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 link https://www.box.net/shared/aboumapp9h9gftunenja/1f7c7a6ef1/rss.xml If 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 time Regards Vijay Project Director Stabicon Life Sciences Pvt Ltd Mobile: +919591974355/080-41714280 www.stabicon.com

1. Stabicon Life Sciences Pvt Ltd
1
Biopharmaceutical Solution
By
Vijay Kumar Ranka

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

3. Chapter –I
3
Introduction

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

5. How different they are ?
5
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

6. What are these?
6
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?

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

8. Effect in the body
8
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.

9. Chapter –II
9
Protein Basic Mechanism

10. Genotype determines phenotype
10

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

12. Eukaryotic cell
12

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

14. Proteome
14
Lipidation Lipid —
GenomeD
Sugar —
Genomics Glycosylation
—
P— P
Phosphorylation
P—
TranscriptomeD
Ubiquitination Ub —
Ub —
Proteomics Cleavage P—
Proteome
~ 300 modifications Many more ?—

15. Chapter –III
15
Therapeutic Protein Production

16. Biopharma
16
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.

17. How are these designed
17

18. Protein Factory
18

19. Bioprocessing Phase
19

20. Examples of Biologics marketed:
20
Insulin
Imiglucerase
Glucagon
Human Growth Hormone
Erythropoietin
G-CSF
Interferon

21. Chapter –IV
–IV
21
Protein Characterization

22. Characterization Step:
22
Intact Mass analysis
Primary structure - peptide mapping
Glycan analysis
Amino acid and media analysis
Data processing

23. How to characterize ?
23
Large scale screening of proteins, their expression, modification and interaction
by using high-throughput approaches

24. Characterization Required for
24
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

25. Why Protein by Mass Spectrometry ?
25
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

26. Isolation and characterization
26

27. Protein Identification Technology
27
Seperation
Mass Analysis
Data processing

28. Mass Spectrometry Schematic Diagram
28

29. MALDI Ionization
29
Protein or Mass Spectrometer Mass/Charge
Peptide (m/z)
Ionization
Matrix assisted laser desorption ionization (MALDI),
Solution Gas
Koichi Tanaka
Phase Phase

30. Data Acquisition from MALDI-MSI
30
Alanine
Valine
Alanine, peptide in plasma Valine, m/z = 1502.7
m/z = 1474.6

31. ESI Ionization
31
Protein or Mass Spectrometer Mass/Charge
Peptide (m/z)
Ionization
Solution Gas
Electrospray ionization (ESI), John B Fenn
Phase Phase

32. Data Acquisition from ESI-MSI
32

33. What is MSE?
33

34. Single protein identification
34
Mass/Charge Mass
(m/z)
How to identify a single protein by MS?
Digest into many peptides Mass of many
peptides
Peptide mass fingerprinting (PMF)
Mass of many peptide fragments
By
Tandem Mass Spectrometry

35. Protein mixture Analysis by LC-MS/MS
35
Digestion HPLC
Protein mixture Peptides 0 10 20 30 min
MS
Database
Searching
LLTTIADAAK
MS/MS
SAGGNYVVFGEAK
EDDVEEAVQAADR
400 800 1200 1600
m/z
1 sequencing attempt per 0.5 sec.
3600 sequencing attempts in 30 min.
All peptide sequences Identification of many proteins

36. Protein structural Seperation
36
Ring Electrodes (Potential Gradient. +ve force)
Detector
Gate
Neutral Buffer Gas (-ve force)
•An ion in a compact-form has a high mobility, and hence shorter drift time,
compact-
•The same ion in a more open conformation has a lower mobility, and hence
and
a longer drift time

37. HDMS FOR STRUCTURAL SEPERATION OF ISOMER
37

38. IMS separation of peptides and lipids
38
No IMS separation IMS selection of peptides IMS selection of lipids

39. Why Accurate mass?
39
Intact Protein Mass
Digested Protein Mass

40. Intact Mass Analysis
40

41. How to identify a single protein by MS/MS?
41
Protein MS spectrum MS/MS spectrum Theoretical
digestion Spectrum
Database
Ionization searching
Fragmentation
m/z m/z m/z
Peptides
Peptide/protein
identification
LIFAGKQLEDGR
b ions y ions
LI F A G K Q L E D G
1: L IFAGKQLEDGR:11
2: LI FAGKQLEDGR:10 D E L Q K G A F
3: LIF AGKQLEDGR :9
4: LIFA GKQLEDGR :8
5: LIFAG KQLEDGR :7
6: LIFAGK QLEDGR :6
7: LIFAGKQ LEDGR :5 Q A
8: LIFAGKQL EDGR :4
9: LIFAGKQLE DGR :3
10:LIFAGKQLED GR :2
11:LIFAGKQLEDG R :1 200 400 600 800 1000 1200 m/z

42. N & C terminal Ions
42
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

43. Peptide Mapping
43

44. Post Translational Identification
44

45. Glycoprotein
45

46. Chapter – V
46
Monitor the Bioreactor Media
&
Protein Synthesis

47. UV Aminoacid Analysis
47

48. Water Purity
48

49. Amylase Protein Expression
49

50. E.Coli Lysate Analysis
50

51. Batch Analysis
51
Batch1a Batch 2aB
Batch1b Batch2b
difference in proteins
Batch1c Batch2c
Proteins (to identify and quantify proteins in multiple samples)
How many proteins ?
The choice of method?
How many samples?
How many variability parameter?

52. Chapter –VI
52
Data processing

53. Data processing
53
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,…

54. What software Does
54
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

55. Intact Protein Chromatogram
55

56. Protein Charge determination
56
The
theoretical
peak
constructe
d with the
isotope
distribution
(purple)
and the
experiment
al peak
(green)
have the
same width
at half
height.

57. Results :Spectra view
57
Mirror
Stack
Overlay
Control :BP_079 non-deglycosylated VICAM
Analyte :BP_092 deglycosylated 19h VICAM

58. Results Spectra view (Intensity filter)
58
Threshold defined
automatically on the spectra
hreshold defined automatically
on the spectra
Threshold value Threshold
value typed in the tablehe
table
Filter applied on Filter
applied on the results
tableesults table

59. Results: Highlight unique peaks
59
Unique peaks highlighting
Glycosylated T022 fragments (control
only)
Deglycosylated T022
fragment (analyte only)
Control :BP_094 non-deglycosylated digested
VICAM
Analyte :BP_097 deglycosylated 2h digested
VICAM

60. Result : Peak match data comparison analyte/control
60

61. Results Peak match data for control (glycosylated)
61
Percentage of each
glycosylation state in control
Control :BP_079 non-deglycosylated VICAM
Analyte :BP_092 deglycosylated 19h VICAM

62. Results Peak match data for analyte (deglycosylated )
62
Percentage of each
glycosylation state in analyte
Control :BP_079 non-deglycosylated VICAM
Analyte :BP_092 deglycosylated 19h VICAM

63. Results Peak match data comparison analyte/control
63
You can add your own
commentsdd your own
comments
Control :BP_079 non-deglycosylated VICAM
Analyte :BP_092 deglycosylated 19h VICAM

64. 64
PEPTIDE MAP ANALYSIS

65. Protein digest Chromatogram
65
Matched peptides annotation
Processed Raw
Control :BP_094 non-deglycosylated digested VICAM
Analyte :BP_097 deglycosylated 2h digested VICAM

66. Results:
Differential view
66
Control :BP_094 non-deglycosylated digested VICAM
Analyte :BP_097 deglycosylated 2h digested VICAM

67. Protein digest Analysis
67
Reprocess the data with another
Set the selected analyte as method
control
Add or remove analyte
List of the raw data file
Selected analyte compared with the control 2h

68. Annotation of the peptides
68
1:T001
First chain of the protein First digest product
of the chain
Trypsin digestion
1:T001* Modified form of 1:T001
1:T001-002 Missed cleavage between
1:T001 and 1:T002
Disulfide bridge between
1:T001-3:T001 1:T001 and 3:T001

69. Results:
Intensity normalisation
69

70. Results:
Highlight unique peaks
70
Control :BP_094 non-deglycosylated digested VICAM
Analyte :BP_097 deglycosylated 2h digested VICAM

71. Results
Coverage map
71

72. Results
Protein digest Mass
72

73. Results
Peak match data comparison analyte/control
73

74. Results
Peak match data advanced table
74
When a mass can correspond to several peptides, the different possibilities can be seen in
the advanced view.

75. Results
Discrimination between two assignments
75
If high energy data are available (acquisition with MSE mode), the
fragmentation data can be used to discriminate several assignment for the
same mass.
The sequence
corresponding to the
fragment 1:T009* of
the LC gave a better
score than the
sequence of 1:T021

76. Future
76

77. Question???
77
Please email to vijay.ranka@stabicon.com
Write to Stabicon Life Sciences Pvt Ltd
3BM-416,3rd Block,
HRBR Extension,
Bangalore – 560043
Karnataka, India
Phone :+9180 – 41714280/81

78. Stabicon Life Sciences Pvt Ltd
78
Thank you