What Oligo Manufacturing Risks Are We Underestimating?

By Anna Rose Welch, Editorial & Community Director, Advancing RNA

Over the past few months, I’ve written a handful of articles on oligo quality — particularly what we’ve been watching and hearing from regulatory agencies on hot topics like comparability, impurity analysis, and stereochemistry. Though we do have some patchwork guidance provided around these topics, what is clear is that our understanding of oligo quality will continue to be shaped by future guidance and ICH guideline revisions. The question then becomes: How well equipped are we and our manufacturing partners to be able to achieve the necessary quality, and what are some of the common stumbling blocks on the road to achieving that quality?

Earlier this year, I attended the OPT Congress, where I had the great fortune of meeting consultant Marc Lemaitre. During his presentation, Lemaitre was a fountain of wisdom, sharing the issues he’s observed in working with sponsors and CDMOs on a variety of different oligo programs. I recently sat down with Lemaitre for a follow-up interview to gauge some of the evolutions he’s been watching within the CMC landscape for oligo development. Here, he digs into the shifts in quality he’s noted, pointing out some of the potential pitfalls we may encounter in our manufacturing that could stand to impact the overall quality of our oligos.  

Anna Rose Welch: You work with a wide array of companies in the oligo space. What areas do you consistently see sponsor companies overlooking or underestimating, and what does this suggest about the current state of the industry?

Marc Lemaitre: Regulators have evolved. They are no longer looking at oligonucleotide INDs in quite the same way that they did 5-10 years ago. I do not think that this shift is primarily due to COVID or changes in political administrations. It reflects the evolution of scientific knowledge and, importantly, the evolution of analytical capabilities.

The latest European guideline on the development and manufacturing of oligonucleotide therapeutics which is in its late-stage approval phase in Europe is a great indication that regulators have more questions about impurities and impurity characterization, including stereochemical impurities. So, we are facing a situation in which what we’ve used before to analyze impurities in a product is not good enough. It calls for additional investigation of diastereomeric composition and the use of state-of-the-art analytical methods to characterize stereochemistry. A conventional purity method may demonstrate that a product is, for example, 95–98% chromatographically pure, but that does not necessarily mean that we understand what constitutes the remaining 2–5%, nor that the method can resolve the relevant product-related impurities.

This creates an important challenge for sponsors. They may underestimate the difficulty that some CDMOs will have to provide the characterization information that is acceptable to the regulatory authorities. Overall, I would say that this points to an ongoing problem within analytical method development and characterization.

I would also argue that there is a significant knowledge gap within many younger biotechnology companies. In particular, there are not enough people who fully appreciate the analytical and manufacturing implications of phosphorothioate stereochemistry and the diastereomeric composition of PS-containing oligonucleotides. Today, the PS-diastereomer issue is not just an analytical curiosity; it is becoming a CMC knowledge issue. Understanding that an ASO is not simply a single chemical entity, but potentially a highly complex population of stereoisomers, is becoming increasingly important for both development and regulatory strategy.

In my view, this is likely to become a significant differentiator between CDMOs. The question will increasingly be not simply, “Can you manufacture the oligonucleotide?” but rather, “Can you demonstrate, analytically and scientifically, exactly what you manufactured?

Welch: I recently attended a panel discussion (and wrote an article) that asked the question, “Does Stereochemistry matter?” How do you see this quality debate taking shape in the oligo sector?

Lemaitre: We can ask that question in two ways. The simplest question is what you asked: Does stereochemistry matter? And the answer is definitely yes.

Now the second question is related to what Wave therapeutics and other companies are trying to do in terms of making stereopure oligonucleotides: Do we need stereopure oligos?

We don’t currently have a definitive answer to that second question. If we want to take a short-sighted approach, we know that the oligos approved today are not stereopure, and there is no demonstrated toxicity. At the same time, none of the clinical trials investigating stereopure products have come with a clear answer so far as to whether a stereopure product will have a better clinical effect in patients.

What we do know is that, in some cases, if you change the stereochemistry of an oligo compared to previous batches, you can see significant variation in the biological activity of the oligo — the best example of this being the notable Mongersen clinical trial failure back in 2017.

Maintaining the RP/SP ratio from batch to batch for any given oligonucleotide is key. Make sure that purification conditions are maintained as similarly as possible from batch to batch. Likewise, keep the bed height of the purification column as similar as possible. And, if you start your selection process using a certain activator for the synthesis of your oligonucleotide, don’t change it from preclinical to commercial. Or, better, but rare, you can compare oligos made with different activators to see if one gives a higher biological activity than another. Normally, CDMOs know that activator should not be changed. But I have seen at least one well-established CDMO make this change, and it did impact the product’s biological activity.

Welch: In a presentation you gave at the OPT Congress earlier this year, you emphasized the importance of due diligence when assessing outsourcing partners — and based on your previous response, it’s clear there can be pitfalls, even with well-established CDMOs. What are some of the issues you’ve seen arise that sponsors should pay close attention to or be aware of when working with their outsourcing partner?

Lemaitre: I’ve seen a handful of issues in the past five years that are, fortunately, preventable.

Firstly — and this is more common with new CDMOs — there can be risks related to water contamination in their system. Water during oligo synthesis is a big enemy because water dramatically reduces the coupling rate. If you are developing a typical oligo, you are attaching 20 different nucleosides using amidite chemistry. Water contamination will translate into a lower level of full-length product in your crude material. If you have something that contains 40% of impurities when you start purification and continue to see that repeatedly from batch to batch, it’ll be incredibly difficult to end up with a product that has an impurity profile that is very comparable from one batch to another. This will likely lead to comparability studies and perhaps some bridging studies, especially if you encounter an impurity that isn’t in your tox batch. Additionally, it will reduce your manufacturing yield that will increase the cost per gram of your drug substance.

Secondly, I have occasionally seen issues in which the solid support has some modified physical parameters that are not tested during the release because nobody has apparently identified that as a critical parameter. In particular, the swelling of the polymeric support may not be as constant as we thought, meaning that, for oligos a little bit longer than the average [say 23 nucleotides], the pressure in the column can increase, leading to an increased percentage of n-1/n-2 impurities. That could be avoided by introducing a “cut-off” test when releasing the batches of solid support.

Finally, I have occasionally seen CDMOs working on what they call “generic processes,” “best practices,” or “internal IP,” and they run everything according to the same parameter. This can occasionally be to the detriment of a slightly higher quality product for the client. My opinion is that every single manufacturing process for every oligo [with possibly the exception of the N-of-1 oligos] should be developed, even minimally, from the start to make sure that coupling and deprotection are as good as possible.

Welch: What about new outsourcing partnerships — what factors do you encourage your oligo clients to weigh when selecting a new CDMO?

Lemaitre: Price is obviously one of the biggest factors; we can see huge price differences between 50%-200% relative to an average value. Quite often on the higher end of that spectrum, it’s a great sign the CDMO did not fully understand the request for proposal and/or the sponsor needs to be much more prescriptive in their RFP (e.g., provide some goals for purity of the drug substance that are initially “reasonable low” [NLT 85% is a good start], limit the scope of release testing for “Tox batch”). If you only provide “skeleton-level” details to a CDMO, it’s possible the CDMO will fill in additional details.

IP is a close second and is often the source of misalignment between sponsors and CDMOs. The sponsor will argue ‘I pay for this, so I own it.’ However, when some CDMOs start developing, they may think of it as their IP, which, in turn, can limit sponsors’ abilities to transfer the process to another CDMO. For example, I have encountered issues transferring a manufacturing process from one CDMO to another competitor CDMO based overseas. In the beginning, it seemed as though it would be impossible, but we came to an agreement where the original CDMO owned the documents, but the sponsor owned the content/results outlined in the document. So, we had to extract all the important data from the documents and write a transfer support document so the new company could develop something that would give us the same results as the initial process. The caveat is that there are always details the sponsor will have difficulties understanding and that will generate differences — albeit hopefully small.

A CDMO’s quality record is also critical — especially today with so many new Chinese newcomers. Prior to COVID, the Chinese authorities did exactly what the European authorities would do. You could register with them as an API manufacturer, and they would come audit your laboratory and issue a GMP certificate for that facility and oligo manufacturing process. This has since changed. Now, the only documentation that exists for newcomers is confidential audit reports that have been made by potential client companies. I’ve recently seen one audit made by a big Pharma company in China of two CDMOs, and they both got a red flag. There were dozens of major or critical observations. So, it’s important to remember when assessing a CDMO: Everybody can claim to be compliant, but just because you may be compliant to make a monoclonal antibody doesn’t mean that you’re compliant to make an oligonucleotide. At the same time, there are also some very good Chinese CDMOs and they have proven track record, like having successfully supported sponsors who got their IND approved by FDA and/or EMA. In this case, geopolitical situation may be an issue.

In conclusion, based on the principle “what you see is what you get,” nothing is better than an on-site audit to make sure that all is good for what you need [phase appropriate], including documentation and SOPs that many companies do not share with potential clients.