Improving downstream processing for viral vectors and viral vaccines

Viral vectors are playing an increasingly important role in the vaccine and gene therapy elds. The broad spectrum of potential applications, together with expanding medical markets, drives the e orts to improve the production processes for viral vaccines and viral vectors. Developing countries, in particular, are becoming the main vaccine market. It is thus critical to decrease the cost per dose, which is only achievable by improving the production process. In particular advances in the upstream processing have substantially increased bioreactor yields, shifting the bioprocess bottlenecks towards the downstream processing. The work presented in this thesis aimed to develop new processes for adenoviruses puri cation. The use of state-of-the-art technology combined with innovative continuous processes contributed to build robust and cost-e ective strategies for puri cation of complex biopharmaceuticals.(...

Improved seed train strategy applied to PER.C6® cells for manufacturing readiness in vaccines production

The prevention of infectious diseases is a major goal for global healthcare and vaccines are still among the best preventive tools. The recent Ebola virus outbreak has challenged the vaccine industry to boost the speed to market and process development activities for new vaccines. To be able to cope with infectious disease outbreaks, our company developed an intensified cell and virus culture process using PER.C6® cells and AdVac™ platform. In our intensified process, perfusion bioreactors are used to increase Adeno vector titers while minimizing capital investments. To further increase readiness and flexibility for manufacturability, we improved the current process by substituting the classical cell culture seed train with an innovative concept called LVHD (Large volume high density) where the seed train can be generated from a single bag of cells at high density reducing significantly the time needed for the final cell culture. In the current study the LVHD expansion train in comparison to the classic roller bottle expansion was evaluated in regard to Cost of Goods, facility footprint and operational flexibility. Moreover, the Adeno vector drug substances obtained from the two processes were compared. Finally, the robustness and consistency of our LVHD seed train will be shown by using Monte Carlo simulation approach

Tackling a capacity bottleneck to permit large-scale downstream processing of an adenovirus-vectored vaccine

We recently described the strategy by which the University of Oxford and AstraZeneca collaboratively scaled up production of our adenovirus-vectored COVID-19 vaccine, using a productive fed batch process and distributed manufacturing approach in twelve countries around the world. Here we will focus on the development of the downstream process used to make this vaccine. In early development, the first tangential flow filtration step in our previously developed process was noted to be a potential obstacle for scale-up beyond 200L. By removing this first tangential flow filtration step, we established a simple purification process capable of handling the increasing quantities and concentrations of viral titers which are becoming a bottleneck for many adenoviral vector manufacturing processes. Product quality was in line with regulatory expectations. This strategy has enabled 2 billion doses of the Oxford/AstraZeneca vaccine to be produced by November 2021, with the majority made and used in low- and middle-income countries

Accelerating and intensifying manufacturing to enable large-scale supply of a new adenovirus-vectored vaccine within 100 days

The Coalition for Epidemic Preparedness Innovations’ ‘100-day mission’ aspires to launch of a new vaccine within 100 days of pathogen identification. We have previously reported a simple fed batch process and strategy of internationally-distributed manufacturing, which enabled 2 billion doses of the ‘Oxford / AstraZeneca’ adenovirus-vectored COVID-19 vaccine to be produced in less than 600 days from publication of the SARS-CoV-2 genome sequence. The majority was made and used in low and middle income countries. Here, after briefly reviewing that previous work, we will describe efforts to further improve adenovirus manufacturing for response to future pathogen outbreaks and variants. Please click Download on the upper right corner to see the full abstract. Please click Additional File below for the presentation

Data from: Evaluation of novel large cut-off ultrafiltration membranes for adenovirus serotype 5 (Ad5) concentration.

The purification of virus particles and viral vectors for vaccine and gene therapy applications is gaining increasing importance in order to deliver a fast, efficient, and reliable production process. Ultrafiltration (UF) is a widely employed unit operation in bioprocessing and its use is present in several steps of the downstream purification train of biopharmaceuticals. However, to date few studies have thoroughly investigated the performance of several membrane materials and cut-offs for virus concentration/diafiltration. The present study aimed at developing a novel class of UF cassettes for virus concentration/diafiltration. A detailed study was conducted to evaluate the effects of (i) membrane materials, namely polyethersulfone (PES), regenerated cellulose (RC), and highly cross-linked RC (xRC), (ii) nominal cut-off, and (iii) UF device geometry at different production scales. The results indicate that the xRC cassettes with a cut-off of approximately 500 kDa are able to achieve a 10-fold concentration factor with 100% recovery of particles with a process time twice as fast as that of a commercially available hollow fiber. DNA and host cell protein clearances, as well as hydraulic permeability and fouling behavior, were also assessed

Evaluation of novel large cut-off ultrafiltration membranes for adenovirus serotype 5 (Ad5) concentration.

The purification of virus particles and viral vectors for vaccine and gene therapy applications is gaining increasing importance in order to deliver a fast, efficient, and reliable production process. Ultrafiltration (UF) is a widely employed unit operation in bioprocessing and its use is present in several steps of the downstream purification train of biopharmaceuticals. However, to date few studies have thoroughly investigated the performance of several membrane materials and cut-offs for virus concentration/diafiltration. The present study aimed at developing a novel class of UF cassettes for virus concentration/diafiltration. A detailed study was conducted to evaluate the effects of (i) membrane materials, namely polyethersulfone (PES), regenerated cellulose (RC), and highly cross-linked RC (xRC), (ii) nominal cut-off, and (iii) UF device geometry at different production scales. The results indicate that the xRC cassettes with a cut-off of approximately 500 kDa are able to achieve a 10-fold concentration factor with 100% recovery of particles with a process time twice as fast as that of a commercially available hollow fiber. DNA and host cell protein clearances, as well as hydraulic permeability and fouling behavior, were also assessed

Figure raw data

The file provides the raw data of the figures 2-

Trade-off between throughput, indicated as liter of feed processed in the unit of time (h), given a defined membrane area (m²), and infective particle recovery yield.

<p>The values referrer at 10 times concentration factor. The orange area on the right top corner depicts the best membranes. RC and xRC membranes showed the highest throughput coupled with high recovery yield. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article)</p

(average SEM) for the different UF membranes.

<p>(A) R&D prototype devices with different materials, namely RC, xRC, and PES. (C) The pilot production devices were only made of xRC and compared against commercially available GE HF 750 kDa (PES UF 7) modules. (B, D) water flux (LMH) at various values of ranging between 0.5 and 2 bar.</p