biopharmaceutical
DESCRIPTION
Dear Customer,Stabicon has helped needs of customers in specific areas of the pharmaceutical Analytical Method Development, Validation and Stability study. As the expansion of our analytical services in new technologies and testing methods to help our clients in characterizing future medicines. We understand biology at each of these levels to advance an integrated view of life processes for Biopharmaceuticals. We realize that future medicines (Biopharma) success requires ability to integrate protein testing capabilities which will provide strategic time and cost advantage for Biopharmaceutical sector.We would like to share our presentation on Biopharma solution, to download the presentation please click on the below linkhttps://www.box.net/shared/aboumapp9h9gftunenja/1f7c7a6ef1/rss.xmlIf you are interested in our Biopharma services, it will be a good idea to interact with our technical team/ commercial team for further clarification, looking forward to your response. Thanking you and assuring our best service at all timeRegardsVijayProject DirectorStabicon Life Sciences Pvt LtdMobile: +919591974355/080-41714280www.stabicon.comTRANSCRIPT
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Biopharmaceutical SolutionBiopharmaceutical Solution
ByBy
Vijay Kumar RankaVijay Kumar Ranka
Stabicon Life Sciences Pvt LtdStabicon Life Sciences Pvt Ltd
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Topics Topics CoveredCovered ::
I.I. IntroductionIntroduction
II.II. Protein Basic MechanismProtein Basic Mechanism
III.III. Step in Protein Production Step in Protein Production
IV.IV. Identification and characterization techniqueIdentification and characterization technique
V.V. Monitoring protein synthesisMonitoring protein synthesis
VI.VI. Data Processing MethodologyData Processing Methodology
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IntroductionIntroduction
Chapter Chapter ––II
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Therapeutic small molecule being dominant for more than 100year Therapeutic small molecule being dominant for more than 100year for for
treatment then why biologics required for therapeutic?treatment then why biologics required for therapeutic?
StabiconStabiconStabiconStabicon
Why???Why???
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How different they are ?How different they are ?
RequiredRequiredNot RequiredNot RequiredStructural FoldingStructural Folding
EndocytosisEndocytosisMetabolismMetabolismEliminationElimination
LowLowHighHighToxicityToxicity
SpecficSpecficNon Non SpecficSpecficRegulationRegulation
Extensive TrialsExtensive TrialsLimited TrialsLimited TrialsDevelopmentDevelopment
Living organismLiving organismChemical synthesisChemical synthesisProductionProduction
HighHigh--low doselow doseLowLow––high dosehigh doseProduct ActivityProduct Activity
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What are these?What are these?
�� What are these large molecule or biomolecule and how similar areWhat are these large molecule or biomolecule and how similar are they to they to
human body?human body?
�� What make them so specific and effective?What make them so specific and effective?
�� What is the correlation of large/ biomolecule molecule to livingWhat is the correlation of large/ biomolecule molecule to living machinery?machinery?
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Mechanism for SignalingMechanism for Signaling
Several types of molecule Several types of molecule
modification are involved in modification are involved in
regulation for a signal regulation for a signal
transfer such as : transfer such as :
glycosylation, acetylation,etcglycosylation, acetylation,etc
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Effect in the bodyEffect in the body
�� Small molecule rarely elict secondary signaling thus effect prevSmall molecule rarely elict secondary signaling thus effect prevail until drug ail until drug
adhere to the target site whereas in case of large molecule alwaadhere to the target site whereas in case of large molecule always elict an ys elict an
secondary signaling hence effect remain even after the drug is esecondary signaling hence effect remain even after the drug is eliminated. liminated.
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Protein Basic MechanismProtein Basic Mechanism
Chapter Chapter ––II II
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Genotype determines phenotypeGenotype determines phenotype
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Central dogmaCentral dogma
Prokaryotic Cell:Prokaryotic Cell:
DNA DNA –– RNA RNA –– PROTEIN PROTEIN
(Transcription) (Translation)(Transcription) (Translation)
Eukaryotic CellEukaryotic Cell::
DNA DNA –– RNA RNA –– PROTEIN PROTEIN -- PROTEIN MODIFIED PROTEIN MODIFIED
(P(Post Translation)ost Translation)
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Eukaryotic cellEukaryotic cell
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Protein StructureProtein Structure
�� PrimaryPrimary
�� SecondarySecondary
�� TertiaryTertiary
�� QuaternaryQuaternary
ACDEFGHIKLMNPQRSTVWY
primary structure
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ProteomeProteome
GenomeDGenomeD
TranscriptomeDTranscriptomeD
ProteomeProteome
~ 300 modifications~ 300 modifications
GenomicsGenomics
ProteomicsProteomics
GlycosylationGlycosylation
LipidationLipidation
UbiquitinationUbiquitination
Many moreMany more
PhosphorylationPhosphorylation
SugarSugar —
LipidLipid —
PP —
CleavageCleavage
?? —
UbUb —
——
PPPP —
PP —
UbUb —
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Therapeutic Protein ProductionTherapeutic Protein Production
Chapter Chapter ––IIIIII
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BiopharmaBiopharma
�� Biopharmaceutical are protein with considerable therapeutic strBiopharmaceutical are protein with considerable therapeutic structural uctural
diversity. They tend to between 100 to 1000 times larger than trdiversity. They tend to between 100 to 1000 times larger than traditional small aditional small
molecule drug .molecule drug .
�� Such complex protein can't be produced using convential chemicaSuch complex protein can't be produced using convential chemical synthesis l synthesis
rather than in a living cell under stringently controlled conditrather than in a living cell under stringently controlled condition.ion.
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How are these designedHow are these designed
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Protein FactoryProtein Factory
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Bioprocessing PhaseBioprocessing Phase
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Examples of Biologics marketed:Examples of Biologics marketed:
�� InsulinInsulin
�� ImigluceraseImiglucerase
�� GlucagonGlucagon
�� Human Growth HormoneHuman Growth Hormone
�� ErythropoietinErythropoietin
�� GG--CSFCSF
�� InterferonInterferon
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Chapter –IVChapter Chapter ––IVIV
Protein CharacterizationProtein Characterization
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Characterization Step:Characterization Step:
�� Intact Mass analysis Intact Mass analysis
�� Primary structure Primary structure -- peptide mappingpeptide mapping
�� Glycan analysisGlycan analysis
�� Amino acid and media analysisAmino acid and media analysis
�� Data processingData processing
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How to characterize ?How to characterize ?
�� Large scale screening of proteins, their expression, modificatiLarge scale screening of proteins, their expression, modification and interactionon and interaction
by using highby using high--throughput approachesthroughput approaches
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Characterization Required for Characterization Required for
� Protein identity (mutant protein)Protein identity (mutant protein)
�� Protein quantity (Expression)Protein quantity (Expression)
�� Protein postProtein post--translational modifications (up or down)translational modifications (up or down)
�� Protein structureProtein structure
�� ProteinProtein--protein interactionprotein interaction
�� Protein localizationProtein localization
�� Change in any protein property may cause functional abnormalitChange in any protein property may cause functional abnormality and y and
might be relevant to pathogenesis.might be relevant to pathogenesis.
ToolsTools
�� Protein ArrayProtein Array
�� Mass SpectrometryMass Spectrometry
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Why Protein by Mass SpectrometryWhy Protein by Mass Spectrometry ??
�� MS can unambiguously identify proteinsMS can unambiguously identify proteins
Gel separated proteinsGel separated proteins
Proteins in mixtureProteins in mixture
Protein: protein associationProtein: protein association
�� Identify precise post translational changesIdentify precise post translational changes
PhosphorylationPhosphorylation
NN-- or Cor C-- terminal modificationterminal modification
Many moreMany more
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Isolation and characterizationIsolation and characterization
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Protein Identification TechnologyProtein Identification Technology
Seperation Seperation
Mass AnalysisMass Analysis
Data processing
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Mass Spectrometry Schematic DiagramMass Spectrometry Schematic Diagram
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MALDI IonizationMALDI Ionization
Protein or
PeptideMass Spectrometer Mass/Charge
(m/z)Ionization
Solution
Phase
Gas
Phase
Matrix assisted laser desorption ionization (MALDI),
Koichi Tanaka
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Data Acquisition from MALDIData Acquisition from MALDI--MSIMSI
Alanine, peptide in plasma
m/z = 1474.6Valine, m/z = 1502.7
Alanine
Valine
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Protein or
PeptideMass Spectrometer Mass/Charge
(m/z)Ionization
Solution
Phase
Gas
Phase
ESI IonizationESI Ionization
Electrospray ionization (ESI), John B Fenn
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Data Acquisition from ESIData Acquisition from ESI--MSIMSI
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What is MSE?What is MSE?
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How to identify a single protein by MS?
Mass/Charge
(m/z)Mass
Digest into many peptides Mass of many
peptides
Peptide mass fingerprinting (PMF)
Mass of many peptide fragmentsBy
Tandem Mass Spectrometry
Single protein identificationSingle protein identification
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Protein mixture Analysis by LCProtein mixture Analysis by LC--MS/MSMS/MS
Protein mixture
Digestion
Peptides
400 800 1200 1600m/z
MS
MS/MS
10 20 30 min0
HPLC
Database
Searching
LLTTIADAAK
SAGGNYVVFGEAK
EDDVEEAVQAADR
All peptide sequences Identification of many proteins
1 sequencing attempt per 0.5 sec.
3600 sequencing attempts in 30 min.
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Protein structural SeperationProtein structural Seperation
••An ion in a compactAn ion in a compact--form has a high mobility, and hence shorter drift time, form has a high mobility, and hence shorter drift time,
••The same ion in a more open conformation has a lower mobility, aThe same ion in a more open conformation has a lower mobility, and hence nd hence
a longer drift timea longer drift time
Gate
Detector
Neutral Buffer Gas (-ve force)
Ring Electrodes (Potential Gradient. +ve force)
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HDMS FOR STRUCTURAL SEPERATION OF ISOMERHDMS FOR STRUCTURAL SEPERATION OF ISOMER
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IMS separation of peptides and lipidsIMS separation of peptides and lipids
No IMS separation IMS selection of peptides IMS selection of lipids
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Why Accurate mass?Why Accurate mass?
Intact Protein Mass
Digested Protein Mass
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Intact Mass AnalysisIntact Mass Analysis
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How to identify a single protein by MS/MS?How to identify a single protein by MS/MS?
PeptidesPeptides
Theoretical
Spectrum
Database Database
searchingsearching
m/z
Ionization
MS spectrum MS/MS spectrum
Fragmentation
Protein
digestion
Peptide/proteinPeptide/protein
identificationidentification
m/z m/z
200 400 600 800 1000 1200 m/z
K G A FD E L Q
LIFAGKQLEDGR
b ions
1: L
2: LI
3: LIF
4: LIFA
5: LIFAG
6: LIFAGK
7: LIFAGKQ
8: LIFAGKQL
9: LIFAGKQLE
10:LIFAGKQLED
11:LIFAGKQLEDG
y ions
IFAGKQLEDGR:11
FAGKQLEDGR:10
AGKQLEDGR :9
GKQLEDGR :8
KQLEDGR :7
QLEDGR :6
LEDGR :5
EDGR :4
DGR :3
GR :2
R :1
GAF DEL GLI K Q
Q A
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N & C terminal Ions
Selected Peptides (parent ions) are fragmented in the of a nebulizing neutral gas.
Energy imparted by collision breaks the covalent bond in parent bonds.
y & b-type ions series thus generated can give us the sequence of the peptide
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Peptide MappingPeptide Mapping
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Post Translational IdentificationPost Translational Identification
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GlycoproteinGlycoprotein
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Monitor the Bioreactor Media Monitor the Bioreactor Media
&&
Protein SynthesisProtein Synthesis
Chapter Chapter –– VV
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UVUV Aminoacid AnalysisAminoacid Analysis
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Water PurityWater Purity
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Amylase Protein ExpressionAmylase Protein Expression
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E.Coli Lysate AnalysisE.Coli Lysate Analysis
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Batch AnalysisBatch Analysis
Batch1aBatch1a Batch 2aBBatch 2aB
Batch1bBatch1b Batch2bBatch2b
Batch1cBatch1c Batch2cBatch2cdifference in proteinsdifference in proteins
�� Proteins (to identify and quantify proteins in multiple samplesProteins (to identify and quantify proteins in multiple samples))
How many proteins ?How many proteins ?
The choice of method?The choice of method?
How many samples?How many samples?
How many variability parameter?How many variability parameter?
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Data processing
Chapter Chapter ––VIVI
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Data processing Data processing
�� Using a software product designed to facilitate MS and LCMS anaUsing a software product designed to facilitate MS and LCMS analysis of lysis of
biopharmaceutical samplesbiopharmaceutical samples
�� Intact proteins: Intact proteins: Comparison of an entire Comparison of an entire protein(sprotein(s) against a well) against a well--
characterized standard. Identification of differences, and variacharacterized standard. Identification of differences, and variants that nts that
require further investigation (some could be contaminants). require further investigation (some could be contaminants).
�� Peptide map:Peptide map: Comparison of the peptides resulting from a digested Comparison of the peptides resulting from a digested
protein against the peptides from the known standard. Identificaprotein against the peptides from the known standard. Identification of tion of
differences in protein coverage, modifications,differences in protein coverage, modifications,……
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What software DoesWhat software Does
�� Automates data processing and annotation of experimental resultsAutomates data processing and annotation of experimental results
Produces annotated spectra, chromatograms, coverage maps and tabProduces annotated spectra, chromatograms, coverage maps and tabular ular
datadata
�� Facilitates comparisons between a reference standard and batcheFacilitates comparisons between a reference standard and batches of s of
experimental samplesexperimental samples
�� Outputs include formal reports, figure copy/paste, and tabular Outputs include formal reports, figure copy/paste, and tabular data exportdata export
Frees users to concentrate on important questionsFrees users to concentrate on important questions
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Intact Protein ChromatogramIntact Protein Chromatogram
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Protein Charge determinationProtein Charge determination
The theoretical peak
constructed with the isotope
distribution (purple) and the
experimental peak (green) have the same width at half height.
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Results :Spectra view Results :Spectra view
Stack
Overlay
Mirror
Control :BP_079 non-deglycosylated VICAMAnalyte :BP_092 deglycosylated 19h VICAM
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Results Spectra view (Intensity filter)Results Spectra view (Intensity filter)
Threshold defined automatically on the spectrahreshold defined automatically
on the spectra
Threshold value Threshold value typed in the tablehe table
Filter applied on Filter applied on the results tableesults table
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Results: Highlight unique peaksResults: Highlight unique peaks
Unique peaks highlighting
Deglycosylated T022 fragment (analyte only)
Glycosylated T022 fragments (control only)
Control :BP_094 non-deglycosylated digested VICAM
Analyte :BP_097 deglycosylated 2h digested VICAM
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Result : Peak match data comparison analyte/controlResult : Peak match data comparison analyte/control
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Results Peak match data for control (glycosylated)Results Peak match data for control (glycosylated)
Percentage of each
glycosylation state in control
Control :BP_079 non-deglycosylated VICAMAnalyte :BP_092 deglycosylated 19h VICAM
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Results Peak match data for analyte (deglycosylated )Results Peak match data for analyte (deglycosylated )
Percentage of each glycosylation state in analyte
Control :BP_079 non-deglycosylated VICAMAnalyte :BP_092 deglycosylated 19h VICAM
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Results Peak match data comparison analyte/controlResults Peak match data comparison analyte/control
You can add your own commentsdd your own comments
Control :BP_079 non-deglycosylated VICAMAnalyte :BP_092 deglycosylated 19h VICAM
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PEPTIDE MAP ANALYSISPEPTIDE MAP ANALYSIS
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Protein digest Chromatogram Protein digest Chromatogram
Processed Raw
Matched peptides annotation
Control :BP_094 non-deglycosylated digested VICAMAnalyte :BP_097 deglycosylated 2h digested VICAM
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Results: Results:
Differential viewDifferential view
Control :BP_094 non-deglycosylated digested VICAM
Analyte :BP_097 deglycosylated 2h digested VICAM
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Protein digest AnalysisProtein digest Analysis
List of the raw data file
Selected analyte compared with the control 2h
Add or remove analyte
Set the selected analyte as control
Reprocess the data with another method
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Annotation of the peptidesAnnotation of the peptidesAnnotation of the peptides
1:T001
First chain of the protein
Trypsin digestion
First digest product of the chain
1:T001* Modified form of 1:T001
1:T001-002 Missed cleavage between 1:T001 and 1:T002
1:T001-3:T001Disulfide bridge between
1:T001 and 3:T001
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Results: Results:
Intensity normalisation Intensity normalisation
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Results: Results:
Highlight unique peaksHighlight unique peaks
Control :BP_094 non-deglycosylated digested VICAM
Analyte :BP_097 deglycosylated 2h digested VICAM
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Results Results
Coverage mapCoverage map
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Results Results
Protein digest MassProtein digest Mass
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ResultsResults
Peak match data comparison analyte/controlPeak match data comparison analyte/control
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ResultsResults
Peak match data advanced tablePeak match data advanced table
When a mass can correspond to several peptides, the different possibilities can be seen in the advanced view.
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ResultsResults
Discrimination between two assignmentsDiscrimination between two assignments
The sequence
corresponding to the
fragment 1:T009* of
the LC gave a better
score than the
sequence of 1:T021
If high energy data are available (acquisition with MSE mode), the
fragmentation data can be used to discriminate several assignment for the
same mass.
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FutureFuture
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Question???Question???
� Please email to [email protected]
� Write to Stabicon Life Sciences Pvt Ltd
3BM-416,3rd Block,
HRBR Extension,
Bangalore – 560043
Karnataka, India
Phone :+9180 – 41714280/81
78
Thank you
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