bioanalysis of sirna and oligonucleotide therapeutics in biological fluids and tissues

595ISSN 1757-6180Bioanalysis (2009) 1(3), 595–60910.4155/BIO.09.66 © 2009 Future Science Ltd

Review

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 cyto-megalovirus retinitis, and pegaptanib, an anti-angiogenic 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 therapeu-tics occupy an increasingly important space in the biopharmaceutical drug-discovery and -development landscape. It is expected that more OGN drugs will reach the market, con-sidering 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 determina-tion in increasingly complex biological matrices, such as skin, colon or brain tissue. In addition, sensitive assays are required for the lower thera-peutic 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:

nsiRNA [2,3]

nASO OGNs [4]

nAptamers [5]

nImmunomodulatory OGNs (IMOs) [6,7]

nmiRNA-blocking OGNs [8]

nRNA decoys [9]

nSplice switching/exon skipping OGNs [10]

nRibozymes and DNA enzymes [11]

Current investigational therapeutic OGNs are typically either siRNA, ASO, IMOs or aptamers. ASOs are complementary to a targeted, disease-associated RNA molecule, generally a mRNA. The classical ASO mechanism is based on trig-gering RNase H cleavage of the ASO–RNA tar-get 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 pattern-recognition 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 molecu-lar evolution technique known as Systematic Evolution of Ligands by Exponential enrich-ment (SELEX) [12]. They are habitually designed to work at the extracellular level.

Recent advances with RNAi and siRNA syn-thetic compounds have fueled interest in thera-peutic OGNs in recent years. Andrew Z Fire and

Bioana lysis 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.

Guy A Tremblay1 & Philip R Oldfield1†

†Author for correspondence 1Immunochemistry, Charles River Preclinical & Clinical Services, 22022 Transcanadienne, Senneville, QC H9X 3R3, Canada, Tel.: +1 514 630 8263 Fax: +1 514 630 8230 E-mail: [email protected]

AptAmeR

Oligonucleotide ligand obtained by in vitro evolution

Antisense

Complement strand of a targeted nucleic acid, typically mRNA

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

Bioanalysis (2009) 1(3)596 future science group

Craig C Mello were awarded the 2006 Nobel Prize for their discovery of RNAi gene silenc-ing by double-stranded RNA [303]. RNAi is a natural mechanism of RNA inhibition medi-ated 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, includ-ing ovarian [13] and bone cancer [14], hypercholes-terolemia [15], liver cirrhosis [16], respiratory syn-cytial 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 transfec-tion efficiency will help to lower the effective dose. For those OGNs that act at the extracel-lular 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 deter-mine the toxicokinetic (TK)/pharmacokinetic (PK) parameters and exposure–response in order to choose the right dosage regimens of therapeutic OGNs in support of their preclini-cal 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 therapeuticsSeveral modifications to investigational thera-peutic 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.

For therapeutic OGNs to be part of clini-cal trials, they are required to be robust, safe and effective. FiguRe 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-genera-tion ASOs intended to increase the nuclease resis-tance 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 5́ end and has an inverted 3 -́3´deoxythymidine residue at the 3´ end. Pegaptanib is 2 -́O-methylated (2Ó-Me) 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 poly-merases 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 2Ó-Me RNAs (FiguRe 1d) are second-generation ASO modifications and confer con-siderable protection from nucleases while having similar hydrogen bonding properties to RNA–RNA hybrids [23]. An interesting property of 2Ó-Me 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-stimu-latory mechanism [31]. Interestingly, the 5-meth-ylcytosine (5mC) nucleobase modification, natu-rally found in CpG motifs, has also been shown to reduce immunogenicity for a PS ODN [32].

Typically used in combination with PS, 2´-O-methox y e thy l (2´MOE ; FiguRe 1e)-modified ODNs are also second-generation ASO modifications [33]. In addition to supporting RNase H cleavage [34], OGNs with the 2´MOE modification have increased affinity

phARmAcokinetics

Describes the relationship between mechanisms of drug absorption, distribution and elimination over time

Review | Tremblay & Oldfield

www.future-science.com 597future science group

towards target RNA and increased nuclease sta-bility relative to unmodified phosphoramidate (PN) ODNs [35].

One example of a second-generation molecule modified with PS and 2´MOE in clinical devel-opment is OGX-011, an ASO designed to block

the production of clusterin, a cell-survival pro-tein that is overproduced in several cancer indi-cations [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].

Figure 1. Chemical modifications developed for investigational therapeutic oligonucleotides.

OO

OHO

O-

O

O

P baseO

O

OO-

S

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P base

O

O F

OO-

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O

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OO-

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O

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baseOO-

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P

A. RNA B. Phosphorothiate oligodeoxynucleotide

C. 2´-fluoro RNA D. 2´-O-methyl RNA

E. 2´-O-methoxy ethyl F. Locked nucleic acid

G. Morpholino oligomer

ON

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H3C

N

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Obase

H3C

Bioana lysis of siRNA & oligonucleotide therapeutics in biological fluids and tissues | Review


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 hybridiza-tion 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 non-ionic phosphorodiamidate groups (FiguRe 1g). The morpholino oligomers are often used as an ASO technology to block translation or inter-fere with RNA processing, including splicing and miRNA maturation [40,41]. They differ from tra-ditional AS ODNs in that they function by ste-ric 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 systemsOligonucleotides 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, includ-ing 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 environ-ment 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].

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-spe-cific 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 drug-delivery system is comprised of positively charged peptides and polymers, formulated with negatively charged nucleic acids, result-ing in stable nanoparticles. PEG can be used to stabilize the nanoparticles and prevent aggrega-tion [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 con-cern regarding the toxicity of PEI; however, dif-ferent 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 con-junction with the transferrin protein for target-ing cancer cells, Heidel et al. delivered a siRNA payload to target the M2 subunit of ribonucleo-tide 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 formula-tion 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 poly-mers, and release the nucleic acids for hybrid-ization without the further need for purification of the OGN for downstream quantification with hybridization assays.

Comparison of recovery of formulated ver-sus unformulated quality control (QC) sam-ples 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.

hyBRidizAtion AssAy

Is a ligand-binding assay used for the determination of the oligonucleotide therapeutic



Administration & PKOligonucleotides have been administered by various routes in support of preclinical and clinical studies, including ocular [50], paren-teral intravenous injection [35], intravenous infusion [51], topical [52], subcutaneous injec-tion [53], intramuscular injection [54], lung inha-lation [55], intranasal inhalation [56], intracere-bral [57] and even oral routes [58]. The choice of drug regimen is determined by indication, intended clinical route of administration, con-siderations for systemic exposure and character-ization 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 fol-lowing systemic administration [23]. Potential toxicological effects exist as a result of comple-ment 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 tox-icity 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 biologi-cal 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 thera-peutic OGNs are also discussed in Chapter 13 of the book [23].

Hybridization assaysn General considerations

Hybridization assays (also known as hybridiza-tion 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

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 sen-sitivity 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 pro-files are expected. Optimization of the (capture and/or detection) probe concentration is of para-mount 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 colorimet-ric 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 stan-dard 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 amine-conjugation 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 phos-phatase or horseradish peroxidase (HRP), with colorimetric or f luorimetric substrates. One

Bioana lysis of siRNA & oligonucleotide therapeutics in biological fluids & tissues | Review


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, purifica-tion 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 bio-logical 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–liq-uid extraction (LLE) with phenol and chloro-form 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 pro-teins. Alternatively, a straightforward extrac-tion procedure is performed on a small amount of tissue sample (e.g., 50 µl of a 200 mg/ml tissue homogenate) using proteinase K, soni-cation 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 compat-ible with both hybridization and proteinase K (Tremblay GA, Unpublished Data).

n Sandwich hybridization assayThe sandwich hybridization method is the sim-plest 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 sec-ond detection probe, modified for downstream signaling, is complementary to the second por-tion 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 micro titer 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 exam-ple, 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 hybrid-ization for the sandwich assay via thermal stabi-lization of short hybrids. Improving the hybrid-ization 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 assayThe 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

BiotinBiotinStreptavidin-coated plate

Streptavidin-coated plate

Oligonucleotidetest article

Detection probe

‘Sandwich complex’

Capture probe

Label Label

Bioanalysis © Future Science Group (2009)

Figure 2. Schematic representation of the sandwich hybridization assay.



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 5́ end. This extension is complementary to a generic 5́ phos-phorylated 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 strep-tavidin-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 5́ -end metabolites will be detected; however, appar-ently, the vast majority of metabolites present in plasma are truncated at the 3´ end via 3´→5́ 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 hybridization assayThe nuclease-based hybridization method, also known as the S1 nuclease cutting assay, takes advantage of the properties of a single strand-specific 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 down-stream 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

for the complete active pharmaceutical ingredi-ent. 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-strand-specific nucleases may help to improve discrimi-nation [67]. Both the nuclease-based and liga-tion–hybridization assays are patented by Isis Pharmaceuticals (CA, USA) [201,202].

n Competitive hybridization assayThe 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, depend-ing on the sensitivity required. Direct labels include radiolabels or fluorescent and chemilu-minescent substrates. Indirect labels, such as anti-gens (e.g., digoxigenin), can selectively interact with reporter enzymes, enabling reaction with an enzymatic substrate, resulting in a colorimetric

Label

Biotin



Template probe

Ligation probe

Ligation

Oligonucleotidetest article

Ligation site

5´PO4

3´OH

Biotin

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.



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 fea-sible. 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 fluorescence assayA method for quantification of single-strand DNA OGNs in solution based on intercala-tion 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 autoradiographyFor distribution of OGN test articles in ani-mals, quantitative whole-body autoradiography (QWBA) has been applied. QWBA enables sub-organ and whole-body distribution with mini-mal 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 quali-tatively examined by micro-autoradiography [70]. The results of QWBA have a limited selectiv-ity due to the fact that metabolites/breakdown products will be measured along with the full-length OGN active pharmaceutical ingredient. Therefore, this technique provides good pre-liminary data but fails to differentiate between the parent compound and its metabolites. In addition, because only the associated radio-activity 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 previ-ously unknown target, QWBA would be likely to detect this.

Nuclear imaging emission tomographyPET and single-photon emission tomography are high-resolution, sensitive imaging tech-niques that can be used for the repeated, non-invasive quantification of OGNs in animals and humans. These techniques have the same limitations as QWBA regarding lack of selectiv-ity for the parent compound versus metabolites. One advantage is their ability to study molecular interactions and functional studies in vivo [71,72].

HPLCThe 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 spectrometryThe 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 compo-nents 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 mobile-phase modifiers and ion-pairing reagents, such as tetraethylammonium acetate [79], hexafluoro-2-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 compo-nents, 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 quantifica-tion of OGNs in biological matrices, especially in tissues.



Recently, quantification of OGNs by LC–MS/MS, where the OGN test article was pre-extracted from the plasma samples using LLE followed by solid-phase extraction, has been reported with promising results [84]. The quan-tification of a phorphorothioate OGN in tissues by LC–MS/MS with a LLE extraction has also been established [85].

Capillary gel electrophoresisCapillary gel electrophoresis (CGE) is an ana-lytical 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

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 proce-dures 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 fluores-cence 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 MSThe development of ESI-MS enabled interfacing to CGE systems in addition to LC systems for the bioana lysis of OGNs [89].

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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0.012

0.014

0.016

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

(n-)metabolites

Full-length PS ODN

(n-)metabolites

ISIS

A B

Retention time (min)

UV

abso

rban

ce (2

60 n

m)



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 12–20-base 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 solu-tion 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 PCRQuantitative PCR (qPCR), also referred to as real-time PCR, is a variation of the PCR per-formed in capillaries, where the product forma-tion of a PCR reaction is monitored in real time using various detection methods. qPCR enables amplification of nucleic acids up to the zepto-mol 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 tech-nique that may be useful for the determina-tion 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 & validationBioanalytical methods for the quantitative deter-mination of OGN test articles are compared in tABle 1. These methods are used in the pharma-ceutical 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 com-ponents 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

Table 1. Comparison of bioanalytical methods for the determination of oligonucleotides.

Method Sample processing

Sensitivity Selective for parent OGN

Metabolite quantification

Robustness Class of OGN

QWBA Extensive tissue preparation

Moderate No No Good All

HPLC Moderate Low No No Good Not good for PEG aptamers

CGE–UV Extensive Moderate Yes Yes Good Not good for PEG aptamers or double-stranded OGN

CGE–LIF Extensive High Yes Yes Poor Not good for PEG aptamersor double-stranded OGN

LC–MS Moderate High Yes Yes Good All

Hybridization assay

None for plasma samples (moderate for tissues)

Very high Yes No (unless a specific probe is designed to that effect)

Good All

qPCR Moderate Highest No 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.

RegulAtoRy Filing

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



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:nAssay format selection (sensitivity vs selectivity)

nOGN class and formulation considerations

nProbe design

nReagent selection

nStandard reference material

nMatrix selection

nMethod optimization

nReagent concentrations

nIncubation conditions

nPrevalidation 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 perspectivesiRNAs 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 bioanalyti-cal 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 predeces-sors, 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 con-tained in the formulation, may become a topic of

interest from a regulatory standpoint; for exam-ple, 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 indica-tions, 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 determina-tion of the two strands of a siRNA molecule can also benefit from multiplexing technologies.

Luminex™ xMAP microsphere-based tech-nology 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 nucle-otide 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 validationn Prove the reliability, robustness and reproducibility of the assayn Acceptance criteria for validation parameters are predefinedn 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

Range of calibration and QC sample rangesInterbatch precision and accuracy

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

Selectivity/specificityn Ten independent lots of matrix (e.g., plasma, urine and tissue samples) assessed

blank and spiked with the analyten Metabolite cross-reactivity

Dilution linearityn Use a concentrated standard in the matrix dilute with a blank sample/matrix and

check for linearity of dilutionn Check for parallelism using an incurred sample with a high concentration and

dilute with a blank sample/matrix and check for linearity

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

Stabilityn In-process: room temperature, 4°C, freeze-thawn Long-term storage (-20/-80°C)

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



Meso Scale Discovery (MSD™) multiplex-ing technology is a promising electrolumines-cence-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 quan-titative 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 amplifi-cation of cDNA ends (RACE) assay has been used for demonstrating RNAi-mediated silenc-ing 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 thera-peutics, that is, the impact of an unforeseen asso-ciation of siRNA to untargeted RNA [105], may also become a subject of concern, since siRNA

drugs are relatively new and considering the importance of mechanisms of action for pharma-cological 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 technologi-cal innovation, which will reduce the burden of sample collection on patients in the course of clinical trials.

AcknowledgementsThe authors would like to thank Helen Legakis for critical review of the manuscript.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a finan-cial 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.

Executive summary

Hybridization-based assays have several key advantagesn High target specificityn 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 costn Low instrumentation costn Equipment is not the limiting factorn Methods are easily automated for high throughputn High sensitivity (pg/ml to ng/ml levels) 100–10000-times more sensitive than capillary gel electrophoresis–UV

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

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303 Fire and Mello win Nobel Prize www.the-scientist.com/news/print/24964


bioanalysis of sirna and oligonucleotide therapeutics in biological fluids and tissues

Documents

n n n n n mirna

aso ogns

immunomodulatory ogns

oligonucleotide test

determination of sirna

ogn drugs

number of ogn chemistries

targeted delivery