Training Course M-2 - 8am to 5:30pm, Monday April 9, 2018

"The Exciting World of Oligonucleotides:
A multidisciplinary complex challenge for multitasking ingenious bioanalysts"

All you need to know to enter in this fascinating field: small & large molecule approaches, LCMS, HRMS and LBA advanced techniques, Hybridization HPLC-FL, expressed protein analysis, Hybridization ELISA, branched DNA (bDNA), RT-qPCR and new technologies

Outline of Training Course M-2

(You can click on each blue topic title below to see details, or simply scroll down to see details)


Course Co-Chair: Dr. Yuanxin, Senior Director Bioanalytical Sciences, Alnylam
Course Co-Chair: Dr. Adrien Musuku, Senior Director Biopharmaceutics, Pharmascience

Part 1: Oligonucleotide Therapies & Bioanalytical Challenges: Past, Present and Future
Part 2: Hybridization ELISA, RT-qPCR and bDNA Assays
Part 3: LCMS, HRMS and Hybridization HPLC-FL Assays


DETAILS of Training Course M-2

Course Co-Chair: Dr. Yuanxin, Senior Director Bioanalytical Sciences, Alnylam
Course Co-Chair: Dr. Adrien Musuku, Senior Director Biopharmaceutics, Pharmascience

Part 1: Oligonucleotide Therapies & Bioanalytical Challenges: Past, Present and Future
  • Lesson 1
    Recent advances in Oligonucleotides, Future trends and Impact on Bioanalysis
    - Dr. Mark Ma, Sr Director, Alexion Pharmaceuticals
    • Bioanalysis of Oligonucleotides is a fast-developing but not well-established yet area even if these drugs have been under clinical development for approximately 30 years
      • Recent increase in Oligonucleotides development activities after recent approvals in 2016 of
        • Nusinersen (INN) for spinal muscular atrophy (SMA)
        • Eteplirsen (AVI-4658) for Duchenne muscular dystrophy (DMD)
      • Focus on the most recent developments in Oligonucleotide drugs
        • Targeted biological processes for Oligonucleotides
      • Successful cases and main failures in Oligonucleotides development history
        • Lesson learnt
          • Ongoing and future challenges for Oligonucleotides in development for Rare & Genetic Diseases
    • Bioanalytical issue/complications
      • Systemic/tissues distribution
      • Drug Administration/Delivery mechanisms
      • Drug breakdown products/elimination
      • Lack of Regulatory Guidance/Guidelines on Oligonucleotides
      • Current Industry Standards:
        • 2017 White Paper Part 2 Recommendations on Oligonucleotides
    • What’s next for oligonucleotides bioanalysis
    • Case Studies: Current and future challenges in Oligonucleotides bioanalysis

  • Lesson 2
    Importance of Having Reliable Bioanalytical Data to Understand the Pharmacokinetic (PK) Properties of Oligonucleotides
    - Dr. Daniel Norris, Director Pharmacokinetics and Clinical Pharmacology, Ionis Pharmaceuticals
    • Reaching the full characterization of the PK profile through
      • Many different analytical techniques
      • In many different matrices
      • At very different level of sensitivity
      • From the intact to the metabolites/fragments
    • Challenges involved in monitoring Oligonucleotides levels after multiple routes of administration
      • Dealing with accumulation in tissues
      • Needs for very sensitive bioanalytical methods for the determination of Oligonucleotides elimination half-life
      • Needs for very selective methods for distinguishing full length, fragments, unconjugated/conjugated molecules
    • Case Studies: Oligonucleotides bioanalysis as an essential component for understanding the PK of these drugs

  • Lesson 3
    Lipid Nanoparticles Containing Small Interfering RNA (siRNA) Payloads: A New Paradigm for Immunogenicity, ADA and PK Assay Development
    - Dr. Uma Kavita, Senior Research Investigator, Bristol-Myers Squibb
    • Immunogenicity of lipid nanoparticles and potential to activate the innate and adaptive arms of the immune system
      • Complexity relative to traditional biologics. Will an assay monitoring antibody response suffice?
      • Will assays have to be developed for all components of the drug?
        • Novel and compendial lipids
        • Targeting moieties
        • siRNA
      • Immunogenicity assay formats
      • Risk-based prioritization of assays needed to address LNP mediated immunogenicity
      • Case study of an anti-LNP ADA assay method development and associated challenges
      • Impact of LNP immunogenicity on PK & PD parameters
    • Pharmacokinetics of lipid nanoparticles
      • What, where and how to measure analytes?
      • Relevance of analytes for drug exposure, toxicity and immunogenicity evaluations
      • Method development and assay validation considerations for an LBA PK assay
      • Beyond a good method-factors that may impact drug exposure levels
      • Immunogenicity and PK/PD correlation
    • Case Studies: Answering the key questions during the method development LNP drugs containing siRNA

  • Lesson 4
    Recent Advancements in the Assessment of Lipid Nanoparticle-Encapsulated mRNA: Enhancing intracellular delivery of the Oligonucleotides & Bioanalytical support
    - Dr. Alex Bulychev, Principal Scientist, Moderna
    • Therapeutic applications of modified mRNA
      • Direct template for protein translation
    • Recent developments in Intracellular delivery of mRNA
      • LNP extended systemic circulation & accumulation at disease sites
      • LNP endocytosis
    • Overcoming bioanalytical method development issue with lipid-based nanoparticles-encapsulated (LNP) mRNA
      • IV Administration
      • LNP delivery technology
      • Encapsulation
      • Distribution from the bloodstream to target tissues
    • Most advanced methods to measure the LNP-mediated mRNA delivery
    • Next generation of LNP-mediated mRNA therapeutics with higher potency and limited toxicity
    • Case Studies: How to address the bioanalytical challenges in LNP-mediated mRNA delivery


Part 2: Hybridization ELISA, RT-qPCR and bDNA Assays
  • Lesson 5
    Hybridization ELISA Assays & LBA Current Developments Focused on Antisense Oligonucleotides (ASO) PK & Immunogenicity determination
    - Dr. Chris Stebbins, Principle Scientist in Translational Medicine, Biogen
    • PK and Immunogenicity assays development in support of several Antisense Oligonucleotides (ASO) studies
      • Blocking translation or increasing degradation of single target mRNA
      • Quantification of ASO parent + metabolites
      • Targeting biodistribution
      • Administration route: IV vs SC vs IM
      • Evaluation of ASO stability in matrix
    • Immunogenicity evaluation of
      • Delivery vector
      • mRNA
      • Expressed protein
    • Advanced application of hybridization ELISA assays
      • Hybridization of Oligonucleotides in sample to complementary capture & detection probes
      • Addition of a detection reagent and substrate
      • Specific design of capture & detection probes to maximize affinity for ASO
    • Pros/Cons of hybridization ELISA formats
      • Ligation hybridization ELISA
      • Nuclease-based hybridization ELISA
      • Dual probe hybridization ELISA
    • Case Studies: Recent data on Bioanalytical support for ASO therapeutics for multiple studies

  • Lesson 6
    Quantitation of siRNAs in Liver & Kidney by stem-loop qPCR & hybridization ELISA
    - Dr. Kirk Brown, Associate Director, Alnylam
    • Advanced bioanalytical strategies to support Enhanced Stabilization Chemistry Plus (ESC+) N-acetylgalactosamine (GalNAc) ligands, covalently attached to the short interfering RNA (GalNAc-siRNA) conjugate platform
      • For systemic and liver-directed delivery
      • Preclinical comparative studies of GalNAc-siRNA and GalNAc-ASO compounds as gene silencing approaches
    • Bioanalytical strategies for quantification of mRNA from liver biopsies
      • Advanced use of RT-qPCR to measure the amount mRNA/pregenomic RNA (pgRNA)
        • Target signal amplification
        • Strategies to meet regulated bioanalysis requirements
        • Current discussions on qPCR validation criteria within the bioanalytical community
        • Sharing method development experiences
    • Combination of RT-qPCR assays & hybridization ELISA assays for full characterization of GalNAc-siRNA
      • Evidence-based scientific discussions
      • Industry standards on how Oligonucleotides are measured
      • What studies need to be performed
    • Complete analysis setup for Hepatocyte-targeted RNAi
      • DNA assay
      • RNA assays
      • Protein assay
    • Case Studies: Advanced bioanalytical applications for RNAi based therapeutic

  • Lesson 7
    Branched DNA (bDNA) & RT-qPCR Assays
    - Dr. Neil Henderson, Associate Principal Scientist, AstraZeneca
    • An in depth understanding of method Development & validation challenges in the quantification of oligonucleotides with specific applications on modified mRNA
      • Advantage/disadvantages and bDNA & RT-qPCR performance in regulated bioanalysis
      • Using bDNA as primary method for assessing modified mRNA drug in samples
        • Use of target-specific probes to amplify signal
        • Simplified bDNA assays performance similar to regular LBA
          • Ability of bDNA assays to meet LBA BMV Guidance/Guidelines criteria
        • No need for sample purification or conversion to cDNA prior to analysis
      • Considerations on bDNA & RT–qPCR applications in a fully regulated environment
        • Validation of mRNA assays
        • Meeting performance expectations for regulated studies
        • Special matrices where bDNA & RT–qPCR cannot meet current regulatory criteria
    • Case Studies: State-of-the-art bioanalytical approaches for quantification of modified mRNA


Part 3: LCMS, HRMS and Hybridization HPLC-FL Assays
  • Lesson 8
    Long-term tissue exposure of a GalNAC-conjugated oligonucleotide in monkeys after acute dosing
    - Dr. Tate Owen, Senior DMPK Scientist, Regulus Therapeutics
    • Pharmacokinetics of a GalNAC-conjugated oligonucleotide (RG-101) in monkey tissue after acute dosing
      • Oligonucleotide concentrations in the liver were monitored over the course of 1 year after a pharmacologically relevant doses ranging from 0.15 to 2 mg/kg
      • Concentrations of therapeutic oligonucleotides were measured from in-life biopsies using HPLC-FL
      • Correlation of liver concentrations with serum biomarkers (e.g., total cholesterol, HDL)
    • Techniques and considerations when taking PK measurements in biopsies
      • Biopsy procedure (e.g., needle gauge, sample area, etc)
      • Limited sample size of biopsy can limit techniques available (e.g., HPLC-FL vs. HPLC-HRMS)
      • Potential bias encountered when measuring tissue concentrations from biopsies
      • Metabolic profile in tissues as monitored through biopsy measurements vs. ‘whole tissue’ measurements
    • Case Studies: Pharmacokinetics and Pharmacodynamics of a Novel Hepatocyte Targeted Therapeutic Oligonucleotide in Cynomolgus Monkeys

  • Lesson 9
    Hybridization HPLC-FL Assays & Advanced use of HRMS
    - Dr. Alexander Behling, Senior Scientist Biomarkers, PPD
    • Mastering Hybridization HPLC-FL technique
      • Successful development and validation of bioanalytical methods for sensitive and specific quantification of Oligonucleotides
      • Strategies to hybridize fluorescent probe to the target
        • Probe-design requirements
      • Developing an anion-exchange HPLC
        • Minimizing non-specific binding
        • Increasing sensitivity
      • Considerations for Validation of Hybridization HPLC-FL assays
    • Quantitative determination of Oligonucleotides in plasma using peptide nucleic acid probe and HPLC with fluorescence detection
    • Additional advanced use of HRMS in support of Oligonucleotides Bioanalysis
      • Achieving selectivity & sensitivity in one single assay between multiple Oligonucleotides metabolites and parent molecule
    • Case Studies: Novel combination of Hybridization HPLC-FL and HRMS data to understand Oligonucleotides biodistribution

  • Lesson 10
    Reliable and Sensitive LCMS Based Bioanalytical Methods to Quantify “Stereocontrolled” Oligonucleotides in multiple Biological Matrices
    - Dr. Meena, Senior Director of Bioanalytical, Pharmacology and Biomarker , Wave Life Sciences
    • Understanding the advantages of LCMS vs. other hybridization-based techniques (HPLC-FL and ELISA) for modified Oligonucleotides
      • Developing high throughput methods
      • Increasing selectivity and improving sensitivity
    • LCMS higher selectivity vs non-chromatographic techniques
    • Solutions to overcome traditional Oligonucleotides issues in LCMS
      • Extreme polarity
      • Non-specific binding
      • Low extraction efficiency
      • Poor chromatography
      • High carryover
    • Using automation to increase throughput
    • Full BMV of LCMS methods according to Regulatory Guidance/Guidelines
    • Case Studies: Quantitative determination of Phosphorodiamidate Morpholino Oligonucleotides (PMO) in plasma by LCMS

  • Lesson 11
    LCMS Quantification of Lipid Containing Oligonucleotides with Phosphorothioamidate Linkage & Comparison with Hybridization ELISA
    - Dr. Lieve Dillen, Principal Scientist Bioanalysis, Janssen
    • Importance of the extraction method to increase selectivity & sensitivity
      • Development of AEX-SPE & Hybridization
        • Hybridization extraction with biotinylated complementary probe
      • Challenges in supporting preclinical & clinical studies
        • Method evolution
      • Chromatographic issues and investigation of specificity of the hybridization probes
    • Comparison of results between LCMS and Hybridization ELISA
      • In preclinical study
      • In clinical study
    • Impact of ion pairing and/or hexafluoroisopropanol (HFIP) concentrations on chromatography of several modified Oligonucleotides
    • Investigation of specificity of the hybridization probes for modified Oligonucleotides
      • Phosphodiester
      • Phosphorothioate
      • GAPmers
      • Truncated Oligonucleotides
    • Case Studies: LCMS method development for a lipid containing Oligonucleotide with Phosphorothioamidate linkage and other modified Oligonucleotides

  • Lesson 12
    PNA-HPLC vs LCMS Assay Bioanalysis of siRNA & sgRNA
    - Dr. Ingo Röhl, Director Analytics and Bioanalytics, Axolabs
    • LCMS method development & validation challenges to allow selective quantification of the intact oligonucleotides and the corresponding metabolites from tissues & plasma
      • LCMS for quantification of therapeutic oligonucleotides at low ng/ml
      • Achieving selectivity between metabolites and parent molecules in a single assay
        • Specific sample clean-up
        • Optimization of the LCMS parameter
        • Avoiding m/z overlap of numerous molecular precursor ions
        • Need for optimal chromatographic separation between interfering analytes
        • Careful monitoring of MS/MS transitions to identify possible interference
    • Specific applications of PNA-HPLC Assay
      • siRNA (short interfering RNA)
      • sgRNA (single guide RNA)
    • Peptide Nucleic Acid (PNA) hybridization-based HPLC-fluorescence assay
      • Unique new method development strategies and approaches
      • Selective & sensitive bioanalysis of Oligonucleotides
      • Why is PNA-HPLC technique different form other approaches?
        • Ability to accurately quantify Oligonucleotides up to 100 units in length
    • Case Studies: Original development and validation of PNA-HPLC and LCMS methods for siRNA & sgRNA

  • Lesson 13
    HRMS Specific Approaches for Quantitation of Double Stranded (DS) Oligonucleotides
    - Dr. Zamas Lam, SVP Preclinical Development, QPS
    • Simultaneously determination of both sense and anti-sense strands using the exact mass of the individual isotopic peaks of the different charge envelopes
    • Method Development and BMV for HRMS vs LCMS
      • Limitation of LCMS (Triple Quad MRM) in selectivity due to
        • Poor fragmentation
        • Metabolites with molecular weight differences of a single nucleotide
        • Metabolites cationization forming multiple pseudo-molecular ions
      • HRMS potentials for Oligonucleotides (QTOF or Orbitrap)
        • Unique characteristic for Oligonucleotides with unmodified backbone
        • Separation the multiply charged multiple pseudo-molecular ions of the parent Oligonucleotides as well as metabolites
    • Case Studies: Accurate quantification of up to 20,000 Daltons DS Oligonucleotides in HRMS




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