Pharmaceutical manufacturing typically uses batch processing at multiple locations. Disadvantages of this approach include long production times and the potential for supply chain disruptions. As a preliminary demonstration of an alternative approach, we report here the continuous-flow synthesis and formulation of active pharmaceutical ingredients in a compact, reconfigurable manufacturing platform. Continuous end-to-end synthesis in the refrigerator-sized [1.0 meter (width) × 0.7 meter (length) × 1.8 meter (height)] system produces sufficient quantities per day to supply hundreds to thousands of oral or topical liquid doses of diphenhydramine hydrochloride, lidocaine hydrochloride, diazepam, and fluoxetine hydrochloride that meet U.S. Pharmacopeia standards. Underlying this flexible plug-and-play approach are substantial enabling advances in continuous-flow synthesis, complex multistep sequence telescoping, reaction engineering equipment, and real-time formulation.

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations, including their reliance on exogenous materials or electrical fields, which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30-80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material, such as carbon nanotubes, proteins, and siRNA, to 11 cell types, including embryonic stem cells and immune cells. When used for the delivery of transcription factors, the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed, its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications.drug delivery | induced pluripotent stem cells | reprogramming | protein delivery | nanoparticle delivery I ntracellular delivery of macromolecules is a critical step in therapeutic and research applications. Nanoparticle-mediated delivery of DNA and RNA, for example, is being explored for gene therapy (1, 2), while protein delivery is a promising means of affecting cellular function in both clinical (3) and laboratory (4) settings. Other materials, such as small molecules, quantum dots, or gold nanoparticles, are of interest for cancer therapies (5, 6), intracellular labeling (7,8), and single-molecule tracking (9).The cell membrane is largely impermeable to macromolecules. Many existing techniques use polymeric nanoparticles (10, 11), liposomes (12), or chemical modifications of the target molecule (13), such as cell-penetrating peptides (CPPs) (14, 15), to facilitate membrane poration or endocytotic delivery. In these methods, the delivery vehicle's efficacy is often dependent on the structure of the target molecule and the cell type. These methods are thus efficient in the delivery of structurally uniform materials, such as nucleic acids, but often ill-suited for the delivery of more structurally diverse materials, such as proteins (16,17) and some nanomaterials (7). Moreover, the endosome escape mechanism that most of these methods rely on is often inefficient; hence, much material remains trapped in endosomal and lysosomal vesicles (18). More effective gene delivery methods, such as viral vectors (19,20), however, often risk chromosomal integration and are limited to DNA and RNA delivery.Membrane poration methods, such as electroporation (21, 22) and sonoporation (23), are an attractive alternative in some applications. Indeed, electroporation has demonstrated its efficacy in a number of DNA (24) and ...

Chemical synthesis generally requires labor-intensive, sometimes tedious trial-and-error optimization of reaction conditions. Here, we describe a plug-and-play, continuous-flow chemical synthesis system that mitigates this challenge with an integrated combination of hardware, software, and analytics. The system software controls the user-selected reagents and unit operations (reactors and separators), processes reaction analytics (high-performance liquid chromatography, mass spectrometry, vibrational spectroscopy), and conducts automated optimizations. The capabilities of this system are demonstrated in high-yielding implementations of C-C and C-N cross-coupling, olefination, reductive amination, nucleophilic aromatic substitution (SNAr), photoredox catalysis, and a multistep sequence. The graphical user interface enables users to initiate optimizations, monitor progress remotely, and analyze results. Subsequent users of an optimized procedure need only download an electronic file, comparable to a smartphone application, to implement the protocol on their own apparatus.

Here we report a fully automated, flow-based approach to solid-phase polypeptide synthesis, with amide bond formation in 7 seconds and total synthesis times of 40 seconds per amino acid residue. Crude peptide purities and isolated yields were comparable to those for standard-batch solid-phase peptide synthesis. At full capacity, this approach can yield tens of thousands of individual 30-mer peptides per year.

We describe the development and application of an improved, membrane-based, liquid-liquid separator. Membrane based separation relies on the exploitation of surface forces and the use of a membrane wetted by one of the phases; however, successful separation requires accurate control of pressures making the operation and implementation cumbersome. Here we present an improved separator design that integrates a pressure control element to ensure that adequate operating conditions are always maintained. Additionally, the integrated pressure control decouples the separator from downstream unit operations. A detailed examination of the controlling physical equations shows how to design the device to allow operation across a wide range of conditions. Easy to implement, multistage separations such as solvent swaps and countercurrent extractions are demonstrated. The presented design significantly simplifies applications ranging from multistep synthesis to complex multistage separations.

Mechanical properties of cells have been shown to have a significant role in disease as in many instances cell stiffness changes when a cell is no longer healthy. We present a high throughput microfluidics based approach that exploits the connection between travel time of a cell through a narrow passage and cell stiffness. The system resolves both cell travel time and relative cell diameter while retaining information on the cell level. We show that stiffer cells have longer transit times than less stiff ones and that cell size significantly influences travel times. Experiments with untreated HeLa cells and cells made compliant with Latrunculin A and Cytochalasin B further demonstrate that travel time is influenced by cell stiffness, with the compliant cells having faster transit time.

A flow-based solid phase peptide synthesis methodology that enables the incorporation of an amino acid residue every 1.8 minutes under automatic control, or every three minutes under manual control, is described. This is accomplished by passing a stream of reagent through a heat exchanger, into a low volume, low backpressure reaction vessel, and through a UV detector. These features enable the continuous delivery of heated solvents and reagents to the solid support at high flow rate, maintaining a maximal concentration of reagents in the reaction vessel, quickly exchanging reagents, and eliminating the need to rapidly heat reagents after they have been added to the vessel. The UV detector enables continuous monitoring of the process. To demonstrate the broad applicability and reliability of this method, it was employed in the total synthesis of a small protein, as well as dozens of peptides. The quality of the material obtained with this method is comparable to traditional batch methods, and, in all cases, the desired material was readily purifiable via RP-HPLC. The application of this method to the synthesis of the 113 residue B. amyloliquefaciens RNase and the 130 residue pE59 DARPin is described in the accompanying manuscript.

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