Towards an integrated continuous manufacturing process of adeno- associated virus (AAVs)

Please click Additional Files below to see the full abstract

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

Funding Information: The authors acknowledge the funding of Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), projects PTDC/EQU-EQU/0142/2020 and EXPL/EQU-EQU/1567/2021; SN is the recipient of an FCT fellowship from the project PTDC/EQU-EQU/0142/2020. Publisher Copyright: Copyright © 2023 Neto, Mendes, Santos, Solbrand, Carrondo, Peixoto and Silva.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.publishersversionpublishe

Addressing the challenges of influenza virus-like particles purification

Virus-like particles (VLPs) have been widely used in vaccine development over the last decades [1]. In fact, there are already several approved human vaccines against viruses that use recombinant VLPs as antigen, e.g. for hepatitis B virus and human papillomavirus [2]. Vaccination remains the most effective way to prevent infection with influenza viruses. However, their constant antigenic drift requires an annual update of the seasonal vaccine to prevent influenza epidemics [3-4]. To use the full potential of VLPs as vaccines efficient upstream processing as well as downstream processing (DSP) trains need to be established. The latter is of particular importance as it often accounts for the major biomanufacturing costs. Here we describe the establishment of an improved DSP unit train platform, adapted from virus particles to influenza VLPs, using pseudo-affinity sulfated cellulose membrane adsorbers (SCMA) [5]. An initial clarification step prepares the bulk for the subsequent purification steps. SCMA performance was optimized using a design of experiments (DoE) approach. More than 80% of the product was recovered with removal of host cell protein and DNA above 89% and 80%, respectively. This represents a significant improvement in performance compared to the traditional use of ion exchangers commercially available. Using this SCMA platform for influenza virus particles purification we were able to speed up the process by decreasing the number of DSP steps, to improve the scale-up and to reduce costs due to the removal of other chromatographic steps. References [1] L. Lua, et al., Biotechnology and Bioengineering, 111(3): p. 425-440 (2014). [2] Q. Zhao, et al., Trends in Biotechnology, 31(11): p. 654-663 (2013). [3] D. Smith, et al., Science, 305(5682): p. 371-376 (2004). [4] C. Thompson, et al., Virology Journal, 10 (2013). [5]M. Wolff, and U. Reichl, Expert Review of Vaccines, 10(10): p. 1451-1475 (2011)

Design of a periodic counter-current chromatography process for efficient oncolytic virus purification

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. These biologicals share between them a high degree of complexity and offer various challenges requiring innovative technologies for their manufacturing. Oncolytic virus manufacturing scale can range from 5L in research and development up to 50L for clinical studies and reach hundreds of liters for commercial scale. The inehrent productivity and high integration potential of periodic counter-current chromatography offers a transversal solution to decrease equipment footprint and the reduction of several non-value-added unit operations. The work to be reported focus on the design of a periodic counter-current chromatography process applied to the intermediate purification of oncolytic adenovirus. Moving away from single-column batch operation towards continuous or semi-continuous, multi-column chromatography creates the opportunity to benefit from synergies of solvent gradients, recycling chromatography, and simulated counter-current movement of the adsorbent and fluid phases, providing substantial reductions in chromatographic resin volume and buffer consumption. The developed ion exchange chromatographic purification method was carried out using a four-column setup, supported by mechanistic mathematical modeling. Obtained virus recoveries (\u3e 60%) and impurity reductions (\u3e 80% DNA, and \u3e 70% total protein) match or overcome batch purification. The impact of column cycling on column capacity will be presented and the steps taken to minimize it will be discussed, highlighting the optimization of the cleaning-in-place step and the need to include organic solvents to promote the stripping of tighter-adsorbing impurities. Moreover, the robustness of the dynamic control strategy and its ability to overcome perturbations originated in precedent stages will be demonstrated using feeds with different impurity profiles and titers, showing that it is possible to generate elution pools with consistent quality and traceability. Additionally, due to the wealth of data generated through the cycling operations, such as historic columns breakthrough and elution peak profiles, a deeper insight on product quality and process knowledge is gained. Moreover, process automation enables the minimization of errors, maximizing process efficiency, uptime, repeatability, and process replication

Slowly rotating charged black holes in anti-de Sitter third order Lovelock gravity

In this paper, we study slowly rotating black hole solutions in Lovelock gravity (n=3). These exact slowly rotating black hole solutions are obtained in uncharged and charged cases, respectively. Up to the linear order of the rotating parameter a, the mass, Hawking temperature and entropy of the uncharged black holes get no corrections from rotation. In charged case, we compute magnetic dipole moment and gyromagnetic ratio of the black holes. It is shown that the gyromagnetic ratio keeps invariant after introducing the Gauss-Bonnet and third order Lovelock interactions.Comment: 14 pages, no figur

Combination of searches for Higgs boson pairs in pp collisions at \sqrts = 13 TeV with the ATLAS detector

This letter presents a combination of searches for Higgs boson pair production using up to 36.1 fb(-1) of proton-proton collision data at a centre-of-mass energy root s = 13 TeV recorded with the ATLAS detector at the LHC. The combination is performed using six analyses searching for Higgs boson pairs decaying into the b (b) over barb (b) over bar, b (b) over barW(+)W(-), b (b) over bar tau(+)tau(-), W+W-W+W-, b (b) over bar gamma gamma and W+W-gamma gamma final states. Results are presented for non-resonant and resonant Higgs boson pair production modes. No statistically significant excess in data above the Standard Model predictions is found. The combined observed (expected) limit at 95% confidence level on the non-resonant Higgs boson pair production cross-section is 6.9 (10) times the predicted Standard Model cross-section. Limits are also set on the ratio (kappa(lambda)) of the Higgs boson self-coupling to its Standard Model value. This ratio is constrained at 95% confidence level in observation (expectation) to -5.0 &lt; kappa(lambda) &lt; 12.0 (-5.8 &lt; kappa(lambda) &lt; 12.0). In addition, limits are set on the production of narrow scalar resonances and spin-2 Kaluza-Klein Randall-Sundrum gravitons. Exclusion regions are also provided in the parameter space of the habemus Minimal Supersymmetric Standard Model and the Electroweak Singlet Model. For complete list of authors see http://dx.doi.org/10.1016/j.physletb.2019.135103</p

Searches for lepton-flavour-violating decays of the Higgs boson in s=13\sqrt{s}=13 TeV pp\mathit{pp} collisions with the ATLAS detector

This Letter presents direct searches for lepton flavour violation in Higgs boson decays, H → eτ and H → μτ , performed with the ATLAS detector at the LHC. The searches are based on a data sample of proton–proton collisions at a centre-of-mass energy √s = 13 TeV, corresponding to an integrated luminosity of 36.1 fb−1. No significant excess is observed above the expected background from Standard Model processes. The observed (median expected) 95% confidence-level upper limits on the leptonflavour-violating branching ratios are 0.47% (0.34+0.13−0.10%) and 0.28% (0.37+0.14−0.10%) for H → eτ and H → μτ , respectively.publishedVersio

Search for flavour-changing neutral currents in processes with one top quark and a photon using 81 fb⁻¹ of pp collisions at \sqrts = 13 TeV with the ATLAS experiment

A search for flavour-changing neutral current (FCNC) events via the coupling of a top quark, a photon, and an up or charm quark is presented using 81 fb−1 of proton–proton collision data taken at a centre-of-mass energy of 13 TeV with the ATLAS detector at the LHC. Events with a photon, an electron or muon, a b-tagged jet, and missing transverse momentum are selected. A neural network based on kinematic variables differentiates between events from signal and background processes. The data are consistent with the background-only hypothesis, and limits are set on the strength of the tqγ coupling in an effective field theory. These are also interpreted as 95% CL upper limits on the cross section for FCNC tγ production via a left-handed (right-handed) tuγ coupling of 36 fb (78 fb) and on the branching ratio for t→γu of 2.8×10−5 (6.1×10−5). In addition, they are interpreted as 95% CL upper limits on the cross section for FCNC tγ production via a left-handed (right-handed) tcγ coupling of 40 fb (33 fb) and on the branching ratio for t→γc of 22×10−5 (18×10−5). © 2019 The Author(s

Comparison between simulated and observed LHC beam backgrounds in the ATLAS experiment at Ebeam =4 TeV

Results of dedicated Monte Carlo simulations of beam-induced background (BIB) in the ATLAS experiment at the Large Hadron Collider (LHC) are presented and compared with data recorded in 2012. During normal physics operation this background arises mainly from scattering of the 4 TeV protons on residual gas in the beam pipe. Methods of reconstructing the BIB signals in the ATLAS detector, developed and implemented in the simulation chain based on the \textscFluka Monte Carlo simulation package, are described. The interaction rates are determined from the residual gas pressure distribution in the LHC ring in order to set an absolute scale on the predicted rates of BIB so that they can be compared quantitatively with data. Through these comparisons the origins of the BIB leading to different observables in the ATLAS detectors are analysed. The level of agreement between simulation results and BIB measurements by ATLAS in 2012 demonstrates that a good understanding of the origin of BIB has been reached