RIBOPRO is not your average CDMO. Instead, we invest heavily in internal R&D with the believe that this is the best way to advance our knowledge, technologies and product quality.
Our customers benefit from this investment by way of a better product and a higher chance of reaching the clinic/patient.
Activity & Quality
There are several challenges with the use of mRNA:
1. intracellular innate immunity that resides in every cell-type, that limits protein expression and can even induce cell-death, that is triggered by impurities (like dsRNA) and physicochemical properties of the mRNA itself.
2. the difficulty of obtaining a pure, mature mRNA, containing a well-defined poly(A)tail and a cap on virtually all molecules, as this is influenced by the sequence (structure)
3. (auto-catalytic) degradation of intact mRNA during production and storage for prolonged periods of time
RIBOPRO has developed several technologies that address these issues:
1. Patented sequence-based de-immunization
This technique reduces (and in some cases prevents) the induction of innate immunity against the mRNA. It uses exchange of synomynous codons throughout the coding sequence following a particular exchange table. The goal of the procedure is to break-up subsequences that are know to have a high affinity for the TLR-7 and -8. As a result TLR-7 and -8 do not activate the IFN-I pathway via MyD88, so protein expression is not reduced and pro-inflammatory cytokines are not released.
The difficulty with this method is that it must work within the context of the evolutionary optimized sequence, as mutations on the protein level are usually not desireable, and for certain codons there is limited or no alternative. Furthermore, by performing codon exchange also secondary structure of the mRNA changes, codon optimality changes and potentially even protein folding dictated by the translational speed changes. Therefore, RIBOPRO has developed an algorithm that uses state-of-the-art models of each of these parameters and performs millions of in silico folding and codon use analyses to arrive at the proposed optimized sequence. In the ideal situation, multiple variants of a construct are tested side-by-side and the results are used for informed evolution of the construct.
The benefit of using this method, over the only alternative; the use of chemically modified nucleotides, is 2-fold:
a. it produces similarly de-immunized mRNA, but does not have the ribosomal misinterpretation of the chemically modified nucleotides, which leads to read-through and possibly auto-immunity-inducing misincorporated aminoacids. This seems to be not a great issue for vaccination, but for (repeated) therapeutic use, this is a major cause for concern. By using only canonical nucleotides, a more safe and unique (patentable by you) sequence is generated, that produces between 5 and 40x more protein than its wild-type counterpart.
b. clarity and cooperation on the IP-situation; you only have to deal with RIBOPRO, who has a vested interest in your success and market entry.
2. In-house developed enzymes
These enzymes contain stabilizing, anti-abortive and/or ds-RNA preventing mutations to ensure high mRNA purity, even before purification. We have developed our own variants of T7 RNAP, SP6 RNAP, Vaccinia capping enzyme, and more are to follow. Subsequent to IVT, we use extensive purification to remove proteins, smaller RNA fragments and degraded DNA fragments. By using extra-ordinarily pure mRNA, protein expression and safety are maximized.
3. mRNA storage technologies.
It is well-known that mRNA is best stored at lower temperatures, especially at prolonged periods of time. We store and ship our mRNA products at -80°C (dry ice) to ensure maximum potency when arriving at your facility. Furthermore, we experiment with lyophilized and dried mRNA to enhance stability. Furthermore, our production process is swift and optimized for minimal in-process degradation. The use of high pure enzymes and the addition of RNAse-inhibitor prevents RNAse-mediated degradation.
Targeting & escape
The delivery of mRNA is a critical step in the overall performance of any mRNA. For in vivo and medicinal purposes, lipid nanoparticles remain the most avanced and often well-performing technology. Challenges remain:
1. Control over bio-distribution
2. Efficient endosomal escape
3. Suitable (lack of) immunogenicity
RIBOPRO has developed several technologies that address these issues:
1. Patented class of ionizable lipids
These lipids have an extra-ordinary low toxicity and immunogenicity, while having a reasonably high endosomal escape activity.
2. Proprietary core lipid system
By using a novel core lipid system, our LNPs do show a substantially lower bio-distribution to the liver, allowing the (active) targeting towards other organs.