12/18/2011
2011 IETF Day 3 Skilton 1
Biosimilars, Biobetters and Beyond:
Coping with an Accelerating
Biotherapeutic Market
©2010 Waters Corporation | COMPANY CONFIDENTIAL
St John Skilton, Ph.D., Ying Qing Yu Ph.D., Weibin Chen Ph.D.
Waters Biopharmaceutical SciencesRakesh KS, Deepalakshmi PD - Waters India Pvt Ltd
India Executive Technology ForumMilford, December 2011
©2010 Waters Corporation | COMPANY CONFIDENTIAL
12/18/2011
2011 IETF Day 3 Skilton 2
Terminology
‘Bio-betters’
‘Bio-generics’
‘subsequent-entry biologicals/biologics’
‘me-too biologicals/biologics’
‘similar biopharmaceuticals’
©2011 Waters Corporation | COMPANY CONFIDENTIAL 3
‘Follow-on Biologics’‘2nd Generation mAbs’
‘noninnovator proteins’
Terminology
‘Bio-betters’
‘Bio-generics’
‘subsequent-entry biologicals/biologics’
‘me-too biologicals/biologics’
‘similar biopharmaceuticals’
BIOSIMILARS
©2011 Waters Corporation | COMPANY CONFIDENTIAL 4
‘Follow-on Biologics’‘2nd Generation mAbs’
‘noninnovator proteins’
12/18/2011
2011 IETF Day 3 Skilton 3
Overview
Market Factors
Regulatory Positions
Deploying
Factors
©2011 Waters Corporation | COMPANY CONFIDENTIAL 5
Deploying New Tools
Return on Investment
Overview
Market Factors
Regulatory Positions
Deploying
Factors
©2011 Waters Corporation | COMPANY CONFIDENTIAL 6
Deploying New Tools
Return on Investment
12/18/2011
2011 IETF Day 3 Skilton 4
Top Prescription Drug Sales: Proteins Increasing
Biomols 20081 of top 5 1 of top 5 5 of top 10
28% of top 100
©2011 Waters Corporation | COMPANY CONFIDENTIAL 7 EvaluatePharma.com
Top Prescription Drug Sales: Proteins Increasing
Biomols 20081 of top 5 1 of top 5 5 of top 10
28% of top 100
©2011 Waters Corporation | COMPANY CONFIDENTIAL 8
Biomols 20145 of top 5 7 of top 10
50% of top 100
EvaluatePharma.com
12/18/2011
2011 IETF Day 3 Skilton 5
Antibodies represent the next biotherapeutic class open to European biosimilar competition (McKinsey Report)
©2011 Waters Corporation | COMPANY CONFIDENTIAL 9
Anti-infectivePEGIntron
Erbitux
Pegasys
5.7AvastinRituxanHerceptin Remicade
mAbs are a major class of biotherapeutic soon open to US biosimilar competition
2009 sales ($Billion)
Oncology
Endocrine
Rheumatology
Erbitux
NovoRapid/NovoLog
4.3Lantus Neulasta
Avonex Rebif
5.7
6.1Enbrel
Humira
Levemir
Norditropin SimpleXx
NovoMix
Humalog1.1
0.8
©2011 Waters Corporation | COMPANY CONFIDENTIAL 10
Blood
CNS
Modified slide from McKinsey and CompanyData Source: Evaluate Pharma
201520142013201220112010
Rebif
NovoSeven KogenateProcrit/Eprex
Tysabri
20202019201820172016
Monoclonal AbOther protein
US Patent Expiration
2021
12/18/2011
2011 IETF Day 3 Skilton 6
World-wide Sale of Biologics Coming Off Patent
20
Significant businessopportunity for
biosimilars
58($B)
17
9
©2011 Waters Corporation | COMPANY CONFIDENTIAL 11
2009 2010 20132011 2012 2014 2015 Total
1 4 2 3 9 167 44# ofmlc
1 2 1
8
From: Patricia Seymour, The Challenges of Demonstrating Comparability of Biosimilar Products
Pharmaceutical Growth Biopharmaceuticals vs. Small molecules vs. Biosimilars
Projected Biosimilar
2015
$3.7 Bn
Biosimilar Market
2010
©2011 Waters Corporation | COMPANY CONFIDENTIAL 12
http://pharmtech.findpharma.com/pharmtech/News/Biosimilars-Market-will-be-Worth-37-Billion-by-201/ArticleStandard/Article/detail/713914?contextCategoryId=35097
$234 M
12/18/2011
2011 IETF Day 3 Skilton 7
Cost Estimates High …But Profitability High
Development Cost estimates:
75 250 Milli USD
©2011 Waters Corporation | COMPANY CONFIDENTIAL 13
— 75 - 250 Million USD
— 7 – 8 years
Highly profitable outcomes
Overview
Market Factors
Regulatory Positions
Deploying
Factors
©2011 Waters Corporation | COMPANY CONFIDENTIAL 14
Deploying New Tools
Return on Investment
12/18/2011
2011 IETF Day 3 Skilton 8
Worldwide Legislation
“Although copy versions of original biopharmaceuticals are
already available in different parts of the world, there are no
consistent worldwide requirements for their registration”.
o (Yet)
©2011 Waters Corporation | COMPANY CONFIDENTIAL 15
http://www.nature.com/nbt/journal/v29/n8/full/nbt.1936.htmlNature Biotechnology; 29, 690–693, 2011. doi:10.1038/nbt.1936
Worldwide Harmonisation is Getting Closer
“Steps toward a global,
harmonized regulatory approach
may already be in sight, with the
World Health Organization (WHO)
scheduled to finalize its guidelines
on the evaluation of similar
biotherapeutic products in 2011.
According to EGA, the guidelines
ill b f d d h
©2011 Waters Corporation | COMPANY CONFIDENTIAL 16
will be founded on the same
basic scientific principles as in
the EU”. Stephanie Sutton, ‘Call for "Workable" Guideline
for Biosimilar mAbs’; September 9, 2010.
PharmTech.com.
12/18/2011
2011 IETF Day 3 Skilton 9
Current EU Legislation is ‘Clear’
EU: biosimilar is a copy version of the reference product
“D t t d i il it i h i h i l h t i ti “Demonstrated similarity in physicochemical characteristics, efficacy and safety, based on a comprehensive comparability exercise”
Acceptance of variability ('microheterogeneity').
©2011 Waters Corporation | COMPANY CONFIDENTIAL 17
EMA – Established Biosimilar Pathway since 2005
Established pathway allows biosimilars with limited Clinical Trials
2009 workshop debated what ‘similarity’ meant
— Agreed with previous model of ‘well-characterised b h d ’
©2011 Waters Corporation | COMPANY CONFIDENTIAL 18
biotherapeutic product’
— Confirmed that ‘comparability’ concept was appropriate
12/18/2011
2011 IETF Day 3 Skilton 10
Regulator Readiness – FDA is Experienced but Only Now Devising guidelines
BPCI act of 2009 established a framework to develop guidelines
FDA outlined a ‘case-by-case’ principle - no formula exists for biosimilars:
— ‘it’s unlikely that a “one size fits all” systematic assessment of biosimilarity can be
developed’
— ‘ a “totality of the evidence” approach by evaluating more attributes and
©2011 Waters Corporation | COMPANY CONFIDENTIAL 19
— …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’.
— ‘Immunogenicity remains a critical factor when assessing biosimilarity’
A Real Biotech Story – Genzyme
Change of reactor led to Change in Glycosylation Pattern
4000L
160L
Reactor
Glycosylation Pattern BGlycosylation
Pattern A
160 L Reactor
©2011 Waters Corporation | COMPANY CONFIDENTIAL 21
Genzyme receives PDUFA date from FDA for its Biologics License Application for Lumizyme - 21. January 2010 23:52 http://www.news-medical.net/news/20100121/Genzyme-receives-PDUFA-date-from-FDA-for-its-Biologics-License-Application-for-Lumizyme.aspx
Reactor
4000L Reactor
12/18/2011
2011 IETF Day 3 Skilton 11
A Real Biotech Story – Genzyme (now Sanofi)
Biotech company had to re-submit as a new product
Glycosylation B Not 4000L 160 L R t Glycosylation B Not
COMPARABLE to Glycosylation A
4000L React
or
Glycosylation Pattern B
Glycosylation Pattern A
Reactor
©2011 Waters Corporation | COMPANY CONFIDENTIAL 22
Genzyme receives PDUFA date from FDA for its Biologics License Application for Lumizyme - 21. January 2010 23:52 http://www.news-medical.net/news/20100121/Genzyme-receives-PDUFA-date-from-FDA-for-its-Biologics-License-Application-for-Lumizyme.aspx
US: New Clinical Trials
US: Resubmission for New Product
EU: (fast) Submission as Biosimilar
EU: Limited Clinical Trials
ESTIMATED COST > 40 M USD
Overview of Types of Biotherapeutic
Size and Complexity increase means that analytical tools need to be able to cope
mAb
150kDaEPO
Conjugated mAb
150kDa+
Blood Factors
250kDa
©2011 Waters Corporation | COMPANY CONFIDENTIAL 23
Insulin
6kDa
GCSF
19kDa
34kDa
PEG-Interferon
20kDa
150kDa+
12/18/2011
2011 IETF Day 3 Skilton 12
Overview
Market Factors
Regulatory Positions
Deploying
Factors
©2011 Waters Corporation | COMPANY CONFIDENTIAL 25
Deploying New Tools
Return on Investment
Biosimilar Screening Workflow
Intact mAb LC/MSE
ReductionAlkylationDigestion
VariantProfiling
Intact mAb LC/MS
LC/MSE
Peptide Map
Reduction
Digestion
PNGaseF DeglycosylationHILIC Glycan SPE2AB Label & CleanupHILIC-FLR (MS?)
Higher OrderStructure
Host CellProteins
Stability/Formulation
©2011 Waters Corporation | COMPANY CONFIDENTIAL 26
Released Glycan (FL)LC & HC
LC/MS
Bioanalysis
QCTesting
12/18/2011
2011 IETF Day 3 Skilton 13
Overview
Market Factors
Regulatory Positions
Deploying
Factors
‘Profile’: EPO Glycan profiling
Similarity: Sequence Variants
©2011 Waters Corporation | COMPANY CONFIDENTIAL 27
Deploying New Tools
Return on Investment
Immunogenicity: Host Cell Proteins
Comparability in Higher Order
Structure
Overview
Market Factors
Regulatory Positions
Deploying
Factors
‘Profile’: EPO Glycan profiling
Similarity: Sequence Variants
©2011 Waters Corporation | COMPANY CONFIDENTIAL 28
Deploying New Tools
Return on Investment
Immunogenicity: Host Cell Proteins
Comparability in Higher Order
Structure
12/18/2011
2011 IETF Day 3 Skilton 14
Critical Tool: UPLC/FL/(MS) methodology -First UPLC 2AB method to Market, Best in Class
Extensive work on complex glycoproteins from Waters and International collaborators
2011 poster
©2011 Waters Corporation | COMPANY CONFIDENTIAL 29
http://www.waters.com/webassets/cms/library/docs/720004019en.pdf
2011 apps note
Waters India EPO Biosimilars Comparison Poster – UPLC/ FL/ MS
EPO poster at NCBS, Bangalore, India, 2nd International Symposium on Mass Spectrometry in Life Sciences
©2011 Waters Corporation | COMPANY CONFIDENTIAL 30
http://www.ncbs.res.in/node/497
12/18/2011
2011 IETF Day 3 Skilton 15
Waters India: Glycan analysis of three biosimilars of EPO using UPLC FLR MS
Biosimilar 1
Biosimilar 2
Biosimilar 3
©2011 Waters Corporation | COMPANY CONFIDENTIAL 31
High reproducibility of UPLC/ FL/ MS allows:— Accurate determination even at low abundance
— Relative quantities in a complex profile
Greater security of information in a challenging marketplace
Overview
Market Factors
Regulatory Positions
Deploying
Factors
‘Profile’: EPO Glycan profiling
Similarity: Sequence Variants
©2011 Waters Corporation | COMPANY CONFIDENTIAL 32
Deploying New Tools
Return on Investment
Immunogenicity: Host Cell Proteins
Comparability in Higher Order
Structure
12/18/2011
2011 IETF Day 3 Skilton 16
Characterisation Workflow as Viewed in 2006 (Barnes et al, Lilly)
No Automated processing existed in 2006existed in 2006— Proteomics tools
predominated
MS/MS was at that time not routinely used in the industry for peptide
©2011 Waters Corporation | COMPANY CONFIDENTIAL 33
Barnes et al, 2006; Fig 2.
mapping
Characterisation Workflow Improvements in the Modern Era – Waters 2008 - 2011
Major Workflow advantages with modern UPLC/MS
Deconvolution Required
Optimised, automated i ith M E t
Protein Automated Intact mass
Denaturation, Reduction, Alkylation
©2011 Waters Corporation | COMPANY CONFIDENTIAL 34
processing with MaxEnt
Provision of Intact Mass kit
12/18/2011
2011 IETF Day 3 Skilton 17
2009 Studies on Marketed Biotherapeutics with BPLx and Intact Mass Kit
©2011 Waters Corporation | COMPANY CONFIDENTIAL 35
http://www.waters.com/webassets/cms/library/docs/720
002911en.pdf
HC
LC
HCSpectrum
LCSpectrum
Natalizumab, IgG4
Method Development Simplicity: GenericSEC/UV/MS Method for Reduced IgGs
ACQUITY UPLC™ BEH200 SEC 1.7 µm
4.6x300 mm Column, p/n: 186005226
IgG2
Adalimumab
Bevacizumab
Infliximab
TIC
©2011 Waters Corporation | COMPANY CONFIDENTIAL 36
HC-HC
Rituximab
PTG1
Trastuzumab 10 min
ASMS2011 Poster
12/18/2011
2011 IETF Day 3 Skilton 18
Major Workflow advantages with modern UPLC/MS
Deconvolution Required
Characterisation Workflow Improvements in the Modern Era – Waters 2008 - 2011
Abandoned?
Protein Automated Intact mass
Denaturation, Reduction, Alkylation
©2011 Waters Corporation | COMPANY CONFIDENTIAL 37
How to QUANTIFY?
Major Workflow advantages with modern UPLC/MS
Deconvolution Required
Characterisation Workflow Improvements in the Modern Era – Waters 2008 - 2011
Efficiency NeededAbandoned?
Protein Automated Intact mass
Enzymatic Digestion
Denaturation, Reduction, Alkylation
One Analysis for
©2011 Waters Corporation | COMPANY CONFIDENTIAL 38
UPLC/ MSE
Automated Disulfide Bond
Mapping
Automated PTM Identification and
Quantification
ymultiple results
Automated Sequence Validation
12/18/2011
2011 IETF Day 3 Skilton 19
Critical Tools: BiopharmaLynx™ and UNIFI™ - First to Market, Best in Class
Purpose-built
Built in collaboration with our partners Built in collaboration with our partners
Ongoing development
©2011 Waters Corporation | COMPANY CONFIDENTIAL 39
Automated EPO comparisons: Protein sequence coverage between Biosimilar 1 and 2
Automated Processing of UPLC/ MSE data
— Early Adopter quote: ‘This has saved us eight weeks of data analysis per sample’
— Performs multiple automated tasks (Intact Mass, Peptide Mapping, Disulfide Bond Mapping)
Colour-coding used to indicate whether ‘control’ and ‘analyte’ have the same match
C t
©2011 Waters Corporation | COMPANY CONFIDENTIAL 40
Line indicates fragment ion data available to validate sequence
Coverage measurement
12/18/2011
2011 IETF Day 3 Skilton 20
UPLC/ MSE: Automated Peptide map Processing of three Biosimilars of EPO
Manual processing would be prohibitively time-consuming
BPLx automates the process for peptide maps— Provides validation of peptide sequence
Biosimilar 1 Sequence Coverage: 96.4%
Biosimilar 2 Sequence Coverage: 95.8%
Provides validation of peptide sequence
©2011 Waters Corporation | COMPANY CONFIDENTIAL 41
Biosimilar 3 Sequence Coverage: 91.1%
Spectrum comparison between Biosimilar 1 and 2-difference plot
Automated, Rapid Visualisation of extent and
location of differences – using objective criteria
justifiable to the regulatorsj g
©2011 Waters Corporation | COMPANY CONFIDENTIAL 42
12/18/2011
2011 IETF Day 3 Skilton 21
Overview
Market Factors
Regulatory Positions
Deploying
Factors
‘Profile’: EPO Glycan profiling
Similarity: Sequence Variants
©2011 Waters Corporation | COMPANY CONFIDENTIAL 44
Deploying New Tools
Return on Investment
Immunogenicity: Host Cell Proteins
Comparability in Higher Order
Structure
Deconvoluted Spectra of the Heavy Chain
INNOVATOR
For each pair of forms the mass difference is consistent— Each (glyco)form of the
Biosimilar sample is approx. 32Da less than the corresponding form in th C t l
©2011 Waters Corporation | COMPANY CONFIDENTIAL 46
the Control
The pairs appear to match up for all variants.
32Da 32Da 32DaBIOSIMILAR
12/18/2011
2011 IETF Day 3 Skilton 22
LCMSE Peptide Map Comparison
Innovator mAb
Result: No obvious, gross differences between antibody maps
©2011 Waters Corporation | COMPANY CONFIDENTIAL 47
“Biosimilar” mAb
MIRROR MAP shows Difference Due to Unknown Peak in Biosimilar Sample
Changes clear in mirror plot INNOVATOR
(chromatogram)
Significant Differences are visually salient
BIOSIMILAR(chromatogram)
INNOVATOR(spectrum)
©2011 Waters Corporation | COMPANY CONFIDENTIAL 48
New, Unknown Peak in BIOSIMILAR sample at
m/z 1872.96
Peptide T34-35 (missed cleavage) detected in INNOVATOR sample not
detected in BIOSIMILAR sample
BIOSIMILAR(spectrum)
12/18/2011
2011 IETF Day 3 Skilton 23
Heavy Chain Sequence Coverage –INNOVATOR mAb Sequence T35: EEMTK
©2011 Waters Corporation | COMPANY CONFIDENTIAL 49
Coverage Map for Heavy Chain for Innovator sample is high and mAb Sequence confirmed
The T35 peptide is correctly identified and sequence confirmed in the Innovator product – but not in the Biosimilar
MSE Spectra of INNOVATOR mAb T35 (EEMTK)
BiopharmaLynx automatically provides sequence confirmation thanks to UPLC/ MSE data/
©2011 Waters Corporation | COMPANY CONFIDENTIAL 50
T35 (EEMTK), RT = 3.86 min, Innovator Sample
12/18/2011
2011 IETF Day 3 Skilton 24
Heavy Chain Sequence – Biosimilar Sample with Drugbank mAb Sequence
Sequence information for the Innovator Sample was copied
from a peer-reviewed paper published after submission of IPfrom a peer reviewed paper published after submission of IP
HT35 sequence is confirmed by MSE
©2011 Waters Corporation | COMPANY CONFIDENTIAL 51
Heavy Chain Sequence – Biosimilar Sample with Drugbank mAb Sequence
Sequence information for the Innovator Sample was copied
from a peer-reviewed paper published after submission of IPfrom a peer reviewed paper published after submission of IP
HT35 sequence is validated
Primary AA sequence in Drugbank for this mAb indicated that
there was a difference at that position
HT35 sequence in Drugbank is –DELTK-
©2011 Waters Corporation | COMPANY CONFIDENTIAL 52
The same dataset was then re-analysed with the
alternative sequence
UPLC/ MSE collected information on ALL ions
12/18/2011
2011 IETF Day 3 Skilton 25
Heavy Chain Sequence – Biosimilar Sample with Drugbank mAb Sequence
Coverage Map for Heavy Chain for Biosimilar sample using Drugbank Sequence
©2011 Waters Corporation | COMPANY CONFIDENTIAL 53
HT35 peptide now correctly identified
No further sample acquisition was necessary as the MSE
data was already acquired – re-processing with the alternative sequence was completed in minutes.
Mirror View of T35s in BiopharmaLynx Processed data
T35 (EEMTK)Innovator SampleInnovator Sample
©2011 Waters Corporation | COMPANY CONFIDENTIAL 55
T35 (DELTK)‘Biosimilar’ SamplePeptides in each sample can
now be correctly attributed to their respective peptide
sequences purely by reprocessing the data with
new information
12/18/2011
2011 IETF Day 3 Skilton 26
Sample Handling is Critical to Accurate Results
mAbs 2:4, 1-16; July/August 2010; © 2010 Landes Bioscience
©2011 Waters Corporation | COMPANY CONFIDENTIAL 56
Overview
Market Factors
Regulatory Positions
Deploying
Factors
‘Profile’: EPO Glycan profiling
Similarity: Sequence Variants
©2011 Waters Corporation | COMPANY CONFIDENTIAL 58
Deploying New Tools
Return on Investment
Immunogenicity: Host Cell Proteins
Comparability in Higher Order
Structure
12/18/2011
2011 IETF Day 3 Skilton 27
Comparability and Biosimilars: BMS and Biogen Idec use of HDX
2011
©2011 Waters Corporation | COMPANY CONFIDENTIAL 59
2011 poster
Biogen Idec Paper on the Advantages of Adopting HDX for Comparability (2011)
Biogen Idec early Adopters of Waters HDX
Fig 6 from paper example of rapid and automated processing of UPLC/ MSE data for a Totality of information not available from traditional MS techniques— LOCALISATION of differences and measure of AMOUNT of difference
simultaneously
The Utility of Hydrogen/Deuterium Exchange Mass Spectrometry
i Bi h ti l C bilit
©2011 Waters Corporation | COMPANY CONFIDENTIAL 60
in Biopharmaceutical Comparability Studies. DAMIAN HOUDE, STEVEN A.
BERKOWITZ, JOHN R. ENGEN. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL.
100, NO. 6, MAY 2011http://onlinelibrary.wiley.com/doi/10.10
02/jps.22432/abstract
12/18/2011
2011 IETF Day 3 Skilton 28
Comparability and Biosimilars: BMS use of HDX for PEGylated GCSF
Application of Waters ‘Butterfly Plot’ to a real-Butterfly Plot to a realworld situation
— Automation of data processing
— MONTHS of time savings over manual workflow
— Objective analysis of
©2011 Waters Corporation | COMPANY CONFIDENTIAL 61
Objective analysis of ‘difference’ vs (subjective) human interpretation
Higher Order Structure and Protein Analytics – Regulatory Authorities
“Our current ability to predict the potency of biologics would be Our current ability to predict the potency of biologics would be
enhanced if we had improved ability to measure and
quantify the correct (major) three-dimensional structure,
aberrant three-dimensional structures (misfolding), and the
distribution of different three-dimensional structures”.
©2011 Waters Corporation | COMPANY CONFIDENTIAL 62
• [TESTIMONY BEFORE THE SUBCOMMITTEE ON TECHNOLOGY AND INNOVATION COMMITTEE ON
SCIENCE AND TECHNOLOGY U.S. HOUSE OF REPRESENTATIVES. SEPTEMBER 24, 2009]
Steven Kozlowski, M.D.Director, Office of Biotechnology Products, Office of Pharmaceutical Science. CDER
12/18/2011
2011 IETF Day 3 Skilton 29
Basics of BiopharmaLynx™ Display for Automated Disulfide Bond Mapping
Each Peak labeled with Peptide Number
©2011 Waters Corporation | COMPANY CONFIDENTIAL 63
Hoverbox for Peptide Information
‘=‘ Symbol represents S-S bond
BSA - Automated assignment of Expected Peptides from Tryptic Digest
With BiopharmaLynx™ All 9 peptides with expected S-S bonds are assigned automatically shown by ‘Peptide1=Peptide2’
‘h b ’ h d k
T07=T07
T08=T10=T12
T14=T21=T22T39=T41=T42
T63=T66=T67
‘hoverboxes’ show sequences at processed peak apex
©2011 Waters Corporation | COMPANY CONFIDENTIAL 64
T28=T37 T44=T50=T51 T54=T61=T62
T69=T76=T77
12/18/2011
2011 IETF Day 3 Skilton 30
Overview
Market Factors
Regulatory Positions
Deploying
Factors
‘Profile’: EPO Glycan profiling
Similarity: Sequence Variants
©2011 Waters Corporation | COMPANY CONFIDENTIAL 65
Deploying New Tools
Return on Investment
Immunogenicity: Host Cell Proteins
Comparability in Higher Order
Structure
Host Cell Protein Work - 2011
©2011 Waters Corporation | COMPANY CONFIDENTIAL 66
http://www.waters.com/webassets/cms/library/docs/720004043en.pdf
http://www.waters.com/webassets/cms/library/docs/thp637.pdf
12/18/2011
2011 IETF Day 3 Skilton 31
Waters HCP analysis with LC/MSWorkflow Overview - Discovery
Protein mixture is digested (Trypsin) to identify the peptides by LC/MS
Complexity of mixture is reduced by using online two-dimensional chromatographychromatography
Uses established Informatics tools (PLGS) to:
— Identify the individual proteins by matching [tryptic] peptides to database entries
— Calculate protein concentration based on signal response
— Display and rank matches in a protein-centric format
©2011 Waters Corporation | COMPANY CONFIDENTIAL 67
Perform online 2D LC/MSE to discover impurity proteins.
Spike mixture with known proteins and enzymatically digest
Identify proteins and calculate concentrations
Key Figure for HCP study
Section of table from HCP study
— Multiple cell lines Specific Proteins are identified
Prot Proteinno Description I II III IV
1 Nascent polypeptide associated complex subunits Mus musculus 50 17682 Nucleolin Mesocricetus auratus 2613 Heterogeneous nuclear ribonucleoprotein isoforms Mus musculus 12274 Elongation factor isoforms Mus musculus 142 665 20495 Procollagen C endopeptidase enhancer 1 Mus musculus
CHO-S Cells
— Multiple purification schemes
— Null cell lines
are identified
Protein concentration is measured
©2011 Waters Corporation | COMPANY CONFIDENTIAL 68
5 Procollagen C endopeptidase enhancer 1 Mus musculus6 Laminin subunits Mus musculus7 Actin isoforms Mesocricetus auratus 287 1298 Clusterin Mus musculus 537 2329 Nidogen 1 Mus musculus
10 Glyceraldehyde 3 phosphate dehydrogenase Mes auratus 153 59 7511 Splicing factor proline and glutamine rich Mus musculus 19812 Glycogen phosphorylase b rabbit - PHO (800 fmoles) 778 778 778 77813 Plasminogen activator inhibitor 1 RNA binding protein Mus musculus 226
Spiked standard with measured recovery
Consistentmeasurement across
all conditions
12/18/2011
2011 IETF Day 3 Skilton 32
High Throughput MRM Monitoring and absolute quantitation
Absolute Quantification with stable isotopically
labelled peptides as internal standards
— Targeted for specific proteins
— Simple transfer of MSE conditions to MRM
methods
Provision for alternative peptides to be chosen
Assay extremely simple to update/ change in
minutes
©2011 Waters Corporation | COMPANY CONFIDENTIAL 69
minutes
MRM Monitoring of an HCP level in MRM Monitoring of an HCP level in 10 PTG1 mAb Batches from CHO10 PTG1 mAb Batches from CHO
mAb Batch (in Triplicate)
1 2 3 4 5 6
7 8
9 10
©2011 Waters Corporation | COMPANY CONFIDENTIAL 70
12/18/2011
2011 IETF Day 3 Skilton 33
Overview
Market Factors
Regulatory Positions
Deploying
Factors
©2011 Waters Corporation | COMPANY CONFIDENTIAL 71
Deploying New Tools
Return on Investment
Engineering Routine High Performance Mass Spectrometry
Automating tuning and calibration Integrated fluidics system
©2011 Waters Corporation | COMPANY CONFIDENTIAL 72
Automated LC/MS system checks
Continuous background monitoring
Updated electronics for diagnostics
No tools needed
12/18/2011
2011 IETF Day 3 Skilton 34
Analytical Platforms across Analytical Platforms across Biotherapeutic OrganizationsBiotherapeutic Organizations
MassLynx Usage
Empower Usage
Discovery
Development
Discovery
Development
MassLynx Usage
Few compliance issues GxP compliance Regulatory Compliance
©2011 Waters Corporation | COMPANY CONFIDENTIAL 73
Production
Post-Approval
QC/QA
Production
Post-Approval
QC/QA
VALIDATIONSTABILITYCHARACTERIZATION
Analytical Platforms across Analytical Platforms across Biotherapeutic OrganizationsBiotherapeutic Organizations
Empower Usage
Discovery
Development
Discovery
Development
Few compliance issues GxP compliance Regulatory Compliance
©2011 Waters Corporation | COMPANY CONFIDENTIAL 74
Production
Post-Approval
QC/QA
Production
Post-Approval
QC/QA
VALIDATIONSTABILITYCHARACTERIZATION
12/18/2011
2011 IETF Day 3 Skilton 35
Biopharmaceutical System Solution
LC and LC/MSE Peptide Mapping
©2011 Waters Corporation | COMPANY CONFIDENTIAL 75
LC/MS Intact Mass
RP, SEC and IEX
Your Success is Our Mission
Satisfy the Regulator
Raise the bar against the Competition
Increase safety for patients
©2011 Waters Corporation | COMPANY CONFIDENTIAL 77
SECURE YOUR POSITION IN THE MARKET
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