Classification and Characterization of Impurities in Phosphoramidites Used in Making Therapeutic Oligonucleotides:

Transcription

1 Tech ote Classification and Characterization of Impurities in hosphoramidites Used in Making Therapeutic ligonucleotides: Risk Mitigation Strategies for Entering Clinical hases He, Kaizhang; Hasan, Ahmad Introduction As advances in gene expression modulation by sira technologies continue, the reality of RAi therapeutics edges closer. ligotherapeutic development programs exist at almost all major pharmaceutical companies, with over $6B invested in oligotherapeutics programs since 2002*. *Asia Tides presentation by Gary Carter of Agilent Technologies DA and RA oligonucleotides are synthesized using nucleic acid monomers called phosphoramidites. hosphoramidites have a dimethoxytrityl (DMT) protection group on the 5 -H and a β-cyanoethyl-, - diisopropylamino-phosphoramidite (CE) on the 3 -H of the dexoyribose (DA) or ribose (RA). RA phosphoramidites have a 2 -H protecting group, most commonly a tert-butyldimethylsilyl (TBDMS) or a methyl group. The nucleobase exocyclic amines of both RA and DA phosphoramidites will be protected by any of several moeities, most commonly benzoyl and isobutyryl. In some circumstances, such as when mild deprotection is required to prevent depurination, other protecting groups are used. ligosynthesis is performed by sequential addition of individual phosphoramidites by coupling of the ribo sugar 3 -H of the new base to the ribo sugar 5 -H of the previous base. The synthesis cycle involves four chemical reactions: detritylation, coupling, capping, and oxidation. These cycles are repeated until the desired nucleotide sequence has been achieved. The repetitive nature of oligosynthesis can amplify the risk of phosphoramidite impurities resulting in poor final oligonucleotide quality. For example, if a single phosphoramidite used in synthesis of a 20-mer contains a critical impurity at a concentration of 0.2%, and this phosphoramidite is added 8 times in the sequence, the resulting oligonucleotide will contain 1.6% of this impurity. The ability of phosphoramidite manufacturers to control phosphoramidite impurities is critical to oligotherapeutic supply chain management. Impurity Classification otential impurities in phosphoramidites are commonly classified in three categories. on-reactive and non-critical Reactive but non-critical Reactive and critical Critical impurities are defined as those which are incorporated into the oligonucleotide during synthesis such that oligos containing these impurities are difficult or impossible to separate from the desired synthesis product. There are also critical impurities which, when incorporated into an oligonucleotide are difficult or impossible to detect. The first class of impurities is non-reactive and non-critical. This class includes compounds having no phosphorus, compounds with phosphorus (V), hydrolyzed nucleoside H-phosphonates, and nucleoside dimer triester. They are not incorporated into oligonucleotides during synthesis and, by definition, are non-critical. The second class of impurities is reactive but non-critical. These are impurities that may become incorporated into oligonucleotides during synthesis but they are easily detected and oligos containing these impurities are easily separated from the desired synthesis product. This class of impurities includes phosphoramidites with modifications on the 5 -H other than DMT, phosphoramidites with different 3 -aminoalkyl protecting groups, and phosphoramidites with different base protecting groups. The third class of impurities is reactive and critical. This class of impurities is of most concern to oligonucleotide manufacturers.

2 Critical Impurity Characterization In an effort to provide further clarification and risk mitigation to our partners, we have further divided critical impurities into four sub-classes. Class 1: Includes amidites with unnatural base modifications. Class 2: Critical impurities include 2-3 bis-ribose or deoxyribose amidites present either as monomers or dimers. Class 3: Critical impurities include DMT-amino nucleobases, unprotected nucleobases, 5 -ribose protection other than DMT and, in the case of RA, ribose 2 -H protection other than TBDMS, 2 --Me or 2 -F. Class 4: Critical impurities are comprised of structural isomers and pose perhaps the greatest therorectical impurity risk. These include alpha-anomers of the nucleobases and inversion of the DMT and amidite sugar protection to create the 3 -DMT-5 -amdite isomer form. An inversion seen in RA is of the 2 and 3 ribose protecting groups to yield 3 -TBDMS-2 -amidites. hosphoramidite Quality and Control of Critical Impurities The high expense associated with failed development programs and rework of key studies necessitates tight supply chain quality and control for raw materials and key components. For these reasons, Thermo Fisher Scientific takes a comprehensive approach to its own supply chain management, quality systems and control and risk mitigation. The intrinsic benefits of this rigorous process are passed on directly to our many partners. Thermo Scientific Theraure DA and RA phosphoramidites are made with the highest standards of supply chain and manufacturing control to minimize or eliminate impurities of concern to oligotherapeutic developers and manufacturers. A core management philosophy of Thermo Scientific phosphoramidite manufacturing is full integration of operations management with quality and business systems. Methods for managing quality are intrinsic to day-today operations and workflow management. The base for the quality systems is the IS standard. Thermo Scientific quality systems significantly exceed IS requirements having the breadth of an FDA registered system and depth, in some cases, cgm compliance. We are not currently registered with the FDA. ur supply chain is controlled at both the supplier level and the item level. Suppliers are regularly audited, subject to change control notification and formal complaint resolution systems. We maintain, wherever possible, multiple, qualified sources of supply and contingency plans for alternate-site manufacturing for supply chain reliability and in the event of catastrophe at any one site. n the item level, key raw materials are qualified prior to use in manufacturing. We employ a risk-based assessment including CoA review, physicochemical analysis and functional performance. We analyze multiple lots for confirmation of lot-to-lot consistency. Theraure amidite manufacturing processes are engineered to minimize or eliminate reaction conditions which could result in amidite impurities. Rigorous, documented process control is maintained. QC testing using advanced analytical technologies including HLC, 31 MR, and LC-UV-MS is performed at intermediate steps. State of the art purification methods are used to obtain the highest amidite purity possible. Theraure DA and RA phosphoramidites are manufactured to provide high overall purity and control of critical impurity levels with excellent batch to batch reproducibility required for synthesis of oligonucleotides as active pharmaceutical ingredients. In summary, the comprehensive process, quality and systems control employed provide a higher level of confidence and reduced risk and exposure for our clients as their oligotherapeutic programs enter critical phases in the clinic. We believe that the combination of all of these measures in totality provide for a better development outcome for our clients and their programs.

3 Table 1. otential Impurities in DA, 2 -Me, and RA hosphoramidites. Compound # ame Classification I 5 -DMT-3 -H-nucleoside on-reactive and non-critical II 3 -DMT-5 -H-nucleoside on-reactive and non-critical III 5,3 -Bis-DMT-nucleoside on-reactive and non-critical IV 5 -TBDMS-2 (3 )-TBDMS-nucleoside on-reactive and non-critical V 5 -DMT-3,2 -bis-tbdms-nucleoside on-reactive and non-critical VI 5 -DMT-bis-CE-phosphite on-reactive and non-critical VII 5 -DMT-3 -CE-H-phosphonate on-reactive and non-critical VIII 5 -DMT-3 -CE-H-phosphonoamidate on-reactive and non-critical I 5 -DMT-3 -amidate on-reactive and non-critical 5 -DMT-3 -phosphonoamidate on-reactive and non-critical I 5 -Modified-trityl-3 -amidite Reactive but non-critical II 5 -DMT-3 -modified-aminoalkyl-amidite Reactive but non-critical III 5 -DMT--modified-base-protection-3 -amidite Reactive but non-critical IV 5 -DMT-3 -methoxy-amidite Reactive and critical V 5 -DMT-(CE-amiditoethyl)-phosphite Reactive and critical VI 5 -DMT-amiditoethyl-amidite Reactive and critical VII Bis-nucleoside-amidite Reactive and critical VIII Bis-nucleoside-CE-phosphite on-reactive and non-critical I Bis-nucleoside-amidinoethyl-amidite Reactive and critical 5 -DMT-3 -ethoxy-ethoxy-amidite Reactive but non-critical I 3,3 -Bis-diisopropylamine-amidite Reactive and critical II CET-pyrimidine-3 -amidite Reactive and critical III 5 -DMT-base-unprotected-3 -amidite Reactive and critical IV 1 -Bis-nucleoside dimer-3 -amidite Reactive and critical V 5,-Bis-DMT-nucleoside-3 -amidite Reactive and critical VI 5,2 -Bis-TBDMS-3 -amidite Reactive and critical VII 3,2 -Bis-TBDMS-5 -amidite Reactive and critical VIII 3 -DMT-5 -amidite Reactive and critical I 5 -DMT-1 -alpha-anomer-3 -amidite Reactive and critical 5 -DMT-3 -TBDMS-2 -amidite Reactive and critical

4 Table 2. Chemical Structures of otential Impurities ( = H, Me, or TBDMS). (I): 5 -DMT-3 -H-nucleoside G DMT (IV): 5 -TBDMS-2 (3 )-TBDMSnucleoside G H G DMT H (VI): 5 -DMT-bis-CE-phosphite G (VIII): 5 -DMT-3 -CE-H-phosphonoamidate H H C (): 5 -DMT-3 -phosphonoamidate (I): 5 -Modified-trityl-3 -amidite (II): 5 -DMT-3 -modified-aminoalkyl-amidite G R2 R3 R1 C DMT C R2 G DMT Cl CH3 C (V): 5 -DMT-(CE-amiditoethyl)-phosphite G DMT (IV): 5 -DMT-3 -methoxy-amidite R1, R2 = CH3, CH2CH3, CH(CH3)2, CH2CH2CH3, etc. R1, R2, R3 = CH3, CH2CH3, Cl, H, H2, etc. (III): 5 -DMT--modified-baseprotection-3 -amidite C G G DMT C R1 DMT C G DMT (I): 5 -DMT-3 -amidate G DMT TBDMS TBDMS G DMT G DMT C (VII): 5 -DMT-3 -CE-H-phosphonate DMT (V): 5 -DMT-3,2 -bis-tbdms-nucleoside TBDMS TBDMS DMT G DMT DMT (III): 5,3 -Bis-DMT-nucleoside G H H DMT (II): 3 -DMT-5 -H-nucleoside C C

5 (VI): 5 -DMT-amiditoethyl-amidite C DMT C G DMT (VIII): Bis-nucleoside-CE-phosphite G DMT G DMT (VII): Bis-nucleoside-amidite DMT G (I): Bis-nucleoside-amiditoethylamidite DMT (II): CET-pyrimidine-3 -amidite DMT C (III): 5 -DMT-base-unprotected-3 amidite DMT DMT C (V): 5,-Bis-DMT-nucleside-3 amidite DMT H DMT (VI): 5,2 -Bis-TBDMS-3 -amidite C (VIII): 3 -DMT-5 -amidite C DMT TBDMS C C (VII): 3,2 -Bis-TBDMS-5 -amidite G TBDMS C G C TBDMS (): 5 -DMT-3 -TBDMS-2 -amidite DMT G G (I): 5 -DMT-1 -alpha-anomer-3 amidite DMT G TBDMS C DMT (IV): 1 -Bis-nucleoside dimer-3 -amidite H2 DMT G (I): 3,3 -Bis-diisopropylamine-amidite DMT G G (): 5 -DMT-3 -ethoxy-ethoxy-amidite DMT G G G TBDMS C

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