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

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Bioprocess intensification for production of a Peste des petits ruminants virus (PPRV) vaccine

Peste des Petites Ruminants Virus (PPRV) is a highly contagious disease affecting small ruminants in Africa and Asian countries, with negative/significant economic impact. Aiming to eradicate the disease, targeted by the Food and Agriculture Organization for 2030, a novel and scalable PPRV vaccine production process is clearly needed. Built upon work previously done at iBET, a new production process is herein proposed using Vero cells growing on microcarriers, serum-free medium (SFM) and stirred-tank bioreactors (STB). This includes a new method for cells detachment from microcarriers, and perfusion culture for reducing turnaround time. The PPRV vaccine production process was developed in the 2L BIOSTAT® DCU-3 and the 20L BIOSTAT® Cplus STB (both from Sartorius) using Nigeria 75/1 strain. Engineering correlations (energy dissipation rate, shear stress and Kolmogorov Eddy size) were used to optimize culture conditions in the 2 L STB and to scale-up the process to the 20 L STB. Vero cells were adapted to grow in ProVeroTM-1 SFM (Sartorius). A new enzymatic and mechanical method for in situ cell detachment from microcarriers was designed. Perfusion was evaluated in the 2 L STB (equipped with internal spin-filter) in order to reduce seed-train preparation time. PPRV were clarified using depth filtration (Sartopure PP3, Sartorius). Process scalability was validated in the 20 L STB. Vero cells were adapted to ProVeroTM-1 SFM, reaching growth rates similar to serum-containing cultures (0.03 h-1). The new in situ cell detachment method was successfully implemented, with yields above 80%. A two-fold increase in maximum cell concentration was obtained using perfusion when compared to batch culture. Combining perfusion with the new in situ cell detachment method enabled the scale-up to 20 L STB directly from a 2 L STB, surpassing the need for a mid-scale platform and thus reducing seed-train preparation time. Infectious PPRV titers increase over culture time in both 2 L and 20 L STBs, reaching maximum values of 4.5-4.9x106 TCID50/mL at day 4-5 post-infection. The potential of depth filtration for PPRV clarification was confirmed; comparable PPRV recovery yields after clarification (85-90%) were obtained in both STBs. Overall, the novel and scalable vaccine production process herein proposed has the potential to assist the upcoming PPR Global Eradication Program (PPR GEP), to which iBET already contributes as partner in the PPR Global Research and Experts Network (PPR GREN), and thus support the One Health concept. Acknowledgments: This work was supported by Sartorius Stedim Biotech GmbH (Germany)

Pseudotyping retrovirus like particles vaccine candidates with Hepatitis C virus envelope protein E2 requires the cellular expression of CD81

Hepatitis C virus (HCV) infects 3% of world population being responsible for nearly half a million deaths annually urging the need for a prophylactic vaccine. Retrovirus like particles are commonly used scaffolds for antigens presentation being the core of diverse vaccine candidates. The immunogenicity of host proteins naturally incorporated in retrovirus was hypothesized to impact the performance of retrovirus based vaccines. In this work, the capacity of engineered retrovirus like particles devoided of host protein CD81 to display HCV envelope antigens was compared to non-engineered particles. A persistent inability of CD81 negative VLPs to incorporate HCV E2 protein as a result from the inefficient transport of HCV E2 to the plasma membrane, was observed. This work enabled the identification of a CD81-mediated transport of HCV E2 while stressing the importance of host proteins for the development of recombinant vaccines.publishe

Impact of Viral Protease Activity in the Production of LV Pseudotypes

Lentiviral vectors (LVs) are excellent tools for gene transfer into mammalian cells. It is noteworthy that the first gene therapy treatment using LVs was approved for commercialization in 2017. The G glycoprotein from rhabdovirus vesicular stomatitis virus (VSV-G) is the glycoprotein most used to pseudotype LVs, due to its high efficiency in transducing several cell types and its resistance to viral vector purification and storage conditions. However, VSV-G expression induces cytotoxicity, which limits LV production to short periods. As alternative to VSV-G, γ-retrovirus glycoproteins (4070A derived, GaLV derived, and RD114 derived) have been used to pseudotype both γ-retroviral vectors (RVs) and LVs. These glycoproteins do not induce cytotoxicity, allowing the development of stable LV producer cells. Additionally, these LV pseudotypes present higher transduction efficiencies of hematopoietic stem cells when compared to VSV-G. Here, new 4070A-, RD114-TR-, and GaLV-TR-derived glycoproteins were developed with the aim of improving its cytoplasmic tail R-peptide cleavage and thus increase LV infectious titers. The new glycoproteins were tested in transient LV production using the wild-type or the less active T26S HIV-1 protease. The GaLV-TR-derived glycoproteins were able to overcome titer differences observed between LV production using wild-type and T26S protease. Additionally, these glycoproteins were even able to increase LV titers, evidencing its potential as an alternative glycoprotein to pseudotype LVs.publishe

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

Improving Influenza HA-Vlps Production in Insect High Five Cells via Adaptive Laboratory Evolution

The use of non-standard culture conditions has proven efficient to increase cell performance and recombinant protein production in different cell hosts. However, the establishment of high-producing cell populations through adaptive laboratory evolution (ALE) has been poorly explored, in particular for insect cells. In this study, insect High Five cells were successfully adapted to grow at a neutral culture pH (7.0) through ALE for an improved production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs). A stepwise approach was used for the adaptation process, in which the culture pH gradually increased from standard 6.2 to 7.0 (ΔPh = 0.2–0.3), and cells were maintained at each pH value for 2–3 weeks until a constant growth rate and a cell viability over 95% were observed. These adapted cells enabled an increase in cell-specific HA productivity up to three-fold and volumetric HA titer of up to four-fold as compared to non-adapted cells. Of note, the adaptation process is the element driving increased specific HA productivity as a pH shift alone was inefficient at improving productivities. The production of HA-VLPs in adapted cells was successfully demonstrated at the bioreactor scale. The produced HA-VLPs show the typical size and morphology of influenza VLPs, thus confirming the null impact of the adaptation process and neutral culture pH on the quality of HA-VLPs produced. This work strengthens the potential of ALE as a bioprocess engineering strategy to improve the production of influenza HA-VLPs in insect High Five cells

Viruses and virus-like particles in biotechnology: Fundamentals and applications

Although viruses are simple biological systems, they are capable of evolving highly efficient techniques for infecting cells, expressing their genomes, and generating new copies of themselves. It is possible to genetically manipulate most of the different classes of known viruses in order to produce recombinant viruses that express foreign proteins. Recombinant viruses have been used in gene therapy to deliver selected genes into higher organisms, in vaccinology and immunotherapy, and as important research tools to study the structure and function of these proteins. Virus-like particles (VLPs) are multiprotein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome. They have been applied not only as prophylactic and therapeutic vaccines but also as vehicles in drug and gene delivery and, more recently, as tools in nanobiotechnology. In this chapter, basic and advanced features of viruses and VLPs are presented and their major applications are discussed. The different production platforms based on animal cell technology are explained, and their main challenges and future perspectives are explored. The implications of large-scale production of viruses and VLPs are discussed in the context of process control, monitoring, and optimization. The main upstream and downstream technical challenges are identified and discussed accordingly