Considerations of manufacturability for AAV based gene therapy products for rare diseases

Passage Bio is developing a robust and efficient manufacturing platform to streamline the development and technology transfer of preclinical and clinical candidates to external manufacturing partners. In this presentation, we highlight Passage Bio’s approach to manufacturability for AAV vectors by advancements in cell line development, optimizing process conditions, implementation of next generation analytics, and viral clearance strategy to ensure a safe and efficacious final product. It is our goal that this work will result in a state-of-the-art process for AAV production to facilitate rapid transition toward pre-commercial development and build out a robust gene therapy pipeline

Next generation manufacturing for biologics: Integration of a hybrid model for continuous manufacturing concepts into a clinical facility

The “one size fits all” concept is rarely applicable in life, this is also true for the concept of continuous manufacturing where specific applications will differ based upon the requirements of the end user. This is the scenario we describe here in which aspects of continuous manufacturing for both upstream and downstream biologics manufacturing are being incorporated to address the current pipeline needs within Bristol-Myers Squibb. The application is for stable, easily expressed monoclonal antibody processes that require moderate volumes and throughputs, such as for most oncology or immuno-oncology therapies. However, this is countered by challenges of an expanding pipeline that necessitates looking beyond the current platform philosophy for how to modify the process with the goal to increase overall productivity in a flexible manner. BMS recently constructed a clinical biologics manufacturing facility on the Devens, Massachusetts campus with operations being initiated in two phases. The first phase start-up aligned with a traditional, but flexible (i.e., based upon disposable technologies) upstream and downstream processes and was rapidly brought on line. The second phase is the design and construction within that same manufacturing building purposely left unfinished to allow for the process development group to design and demonstrate a next generation concept for manufacturing. With respect to the upstream process, the decision was made to maintain a fed-batch production bioreactor philosophy, but to employ much higher inoculation densities through use of perfusion culture at the seed bioreactor stage generating the inoculum. This results in cultures with shorter durations and opportunities for increased titer. Selection of the overall cycle time is an optimization between cadence and bioreactor throughput. With respect to the downstream processes, numerous continuous manufacturing technologies were evaluated to handle the increased titers being generated in the bioreactors. These downstream technologies include continuous harvest technologies, multicolumn continuous chromatography for capture, integrated pool-less polishing steps, automated viral inactivation, single pass TFF and in-line diafiltration. The advantages for manufacturing cadence and overall throughput, as well as other outcomes including efforts to decrease perfusion media usage, and a significant reduction in downstream resin costs will be presented. Once the second phase is implemented, the facility will accommodate both traditional as well as this hybrid model for continuous manufacturing interchangeably. The overall benefit to support multiple clinical products and the higher titer/throughputs are expected to reduce the number of batches as well as eliminate resupply batches for clinical supply

Thermodynamic Adsorption Studies of Peptides on Well-defined, Surface-Confined Polymers with Applications in Membrane Bioseparations

This dissertation deals with the fundamental studies of peptide adsorption on surfaces modified by surface-confined polymer films, with an application emphasis on membrane bioseparations. In order to understand protein adsorption at a fundamental level, it is important to study the specific residue-level interactions with surfaces. Keeping this idea in view, the present work describes experimental measurements of submolecular-level interaction energies involved in the process of peptide adsorption on polymer films using surface plasmon resonance spectroscopy. Gibbs energy change on adsorption (ΔGad) for tyrosine, phenylalanine, and glycine homopeptides were measured at 25 °C and pH 7 on highly uniform, nanothin polymer films, and the results were used to predict ΔGad for homologous homopeptides with a larger number of residue units. Nanothin poly(2-vinylpyridine), poly(styrene) and poly(1-benzyl-2-vinylpyridinium bromide) films were used for the adsorption studies; they were prepared using a graft polymerization methodology. In-situ swelling experiments were done with ellipsometry to examine the uniformity of the surfaces and to ensure that the graft densities of the different polymer films were similar to facilitate the comparison of adsorption results on these surfaces. To extend this approach to a mixed-residue peptide, measurements were made for glycine, phenylalanine, and tyrosine-leucine subunits found in leucine enkephalin. It was found that combining ΔGads values for adsorption of the individual peptide units in a short- chain peptide allows us to predict its overall ΔGads value with reasonable estimates. Calculations for uncharged surfaces (poly(2-vinylpyridine) and poly(styrene)) gave estimates deviating by no more than |9%| from experiment. Deviations between measured and predicted adsorption energies were larger for the charged poly(1-benzyl-2-vinylpyridinium bromide) surface relative to uncharged surfaces, and, generally speaking, measured uncertainty values were slightly larger for the charged surface. Nevertheless, the adsorption energies were found to be additive within experimental uncertainties for the charged surface as well. One of the central parts of my dissertation is the fabrication of uniform polymer nanolayers with independent control of the layer thickness and chain grafting density using surface-initiated polymerization. Surface-tethered polymer brushes with independently variable grafting densities and layer thicknesses were fabricated for peptide adsorption and cell-adhesion studies. Surface-initiated atom transfer radical polymerization (ATRP) was used together with thiol self-assembly to generate these nanothin polymer brush layers of poly((polyethylene glycol) methacrylate). A kinetic study was done to measure the layer thickness growth rate at room temperature from flat gold substrates presenting different polymerization initiator molecule surface densities. The polymer brush layers transition from mushroom to brush regimes with increasing grafting density. The results showed that layer properties such as wettability and dry layer thickness depend strongly on initiator surface density. Ultimately, the interaction energy of an RGD-containing synthetic peptide Gly-Arg-Gly-Asp-Ser and the adhesion and spreading of cells were correlated with surface properties, which continues to be a major research theme in biomedical and biomaterials research. Finally, the work was extended to the surface modification of polymeric membranes to tune the physical and chemical properties of the membranes. I describe a methodology to surface modify commercially available membranes with various functionalities to prepare ion-exchange membranes using graft polymerization from the surfaces of the membranes. ATRP was used to modify the membranes with pyridinium exchange groups and carboxylic acid groups. Polymerization time was used as the independent variable to manipulate the amount of grafted polymer on the membrane surface. ATRP was used to make adsorptive (ion-exchange) membranes with among the highest static and dynamic protein binding capacities, and in a way that allowed us to control the impact on membrane permeability. Confocal laser scanning microscopy was used to visualize membrane pore structure of the unmodified and modified membranes to prove that modification by ATRP did not impact the membrane pore structure detrimentally and also to visualize binding of fluorescently labeled lysozyme

Challenges in downstream purification of gene therapy viral vectors

The diversity and application of viral products has continued to explode. These modalities can be vaccines, cancer therapies, and gene therapies. However, their diversity is also the challenge in the downstream processing. Viral particles can be very labile, thus requiring intimate knowledge of biology to create environments where they are stable. However, despite these challenges, we have created many processes that produce large amounts of viral products. Different purification methods are utilized throughout the process. New modalities of chromatography are overcoming many of the challenges of diffusion-limited beads. Of special concern for gene therapy vectors is the need to separate the empty capsids from the full capsids, which contain the therapeutic gene of interest. With the discovery of novel therapuetic modalities that could revolutionize care by finding cures, the downstream processing of viral therapies needs to find solutions to make these therapies and cures affordable

Energy generation from water flow over a reduced graphene oxide surface in a paper–pencil device

Energy generation using liquid movement over a graphene surface generally demands a very high rate of flow (e.g. ∼100 ml min−1). In addition, a continuous flow of liquid is unable to generate a desired voltage, as it needs modification of the substrate such as development of nanopores and criss-cross network structures. Here, we report an ultra-low-cost yet highly efficient portable device for energy conversion, by exploiting the capillary flow of an electrolyte on a filter paper matrix in which a naturally deposited gradient of reduced graphene oxide is induced through chemical synthesis. In addition, the fibres and pores present in the paper offer a criss-cross network, acting as a natural splitter of a continuous flow into tiny droplets. Our methodology thus obviates the need for any elaborate procedure for pre-generation of droplets. Further, we fabricate the necessary electrodes on filter paper by simply scribing pencil tips on the same filter paper, which facilitates the necessary electrochemical reactions. Effectively, at the anode, electrons are released, which travel through the outer circuit for cation reduction at the cathode and deliver an electrical output (voltage/current), realizing the conversion of the chemical form of energy to the electrical form in the filter paper. An absorbent pad at the channel outlet ensures continuous flow of fresh solution in the device, resulting in an inexpensive platform for power generation over a prolonged period of time. A maximum current density of 325 mA cm−2 and a maximum power density of 53 mW cm−2 have been observed, which significantly outweigh the capabilities of other reported devices fabricated for similar purposes

Novel synthesis of a mixed Cu/CuO–reduced graphene oxide nanocomposite with enhanced peroxidase-like catalytic activity for easy detection of glutathione in solution and using a paper strip

A reduced graphene oxide (rGO) based mixed copper nanocomposite, Cu/CuO–rGO is prepared through a novel synthetic approach: a simple one-step oxidation–reduction reaction between aqueous graphene oxide (GO) and copper(II) chloride (CuCl2) solutions at ambient temperature and pressure. The nanocomposite shows enhanced peroxidase-like catalytic activity by rapidly catalyzing a TMB (3,3′,5,5′-tetramethyl benzidine)–H2O2 reaction that develops a visible blue color in solution due to the oxidation of TMB. The catalyst follows a Michaelis–Menten reaction mechanism and exhibits strong affinity towards both H2O2 and TMB. The blue color developed by the Cu/CuO–rGO–TMB–H2O2 system becomes colorless in solution when glutathione is present even at a very low concentration (0.032 μM). This distinct color change provides the basis of the present colorimetric method for highly sensitive and selective detection of GSH in solution as well as on a paper-strip within a <5 min time period. The use of Cu/CuO–rGO as an enzyme-like catalyst in TMB–H2O2 mediated GSH sensing process shows the benefits of simplicity, cost-effectiveness and provides an alternative non-enzymatic way of glutathione estimation in real samples such as commercially available tablets and human blood plasma

Selection of Graphite Pencil Grades for the Design of Suitable Electrodes for Stacking Multiple Single‐Inlet Paper‐Pencil Fuel Cell

We present a single‐inlet paper‐pencil based microfluidic fuel cell which works on the capillary driven fluid flow of a mixture of fuel (formic acid) and electrolyte (dilute sulfuric acid) solutions as a single entity. Different types of pencils are used to prepare the graphite electrodes to observe their effect on the fuel cell performance. The graphite electrodes are porous and act as a gas diffusion electrode which breaths in oxygen directly from the quiescent air and out CO2 from the cell. The paper fuel cell also fabricated in different dimensions and connected as cell‐stacks to enhance its performance. The paper‐pencil device generates maximum open circuit potential of ∼2.5 V in series combination and maximum current density of ∼ 82 mA cm−2 and power density of ∼ 100 mW cm−2 in parallel combination. Later, rGO−Cu/CuO nanocomposite embedded electrodes are used to observe their effect on the device performance

A spiral shaped regenerative microfluidic fuel cell with Ni‐C based porous electrodes

Microchannel geometry, electrode surface area, and better fuel utilization are important aspects of the performance of a microfluidic fuel cell (MFC). In this communication, a membraneless spiral‐shaped MFC fabricated with Ni as anode and C as a cathode supported over a porous filter paper substrate is presented. Vanadium oxychloride and dilute sulfuric acid solutions are used as fuel and electrolyte, respectively, in this fuel cell system. The device generates a maximum open‐circuit voltage of ~1.2 V, while the maximum energy density and current density generated from the fuel cell are ~10 mW cm−2 and ~51 mA cm−2, respectively. The cumulative energy density generated from the device after five cycles are measured as ~200 mW after regeneration of the fuel by applying external voltage. The spiral design of the fuel cell enables improved fuel utilization, rapid diffusive transport of ions, and in‐situ regeneration of the fuel. The present self‐standing spiral‐shaped MFC will eliminate the challenges associated with two inlet membrane‐less fuel cells and has the potential to scale up for commercial application in portable energy generation

Water desalination using graphene oxide-embedded paper microfluidics

The need for the removal of salt constituents is very critical in several downstream processes of biological materials and saltwater purification. Substantial efforts to drive low cost-effective techniques for desalination are ongoing, and it is hopeful that novel nanomaterials could provide useful insight to a new paradigm in salt capturing both in biogenic fluids and complex solutions like seawater. In this report, we demonstrate a microfluidic proof-of-concept for a desalination system, in which graphene oxide deposited on the paper substrate was used to remove salt-ion concentration. Our investigation suggests that the optimal modification of paper with the five-time deposition of graphene oxide (paper@5GO) shows the best salt removal performance with the salt-rejection efficiency of ~ 97.0%. The salt rejection occurs by the phenomenon of surface adsorption on the GO-modified paper membrane which is confirmed by the detailed analytical studies of pre- and post-treatment. The system presented does not require additional energy input in the process and thus would become cost-effective and scalable with high salt removal efficiency which may be useful in bioanalysis and saltwater purification for sustainable development