Biopharmaceutical

Biologic Traditional Drug
1www.stabicon.com
Biopharma
vs
Small Molecules
Therapeutic
bd@stabicon.com

Topics Covered :
I. Introduction
II. Protein Basic Mechanism
III. Steps in Protein Production
IV.Identification and characterization technique
V. Monitoring protein synthesis
VI.Data Processing Methodology
2www.stabicon.com bd@stabicon.com

Introduction
Chapter –I

Therapeutic small molecule are being dominant for more than 100 years
for treatment then why biologics required for therapeutics ?
Why???

How different they are ?
Product Activity Low–high dose High-low dose
Production Chemical synthesis Living organism
Development Limited Trials Extensive Trials
Regulation Non Specific Specific
Toxicity High Low
Elimination Metabolism Endocytosis
Structural Folding Not Required Required

What are these?
 What are these large molecule or biomolecules and how similar are they
to human body?
 What makes them so specific and effective?
 What is the correlation of large molecule / biomolecule to living
machinery?

Mechanism for Signaling
Several types of molecular
modification are involved in
regulation for a signal transfer
such as : glycosylation,
acetylation,etc

Effect in the body
 Small molecules rarely elicit secondary signaling, thus effect prevails
until drug adheres to the target site whereas large molecules always
elicit a secondary signaling hence effect remains even after the drug
is eliminated.

Protein Basic Mechanism
Chapter –II

Genotype determines phenotype

Central dogma
Prokaryotic Cell:
DNA –
(Transcription)
RNA – PROTEIN
(Translation)
Eukaryotic Cell:
DNA – RNA – PROTEIN - PROTEIN MODIFIED
(Post Translation)

Eukaryotic cell

Protein Structure
 Primary
 Secondary
 Tertiary
 Quaternary
ACDEFGHIKLMNPQRSTVWY
primary structure

Therapeutic Protein Production
Chapter –III

Biopharma
 Biopharmaceuticals are protein with considerable therapeutic structural
diversity. They tend to be between 100 to 1000 times larger than traditional
small molecule drug .
 Such complex proteins can't be produced using conventional chemical
synthesis rather than in a living cell under stringently controlled condition.

How are these designed

Protein Factory

Bioprocessing Phase

Examples of Biologics marketed:
 Insulin
 Imiglucerase
 Glucagon
 Human Growth
Hormone
 Erythropoietin
 G-CSF
 Interferon

Chapter –IV
Protein Characterization

Characterization Step
 Primary structure - peptide mapping
 Glycan analysis
 Intact Mass analysis
 Amino acid and media analysis
 Data processing

How to characterize ?
 Large scale screening of proteins, their expression, modification and
interaction by using high-throughput approaches

Characterization Required for
 Protein identity (mutant protein)
 Protein quantity (Expression)
 Protein post-translational modifications (up or down)
 Protein structure
 Protein-protein interaction
 Protein localization
 Change in any protein property may cause functional
abnormality and might be relevant to pathogenesis.
Tools
 Protein Array
 Mass Spectrometry

Why Protein by Mass Spectrometry?
 MS can unambiguously identify proteins Gel
separated proteins
 Proteins in mixture
 Protein: protein association
 Identify precise post translational changes
Phosphorylation
 N- or C- terminal modification
Many more

Isolation and characterization

Protein Identification Technology
Separation
Mass Analysis
Data processing

MALDI Ionization
Protein or
Peptide
Mass Spectrometer Mass/Charge
(m/z)
Ionization
Solution
Phase
Gas
Phase
Matrix assisted laser desorption ionization (MALDI),
Koichi Tanaka

Data Acquisition from MALDI-MSI
Alanine, peptide in
plasma
m/z = 1474.6
Valine, m/z = 1502.7
Alanine
Valine

Protein or
Peptide
Mass Spectrometer Mass/Charge
(m/z)
Ionization
Solution
Phase
Gas
Phase
ESI Ionization
Electrospray ionization (ESI), John B Fenn

Data Acquisition from ESI-MSI

What is MSE?

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 fragments
By
Tandem Mass Spectrometry
Single protein identification

Protein mixture Analysis by LC-MS/ MSy
Protein mixture
Digestion
Peptides
400 800 1200 1600
m/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.

Protein structural Separation
•An ion in a compact-form has a high mobility, and hence shorter drift time,
•The same ion in a more open conformation has a lower mobility, and hence a
longer drift time
Gate
Detector
Neutral Buffer Gas (-ve force)
Ring Electrodes (Potential Gradient. +ve force)

HDMS FOR STRUCTURAL SEPERATION OF ISOMER

IMS separation of peptides and lipids
No IMS separation IMS selection of peptides IMS selection of lipids

Why Accurate mass?
s
Intact Protein Mas
Digested Protein Mass

Intact Mass Analysis

How to identify a single protein by
MS/MS?
Peptides
Theoretical
Spectrum
Database
searching
m/z
Ionization
MS spectrum MS/MS spectrum
Fragmentation
Protein
digestion
Peptide/protein
identification
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: LI3: LIF
4: LIFA
5: LIFAG
6: LIFAGK
7: LIFAGKQ
8: LIFAGKQL
9: LIFAGKQLE
10:LIFAG
KQLED11: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
A GF DEL GLI K Q
Q A

N & C terminal Ions
 Selected Peptides (parent ions) are fragmented in the mass 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

Peptide Mapping

Post Translational Identification

Glycoprotein

Monitor the Bioreactor
Media &
Protein Synthesis
Chapter – V

UV AminoacidAnalysis

Water Purity

Amylase Protein Expression

E.Coli Lysate Analysis

Batch Analysis
Batch 1a Batch 2aB
Batch1b Batch2b
Batch1c Batch2c
difference in proteins
 Proteins (to identify and quantify proteins in multiple
samples) How many proteins ?
 The choice of
method? How
many samples?
 How many variability parameter?

Data processing
Chapter –VI

Data processing
 Using a software product designed to facilitate MS and LCMS analysis of
biopharmaceutical samples
 Intact proteins: Comparison of an entire protein(s) against a well-
characterized standard. Identification of differences, and variants that
require further investigation (some could be contaminants).
 Peptide map: Comparison of the peptides resulting from a digested
protein against the peptides from the known standard. Identification of
differences in protein coverage, modifications,…

What software Does
 Automates data processing and annotation of experimental
results Produces annotated spectra, chromatograms,
coverage maps and tabular data
 Facilitates comparisons between a reference standard and
batches of experimental samples
 Outputs include formal reports, figure copy/paste, and tabular
data export Frees users to concentrate on important questions

Intact Protein Chromatogram

54www.stabicon.com
Protein Charge determination
The
theoretical
peak
constructe
d with the
isotope
distribution
(purple)
and the
experiment
al peak
(green)
have the
same width
at half
height.
bd@stabicon.com

Results :Spectra view
Stack
Overlay
Mirror
Control :BP_079 non-deglycosylated
VICAM Analyte :BP_092
deglycosylated 19h VICAM

Results Spectra view (Intensity
filter)
Threshold defined
automatically on the
spectra
T
hreshold value
typed in the table
Filt
er applied on the
results table

Results:
Highlight unique peaks
Unique peaks highlighting
Deglycosylated T022
fragment (analyte only)
Glycosylated T022 fragments (control
only)
digested VICAM
Analyte :BP_097 deglycosylated 2h
digested VICAM

Result : Peak match data
comparison analyte/control

Results Peak match data
control (glycosylated)
Percentage of each
glycosylation state in control

Results: Peak match data for
analyte (deglycosylated )
Percentage of each
glycosylation state in analyte
Control :BP_079 non-deglycosylated VICAM
Analyte :BP_092 deglycosylated 19h VICAM

Results: Peak match data
comparison analyte/control
You can add your own
comments

PEPTIDE MAP ANALYSIS

Protein digest Chromatogram
Processed
digested VICAM Analyte :BP_097
deglycosylated 2h digested VICAM
Raw
Matched peptides annotation

Results: Differential view
Control :BP_094 non-deglycosylated digested VICAM
Analyte :BP_097 deglycosylated 2h digested VICAM

Protein digest Analysis
List of the raw data file
Selected analyte compared with the control
Add or remove analyte
Set the selected analyte as
control
Reprocess the data with another
method

Annotation 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:T001
Disulfide bridge between
1:T001 and 3:T001

Results: Intensity normalization

Results: Highlight unique peaks
digested VICAM Analyte :BP_097
deglycosylated 2h digested VICAM

Results: Coverage map

Results: Protein digest Mass

Results :Peak match data comparison
analyte /control

Results: Peak match data
advanced table
When a mass can correspond to several peptides, the different possibilities can be
seen in the advanced view.

Results: Discrimination 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.

Future

Thank you
75www.stabicon.com
Stabicon Life Sciences, #4M-413,1st Floor, H R B R
3rd Block, Kammanahalli Main Road, Bangalore –
560043, Karnataka, India.
Tel: +91 80 41250324
bd@stabicon.com

Biopharmaceutical

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