Optical pH Sensor Based on a Long-Period Fiber Grating Coated with a Polymeric Layer-by-Layer Electrostatic Self-Assembled Nanofilm.

An optical fiber pH sensor based on a long-period fiber grating (LPFG) is reported. Two oppositely charged polymers, polyethylenimine (PEI) and polyacrylic acid (PAA), were alternately deposited on the sensing structure through a layer-by-layer (LbL) electrostatic self-assembly technique. Since the polymers are pH sensitive, their refractive index (RI) varies when the pH of the solution changes due to swelling/deswelling phenomena. The fabricated multilayer coating retained a similar property, enabling its use in pH-sensing applications. The pH of the PAA dipping solution was tuned so that a coated LPFG achieved a pH sensitivity of (6.3 ± 0.2) nm/pH in the 5.92-9.23 pH range. Only two bilayers of PEI/PAA were used as an overlay, which reduces the fabrication time and increases the reproducibility of the sensor, and its reversibility and repeatability were demonstrated by tracking the resonance band position throughout multiple cycles between different pH solutions. With simulation work and experimental results from a low-finesse Fabry-Perot (FP) cavity on a fiber tip, the coating properties were estimated. When saturated at low pH, it has a thickness of 200 nm and 1.53 ± 0.01 RI, expanding up to 310 nm with a 1.35 ± 0.01 RI at higher pH values, mostly due to the structural changes in the PAA.

Extending AAV Packaging Cargo through Dual Co-Transduction: Efficient Protein Trans-Splicing at Low Vector Doses.

Adeno-associated viral (AAV) vectors represent one of the leading platforms for gene delivery. Nevertheless, their small packaging capacity restricts their use for diseases requiring large-gene delivery. To overcome this, dual-AAV vector systems that rely on protein trans-splicing were developed, with the split-intein Npu DnaE among the most-used. However, the reconstitution efficiency of Npu DnaE is still insufficient, requiring higher vector doses. In this work, two split-inteins, Cfa and Gp41-1, with reportedly superior trans-splicing were evaluated in comparison with Npu DnaE by transient transfections and dual-AAV in vitro co-transductions. Both Cfa and Gp41-1 split-inteins enabled reconstitution rates that were over two-fold higher than Npu DnaE and 100% of protein reconstitution. The impact of different vector preparation qualities in split-intein performances was also evaluated in co-transduction assays. Higher-quality preparations increased split-inteins' performances by three-fold when compared to low-quality preparations (60-75% vs. 20-30% full particles, respectively). Low-quality vector preparations were observed to limit split-gene reconstitutions by inhibiting co-transduction. We show that combining superior split-inteins with higher-quality vector preparations allowed vector doses to be decreased while maintaining high trans-splicing rates. These results show the potential of more-efficient protein-trans-splicing strategies in dual-AAV vector co-transduction, allowing the extension of its use to the delivery of larger therapeutic genes.

Efficient adeno-associated virus serotype 5 capture with affinity functionalized nanofiber adsorbents.

Adeno-associated viruses (AAVs) are one of the most promising tools for gene therapy applications. These vectors are purified using affinity and ion exchange chromatography, typically using packed beds of resin adsorbents. This leads to diffusion and pressure drop limitations that affect process productivity. Due to their high surface area and porosity, electrospun nanofiber adsorbents offer mass transfer and flow rate advantages over conventional chromatographic media. The present work investigated the use of affinity cellulose-based nanofiber adsorbents for adeno-associated virus serotype 5 (AAV5) capture, evaluating dynamic binding capacity, pressure drop, and AAV5 recovery at residence times (RT) less than 5 s. The dynamic binding capacity was found to be residence time-dependent, but nevertheless higher than 1.0 × 1014 TP mL-1 (RT = 1.6 s), with a pressure drop variation of 0.14 MPa obtained after loading more than 2,000 column volumes of clarified AAV5 feedstock. The single affinity chromatography purification step using these new affinity adsorbents resulted in 80% virus recovery, with the removal of impurities comparable to that of bead-based affinity adsorbents. The high binding capacity, virus recovery and reduced pressure drop observed at residence times in the sub-minute range can potentially eliminate the need for prior concentration steps, thereby reducing the overall number of unit operations, process time and costs.

Spectral Analysis Methods for Improved Resolution and Sensitivity: Enhancing SPR and LSPR Optical Fiber Sensing.

Biochemical-chemical sensing with plasmonic sensors is widely performed by tracking the responses of surface plasmonic resonance peaks to changes in the medium. Interestingly, consistent sensitivity and resolution improvements have been demonstrated for gold nanoparticles by analyzing other spectral features, such as spectral inflection points or peak curvatures. Nevertheless, such studies were only conducted on planar platforms and were restricted to gold nanoparticles. In this work, such methodologies are explored and expanded to plasmonic optical fibers. Thus, we study-experimentally and theoretically-the optical responses of optical fiber-doped gold or silver nanospheres and optical fibers coated with continuous gold or silver thin films. Both experimental and numerical results are analyzed with differentiation methods, using total variation regularization to effectively minimize noise amplification propagation. Consistent resolution improvements of up to 2.2× for both types of plasmonic fibers are found, demonstrating that deploying such analysis with any plasmonic optical fiber sensors can lead to sensing resolution improvements.

AAV process intensification by perfusion bioreaction and integrated clarification.

Adeno-associated viruses (AAVs) demand for clinical trials and approved therapeutic applications is increasing due to this vector's overall success and potential. The high doses associated with administration strategies challenges bioprocess engineers to develop more efficient technologies and innovative strategies capable of increasing volumetric productivity. In this study, alternating tangential flow (ATF) and Tangential Flow Depth filtration (TFDF) techniques were compared as to their potential for 1) implementing a high-cell-density perfusion process to produce AAV8 using mammalian HEK293 cells and transient transfection, and 2) integrating AAV harvest and clarification units into a single step. On the first topic, the results obtained demonstrate that AAV expression improves with a medium exchange strategy. This was evidenced firstly in the small-scale perfusion-mocking study and later verified in the 2 L bioreactor operated in perfusion mode. Fine-tuning the shear rate in ATF and TFDF proved instrumental in maintaining high cell viabilities and, most importantly, enhancing AAV-specific titers (7.6 × 104 VG/cell), i.e., up to 4-fold compared to non-optimized perfusion cultures and 2-fold compared with batch operation mode. Regarding the second objective, TFDF enabled the highest recovery yields during perfusion-based continuous harvest of extracellular virus and lysate clarification. This study demonstrates that ATF and TFDF techniques have the potential to support the production and continuous harvest of AAV, and enable an integrated clarification procedure, contributing to the simplification of operations and improving manufacturing efficiency.

Continuous Affinity Purification of Adeno-Associated Virus Using Periodic Counter-Current Chromatography.

Replacing batch unit operations of biopharmaceuticals by continuous manufacturing is a maturing concept, with periodic counter-current chromatography (PCC) favoured to replace batch chromatography. Continuous affinity capture of adeno-associated virus (AAV) using PCC has the potential to cope with the high doses required for AAV therapies thanks to its inherent high throughput. The implementation of continuous AAV affinity capture using a four-column PCC process is described herein. First, elution buffer screening was used to optimize virus recovery. Second, breakthrough curves were generated and described using a mechanistic model, which was later used to characterize the loading zone of the PCC. The experimental runs achieved a stable cyclic steady state yielding virus recoveries in line with the optimized batch process (>82%), with almost a three-fold improvement in productivity. The PCC affinity capture process developed here can bolster further improvements to process economics and manufacturing footprint, thereby contributing to the integrated continuous manufacturing concept.

Differential Refractometric Biosensor for Reliable Human IgG Detection: Proof of Concept.

A new sensing platform based on long-period fiber gratings (LPFGs) for direct, fast, and selective detection of human immunoglobulin G (IgG; Mw = 150 KDa) was developed and characterized. The transducer's high selectivity is based on the specific interaction of a molecularly imprinted polymer (MIPs) design for IgG detection. The sensing scheme is based on differential refractometric measurements, including a correction system based on a non-imprinted polymer (NIP)-coated LPFG, allowing reliable and more sensitive measurements, improving the rejection of false positives in around 30%. The molecular imprinted binding sites were performed on the surface of a LPFG with a sensitivity of about 130 nm/RIU and a FOM of 16 RIU-1. The low-cost and easy to build device was tested in a working range from 1 to 100 nmol/L, revealing a limit of detection (LOD) and a sensitivity of 0.25 nmol/L (0.037 µg/mL) and 0.057 nm.L/nmol, respectively. The sensor also successfully differentiates the target analyte from the other abundant elements that are present in the human blood plasma.

Analysis of the Relative Humidity Response of Hydrophilic Polymers for Optical Fiber Sensing.

Relative humidity (RH) monitorization is of extreme importance on scientific and industrial applications, and optical fiber sensors (OFS) may provide adequate solutions. Typically, these kinds of sensors depend on the usage of humidity responsive polymers, thus creating the need for the characterization of the optical and expansion properties of these materials. Four different polymers, namely poly(vinyl alcohol), poly(ethylene glycol), Hydromed™ D4 and microbiology agar were characterized and tested using two types of optical sensors. First, optical fiber Fabry-Perot (FP) tips were made, which allow the dynamical measurement of the polymers' response to RH variations, in particular of refractive index, film thickness, and critical deliquescence RH. Using both FP tips and Long-Period fiber gratings, the polymers were then tested as RH sensors, allowing a comparison between the different polymers and the different OFS. For the case of the FP sensors, the PEG tips displayed excellent sensitivity above 80%RH, outperforming the other polymers. In the case of LPFGs, the 10% (wt/wt) PVA one displayed excellent sensitivity in a larger working range (60 to 100%RH), showing a valid alternative to lower RH environment sensing.

Oncolytic virus purification with periodic counter-current chromatography.

Virus-based biologicals are one of the most promising biopharmaceuticals of the 21st century medicine and play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. Oncolytic virus manufacturing scale can range from 5 L in research and development up to 50 L for clinical studies and reach hundreds of liters for commercial scale. The inherent productivity and high integration potential of periodic counter-current chromatography (PCC) offer a transversal solution to decrease equipment footprint and the reduction of several non-value-added unit operations. We report on the design of an efficient PCC process applied to the intermediate purification of oncolytic adenovirus. The developed ion-exchange chromatographic purification method was carried out using a four-column setup for three different scenarios: (i) variation in the feedstock, (ii) potential use of a post-load washing step to improve virus recovery, and (iii) stability during extended operation. Obtained virus recoveries (57%-86%) and impurity reductions (>80% DNA, and >70% total protein) match or overcome batch purification. Regarding process stability and automation, our results show that not only the dynamic control strategy used is able to suppress perturbations in the sample inlet but also allows for unattended operation in the case of ion exchange capture.

Continuous Chromatography Purification of Virus-Based Biopharmaceuticals: A Shortcut Design Method.

Novel biopharmaceutical products, such as vaccines and viral vectors, play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. However, several challenges are posed when manufacturing these products. The diversity of cell lines and the different physical and chemical properties of these biologicals require the use of different production and processing technologies. Alternative purification strategies that can improve the purification yield, such as continuous chromatography, are regarded nowadays as enabling technologies to overcome some of the bottlenecks in biomanufacturing. This chapter offers a shortcut approach to implement a semi-continuous chromatography purification of hepatitis C virus-like particles produced in insect cells with recombinant baculovirus. Although the purification is based on ion exchange chromatography, the present methodology can be extended to other types of chromatography.

Comparative biochemical profiles, production and reproduction status of the post-partum dairy cows with and without purulent vaginal discharge.

Purulent vaginal discharge (PVD) is a prevalent uterine disease of dairy cows during the puerperium that affects the milk production and affects the profitability of farms. The objectives of this study were to evaluate the biochemical profile, the body condition score, the milk production of cows with PVD and the effects PVD on reproductive performance. A total of 338 Holstein dairy cows aged from 3 to 5 years, from three commercial dairy farms, from Brazil, were used. Blood samples were collected within 25 ± 3 days post-partum from Holstein dairy cows without PVD (control cows, n = 242) and cows with PVD (n = 96), based on scoring of the vaginal discharge. The body condition score and milk production were recorded on the day of sampling. The biochemical profile encompassed albumin, urea, gamma-glutamyl transferase, calcium, fibrinogen and cholesterol concentrations. The number of services per pregnancy was lower (p < 0.01), and the number of days until first insemination and the median time to pregnancy were higher in cows with PVD (p < 0.01) when compared with control cows. Milk production and body condition score were lower (p < 0.01) in cows with PVD than in control group. Cows with PVD had lower (p < 0.05) serum albumin, urea, calcium and cholesterol concentrations, and higher serum gamma-glutamyl transferase activity and fibrinogen concentration than cows without PVD. Our results show that cows with PVD have changes in the biochemical profile and negative effects on production and reproduction performance.