After Cytiva, a Danaher company, unveiled Fibro dT platform for enhanced mRNA purification, European Pharmaceutical Manufacturer spoke to Emmanuel Abate, President of Genomic Medicine and head of sustainability at Cytiva to gain more information.
Cytiva
How does the electrospun cellulose in the Cytiva fibro dT differ structurally and functionally from traditional resin beads used in mRNA purification?
In mRNA purification, structural design directly impacts process speed and capacity. Traditional resin beads contain dead-end pores that slow mass transfer and restrict flow rates, creating longer cycle times.
The fibro dT platform is made of electrospun cellulose which is a porous matrix with an open and well-defined structure without dead-end pores. The high porosity and high mechanical strength of the fibro adsorber matrix allows for operation under high flow rates, allowing rapid chromatography cycle time.
The relatively large surface area allows for high binding capacity of large mRNA as compared to the binding capacity of mRNA in chromatographic beads.
In summary, the porosity and rapid mass transfer allows for high binding capacity at short residence times. The result is faster chromatography cycles and higher mRNA capacity compared to bead-based systems.
Are there limitations in RNA size or sequence complexity for which Cytiva fibro dT is most effective?
Fibro dT is broadly effective across RNA sizes and sequence complexities, but optimisation is key for best performance. Each sequence is unique, and optimisation is needed to maximise recovery and impurity removal.
Work has primarily used a 2000 nucleotide (nt) long mRNA in the development of Cytiva fibro dT. We have observed that the mRNA capacity increases for shorter sequences, and capacity decreases for longer sequences. The drop in capacity for longer sequences is expected and fibro dT can be used for purification of long sequences.
The RNA sequences missing the poly(A)-tail shall ideally be removed when purifying mRNA with an dT adsorber. For sequences with long internal A-rich stretches one can seek to optimise buffer and running conditions to limit undesired binding in such cases, as an example, by exploring a low-salt wash step in the chromatography method. In short, Fibro dT offers flexibility across diverse mRNA designs, with method optimisation ensuring high recovery and purity.
How does fibro dT maintain high reproducibility across batches—what specific quality control measures are in place?
The specifications of the electrospun cellulose raw material, the functionalisation of Cytiva fibro dT membrane and the moulding into the 0.4 mL units are key for low batch-to-batch variation.
In addition, the manufacturing quality control assesses that the oligo dT ligand density, flow properties and other parameters within specification to ensure consistent quality.
Can fibro dT handle high-throughput workflows, or is it primarily suited for smaller-scale purification?
Cytiva fibro dT is well suited for high-throughput workflows.
The mRNA purification cycle time, including regeneration, is short. The ÄKTA chromatography instruments with UNICORN software have scouting methods if combined with an autosampler, for high-throughput screening of various parameters and samples.
What is the expected lifespan of a Fibro dT unit under routine use?
In the data file and other external documents, we show consistent performance over 20 cycles. The lifespan can maintain a high level of performance provided the fibro dT unit is cleaned and stored according to instructions.
Are there any compatibility considerations for integrating fibro dT into existing mRNA production pipelines beyond ÄKTA systems?
We have exposed the Cytiva fibro dT membranes to harsh sanitisation conditions, and they withstand at least one sanitisation cycle with performance within specifications.
The 0.4 mL unit uses threaded connections for finger-tight fittings, and we haven’t tested sanitisation on this format.
The launch of the Cytiva fibro dT 0.4 mL unit for R&D use is an opportunity to make the product available for labs purifying mRNA and provide feedback about ideal complementary fibro dT products.
We are developing other GMP-compliant formats for Cytiva fibro dT to manufacture clinical mRNA. We will follow up with more information regarding availability of future Cytiva fibro dT products suitable for the GMP environment.
How does the platform’s low-salt loading capability specifically reduce precipitation risk compared with other purification methods?
The Cytiva fibro dT adsorber has a relatively high capacity and efficient binding even when loading at lower salt concentrations. This flexibility to load at low salt concentrations allows for purification of sequences that are precipitation prone. In addition, loading at a reduced salt concentration is advantageous and promotes selectivity but will negatively impact capacity. Optimisation may be required for specific mRNA constructs to balance selectivity, capacity, productivity and the achievement of desired critical quality attributes (CQAs).
How did working with Aldevron shape the development of Fibro dT, and what were the most important customer insights gained?
The early work with Aldevron using prototypes with dT20-ligand was crucial for evaluation of our prototypes using many different mRNA and saRNA sequences. In addition, Aldevron's analytical capabilities and experience from many purification protocols were helpful for guidance to set the product specifications.
At that point, we also evaluated different membrane technologies. The data from Aldevron contributed to the decision-making about prototypes to proceed with. The ligand is no longer dT20. The dT ligand design has been modified after the initial collaboration with Aldevron, and with Merck.
The work with Merck was also helpful and resulted in a peer-reviewed article based on data using the early dT20 prototypes.
Are there plans to expand fibro dT applications beyond mRNA, such as other RNA therapeutics or DNA-based products?
Yes, the fibro adsorber is a chromatography platform for Cytiva, complementing resins and membrane adsorbers (Mustang products). Currently, we have the Cytiva fibro dT product and Hitrap and HiScreen Fibro PrismA products supporting mRNA and mAb purification on the market. This demonstrates the versatility of the platform, and it will expand with other format sizes, suitable for GMP manufacturing and with other ligands.
How does fibro dT help minimise RNA loss compared with conventional methods, and what does that mean for overall therapeutic yield?
The predictable and robust performance between cycles and between batches of Cytiva fibro dT is intended to ensure consistent and selective purification of mRNA. The high-performance over a range of chromatography running conditions including low salt conditions, is favourable to have good recovery in the purification of various poly(A)-tailed RNA sequences.
High recovery of the RNA is important from a cost and sustainability perspective. Another sustainability advantage with fibro dT 0.4 mL units is that they, in most regions and after NaOH cleaning for >30 min, can be recycled with other plastic recyclables.