Biopharma Solution
Upcoming SlideShare
Loading in...5
×
 

Biopharma Solution

on

  • 634 views

Dear Customer, ...

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

Director Operations
Stabicon Life Sciences Pvt Ltd
Mobile: +919591974355/080-41714280
www.stabicon.com

Statistics

Views

Total Views
634
Views on SlideShare
634
Embed Views
0

Actions

Likes
0
Downloads
9
Comments
0

0 Embeds 0

No embeds

Accessibility

Upload Details

Uploaded via as Adobe PDF

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment

Biopharma Solution Biopharma Solution Presentation Transcript

  • Stabicon Life Sciences Pvt Ltd1 Biopharmaceutical Solution By Vijay Kumar Ranka
  • 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
  • Chapter –I3 Introduction
  • Why???4 Therapeutic small molecule being dominant for more than 100year for treatment then why biologics required for therapeutic? Stabicon
  • 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
  • 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?
  • Mechanism for Signaling7 Several types of molecule modification are involved in regulation for a signal transfer such as : glycosylation, acetylation,etc
  • Effect in the body8 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.
  • Chapter –II9 Protein Basic Mechanism
  • Genotype determines phenotype10
  • Central dogma11 Prokaryotic Cell: DNA – RNA – PROTEIN (Transcription) (Translation) Eukaryotic Cell: DNA – RNA – PROTEIN - PROTEIN MODIFIED (Post Translation)
  • Eukaryotic cell12
  • Protein Structure13 primary structure Primary ACDEFGHIKLMNPQRSTVWY Secondary Tertiary Quaternary
  • Proteome14 Lipidation Lipid — GenomeD Sugar —Genomics Glycosylation — P— P Phosphorylation P— TranscriptomeD Ubiquitination Ub — Ub —Proteomics Cleavage P— Proteome ~ 300 modifications Many more ?—
  • Chapter –III15 Therapeutic Protein Production
  • Biopharma16 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 cant be produced using convential chemical synthesis rather than in a living cell under stringently controlled condition.
  • How are these designed17
  • Protein Factory18
  • Bioprocessing Phase19
  • Examples of Biologics marketed:20 Insulin Imiglucerase Glucagon Human Growth Hormone Erythropoietin G-CSF Interferon
  • Chapter –IV –IV21 Protein Characterization
  • Characterization Step:22 Intact Mass analysis Primary structure - peptide mapping Glycan analysis Amino acid and media analysis Data processing
  • How to characterize ?23 Large scale screening of proteins, their expression, modification and interaction by using high-throughput approaches
  • Characterization Required for24 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 ?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
  • Isolation and characterization26
  • Protein Identification Technology27 Seperation Mass Analysis Data processing
  • Mass Spectrometry Schematic Diagram28
  • MALDI Ionization29 Protein or Mass Spectrometer Mass/Charge Peptide (m/z) Ionization Matrix assisted laser desorption ionization (MALDI), Solution Gas Koichi Tanaka Phase Phase
  • Data Acquisition from MALDI-MSI30 Alanine Valine Alanine, peptide in plasma Valine, m/z = 1502.7 m/z = 1474.6
  • ESI Ionization31 Protein or Mass Spectrometer Mass/Charge Peptide (m/z) Ionization Solution Gas Electrospray ionization (ESI), John B Fenn Phase Phase
  • Data Acquisition from ESI-MSI32
  • What is MSE?33
  • Single protein identification34 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
  • Protein mixture Analysis by LC-MS/MS35 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
  • Protein structural Seperation36 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
  • HDMS FOR STRUCTURAL SEPERATION OF ISOMER37
  • IMS separation of peptides and lipids38 No IMS separation IMS selection of peptides IMS selection of lipids
  • Why Accurate mass?39 Intact Protein Mass Digested Protein Mass
  • Intact Mass Analysis40
  • 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
  • N & C terminal Ions42 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
  • Peptide Mapping43
  • Post Translational Identification44
  • Glycoprotein45
  • Chapter – V46 Monitor the Bioreactor Media & Protein Synthesis
  • UV Aminoacid Analysis47
  • Water Purity48
  • Amylase Protein Expression49
  • E.Coli Lysate Analysis50
  • Batch Analysis51 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?
  • Chapter –VI52 Data processing
  • Data processing53 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 Does54 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 Chromatogram55
  • Protein Charge determination56 The theoretical peak constructe d with the isotope distribution (purple) and the experiment al peak (green) have the same width at half height.
  • Results :Spectra view57 Mirror Stack Overlay Control :BP_079 non-deglycosylated VICAM Analyte :BP_092 deglycosylated 19h VICAM
  • 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
  • Results: Highlight unique peaks59 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
  • Result : Peak match data comparison analyte/control60
  • 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
  • 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
  • Results Peak match data comparison analyte/control63 You can add your own commentsdd your own comments Control :BP_079 non-deglycosylated VICAM Analyte :BP_092 deglycosylated 19h VICAM
  • 64 PEPTIDE MAP ANALYSIS
  • Protein digest Chromatogram65 Matched peptides annotation Processed Raw Control :BP_094 non-deglycosylated digested VICAM Analyte :BP_097 deglycosylated 2h digested VICAM
  • Results: Differential view66 Control :BP_094 non-deglycosylated digested VICAM Analyte :BP_097 deglycosylated 2h digested VICAM
  • Protein digest Analysis67 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
  • Annotation of the peptides68 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
  • Results: Intensity normalisation69
  • Results: Highlight unique peaks70 Control :BP_094 non-deglycosylated digested VICAM Analyte :BP_097 deglycosylated 2h digested VICAM
  • Results Coverage map71
  • Results Protein digest Mass72
  • Results Peak match data comparison analyte/control73
  • Results Peak match data advanced table74 When a mass can correspond to several peptides, the different possibilities can be seen in the advanced view.
  • Results Discrimination between two assignments75 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
  • Future76
  • 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
  • Stabicon Life Sciences Pvt Ltd78 Thank you