"LBA Novel Technologies, Novel Modalities, and Novel Challenges"
Innovative Tools/Instrumentation/Approaches; Advanced Strategies in Automation; Advancements in Critical Reagents Deep Functional Characterization; Problem Solving for Novel Modalities, Rare Matrices and Complex NAb Assays
(You can scroll down to see the details of each topic)
Part 1: Novel Technologies & Automation in LBA
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Topic 1:
Impact of Novel Technologies in Bioanalysis for advanced PK Assays and Biomarker Assays Applications
Dr. Sally Fischer, Senior Director Bioanalytical Sciences, Genentech -
Topic 2:
Advanced Strategies in Automation of Large Molecule Bioanalysis
Dr. Mark Ware, Scientific Director, Janssen -
Topic 3:
Automated Bioanalytical Workflow for PK & ADA LBA Development
Dr. Linlin Luo, Director Immunogenicity & PK Assay Group, Merck -
Topic 4:
Automation or "industrialization" for sample pre-treatment/ADA enrichment for Immunogenicity assays
Ms. Alison Joyce, Director, Discovery Bioanalytical & Critical Reagents, Pfizer
Part 2: Novel Modalities & Novel Method Development/Validation Challenges
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Topic 5:
Challenges in Enzyme Assays Development/Validation for Gene Therapy: How to overcome lacks enzyme reference material, to establish QC ranges and manage long term assay performance
Dr. Yanmei Lu, Vice President Biomarker and BioAnalytical Sciences, Sangamo -
Topic 6:
Complex LBA Method Development/Fit for Purpose Validation for Cas9: From Critical Reagents selection to different molecule recognition, Step by step method development challenges & solutions
Dr. Neil Henderson, Associate Director Nucleotide Bioanalysis Group, AstraZeneca -
Topic 7:
Challenges with Development/Validation of a PK assay to Measure a Variably Glycosylated Fusion Proteins
Dr. Gizette Sperinde, Principal Scientist Bioanalytical Sciences, Genentech
Part 3: Novel Modalities & Rare Matrices
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Topic 8:
PK & ADA Assays for Drugs Administered through Nonstandard Routes (Intravitreal & inhaled): Is there benefit to analyze in these matrices?
Ms. Kelly Coble, Director of Bioanalytical Sciences, Boehringer Ingelheim -
Topic 9:
Novel Strategies in Method Development/Validation and Sample Collection/Analysis for LBA Ophthalmology Bioanalysis: Special focus on Novel Modalities and Limitations in Sample Availability
Dr. Kay-Gunnar Stubenrauch, Strategy Area Lead Genomic Medicine Bioanalytics & Biomarkers, Roche
Part 4: Problem Solving for Complex NAb Assays
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Topic 10:
A Brand-new Idea to Overcome Drug Interference in NAb Assay: “The PEG precipitation of the drug/NAb complex”
Dr. Weifeng Xu, Director, Merck -
Topic 11:
Novel Duplex Assay to Assess NAb Response to Bispecific Biotherapeutic with Weak Binding to T Cell Receptor
Dr. Bonnie Wu, Associate Scientific Director Biologics Development Sciences, Janssen
Part 5: Critical Reagents Deep Characterization
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Topic 12:
Importance of Assay Reagent Binding Kinetics for Bioanalysis: What Factors Drives the Bioanalytical Assay Strategy and will we be able to Predict the "Best Assay Protocol"?
Dr. Gregor Jordan, Lab Head Bioanalytics & Biomarkers, Roche -
Topic 13:
Importance to Develop of an Appropriate Target-Antigen Binding Assay to evaluate ADA Impact on Drug-to-Target Interaction
Dr. Gregor Lotz, Group Leader Bioanalytics & Biomarkers, Roche -
Topic 14:
Evolved Applications of Biolayer Interferometry for Bioanalytical Assay Development
Dr. Florian Neff, Lab Head Bioanalytics & Biomarkers, Roche
Part 5: White Paper in Bioanalysis
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2022 White Paper on LBA Topics:
Consensus & Conclusions on LBA Topics for 2022 White Paper
Topic DETAILS of Th1
Part 1: Novel Technologies & Automation in LBA
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Topic 1
Impact of Novel Technologies in Bioanalysis for advanced PK Assays and Biomarker Assays Applications
Dr. Sally Fischer, Senior Director Bioanalytical Sciences, Genentech-
Novel Technologies and their huge potential in bioanalysis
- Enabling more informed drug development decisions
- Interrogation of drug and biomarkers in serum/plasma as surrogate for site of action
- Evaluations at the site of action when needed
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Multiplexing technologies
- Quantification of multiple biomarkers for evaluation of complex diseases
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Evaluation of PK and Biomarkers at lower volumes and concentrations
- Novel technologies for nasal samples for asthma indications
- PK assay for an oral antigen binding fragment of heavy chain only antibodies (VHH drug) at very low concentration in serum
- Quantification of neuro biomarkers in serum and/or plasma instead of CSF to eliminate invasive spinal tap collections
- Specific technologies for biomarkers quantification for ophthalmology indications in vitreous fluid
- Case Studies: Illustration of the capabilities of promising Novel Technologies in bioanalytical field
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Novel Technologies and their huge potential in bioanalysis
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Topic 2
Advanced Strategies in Automation of Large Molecule Bioanalysis
Dr. Mark Ware, Scientific Director, Janssen-
Current developments in the fully automated processes in assay development and bioanalysis of biologics
- When and how to apply automation
- Application of automation highly suitable to large molecule bioanalysis
- Degree of automation and standardized processes
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Expectations of outcomes of automation
- Benefit such investments and disruptions as to how operate
- Infrastructure changes and workspace defined layouts
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Impact upon workforce
- Adaptation of laboratory scientists to this new environment
- Right skill sets
- Needs for IT infrastructure
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Implementation, execution, and monitoring
- Hurdles of the on-site testing before implementation
- Computer systems validation (CSV)
- Monitoring /maintenance to ensure long term benefits
- Regulatory considerations
- Case Studies: Integrating automated approaches in bioanalysis
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Current developments in the fully automated processes in assay development and bioanalysis of biologics
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Topic 3:
Automated Bioanalytical Workflow for PK & ADA LBA Development
Dr. Linlin Luo, Director Immunogenicity & PK Assay Group, Merck-
Need for implementing Automation into LBA
- Meeting the increasing demand for project support
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Innovative workflows using automation
- Implementation for both PK & ADA assay development
- Comprehensive design of experiment (DOE)
- Choosing the right Assay Format for robust PK assays
- Using DOE and automation t to optimize the ADA assay conditions
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Automation LBA improvements
- Robustness
- Reproducibility & Reliability
- Throughput
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Screening & Selecting Optimal Reagents
- Quick ranking of candidate anti-IDs
- Identification the best anti-ID pairing
- Case Studies: Automated bioanalytical workflow consisting of automated assay implementation and data analysis
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Need for implementing Automation into LBA
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Topic 4:
Automation or “industrialization” for sample pre-treatment/ADA enrichment for Immunogenicity assays
Ms. Alison Joyce, Director, Discovery Bioanalytical & Critical Reagents, Pfizer-
Need for efficient sample pre-treatment steps for ADA assays & NAb assays to address issues such as
- Drug Tolerance
- Matrix Interference
- Multiple acid steps and drug-coated magnetic beads used on pre-treatment steps
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Current problems in sample pre-treatment methods
- Method Complexity
- Time-consuming
- Difficulty to transfer to CRO
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Innovative use of automation as alternative methods to magnetic bead approach
- Using tips pre-loaded with streptavidin resin for automated ADA enrichment
- Case Studies: Automated pre-treatment/ADA enrichment for Immunogenicity assays
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Need for efficient sample pre-treatment steps for ADA assays & NAb assays to address issues such as
Part 2: Novel Modalities & Novel Method Development/Validation Challenges
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Topic 5:
Challenges in Enzyme Assays Development/Validation for Gene Therapy: How to overcome lacks enzyme reference material, to establish QC ranges and manage long term assay performance
Dr. Yanmei Lu, Vice President Biomarker and BioAnalytical Sciences, Sangamo-
Considerations on enzymes and inherited metabolic disorders
- Therapeutic approaches for treating hereditary monogenic disorders with enzyme deficiency
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Enzyme Assays vs LBA
- Utility of enzyme activity readout during preclinical and clinical drug development
- Advantage of using enzyme activity assay over protein level quantification technologies such ligand binding assays (LBA)
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Enzyme Assay Technologies
- Different enzyme assay technologies
- Assay designs
- Recording modes
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Unique challenges of enzyme assay for Gene Therapy
- Enzyme assay for Gene Therapy vs Enzyme Replacement Therapy (ERT)
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Fluorometric enzyme assay Development/Validation
- Reading 4-Methylumbelliferone (4-MU) product conversion for Gene Therapy Biomarker Analysis
- Selection of instrument
- Quantitation strategy of using 4-MU product curve instead of recombinant enzyme calibration curve
- Assay development considerations and steps
- Assay validation parameters
- Case Studies: Recommended practice in enzyme assay development and validation to quantify the activity of transgene expressed enzyme in gene and cell therapy as a biomarker readout during preclinical and clinical drug development.
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Considerations on enzymes and inherited metabolic disorders
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Topic 6:
Complex LBA Method Development/Fit for Purpose Validation for Cas9: From Critical Reagents selection to different molecule recognition, Step by step method development challenges & solutions
Dr. Neil Henderson, Associate Director Nucleotide Bioanalysis Group, AstraZeneca-
Bioanalytical challenges in the quantification of Cas9 endonuclease bacterial protein
- Cas9 technology characterization & bioanalytical assays
- Efficacy assessment & Safety considerations
- Specificity evaluation
- Delivery to the target cells
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LBA approaches to measuring CRISPR Cas9 RNP complex components
- Method Development strategies
- Historical and future landscape of Critical Reagent sourcing
- Overcoming cell extract DNA interference
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Need for different selective methods for
- spCas9
- SaCas9
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Helpful information coming from bDNA for guide RNA
- Guide RNA probe design
- Dual guide delivery
- Extraction efficiency
- Cross reactivity with transfected DNA when trying to measure expressed RNA transcript
- Case Studies: LBA Method Development/Fit for Purpose Validation for Cas9
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Bioanalytical challenges in the quantification of Cas9 endonuclease bacterial protein
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Topic 7:
Challenges with Development/Validation of a PK assay to Measure a Variably Glycosylated Fusion Proteins
Dr. Gizette Sperinde, Principal Scientist Bioanalytical Sciences, Genentech-
Protein Glycosylation for Large Molecule therapeutic drug candidates
- Significant impact on PK
- Difficulty in controlling Protein glycosylation during the manufacturing process of large molecules
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Highlighting the challenges in quantification of a of highly glycosylated product
- Sialic acid variability
- Different assay development strategies
- Ensuring robust and unbiased detection of the therapeutic candidate
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Detection of variably glycosylated proteins
- Impact from both critical reagents and platforms used in bioanalysis
- Evaluation of assay platform and formats to ensure detectability of a wide set of sialic acid variants
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Identification of the best assay platform able to meet requirements
- Simoa assay
- Hybrid assay
- Case Studies: Development/Validation of a PK assay to Measure Variably Glycosylated Fusion Proteins
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Protein Glycosylation for Large Molecule therapeutic drug candidates
Part 3 Novel Modalities & Rare Matrices
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Topic 8:
PK & ADA Assays for Drugs Administered through Nonstandard Routes (Intravitreal & inhaled): Is there a benefit to analyze in these matrices?
Ms. Kelly Coble, Director of Bioanalytical Sciences, Boehringer Ingelheim-
Influence of Route of Administration on Systemic Exposure and Immunogenicity of biotherapeutics
- What is known about absorption into systemic circulation after administration?
- Biotherapeutic agents administered via a nonstandard routes
- What are the gaps with ocular or inhaled administration of biotherapeutics?
- Impact of the specific properties of the different tissue matrices considered for nonstandard routes of administration
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Challenges for sample collection & sample processing of human tissue matrices
- Bronchoalveolar lavage fluid (BALF)
- Ocular tissues
- Tumor tissues
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Bioanalytical assay considerations for development of assays for non-standard tissue matrices
- PK assays and ADA assays from matrices collected from nonstandard routes of administration
- Case Studies: Bioanalysis of biotherapeutics administered via nonstandard routes and specific tissue bioanalysis
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Influence of Route of Administration on Systemic Exposure and Immunogenicity of biotherapeutics
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Topic 9:
Novel Strategies in Method Development/Validation/Sample Collection/Analysis for LBA Ophthalmology Bioanalysis: Special focus on Novel Modalities and Limitations in Sample Availability
Dr. Kay-Gunnar Stubenrauch, Strategy Area Lead Genomic Medicine Bioanalytics & Biomarkers, Roche- Bioanalytical Lesson Learned from VABYSMO (faricimab-svoa) for the treatment of adults with Neovascular (Wet) Age related Macular Degeneration (AMD) and Diabetic Macular Edema (DME)
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Understanding how the molecular structure and route of administration (Intravitreal - IVT) of engineered bi-specific antibody drive the selection of appropriate assays for PK, ADA and Soluble Targets analysis in
- Pre-clinical Development
- Clinical Studies
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Challenges, solutions, and learnings over a decade of method development, validation and sample collection/analysis for PK and ADA Assays
- Overcoming the limitations in sample availability by using ultra-sensitive analysis
- Drug Interference and Soluble Target Inference (against two multimeric soluble targets, VEGF and Ang2) for ADA testing with special respect to ophthalmology specific issues
- Case Studies: Novel data on the relationship of the ADA response in different matrices (plasma vs ocular fluids) and use of Ultra-sensitive platforms to overcome sample availability
Part 4: Problem Solving for Complex NAb Assays
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Topic 10:
A Brand-new Idea to Overcome Drug Interference in NAb Assay: “The PEG precipitation of the drug/NAb complex”
Dr. Weifeng Xu, Director, Merck-
Drug Interference is one of the biggest challenges for NAb assay
- Major impact especially for mAb therapeutics in oncologic indication with high dose and long half-life
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Considerations on the most popular sample pre-treatment used to mitigate drug interference
- Acid dissociation followed by biotinylated-drug to extract NAb (BEAD)
- Known impact of harsh acid which often denature acid sensitive NAb PC
- Need for large amount of biotin-drug
- Need for expensive SA-magnetic beads
- Overall tedious and time-consuming process involving beads
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Applications of a novel use of PEG to selectively precipitate drug/NAb complexes and wash away free drug
- Very small amount of biotin-drug is added to dissociated drug and NAb
- Much mild acid to form biotin-drug/NAb complex.
- Complex binds to MSD plate and is detected with a sulfo-tagged drug target
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Advantage of this PEG precipitation and acid dissociation with biotin-drug as assay drug (PABAD)
- One step mild acid treatment instead of two-step harsh acid in BEAD;
- Less than 1/1000 of biotin-drug needed
- No magnetic beads needed
- No additional assay drug needed
- Case Studies: Novel approaches to overcome Drug Interference in NAb Assay
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Drug Interference is one of the biggest challenges for NAb assay
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Topic 11:
Novel Duplex Assay to Assess NAb Response to Bispecific Biotherapeutic with Weak Binding to T Cell Receptor
Dr. Bonnie Wu, Associate Scientific Director Biologics Development Sciences, Janssen-
Considerations on the Bioanalytical strategy for Bispecific Therapeutic Proteins
- Customization of the Bioanalytical strategy
- MOA and impact on assay selection
- Cell-based vs Non-cell based assays
- Target biology
- Availability of the critical reagents
- Positive Control (PC) used
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NAb assay format for bispecific therapeutic proteins
- Selection of Non-cell based assay format for assessment of NAb to bispecific therapeutic proteins
- Challenge to develop NAb assays for bispecific MAb biotherapeutic that is engaged in T Cell redirected cancer cell killing but has weak binding to T cell receptor
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Method development using an AlphaLisa technology platform
- Optimization of duplex AlphaLisa assay
- Strategies for a robust NAb detection
- Assay sensitivity, robustness, and drug tolerance
- Key assay parameters for the duplex AlphaLisa-based NAb assay
- Case Studies: Development of a movel Duplex Assay to assess NAb response to Bispecific Biotherapeutic with Weak Binding to T Cell Receptor
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Considerations on the Bioanalytical strategy for Bispecific Therapeutic Proteins
Part 5: Critical Reagents Deep Characterization
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Topic 12:
Importance of Assay Reagent Binding Kinetics for Bioanalysis: What Factors Drives the Bioanalytical Assay Strategy and will we be able to Predict the "Best Assay Protocol"?
Dr. Gregor Jordan, Lab Head Bioanalytics & Biomarkers, Roche-
Modelling & Simulation Strategies:
- What are the minimum parameters we need to select a platform as suitable?
- Use of mathematical assay model to support the decision for when the soluble target could be bound by several soluble binding partners
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Approaches for selecting an Assay Platform/Protocol
- Relevant parameters for Free or Target Engagement analysis.
- KD value, Rate Constants or Reagent Concentrations
- Need to evaluate the KD value
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Critical Reagents Selection
- Has the Capture Reagent always to be less affine to the soluble target compared to the drug?
- Do Critical Reagents (Capture and Detection) have to be in terms of binding properties comparable, and in case not, what can be done to optimize the assay protocol?
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How to optimize assays protocols
- Equilibrium Based or Kinetic Based
- Considering a Total assay protocol:
- Shall we skip Target Engagement(TE) assays and go for Total assay analysis and then calculate the Free portion?
- Case Studies: Bioanalytical assay strategies to predict the most suitable assay platforms/protocols
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Modelling & Simulation Strategies:
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Topic 13:
Importance to Develop an Appropriate Target-antigen Binding Assay to evaluate ADA Impact on Drug-to-target Interaction
Dr. Gregor Lotz, Group Leader Bioanalytics & Biomarkers, Roche-
Importance to develop proper target-antigen assay Critical Reagents able to
- Be a mimicry of the target antigen in patients to detect functional drugs
- Assess ADA interference on target binding
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Need for a deeper functional characterization of recombinant target-antigen critical reagents for clinical assays to use them as an adequate reagent tool for PK assay development
- Biochemical binding
- Biological activity
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Patient-derived ADAs interference with the drug binding to recombinant and cell membrane target
- Evaluation of adequate comparability of loss of drug binding function
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Use of PK assay with the characterized recombinant target-reagent tool to show
- Correlation of ADA response
- Loss of exposure in clinical trial
- Case Studies: Development challenges and strategies for Target-antigen Binding Assay Critical Reagents
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Importance to develop proper target-antigen assay Critical Reagents able to
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Topic 14:
Evolved Applications of Biolayer Interferometry for Bioanalytical Assay Development
Dr. Florian Neff, Lab Head Bioanalytics & Biomarkers, Roche-
Characterization of bioanalytical assay Critical Reagents:
- Biolayer Interferometry (BLI) vs
- Surface Plasmon Resonance (SPR)
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Head-to-head evaluation to measure
- Binding kinetics
- Affinity of molecular interactions
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Method comparison for quality determination of
- Anti-idiotypic antibodies
- Antibody conjugates
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Use of Biolayer Interferometry to accelerate screening for optimized sample pre-treatment conditions
- Evaluation of Drug/Target complex dissociation
- High potential of Biolayer Interferometry for fast characterization of ADA responses
- Case Studies: Recent advanced applications of Biolayer Interferometry (BLI) vs SPR for LBA Critical Reagents development in bioanalysis
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Characterization of bioanalytical assay Critical Reagents: