intelligent disulfide bond analysis · ask us for the technical note on performing disulfide bond...
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Intelligent Disulfide Bond Analysis
Why Study Disulfide Bonds?
Regulations Process Control
Protect from Competition
Efficacy
Save Money by making cell cultures more efficient!
Protect IP by making biotherapeuticsharder to copy
Higher Quality biotherapeutics
Why Study Disulfide Bonds?
Regulations Process Control
Protect from Competition
Efficacy
Save Money by making cell cultures more efficient!
Protect IP by making biotherapeuticsharder to copy
Higher Quality biotherapeutics
Why Study Disulfide Bonds?
Regulations Process Control
Protect from Competition
Efficacy
Save Money by making cell cultures more efficient!
Protect IP by making biotherapeuticsharder to copy
Higher Quality biotherapeutics
Why Study Disulfide Bonds?
Regulations Process Control
Protect from Competition
Efficacy
Save Money by making cell cultures more efficient!
Protect IP by making biotherapeuticsharder to copy
Higher Quality biotherapeutics
Regulatory Guidelines
HIGHER ORDER STRUCTURE IS CRITICAL!
Regulators Are Asking….
• ‘Higher Order Structure’ is specifically mentioned in USFDA, EMEA and ICH guidelines
Newer Guidelines ADDED Demand for Analysis of Biotherapeutics
• USA BPCI act of 2009 established a framework and in 2012 the USFDA guidelines
for biosimilars:
• ‘…a “totality of the evidence” approach, … by evaluating more attributes and
combinations of attributes at greater sensitivities with multiple complementary
methods.
• ‘… the scope and extent of [clinical] studies may be reduced further if more
extensive fingerprint-like characterization is used’.
FDA desire for specific innovations in biotherapeutic analysis
Steven Kozlowski, M.D.Director, Office of Biotechnology ProductsOffice of Pharmaceutical ScienceCenter for Drug Evaluation and ResearchFood and Drug Administration
Three Specific Properties Needing Improved Measurement
[The] FDA has identified three properties of therapeutic proteins that cannot be sufficiently measured at this time but that are very important for understanding the behavior of protein drugs. …..
1. Post-translation Modifications …2. Three-dimensional Structure …3. Protein Aggregation …
TESTIMONY BEFORE THE SUBCOMMITTEE ON TECHNOLOGY AND INNOVATION COMMITTEE ON SCIENCE AND TECHNOLOGY.U.S. HOUSE OF REPRESENTATIVES. SEPTEMBER 24, 2009
FDA desire for specific innovations in Biotherapeutic analysis
Steven Kozlowski, M.D.Director, Office of Biotechnology ProductsOffice of Pharmaceutical ScienceCenter for Drug Evaluation and ResearchFood and Drug Administration
Three Specific Properties Needing Improved Measurement
[The] FDA has identified three properties of therapeutic proteins that cannot be sufficiently measured at this time but that are very important for understanding the behavior of protein drugs. …..
1. Post-translation Modifications …2. Three-dimensional Structure …3. Protein Aggregation …
TESTIMONY BEFORE THE SUBCOMMITTEE ON TECHNOLOGY AND INNOVATION COMMITTEE ON SCIENCE AND TECHNOLOGY.U.S. HOUSE OF REPRESENTATIVES. SEPTEMBER 24, 2009
Protein Metrics Higher Order Structure
Higher Order Structure Covered in Multiple Ways
Higher Order
Structure
Disulfide Bonds (Secondary Structure)
Cross-Linking StudiesEpitope Mapping (Oxidative Footprinting)
Ask for our posters at: https://www.proteinmetrics.com/resources/
Why are Disulfide Bonds so Hard to Study?
Maths• VERY Large
search space
• VariabilitySample Issues
• Batch-to-batch changes
• Sample prep/ handling
Mass Spec
issues
• Ionization
• Fragmentation
• LC and CE challenges
• DIFFICULT!
• Size
Protein Variation
• ‘Family’ of molecules
• Dynamic system
Why are Disulfide Bonds so Hard to Study?
Maths• VERY Large
search space
• VariabilitySample Issues
• Batch-to-batch changes
• Sample prep/ handling
Mass Spec
issues
• Ionization
• Fragmentation
• LC and CE challenges
• DIFFICULT!
• Size
Protein Variation
• ‘Family’ of molecules
• Dynamic system
Why are Disulfide Bonds so Hard to Study?
Maths• VERY Large
search space
• VariabilitySample Issues
• Batch-to-batch changes
• Sample prep/ handling
Mass Spec
issues
• Ionization
• Fragmentation
• LC and CE challenges
• DIFFICULT!
• Size
Protein Variation
• ‘Family’ of molecules
• Dynamic system
Why are Disulfide Bonds so Hard to Study?
Maths• VERY Large
search space
• VariabilitySample Issues
• Batch-to-batch changes
• Sample prep/ handling
Mass Spec
issues
• Ionization
• Fragmentation
• LC and CE challenges
• DIFFICULT!
• Size
Protein Variation
• ‘Family’ of molecules
• Dynamic system
How do I Simplify Disulfide Bond Analysis?
Workflow Automation – Disulfide Bond Identification and Presentation
FIND the BondsDivide between
‘Expected’ and ‘Shuffled’Report
Byonic: Added Scoring Regime
PEPTIDEABCDEF
PEPTIDECXYZ
• Score fragments of PeptideA AND PeptideX …
• AND fragments of PeptideA+PeptideX
• Provide a combined ‘X-Link Score’
Dedicated Byonic Tab – Crosslinking and Disulfide Bonds
• Simple ‘check box’ for users
• Same process as for cross-linked proteins
Dedicated Byonic Tab – Crosslinking and Disulfide Bonds
• Links BETWEEN and INTRA-chain can be controlled – e.g. Chain 1 links to Chain 2
Dedicated Byonic Tab – Crosslinking and Disulfide Bonds
• Links BETWEEN and INTRA-chain can be controlled – e.g. Chain 1 links to Chain 2
• Use “;” means 1 and 2 DO NOT LINK TO CHAIN 3 – Intra-chain bonds only
Chain1 Chain2 Chain3xxxxxCx
Dedicated Byonic Tab – Crosslinking and Disulfide Bonds
• All theoretical bonds can be searched
• ‘Generate Details’ button displays which bonds will be searched
SORT the Bonds in Byologic® - Automated IgG Function
• Dedicated ‘Disulfides’ Tab in Byologic® allows user to select ‘IgG’ function
SORT the Bonds in Byologic® - Automated IgG Function
• Bonds are automatically assigned for IgG molecules
• All these bonds are ‘EXPECTED’
SORT the Bonds in Byologic® - Automated IgG Function
• Table is editable!
• User can select specific bond locations
• Useful for Engineered IgGs/ ADCs/ multi-specifics etc.
SORT the Bonds in Byologic® - List for User Review
• Bonds are ‘sorted’ into categories: • all bonds in the table are ‘Expected’
• All other bonds are ‘Shuffled’
REPORT the Bonds in Byologic® - Automated Graphing
• Protein Metrics has provided a pre-set report for Disulfide Bonds
Byologic® Report Automated Graphs
• Report tables and graphs are generated at the click of a button
• Data in the report reflects what is in the project
Poster Presented at ASMS
• XIC for quantitation
• Unambiguous identification of disulfide and trisulfide bonds
• Copy available on request from [email protected]
Ask us for the Technical Note on Performing Disulfide Bond Analysis
• Comprehensive overview of the use of Byonic™ and Byologic® for Disulfide Bonds
• For a copy contact us at www.proteinmetrics.com/resources
Protein Metrics Product Promise
Quality of Results
Intuitive User Experience
Vendor Neutral Solutions
Workflow Automation
Cost & Time Savings
Intact Mass™Sample ID and
Comparison
Byomap™Chromatogram
Analysis
Byonic™Protein and Peptide ID
Byologic®Inspection, Quant,
Reporting
Supernovo™mAb de novo sequencing
Pfizer Sequence Variant Webinar• Pfizer needs to monitor all sequence variants as part of development
• All potential sequence variants need to be accurately assessed and quantified
• Protein Metrics’ Byonic™ and Byologic® tools provide an ideal workflow
• Ability to dig very deep into the data and AUTOMATE the analysis
http://www.biopharminternational.com/bp/biotherapeutic
Old way was slow, inefficient, manual
New way with Protein Metrics is fast, efficient and automated - and MORE SAMPLES can be analyzed.
ADC webinar StemCentrx (BRK and PMI)
• Ability to use ALL of the data, no matter how complex
• Bruker data on Antibody-Drug-Conjugates (ADCs) in a StemCentrx webinar shows complex data
• Need to calculate ‘Drug-to-Antibody Ratio’ (DAR)
• StemCentrx, Bruker and Protein Metrics worked to improve the workflow in a real-world environment with HIGHLY COMPLEX data
http://worldadc-webinars.com/view-webinars/webinar-archive/
Bi-Specific webinar with Genentech
• Moving data processing to the next level of efficiency
• Software to quickly identify and report desired, undesired and unknown species
• High throughput capabilities
https://www.technologynetworks.com/tn/webinars/high-throughput-quantitative-analysis-of-bispecific-antibody-moieties-and-glycosylation-290703
