Liquid Chromatography Methods for the Separation of

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Liquid Chromatography Methods for the Separation of Short RNA Oligonucleotides

Synthetic oligonucleotides have become increasingly popular as a result of the recent discovery of ribonucleic acid interference (RNAi), a natural process for silencing gene expression. As biomedical researchers evaluate the use of antisense and small interfering RNAs (siRNAs) as potential therapies for the treatment of disease, the need for improved methods for the chromatographic separation and analysis of oligonucleotides has become apparent. This article presents a review of different liquid chromatography (LC) methods and strategies for the chromatographic separation of short RNA oligonucleotides.

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Mirlinda Biba*,†, Bing Mao*, Christopher J. Welch*, and Joe P. Foley† *Department of Analytical Chemistry, Merck Research Laboratories, Rahway, New Jersey; †Department of Chemistry, Drexel University, Philadelphia, Pennsylvania. Direct correspondence to: [email protected]

here has been considerable interest molecule inactivates multiple mRNAs). An recently in the use of synthetic oligo- overview of the RNAi pathway for targeted nucleotides as potential therapeutic gene silencing is illustrated in Figure 1. agents capable of suppressing the synthesis The discovery of siRNA gene silencing in of specific proteins (1–3). Targeted “knock- animals (8) and human cells (9) has led to down” of specific gene products using an a surge of interest in the use of siRNA for antisense ribonucleic acid (RNA) strategy biomedical and drug development research. dates to the late 1990s (4). In this approach, Many biomedical and pharmaceutical coma single-stranded oligonucleotide comple- panies have become involved in the exploramentary to the messenger RNA (mRNA) tion of the preparation and use of siRNAs encoding a targeted protein leads to disrup- as potential therapies for the treatment of tion of ribosomal transcription and protein diseases such as cancer, macular degenerasynthesis. In theory, antisense oligonucle- tion, and viral infections (10). otides can be applied to any disease in which protein overexpression is detrimental, Oligonucleotide and siRNA and a number of antisense oligonucleotides Structure and Preparation have been evaluated as potential therapies RNA is a biologically important molecule (5). The need for long complementary oli- that consists of a long chain of nucleotide gonucleotides and the stoichiometric nature units. Each nucleotide contains a ribose of mRNA inactivation (1 antisense mole- sugar, a nitrogenous base, and a phosphate cule:1 mRNA inactivation) places consider- group. There are four bases in RNA: adeable constraints on developing cost-effective nine (A), guanine (G), cytosine (C), and antisense drugs. uracil (U) (Figure 2). Oligonucleotides are The more recently discovered small short, single-stranded RNA or deoxyriinterfering RNA (siRNA) mechanism for bonucleic acid (DNA) molecules that can silencing gene expression involves a short readily bind, in a sequence-specific manner, double-stranded RNA molecule of about to their respective complementary oligonu21 base pair length, which activates the cleotides to form duplexes. Small interferRNA interference (RNAi) silencing path- ing RNA is a small double-stranded RNA way (6,7), thereby achieving catalytic deg- (usually 21 nucleotides) with two nucleoradation of the target mRNA (one siRNA tide overhangs on each 3′-end. Each strand

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final oligonucleotide product typically contains a variety of closely related impurities that can be very difficult to separate and remove during final product purification (15). Some of the most common impurities include sequence deletions, such as n–1, n–2, and so on, where one or more nucleotide fails to attach to the sequence during synthesis. Additionally, depurination, oxidation, and other chemical modification or degradation of the nucleotide bases can lead to a variety of closely related impurities that can be very challenging to resolve from the desired product. When dealing with double-stranded siRNAs, the sample mixtures can become even more complex with each strand introducing its own set of impurities. These impurities include mismatched sequences and noncomplementary single stranded sequences. The presence of these impurities in a therapeutic mixture can lead to unwanted, nontargeted gene silencing, while the presence of any nonhybridized single-stranded RNAs can also lead to a decrease in therapeutic potency (40). Therefore, when developing siRNA therapeutics, one of the major challenges is ensuring good purity to minimize offtarget gene silencing effects. Consequently, developing good chromatographic techniques is often critically important in the oligonucleotide drug development process. Figure 1: RNA interference mechanism. Long dsRNA in the cytoplasm is cleaved into 21-mer strands (siRNA) by the protein, Dicer. Small interfering RNA is incorporated into the RNA-induced silencing complex (RISC), where the passenger strand is unwound and degraded leaving the guide strand bound to RISC. The RISC-guide strand complex base-pairs with a complementary sequence of the mRNA and induces cleavage of the mRNA, thereby preventing protein translation. Synthetic siRNAs can be introduced into the cell and achieve the same action in the RNAi mechanism.

has a 5′-phosphate group and a 3′-hydroxyl group (Figure 3). Various chemical modifications are often made to synthetic oligonucleotides to prevent attack by nucleases, which can lead to siRNA degradation and instability (11,12). Incorporation of either a fluoro or methoxy group into the 2′ position of the sugar or the use of a phosphothioate linkage is commonly used to improve siRNA stability (Figure 2) (13). In the phosphothioate modification, oxygen in the phosphodiester linkage is replaced with a sulfur atom. This introduces an additional stereocenter into the molecule giving rise to two possible diastereomers for every phosphothioate linkage, and making the resulting oligonucleotide sample mixtures highly complex and very difficult to chromatographically

Chromatographic Separation of Oligonucleotides

When developing analytical methods for the separation of oligonucleotides, some of the unique features of these molecules need to be considered. First, the pKa of resolve. All of these modifications help to the phosphodiester linkage is 2, meaning improve oligonucleotide stability while that in aqueous solution above pH 4 these retaining, and sometimes even increasing, molecules contain one negative charge for their silencing activity. These modifications every phosphodiester linkage — that is, a also tend to increase the hydrophobicity of 21-mer oligonucleotide contains 21 negathe oligonucleotides, while also increasing tive charges at pH 7. Therefore, traditional the temperature at which the duplex melts reversed-phase liquid chromatography (LC) (Tm) into its corresponding single strands. conditions tend not to work well, and ionOligonucleotides are readily synthesized pair reversed-phase LC or anion-exchange via stepwise synthesis using phosphorami- chromatography techniques need to be dite chemistry with automated solid-phase considered. Secondly, many single-stranded synthesizers (14). Although the individual RNA oligonucleotides can form higher synthetic process reactions can be very order (tertiary) structures including bends, efficient and provide high yields, the total loops, dimers, or other aggregates, and elenumber of synthetic steps for making a vated chromatographic temperatures must 21-mer RNA can be more than 80 chemi- be used to “melt” such structures, allowing cal steps (with about four chemical steps for for efficient analysis (16). Also, when anaeach cycle). Consequently, because of the lyzing single-strands versus duplex samples, accumulation of many small errors, the different methods must be considered, with


APRIL 2014 RECENT DEVELOPMENTS IN LC COLUMN TECHNOLOGY

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anion-exchange chromatography is used with positively charged stationary phases that exchange the negatively charged oligonucleotides through competition with anions from the mobile phase. As a result, longer oligonucleotides with greater charge are more strongly retained on the column and shorter oligonucleotides, which have progressively fewer negative charges, have progressively shorter retention. This method is especially useful for the separation of the very common N–x deletions of oligonucleotides (Figure 5a), but is less useful for detecting subtle changes on the fulllength sequence that do not alter the total number of charges (Figure 5b). Successful separation of small oligonucleotides and their sequential analysis by anionexchange HPLC was originally reported more than 30 years ago (21,22). In these studies, anion-exchange columns, such as Permaphase AEX (DuPont) or Partisil SAX Figure 2: Oligonucleotide structure. Different modifications include phosphothioate (HiChrom), with a salt gradient using phosbackbone modification where one oxygen atom on the phosphodiester backbone is phate or acetate buffers were used. Later, replaced with a sulfur atom, and 2’-sugar modifications, such as 2’-F and 2’-O-Me. Pingoud and colleagues (23) showed the use The four bases in RNA are adenine (A), guanine (G), cytosine (C), and uracil (U). of strong-anion-exchange LC for the excellent resolution of longer oligonucleotides (up to 64 bases) from their N–1 deletion products, using a Whatman SAX column and a phosphate salt gradient. Over the years, improved types of anion-exchange resins were developed to improve the resolution of oligonucleotides with strong-anionexchange LC. Alkylamine derivatized (24) and polyethyleneimine (PEI) coated (25,26) Figure 3: Small interfering RNA (siRNA) structure. A 21-mer siRNA is shown with two porous silica phases were prepared for the nucleotide overhangs on each 3’-end. Each strand has a 5’-phosphate group and a anion-exchange HPLC separation of oligo3’-hydroxyl group. The siRNA duplex consists of two complementary strands, the nucleotides, where resolution of oligonuclesense (or passenger) and antisense (or guide) strands. In RNA, adenine base-pairs otides of up to 30 bases from their N–1 with uracil by forming two intermolecular hydrogen bonds (A–U) and guanine basepairs with cytosine by forming three intermolecular hydrogen bonds (G–C). deletion products was obtained. Optimization of the PEI bonding chemistry with single-strand analysis typically taking place ion-pair reversed-phase LC (34–51), and quaternization of the ion-exchange matrix at temperatures >60 °C and duplex samples mixed-mode LC (52–56). These different was shown to further increase the resolution typically being analyzed at or below room LC techniques are reviewed in the follow- of N–1 deletion products for up to 50-mer temperature. A typical siRNA duplex LC ing sections. oligonucleotides (27). analysis is shown in Figure 4. Finally, since For chromatographic separations using RNA and DNA molecules both have a Ion-Exchange harsher conditions such as elevated colstrong absorbance at 260 nm, these sam- Liquid Chromatography umn temperatures and neutral-to-high pH ples are often analyzed using UV detection, Ion-exchange chromatography is an mobile phases, the relative chemical instaalthough fluorescence and especially mass excellent method for separating charged bility of silica-based stationary phases can spectrometry (MS) detection (17) are also molecules, and is a commonly used chro- be a significant disadvantage, because parquite important for RNA analysis. matographic method for the separation of ticle erosion and column degradation can A number of chromatographic methods multiply charged oligonucleotides (20). In lead to a significant loss of performance have been reported for the analysis and ion-exchange chromatography, separation over time (28). Consequently, PEI-coated purification of oligonucleotides, includ- is based on the differential electrostatic porous zirconia stationary phases were proing capillary gel electrophoresis (CGE) affinities of charged molecules for a charged vided by Professor Carr for use as anion (18,19), anion-exchange high performance stationary phase. For the separation of exchangers in the strong-anion-exchange liquid chromatography (HPLC) (20–33), highly negatively charged oligonucleotides, LC of oligonucleotides (29). These stationary

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Figure 4: A typical UHPLC analysis of duplex 21-mer siRNA. Column: 100 mm × 2.1 mm, 1.7-µm dp Waters CSH C18; mobile-phase A: 400 mM HFIP–16.3 mM TEA in water (pH 7.9); mobile-phase B: methanol; segmented linear gradient: 25–33% B in 10 min, 33–36% B in 20 min, 36–60% B in 28 min, 3-min equilibration at 25% B; total run time: 31 min; flow rate: 0.3 mL/min; detection: UV absorbance at 260 nm; column temperature: 15 °C.

phases were able to provide single-nucleo- been developed and successfully used for tide unit resolution for oligonucleotides of oligonucleotide separations (32–34). up to 50-nucleotide length. Furthermore, the zirconia-based stationary phases can be Ion-Pair Reversed-Phase operated at elevated column temperatures Liquid Chromatography of 75 °C, which allow for the highly advan- Ion-pair reversed-phase LC is another very tageous elution of oligonucleotides with commonly used LC technique for the anala low ionic strength mobile phase; those ysis and separation of oligonucleotides (35). conditions lead to loss of performance and In ion-pair reversed-phase LC, negatively bed collapse with conventional silica-based charged oligonucleotides interact with posicolumns. tively charged alkylammonium ions in a The development of nonporous anion way that permits the chromatographic sepexchangers for use in strong-anion- aration of the former on a reversed-phase exchange LC of proteins (30) and oli- stationary phase. Different mechanisms gonucleotides (31) is another important regarding the ion pair interaction have been milestone. These nonporous columns, such proposed, depending on whether the ionas TSKgel (Tosoh Biosciences), were pre- pair process occurs in the mobile phase (36) pared by introducing diethylaminoethyl or in the stationary phase (37). In the first (DEAE) groups into nonporous spherical proposed mechanism, ion-pair formation hydrophilic resins with 2.5-µm diameter occurs in the aqueous mobile phase, and particles. The effects of some of the criti- the neutralized ion pair is then adsorbed cal chromatographic parameters in strong- onto the hydrophobic stationary phase, anion-exchange LC for oligonucleotides with retention controlled by the overall were also studied. Examination of the elu- hydrophobicity of the ion pair. In the secent pH showed that pH should ideally be ond mechanism, the unpaired ion (such as ≥8.5 for the separation of oligonucleotides. an alkylammonium ion) from the mobile Furthermore, the range of pH 8.5–9.5 was phase is adsorbed onto the stationary phase, shown to be better suited for separations which then acts as an ion-exchange stationbased on differences in chain length, while ary phase for the separation of charged olia pH of 10.5 was better suited for separa- gonucleotides. tion based on differences in base compoIn ion-pair reversed-phase LC, in addisition. Because these stationary phases are tion to the charge–charge interactions, chemically very stable, operating at high hydrophobic interactions from the indipH would not cause any problems. The vidual bases also significantly contribute addition of salts, such as sodium chloride or to the overall oligonucleotide retention. sodium perchlorate, also has an influence The hydrophobicity of oligonucleotide on chromatographic separation. Finally, bases follows the order C < G < A < T other useful methacrylate-based (polymeric) (for DNA based oligonucleotides), with anion-exchange stationary phases have also cytosine being the least hydrophobic base


(38,39). Therefore, in addition to the oligonucleotide length, the retention of oligonucleotides also depends on the specific base composition, where the overall hydrophobicity is the sum of all the bases in the sequence. As such, ion-pair reversed-phase LC can be especially useful for separating impurities from changes in the full-length sequence such as depurinations and other chemical modifications on the bases. Separation of an oligonucleotide and a series of its deletions (N–1 to N–15) are shown in Figure 6a. This illustrates the retention of oligonucleotides by ion-pair reversed-phase LC in which the longer oligonucleotides are more retained, but overall retention is mostly governed by the base composition, where cytosine (the least hydrophobic) base deletions resulted in increased retention. Additionally, the ion-pair reversed-phase LC method provided some separation of very subtle differences on the oligonucleotide sequence, such as the reversal of two neighboring bases at different locations along the sequence (Figure 6b). Triethylammonium acetate (TEAA) is one of the most commonly used ion-pair reagents for the separation of oligonucleotides because of its good separation efficiency (40,41). Typical TEAA concentrations in the aqueous solution are 100 mM at pH 7 with acetonitrile as the organic solvent. Gilar and colleagues (42) did an extensive study of the prediction of retention for oligonucleotides with ion-pair reversed-phase LC using TEAA at pH 7 as the ion-pairing reagent. Using 39 different oligonucleotides, they demonstrated the successful application of a model for the prediction of the mobile phase strength required to elute the oligonucleotides. Shallow linear gradients of organic modifiers are typically used for these separations because studies have shown that there can be a sharp change in the retention factor (k) of oligonucleotides with a small change in the mobile phase strength. Gilar and colleagues (42) reported that the retention factor for a 15-mer oligonucleotide decreased from 100 to 13.5 to 3.2 with a very small change of mobile-phase composition from 8% to 9% to 10% acetonitrile. Another very useful ion-pairing mobile phase is the combined hexafluoroisopropanol (HFIP) and triethylamine (TEA) buffer system proposed by Apffel and colleagues (43). This HFIP-based separation uses methanol as the organic modifier


Figure 5: Chromatographic separation of oligonucleotide standards by strong-anionexchange LC: (a) Separation of N–x deletion series, (b) separation of “base-flip” isomer standards (25–29 min portion of chromatogram shown). Column: 150 mm × 4.6 mm, 3-µm dp Proteomix SAX-NP3 (Sepax Technologies); mobile-phase A: 80:20 (v/v) 10 mM Tris (pH 8)–ethanol; mobile-phase B: 600 mM sodium bromide salt concentration in mobile-phase A; linear gradient: 20–80% B over 30 min with 5-min column equilibration at 20% B; flow rate: 0.5 mL/min; column temperature: 60 °C; injection volume: 15 µL; detection: UV absorbance at 260 nm. Adapted with permission from reference 59.

because HFIP is immiscible with aceto- by evaporation. First, comparing the volanitrile and miscible with water, methanol, tilities for the two different buffer systems, isopropanol, and hexane. Using 400 mM HFIP (boiling point [bp] = 57 °C) is more HFIP and adjusting the solution to pH volatile than TEA (bp = 89 °C), with acetic 7.0 with TEA, comparable separations acid being the least volatile (bp = 118 °C). were obtained as those with the 100 mM Secondly, the weak acid and base system TEAA mobile phases. The main difference, with HFIP and TEA maintains a stable pH however, was in the electrospray ionization at ~7.0. The pKa values of acetic acid, HFIP, (ESI) performance for the MS detection of and TEA are 4.75, ~9, and 11.01. Therefore, oligonucleotides. Comparison of different at pH 7.0, acetic acid is completely dissocisolvent systems, such as 400 mM HFIP ated and it cannot be removed by evaporaadjusted to pH 7.0 with TEA, water, 100 tion on the MS source, whereas HFIP is mM TEAA (pH 7), and 25 mM TEA (pH not charged and it can be evaporated freely. 10), showed superior MS signal with the Furthermore, the mechanism proposed by 400 mM HFIP buffer system, compared to Apffel suggests that during the separation, significantly suppressed MS signal with the the TEA ions ion-pair with the negatively 100 mM TEAA buffer. This significant charged phosphates on the oligonucleotide difference in MS detection was attributed backbone, because the more volatile HFIP to the dynamic adjustment of the pH in the is evaporated from the droplet surface causESI droplet as a function of the removal of ing the pH on the surface to rise (~ pH 10). the anionic counterion from the droplet As the pH rises, the oligonucleotide–TEA


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ion pair dissociates, and the oligonucleotide can be desorbed into the gas phase. Additionally, the role of TEA is also very important in this mechanism because, in general, oligonucleotides have a high binding affinity for Na+ and K+ cations on the polyanionic phosphate backbone and these cation adducts can diminish the sensitivity for electrospray ionization. The use of a strong base such as TEA effectively suppresses the sodium and potassium adducts formation by a displacement mechanism and consequently dramatically increases the ESI sensitivity. This HFIP and TEA mobile-phase system is now routinely used for oligonucleotide analysis with ion-pair reversed-phase LC and ESI-MS detection (43–50). Gilar and colleagues (47) further evaluated the HFIP and TEA buffer system and showed that concentration of the ion-pairing TEA ion, rather than the concentration of HFIP, in the mobile phase plays a critical role in the separation. They extensively studied the effect of the concentration of both, TEA and HFIP in the mobile phase by varying the TEA concentration from 0.56 to 31.4 mM range (pH 7–9), and the HFIP concentration from 12.5 to 400 mM, using oligonucleotides up to 30-mer length. They showed that the HFIP and TEA buffer system was most effective at 400 mM HFIP and 16.3 mM TEA concentration (where 16.3 mM TEA was the highest concentration possible to dissolve in 400 mM HFIP at room temperature). Interestingly, they also showed that HFIP effectively disrupts any oligonucleotide secondary structures and this buffer can be an efficient denaturant that allows for more efficient oligonucleotide separations. As reported by Huber, Oefner, and Bonn (39,51–53), nonporous poly(styrene–divinylbenzene) (PS-DVB) particles with a diameter of 2.1 µm were prepared and successfully used for the ion-pair reversedphase LC separation of oligonucleotides with methods using 100 mM TEAA as the ion-pairing reagent and a column temperature of 50 °C. Other reversed-phase media such as porous C18 columns can also be used. The mass transfer of relatively larger molecules such as oligonucleotides is one of the major factors contributing to peak broadening on porous C18 stationary phases. A study using 50 mm × 4.6 mm columns packed with 5-, 3.5-, and 2.5-µm particles

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typical oligonucleotide impurities, such as N–x deletions, which can be well separated by either strong-anion-exchange LC or ion-pair reversed-phase LC alone, these columns also showed an excellent separation of very challenging isomeric oligonucleotides where one single nucleotide base was reversed with its neighboring base, affording separations that could not be achieved by either strong-anion-exchange LC or ion-pair reversed-phase LC alone. A mixed-mode chromatography separation of oligonucleotides with the Scherzo SM-C18 column and sodium chloride salt gradient is shown in Figure 7. Future Approaches to the Chromatographic Separation of Oligonucleotides

Figure 6: Chromatographic separation of oligonucleotide standards by ion-pair reversed-phase LC: (a) Separation of N–x deletion series, (b) separation of “base-flip” isomer standards (11–15 min portion of chromatogram shown). Column: 150 mm × 4.6 mm, 3.5-µm dp XBridge C18 (Waters); mobile-phase A: 100 mM TEAA in water; mobile-phase B: 100 mM TEAA in acetonitrile; linear gradient: 5–10% mobile phase B over 15 min with 5-min column equilibration at 5%; flow rate: 1.5 mL/min; column temperature: 65 °C; injection volume: 15 µL; detection: UV absorbance at 260 nm. Adapted with permission from reference 59.

showed that the mass transfer in the sta- exchange LC and ion-pair reversed-phase tionary phase had a major impact on the LC methods, these approaches are someseparation (42). To overcome this problem, what complementary and often both columns containing smaller particles (≤2.5 methods are used for complete analysis µm in diameter) and the use of ultrahigh- and characterization of oligonucleotide pressure liquid chromatography (UHPLC) mixtures (55,56). As a result, mixed-mode instruments can be used to improve overall chromatography columns possessing both separation performance (40). Additionally, reversed-phase and ion-exchange properthe use of core–shell C18 particle columns ties have been prepared and evaluated for has been shown to significantly improve use in oligonucleotide separations (57,58). oligonucleotide separations compared to When using mixed-mode columns, oligofully porous particles (54). Systematic eval- nucleotides can experience ionic (such as in uation of different core–shell C18 columns strong-anion-exchange LC) and hydrophoshowed the best separation with a sub- bic (such as in reversed-phase LC) interac2-µm core–shell particle column. However, tions simultaneously. The dominating the long-term stability of these silica-based mode of interaction can be significantly columns when operating at neutral pH and influenced by the type of mobile phase used. elevated column temperatures of >60 °C More recently, an evaluation of differcan be an issue. ent Scherzo C18 mixed-mode (Imtakt USA) columns showed a significant benMixed-Mode Liquid Chromatography efit when using mixed-mode columns Because of differences in the separation for separation of oligonucleotides (59). In mechanism between the strong-anion- addition to providing good separation of

As research on the biomedical uses of short RNA oligonucleotides continues, we can expect ongoing development of improved methods to chromatographically analyze and purify these fascinating compounds. Given the readiness with which these molecules engage in Watson-Crick base pairing with complementary oligonucleotide strands, affinity chromatography–like approaches in which a complementary oligonucleotide stationary phase is used for the targeted retention and chromatographic separation of particular oligonucleotide products may be possible for the analysis, and especially purification, of RNA oligonucleotides. Affinity chromatography purification of oligonucleotide binding proteins has been carried with an oligonucleotide affinity column (oligo (dT)12-18 cellulose) (60) and more recently, with a stationary phase consisting of 2′-fluoro modified RNA covalently linked to agarose beads (61). Oligonucleotide hybridization has long been used in the formation of DNA microarrays and similar technologies (62,63), and can reliably be used for the selective capture of particular oligonucleotide sequences. While these experiments involve only simple binding at lower temperature with release at elevated temperature, true chromatographic separation may be possible on complementary oligonucleotide stationary phases operated at elevated temperatures, or in the presence of mobile phase additives that make adsorption or desorption fast on the “HPLC time scale.” Such stationary phases could, in principle, be tailor-made for analytical or purification tasks such as the selective binding of desired



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another useful method for selective separation and purification of these important compounds. References

Figure 7: Chromatographic separation of RNA oligonucleotide samples by mixed mode chromatography using a Scherzo SM-C18 column with a sodium chloride gradient: (a) Separation of N–x deletion series, (b) separation of “base-flip” isomer standards. Mobile-phase A: 100 mM Tris (pH 7.4) in water; mobile-phase B: 90:10 (v/v) 2 M sodium chloride in 100 mM Tris (pH 7.4) water–acetonitrile; linear gradient: 50–85% B over 35 min; flow rate: 1 mL/min; column temperature: 50 °C; injection volume: 3 µL; detection: UV absorbance at 260 nm. Adapted with permission from reference 59.

target oligonucleotides, or of an otherwise difficult-to-remove isomeric or closely related impurity. A preliminary investigation showed that the use of RNA-based stationary phases for selective purification of short RNA sequences suffers from rapid degradation of the RNA stationary phase at elevated column temperatures (64), a problem that could potentially be solved by the use of more thermostable DNA-based stationary phases or even the use of complementary phases based on peptide nucleic acids (PNAs), which are significantly more thermostable than either RNA or DNA, but retain the ability to form complementary duplexes (65). Conclusions

Developing good chromatographic methods for the accurate and sensitive analysis and separation of oligonucleotides is a critical part in biomedical investigations involving antisense and siRNA oligonucleotides. In this review, we have surveyed different

LC approaches including strong-anionexchange, ion-pair reversed-phase, mixedmode, and affinity liquid chromatography. Strong-anion-exchange LC is one of the most often used methods in which separation of oligonucleotides is mainly based on the different charges, making this technique especially useful for the separation of the very common N–x deletion impurities. Ion-pair reversed-phase LC is also another commonly used method where, in addition to charge–charge interactions, hydrophobic interactions mainly govern the retention and separation mechanism, making this technique useful for detecting small chemical changes on the full-length sequence. Mixed-mode chromatography, consisting of both reversed-phase and ion-exchange separation modes, provides additional benefits where a single column and method can be used for complete oligonucleotide analysis. Finally, preliminary studies suggest that oligonucleotidebased chromatographic separation may be

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Bing Mao

Mirlinda Biba

Direct correspondence to: [email protected] ◾

is an Associate Principal Scientist in the Analytical Chemistry Department at Merck in Rahway, New Jersey. She is also a part-time PhD student at Drexel University in Philadelphia, Pennsylvania, studying under the direction of Professor Joe Foley and Dr. Chris Welch. Her research focuses on analysis and separation of short RNA oligonucleotides by different liquid chromatography techniques.

is currently Director, Analytical Chemistry within Process and Analytical Chemistry Department at Merck Research Laboratories in Rahway. He has more than 17 years of experience at Merck with analytical characterization and development to support pharmaceutical small molecule, peptide, and oligonucleotide drug substance manufacturing process development and optimization.

Christopher J. Welch

is a science lead for analytical chemistry within the Process and Analytical Chemistry area at Merck Research Laboratories in Rahway. Chris also co-chairs the New Technologies Review and Licensing Committee (NT-RLC), the organization that oversees identification, acquisition, and evaluation of new technologies of potential value to Merck Research Laboratories. Chris also co-chairs the MRL Postdoctoral Research Fellows Program.

Joe P. Foley

is Professor of Chemistry and Associate Department Head at Drexel University, and a lifetime member of the Chromatography Forum of the Delaware Valley (CFDV). He received his PhD in Chemistry from the University of Florida, and followed it with a two-year NRC postdoc at NIST. His research interests are in the fundamental and applied aspects of analytical chemistry and separation science, and he has authored or co-authored more than 110 articles, book chapters, reviews, and one patent pertaining to pressure- and voltage-driven liquid-phase chiral and achiral separations (that is, HPLC, UHPLC, SFC, and CE/EKC).

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