Therapeutic Oligonucleotides, Impurities, Degradants, and Their Characterization by Mass Spectrometry

Abstract

Oligonucleotides are an emerging class of drugs that are manufactured by solid‐phase synthesis. As a chemical class, they have unique product‐related impurities and degradants, characterization of which is an essential step in drug development. The synthesis cycle, impurities produced during the synthesis and degradation products are presented and discussed. The use of liquid chromatography combined with mass spectrometry for characterization and quantification of product‐related impurities and degradants is reviewed. In addition, sequence determination of oligonucleotides by gas‐phase fragmentation and indirect mass spectrometric methods is discussed. © 2019 John Wiley & Sons Ltd. Mass Spec Rev

Regulatory guidelines and preclinical tools to study the biodistribution of RNA therapeutics

“…For example, the European Public Assessment Reports of siRNA drugs Onpattro® and Givlaari® state that LC-MS/MS was used for quantification of siRNA in plasma and/or tissue samples [22] , [54] . The various MS modalities for detailed analysis of oligonucleotides beyond biodistribution have recently been extensively reviewed [78] , [79] and is beyond the scope of this review. In contrast to siRNA, mRNA molecules can exhibit a molecular weight into the MDa range.…”

Scalable convergent synthesis of therapeutic oligonucleotides

“…18,19 This technique is insufficient to resolve and quantify all of the product related impurities. 20 Therefore, it is difficult to know whether they will provide oligonucleotides with purities high enough to support clinical trials. 21,22 Quantitation of certain types of product related impurities can currently only be achieved by LC-UV coupled to mass spectrometry (LC-UV-MS).…”

Tandem mass spectrometric sequence characterization of synthetic thymidine‐rich oligonucleotides

“…Tandem mass spectrometric sequencing of oligonucleotides, however, is affected by multiple factors, including intrinsic properties of oligonucleotides (e.g., nucleotide composition, sequence, and structural modifications) and experimental parameters (e.g., precursor ion polarity, charge state, and ion activation method).10-14 Thymidine (T)-rich oligonucleotides are notoriously difficult to sequence because of unfavorable bond cleavages at thymidine residues by MS/MS. 5,14,15 In addition, structural modifications of oligonucleotides, such as internucleotide phosphodiester linkages replaced by phosphorothioate (PS) linkages, may pose potential challenges to the sequence elucidation by MS/MS because of the added resistance to bond cleavages by the modified linkages. 7,8,13,14,16 In this study, sequence characterization of custom synthesized DNA oligonucleotides of the same nucleotide sequence as nusinersen (an 18-mer with seven T units, marketed as Spinraza™ and approved by FDA in 2016) with or without PS modification was investigated using two ion activation techniques: resonant or trap-type collisioninduced dissociation ("CID") and beam-type CID or higher-energy collisional dissociation ("HCD").…”

“…MS/MS of DNA (unmodified): CID versus HCDSequence characterization of oligonucleotides by MS/MS fragmentation of multiply charged oligonucleotide precursor ions in negative ion mode has been intensively studied 5,7. Four possible cleavage sites along the phosphodiester backbone through corresponding dissociation channels can produce four series of sequence-defining complementary ion pairs.…”