Chromatography focus on


Oligonucleotide Biopolymers – Future Challenges for Chromatography George Okafo1 , Daren S Levin2 and David Elder1 1. GlaxoSmithKline, Scinovo, Ware, Hertfordshire, United Kingdom. 2.GlaxoSmithKline, Exploratory Development Sciences, RTP, North Carolina, USA Synthetic Oligonucleotides as Therapeutic Medicines


Synthetic oligonucleotides are an exciting new class of biomolecules capable of treating many disorders, which are currently not amenable to existing drugs, including viral infections [1], respiratory disorders [2], cancers [3] and rare diseases [4]. Current interest has been largely fuelled by two key events: firstly, Fire and Mello’s Nobel-prize winning discovery of gene silencing by RNA interference (which helped to improve our understanding of the genetic basis of many diseases) [5]; and secondly, the regulatory approval of two oligonucleotide-based drugs, namely Vitravene® for cytomegalovirus infections) and Macugen®


[6] (a 21-base single stranded antisense oligonucleotide approved by the FDA in 1998 [7] (a pegylated aptamer approved in 2005 for treating wet macular degeneration).


Synthetic oligonucleotides currently in clinical development are comprised of single or double stranded DNA [8], RNA [9], locked nucleic acid (LNA) sequences [10], aptamers [11], speigelmers [12] and oligonucleotides conjugated to polymers [13]. Little is known about their exact mechanism of action, but they are thought to involve protein biosynthesis control via immunostimulation [14] or via interference with gene transcription/translation processes to inhibit production of potentially harmful proteins [15]. Alternatively, oligonucleotides could work via an exon-skipping mechanism partially restoring the functional properties of a defective protein [4].


Structural and Regulatory Considerations


The unique chemical/structural properties of oligonucleotide therapeutics has left them in somewhat of a grey area when it comes to regulatory guidance for the development and control of drug substance and product. Oligonucleotide therapeutics are macromolecules derived from DNA and RNA building blocks with a suggested optimal length in the range of about 20 bases (or 20-mer) [16]. They are manufactured in a stepwise fashion using solid-phase synthesis (for example, one nucleotide at a time) and are specifically excluded from the current ICH guidelines Q6A [17], Q3A(R2) [18], and Q3B(R2) [19].


Whilst there is currently no specific guidance from regulatory agencies for the Chemistry, Manufacturing, and Controls (CMC) of oligonucleotide based therapeutics, the spirit of the existing guidance, particularly Q6A, around quality and safety can certainly be followed. In addition, many of the concepts discussed in ICH Q6B [20], for the analysis of biologically engineered products, are applicable to oligonucleotides as both share similar analytical challenges due to their size, polymeric and secondary structure and closely related impurities or variants.


This short review identifies the source of many impurities and focuses on the application of chromatographic methods using UV and mass spectrometric detection to characterise single and double stranded oligonucleotides and their related impurities.


Chromatographic Analysis of Oligonucleotides Source of Impurities


Oligonucleotides are synthesised using either H-phosphonate [21] or phosphoramidite chemistry [22] on automated commercial synthesisers. The chemistry starts with an amidite starting material chemically bonded to a solid support, typically glass [23] or a polymeric resin [24]. The chain length is then extended by repeated cycles of deprotection, coupling, oxidation (or sulphurisation) and capping. When the desired chain length is achieved, the crude oligonucleotide is cleaved from the solid support and purified via preparative ion exchange and reverse phase chromatography. Figure 1 shows a short fragment of a typical oligonucleotide chemically modified on the phosphate backbone and on the ribose sugar. The overall quality of the oligonucleotide is assured through control of the raw materials (primarily the amidite starting materials). However, the large number of synthetic steps (up to 120 steps for a typical 20-mer) coupled with the relative inefficiencies of each reaction step can give rise to a significant number of impurities. Impurities can arise from many sources including manufacturing conditions, from raw materials and from degradation. Most of these compounds fall into the following categories [25]:


• Shortmers, deletion or failure impurities – oligonucleotide sequences where the chain length is shorter by n-x.


• Longmers or extension impurities – oligonucleotides sequence where the chain length has been extended by n+x.


• Adducts– these are modified full-length sequences which arise from incomplete deprotection of the oligonucleotides. Examples of these adducts include isobutyl, benzoyl or cyanoethyl derivatives.


• Oxidised phosphodiesters – oligonucleotide impurities found only in phosphorothioate oligonucleotides where incomplete sulphurisation of the phosphate linkage leads to trace levels of phosphodieser linkages (P=O).


• Depurination – oligonucleotide impurities where the nucleoside has lost a purine base.


• Degradants – impurities formed from degradation of the oligonucleotide during synthesis or under storage conditions (heat, humidity, oxidative, etc.).


• Non-hybridised single strand– excess single strand that remain unreacted during the annealing process to form the double stranded oligonucleotide.


• Derived from structural modification – such as phosphothiolation of the phosphate linkages [26] and the addition of alky and alkyloxy groups at the C-2 position of ribose sugars [27], peptides conjugates [13], bridging riboses [28] and morpholino ring derivatives [29].


Chromatographic Methodology


Figure 2. IP-RP-HPLC Analysis of a 21-mer Oligonucleotide in its crude and purified states. See reference [39] for more details.


Figure 1. Short Fragment of a typical tetramer oligonucleotide showing chemical modifications.


The primary objective of any chromatographic method is to separate impurities from the desired product. This task is made more challenging for oligonucleotides because of their complex structure, multiply charged nature and the presence of myriads of structurally similar impurities. Over the years, methods for separating oligonucleotides have evolved from traditional slab gel electrophoresis [30] and P-31 NMR [31] to modern high performance liquid chromatographic (HPLC) [32] and capillary electrophoretic (CE) [33] approaches.


Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52