Lecture DETAILS of Th1 – Conventional & Novel Critical Reagents

• Lecture 1
  Impact of Restrictions on Animal-Derived Antibodies on Critical Reagents: Current Regulatory Status and Industry "Thinking out of the Box"
  - Dr. Boris Gorovits, Vice President Biomarker Discovery and Bioanalysis, Sana Biotechnologies
  • Non-animal-derived antibodies
    • Mature technology for critical reagents generation
    • Well-defined reagents equivalent to animal-derived antibodies
    • Better reagents with performance superior to animal-derived antibodies
    • Polypeptide sequences established as part of the generation process
    • Improved Reproducibility
    • Better supply sustainability of critical reagents
  • Thinking out of the box and alternative proposed solutions
    • Phage-display produced monoclonal antibodies
      • Current data on phage-display technique and its development as alternative antibody production
      • Potential issues with production of polyclonal antibody
      • Selection of antibodies using a universal recombinant library
    • DARPins (Designed Ankyrin Repeat Proteins)
      • Genetically engineered antibody mimeticnon-immuno globulinproteins
      • Derived from natural ankyrin repeat proteins
      • Highly specific and high-affinity target protein binding
      • Already successfully used and binders against several hundred targets have been created
      • Low-cost production in bacteria and the rapid generation of many target-specific reagents
    • Affimers
      • Small proteins that bind to target molecules with similar specificity and affinity to that of antibodies
      • Applications of non-antibody binding proteins to mimic the molecular recognition characteristics of monoclonal antibodies
    • Affibody
      • Small proteins engineered to bind to a large number of target proteins or peptides
      • Applications of these high affinity proteins and their ability to imitate monoclonal antibodies
  • Multiclonal non-animal-derived antibodies
    • Recent results showing exceeding performance vs monoclonal antibodies
    • Lower unspecific reactions and higher reproducibility vs polyclonal antibodies
  • Industry concern on the proposal by the European Commission to eliminate the use of antibodies derived from animals
    • Level of impact on LBA practice
    • Strong progress in reduction of animal use in the drug discovery and development process
    • Expected significant reduction of animal use in production of antibody reagents
    • Need to generate more data on non-animal derived antibodies
  • Case Studies: New ideas and methods, approaches currently tested. Use offully synthetic libraries and potential risk to produce lower affinity reagents

 

• Lecture 2
  Novel Critical Reagent Modalities: Pfizer positive/negative experience with Aptamers, Peptides, Virus-Like Particles (VLPs) and other non-antibody reagents
  - Ms. Beth Leary, Principal Scientist Bio Medicine Design Group, Pfizer
  • Investigations on novel critical reagents modalities to demonstrate that non-antibody reagents can perform as substitutes for antibody reagents
    • Why consider alternatives to antibodies for ligand-binding assay (LBA) reagents?
  • Aptamers
    • Synthetic oligonucleotides that adopt a 3D structure and bind to target molecules
    • Major benefit of aptamers as synthetic reagents
    • Less lot-to-lot variability
    • No issues with loss of antibody producing cell lines
  • Pros and cons for using Aptamers routinely in LBA
    • Advantages of Aptamers, reagents versus traditional antibody approaches
    • Considerations to be taken into account for substitution of the IgG protein framework
    • Non-antibody reagents costs vs traditional antibodies
    • Availability and companies able to produce them
  • Use of Critical Reagents generated from a fully synthetic gene library
    • Protein scaffolds able to provide certain advantages in particular applications
    • New alternative binders introduced in the market
  • Case Studies: Direct comparison of antibody and non-antibody reagents to determine if performance is comparable and can be used in LBA

 

• Lecture 3
  Recent Issues in Conventional & Novel Critical Reagents
  - Dr. Andrew Mayer, Group Leader Bioanalysis, GlaxoSmithKline
  • Issues in Conventional Critical Reagents
    • Variability between different batches produced in different animals at different times
    • Difficulty in reproducing a well-performing batch
    • Current approaches to minimized batch-to-batch variability
  • Potentially intrinsic lack/low of specificity
    • Considerations on the impact of additional light chains from various origins on specificity
  • Novel Reagents with improved affinity and specificity
    • Optimization of the binding kinetics
    • Directed evolution the association and dissociation rates
    • Ability to successfully fulfil the requirements of demanding applications as the natural immune response
    • Reduced cross-reactivity
    • Improved stability, affinity, avidity specificity and functionality
    • Multi-epitope recognition
  • Comparing the use of animal and non-animal derived antibodies as critical reagents
    • Impact on in bioanalytical assays
    • Non-animal derived critical reagents can be fit for purpose
    • Flexibility to use animal derived reagents when necessary.
  • Case Studies:Challenges & solutions in the application of conventional &novel critical reagents

• Lecture 4
  Advanced Approaches in Selection of Critical Reagents for Sensitive Clinical PK Assays
  - Ms. Kelly Coble, Director Bioanalytical Sciences, Boehringer Ingelheim
  • High Throughput Screening
    • Use of a fully automated system to perform high throughput screening of potential critical reagent pairs for use in early clinical PK method development
    • Evolution in process for assessment of reagent binding specificity and binding potency
      • Establishing blocking, Partial blocking andNnon-blocking reagents
  • Increasing ruggedness using Multiple Platforms
    • Use of high sensitivity platforms including SMCxPro, GyroLab and MSD during critical reagent evaluation and selection
    • Use of Octet and LBA assessment of reagent binding specificity
    • Use of Biacorefor binding potency assessment
    • Generation, screening and pairing of multiple anti-Ids
    • Risk of missing possible pair candidates
  • Evaluation of optimal performance
    • Sensitivity, Range, Robustness and Reproducibility
    • Critical Reagents and major impact on assays performance
    • Strategic reagent screening and characterization to minimize assays failure
    • Insights on strategies for selection of candidate pairs
  • Case Studies: Highlight approaches as well as some key lessons learned during the evaluation of Critical Reagents

 

• Lecture 5
  Development of Novel Technologies to Enable Efficient Generations of High-Quality Critical Reagents: AstraZeneca’s experience
  - Dr. Ellen O’Connor, Associate Director Biopharmaceutical Development, AstraZeneca
  • Novel Technologies and applications in bioanalytical reagents characterization
    • Improvement of critical reagent controls with new orthogonal techniques
    • High-throughput kinetic information
    • Assay failures based on lack of in-depth biophysical characterization
    • Using automated platforms to make reagent generation more efficient and improve turn-around times
  • Application of Immobilized Metal Affinity Chromatography (IMAC) in critical reagent selection
    • Applications of IMAC to show how the isoelectric point of antibodies influence the conjugation reaction and formulation conditions
    • Affinity chromatography as preferred method to capture proteins from cell culture harvest
    • Highly selective ligand to bind a Fc-containing in monoclonal antibodies
  • State-of-the art strategies in Critical Reagent purification
    • Development of new technologies to enable efficient reagent generations
    • Strategic reagent screening and characterization to minimize assays failure
  • Case Studies: Lesson learned in critical reagent sciences from recent biotherapeutic programs and solution to generate high quality reagent to drive the R&D pipeline

 

• Lecture 6
  Importance of Affinity Determination in Critical Reagent Development: Pfizer experience based on multiple case studies
  - Ms. Teresa Caiazzo, Senior Principal Scientist BioMedicine Design Group, Pfizer
  • Current approaches for selection of high-affinity, stable and well-characterized Critical Reagents
    • Importance of well-characterized critical reagents as essential requirements for high performing ligand binding assays (LBA)
    • Identification and understanding of key characteristics of mAb reagents related to LBA performance to provide a strategic advantage to improved LBA performance
    • Issues with specific antidrug and/or anti-target Critical Reagents
    • Impact of Critical Reagents on the assay design and format
    • Critical Reagents cross reactivity
    • Affinity Determination by Biacore, Octet or similar applications
  • Assessment of binding properties
    • Critical Reagent Affinity and relation to bioanalytical assay function
    • Cross-reactivity to matrix components
    • Binding kinetics
    • Altered binding issues due to labeling or storage
  • Case Studies: Lesson learned on Importance of Affinity Determination from numerous biotherapeutic programs. Exploitation of the kinetic attributes of monoclonal antibody (mAb) reagents to facilitate fit-for-purpose applications in LBA and minimize development times

 

• Lecture 7
  The Effect of Degree of Labeling (DoL) & Aggregation on Pharmacokinetic Ligand Binding Assays (PK LBA)
  - Dr. Shannon Chilewski, Principal Scientist, Bristol-Myers Squibb
  • Consideration on Labeling of Capture & Detection Reagents for PK LBA
    • Antibodies conjugation with a chemical moiety (Tag) for
      • Being coated onto the plate (capture)
      • Signal generation in the assay (detection)
  • Impact of Degree of Labeling (DOL) of Tags on the antibodies
    • Influence on stability and efficacy
    • Binding alteration of the physical and chemical properties of both antibody and conjugate
  • Issues with the Aggregation propensity of unconjugated antibody
    • Compounding or reduction during labeling
    • Affect reagent stability over time
  • Vital importance of effects DOL & Aggregation on different PK LBA to ensure high assay performance
    • Understanding the effects of DOL & Aggregation
    • Application of a systematic approach to evaluate different levels of DOL & Aggregation
    • Testing on three types of commonly used PK LBA
      • Chemiluminescent ELISA
      • MSD-ECL assay
      • Gyrolab assay
  • Case Studies: LDOL & Aggregation impact on PK LBA and determination of assay characteristics including an effect on binding, assay sensitivity, and assay range

• Lecture 8
  Selection and Application of "Clinically Relevant" Positive Control (PC) anti-Ids for ADA Assays
  - Dr. Christine Grimaldi, Distinguished Research Fellow, Boehringer Ingelheim
  • Current challenges in the development of drug-related surrogate Positive Control
    • Anti-ID PC as a surrogate for ADA
    • Clinical significance/relevance of binding epitopes, binding potency and isotypes for patient ADA vs lab generated anti-ids
  • Screening paradigms to PC selection and impact on ADA assay method
    • Assessment of affinity/avidity and relevance to assay sensitivity
    • Neutralizing vs binding anti-id PCs
    • Selection of PCs for multidomain molecules
    • Impact of acid treatments on PCs
  • Impact of PC on ADA Assays performance
    • Assay performance using polyclonal vs monoclonal vs pooled monoclonal anti-ids
    • Impact of PC binding potency on assay sensitivity
  • Are improved Positive Controls representative of patients’ immune response?
    • Strategies to overcome the risk of developing ADA assays with high affinity PC that are not reflective of the patients’ immune response
  • Case Studies: Importance of selecting clinically relevant Positive Controls for improving the performance ofADA assays and of testing multiple PCs and assay conditions

 

• Lecture 9
  Determination of Binding Affinity for Positive Controls (PC) and Critical Reagents for ADA Assays
  - Dr. Yang Xu, Senior Principal Scientist, Merck
  • Improved approach in the development of PC
    • Specificity
    • Acid tolerability
    • mAb PC more consistent from lot-to-lot
    • pAb PC considered more representative
  • Importance of characterization of suitable Positive Controls
    • Best Positive Control for assay performance and determination of Assay Sensitivity
    • Altered Drug Tolerance in ADA assays due to Positive Control and Critical Reagents issues
    • Positive Control influence on Cut Point Determination
  • Novel strategies
    • Current challenges in the development of drug-related surrogate Positive Control
    • Well-characterized Positive Controls and ADA Assays performance
    • Purity and stability
  • Case Studies: Recent improvementsin the Generation & Characterization of Positive Controls (PCs) for ADA Assays

 

• Lecture 10
  Reagent Troubleshooting for ADA Assay - Lesson learned from recent biotherapeutic programs
  - Dr. Hanjo Lim, Scientific Manager BioAnalytical Sciences, Genentech
  • Current strategies for conjugation and BPC capabilities
    • Streamlined reagent request and report systems/processes
  • ADA Assays - complete investigations to find the rootcause
    • Method Troubleshooting using characterization strategies
    • Follow‐up analytical investigation
    • Strategy to monitor the quality of ADA LBA critical reagents
  • Overall consideration on how to perform Bioanalytical troubleshooting of recent LBA impacted by the characteristic of the Critical Reagents
    • Step-by-step investigation
    • Tools for assuring reagent consistency for long‐term LBA performance and high‐quality data generated
    • Issues with conjugate incorporation ratios that affected assay performance
    • Issues with buffer component that affected conjugation efficiency
    • Troubleshooting binding affinity that affected assay performance
  • Case Studies: Lesson learned from recent biotherapeutic programs involving ADA assays. In depth investigations on the Impact of LBA Critical Reagent Quality on ADA Assay performance

• Lecture 11
  Vaccine LBA - Strategies for a successful Critical Reagent Management
  - Dr. Francis Dessy, Clinical Assays Science Advisor, GlaxoSmithKline
  • Influence of Critical Reagents Life Cycle Management on Vaccine LBA
  • How to minimize the risk of interruptions/delays to/of the supply chain of Critical Reagents for Vaccine Clinical Assays
    • Bad planning, forecasting and management
    • Unavailability of the right reagents at the right time
    • Delays for method development and validation
    • Impact on program decisions, time and resources
  • Common failures inCritical Reagents Life Cycle Management
    • Inefficient and insufficient implementation of adequate characterization technologies
    • Production of low-quality reagents
  • Strategies for new Lots of Critical Reagentsand need to
    • Re-develop
    • Re-validate
    • Cross-validate Vaccine LBA
  • Case Studies: Considerations to ensure a well-designed Critical Reagent Management for long term support of Vaccine LBA

 

• Lecture 12
  Critical Reagent Management for Lot-to-Lot Bridging and Assay Comparability: Janssen’s Experience & Perspective
  - Dr. Mark Ware, Scientific Director, Janssen
  • Consistency of Performance of Critical Reagents
    • Foundation of maintaining accurate and precise assay results of sample analysis throughout the lifespan of its use
    • Strategies to control production, labeling, storage and use of critical reagents
  • Considerations on lot-to-lot bridging
    • Lot-to-lot bridging cannot always be expected to be a replicate of current performance
    • Variability within acceptance criteria may be observed throughout the lifecycle of an assay
  • Variability of Performance of assays over time
    • Presence of variability also when additional control parameters are in place
    • Variability can exist across and within studies, with no determined singular root cause
    • Considerations impactful differences in sample accuracy or precision
    • Considerations on Trend Analysis over the years
  • Case Studies: Proper design ofbridging methods for Critical Reagents. Influence of Critical Reagents Life Cycle Management on LBA for successful biotherapeutics discovery & development