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Bioanalysis of siRNA and oligonucleotide therapeutics in biological fluids and tissues

Bioanalysis of siRNA and oligonucleotide therapeutics in biological fluids and tissues

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This article summarizes bioanalytical avenues for the determination of siRNA and oligonucleotide therapeutics, with an emphasis on hybridization methods. Aspects of the chemistry and delivery of investigational oligonucleotide therapeutics are considered. The nature of the oligonucleotide under investigation will dictate the best analytical course of action; each method has its advantages and disadvantages, depending upon the oligonucleotide test article and the anticipated toxicokinetic and pharmacokinetic study parameters. Stringent method development and specific validation criteria are essential to attain the best quality results in support of a regulatory filing.
This article summarizes bioanalytical avenues for the determination of siRNA and oligonucleotide therapeutics, with an emphasis on hybridization methods. Aspects of the chemistry and delivery of investigational oligonucleotide therapeutics are considered. The nature of the oligonucleotide under investigation will dictate the best analytical course of action; each method has its advantages and disadvantages, depending upon the oligonucleotide test article and the anticipated toxicokinetic and pharmacokinetic study parameters. Stringent method development and specific validation criteria are essential to attain the best quality results in support of a regulatory filing.

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Review

Bioanalysis of siRNA and oligonucleotide therapeutics in biological fluids and tissues
This article summarizes bioanalytical avenues for the determination of siRNA and oligonucleotide therapeutics, with an emphasis on hybridization methods. Aspects of the chemistry and delivery of investigational oligonucleotide therapeutics are considered. The nature of the oligonucleotide under investigation will dictate the best analytical course of action; each method has its advantages and disadvantages, depending upon the oligonucleotide test article and the anticipated toxicokinetic and pharmacokinetic study parameters. Stringent method development and specific validation criteria are essential to attain the best quality results in support of a regulatory filing.
Oligonucleotide (OGN) biopharmaceuticals are currently being investigated at preclinical and clinical stages for various disease indications. To date, fomivirsen, an antisense oligonucleotide (ASO) phosphorothioate (PS) targeted at cytomegalovirus retinitis, and pegaptanib, an antiangiogenic pegylated aptamer for the treatment of neovascular age-related macular degeneration, have been approved by the US FDA in 1998 and 2004, respectively [301,302] . Investigational nucleic acid-based therapeutics occupy an increasingly important space in the biopharmaceutical drug-discovery and -development landscape. It is expected that more OGN drugs will reach the market, considering the relative potency of later generation compounds, such as siRNA [1] , the capacity for controlled manufacturing of OGN and the appealing prospect of simple and effective drug design. Oligonucleotide drug candidates require robust bioanalytical assays for their determination in increasingly complex biological matrices, such as skin, colon or brain tissue. In addition, sensitive assays are required for the lower therapeutic doses that more effective drug regimens and targeted delivery will bring about. The bioanalytical method platform is also carefully selected based on the structure and function of the therapeutic OGN. Numerous classes of therapeutic OGN compounds exist, including:
n n n n n

miRNA-blocking OGNs [8]

RNA decoys [9] Splice switching/exon skipping OGNs [10] Ribozymes and DNA enzymes [11]

siRNA [2,3] ASO OGNs [4] Aptamers [5] Immunomodulatory OGNs (IMOs) [6,7]

n

n

n

Current investigational therapeutic OGNs are typically either siRNA, ASO, IMOs or aptamers. ASOs are complementary to a targeted, diseaseassociated RNA molecule, generally a mRNA. The classical ASO mechanism is based on triggering RNase H cleavage of the ASO–RNA target duplex [4] . ASOs pioneered the therapeutic OGNs, and a number of OGN chemistries were developed for ASO that have been adapted for use with other OGNs. Toll-like receptors (TLRs) are patternrecognition receptors and recognize molecules common to bacteria and viruses. TLR9 is expressed in two types of cells of the immune system: plasmacytoid dendritic cells and B cells. IMOs containing unmethylated CpG motifs mimic nonmammalian DNA and therefore bind to the TLR9, thereby inducing proinflammatory cytokines and Th1-type immune responses [6,7] . They are agonists of the innate immune system. Aptamers are OGNs that have been selected via in vitro molecular evolution techniques [5] . They are ligands for specific molecular targets, mostly proteins of therapeutic relevance. Aptamers are typically selected using an iterative molecular evolution technique known as Systematic Evolution of Ligands by Exponential enrichment (SELEX) [12] . They are habitually designed to work at the extracellular level. Recent advances with RNAi and siRNA synthetic compounds have fueled interest in therapeutic OGNs in recent years. Andrew Z Fire and
Bioanalysis (2009) 1(3), 595–609

Guy A Tremblay1 & Philip R Oldfield1† † Author for correspondence 1 Immunochemistry, Charles River Preclinical & Clinical Services, 22022 Transcanadienne, Senneville, QC H9X 3R3, Canada, Tel.: +1 514 630 8263 Fax: +1 514 630 8230 E-mail: philip.oldfield@crl.com
Antisense Complement strand of a targeted nucleic acid, typically mRNA AptAmeR Oligonucleotide ligand obtained by in vitro evolution
siRnA

19–25-nucleotide long double-stranded RNA molecules involved in the RNAi pathway theRApeutic oligonucleotide A class of oligonucleotides consisting of approximately 10–40 DNA, RNA or modified nucleic acid monomers used for therapeutic applications

10.4155/BIO.09.66 © 2009 Future Science Ltd

ISSN 1757-6180

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Craig C Mello were awarded the 2006 Nobel Prize for their discovery of RNAi gene silencing by double-stranded RNA [303] . RNAi is a natural mechanism of RNA inhibition mediated by small, double-stranded RNA molecules of 19–25 nucleotides. The RNAi field expanded fast within academia, where siRNA molecules were designed to knock-down the expression of a selected gene in cell culture. It has been reported that a proper selection of the target sequence may typically lead to a knockdown of approximately 75% of a given mRNA and its protein product thereof [1] . Synthetic siRNAs have been demonstrated to target genes in vivo for multiple diseases, including ovarian [13] and bone cancer [14] , hypercholesterolemia [15] , liver cirrhosis [16] , respiratory syncytial virus [17] , hepatitis B virus [18] and human papillomavirus [19] . Delivery and modification are important areas of focus for OGN drug development, since they can improve upon aspects of tissue-specific targeting, cell entry, stability and potency. Improved cell-specific targeting and transfection efficiency will help to lower the effective dose. For those OGNs that act at the extracellular level, robust modification chemistries that enhance the half-life and solubility of the OGN in plasma will be necessary in order to prevent degradation by circulating nucleases. Quantitative, highly specific and sensitive bioanalytical methods are required to determine the toxicokinetic (TK)/pharmacokinetic (PK) parameters and exposure–response in order to choose the right dosage regimens of therapeutic OGNs in support of their preclinical and clinical development. The methods will be used to measure OGN concentrations in plasma, urine, bile, feces and solid tissues, typically kidney, liver, brain and spleen; but other target tissues, such as skin and vitreous humor, have also been investigated. Chemistry of investigational OGN therapeutics Several modifications to investigational therapeutic OGNs have been introduced over the years. They can involve the phosphodiester linkage group, the ribose sugar moiety or the nucleotide bases. Termini can be modified as well; for example, additions to the 5´ or 3´ ends of OGNs with polyethylene glycol (PEG) for aptamers [20] , cholesterol for siRNA [21] or peptides for exon skipping OGNs [22] have been developed.
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phARmAcokinetics Describes the relationship between mechanisms of drug absorption, distribution and elimination over time

For therapeutic OGNs to be part of clinical trials, they are required to be robust, safe and effective. F iguRe 1 depicts native RNA (FiguRe 1A) and modification chemistries used with investigational therapeutic OGNs. The approved ASO therapeutic fomiversen has a phosphorothioate (PS) oligodeoxynucleotide (ODN) chemistry. PS ODNs are the first-generation ASOs intended to increase the nuclease resistance and cellular uptake of the phosphodiester backbone in vivo [23] . The PS modification, where a nonbridging oxygen in the phosphodiester link is substituted with sulfur (FiguRe 1B) , imparts considerable stability to PS ODNs in vivo. The PS group also confers binding affinity towards proteins [24] , which may help to protect the OGN from circulating nucleases [25] . The pegaptanib aptamer, the other approved therapeutic OGN, is pegylated at the 5´ end and has an inverted 3´-3´deoxythymidine residue at the 3´ end. Pegaptanib is 2´-O-methylated (2´OMe) on purines and 2´-fluorine-modified (2´F) on pyrimidines [26] . The 2´-F-modified (FiguRe 1c) nucleic acid derivatives adopt the A-form typically found in RNA, bind targets with high affinity and are more resistant to nucleases in vivo [27] . The 2´F is a useful modification for aptamers, since the 2´F residues can be incorporated by RNA polymerases used with in vitro molecular evolution techniques [28] . Of note, the RNAi pathway is tolerant to 2´F derivatives, which also makes the modification useful for siRNA therapeutic applications [29] . The 2´O-Me RNAs (FiguRe 1d) are secondgeneration ASO modifications and confer considerable protection from nucleases while having similar hydrogen bonding properties to RNA– RNA hybrids [23] . An interesting property of 2´OMe is related to their propensity to abrogate the inherent immunostimulatory characteristics of OGNs when added selectively to guanosine or uridine residues of a siRNA [30] , considering the observation that the siRNA under investigation may function via an unspecific immune-stimulatory mechanism [31] . Interestingly, the 5-methylcytosine (5mC) nucleobase modification, naturally found in CpG motifs, has also been shown to reduce immunogenicity for a PS ODN [32] . Typically used in combination with PS , 2´- O -me t hox y e t hy l ( 2´MOE ; F iguRe 1e )-modified ODNs are also secondgeneration ASO modifications [33] . In addition to supporting RNase H cleavage [34] , OGNs with the 2´MOE modification have increased affinity
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Bioanalysis of siRNA & oligonucleotide therapeutics in biological fluids and tissues
A. RNA B. Phosphorothiate oligodeoxynucleotide

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O OP O O base O O-

O P S O base O

O C. 2´-fluoro RNA O OP O O

OH

O D. 2´-O-methyl RNA O base OP O O base O

O

O E. 2´-O-methoxy ethyl

F F. Locked nucleic acid

O

O CH3

O O
-

O O O base OP O O base O

P O

O

O

O

CH3

O

O

G. Morpholino oligomer O N H3C P O O O base

H3C

N O

Figure 1. Chemical modifications developed for investigational therapeutic oligonucleotides.

towards target RNA and increased nuclease stability relative to unmodified phosphoramidate (PN) ODNs [35] . One example of a second-generation molecule modified with PS and 2´MOE in clinical development is OGX-011, an ASO designed to block
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the production of clusterin, a cell-survival protein that is overproduced in several cancer indications [36] . OGX-011 is complementary to the mRNA translation initiation site and has been shown to inhibit clusterin expression in in vitro and in vivo laboratory models [37] .
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Also currently in clinical trials, EZN-2968 is a synthetic antisense (AS) ODN-targeting hypoxia-inducible factor (HIF), with potential antineoplastic activity resulting in inhibition of angiogenesis, the inhibition of tumor cell proliferation and apoptosis [38] . EZN-2968 is a mixed polymer consisting of a PS core with locked nucleic acid (LNA) components at the 5´ and 3´ ends. Along with protection from degradation in vivo, the LNA modification (FiguRe 1F) keeps the ribose moiety in a C3´-endo conformation and locks the OGN in the A-form. This results in improved nucleobase stacking interactions, as well as higher affinity and specificity [39] . Designing probes with LNA nucleotides will also increase hybridization kinetics and impart stability to duplexes by effectively increasing the melting temperature. In morpholino OGNs, the ribose sugar moiety is replaced with morpholine rings and the anionic phosphodiester linkage is replaced with nonionic phosphorodiamidate groups (FiguRe 1g) . The morpholino oligomers are often used as an ASO technology to block translation or interfere with RNA processing, including splicing and miRNA maturation [40,41] . They differ from traditional AS ODNs in that they function by steric hindrance of the target sequence rather than ASO-mediated RNase H degradation [42] . One example is AVI-4658, an exon-skipping OGN in clinical development, designed to skip exon 51 of the dystrophin gene, allowing for restoration of the reading frame in the mRNA sequence in patients with Duchenne muscular dystrophy [43] . Delivery systems Oligonucleotides can be formulated to increase their half-life in vivo and to modulate delivery to specific organs and tissues. Delivery vehicles will also help the intrinsically negatively charged and bulky OGN to cross the cell membrane. Thus, although a number of OGN compounds, including siRNA, have been shown to be active ‘naked’, or unformulated, further OGN drugs will benefit from safe and effective delivery systems [44] . Liposomes and lipid-like particles can be charged with pharmaceuticals in their aqueous center, protected from the extracellular environment by a spherical lipid bilayer [45] . Liposomes are divided into three classes: multilamellar vesicles, small unilamellar vesicles and large unilamellar vesicles. The latter class is preferred for OGN drugs because of its ability to achieve favorable drug–lipid ratios and more predictable drug-release kinetics [23] .
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hyBRidizAtion AssAy Is a ligand-binding assay used for the determination of the oligonucleotide therapeutic

Several lipid-based technologies for nucleic acid delivery exist. Recently, stable nucleic acid– lipid particle formulations have been used for a number of disease models in vivo. Zimmermann et al. were the first to demonstrate sequence-specific RNAi in nonhuman primates using a stable nucleic acid–lipid particle formulation [46] . A single injection of siRNA resulted in a maximal silencing of more than 90% of the ApoB mRNA expression in the liver 48 h after administration. In addition, silencing persisted for 11 days at the highest administered dose [46] . Another important class of in vivo drugdelivery system is comprised of positively charged peptides and polymers, formulated with negatively charged nucleic acids, resulting in stable nanoparticles. PEG can be used to stabilize the nanoparticles and prevent aggregation [23] . Polyethyleneimine (PEI) is perhaps the most widely used cationic polymer for in vivo nucleic acid drug delivery. PEI can be either branched or linear. There has been some concern regarding the toxicity of PEI; however, different chemistries are implemented to minimize those effects [47,48] . Yet another cationic polymer, cyclodextrin, is a circular polysaccharide that has been used for siRNA-mediated gene knockdown. Used in conjunction with the transferrin protein for targeting cancer cells, Heidel et al. delivered a siRNA payload to target the M2 subunit of ribonucleotide reductase, making it the first nonhuman primate study on targeted, systemic delivery of siRNA [49] . The ideal bioanalytical method will take into consideration the delivery vehicle and formulation specifics of an investigational OGN. The use of mild nonionic detergents that disrupt the lipid bilayer, such as Triton® X-100, alone or in combination with thermal denaturation will enable solubilization of most lipids and polymers, and release the nucleic acids for hybridization without the further need for purification of the OGN for downstream quantification with hybridization assays. Comparison of recovery of formulated versus unformulated quality control (QC) samples will show whether the OGN is completely released from the delivery vehicle in the course of the bioanalytical method development. In addition, the routine use of formulated QC samples determined from an unformulated standard curve will ascertain whether release of the test article is representative of any generated study samples.
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Administration & PK Oligonucleotides have been administered by various routes in support of preclinical and clinical studies, including ocular [50] , parenteral intravenous injection [35] , intravenous infusion [51] , topical [52] , subcutaneous injection [53] , intramuscular injection [54] , lung inhalation [55] , intranasal inhalation [56] , intracerebral [57] and even oral routes [58] . The choice of drug regimen is determined by indication, intended clinical route of administration, considerations for systemic exposure and characterization of toxicity. Parenteral administration is clinically the most common way to administer OGNs and is typically the route of choice for most oncology indications. Administration of OGNs via the inhalation route is also an ideal way to locally administer therapeutics to the upper airway and lungs. Accumulation of the OGN test article is found primarily in kidney and liver tissues following systemic administration [23] . Potential toxicological effects exist as a result of complement activation and stimulation of the immune system (which is desirable for IMO OGNs). Additional clinical pathology parameters related to the kidney and liver, histological pathology staining, immunology assessments and liver enzymes can be included in the study design to characterize these effects. The systemic toxicity can be minimized by local administration of the material to the tissue of interest. Peak concentration-related toxicity, often associated with large intravenous bolus doses, can also be minimized or avoided by administration via intravenous infusion, where lower steady states can be achieved and exposure controlled by the duration of the infusion period. Infusion is also ideal for OGNs with a short biological half-life [23] . A thorough overview of PK, as well as routes and formulations for OGNs can be found in Chapters 7 and 8 of Antisense Drug Technology. Principles, Strategies and Applications; toxicity-related aspects of therapeutic OGNs are also discussed in Chapter 13 of the book [23] .
n General

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Hybridization assays considerations Hybridization assays (also known as hybridization immunoassays or hybridization ELISAs) are carried out in a microtiter plate format, such as in 96-well plates, with an OGN analyte instead of an antigen analyte, as is the case with a typical ELISA. Hybridization assays typically involve
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the hybridization of the OGN to a capture probe (immobilization) and/or to a detection probe (signaling), the detail of which is described in the following sections. Hybridization-based immunoassay methods, in general, provide the best reported assay sensitivity and throughput compared with other bioanalytical methods for OGNs. Hybridization assays require little or no sample clean-up and are therefore less time consuming. They have been used widely for the quantitative ana lysis of OGN in support of TK/PK evaluation and are particularly useful for the terminal-phase PK assessment [59,60] . A number of parameters can be optimized to increase the sensitivity of hybridization assays, if and when samples contain very low amounts of the OGN test article or if high metabolite profiles are expected. Optimization of the (capture and/or detection) probe concentration is of paramount importance, as this will directly impact the signal-to-noise ratio; the noise being derived mainly from the plasma or tissue matrices found in hybridization mixtures. The range of the standard curve between the lower limits of quantifications (LLOQ) versus the upper limits of quantification (ULOQ) can also be optimized. Customary calibration curve ranges (ULOQ:LLOQ ratio) of 50:1 with fluorimetric detection and 30:1 with colorimetric detection are typically attained. The use of nonlinear logistic regression ana lysis with four parameters (4-PL) or, more recently, with five parameters (5-PL; reviewed in [61]), as opposed to linear regression, is typical for generating the standard curve in ligand-binding assays as it helps the range, the LLOQ and the concentration accuracy of PK/TK samples. Chemically modified probes, such as LNA, can also be used to increase the hybridization kinetics [62] . Commercially available streptavidin clear plates for colorimetric analysis or opaque black plates for fluorescent detection coated with streptavidin can be used. Primary amineconjugation plates for immobilization of 5´ amino-derived capture probes can also be used for immobilization. The detection probe can be labeled with digoxigenin for detection with commercially available antidigoxigenin conjugates, for example. Signaling is produced via the reporter enzymes typically used for ELISA, such as alkaline phosphatase or horseradish peroxidase (HRP), with colorimetric or fluorimetric substrates. One
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advantage of HRP is lower noise compared with alkaline phosphatase, whose substrates may have labile phosphate groups. For the determination of OGN in plasma samples with a hybridization assay, purification of the OGN from biological samples is not necessary. If the assay involves a denaturation/ renaturation step for capture of the OGN test article by a complementary probe, for example, the fibrin and other blood components will also denature when heated at 94°C for a few minutes. However, since a relatively small amount of biological sample material (e.g., 40 µl) is required in the hybridization mixture, the denatured components of plasma will not interfere with the hybridization per se or the assay in general (Tremblay GA, Unpublished Data) . For the determination of OGNs in tissues with a hybridization assay, an initial step of extraction can be carried out using liquid–liquid extraction (LLE) with phenol and chloroform in order to remove the proteins and other organic phase-soluble contaminants from the tissues samples. This may be useful for OGNs such as PS ODNs, well known to bind proteins. Alternatively, a straightforward extraction procedure is performed on a small amount of tissue sample (e.g., 50 µl of a 200 mg/ml tissue homogenate) using proteinase K, sonication and nonionic detergents for disruption of lipid bilayers, making the extraction much easier than a LLE. With hydrophobic tissues, such as the skin, the nonionic detergents can be replaced with sodium dodecyl sulphate, an ionic denaturing detergent that is compatible with both hybridization and proteinase K (Tremblay GA, Unpublished Data) .
n Sandwich

Label

Label

Detection probe

Oligonucleotide test article

‘Sandwich complex’

Capture probe

hybridization assay The sandwich hybridization method is the simplest version of a hybridization assay (FiguRe 2) . In this dual hybridization method, a capture probe is complementary to the first portion of the OGN test article and modified to allow it to be immobilized to a solid support, while a second detection probe, modified for downstream signaling, is complementary to the second portion of the test article. The sandwich assay is simple, straightforward and may be the method of choice when dealing with complex or highly modified OGN drugs. Efler et al. used an amino-modified 5´ end to immobilize the capture probe to a microtiter plate and a 3´-labeled biotin detection probe for colorimetric detection using a substrate of HRP coupled to streptavidin [63] . They obtained very good sensitivity with a LLOQ of 10 pg/ml. However the sensitivity is largely dependent upon the sequence of the probes and the OGN test article, and it is more typical to achieve LLOQs of approximately 100 pg/ml with the hybridization sandwich assay. Apart from aptamers, which may be over 30 nucleotides long, therapeutic OGNs will often range between 16 and 25 nucleotides. This means that, for a 16-mer OGN drug, for example, each detection and capture probe will only be eight nucleotides long. Locked nucleic acid probes can be used to improve the sensitivity and kinetics of hybridization for the sandwich assay via thermal stabilization of short hybrids. Improving the hybridization kinetics is useful if the capture probe is prebound to the microtiter plate and also for hybridization of the detection probe to the immobilized OGN test article–capture probe duplex [39] .
n Hybridization–ligation

Biotin Streptavidincoated plate

Biotin Streptavidincoated plate

Bioanalysis © Future Science Group (2009)

Figure 2. Schematic representation of the sandwich hybridization assay.

assay The hybridization–ligation assay [60] , also known as the ligation-based hybridization assay, is a specific and sensitive method for measuring an OGN test article. The ligation assay requires that the 3´-hydroxyl end group of the test article is free and accessible for ligation to a phosphorylated
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ligation probe, as is the case for many, but not all, OGN test articles. A schematic of the ligation assay is shown in FiguRe 3. In the ligation assay, a template probe is designed to be complementary to the OGN test article. However, in addition to the OGN test article complementary sequence, the template probe has a generic extension at its 5´ end. This extension is complementary to a generic 5´ phosphorylated ligation probe. The 3´ end of a nine nucleotide-long ligation probe is labeled with, for example, digoxigenin, whereas the 5´ end of the template probe can be biotinylated for immobilization to the plate. The OGN test article is first denatured and then hybridized to the template probe. The hybridization mixture is then bound to a streptavidin-coated microtiter plate. The ligation ensues on a solid support (FiguRe 3) . T4 DNA ligase serves to bind the ligation probe onto the intact 3´ end of the hybridized OGN test article. After stringent washes to remove the nonligated ligation probe, the amount of signal is measured downstream of digoxigenin via reporter enzymes and substrates. The main advantage of the ligation-based assay is that 3´ end, n-truncated metabolites are hardly detectable since the efficiency of T4 DNA ligase is very low in the presence of a gap. The 5´-end metabolites will be detected; however, apparently, the vast majority of metabolites present in plasma are truncated at the 3´ end via 3´→5´ exonuclease activity [64,65] . The method is also very sensitive and compares favorably with the simpler sandwich hybridization method in this regard. The ligation reaction is versatile enough to enable a number of nucleotide chemistries, such as PS or LNA. Also, in addition to DNA, the ligation reaction is compatible with RNA OGN test article substrates, such as siRNA.
n Nuclease-based

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Label Ligation probe 3´OH Ligation site Ligation Oligonucleotide test article 5´PO4

Template probe

Biotin Streptavidincoated plate

Biotin Streptavidincoated plate Bioanalysis © Future Science Group (2009)

Figure 3. Schematic representation of the ligation step for the hybridization–ligation assay. Briefly, the ligation probe is ligated to the 3’ end of the oligonucleotide test article with the 5’ end of the template probe serving as a template for ligation.

for the complete active pharmaceutical ingredient. However, in practice, the sequence context and the intrinsic characteristics of S1 nuclease commonly used with the cutting assay may also allow the detection of n-1-truncated OGN, since the enzyme only partially degrades nicked DNA hybrids, for example [66] . Optimization of conditions and use of different single-strandspecific nucleases may help to improve discrimination [67] . Both the nuclease-based and ligation–hybridization assays are patented by Isis Pharmaceuticals (CA, USA) [201,202] .
n Competitive

hybridization assay The nuclease-based hybridization method, also known as the S1 nuclease cutting assay, takes advantage of the properties of a single strandspecific nuclease, namely S1 nuclease, to degrade the free capture probe and nonfully matched hybrids, leaving only the full-length capture probe–OGN test article duplex intact for downstream quantification. It is a variant of the classic nuclease protection assay, developed for OGNs and on a microtiter plate format. The key advantage of the cutting assay is that, in theory, only the full-length OGN test article should be detected, making the assay specific
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hybridization assay The competitive hybridization assay format involves the competition between the OGN test article and a tracer OGN (i.e., label probe), bearing the same sequence composition as that of the OGN test article for hybridization to a solid phase-tethered complementary OGN [59] . The tracer OGN can be labeled at the 3´ end with either a direct or an indirect label, depending on the sensitivity required. Direct labels include radiolabels or fluorescent and chemiluminescent substrates. Indirect labels, such as antigens (e.g., digoxigenin), can selectively interact with reporter enzymes, enabling reaction with an enzymatic substrate, resulting in a colorimetric
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or fluorescent signal. As the concentration of the test article increases, the amount of competitively bound label probe decreases, thereby resulting in a signal profile (calibration curve) that is inversely proportional to the concentration of OGN test article in the matrix. The competitive assay can be used as a third option if sandwich or ligation assays are not feasible. For example, it may be difficult to develop an assay using a sandwich hybridization for a short 12-mer OGN because the capture and detection probes will be too short and will not enable sufficient hybridization specificity, or the 3´ end of the OGN test article may be blocked or inaccessible, in addition to it being very short.
n Hybridization-based

Nuclear imaging emission tomography PET and single-photon emission tomography are high-resolution, sensitive imaging techniques that can be used for the repeated, noninvasive quantification of OGNs in animals and humans. These techniques have the same limitations as QWBA regarding lack of selectivity for the parent compound versus metabolites. One advantage is their ability to study molecular interactions and functional studies in vivo [71,72] . HPLC The challenges encountered with quantification of OGN using HPLC have been to improve the resolution between parent and metabolites and to increase the sensitivity of the technique. Improvement in separation has been performed mainly using ion-pair reversed-phase HPLC [73] but also anion-exchange HPLC [74] . Fluorescent detection has improved the sensitivity of HPLC. Sensitivities reported were 250 ng/ml by UV detection and 40 ng/ml with fluorescent detection [75] . Liquid chromatography coupled to mass spectrometry The introduction of novel soft-ionization techniques, such as electrospray ionization (ESI), enabled the ana lysis of both low- and high-molecular-mass, nonvolatile and fragile compounds by LC–MS. LC–MS combines the chromatographic separation of the components of an OGN mixture with the selectivity and sensitivity of detection found in MS, along with molecular mass and molecular structure information [76–78] . The use of ESI-LC–MS has been applied to the quantitative ana lysis of OGN. LC mobilephase modifiers and ion-pairing reagents, such as tetraethylammonium acetate [79] , hexafluoro2-isopropanol and triethylamine [80] , have been used to improve chromatography and signal intensity with MS. This approach has also been used for the ana lysis of PN-modified OGNs using LC–MS [81] . Matrix effects, including ion suppression from salts, small organic and inorganic components, proteins and nonprotein macromolecules, have detrimental effects on the ESI signal [82,83] . Therefore, purification steps are usually carried out using phenol/chloroform-based LLE and solid-phase extractions. Purification steps limit the applicability of LC–MS for the quantification of OGNs in biological matrices, especially in tissues.
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fluorescence assay A method for quantification of single-strand DNA OGNs in solution based on intercalation of fluorochromes, such as Hoechst 33258 or ethidium bromide, has been developed [68] . It does not rely on enzymatic amplification, as the usual hybridization assays do. The method makes use of the relative affinity of fluorescent intercalating agents towards double-stranded DNA hybrids as opposed to single-stranded DNA. It is claimed that the method reaches the low nanomolar range. Quantitative whole-body autoradiography For distribution of OGN test articles in animals, quantitative whole-body autoradiography (QWBA) has been applied. QWBA enables suborgan and whole-body distribution with minimal sample disruption. Labeling of the OGN can be performed, for example, with 3H or 14C. The elimination phase of the OGN can be studied with QWBA [69] . The OGN test article penetration of cells in tissues can also be qualitatively examined by micro-autoradiography [70] . The results of QWBA have a limited selectivity due to the fact that metabolites/breakdown products will be measured along with the fulllength OGN active pharmaceutical ingredient. Therefore, this technique provides good preliminary data but fails to differentiate between the parent compound and its metabolites. In addition, because only the associated radioactivity is measured, should there be any (e.g., tritium exchange with the biological medium), it would invalidate the results obtained. However, should the OGN test article bind to any previously unknown target, QWBA would be likely to detect this.

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Recently, quantification of OGNs by LC– MS/MS, where the OGN test article was preextracted from the plasma samples using LLE followed by solid-phase extraction, has been reported with promising results [84] . The quantification of a phorphorothioate OGN in tissues by LC–MS/MS with a LLE extraction has also been established [85] . Capillary gel electrophoresis Capillary gel electrophoresis (CGE) is an analytical technique with high resolution suitable for the ana lysis of therapeutic OGNs and their metabolites in biological matrices in support of TK/PK studies [86] . The capillary in CGE is filled with a gel that separates molecules by size and charge with the application of a voltage. Small, negatively charged OGN analytes move away from the anode faster than larger OGN analytes. The results are expressed as an electropherogram (similar to a chromatogram) with separation peaks and can usually resolve the parent compound from the
A 0.016 0.014 0.012 0.010 IS 0.008 0.006 0.004 0.002 0.000 7.5 10.0 Retention time (min) (n-) metabolites 0.012 Full-length PS ODN 0.014

| Review

n-1 metabolite right down to the n-9 metabolite. An example of results obtained with CGE–UV performed on human samples of a single-stranded PS ODN is shown in FiguRe 4 [87] . Similar to chromatographic methods, study samples require elaborate pre-extraction procedures prior to injection into the system, hence the need for an internal standard. In addition, the exact nature of size-separated peaks cannot always be determined, especially when it is not possible to distinguish comigrating OGN. Typically, the UV detection lower limits of quantitation are in the order of 30–100 ng/ml. However, the use of laser-induced fluorescence detection [88] has enabled a lower limit of quantification of approximately 250 pg/ml in human blood plasma with an acceptable signal-to-noise ratio. CGE coupled to MS The development of ESI-MS enabled interfacing to CGE systems in addition to LC systems for the bioana lysis of OGNs [89] .
B 0.016

0.010 UV absorbance (260 nm) IS (n-) metabolites

UV absorbance (260 nm)

0.008

0.006

0.004

0.002

0.000 10.0 12.5 Retention time (min)

Figure 4. Example of a capillary gel electrophoresis–UV electropherogram of a PS ODN in human plasma sample following (A) 4 h of infusion, or (B) in human urine at day 15 of the study, pre-end of infusion. CE system: Beckman P/ACE System MDQ; Injection: electrokinetic; 10 kV; 5–20 s; IS: Internal standard; ODN: Oligodeoxynucleotide; PS: Phosphorothioate.

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Capillary gel electrophoresis is considered to be less expensive and faster than HPLC. Smaller sample volumes and higher separation efficiency can be achieved. CGE–MS therefore combines the advantages of both techniques [90] . Freudeman et al. successfully separated and detected 12–20base long homo-oligodeoxyribonucleotides and their PN counterparts using CGE interfaced with ESI-MS [89] . The coated capillaries used for the separation contained an entangled polymer solution consisting of PEG. One problem with the use of CGE–MS is the need for high-concentrated buffer solutions, which dramatically decrease the sensitivity of MS detection. Also, and again, elaborate extraction methods are required [90] . Quantitative PCR Quantitative PCR (qPCR), also referred to as real-time PCR, is a variation of the PCR performed in capillaries, where the product formation of a PCR reaction is monitored in real time using various detection methods. qPCR enables amplification of nucleic acids up to the zeptomol range [91] , compared with the femtomol range found with immunoassays, for example. Different methods have been implemented for the quantitative determination of miRNA and siRNA, including primer extension [92] , invader assay [93] , ligation assay [94] , stem–loop reverse transcriptase PCR assay [95] and competitive qPCR [96] . Quantitative PCR is a very sensitive technique that may be useful for the determination of therapeutic OGNs in small amounts of samples, such as human biopsies obtained in the course of clinical trials. One advantage of qPCR is that there may be little or no need for sample processing and purification [96] . On the other hand, owing to the inherent nature of the PCR and due to the background that can be obtained with the formation of primer dimers, extensive optimization of the method may be required to improve precision and accuracy. In addition, the method should be selective enough to mitigate the interference of truncated OGN metabolites. Bioanalytical method development & validation Bioanalytical methods for the quantitative determination of OGN test articles are compared in tABle 1. These methods are used in the pharmaceutical industry to generate results and evaluate TK/PK profiles and the bioequivalence of the test article. The quality of these studies, which are often used to support regulatory filings, is directly related to the quality of the underlying bioanalytical data and therefore to the quality of the method-validation process. Unlike proteins, OGNs may be much less prone to lot-to-lot variability in purity and potency, which compares in this regard to small molecules. Specificity is the ability to measure the therapeutic OGN unequivocally in the presence of other components in the assay matrix. Both ligand-binding assays and/or chromatographic methods may be used dependent upon the therapeutic OGN of interest. Prior to any validation, it is always

RegulAtoRy Filing Application filed with the US FDA and/or other regulatory authorities prior to the evaluation of a medicinal therapeutic in humans

Table 1. Comparison of bioanalytical methods for the determination of oligonucleotides.
Method
QWBA HPLC CGE–UV CGE–LIF LC–MS Hybridization assay

Sample processing
Extensive tissue preparation Moderate Extensive Extensive Moderate None for plasma samples (moderate for tissues) Moderate

Sensitivity Selective for parent OGN
Moderate Low Moderate High High Very high No No Yes Yes Yes Yes

Metabolite quantification
No No Yes Yes Yes No (unless a specific probe is designed to that effect) No

Robustness Class of OGN
Good Good Good Poor Good Good All Not good for PEG aptamers Not good for PEG aptamers or double-stranded OGN Not good for PEG aptamers or double-stranded OGN All All

qPCR

Highest

No

Poor

All; ligation method required if the 3’ end is blocked

CGE: Capillary gel electrophoresis; LIF: Laser-induced fluorescence; OGN: Oligonucleotide; q: Quantitative; QWBA: Quantitative whole-body autoradiography; UV: Ultraviolet.

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important to ensure that the bioanalytical method is fully developed and that any major pitfalls are resolved before the actual validation starts. First, one determines what is expected from the method in order to choose the right assay platform, that is, hybridization, HPLC, MS, CGE and so forth. Recommended development parameters will include specificity and selectivity, accuracy and precision, and in-process stability. The method can be validated once these parameters are tackled within a fine-tuned experimental procedure. For a ligand-binding method development, as would be the case for a hybridization assay, the following items should be considered: n Assay format selection (sensitivity vs selectivity)
n n n n n n n n n

| Review

OGN class and formulation considerations Probe design Reagent selection Standard reference material Matrix selection Method optimization Reagent concentrations Incubation conditions

interest from a regulatory standpoint; for example, to verify the selective delivery of a siRNA to tissues and organs and to document the extent of nonspecific siRNA release in plasma. For a number of applications, including the determination of multiple OGN indications, multiplexed determination of therapeutic OGNs is currently implemented. For instance, not only can multiple siRNA indications be determined in one study sample, but so can cytokine expression, biomarkers, therapeutic target(s) and delivery vehicles. The determination of the two strands of a siRNA molecule can also benefit from multiplexing technologies. Luminex™ xMAP microsphere-based technology enables the simultaneous quantification of up to 100 analytes, such as cytokines. Nucleic acid quantification methods have been developed for the Luminex platform, including single nucleotide polymorphism genotyping, genetic disease screening, gene-expression profiling, HLA DNA typing and microbial detection [102] . Analysis of siRNA in multiplex with the Luminex platform has also been reported [103] .
Box 1. Validation parameters of quantitative methods for oligonucleotides.
Method validation

Prevalidation assessments For the validation itself, there are many extensive Guidance Documents and White Papers already published [97–99] . Generally, the parameters listed in Box 1 are assessed.

Future perspective siRNAs have probably become the leading class of investigational therapeutic OGNs owing to their relatively high potency [1] . New types of OGN and RNAi-related therapeutics, such as miRNA [100] or piwi-interacting (pi)RNA [101] , are expected to emerge from the wave of small RNA therapeutics. Current bioanalytical methods will need to evolve according to these new compounds and the new regulatory paradigm that it entails. Nevertheless, siRNA will benefit from the experience of its predecessors, as current quantification methods of AS ODNs are, for the most part, readily adaptable to siRNA indications. In parallel to siRNA therapeutics, several nanotechnology delivery vehicles are currently under development [44] . The qualitative and quantitative determination of delivery vehicles, in addition to the active therapeutic OGNs contained in the formulation, may become a topic of
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Prove the reliability, robustness and reproducibility of the assay Acceptance criteria for validation parameters are predefined n Accuracy should be within ±20% compared with the nominal concentration, and precision should be ≤20% n QC samples (QC1, QC2 and QC3) and standard curve prepared in the same biological matrix at anticipated study samples
n n

Range of calibration and QC sample ranges Interbatch precision and accuracy
n

Five QC levels (LLOQ, QC1, QC2, QC3 and ULOQ) in replicates of three performed on six occasions

Selectivity/specificity

Ten independent lots of matrix (e.g., plasma, urine and tissue samples) assessed blank and spiked with the analyte n Metabolite cross-reactivity
n

Dilution linearity

Use a concentrated standard in the matrix dilute with a blank sample/matrix and check for linearity of dilution n Check for parallelism using an incurred sample with a high concentration and dilute with a blank sample/matrix and check for linearity
n

Prozone (hook effect)
n

Use a concentrated standard at least 100-times the concentration of the highest calibration standard to be assayed without dilution to ensure the absence of a prozone effect In-process: room temperature, 4°C, freeze-thaw Long-term storage (-20/-80°C)

Stability
n n

LLOQ: Lower limit of quantification; QC: Quality control; ULOQ: Upper limit of quantification.

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Meso Scale Discovery (MSD™) multiplexing technology is a promising electroluminescence-based detection system making use of the microtiter plate format, with favorable sensitivity and range of detection. Also, the volume of study samples can be reduced using MSD due to a lower volume requirement for each well of the plate. The throughput with hybridization assays can be scaled-up using new miniaturization and automated liquid handling technologies. For example, the Gyrolab™ bioanalytical system miniaturizes and integrates assay steps for quantitative immunoassays on CD-microlaboratories processed with automated workstations. The assessments of immunogenicity and/or mechanism of action of siRNA therapeutics can be developed in support of regulatory filing. For a siRNA aimed at PCSK9, a 5´-rapid amplification of cDNA ends (RACE) assay has been used for demonstrating RNAi-mediated silencing activity by investigating the cleavage site of the target mRNA extracted from dosed animal tissues [15] . 5´-RACE has also been applied to monitor the duration of siRNA activity targeted at PLK1 in mice with hepatic tumors [104] . Off-target effect assessment of siRNA therapeutics, that is, the impact of an unforeseen association of siRNA to untargeted RNA [105] , may also become a subject of concern, since siRNA
Executive summary
Hybridization-based assays have several key advantages

drugs are relatively new and considering the importance of mechanisms of action for pharmacological entities and the potential unforeseen side effects associated with this class of compounds. In conclusion, bioanalytical methods will need to adapt to multiple indications for a variety of therapeutic OGNs formulated with increasingly complex delivery vehicles. Delivery targeted at specific organs and tissues will require sensitive methods for lower therapeutic doses. Sample volume can also be reduced with technological innovation, which will reduce the burden of sample collection on patients in the course of clinical trials.
Acknowledgements
The authors would like to thank Helen Legakis for critical review of the manuscript.

Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

High target specificity In general, no sample cleanup is required (e.g., plasma samples) n Minimal cleanup for tissue samples (extraction) n Good accuracy and precision/reproducibility (15–25%) n Assay designs selective for parent oligonucleotide (OGN) n Low reagent cost n Low instrumentation cost n Equipment is not the limiting factor n Methods are easily automated for high throughput n High sensitivity (pg/ml to ng/ml levels) 100–10000-times more sensitive than capillary gel electrophoresis–UV
n n

Key limitations

Quantification of parent/total detectable OGN metabolites (shortmers) not quantifiable in parent assay Narrower calibration range than chromatographic methods (20–50-fold) n High reagent quality imperative (assay robustness) n Cannot detect intact double-stranded OGNs
n n

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