Quantitation of Oligonucleotides Using a Hybridization Method in Various Tissues and Species
for Accurate Pharmacology and Toxicology EvaluationsDANIELLE SALHA
DIRECTOR, LIGAND BINDING ASSAYS
Oligonucleotides (ONs) and their ApplicationsOligonucleotides (ONs) and their Applications
Hybridization-Based Assays to Quantitate ONsHybridization-Based Assays to Quantitate ONs
Quantitations of ONs in TissuesQuantitations of ONs in Tissues
Challenges and Solutions when Quantitating ONs in TissuesChallenges and Solutions when Quantitating ONs in Tissues
ConclusionsConclusions
Take Home MessagesTake Home Messages
Agenda
Oligonucleotides (ONs)
Usually consist of 15 to 20 nucleotides (complementary to target mRNA sequence)Unmodified oligonucleotides are rapidly degraded by nucleases, and RNA transcripts do not easily cross biological membranesChemically modified ribonucleotides are used to protect against nuclease degradation, improve target affinity and delivery to the intended target/tissue/regionCovalent attachment of ON to various ligands are designed to improve biodistribution and cellular uptake, or targeting of specific tissues– Peptides, proteins, carbohydrates, aptamers, and small molecules;
including cholesterol, tocopherol or folic acid
ONs — Past and Present
Lundin K.E. et al. 2015
ONs — Broad Range of Applications
Antisense oligonucleotides (ASOs)– Inhibitors of RNA activity: siRNAs, miRNA mimics, splicing modulators and RNAseH-
dependent ASOsModulators of protein activity (Aptamers)mRNAs encoding for therapeutic proteins or vaccine agentsImmunostimulatory oligonucleotides Genetic information-reprogramming nucleic acids (CRISPR)
siRNA: small interfering RNA
miRNA: micro RNA
CRISPR: Clustered Regulatory Interspaced Short Palindromic Repeats
Four Different Mechanisms of Action for ONs
2016, Mariana Lagos QuintanaMaster of Drug Regulatory Affairs
RISC: RNA induced silencing complex
Regulatory Considerations
Most ONs are evaluated as synthetically manufactured drugs, and follow ICH S2A and S2B guidelinesNon-clinical development and safety evaluations of ON therapeutics have generally followed the regulatory guidelines for small molecules There are no ICH or FDA regulatory guidelines that specifically address the quality expectations/standards for oligonucleotide productsON diversity with different mechanisms of action can cause diverse toxicology profiles/concerns– ON-based therapeutic toxicities can be due to interactions between the ON molecule and
proteins as a result of Watson and Crick base-pairing to unintended nucleic acids or through an independent mechanism.
Distribution of ONs in Tissues and Cellular Uptake
Following different routes of administration, ONs are found at highest concentrations in the liver and kidneys; the spleen follows as the tissue with highest concentrationONs do not cross the blood-brain barrier to target the central nervous system– Therapeutic ONs need to be administered intrathecally — some successes reportedTherapeutic ONs are mostly taken up through endocytosis, indicating that the ON must leave the endosome to reach its target in the cytosol or the nucleus– Thus, to improve ON design leading to more efficient drugs, it is important to consider all
endocytosis routes and trafficking mechanisms in the target cells
Immunogenicity and Safety Assessments of ONs
As ONs are also covalently linked to various ligands, immunogenicity to the linker and ONs can be enhancedWith ONs having different functions and applications, their immunogenicity characteristics are likely to be different Very limited publications on anti-RNA antibodies ASO can bind to proteins, such as human serum albumin, which can serve as a carrier to induce immune responses to the ASO
ASO: Antisense Oligonucleotide
Hybridization-Based Assays to Quantitating ONs
Hybridization-Based Assays (ELISA)
Method specific to the ON– Sequence-specific (complementary nucleic acid probes: capture/detection)Widely used in the industryBioanalytical methods support preclinical and clinical programs– Toxicokinetic (TK)/pharmacokinetic (PK) distribution and elimination studiesValidated assays in accordance with FDA guidance1 and Crystal City 2,3 recommendations
1. FDA. Guidance for Industry: Bioanalytical Method Validation. Rockville, MD:US Department of Health and Human Services, FDA, CDER; 2001.
2. DeSilva B. et al. (2003) Pharm. Res. 20 (3) 1885-1900
3. Viswanathan CT et al. (2007) AAPS J. 2007;9: Article 4.
Variety of Hybridization Assay Formats for Bioanalysis
There are three hybridization ELISA formats:Dual-hybridization ELISALigation-hybridization ELISA (patent protected)Nuclease-dependent hybridization ELISA (patent protected)
Dual Hybridization ELISAOligonucleotide Bioanalytical Services
Detection probe
Capture probe
Single-StrandedOligonucleotide
Enhanced sensitivity over Ligation hybridization ELISA
Wider dynamic range
Disadvantage: Cross-hybridization with 3’ and 5’ metabolites
Efler, SM et al.(2005) Oligonucleotides,.15 (2) 119–131
Ligation Hybridization ELISAOligonucleotide Bioanalytical Services
Ligation to test ON with T4 DNA ligase/ATP
Non-Ligated probe will be washed away
Advantage: Minimal 3’-metabolite cross-reactivity
Detection probe
Single-StrandedOligonucleotide Capture probe
Yu RZ et al.(2002) Anal. Biochem. 304, 19-25
Capture probe: complementary nucleic acid sequence to the ON to capture the ON
Detection probe: complementary nucleic acid sequence to the ON for detection through the label
Nuclease Dependent Hybridization ELISAOligonucleotide Bioanalytical Services
The S1 nuclease is a single-strand-specific enzyme cleaving all free capture probe and imperfectly formed duplexes.
Advantage: Only full-length test ON remains intact and is quantitated
Wei X et al.(2006) Pharmaceutical Research, Vol 23, No.6
Capture and
detection probe
Single-StrandedOligonucleotide
Hybridization ELISA can be Used with Different Tissues and Matrices to Quantitate ONs
Tissues
Matrices
BrainLiver EyesGut
Spinal CordKidneyFeces
Plasma CSF
Other Bioanalytical Tools Compared to Hybridization AssayMethod Advantages Limitations
Capillary Gel Electrophoresis (CGE)
High resolution for parent and metabolites Need of internal standard
Widely used to supportpre clinical and clinical studies
Elaborate pre-extraction procedure
The exact nature of size-separated peaks cannot always be determined
Extensive sample clean upLow sensitivity
Quantitative PCRHighest sensitivity Extensive optimization of the method is required
Little to no sample processing requiredLacks precision and accuracy
No resolution for metabolite quantification
High Resolution MSImproved sensitivity compared to CGE Time-consuming
Good resolution of parent and metabolites Extract-dependent and complex spectra
Hybridization ELISA
No sample clean up (plasma) or minimum sample cleanup (Tissue)
Narrower calibration range than chromatographic methods (10- to 50-fold)Low reagents cost
Very high sensitivity, precision and accuracy
Highly selective of parent Quantitation of parent / total detectable oligonucleotide metabolites
(shortmers) not quantifiable in parent assayWidely used to support pre clinical and clinical studies
High target specificity
High SensitivityOligonucleotide Bioanalytical Services
Hybridization ELISA
Double Ligation
Capture/Detection Probes DNA Template Probe
0.2500 ng/mL 1.00 ng/mL
50.00 ng/g 50.00 to 62.50 ng/g
LLOQ (Plasma)
LLOQ (Tissues)
CGE-UV
3.00 to 100.00 ng/mL
300.00 to 12000 ng/g
CGE-UV: Capillary gel electrophoresis of single-stranded DNA fragments with UV detection
Other Bioanalytical Tools Compared to Hybridization Assay
Method Sensitivity Selectivityfor Parent
Resolution of Parent and Metabolites
limited SampleClean up Specificity
Accuracyand
Precision
Wide DynamicRange
Total Top Performance
Capillary Gel Electrophoresis
(CGE)+ + ++++ + ++ ++++ ++++ 3
Quantitative PCR +++++ + + ++++ ++++ + ++ 3
High ResolutionMS ++ + ++++ + ++ ++++ ++++ 3
Hybridization ELISA ++++ ++++ + ++++ ++++ ++++ ++ 5
Time Associated with Hybridization Assays
Method development: 12 -15 days for plasma and 5 extra days for tissueValidation: 10-15 days for validationSample analysis: 60-90 samples per dayTurnaround: 2 weeks for first-in-human studies (sample analysis, QC and QA’ed data transferred to sponsor)
Method Validation
Goal: Prove the reliability, robustness and reproducibility of the assay– Acceptance criteria for validation parameters predefined– Generally 20-25% for ligand-binding assaysQC samples prepared in the same biological matrix anticipated in study samplesDetermine range of calibration and QC samplesInter-batch precision and accuracy 5 QC levels, n=3, 6 batchesSelectivity/Specificity: 10 independent lots of matrix assessed blank as well as spiked with analyteConfirm dilution linearityDetermine metabolite cross-reactivity– Stability In process: room temperature, 4 oC, freeze-thaw– Long-term storage (-20 oC / -80 oC)
Quantitations of ONs in Tissues
Extraction Method for Tissues
Homogenization of tissues using PolytronUse of detergent to disrupt non-specific protein binding to ONUse of organic solvents to separate the ON and eliminate the proteins (Phenol/Chloroform extraction )Quantitation is performed over two days due to evaporationPrecision between replicates is goodQuantitation procedure is similar to plasma after these steps
2-ml tube
500 µl homogenized tissue spiked (Calibrants and QCs)
or 25-75 mg tissue
+ 425-475 µl of homogenization buffer
200-300 µl of 2-mm beads
Percellys6000 rpm 40 sec.May vary for each tissue
Extraction Method for Tissues
Extraction Method for Tissues
Homogenization in the PercellysAddition of ammonium hydroxide and Phenol: Chloroform: Isoamyl alcohol 25:24:1Extraction in the PercellysCentrifugationTransfer of aqueous layer (top) into labeled empty tubesAddition of ultra pure water to original tube for second extraction Extraction in the PercellysCentrifugationTransfer the aqueous layer (top) into labeled tubes
Percellys Evolution Homogenizer
Dry samples in speed vac.18-24 hours
Reconstitute the dried sample by using the multi-tube vortexerfor 20 min.
Run hybridization ELISA
Extraction Method for Tissues
Challenges with ON Quantitation in TissuesThe ONs are not evenly distributed in a given tissue– The ON concentration may differ depending on where the extraction is completed– Therefore, the incurred samples’ reproducibility may failA whole sample tissue homogenate is highly recommended to ensure the homogeneity between sample extractions
Eyes, skin and bones are challenging to homogenize Different matrix beads should be tested for optimal homogenization
Tissues with a high concentration of salt (e.g., feces) were associated with a higher background in the hybridization assay, suggesting that the concentration of salt may be the causeThe assay is 3- to 5- fold less sensitive in feces than in plasma
Metabolite Interference
The metabolite concentrations evaluated should reflect the maximum and 4 times the maximum potential metabolite concentrations in preclinical and clinical samples, respectivelyMetabolite interference with the quantitation of the ON is typically assessed at Low and High QC concentrationsThere is no metabolite interference if the %RE is within ± 20% of the nominal concentration at each QC levelIf the accuracy of the metabolite interference sample exceeds ± 20% of the nominal concentration at any QC level, the metabolite will interfere in the study sample quantitation
Interference due to Immunogenicity of ONs
Chemically modified ribonucleotides or covalently bound ligands are used to protect ON against nuclease degradation and improve distribution– Consequence: with longer half-life of ONs, immunogenicity to ONs can increase– ADAs interfere with accurate ON quantitation as antibodies and ONs form complexes which are
not detected by the hybridization assay
Interference due to Immunogenicity of ONs
The potential interference of anti-drug antibody (ADA) on study sample quantitation is typically evaluated at Low, High, and Dilution QC (DQC) levels in the presence of 0 (zero) and the expected maximum antibody level in study samples, respectively. ADA will not interfere with the quantitation of the ON in human plasma if the %RE of the replicate determinations is within ± 20% of the nominal concentrations at any QC level.If the ADA is interfering with the quantitation of the ONs, conditions need to be optimized to reduce interference and regain accurate quantitation.
Challenges and Solutions when Quantitating ONs in Tissues(In-Life Collaboration with Janssen Pharmaceutical Companies of Johnson & Johnson)
Issue Encountered and Hypothesis
Issue: During Method Development in which the concentration of the ON needs to be determined in human feces homogenate, high blank signals were observed which impacted selectivity– Difference in signal between lots was so high
(up to fivefold) that the selectivity evaluation did not meet the acceptance criteria of 80%
Hypothesis: – Matrix effect due to the nature of the matrix
(feces)– Hybridization conditions not optimal
Nominal ON Concentration
(ng/mL)
Blank Low High
0.0000 0.3500 75.00
Individual Lot #
Observed Observed
%RE
Observed
%REON Concentration (ng/mL)
ON Concentration (ng/mL)
ON Concentration (ng/mL)
1 0.3782 * 0.7640 * 118.3 84.32 12.42 0.3665 * 0.6617 * 89.1 89.47 19.33 BLQ 0.3894 11.3 79.18 5.64 BLQ 0.3963 13.2 80.28 7.05 BLQ 0.4075 16.4 89.21 18.9
LEGENDBLQ: Below Lower Limit of Quantitation (<0.3500 ng/mL)
*: % Deviation Unacceptable for QCs
Tests and Results
Hypothesis: Matrix effect due to the nature of the matrices
To eliminate the variability between individual matrices, a larger pool was prepared and tested(12 lots were pooled instead of 4) for the test curve and QCs– The selectivity still did not meet acceptance
criteria (80%), but the results were much better
Nominal ON Concentration
(ng/mL)
Blank Low High
0.0000 0.3500 75.00
Individual Lot #
Observed Observed
%RE
Observed
%REON Concentration (ng/mL)
ON Concentration (ng/mL)
ON Concentration (ng/mL)
1 BLQ 0.4440 * 26.9 86.32 15.1
2 BLQ 0.4607 * 31.6 84.27 12.43 BLQ 0.3674 5.0 76.98 2.64 BLQ 0.3691 5.5 78.24 4.35 BLQ 0.4129 18.0 87.67 16.9
LEGENDBLQ: Below Lower Limit of Quantitation (<0.3500 ng/mL)
*: % Deviation Unacceptable for QCs
Tests and Results
Hypothesis: Hybridization conditions not optimal
To better eliminate the matrix effect, the sample volume was reduced by half– Unfortunately, the signal was compromised Therefore, the LOQ was raised threefold
Resolution: The matrix effect was completely eliminated by: – Creating a larger pool for Test curve and QCs– Reducing sample volume – Raising the LOQ 3X
Nominal ON Concentration (ng/mL)
Blank Low High
0.0000 1.0000 75.00
Individual Lot #
Observed Observed
%RE
Observed
%REON
Concentration
(ng/mL)
ON Concentra
tion (ng/mL)
ON Concentra
tion (ng/mL)
1 BLQ 1.108 10.8 80.57 7.4
2 BLQ 1.083 8.3 72.57 -3.23 BLQ 1.068 6.8 86.85 15.84 BLQ 1.198 19.8 74.82 -0.25 BLQ 1.073 7.3 85.12 13.5
LEGENDBLQ: Below Lower Limit of Quantitation (<1.000 ng/mL)
Conclusions
Conclusions
Hybridization assays offer several advantages and are a useful tool to consider for quantitation of ON in various biological matrices (plasma, CSF, tissues, urine, feces, etc.)The Dual-Hybridization ELISA, the Ligation-Hybridization ELISA and the Nuclease-Dependent Hybridization ELISA all have advantages for ON quantitation, and are used for different purposesThe assays described have high target specificityNo sample cleanup generally required for plasma, and minimal for tissueAssay design is selective for the parent ONHybridization ELISA methods are easily validated in accordance with the applicable principles of GLPs as defined by the OECD and the FDAHigh sensitivity (up to 0.2500ng/mL in plasma and 50.00 ng/g in tissues)
Putting It All Together
Altasciences’ Experience in Oligonucleotide Quantitation in the Last 2 Years
MD in Plasma / CSF
19
MD in Tissues
11
Validation in Plasma / CSF
18
Validation in Tissues
7
Production in Plasma/CSF
10
Production in Tissues
7
Acknowledgements
The Altasciences Clinical Research Team:– Martin Clément– Mira Sassin– Aude Carine Ndoti– Thu Bich Vu– Djahida Dejahir– Alexandra Michaux
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