Intelligent Disulfide Bond Analysis · Ask us for the Technical Note on Performing Disulfide Bond...

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 info@proteinmetrics.com

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

For a live demo contact info@proteinmetrics.com

www.proteinmetrics.com

Thank You