CMC Control Strategies and Impurity Evaluation for Complex and Emerging Oligonucleotide Therapeutics

Hagen Cramer, PhD chief Production officer, QurAlis
Marc Lemaitre, PhD Consultant ML Consult
Thomas Rupp, Owner and Principal, Thomas Rupp Consulting UG

In the session “CMC Control Strategies and Impurity Evaluation for Complex and Emerging Oligonucleotide Therapeutics”, speakers Hagen Cramer from QurAlis, Marc Lemaitre from ML Consult, and Thomas Rupp from Thomas Rupp Consulting UG engaged in a comprehensive discussion with the audience about oligonucleotide synthesis.

Cramer, an expert in chemistry-related aspects of the development of oligonucleotide therapeutics, obtained his PhD in chemistry and continued his postdoctoral work at the National Institutes of Health. He has been involved in the biotech industry for more than 20 years and focuses on the discovery, development, and manufacturing of therapeutic oligonucleotides. Dr. Lemaitre specializes in DNA repair and cancer and is involved in pioneering antisense and oligo delivery studies. After holding several positions in R&D, operational management, business development, and general management within CMOs, pharma and biotech companies, he currently works as an independent consultant helping companies on various strategic and/or technical projects. Dr. Rupp has more than 30 years of technical experience in oligonucleotide manufacturing and analytics. Since 2013, he has been working as an independent technical and CMC consultant focusing on oligonucleotide-based therapeutics. He also has experience in the solid-phase manufacturing of a wide range of oligonucleotides. During the Q&A session, certain phases and components of the oligonucleotide synthesis were addressed. The speakers clarified the concerns about the importance of controlled pore glass (CpG) and its batch-to-batch repeatability, the use of size exclusion chromatography (SEC) for blunt ended siRNAs, and impurities with an emphasis on degradation impurities and depurination. The audience also asked whether size exclusion chromatography, high-resolution mass spectrometry (HRMS), and capping are necessary regarding the analytics in the speakers’ presentations. First, the speakers explained CpGs as glass that undergoes the process of leaching. Leaching is not a well-defined process and might end up with different qualities that will affect the results. Pore size is an important parameter in oligosynthesis. According to the speakers, one of the key points is to have a quality agreement with the vendor. Any change in the process needs to be communicated immediately because they could make a significant difference. It is not only about the process of creating pores. It is also important to know where the CpG manufacturer gets the glass from and whether it is a constant supplier.

Regarding size exclusion chromatography, they said that one does not absolutely need size exclusion. It is also possible to use a nondenaturing IPRP method. Dr. Lemaitre found it easier to get a good SEC protocol separating the duplex from sense and antisense strands in a way that does not denature the duplex during migration. However, he said it is possible to use a nondenaturing or denaturing IPRP method as part of a control strategy. Dr. Lemaitre gave an example from siRNAs developed by one of his clients. Both strands had a similar content except for one strand is a little bit rich in purines, while the other is in pyrimidines, so they have always found a small separation between the sense and antisense strand. They underlined that it is critical to choose the proper size for the size exclusion columns among the options. For a blunt ended oligonucleotide, the only difference between two strands is the molecular weight because of the different sequences. QurAlis’ Cramer concluded by underlining the importance of method development for these cases without assuming a standard method will be applicable for every duplex. Impurities during the synthesis were another issue that the audience was concerned about. The speakers stated that some of those impurities, such as IBU and CNET, can be well-separated. However, if someone is not able to resolve those impurities (i.e., excess capping), the only possibility is to use a mass spec and to do an ion extraction. They underlined the following points about impurities:

Pay attention to the capabilities of your CMO in terms of LC-MS analysis when selecting a CMO.

There are two types of depurination. One type is produced during the manufacturing of the oligonucleotide, and those are the real impurities. The other type is depurination that can happen during the ionization of the oligonucleotide inside the mass spec system. Those are not real impurities. It is good to pay attention to the 18 Da mass difference between the two types of depurination.

There are considerable differences between one MS system to another.

The speakers also discussed the advantages of Ultra Performance Liquid Chromatography (UPLC). They found the UPLC-UV method a good strategy to resolve everything because quantification is easier. UPLC can also separate class IV impurities. Only the depurination cannot be solved by UPLC; however, those can be degraded in high pH conditions. Additionally, the speakers explained that the degradants rather than process-related impurities are already existing throughout the synthesis but will become more visible in certain phases such as basic/acidic conditions or oxidation stress. As opposed to small molecules that might be toxic or more potent, degradants of oligo synthesis are major metabolites such as DNA or RNA, which have a similar safety profile, efficacy, and risk of being off target. There is not a significant difference between degradants coming from the synthesis of 20-mer and 90-mer. Regarding capping, Dr. Rupp defined it as an “insurance policy.” Capping also reverts to branching of oligonucleotides. If one can manage the synthesis process well enough to control the formation of branching or to increase coupling efficiency, then capping might not be necessary. However, risk assessment is important at this point. Their suggestion is that even if all other parameters are the same, the sequence can change the result of the process. Making developments based on the output of interest cannot be avoided. Lastly, Dr. Lemaitre briefly mentioned High Resolution Mass Spectrometry (HRMS). To differentiate a T and a methylated C, base composition analysis can be used if it’s not possible to access HRMS. However, HRMS becomes important when peaks, shoulders displaying start to show the same MW as your expected full-length product. It would be helpful when authorities want to see more detail about those close signals. The strategy explained by the speaker which is commonly used is swap sequencing. Together with the desired product, one can synthesize swap sequence, which has small alterations such as replacement of C/T or C/U inverted and can ask CRO to spot the differences demonstrating that the method used is sensitive to detect even a small difference between the two oligonucleotides.