What’s Next in Oligo Manufacturing: Modified Phosphorodiamidate, Blockmers™ and High Purity, Reliable, Stable Raw Materials

Written by Kimo Sanderson

Since the invention of oligonucleotide synthesis in 1970s and the launch of the first oligotherapeutics in the early 2000s, research on the development of effective oligonucleotide-based medicines has continually progressed. Challenges have included in vivo degradation of oligo drugs by exo- and endonucleases, low cellular uptakes, low binding efficacy to mRNA targets, cellular cytotoxicity, and off-target effects. These challenges are associated with the structural nature of oligo ASOs and siRNAs themselves.

Over the last two decades, extensive research to modify oligonucleotide structures has been done to make ASOs and siRNAs effective against disease targets. In this article, we look at new tools and services that are being evaluated by the oligo industry to improve the effectiveness of oligos.

Modification Tools for Oligos

Broad and deep knowledge has been gained by modifying the phosphorodiamidate backbone, nucleotide bases and ribose sugars with unnatural moieties. An extensive library of moieties has been discovered and developed in several categories. It is anticipated that this toolbox of modified monomers and oligo analogs will be increasingly required in the large-scale synthesis of pipeline oligos targeting broad disease areas to treat previously untreatable or unreachable tissues.

The modified monomer and oligo analog toolbox will be increasingly required in the large-scale synthesis of pipeline oligos targeting broad disease areas.

The table below summarizes the different categories of modifications and areas where suppliers in the oligotherapeutics manufacturing segment can build some niche strengths:

Considering the above modifications, here are opportunities for suppliers to build niche strengths.

• Reliable GMP grade bulk supply of modified monomers 
• Expertise in synthesis of these sugar, base, backbone modified oligomers and chains 
• Know-how on alternative chemistries and sulfurization reagents e.g. Fmoc/Bhoc protected phosphorodiamidate, H-phosphonates, Beaucage, PADS, DDTT, TETD, Bu4NBr catalyst, alkyl piperazine groups
• Solubilization and mixing of high sulfur and cysteine-guanine containing reactants
• Capabilities of peptide/ GalNAc conjugation with linkers e.g. phthalimide, ethylene glycol, UNILINKER on CPG support
• Stereospecific chiral synthesis of oligonucleotides e.g. stereocontrolled ON synthesis with iterative capping and sulfurization (SOSICS), Staudinger reaction
• Quick on-demand in-situ synthesis of PMOs e.g. flow chemistry, flow bed reactors, continuous mixing systems, solid support CGG resin based
• Learning and adoption from modified phosphoramidites based oligosynthesis at scale well established for diagnostics industry e.g. gene array, FISH
• Analytical methods for detailed characterization e.g. 2D-NMR, SEC-HPLC-UV-MS, CGE, AX-LC, DSC, XRD, GPC

With half of the nineteen (19) FDA-approved oligos already utilizing modified sugars, bases and backbones by PMOs, sulfurization or methoxyl, and with a growing number in preclinical and clinical pipelines, the equipment vendors, CDMOs and CROs should not only consider these as areas of building capabilities, but recognize these as trends and opportunities for second and third generation oligo therapeutics which are already happening in the oligo manufacturing industry. Therefore, the suppliers and service providers can build these as niche strengths to differentiate themselves and partner with emerging innovative oligo discovery companies working on these different chemistries.

From Monomer to Blockmer™

In parallel, another key area which should not be overlooked include blockmers. A blockmer is a short form of oligonucleotide intermediate with, for example, four to eight residues. Several ready-to-use blockmer intermediates can be ligated by enzymatic reactions in a liquid solution phase synthesis. Their primary advantage is a simpler, faster and easier to scale manufacturing compared to traditional solid phase synthesis. In addition, chiral blockmers can be readily available, e.g. from NATiAS.

Fewer impurities and byproducts may form with fewer orders of reactions e.g. P-S error, GGGG repeat sequence errors, systemic yield losses with chain length, and so on. In addition, energy and solvent usage can also be significantly reduced – important for green chemistry initiatives. Such blockmer technologies have also been successfully attempted to produce siRNA at high purity using Almac’s RNA ligase enzyme, polynucleotide kinase phosphorylation and crude starting materials. Therefore, blockmers may enable higher-efficiency oligo manufacturing in the near future for the specialized oligo formats above, as well as future generic oligos.

Blockmers may enable higher-efficiency oligo manufacturing in the near future.

Considering this, equipment vendors, CDMOs and CROs should start taking a hybrid approach to the next generation of scaled up oligo manufacturing. Raw materials and reagent suppliers can explore providing optimal enzyme libraries and GMP-grade enzymes at bulk together with blockmers and solubilizing agents. Equipment vendors should consider mixing vessels, temperature conditions and materials of construction chemical compatibilities.

CDMOs might accommodate dedicated areas for bio catalytic liquid phase production systems during facility expansions as well as nurturing separate skilled technical personnel. CROs may offer process and analytical development services, as well as build awareness to guide and lead the oligo innovators towards innovative and highly manufacturing platforms with the availability of blockmers and enzymes.

Higher Quality Requirements for Starting Materials

Last but not least, oligonucleotide manufacturing is sensitive to impurities and resulting yield losses from the quality of raw materials, reagents and solvent mixtures. To address this serious issue, an European Pharma Oligonucleotide Consortium, Starting Materials (EPOC SM) working group has been formed. The expert group will help set the criteria, taking ICHQ6A and ICHQ11 as examples for critical starting materials, identifying impurities, establishing analytical methods and performing risk assessments to encourage the adoption of robust, consistent manufacturing practices. This includes covering expected impurities for phosphoramidites, methyl oxy amidites, deoxyamidites, blockmers, LNAs, solvents, sulfurization agents, and so on using 2D NMR and HPLC-UV-MS. The group also mentions the importance of defined chemical structure, similarity with fragment impurities and cross-over contamination.

In general, the recommendation is to treat the starting materials as a commodity offered by vendors at high quality with CoAs. Thus, suppliers to the oligo industry should take these into consideration and follow the recommendations from the EPOC working group when developing documentation packages for starting material offerings. Some suppliers who are becoming key players in the phosphoramidite, reagents and dehydrated acetonitrile market have already made efforts to meet these quality expectations and therefore differentiate themselves as leading suppliers. Others can follow similarly in this new pharma segment of oligotherapeutics, as there are ample niche opportunities of innovation in terms of raw materials, fine chemicals and synthesis techniques.

Read our previous article, “What’s Next in Oligo Manufacturing: From Lyophilization Bottleneck To Liquid API“.

To Summarize:

The increasing chemical complexity of pipeline oligonucleotides will require the use of oligo modification tools, blockmers, and other starting materials with increasing quality requirements. Suppliers and service provides have ample opportunity to develop products, manufacturing services, and testing services to address this emerging market.

Are you interested in custom market research services to gain deeper insights into future oligonucleotide manufacturing trends? Reach out for a consultation.

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