Biopharma VS Small Molecules Therapeutic

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

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

Introduction
Chapter –I
3

Therapeutic small molecule being dominant for more than 100 year for
treatment then why biologics required for therapeutic ?
Why???
4

How different they are ?
Product Activity Low–high dose High-low dose
Production Chemical synthesis Living organism
Development Limited Trials Extensive Trials
Regulation Non Specfic Specfic
Toxicity High Low
Elimination Metabolism Endocytosis
Structural Folding Not Required Required
5

What are these?
 What are these large molecule or biomolecule and how similar are they to
human body?
 What make them so specific and effective?
 What is the correlation of large/ biomolecule molecule to living machinery?
6

Mechanism for Signaling
Several types of molecule
modification are involved in
regulation for a signal
transfer such as :
glycosylation, acetylation,etc
7

Effect in the body
 Small molecule rarely elict secondary signaling thus effect prevail until
drug adhere to the target site whereas in case of large molecule
always elict an secondary signaling hence effect remain even after
the drug is eliminated.
8

Protein Basic Mechanism
Chapter –II
9

Genotype determines phenotype
10

Central dogma
Prokaryotic Cell:
DNA –
(Transcription)
RNA – PROTEIN
(Translation)
Eukaryotic Cell:
DNA – RNA – PROTEIN - PROTEIN MODIFIED
(Post Translation)
11

Eukaryotic cell
12

Protein Structure
 Primary
 Secondary
 Tertiary
 Quaternary
ACDEFGHIKLMNPQRSTVWY
primary structure
13

Therapeutic Protein Production
Chapter –III
14

Biopharma
 Biopharmaceutical are protein with considerable therapeutic structural
diversity. They tend to between 100 to 1000 times larger than traditional
small molecule drug .
 Such complex protein can't be produced using convential chemical
synthesis rather than in a living cell under stringently controlled condition.
15

How are these designed
16

Protein Factory
17

Bioprocessing Phase
18

Examples of Biologics marketed:
 Insulin
 Imiglucerase
 Glucagon
 Human Growth
Hormone
 Erythropoietin
 G-CSF
 Interferon
19

Chapter –IV
Protein Characterization
20

Characterization Step
 Primary structure - peptide mapping
 Glycan analysis
 Intact Mass analysis
 Amino acid and media analysis
 Data processing
21

How to characterize ?
 Large scale screening of proteins, their expression, modification and
interaction by using high-throughput approaches
22

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
23

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
24

Isolation and characterization
25

Protein Identification Technology
Seperation
Mass Analysis
Data processing
26

MALDI Ionization
Protein or
Peptide
Mass Spectrometer Mass/Charge
(m/z)
Ionization
Solution
Phase
Gas
Phase
Matrix assisted laser desorption ionization (MALDI),
Koichi Tanaka
27

Data Acquisition from MALDI-MSI
Alanine, peptide in
plasma
m/z = 1474.6
Valine, m/z = 1502.7
Alanine
Valine
28

Protein or
Peptide
Mass Spectrometer Mass/Charge
(m/z)
Ionization
Solution
Phase
Gas
Phase
ESI Ionization
Electrospray ionization (ESI), John B Fenn
29

Data Acquisition from ESI-MSI
30

What is MSE?
31

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
32

Protein mixture Analysis by LC-MS/MSy
Protein mixture
Digestion
Peptides
400 800 1200 1600
m/z
MS
MS/MS
10 20 30 min
0
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.
33

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)
34

HDMS FOR STRUCTURAL SEPERATION OF ISOMER
35

IMS separation of peptides and lipids
No IMS separation IMS selection of peptides IMS selection of lipids
36

Why Accurate mass?
s
Intact Protein Mas
Digested Protein Mass
37

Intact Mass Analysis
38

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 F
D 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:LIFAG
KQLED
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
A G
F D
E
L G
LI K Q
Q A
39

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
40

Peptide Mapping
41

Post Translational Identification
42

Glycoprotein
43

Monitor the Bioreactor
Media &
Protein Synthesis
Chapter – V
44

UV AminoacidAnalysis
45

Water Purity
46

Amylase Protein Expression
47

E.Coli Lysate Anal ysis
48

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?
49

Data processing
Chapter –VI
50

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,…
51

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
52

Intact Protein Chromatogram
53

54
www.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
55

Results Spectra view (Intensity
filter)zzzz
Threshold defined
automatically on the
spectra
T
hreshold value
typed in the table
Filt
er applied on the
results table
56

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
57

Result : Peak match data
comparison analyte/control
58

Results Peak match data for
control (glycosylated)
Percentage of each
glycosylation state in control
59

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
60

Results: Peak match data
comparison analyte/control
You can add your own
comments
61

PEPTIDE MAP ANALYSIS
62

Protein digest Chromatogram
Processed
digested VICAM Analyte :BP_097
deglycosylated 2h digested VICAM
Raw
Matched peptides annotation
63

Results: Differential view
Control :BP_094 non-deglycosylated digested VICAM
Analyte :BP_097 deglycosylated 2h digested VICAM
64

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
65

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
66

Results: Intensity normalisation
67

Results:Highlight unique peaks
digested VICAM Analyte :BP_097
deglycosylated 2h digested VICAM
68

Results: Coverage map
69

Results: Protein digest Mass
70

Results :Peak match data comparison
analyte /control
71

Results: Peak match data
advanced table
When a mass can correspond to several peptides, the different possibilities can be
seen in the advanced view.
72

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.
73

Future
74

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

Biopharma VS Small Molecules Therapeutic

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