Unveiling process knowledge for plasmid DNA fermentation across upstream scales

Plasmids DNA (pDNA) are small, circular pieces of DNA used to deliver genetic information and are key inputs in gene therapies, cell therapies, gene editing, and in particular mRNA vaccines and therapies. There are currently ~1,400 therapeutic assets in development across these modalities, with the pipeline expected to grow rapidly in the next years given the pandemic scenario. Developing biopharmaceuticals derived from microbial fermentation such as pDNA relies upon performant bioreactors to allow a rapid scale up to commercial batches. For this it is relevant to minimize any possible risks while developing a process that adheres to industry quality standards. The choice of a well characterized system plays an important role from R&D through to production stages in accelerating development timelines and ensuring process success. The aim of this poster is to provide evidence to demonstrate the advantages of a microbial process developed using Sartorius scalable microbial solutions, DoE software and analytical methods for pDNA production. Please click Download on the upper right corner to see the full abstract

Use of sulfated-cellulose membrane adsorbers to intensify purification of cell culturederived influenza A and B viruses

New generation of cell culture-based vaccines enables faster response to pandemic outbreaks and helps coping with the increasing demand for seasonal vaccines. Besides efficient upstream processing technologies, appropriate economic and robust downstream processing is key to consistently achieve high yields. Chromatography membranes have been extensively evaluated to capture viruses at laboratory scale, for influenza, adenovirus or virus like particles. They have shown great potential to intensify processes thanks to a high binding capacity, high flow-rate and ease of use and are now commonly used in new generation of vaccine processes. We present here Sartobind SC, a novel sulfated cellulose membrane adsorber, for the capture of Influenza. In a recent study, the binding capacity and the purification performance of two bead-based resins and Sartobind SC was compared for three influenza virus strains (H1N1, H3N2 and B) produced in MDCK suspension cells in a chemically defined medium. The dynamic binding capacity for the sulfated cellulose membrane adsorbers was consistently higher than for the resins (8 to 22-fold). Overall, recovery of virus varied between 66% and 81%. Total protein and DNA removal were\u3e74% and\u3e96%, respectively. Due to the higher operating flow rate and binding capacity, the productivity with the membrane adsorbers was on average 25-times higher than with the resins. This purification platform based on sulfated cellulose can intensify processes and therefore reduce the cost of influenza purification. This ligand is also a promising candidate for other viruses such as vaccinia, RSV, HSV or measles

Implementation of a process-scale adenovirus purification with a single-use platform

Adenovirus vectors are finding increasing application within the vaccine and gene therapy industries. Companies developing adenovirus-based biopharmaceuticals will benefit from single-use process platforms that are quick and easy to install and have been demonstrated previously to produce purified adenovirus. The platform can be adapted to new adenovirus products with a minimum investment of time and resource in process development activities. We have assembled a platform from existing and proven technologies, available from lab to production scale, that meet the quality requirements of the vaccine industry. The platforms includes clarification, ultrafiltration/diafiltration, chromatography and sterile filtration steps. The approach has been successfully demonstrated for the purification of an Adenovirus serotype 5 vector, at the 20 L scale. It allows the complete purification of the vaccine in one day

Membrane chromatography cassettes for bind and elute applications of viruses and large proteins

For flow-through polishing applications, membrane adsorbers have become a well-established technology. However, there is an increasing demand for bind and elute purifications for larger targets as adeno- and lentiviruses, virus like particles (VLP) and influenza. The reason is the higher binding capacity of macroporous membranes compared to conventional resins having much smaller pores and excluding them by size. But capture applications with such devices suffered from the current size limitation of 5 liters. Here we describe a modular cassette system which has been tested for scale-up and flow performance in comparison with void volume optimized capsules. The goals were to create a system up to 20 L membrane volume which can be optionally expanded to ~100 liter and, be able adapt exactly to the size needed (modular), using the same 4 and 8 mm bed height as the capsules and membranes for single- or intra batch re-use

Chromatographic tools for optimization of IVT reaction and improving mRNA purification process

The recently demonstrated efficacy of mRNA-based Covid-19 vaccines has shown the promise of this therapeutic format, but also highlighted the need for higher efficiency of mRNA production to meet enormous needs for global vaccine supply. Typical mRNA production process involves three key steps: 1) plasmid DNA (pDNA) production in supercoiled (sc) isoform, linearization and purification, 2) in-vitro transcription (IVT) reaction and 3) mRNA purification. Here we present a chromatographic toolbox for integrated mRNA production from pDNA to mRNA purification, including in-process analytics. The pDNA purification approach presented here was designed to fit the specific requirements of mRNA vaccines. It integrates a linearisation step before polishing (removal of unwanted isoforms) of plasmid DNA. The polishing step after enzymatic linearisation, separates linear pDNA from enzyme and other unwanted products. Supporting in-process analytical tools are presented. IVT reaction monitoring with novel HPLC approaches includes CIMac PrimaS analysis of mRNA content as a function of time, with concomitant monitoring of NTP consumption. With information on NTPs, capping reagent and mRNA content, IVT reaction can be rapidly optimized for maximum productivity, in near real-time. Advantage of at-line monitoring is to prevent degradation of mRNA in IVT mixture which would occur after maximum productivity is reached. Purification of mRNA from IVT reaction mixture can be achieved using selective binding to polyA tail (using OligodT chromatography) or multimodal chromatography (PrimaS) which separates the ssRNA product from DNA template and IVT reaction mixture. Polishing approaches for final separation of ssRNA and dsRNA using hydrophobic interaction chromatography (HIC) and reverse-phase (RP) chromatography achieve high purity of final product. Supporting in-process analytical HPLC tools, including multimodal chromatography resin, facilitate a rapid read-out of mRNA concentration and purity profile

High Density HEK293T culture for high yield, high quality, stable adenoviral vector production in Ambr® 250 stirred tank reactors

Adenovirus vectors (AdV) present high safety and immunogenicity for drug development, allowing more and more vaccines to adopt this technology platform in recent years. Due to the current COVID-19 pandemic, the global demand for AdV has experienced significant growth. Therefore, to optimize the upstream process in order to obtain high yields, good quality and stable viral vectors, it becomes critical that processes are stable and easy to scale-up, which has become a key focus of pharmaceutical companies in the field. Please click Download on the upper right corner to see the full abstract

Influenza virus capture using membrane chromatography: Improving selectivity by matrix design and pseudo-affinity ligand interactions

Because next generation cell-based influenza vaccines have to be produced faster and in greater quantities than traditional vaccines, future purification processes will require more efficient unit operations for their isolation and purification. Membrane chromatography has already demonstrated a number of positive characteristics for the bind&elute purification of viral particles like e.g. adenoviruses or influenza viruses. The technology not only addresses the diffusion limitations of porous particle media but also offers dramatic advantages in binding capacity in a disposable format. Therefore, the last remaining challenge for the easy adoption of this technology in the vaccine industry represents selectivity and recovery. We present here a novel cellulose based stationary phase whose active specific surface area is designed for maximum virus accessibility. The resulting gain in selectivity and recovery but also in binding capacity is further maximized by using highly selective pseudo affinity ligands for influenza viruses