Biopharma Solution

Click here to load reader

Embed Size (px)

description

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

Transcript of Biopharma Solution

  • 1. Stabicon Life Sciences Pvt Ltd1 Biopharmaceutical Solution By Vijay Kumar Ranka

2. Topics Covered :2I. IntroductionII.Protein Basic MechanismIII. Step in Protein ProductionIV.Identification and characterization techniqueV. Monitoring protein synthesisVI.Data Processing Methodology 3. Chapter I3 Introduction 4. Why???4 Therapeutic small molecule being dominant for more than 100year fortreatment then why biologics required for therapeutic?Stabicon 5. How different they are ?5Product Activity Lowhigh doseHigh-low doseProduction Chemical synthesis Living organismDevelopmentLimited Trials Extensive TrialsRegulation Non SpecficSpecficToxicity High LowEliminationMetabolism EndocytosisStructural Folding Not Required Required 6. What are these?6What are these large molecule or biomolecule and how similar are they tohuman body?What make them so specific and effective?What is the correlation of large/ biomolecule molecule to living machinery? 7. Mechanism for Signaling7Several types of moleculemodification are involved inregulation for a signaltransfer such as :glycosylation, acetylation,etc 8. Effect in the body8Small molecule rarely elict secondary signaling thus effect prevail until drugadhere to the target site whereas in case of large molecule always elict ansecondary signaling hence effect remain even after the drug is eliminated. 9. Chapter II9 Protein Basic Mechanism 10. Genotype determines phenotype10 11. Central dogma11 Prokaryotic Cell: DNARNA PROTEIN (Transcription)(Translation) Eukaryotic Cell:DNA RNA PROTEIN - PROTEIN MODIFIED(Post Translation) 12. Eukaryotic cell12 13. Protein Structure13 primary structurePrimary ACDEFGHIKLMNPQRSTVWYSecondaryTertiaryQuaternary 14. Proteome14 Lipidation Lipid GenomeDSugar Genomics Glycosylation P P PhosphorylationPTranscriptomeD UbiquitinationUb Ub Proteomics CleavageP Proteome ~ 300 modifications Many more ? 15. Chapter III15Therapeutic Protein Production 16. 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. 17. How are these designed17 18. Protein Factory18 19. Bioprocessing Phase19 20. Examples of Biologics marketed:20InsulinImigluceraseGlucagonHuman Growth HormoneErythropoietinG-CSFInterferon 21. Chapter IV IV21 Protein Characterization 22. Characterization Step:22Intact Mass analysisPrimary structure - peptide mappingGlycan analysisAmino acid and media analysisData processing 23. How to characterize ?23Large scale screening of proteins, their expression, modification and interaction by using high-throughput approaches 24. Characterization Required for24Protein identity (mutant protein)Protein quantity (Expression)Protein post-translational modifications (up or down)Protein structureProtein-protein interactionProtein localization Change in any protein property may cause functional abnormality and might be relevant to pathogenesis. ToolsProtein ArrayMass Spectrometry 25. Why Protein by Mass Spectrometry ?25MS can unambiguously identify proteins Gel separated proteins Proteins in mixtureProtein: protein associationIdentify precise post translational changesPhosphorylationN- or C- terminal modification Many more 26. Isolation and characterization26 27. Protein Identification Technology27SeperationMass AnalysisData processing 28. Mass Spectrometry Schematic Diagram28 29. MALDI Ionization29Protein or Mass Spectrometer Mass/Charge Peptide(m/z)Ionization Matrix assisted laser desorption ionization (MALDI), Solution GasKoichi TanakaPhasePhase 30. Data Acquisition from MALDI-MSI30 Alanine Valine Alanine, peptide in plasmaValine, m/z = 1502.7m/z = 1474.6 31. ESI Ionization31 Protein or Mass Spectrometer Mass/ChargePeptide(m/z) IonizationSolution GasElectrospray ionization (ESI), John B Fenn PhasePhase 32. Data Acquisition from ESI-MSI32 33. What is MSE?33 34. Single protein identification34Mass/ChargeMass (m/z) How to identify a single protein by MS?Digest into many peptidesMass of many peptides Peptide mass fingerprinting (PMF)Mass of many peptide fragments By Tandem Mass Spectrometry 35. Protein mixture Analysis by LC-MS/MS35Digestion HPLCProtein mixturePeptides0 1020 30 minMSDatabaseSearching LLTTIADAAK MS/MS SAGGNYVVFGEAK EDDVEEAVQAADR 400 8001200 1600 m/z1 sequencing attempt per 0.5 sec.3600 sequencing attempts in 30 min.All peptide sequences Identification of many proteins 36. Protein structural Seperation36Ring Electrodes (Potential Gradient. +ve force)Detector GateNeutral 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 anda longer drift time 37. HDMS FOR STRUCTURAL SEPERATION OF ISOMER37 38. IMS separation of peptides and lipids38 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 Analysis40 41. How to identify a single protein by MS/MS?41Protein MS spectrumMS/MS spectrum Theoretical digestion Spectrum Database Ionizationsearching Fragmentation m/zm/z m/zPeptidesPeptide/protein identificationLIFAGKQLEDGR b ionsy ionsLIFA G KQ LED G 1: LIFAGKQLEDGR:11 2: LIFAGKQLEDGR:10D EL Q KG AF 3: LIFAGKQLEDGR :9 4: LIFAGKQLEDGR :8 5: LIFAGKQLEDGR :7 6: LIFAGKQLEDGR :6 7: LIFAGKQLEDGR :5Q A 8: LIFAGKQLEDGR :4 9: LIFAGKQLEDGR :3 10:LIFAGKQLEDGR :2 11:LIFAGKQLEDGR :1 200 400 600 800 10001200m/z 42. N & C terminal Ions42Selected 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 Mapping43 44. Post Translational Identification44 45. Glycoprotein45 46. Chapter V46Monitor the Bioreactor Media& Protein Synthesis 47. UV Aminoacid Analysis47 48. Water Purity48 49. Amylase Protein Expression49 50. E.Coli Lysate Analysis50 51. Batch Analysis51Batch1aBatch 2aBBatch1b Batch2bdifference 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 VI52 Data processing 53. 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, 54. What software Does54Automates 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 Chromatogram55 56. Protein Charge determination56 Thetheoretical peakconstructe d with the isotopedistribution(purple)and theexperiment al peak(green)have thesame widthat half height. 57. Results :Spectra view57MirrorStackOverlay 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 tableFilter applied on Filterapplied on the resultstableesults table 59. Results: Highlight unique peaks59 Unique peaks highlighting Glycosylated T022 fragments (controlonly) Deglycosylated T022 fragment (analyte only)Control :BP_094 non-deglycosylated digestedVICAMAnalyte :BP_097 deglycosylated 2h digestedVICAM 60. Result : Peak match data comparison analyte/control60 61. Results Peak match data for control (glycosylated)61 Percentage of each glycosylation state in controlControl :BP_079 non-deglycosylated VICAMAnalyte :BP_092 deglycosylated 19h VICAM 62. Results Peak match data for analyte (deglycosylated )62Percentage of eachglycosylation state in analyte Control :BP_079 non-deglycosylated VICAM Analyte :BP_092 deglycosylated 19h VICAM 63. 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. 64 PEPTIDE MAP ANALYSIS 65. Protein digest Chromatogram65 Matched peptides annotationProcessedRaw Control :BP_094 non-deglycosylated digested VICAM Analyte :BP_097 deglycosylated 2h digested VICAM 66. Results:Differential view66 Control :BP_094 non-deglycosylated digested VICAM Analyte :BP_097 deglycosylated 2h digested VICAM 67. Protein digest Analysis67Reprocess the data with another Set the selected analyte asmethod controlAdd or remove analyteList of the raw data file Selected analyte compared with the control 2h 68. Annotation of the peptides68 1:T001 First chain of the proteinFirst digest product of the chain Trypsin digestion 1:T001* Modified form of 1:T0011:T001-002 Missed cleavage between1:T001 and 1:T002Disulfide bridge between1:T001-3:T0011:T001 and 3:T001 69. Results: Intensity normalisation69 70. Results: Highlight unique peaks70 Control :BP_094 non-deglycosylated digested VICAM Analyte :BP_097 deglycosylated 2h digested VICAM 71. Results Coverage map71 72. Results