Biopharma Solution

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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

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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


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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


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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


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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



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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



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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???

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HRBR Extension,

Bangalore – 560043

Karnataka, India

Phone :+9180 – 41714280/81

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Thank you

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Biopharma Solution

Health & Medicine

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