Biological Therapeutics Research Articles

An Adhesive Hydrogel Technology for Enhanced Cartilage Repair: A Preliminary Proof of Concept.

Knee cartilage has limited natural healing capacity, complicating the development of effective treatment plans. Current non-cell-based therapies (e.g., microfracture) result in poor repair cartilage mechanical properties, low durability, and suboptimal tissue integration. Advanced treatments, such as autologous chondrocyte implantation, face challenges including cell leakage and inhomogeneous distribution. Successful cell therapy relies on prolonged retention of therapeutic biologicals at the implantation site, yet the optimal integration of implanted material into the surrounding healthy tissue remains an unmet need. This study evaluated the effectiveness of a newly developed photo-curable adhesive hydrogel for cartilage repair, focusing on adhesion properties, integration performance, and ability to support tissue regeneration. The proposed hydrogel design exhibited significant adhesion strength, outperforming commercial adhesives such as fibrin-based glues. An in vivo goat model was used to evaluate the hydrogels' adhesion properties and long-term integration into full-thickness cartilage defects over six months. Results showed that cell-free hydrogel-treated defects achieved superior integration with surrounding tissue and enhanced cartilage repair, with notable lateral integration. In vitro results further demonstrated high cell viability, robust matrix production, and successful cell encapsulation within the hydrogel matrix. These findings highlight the potential of adhesive hydrogel formulations to improve the efficacy of cell-based therapies, offering a potentially superior treatment for knee cartilage defects.

How Biologics Have Changed the Drug Discovery Landscape.

Advances in molecular biology and molecular genetics as well as major scientific breakthroughs in immunology and oncology have led to the rapid growth of biologic therapeutics. Their success has resulted in significant changes to virtually every step in the drug discovery and development process. Biologics are produced by living organisms, and screening libraries are generated by immunization or phage display. Lead optimization utilizes sophisticated protein engineering to improve drug-like properties and targeting specificity. The manufacturing process for biologics is complex and requires highly specialized facilities. Determination of pharmacology and safety must overcome the complications associated with species specificity. Initial clinical testing must proceed more slowly and carefully due to the limited predictive utility of preclinical data. In summary, the drug discovery and development process has been dramatically altered by biologic therapeutics and will continue to evolve with the introduction of messenger RNA-based therapeutics and the application of artificial intelligence.

Stellabody: A novel hexamer-promoting mutation for improved IgG potency.

Advances in antibody engineering are being directed at the development of next generation immunotherapeutics with improved potency. Hexamerisation of IgG is a normal physiological aspect of IgG biology and recently described mutations that facilitate this process have a substantial impact upon monoclonal antibody behavior resulting in the elicitation of dramatically enhanced complement-dependent cytotoxicity, Fc receptor function, and enhanced antigen binding effects, such as targeted receptor agonism or microbe neutralization. Whereas the discovery of IgG hexamerisation enhancing mutations has largely focused on residues with exposure at the surface of the Fc-Fc and CH2-CH3 interfaces, our unique approach is the engineering of the mostly buried residue H429 in the CH3 domain. Selective substitution at position 429 forms the basis of Stellabody technology, where the choice of amino acid results in distinct hexamerisation outcomes. H429F results in monomeric IgG that hexamerises after target binding, so called "on-target" hexamerisation, while the H429Y mutant forms pH-sensitive hexamers in-solution prior to antigen binding. Moreover, Stellabody technologies are broadly applicable across the family of antibody-based biologic therapeutics, including conventional mAbs, bispecific mAbs, and Ig-like biologics such as Fc-fusions, with applications in diverse diseases.

Widespread Impact of Natural Genetic Variations in CRISPR-Cas9 Outcomes.

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a genome editing tool widely used in biological research and clinical therapeutics. Natural human genetic variations, through altering the sequence context of CRISPR-Cas9 target regions, can significantly affect its DNA repair outcomes and ultimately lead to different editing efficiencies. However, these effects have not been systematically studied, even as CRISPR-Cas9 is broadly applied to primary cells and patient samples that harbor such genetic diversity. Here, we present comprehensive investigations of natural genetic variations on CRISPR-Cas9 outcomes across the human genome. The utility of our analysis is illustrated in two case studies, on both preclinical discoveries of CD33 knockout in chimeric antigen receptor-T cell therapy and clinical applications of transthyretin (TTR) inactivation for treating TTR amyloidosis. We further expand our analysis to genome-scale, population-stratified common variants that may lead to gene editing disparity. Our analyses demonstrate pitfalls of failing to account for the widespread genetic variations in Cas9 target selection and how they can be effectively examined and avoided using our method. To facilitate broad access to our analysis, a web platform CROTONdb is developed, which provides predictions for all possible CRISPR-Cas9 target sites in the coding and noncoding regulatory regions, spanning over 5.38 million guide RNA targets and 90.82 million estimated variant effects. We anticipate CROTONdb having broad clinical utilities in gene and cellular therapies.

Material Trends and Clinical Costings in Systematically Identified CDER‐Approved Nanomedicines

AbstractResearch into mechanisms and potential applications of nanomaterials in medicine has expanded steadily in recent decades, with increasing translational focus. Development costs, including for clinical trials, are often cited as factors limiting the translational viability of novel nanomedicines, especially compared to small new molecular entities (NMEs) and therapeutic biologics. Yet, to date, there has been no systematic investigation into the clinical programs or costs of translating and commercializing nanomedicines, nor a comparison to NMEs and therapeutic biologics, to support these claims. Here, nanomedicines approved by the United States Food and Drug Administration's Center for Drug Evaluation and Research (CDER) from 2011 to 2023 are systematically identified and categorized. Nanomedicines were identified as formulations where submicron particle size contributes to product function. Like biologics and NMEs, nanomedicines are most frequently approved for oncological indications. Notably, all anti‐cancer nanomedicines are indicated to treat orphan diseases, which is representative of the potential for nanomedicines to occupy pharmaceutical niches in treating diseases affecting smaller patient cohorts. The median estimated cost of pivotal trials for nanomedicines is 47% that of biologics and NMEs approved in the same timeframe. The findings indicate higher manufacturing costs in nanomedicine development may be mitigated by savings elsewhere, increasing translational viability.

Improving Reliability of Immunological Assays by Defining Minimal Criteria for Cell Fitness.

Human PBMC-based assays are often used as biomarkers for the diagnosis and prognosis of disease, as well as for the prediction and tracking of response to biological therapeutics. However, the development and use of PBMC-based biomarker assays is often limited by poor reproducibility. Complex immunological assays can be further complicated by variation in cell handling before analysis, especially when using cryopreserved cells. Variation in postthaw viability is further increased if PBMC isolation and cryopreservation are done more than a few hours after collection. There is currently a lack of evidence-based standards for the minimal PBMC viability or "fitness" required to ensure the integrity and reproducibility of immune cell-based assays. In this study, we use an "induced fail" approach to examine the effect of thawed human PBMC fitness on four flow cytometry-based assays. We found that cell permeability-based viability stains at the time of thawing did not accurately quantify cell fitness, whereas a combined measurement of metabolic activity and early apoptosis markers did. Investigation of the impact of different types and levels of damage on PBMC-based assays revealed that only when cells were >60-70% live and apoptosis negative did biomarker values cease to be determined by cell fitness rather than the inherent biology of the cells. These data show that, to reproducibly measure immunological biomarkers using cryopreserved PBMCs, minimal acceptable standards for cell fitness should be incorporated into the assay protocol.

Imaged capillary isoelectric focusing method development for charge variants of high DAR ADCs

BackgroundCharge heterogeneity is a critical quality attribute for therapeutic biologics including antibody-drug conjugates (ADCs). Developing an ion exchange chromatography (IEX) or an imaged capillary isoelectric focusing (icIEF) method for ADCs with high drug-to-antibody ratio (DAR) is challenging because of the increased hydrophobicity from the payload-linker, DAR heterogeneity, and payload-linker instability. A sub-optimal method can be poorly stability-indicating due to the inability to discern contributions from charge and size variants conjugated with different number of drugs/payloads. Systematic strategy and guidance on charge variant method development is highly desired for high DAR ADCs with various complex structures. ResultsThis work encompasses the development and optimization of icIEF methods for high DAR ADCs of various DAR values (4–8) and payload linker chemistry. Method optimization focuses on improving resolution and stability indicating capabilities and differentiating contributions from the protein and payload-linker. Types, proportion, and combination of solubilizers and carrier ampholytes, as well as focusing parameters were interrogated. Our findings show that the structural units of the linker, the DAR, and the payload chemistry prescribe the selection of buffer, solubilizer, and ampholyte. We demonstrate that a stronger denaturant or solubilizer is needed for high DAR ADCs with polyethylene glycol (PEG)-containing linker structure compared to peptide linker. For unstable payload-linker, buffer system enhances sample stability which is vital to method robustness. In addition, a longer isoelectric focusing time is necessary for an ADC than its corresponding antibody to reach optimal focusing. SignificanceTo the best of our knowledge, this is the first comprehensive study on icIEF method development for charge variant determination of high DAR ADCs with unique physicochemical properties.

Intestinal Lymphatic Biology, Drug Delivery and Therapeutics: Current status and Future directions.

Historically, the intestinal lymphatics were considered passive conduits for fluids, immune cells, dietary lipids, lipid soluble vitamins and lipophilic drugs. Studies of intestinal lymphatic drug delivery in the late 20th century focussed primarily on the drug physicochemical properties, especially high lipophilicity, that resulted in intestinal lymphatic transport. More recent discoveries have changed our traditional view by demonstrating that the lymphatics are active, plastic and tissue-specific players in a range of biological and pathological processes, including within the intestine. These findings have, in turn, inspired exploration of lymph-specific therapies for a range of diseases, as well as the development of more sophisticated strategies to actively deliver drugs or vaccines to the intestinal lymph, including a range of nanotechnologies, lipid prodrugs and lipid-conjugated materials that 'hitchhike' on lymphatic transport pathways. With the increasing development of novel biological therapeutics there has been interest in whether these novel therapeutics are absorbed and transported through intestinal lymph after oral administration. Here we review the current state of understanding of the anatomy and physiology of the gastrointestinal lymphatic system in health and disease, with a focus on aspects relevant to drug delivery. We summarise the current state-of-the-art approaches to deliver drugs and quantify their uptake into the intestinal lymphatic system. Finally, and excitingly, we discuss recent examples of significant pharmacokinetic and therapeutic benefits achieved via intestinal lymphatic drug delivery. We also propose approaches to advance the development and clinical application of intestinal lymphatic delivery strategies into the future. Significance Statement This comprehensive review details understanding of the anatomy and physiology of the intestinal lymphatic system in health and disease, with a focus on aspects relevant to drug delivery. It highlights current state-of-the-art approaches to deliver drugs to the intestinal lymphatics and the shift toward the use of these strategies to achieve pharmacokinetic and therapeutic benefits for patients.

Adeno-Associated Virus (AAV)-Delivered Exosomal TAT and BiTE Molecule CD4-αCD3 Facilitate the Elimination of CD4 T Cells Harboring Latent HIV-1.

Combinatorial antiretroviral therapy (cART) has transformed HIV infection from a death sentence to a controllable chronic disease, but cannot eliminate the virus. Latent HIV-1 reservoirs are the major obstacles to cure HIV-1 infection. Previously, we engineered exosomal Tat (Exo-Tat) to reactivate latent HIV-1 from the reservoir of resting CD4+ T cells. Here, we present an HIV-1 eradication platform, which uses our previously described Exo-Tat to activate latent virus from resting CD4+ T cells guided by the specific binding domain of CD4 in interleukin 16 (IL16), attached to the N-terminus of exosome surface protein lysosome-associated membrane protein 2 variant B (Lamp2B). Cells with HIV-1 surface protein gp120 expressed on the cell membranes are then targeted for immune cytolysis by a BiTE molecule CD4-αCD3, which colocalizes the gp120 surface protein of HIV-1 and the CD3 of cytotoxic T lymphocytes. Using primary blood cells obtained from antiretroviral treated individuals, we find that this combined approach led to a significant reduction in replication-competent HIV-1 in infected CD4+ T cells in a clonal in vitro cell system. Furthermore, adeno-associated virus serotype DJ (AAV-DJ) was used to deliver Exo-Tat, IL16lamp2b and CD4-αCD3 genes by constructing them in one AAV-DJ vector (the plasmid was named pEliminator). The coculture of T cells from HIV-1 patients with Huh-7 cells infected with AAV-Eliminator viruses led to the clearance of HIV-1 reservoir cells in the in vitro experiment, which could have implications for reducing the viral reservoir in vivo, indicating that Eliminator AAV viruses have the potential to be developed into therapeutic biologics to cure HIV-1 infection.

Region-selective and site-specific glycation of influenza proteins surrounding the viral envelope membrane

Analysis of protein modifications is critical for quality control of therapeutic biologics. However, the identification and quantification of naturally occurring glycation of membrane proteins by mass spectrometry remain technically challenging. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. Straightforward sequencing was enhanced by MS/MS fragmentation of small peptides. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase. Topological analysis provides insights into the site-specific lysine glycation, localizing in the distinct structural regions of proteins surrounding the viral envelope membrane. Our finding highlights the proteome-wide discovery of lysine glycation of influenza membrane proteins and potential effects on the structural assembly, stability, receptor binding and enzyme activity, demonstrating that the impacts of accumulated glycation on the quality of products can be directly monitored by mass spectrometry-based structural proteomics analyses.

Urine-derived podocytes from steroid resistant nephrotic syndrome patients as a model for renal-progenitor derived extracellular vesicles effect and drug screening

BackgroundPersonalized disease models are crucial for evaluating how diseased cells respond to treatments, especially in case of innovative biological therapeutics. Extracellular vesicles (EVs), nanosized vesicles released by cells for intercellular communication, have gained therapeutic interest due to their ability to reprogram target cells. We here utilized urinary podocytes obtained from children affected by steroid-resistant nephrotic syndrome with characterized genetic mutations as a model to test the therapeutic potential of EVs derived from kidney progenitor cells (nKPCs).MethodsEVs were isolated from nKPCs derived from the urine of a preterm neonate. Three lines of urinary podocytes obtained from nephrotic patients’ urine and a line of Alport syndrome patient podocytes were characterized and used to assess albumin permeability in response to nKPC-EVs or various drugs. RNA sequencing was conducted to identify commonly modulated pathways after nKPC-EV treatment. siRNA transfection was used to demonstrate the involvement of SUMO1 and SENP2 in the modulation of permeability.ResultsTreatment with the nKPC-EVs significantly reduced permeability across all the steroid-resistant patients-derived and Alport syndrome-derived podocytes. At variance, podocytes appeared unresponsive to standard pharmacological treatments, with the exception of one line, in alignment with the patient’s clinical response at 48 months. By RNA sequencing, only two genes were commonly upregulated in nKPC-EV-treated genetically altered podocytes: small ubiquitin-related modifier 1 (SUMO1) and Sentrin-specific protease 2 (SENP2). SUMO1 and SENP2 downregulation increased podocyte permeability confirming the role of the SUMOylation pathway.ConclusionsnKPCs emerge as a promising non-invasive source of EVs with potential therapeutic effects on podocytes with genetic dysfunction, through modulation of SUMOylation, an important pathway for the stability of podocyte slit diaphragm proteins. Our findings also suggest the feasibility of developing a non-invasive in vitro model for screening regenerative compounds on patient-derived podocytes.

AAV-aMTD-Parkin, a therapeutic gene delivery cargo, enhances motor and cognitive functions in Parkinson's and Alzheimer’s diseases

Neurodegenerative disorders, such as Parkinson's disease (PD) and Alzheimer's disease (AD), have a global prevalence and profoundly impact both motor and cognitive functions. Although adeno-associated virus (AAV)-based gene therapy has shown promise, its application for treating central nervous system (CNS) diseases faces several challenges, including effective delivery of AAV vectors across the blood-brain barrier, determining optimal dosages, and achieving targeted distribution. To address these challenges, we have developed a fusion delivery therapeutic cargo called AAV-aMTD-Parkin, which combines a hydrophobic cell-penetrating peptide sequence with the DNA sequences of AAV and Parkin. By employing this fusion delivery platform at lower dosages compared to zolgensma, we have achieved significant enhancements in cell and tissue permeability, while reducing the occurrence of common pathological protein aggregates. Consequently, motor and cognitive functions were restored in animal models of PD and AD. With its dual functionality in addressing PD and AD, AAV-aMTD-Parkin holds immense potential as a novel class of therapeutic biologics for prevalent CNS diseases.

Polyurea-urethane Temperature-responsive Hydrogels for Sustained Delivery of Anti-VEGF Therapeutics.

Temperature-responsive hydrogels, or thermogels, have emerged as a leading platform for sustained delivery of both small molecule drugs and macromolecular biologic therapeutics. Although thermogel properties can be modulated by varying the polymer's hydrophilic-hydrophobic balance, molecular weight and degree of branching, varying the supramolecular donor-acceptor interactions on the polymer remains surprisingly overlooked. Herein, to study the influence of enhanced hydrogen bonding on thermogelation, we synthesized a family of amphiphilic polymers containing urea and urethane linkages using quinuclidine as an organocatalyst. Our findings showed that the presence of strongly hydrogen bonding urea linkages significantly enhanced polymer hydration in water, in turn affecting hierarchical polymer self-assembly and macroscopic gel properties such as sol-gel phase transition temperature and gel stiffness. Additionally, analysis of the sustained release profiles of Aflibercept, an FDA-approved protein biologic for anti-angiogenic treatment, showed that urea bonds on the thermogel were able to significantly alter the drug release mechanism and kinetics compared to usage of polyurethane gels of similar composition and molecular weight. Our findings demonstrate the unrealized possibility of modulating gel properties and outcomes of sustained drug delivery through judicious variation of hydrogen bonding motifs on the polymer structure.

Rapid protein evolution by few-shot learning with a protein language model.

Directed evolution of proteins is critical for applications in basic biological research, therapeutics, diagnostics, and sustainability. However, directed evolution methods are labor intensive, cannot efficiently optimize over multiple protein properties, and are often trapped by local maxima. In silico- directed evolution methods incorporating protein language models (PLMs) have the potential to accelerate this engineering process, but current approaches fail to generalize across diverse protein families. We introduce EVOLVEpro, a few-shot active learning framework to rapidly improve protein activity using a combination of PLMs and protein activity predictors, achieving improved activity with as few as four rounds of evolution. EVOLVEpro substantially enhances the efficiency and effectiveness of in silico protein evolution, surpassing current state-of-the-art methods and yielding proteins with up to 100-fold improvement of desired properties. We showcase EVOLVEpro for five proteins across three applications: T7 RNA polymerase for RNA production, a miniature CRISPR nuclease, a prime editor, and an integrase for genome editing, and a monoclonal antibody for epitope binding. These results demonstrate the advantages of few-shot active learning with small amounts of experimental data over zero-shot predictions. EVOLVEpro paves the way for broader applications of AI-guided protein engineering in biology and medicine.

Sequence-specific targeting of intrinsically disordered protein regions.

A general approach to design proteins that bind tightly and specifically to intrinsically disordered regions (IDRs) of proteins and flexible peptides would have wide application in biological research, therapeutics, and diagnosis. However, the lack of defined structures and the high variability in sequence and conformational preferences has complicated such efforts. We sought to develop a method combining biophysical principles with deep learning to readily generate binders for any disordered sequence. Instead of assuming a fixed regular structure for the target, general recognition is achieved by threading the query sequence through diverse extended binding modes in hundreds of templates with varying pocket depths and spacings, followed by RFdiffusion refinement to optimize the binder-target fit. We tested the method by designing binders to 39 highly diverse unstructured targets. Experimental testing of ∼36 designs per target yielded binders with affinities better than 100 nM in 34 cases, and in the pM range in four cases. The co-crystal structure of a designed binder in complex with dynorphin A is closely consistent with the design model. All by all binding experiments for 20 designs binding diverse targets show they are highly specific for the intended targets, with no crosstalk even for the closely related dynorphin A and dynorphin B. Our approach thus could provide a general solution to the intrinsically disordered protein and peptide recognition problem.

Advancing Vaccinology Capacity: Education and Efforts in Vaccine Development and Manufacturing across Africa.

Africa, home to the world's second-largest population of approximately 1.3 billion, grapples with significant challenges in meeting its medical needs, particularly in accessing quality healthcare services and products. The continent faces a continuous onslaught of emerging infectious diseases, exacerbating the strain on its already fragile public health infrastructure. The COVID-19 crisis highlighted the urgency to build local vaccine production capacity and strengthen the health infrastructure in general. The risks associated with a heavy reliance on imported vaccines were exposed during the COVID-19 pandemic, necessitating the need to nurture and strengthen the local manufacturing of vaccines and therapeutic biologics. Various initiatives addressing training, manufacturing, and regulatory affairs are underway, and these require increasing dedicated and purposeful financial investment. Building vaccine manufacturing capacity requires substantial investment in training and infrastructure. This manuscript examines the current state of education in vaccinology and related sciences in Africa. It also provides an overview of the continent's efforts to address educational needs in vaccine development and manufacturing. Additionally, it evaluates the initiatives aimed at strengthening vaccine education and literacy, highlighting successful approaches and ongoing challenges. By assessing the progress made and identifying the remaining obstacles, this review offers insights into how Africa can enhance its vaccine manufacturing capacity to respond to vaccine-preventable disease challenges.

Extracellular vesicle surface display of αPD-L1 and αCD3 antibodies via engineered late domain-based scaffold to activate T-cell anti-tumor immunity.

Extracellular vesicles (EVs) are emerging as promising carriers for the delivery of therapeutic biologics. Genetic engineering represents a robust strategy for loading proteins of interest into EVs. Identification of EV-enriched proteins facilitates protein cargo loading efficiency. Many EV-enriched proteins are sorted into EVs via an endosomal sorting complex required for transport (ESCRT)-dependent pathway. In parallel, viruses hijack this EV biosynthesis machinery via conserved late domain motifs to promote egress from host cells. Inspired by the similarity of biogenesis between EVs and viruses, we developed a synthetic, Late domain-based EV scaffold protein that enables the display of a set of single chain variable fragments (scFvs) on the EV surface. We named this scaffold the Late domain-based exosomal antibody surface display platform (LEAP). We applied the LEAP scaffold to reprogramme HEK293T cell-derived EVs to elicit T-cell anti-tumor immunity by simultaneously displaying αPD-L1 and αCD3 scFvs on the EV surface (denoted as αPD-L1×αCD3 bispecific T-cell engaging exosomes, BiTExos). We demonstrated that αPD-L1×αCD3 BiTExos actively redirected T cells to bind to PD-L1+ tumor cells, promoting T-cell activation, proliferation and tumoricidal cytokine production. Furthermore, the αPD-L1×αCD3 BiTExos promoted T-cell infiltration into the tumor microenvironment to mitigate the tumor burden in vivo. Our study suggested that the LEAP scaffold may serve as a platform for EV surface display and could be applied for a broad range of EV-based biomedical applications.

An Amphiphilic Cell-Penetrating Macrocycle for Efficient Cytosolic Delivery of Proteins, DNA, and CRISPR Cas9.

The discovery of safe platforms that can circumvent the endocytic pathway is of great significance for biological therapeutics that are usually degraded during endocytosis. Here we show that a self-assembled and dynamic macrocycle can passively diffuse through the cell membrane and deliver a broad range of biologics, including proteins, CRISPR Cas9, and ssDNA, directly to the cytosol while retaining their bioactivity. Cell-penetrating macrocycle CPM can be easily prepared from the room temperature condensation of diketopyrrolopyrrole lactams with diamines. We attribute the high cellular permeability of CPM to its amphiphilic nature and chameleonic properties. It adopts conformations that partially bury polar groups and expose hydrophobic side chains, thus self-assembling into micellar-like structures. Its superior fluorescence makes CPM trackable inside cells where it follows the endomembrane system. CPM outperformed commercial reagents for biologics delivery and showed high RNA knockdown efficiency of CRISPR Cas9. We envisage that this macrocycle will be an ideal starting point to design and synthesize biomimetic macrocyclic tags that can readily facilitate the interaction and uptake of biomolecules and overcome endosomal digestion.

An Amphiphilic Cell‐Penetrating Macrocycle for Efficient Cytosolic Delivery of Proteins, DNA, and CRISPR Cas9

AbstractThe discovery of safe platforms that can circumvent the endocytic pathway is of great significance for biological therapeutics that are usually degraded during endocytosis. Here we show that a self‐assembled and dynamic macrocycle can passively diffuse through the cell membrane and deliver a broad range of biologics, including proteins, CRISPR Cas9, and ssDNA, directly to the cytosol while retaining their bioactivity. Cell‐penetrating macrocycle CPM can be easily prepared from the room temperature condensation of diketopyrrolopyrrole lactams with diamines. We attribute the high cellular permeability of CPM to its amphiphilic nature and chameleonic properties. It adopts conformations that partially bury polar groups and expose hydrophobic side chains, thus self‐assembling into micellar‐like structures. Its superior fluorescence makes CPM trackable inside cells where it follows the endomembrane system. CPM outperformed commercial reagents for biologics delivery and showed high RNA knockdown efficiency of CRISPR Cas9. We envisage that this macrocycle will be an ideal starting point to design and synthesize biomimetic macrocyclic tags that can readily facilitate the interaction and uptake of biomolecules and overcome endosomal digestion.

KT-Biologics I (KTB1): A dynamic simulation model for continuous biologics manufacturing

The pharmaceutical industry's shift towards biological therapeutics has led to a transition from conventional batch production to continuous manufacturing. This change highlights the crucial need for effective process monitoring and control strategies to ensure consistent product quality and stability. Open-source benchmark simulation models have become essential tools for refining these processes, offering a platform for testing research hypotheses. This study uses the production of Lovastatin as a case study for continuous biopharmaceutical production. A comprehensive dynamic model covering upstream and downstream components provides an integrated perspective of the production process. The study introduces a basic control system emphasizing realistic sensor and actuator integration to enhance simulation accuracy. It assesses the benchmark through open-loop and closed-loop simulations, offering an in-depth analysis of the KTB1 model's dynamic response and functionality. KTB1 represents a model-driven decision support tool that enables the evaluation of monitoring strategies, process design, process optimization, and control for biomanufacturing.