Auto-transduction in lentiviral vector bioprocessing: A quantitative assessment and a novel inhibition strategy.

Lentiviral vectors are highly efficient gene delivery vehicles used extensively in the rapidly growing field of cell and gene therapy. Demand for efficient, large-scale, lentiviral vector bioprocessing is growing as more therapies reach late-stage clinical trials and are commercialized. However, despite substantial progress, several process inefficiencies remain. The unintended auto-transduction of viral vector-producing cells by newly synthesized lentiviral vector particles during manufacturing processes constitutes one such inefficiency which remains largely unaddressed. In this study, we determined that over 60% of functional lentiviral vector particles produced during an upstream production process were lost to auto-transduction, highlighting a major process inefficiency likely widespread within the industry. Auto-transduction of cells by particles pseudotyped with the widely used vesicular stomatitis virus G protein was inhibited via the adoption of a reduced extracellular pH during vector production, impairing the ability of the vector to interact with its target receptor. Employing a posttransfection pH shift to pH 6.7-6.8 resulted in a sevenfold reduction in vector genome integration events, arising from lentiviral vector-mediated transduction, within viral vector-producing cell populations and ultimately resulted in improved lentiviral vector production kinetics. The proposed strategy is scalable and cost-effective, providing an industrially relevant approach to improve lentiviral vector production efficiencies.

Degradation of specific glycosaminoglycans improves transfection efficiency and vector production in transient lentiviral vector manufacturing processes.

Both cell surface and soluble extracellular glycosaminoglycans have been shown to interfere with the exogenous nucleic acid delivery efficiency of non-viral gene delivery, including lipoplex and polyplex-mediated transfection. Most gene therapy viral vectors used commercially and in clinical trials are currently manufactured using transient transfection-based bioprocesses. The growing demand for viral vector products, coupled with a global shortage in production capability, requires improved transfection technologies and processes to maximise process efficiency and productivity. Soluble extracellular glycosaminoglycans were found to accumulate in the conditioned cell culture medium of suspension adapted HEK293T cell cultures, compromising transfection performance and lentiviral vector production. The enzymatic degradation of specific, chondroitin sulphate-based, glycosaminoglycans with chondroitinase ABC was found to significantly enhance transfection performance. Additionally, we report significant improvements in functional lentiviral vector titre when cultivating cells at higher cell densities than those utilised in a control lentiviral vector bioprocess; an improvement that was further enhanced when cultures were supplemented with chondroitinase ABC prior to transfection. A 71.2% increase in functional lentiviral vector titre was calculated when doubling the cell density prior to transfection compared to the existing process and treatment of the high-density cell cultures with 0.1 U/mL chondroitinase ABC resulted in a further 18.6% increase in titre, presenting a method that can effectively enhance transfection performance.

Lentiviral vector determinants of anion-exchange chromatography elution heterogeneity.

The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatography remains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G]) did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1).

Development of novel lipoplex formulation methodologies to improve large-scale transient transfection for lentiviral vector manufacture.

Large-scale transient transfection has advanced significantly over the last 20 years, enabling the effective production of a diverse range of biopharmaceutical products, including viral vectors. However, a number of challenges specifically related to transfection reagent stability and transfection complex preparation times remain. New developments and improved transfection technologies are required to ensure that transient gene expression-based bioprocesses can meet the growing demand for viral vectors. In this paper, we demonstrate that the growth of cationic lipid-based liposomes, an essential step in many cationic lipid-based transfection processes, can be controlled through adoption of low pH (pH 6.40 to pH 6.75) and in low salt concentration (0.2× PBS) formulations, facilitating improved control over the nanoparticle growth kinetics and enhancing particle stability. Such complexes retain the ability to facilitate efficient transfection for prolonged periods compared with standard preparation methodologies. These findings have significant industrial applications for the large-scale manufacture of lentiviral vectors for two principal reasons. First, the alternative preparation strategy enables longer liposome incubation times to be used, facilitating effective control in a good manufacturing practices setting. Second, the improvement in particle stability facilitates the setting of wider process operating ranges, which will significantly improve process robustness and maximise batch-to-batch control and product consistency.

Time-dependent sorption behavior of lentiviral vectors during anion-exchange chromatography.

Use of lentiviral vectors (LVs) in clinical Cell and Gene Therapy applications is growing. However, functional product loss during capture chromatography, typically anion-exchange (AIEX), remains a significant unresolved challenge for the design of economic processes. Despite AIEX's extensive use, variable performance and generally low recovery is reported. This poor understanding of product loss mechanisms highlights a significant gap in our knowledge of LV adsorption and other types of vector delivery systems. This work demonstrates HIV-1-LV recovery over quaternary-amine membrane adsorbents is a function of time in the adsorbed state. Kinetic data for product loss in the column bound state was generated. Fitting a second order-like rate model, we observed a rapid drop in functional recovery due to increased irreversible binding for vectors encoding two separate transgenes ( t Y 1 / 2 ${t}_{{Y}_{1/2}}$ = 12.7 and 18.7 min). Upon gradient elution, a two-peak elution profile implicating the presence of two distinct binding subpopulations is observed. Characterizing the loss kinetics of these two subpopulations showed a higher rate of vector loss in the weaker binding peak. This work highlights time spent in the adsorbed state as a critical factor impacting LV product loss and the need for consideration in LV AIEX process development workflows.

Machine learning and metabolic modelling assisted implementation of a novel process analytical technology in cell and gene therapy manufacturing.

Process analytical technology (PAT) has demonstrated huge potential to enable the development of improved biopharmaceutical manufacturing processes by ensuring the reliable provision of quality products. However, the complexities associated with the manufacture of advanced therapy medicinal products have resulted in a slow adoption of PAT tools into industrial bioprocessing operations, particularly in the manufacture of cell and gene therapy products. Here we describe the applicability of a novel refractometry-based PAT system (Ranger system), which was used to monitor the metabolic activity of HEK293T cell cultures during lentiviral vector (LVV) production processes in real time. The PAT system was able to rapidly identify a relationship between bioreactor pH and culture metabolic activity and this was used to devise a pH operating strategy that resulted in a 1.8-fold increase in metabolic activity compared to an unoptimised bioprocess in a minimal number of bioreactor experiments; this was achieved using both pre-programmed and autonomous pH control strategies. The increased metabolic activity of the cultures, achieved via the implementation of the PAT technology, was not associated with increased LVV production. We employed a metabolic modelling strategy to elucidate the relationship between these bioprocess level events and HEK293T cell metabolism. The modelling showed that culturing of HEK293T cells in a low pH (pH 6.40) environment directly impacted the intracellular maintenance of pH and the intracellular availability of oxygen. We provide evidence that the elevated metabolic activity was a response to cope with the stress associated with low pH to maintain the favourable intracellular conditions, rather than being indicative of a superior active state of the HEK293T cell culture resulting in enhanced LVV production. Forecasting strategies were used to construct data models which identified that the novel PAT system not only had a direct relationship with process pH but also with oxygen availability; the interaction and interdependencies between these two parameters had a direct effect on the responses observed at the bioprocess level. We present data which indicate that process control and intervention using this novel refractometry-based PAT system has the potential to facilitate the fine tuning and rapid optimisation of the production environment and enable adaptive process control for enhanced process performance and robustness.

Manufacturing a chimpanzee adenovirus-vectored SARS-CoV-2 vaccine to meet global needs.

Manufacturing has been the key factor limiting rollout of vaccination during the COVID-19 pandemic, requiring rapid development and large-scale implementation of novel manufacturing technologies. ChAdOx1 nCoV-19 (AZD1222, Vaxzevria) is an efficacious vaccine against SARS-CoV-2, based upon an adenovirus vector. We describe the development of a process for the production of this vaccine and others based upon the same platform, including novel features to facilitate very large-scale production. We discuss the process economics and the "distributed manufacturing" approach we have taken to provide the vaccine at globally-relevant scale and with international security of supply. Together, these approaches have enabled the largest viral vector manufacturing campaign to date, providing a substantial proportion of global COVID-19 vaccine supply at low cost.

High-throughput screening techniques for rapid PEG-based precipitation of IgG4 mAb from clarified cell culture supernatant.

Locating optimal protein precipitation conditions for complex biological feed materials is problematic. This article describes the application of a series of high-throughput platforms for the rapid identification and selection of conditions for the precipitation of an IgG(4) monoclonal antibody (mAb) from a complex feedstock using only microliter quantities of material. The approach uses 96-microwell filter plates combined with high-throughput analytical methods and a method for well volume determination for product quantification. The low material, time and resource requirements facilitated the use of a full factorial Design of Experiments (DoE) for the rapid investigation into how critical parameters impact the IgG(4) precipitation. To aid the DoE, a set of preliminary range-finding studies were conducted first. Data collected through this approach describing Polyethylene Glycol (PEG) precipitation of the IgG(4) as a function of mAb concentration, precipitant concentration, and pH are presented. Response surface diagrams were used to explore interactions between parameters and to inform selection of the most favorable conditions for maximum yield and purification. PEG concentrations required for maximum yield and purity were dependant on the IgG(4) concentration; however, concentrations of 14 to 20% w/v, pH 6.5, gave optimal levels of yield and purity. Application of the high-throughput approach enabled 1,155 conditions to be examined with less than 1 g of material. The level of insights gained over such a short time frame is indicative of the power of microwell experimentation in allowing the rapid identification of appropriate processing conditions for key bioprocess operations.