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Process development of a serum-free and scalable lentiviral vector production in a single-use bioreactor
Get PDFLentiviral vectors (LV) have played a critical role in gene delivery for ex vivo gene-modified cell therapies, including T cells and NK cells; however, a large-scale manufacturing process is needed to cope with the increasing demand from the cell and gene therapy sector. Single-use fixed-bed bioreactors, such as the iCELLis bioreactor, provide a promising automated, scalable platform for the generation of large volumes of GMP-grade LV. Fixed-bed bioreactors have already been shown to generate high LV titers; nevertheless, most of the work currently undertaken in the field relies on serum-containing medium formulations. This is largely because to date serum-containing media produces higher viral titer yields and an increased LV stability during the down-stream processing of the product. However, relying on serum-based processes adds significant supply chain and manufacturing risk to the process.
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Integrated upstream and midstream processing with scalable pre-configurated single- use assemblies for accelerated commercial reach
Get PDFThe growth of single-use technologies (SUT) has revolutionized the bioprocess industry. The implementation of such technologies was motivated by a substantial reduction in processing time and operational costs compared to their stainless-steel counterparts. Integrated within automated platforms such as the NevoLine™ Upstream, SUT reveal their full potential by offering an increased flexibility and supporting continuous processing. With 100s of different SU configurations the NevoLine platform can accommodate most upstream and midstream processes by integrating four unit-operations in a single manufacturing solution. Cell culture and viral production steps lie in scalable SU bioreactors (SUBs) available in various sizes optimal for process development, as well as clinical and commercial GMP production of viral vaccines, viral vectors for gene therapy and other emerging applications. The SUBs are featured in a range of scale-down automated manufacturing systems that ease scalability. The improved sterility assurance and controls offered by SUT enable predictable batch-to-batch performances and release. The pre-gamma eradiated SU assemblies integrated in Univercells Technologies’ manufacturing systems open the door to ready-to-use solutions reducing installation time and easing sterile continuous processing.
This study will demonstrate how the NevoLine Upstream integrated platform relying on SU assemblies can support continuous processing and accelerate commercial reach.
It will focus on the following points: Intensified cell growth and viral production in the SU scale-X™ bioreactor with up to 100-times increase in titer compared to traditional technologies with experimental results in various gene therapy and vaccine applications. Bioreactor characterization with scalability demonstration from scale-X hydro 2.4 m² to nitro 200 m² cell growth surface by maintaining fluid flow homogeneity, gradient, and volume to surface ratio across scales. Homogeneity within the fixed-bed will be demonstrated by sampling experiments with confident results. The scalability will be demonstrated by similar cell growth and viral titers per m² of growth surface profiles across scales. Increased process flexibility at commercial scale with the integrated and automated NevoLine Upstream platform adapted to multi-product facilities and variability in production capacity demand. A significant reduction in operational footprint (up to 3-times) enabled by intensified and integrated SU assemblies
A fluorescent optical ammonia sensor - Suitable for online bioprocess monitoring
Get PDFOptical sensors have found numerous applications in the last decades, e.g. optical sensors for oxygen and pH are established in bioprocess monitoring. In bio processing ammonia is another important analyte due to its toxicity at certain concentration levels.[1]. Since this compound is often a by-product in bioprocessing, online monitoring is desired. However, sensors for monitoring ammonia or ammonium in bioreactors are rare. We present a new ammonia sensor (Fig. 1 (b)) suitable for bioprocess monitoring. Our system is based on an acid-base concept including a fluorescent pH-sensitive dye.[2] The sensing layer is covered by a hydrophobic porous membrane, which excludes hydrophilic interfering materials. Our target analyte, ammonia (NH3), diffuses through the barrier to the protonated dye whereby it deprotonates the dye and switches off the NIR-emission. Read-out is performed with a commercially available compact phase fluorimeter combined with optical fibers. Dual-lifetime referencing (DLR) acts as detection method and Egyptian blue as reference material. A sensor performance in the range of total ammonia concentration (TAC, NH3 + NH4+) from 1 to 100 mmol L-1 is demanded. Depending on temperature and ammonia concentration the response time t90 and the recovery time vary from 20 s up to 4 min (Fig 1 (a)). The sensor performance is not influenced sufficiently by increasing temperature (Fig. 1 (c)).
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Process intensification: Impact on cost, facility footprint and sustainability matrix
Get PDFAs current biological product pipelines become more diverse, product demand and cost pressures are increasing. To meet these demands, manufacturers are moving towards process intensification. By making changes to unit operations, or indeed the full process, our industry can identify areas for process efficiency gains that shorten timelines, reduce process and thus facility footprint , reduce energy & utility requirements, lower cost of goods, and/or unlock additional manufacturing flexibility.
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The science behind the integrity of single-use system: Investigating liquid leak and microbial ingress mechanisms to determine the maximum allowable leakage limit
Get PDFAs single-use systems (SUS) are increasingly expanding into all process steps of commercial manufacturing, integrity failure can significantly impact drug safety, availability, and costs. Consequently, growing industry scrutiny of single-use system integrity (SUSI) is raising the need to develop good science behind reliable determination of liquid leakage and microbial ingress, as well as the appropriate physical integrity testing technologies. In the current study, the results of the experiments performed at Sartorius to understand the mechanisms of liquid leakage and microbial ingress as the foundation for SUSI are presented. This study establishes that there is a relation between liquid leakage and microbial ingress mechanisms in single- use plastic containers. Microbial ingress testing by the aerosolization method and the liquid leakage method are both used to determine the maximum allowable leakage limit (MALL) below which product leakage and bacteria ingress cannot occur in SUS, regardless of the various fluid and process conditions.
To define the MALL, it is generally assumed that a system or a product will not show any microbial ingress or leakage under a certain defect size. However, this study revealed that statistical analysis of the experimental data indicated the probability of MALL encountered at a certain defect size for each system. Finally, a mathematical model was generated to predict the MALL for any use-case of a SUS.
As most physical integrity testing technologies are based on measuring a gas flow rate, additional experiments were performed to find a correlation between a certain defect size and the corresponding gas flow rate. Therefore, the MALL defined at a gas flow rate can be directly linked to established physical integrity testing systems.
As a result, the methods studied provide a more accurate way of predicting ingress, increasing safety down the line for drug manufacturers and patients alike
Development of a thermostable, multivalent filovirus vaccine based on recombinant subunit proteins
Get PDFEbola Virus Disease (EVD) is the most prominent example of filovirus disease but despite being characterized as a Category A Priority Pathogen by NIH/NIAID over a decade ago, it lacked public and private research resources due to the absence of a commercial market. Transmission from wild animals into the human population typically causes outbreaks of limited scale in endemic areas located in the forested regions of Central Africa and the Philippines (for Reston ebolavirus). In the past decade, a Zaire Ebolavirus (EBOV) outbreak causing more than 11,000 deaths in several West African countries started to reveal the true epidemic potential that filovirus infections can have when entering an urban setting in a highly mobile society. In addition a persistent outbreak in the Democratic Republic of the Congo has continued since August 2018 despite significant progress with the clinical development of several EBOV vaccine candidates (one of which recently gained regulatory approvals in Europe, the U.S. and several African countries) and the advanced testing of promising EBOV specific therapeutics. Despite this significant progress, additional research is needed in particular on understanding the mechanism of protection and defining immune correlates of protection for Ebola and other filoviruses do develop fast and efficacious strategies for outbreak control as the incidence of outbreaks and total case numbers has significantly increased over the last decadesPlease click Download on the upper right corner to see the full abstract
Workshop: Innovation in Global Health
Get PDFVaccination is considered the most successful healthcare initiative in disease prevention, but achieving universal coverage is still beyond reach. According to the WHO, 19.4million did not receive routine life-saving vaccinations in 2018 – many of them children. To address this issue, research from both academia and industry partners are required to provide innovative solutions to problems in Global Health. This workshop will focus on the needs for the Global Health community and will be sub-divided into two sessions. The first session will provide examples of innovative solutions for persons in low and middle income countries, that seek to aid the development, distribution and uptake of vaccines in these very low cost markets. The second session will give contextual information of funding priorities and what funds are available to researchers interested in contributing to this valuable area.
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Driving change in dtap batch release testing
Get PDFThe complexity of vaccine manufacturing has raised the need to drive standardization and quality control requirements as well as batch release of vaccines. The purpose of release testing is to ensure that efficacy and safety of the vaccine product are maintained in all batches. Classical testing includes challenge experiments in animals that provide proof of vaccine potency and identify subpotent vaccines. However, novel concepts such as “consistency testing” question the continued need for in vivo experiments and propose to implement rigorous QC for lot-to-lot consistency testing with other methods at an earlier stage.
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Test tubes and turnaround times: An accelerated biosafety testing approach for new vaccines against emerging pathogens
Get PDFRecent outbreaks of infectious viral disease such as those caused by SARS-CoV-2 and Ebola have led to the successful development and approval of vaccines at unprecedented speed. These achievements are possible by using an accelerated approach to vaccine development. For example, vaccine development under an accelerated or pandemic approach could take 18-24 months compared to the traditional approach of 5-10 years.
A critical part of vaccine development is clinical trials and a key step in this pathway is Quality Control (QC) biosafety testing of materials used in the manufacture of clinical trial material. The traditional approach for applying and performing such QC tests does not align with the accelerated / pandemic development approach. Take for example, identity and adventitious agent testing of cell banks and vaccine seeds used to produce clinical trial material. Using a traditional approach, these alone can each take up to 8–10 weeks even with the best planning, resulting in a total time of 20 weeks if cell line and virus seed stock are characterised sequentially.
Here we describe accelerated biosafety testing strategies used in industry to expedite pre-clinical and first in human clinical studies for a variety of SARS-CoV-2 candidate vaccine modalities without compromising patient safety. We will present examples and data from actual scenarios on approaches and watch-outs for rapidly producing a Chemistry Manufacturing and Control (CMC) data package, that meet regulatory requirements. This will include parallel testing approaches, use of rapid test methods such as next generation sequencing, challenges with neutralising virus seeds and scaling QC testing capacity
Towards updatable, multivalent Covid vaccines: A platform process to produce trimeric spike protein of SARS-COV-2 variants expressed in HEK293 stable cell clones
Get PDFWithin 2.5 years since SARS-COV-2 emergence, COVID-19 pandemic has caused more than 6.3 million registered deaths and 530 million registered cases. The quick development of safe and effective vaccines was very important to mitigate the sanitary crisis. However, the continuous emergence of virus variants with increasing transmissibility continues to cause periodic outbreaks worldwide. The original vaccines keep protecting from severe disease and death caused by variants, but not from transmission and mild disease. Thus, new and improved vaccines are necessary, and updatable, multivalent pan-variant vaccines might be one way to control SARS-COV-2.
The spike (S) protein of SARS-COV-2, a highly glycosylated and very large protein (1380 amino acids), is a key target for diagnostics, therapeutics (e.g. monoclonal antibodies) and vaccines. Very early in the pandemic, Wrapp et al. (https://doi.org/10.1126/science.abb2507) produced the recombinant spike protein as a trimer stabilized in the prefusion conformation and determined its structure by cryoelectron microscopy, providing evidence that it binds to the ACE-2 receptor. The stabilized spike trimer forms the basis of most approved COVID vaccines, across vaccine platforms.
In the present work, we studied the expression and purification of the trimeric prefusion-stabilized spike protein in HEK293 cell lines and developed a platform process applicable to SARS-COV-2 variants (current - and potentially future - ones). Parental HEK293 cells (NRC, Canada or Thermofisher, USA) were transfected by lipofection using Lipofectamine 3000 (Thermofisher Scientific, USA) with a plasmid containing the gene encoding the spike protein. At first, for the ancestral strain (Wuhan), we used a plasmid intended for transient expression kindly provided by VRC/NIAID/NIH (USA). However, for the variants, we ordered synthetic genes (Genscript, USA) that were subcloned in a plasmid intended for stable expression. After genetic modification, stably transfected cells were maintained in the presence of G418 sulfate selection agent. After 3-4 weeks, stable cell pools were obtained and submitted to single cell deposit (FACS Aria, BD Biosciences) in order to obtain clonally-derived cell lines. Documented research cell banks of selected clones were cryopreserved. Batch and fed-batch cultivations were investigated in shake flasks and bioreactors, using the chemically defined HEK TF culture medium and HEK FS feed solution (both Sartorius Xell, Germany). For protein purification, different chromatographic techniques were investigated using Akta Purifier and Akta Pilot systems. Detection of the spike protein secreted in cell culture supernatant was performed by immunoblot, whereas UV280 (Nanodrop, Thermofisher, USA) was used for protein quantitation in purified samples.
After first expressing the spike protein in February 2020 by transient transfection, we developed a stable cell pool by co-transfecting the same transient expression plasmid and an empty stable expression plasmid. This stable cell pool allowed the generation of Wuhan protein that was used to develop serological tests and a hyperimmune equine serum (Cunha et al., doi: 10.1016/j.isci.2021.103315; Alvim et al., in press) and was donated so far to over 90 laboratories in Brazil for basic or applied research.
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