Yu Zhang

The rapid growth of recombinant protein vaccines underscores the need for manufacturing platforms that are efficient, stable, and scalable. We present a biomanufacturing approach integrating bioengineered Pichia pastoris with sodium alginate bioinks using the patented Sprayed Multi Adsorbed-Droplet Reposing Technology (SMART, US20250288524A1) to fabricate uniform microbiofactories. Engineered Pichia pastoris X33 strains expressing the truncated Epstein–Barr virus nuclear antigen 1 (EBNA1, aa 379–641) from the pPICZαA-EBNA1 vector were encapsulated and induced with methanol for antigen production. SMART-assisted encapsulation generated microcarriers averaging ∼50 μm in diameter with a postencapsulation yeast recovery of 87% as assessed by the Alamar Blue viability assay. Across three independent biological replicates, hydrogel-encapsulated yeast consistently produced higher EBNA1 titers than free yeast, corresponding to a 1.70 ± 0.37-fold enhancement, as quantified by ELISA and confirmed by Western blot. Following lyophilization, in a single-experiment proof-of-concept storage study, the microbiofactories sustained EBNA1 production at 392 ± 11 ng/mL over 28 days at ambient temperature (24 °C), compared to 154 ± 18 ng/mL for free yeast; the sustained ELISA signal at day 28 constitutes functional evidence of metabolically active yeast upon reconstitution. These findings demonstrate the in vitro proof-of-concept feasibility of SMART-assisted hydrogel encapsulation as an ambient-temperature-stable platform for microbial antigen production. This approach provides a foundation for future development of biomanufacturing systems that may help reduce cold-chain dependence, pending further scale-up and immunological validation.

The commercialization of additive manufacturing (AM) in pharmaceuticals manufacturing has attracted significant attention for its potential to produce customized products. However, the process is slow and hindered by the lack of designated regulatory guidelines tailored to 3D-printed pharmaceutical products (3DPPs). The 3D-printing technology has paved the way for personalized medicine, enabled treatment of rare genetic disorders, and offered many other possibilities for patients. Despite the US Food and Drug Administration (FDA) approval of Spritam ^® , a clear regulatory framework for licensing 3DPPs by the FDA or EMA remains unavailable. The current practice considers all products the same, regardless of their manufacturing method and/or complexity. While this approach has been generally accepted, it frequently fails to evaluate the unique quality attributes of 3DPPs. The lack of a harmonized regulatory framework tailored to the 3DPPs presents a major barrier to the widespread adoption of AM and other innovative technologies. To bridge this gap, this review highlights the most critical parameters related to the feedstock materials and 3D-printing processes, emphasizing their impact on the quality attributes of finished 3DPPs. Numerous scenarios have been proposed to encourage regulatory authorities to establish robust regulatory guidance for the 3D-printing technology at either industrial or point-of-care (PoC) settings. Coordinated efforts between regulatory authorities, industry partners and other stakeholders are necessary to define product specifications and identify appropriate analytical techniques for evaluating finished 3DPPs. By developing a harmonized regulatory framework and establishing quality control measures, the full potential of AM can be realized. This will ultimately ensure that novel 3DPPs and personalized medicines adhere to rigorous regulatory standards of quality, safety and efficacy. Graphical abstract

Electrospinning has become a widely used and efficient method for manufacturing nanofibers from diverse polymers. This study introduces an advanced electrospinning technique, Xspin - a multi-functional 3D printing platform coupled with electrospinning system, integrating a customised 3D printhead, MaGIC - Multi-channeled and Guided Inner Controlling printheads. The Xspin system represents a cutting-edge fusion of electrospinning and 3D printing technologies within the realm of pharmaceutical sciences and biomaterials. This innovative platform excels in the production of novel fiber with various materials and allows for the creation of highly customized fiber structures, a capability hitherto unattainable through conventional electrospinning methodologies. By integrating the benefits of electrospinning with the precision of 3D printing, the Xspin system offers enhanced control over the scaffold morphology and drug release kinetics. Herein, we fabricated a model floating pharmaceutical dosage for the dual delivery of curcumin and ritonavir and thoroughly characterized the product. Fourier transform infrared (FTIR) spectroscopy demonstrated that curcumin chemically reacted with the polymer during the Xspin process. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) confirmed the solid-state properties of the active pharmaceutical ingredient after Xspin processing. Scanning electron microscopy (SEM) revealed the surface morphology of the Xspin-produced fibers, confirming the presence of the bifiber structure. To optimize the quality and diameter control of the electrospun fibers, a design of experiment (DoE) approach based on quality by design (QbD) principles was utilized. The bifibers expanded to approximately 10-11 times their original size after freeze-drying and effectively entrapped 87% curcumin and 84% ritonavir. In vitro release studies demonstrated that the Xspin system released 35% more ritonavir than traditional pharmaceutical pills in 2 h, with curcumin showing complete release in pH 1.2 in 5 min, simulating stomach media. Furthermore, the absorption rate of curcumin was controlled by the characteristics of the linked polymer, which enables both drugs to be absorbed at the desired time. Additionally, multivariate statistical analyses (ANOVA, pareto chart, etc.) were conducted to gain better insights and understanding of the results such as discern statistical differences among the studied groups. Overall, the Xspin system shows significant potential for manufacturing nanofiber pharmaceutical dosages with precise drug release capabilities, offering new opportunities for controlled drug delivery applications.

Cancer is a complex disease with global significance, necessitating continuous advancements in diagnostics and treatment. 3D printing technology has emerged as a revolutionary tool in cancer diagnostics, offering immense potential in detection and monitoring. Traditional diagnostic methods have limitations in providing molecular and genetic tumor information that is crucial for personalized treatment decisions. Biomarkers have become invaluable in cancer diagnostics, but their detection often requires specialized facilities and resources. 3D printing technology enables the fabrication of customized sensor arrays, enhancing the detection of multiple biomarkers specific to different types of cancer. These 3D-printed arrays offer improved sensitivity, allowing the detection of low levels of biomarkers, even in complex samples. Moreover, their specificity can be fine-tuned, reducing false-positive and false-negative results. The streamlined and cost-effective fabrication process of 3D printing makes these sensor arrays accessible, potentially improving cancer diagnostics on a global scale. By harnessing 3D printing, researchers and clinicians can enhance early detection, monitor treatment response, and improve patient outcomes. The integration of 3D printing in cancer diagnostics holds significant promise for the future of personalized cancer care.

Bone regeneration using inorganic nanoparticles is a robust and safe approach. In this paper, copper nanoparticles (Cu NPs) loaded with calcium phosphate scaffolds were studied for their bone regeneration potential in vitro. The pneumatic extrusion method of 3D printing was employed to prepare calcium phosphate cement (CPC) and copper loaded CPC scaffolds with varying wt% of copper nanoparticles. A new aliphatic compound Kollisolv MCT 70 was used to ensure the uniform mixing of copper nanoparticles with CPC matrix. The printed scaffolds were studied for physico-chemical characterization for surface morphology, pore size, wettability, XRD, and FTIR. The copper ion release was studied in phosphate buffer saline at pH 7.4. The in vitro cell culture studies for the scaffolds were performed using human mesenchymal stem cells (hMSCs). The cell proliferation study in CPC-Cu scaffolds showed significant cell growth compared to CPC. The CPC-Cu scaffolds showed improved alkaline phosphatase activity and angiogenic potential compared to CPC. The CPC-Cu scaffolds showed significant concentration dependent antibacterial activity in Staphylococcus aureus. Overall, the CPC scaffolds loaded with 1 wt% Cu NPs showed improved activity compared to other CPC-Cu and CPC scaffolds. The results showed that copper has improved the osteogenic, angiogenic and antibacterial properties of CPC scaffolds, facilitating better bone regeneration in vitro.

Selective laser sintering (SLS) is a single-step, three-dimensional printing (3DP) process that is gaining momentum in the manufacturing of pharmaceutical dosage forms. It also offers opportunities for manufacturing various pharmaceutical dosage forms with a wide array of drug delivery systems. This research aimed to introduce carbonyl iron as a multifunctional magnetic and heat conductive ingredient for the fabrication of oral tablets containing isoniazid, a model antitubercular drug, via SLS 3DP process. Furthermore, the effects of magnetic iron particles on the drug release from the SLS printed tablets under a specially designed magnetic field was studied. Optimization of tablet quality was performed by adjusting SLS printing parameters. The independent factors studied were laser scanning speed, hatching space, and surface/chamber temperature. The responses measured were printed tablets' weight, hardness, disintegration time, and dissolution performance. It has been observed that, for the drug formulation with carbonyl iron, due to its inherent thermal conductivity, sintering tablets required relatively lower laser energy input to form the tablets of the same quality attributes as the other batches that contained no magnetic particles. Also, printed tablets with carbonyl iron released 25% more drugs under a magnetic field than those without it. It can be claimed that magnetic nanoparticles appear as an alternative conductive material to facilitate the sintering process during SLS 3DP of dosage forms.

Recently, various innovative technologies have been developed for the enhanced delivery of biologics as attractive formulation targets including polymeric micro and nanoparticles. Combined with personalized medicine, this area can offer a great opportunity for the improvement of therapeutics efficiency and the treatment outcome. Herein, a novel manufacturing method has been introduced to produce protein-loaded chitosan particles with controlled size. This method is based on an additive manufacturing technology that allows for the designing and production of personalized particulate based therapeutic formulations with a precise control over the shape, size, and potentially the geometry. Sprayed multi adsorbed-droplet reposing technology (SMART) consists of the high-pressure extrusion of an ink with a well determined composition using a pneumatic 3D bioprinting approach and flash freezing the extrudate at the printing bed, optionally followed by freeze drying. In the present study, we attempted to manufacture trypsin-loaded chitosan particles using SMART. The ink and products were thoroughly characterized by dynamic light scattering, rheometer, Scanning Electron Microscopy (SEM), and Fourier Transform Infra-Red (FTIR) and Circular Dichroism (CD) spectroscopy. These characterizations confirmed the shape morphology as well as the protein integrity over the process. Further, the effect of various factors on the production were investigated. Our results showed that the concentration of the carrier, chitosan, and the lyoprotectant concentration as well as the extrusion pressure have a significant effect on the particle size. According to CD spectra, SMART ensured Trypsin’s secondary structure remained intact regardless of the ink composition and pressure. However, our study revealed that the presence of 5% (w/v) lyoprotectant is essential to maintain the trypsin’s proteolytic activity. This study demonstrates, for the first time, the viability of SMART as a single-step efficient process to produce biologics-based stable formulations with a precise control over the particulate morphology which can further be expanded across numerous therapeutic modalities including vaccines and cell/gene therapies.