the history of developing insect cell lines, presents various recombinant protein production systems utilizing these cells, and summarizes modifications aimed at improving insect cell lines for recombinant protein biomanufacturing. Finally, we explore their implications in pharmaceutical production, particularly on Nuvaxovid^®/Covovax, which is the latest approved vaccine developed using insect cell BEVSs for protection against SARS-CoV-2. Full article

Re-engineering of CHO cells using genome editing and the overexpression of multiple helper genes is the central track for obtaining better cell lines for the production of biopharmaceuticals. Using two subsequent rounds of genome editing of the CHO S cells, we have developed the cell line CHO 4BGD with four knockouts of two pro-apoptotic genes bak1 and bax, and two common selection markers genes—glul (GS) and dhfr, and additional copies of genes bcl-2 and beclin-1 used for enhancement of macroautophagy. The NGS sequencing of 4BGD cells revealed that all eight targeted alleles were successfully disrupted. Two edited loci out of eight contained large inserts of non-relevant DNA. Further data analysis shows that cells have no off-target DNA editing events, and all known CHO genes are preserved. The cells obtained are completely resistant to the induction of apoptosis, and they are suitable for the generation of stably transfected cell lines with the dhfr selection marker. They also properly undergo the target gene amplification. The 4BGD-derived clonal cell line that secretes the monoclonal antibody retains the ability for prolonged fed-batch culturing. The method of obtaining multiply edited CHO cells using the multiplex CRISPR/Cas9 editing and simultaneous stable transfection of plasmids, coding for the housekeeping genes, is suitable for the rapid generation of massively edited CHO cells. Full article

Fed-batch and perfusion bioreactors are commonly used in biopharmaceutical production. This study applies mathematical models to investigate the influence of substrate concentration in the media added ($S_m$), operating substrate concentration in the bioreactor ($S$), and bioreaction time on the performance of both bioreactors. The performance parameters are titer, productivity, product yield, wasted substrate, and mean product residence time. The difference between the substrate concentration in the media and the operating substrate concentration has a major impact on performance parameters. For a fixed $S$, operating at higher values of $S_m$ is more beneficial to both fed-batch and perfusion performance. Higher productivities are obtained in perfusion, and mean product residence times are shorter. Furthermore, perfusion can obtain titers comparable to fed-batch when operated at similar substrate concentrations. All this suggests that perfusion is more advantageous. It is advantageous to operate the bioreactors over a longer bioreaction time. However, for fed-batch bioreactors, there exists an optimal time after which there is a major progressive reduction in productivity. Full article