Preparation and LC/MS Analysis of Oligonucleotide ... - Phenomenex

MaterialsAll chemicals and reagents were purchased from SigmaChemicals (St. Louis, MO, USA) unless otherwise stated.Oligonucleotide samples were either purchased fromIntegrated DNA Technologies (Coralville, IA, USA) orgenerously provided by various industry and academicsources (ISIS: Carlsbad, CA, USA; USC OligonucleotideLaboratory: Los Angeles, CA, USA). HPLC solventswere purchased from EMD (San Diego, CA, USA).Serum, plasma, and specific mouse organ tissues werepurchased from Bioreclamation (Liverpool, NY, USA).

MethodsOligonucleotides were spiked into plasma and tissueto show the utility of the protocol. Equal aliquots of theClarity ® OTX loading buffer (Phenomenex, Torrance,CA, USA) and serum/plasma samples were mixedtogether prior to loading on the SPE cartridge. TheSPE isolation cartridge (Clarity OTX 100 mg/ 3 mL tube)was first “wetted” with methanol then equilibrated withClarity OTX equilibration buffer (10 mM Phosphate pH5.5) prior to sample loading. After sample loading thecartridge was rinsed twice with equilibration bufferfollowed by rinses with Clarity OTX wash buffer (10 mMPhosphate pH 5.5/ 50 % acetonitrile). The oligonucleotidewas eluted from the cartridge using elution buffer (100mM ammonium bicarbonate pH 8.0/ 40 % acetonitrile/10 % tetrahydrofuran). Samples can be either lyophilized orspeed vac evaporated before reconstitution for LC analysis.For tissue samples either proteinase K digestion (3 hours)or a combination of mechanical homogenization anddigestion was used to break intercellular and extracellularmatrices prior to mixing with loading buffer.For LC-UV analysis samples were injected on an Agilent ®HP1100 HPLC (Palo Alto CA, USA) using a Clarity 3 µmOligo-RP ® or Clarity 2.6 µm Oligo-MS HPLC column(Phenomenex, Torrance, CA, USA). For LC/MS analysis,samples were either detected using a AB Sciex API3000 (AB Sciex, Foster City, CA, USA) at Phenomenexor analyzed at Novatia using a Novatia Oligo HTCS HPLCsystem (Monmouth Junction, NJ, USA) connected to aLTQ ® Orbitrap ® mass spectrometer (San Jose, CA, USA).Oligonucleotide ion spectra were reconstructed using theproMass ® software (Novatia). A gradient separation methodusing an aqueous mobile phase A of 8 mM triethylamine/200 mM Hexafluoroisopropanol (pH 8.0) and organicmobile phase B of acetonitrile. Various gradients wereused depending on the column and instrument being usedas well as the specific oligonucleotide being analyzed.

Figure 1. General Schematic for OligonucleotideIsolation from Different Biological SamplesPlasmaSampleLoad (up to 1mL)1:1 Biological Fluid/Loading-Lysis bufferCartridgeEquilibrationSampleLoadWash 1Wash 2ElutionMethanol(1 mL)Equilibrationbuffer (1 mL)Load (up to 1 mL) 1:1tissue homogenate/Loading-Lysis bufferEquilibration buffer(1 ml)Salt removalSugar removalWash buffer (1 mL)Protein removalLipid removalElution buffer(0.5 mL)Oligo elutionTissueHomogenizationReconstitution100 mg liver tissue3 hr Protease K digestLiver TissueMechanicalHomogenizationMuscleor LungTissueLC/MSAnalysis

Figure 2. Recovery Studies from PlasmamAUArea: 53280Area: 532Oligonucleotide Extracted from From Plasma4000 5 10 15 20Area: 546mAUmin80Control OligonucleotideControl OligonucleotideArea: 5464000 5 10 15 20minFigure 2: Recovery and cleanup of a 27mer DNA phosphorothioate oligonucleotide from plasmausing the Clarity OTX protocol. Oligonucleotide was spiked into a plasma sample, extracted, andcompared to a control. Recovery is estimated at 97 % with only minor plasma contaminants.

Figure 3. Linearity Studies from TissuemAU10010 μg oligo in 1 g liver tissue19merOligoInternalStd.50mAU100502 4 6 8100 µg oligo in 1 g liver tissueminmAU100502 4 6 8100 µg oligo and 100 µg internalstandardmin2 4 6 8minFigure 3: UV chromatograms of oligonucleotide extracted from liver tissue using Clarity OTX. The19mer extracted phosphorothioate oligonucleotide was spiked with 10 µg of a oligonucleotideinternal standard before LC/MS analysis. The top two chromatograms represent different levels ofthe incubated P-S oligo. The bottom chromatogram is a external standard of equal amounts of the19mer oligo and internal standard. Note the high recovery of the oligonucleotide and low level ofplasma contaminants from the incubated samples.

Figure 4. Clarity OTX: Speed-Vac Oligo Recovery19mermAU250200150100Control Oligo5.2965004.7230 2 4 6 8 10 12minmAU250200150100Near Dryness5.232504.66200 2 4 6 8 10 12minmAU250Speed Vac Dry200150100504.5405.206App ID 1971100 2 4 6 8 10 12minColumn: Clarity 3 µm Oligo-RPDimensions: 50 x 2.0 mmPart No. 00B-4441-B0Mobile Phase: A: 8 mm Tetraethylammonium / 200 mm HFIPB: AcetonitrileGradient:Time (min) % B0 510 350.3 mL/minFlow Rate:Temperature. AmbientDetection: UV @ 260 nmBackpressure: 200 barSample: 19 mer RNA oligonucleotideFigure 4: Speed-vac effect on oligonucleotide recovery. An oligonucleotide standard (top chromatogram)is compared to samples that are concentrated using a speed-vac evaporator. The middlechromatogram is of the oligonucleotide evaporated to near dryness and little loss in recovery is observed.In the bottom chromatogram the oligonucleotide is evaporated to dryness. Significant loss inrecovery is observed when an oligonucleotide is speed-vac evaporated to dryness.

Figure179655. Balancing LC Retention Versus IonpairingIon Suppression2.8e72.4e7Intensity, cps2.0e71.6e71.2e78.0e6App ID 179654.0e68 1012 14 16 18 20 22 minFigure 5: Ion-pairing concentration effects on LC/MS sensitivity and resolution. A 12-18 poly dToligonucleotide standard is run on a Clarity Oligo-RP column using different ion-pairing concentrations:Black trace = 15 mM TEA/ 400 mM HFIP, Green trace= 2.8 mM TEA/ 280 mM HFIP, Red trace= 4 mM TEA/ 100 mm HFIP, Blue trace = 2 mM TEA/ 50 mM HFIP. Retention, resolution and MSsignal intensity appear optimal between 4-8 mM TEA and 200-300 mM HFIP.

Figure 6. Sensitivity Studies with RNATIC of a 500 ng extractionXIC of a 50 ng extraction1.3E+0081.1E+008A1.4E+0061.1E+006B14.31: 66158.0E+0078.4E+005Intensity5.4E+007Intensity5.6E+0052.7E+00714.31: 66162.8E+0054.8 9.614.4 19.224 min4.8 9.614.4 19.224 minFigure 6: Sensitivity and MS analysis parameters. The total ion chromatogram of 500 ng/mL ofa 19mer oligonucleotide extracted from plasma is shown in chromatogram 6A. Note the low levelpeak corresponding to the oligonucleotide at RT of 14.3 minutes. The extracted ion chromatogram(-7 charge state ion at m/z of 944) of a 50 ng/mL sample is shown in chromatogram 6B. Selectedanalysis of specific ions can realize large increases in sensitivity compared to standard MS methods.

Figure 7. Spectra of Extracted P-S Oligonucleotide4.3E+007944.2-7A3.4E+007A Raw SpectraIntensity2.6E+0071.7E+007826.0-8A8.5E+0061101.7-6A724.1-9A0.0E+000500 6808591039 1218 1398 min6.0E+0076616.34.8E+007Intensity3.6E+007BReconstructed Spectra2.4E+0071.2E+0076638.86482.80.0E+0005860 616064606760 7060 7360 minFigure 7: Using deconvolution software to identify metabolites. The raw spectra from 19meroligonucleotide in A is compared to the reconstructed spectra in B. Note reconstructed massesfor the oligonucleotide of interest at MW=6616 as well as a minor component corresponding to adepurinated oligonucleotide at MW=6482. Deconvolution software is required to identify low levelmetabolites of oligonucleotides.

Results and DiscussionOligonucleotide IsolationA simple mixed-mode SPE-based protocol hasbeen developed (Clarity OTX) to isolate therapeuticoligonucleotides from biological matrices (Figure 1) 2,3 .Oligonucleotide-containing serum or plasma samplestreated with the chaotrope/detergent buffer are loadedon to a mixed-mode SPE sorbent around pH 5.5. Fortissue samples a proteinase K digest and/or mechanicalhomogenization was used to break up tissues prior tochemical disruption. After low pH washes, elution of theoligonucleotide is achieved with moderate organic at anelevated pH (pH ~8). Tight pH control is critical throughoutthe process; DNA extended exposure below pH 5 resultsin depurination and RNA exposure above pH 9 can leadto 2’-3’ isomerization of the ribose sugar.An example chromatogram of the effectiveness ofcleanup is shown in figure 2. HPLC chromatograms ofa phosphorothioate 27mer DNA oligonucleotide spikedinto plasma and purified using the Clarity OTX protocolis compared against a control oligonucleotide. Almostcomplete recovery (97 %) of the oligonucleotide isobtained with only small amounts of matrix contaminantseen in the chromatogram at retention times far awayfrom most oligonucleotides and their metabolites. Aexample of isolating oligonucleotide out of liver is shownin figure 3, where different levels of a 19mer oligo wasspiked along with an internal standard into a liver sample.Recoveries have ranged between 65-99 % dependingon the oligonucleotide and biological matrix used for theisolation with good linearity for a specific oligonucleotideand biological matrix type 4 . While for some “soft” tissuelike liver, simple digestion is sufficient to disrupt a tissue,“hard” tissue like muscle and lung require additionalhomogenization. Initial studies suggest that mechanicaldisruption with detergent is required either with or withoutproteinase K digestion (data not shown).Additional studies were undertaken to better understandsome of the factors in sample recovery. A speedvacevaporator is typically used to concentrate theoligonucleotide prior to injection on HPLC. Commonpractice is to evaporate a sample to dryness beforereconstitution in mobile phase. However, this appearsto affect the recovery of the oligonucleotide whencompared to a sample that is only evaporated to neardryness. Figure 4 demonstrates this convincingly.Sample evaporated to “full dryness” showed significantloss in recovery versus the “near-dryness” sample whichdemonstrated good recovery 5 . Based on such results,speed-vac evaporation should be avoided for low-leveloligonucleotide isolation techniques. Lyophilizationappears to be robust alternative for concentratingsamples without similar deleterious effects.LC/MS Analysis of OligonucleotidesOligonucleotides are typically analyzed by LC/MSusing an ion-pairing reversed phase method where amixture of HFIP and TEA is used to elicit retention of thepolar polyanionic molecule 6,7,8 . In LC/MS applications,ion-pairing retention must be balanced against ionsuppression to maximize MS sensitivity. An example ofthis effect is shown in figure 5 where different levels ofion-pairing buffer were used in the mobile phase of aLC/MS run of a mixture of oligonucleotide standards.Maximizing MS sensitivity becomes a balance betweenion suppression and retention; higher concentrations ofion-pairing buffer result in greater retention and resolutionof oligonucleotides, but only to a certain point.While optimizing mobile phase conditions is important,the mass spectrometer and data collection parameterscan have a much larger influence. An example of thisis shown in figure 6 where different levels of a 19merphosphorothioate RNA oligonucleotide isolated fromplasma samples using the Clarity OTX protocol were runon the Oligo HTCS system connected to an Orbitrap MS.Using extracted ions shows the 50 ng/mL level is far fromthe detection limit for this application.Oligonucleotide analysis requires deconvolutionsoftware for the identification of oligonucleotides. Thiscan be especially important when looking for low-levelmetabolites of oligonucleotide therapeutics, whichcorrespond to unique molecular weights. An exampleof this is shown in figure 7 where the spectrum of the19mer P-S RNA oligonucleotide is displayed in rawand reconstructed mode. The raw spectra in figure 7Ashow the predominant -6, -7, and -8 of the expectedoligonucleotide; however, one cannot discern thepresence of any metabolites in the sample based on thespectra. However, when the spectra is reconstituted usingthe ProMass software (Figure 7B), one can identify lowlevelmasses that correspond to a salt adduct as well asa depurinated oligonucleotide. Such results demonstratethe utility of deconvolution software for oligonucleotideanalysis.

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