Analytical technique to separate and quantify full length mRNAs that are greater than 10 kb long

GSK is seeking analytical techniques for mRNA-based vaccine products that can separate full-length mRNAs from non-full-length mRNAs and quantify them. Analytical techniques or lab tests with high resolving power for mRNAs 10-18 kB long (molecular weight greater than 3-4 MDa), that can distinguish the full-length mRNA products from truncated mRNA species are welcome.

Long mRNAs are becoming increasingly important as therapeutic and prophylactic drug products. GSK uses mRNA that are 10-18kb in length for the vaccine development. The length of the construct is different for different drug products.

The long mRNAs must be manufactured and purified. These processes must be optimized to ensure that the maximum yield of full-length mRNA drug product is obtained.

GSK is seeking analytical techniques that can be used to separate and quantify the amount of full-length mRNA, and truncated variants of the mRNA. Such analytical technique will be used to characterize the drug product itself, and can also be used in process optimization experiments to provide information for each step of the manufacturing and purification process.

Large mRNA-based vaccine products have unique structural features that challenge the current RNA analytical techniques. Each drug product will have a different length, weight and structural profile. Routine separation techniques such as HPLC or CE can analyze much smaller mRNA products but lack sufficient separation power for this size of mRNA.   Molecular biology-based techniques can have higher resolving power but can lack coverage of the full molecular sequence and lack the capability to quantify different mRNA species.  

Analytical methods, which can be a combination of different methods, that:

• can separate large full-length mRNA from other non-full-length mRNA species. The full-length RNA is the mRNA molecule which has the full-length nucleotide sequence from capped 5’ end to the 3’ end with polyA tail
• work with mRNAs with 10,000 – 18,000 bases
• can quantify the amount / proportion of full-length mRNA
• work with samples containing up to 0.1 mg mRNA
• Ideally, GSK would like a technique that can distinguish between the full-length mRNA and truncated species that have lost 1 or 2 nucleotides. We realise this is challenging and we are open to techniques that can’t achieve this but offer a significant improvement in resolution over the existing methods.

Desirable, but not essential:

• A method / technique that can be scaled up as a semi-preparation tool to fractionate mRNA samples

Possible approaches include, but are not limited to:

• Size separation
• Third-generation gene sequencing with quantification capability
• LC-MS

Current, unoptimized analytical techniques that cannot resolve large mRNAs, for example:

• CGE
• DNA/RNA Gel based methods
• HPLC

PCR based methods

• A proposal describing the solution and how the success criteria will be met with the proposed solution
• A non-confidential description of the technology and a schematic of the solution