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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