Training Course F-2 for LBA scientists - 8am to 5:30pm, Friday April 13, 2018

"Large Molecules Bioanalytical Method Development by LCMS Specifically Designed
for Scientists with LBA Background"

Learn LCMS Step by Step to Discover the Potential of LCMS as an Alternative / Complementary Technique to LBA

Course Outline

Course Syllabus

  • Lesson 1
    LCMS Inlets: Coupling HPLC with a Mass Spectrometer
    • Introduction to Mass Spectrometry
      • What is a Mass Spectrometer?
      • Mass Spectrum
    • Main components of a Mass Spectrometer
    • Arpino's Symbolism
    • Soft Ionization Techniques
      • Ion production and fragmentation
        • Hard vs. soft ionization
      • Atmospheric Pressure Ionization (API)
        • Analytical domains
      • Electrospray (ESI)
        • Introduction to Ion Evaporation
        • Strengths and weaknesses
        • Heated capillary (Thermo) vs. curtain gas (Sciex)
        • Ionspray (IS): pneumatically assisted ESI
        • Turboionspray (TIS): effects of the drying gas
        • Formation of adducts and clusters
        • Needle voltage, nebulizer flow
      • Advantages and disadvantages of soft ionization sources
      • Contaminants
  •  

  • Lesson 2
    Mass Analyzers used for Large Molecule Bioanalysis
    • How mass analyzers work: Mass filter
    • Quadrupole Mass Analyzer
      • Schematic diagram of a quadrupole mass spectrometer
      • RF in a quadrupole
      • Quadrupole Theory
      • Types of voltages present on a quadrupole: RF and DC
        • What is RF?
      • "RF only" mode
      • Ion trajectories
      • Ion Stability
      • Quadrupole settings
      • Mathieu Equation and stability diagram
      • Profile scan
      • Bandwidth and peak width
    • Triple Quadrupole Operations
      • MS calibration
        • Peak width
        • Peak shapes
        • Peak assignments
        • Resolution vs. sensitivity
        • Scan line
        • Selected Ion Monitoring (SIM)
      • Tandem Mass Spectrometry (MS/MS)
        • Fragmentation and why is important
        • Collision Cell and CID
      • Understanding Triple Quadrupole (QqQ) Operations:
        • Precursor Ion Scan
        • Multiple or Selected Reaction Monitoring (MRM or SRM)
        • Product Ion Scan
        • SIM vs. SRM
    • High Resolution Mass Spectrometry (HRMS): QTOF & Orbitrap
      • What is High Resolution Accurate Mass MS?
      • How much resolution do you need?
      • Why HRMS for Large Molecules?
      • QTOF
        • Drift region
        • Spatial spread
        • Reflector
        • TOF analyzer process
        • TOF data acquisition
      • Orbitrap
        • CD Collision Cell
        • C-Trap
        • Duty cycle
        • Mass resolution and scan time
        • Molecular size
    • What are the advantages and disadvantages of various mass analyzers in the bioanalysis of Large Molecules
      • m/s range vs. Resolution vs. Accuracy
      • Triple Quad vs. HRMS
  •  

  • Lesson 3
    ESI Optimization for Large Molecules
    • Problems with On axis spray
    • Optimization of sheath/nebulizer gas and auxiliary/make up gas
    • Use of Make up liquid for optimizing the ionization
    • ESI main components in Sciex, Agilent, Thermo and Waters sources
    • Common problems with ESI
      • Signal instability
      • Arching
    • Advantages of Orthogonal spray
    • The V.I.C. Rule: Volatility, Ion Paring and Conductivity
    • ESI Additives & Buffers to use for the best sensitivity
      • Proton donors
      • Proton acceptors
      • Buffers
      • Ion pairing reagents: compromising sensitivity vs. chromatographic resolution
      • Ion pairing efficiency
        • Phosphate, sulfate and borate buffers
      • Negative ion formation
      • Compatibility with HPLC technique
    • The infusion experiment to enhance the sensitivity in ESI
      • Experiment design: HPLC pumps + Infusion pump
      • Solvents preparation: 50/50 MeOH/Water vs. 50/50 ACN/Water
      • Modifiers preparation: 1% Acetic acid; 0.1% formic acid; 5-10mM Ammonium acetate; 0.1-0.02% TFA; 0.1% Ammonium hydroxide
      • Case study: Achieving the best sensitivity for a pentapeptide
    • The 5 Gold Practical rules in ESI
      • Final considerations and strategies on LC mobile phases
    • Tricks and tips of using divert valve with isocratic/gradient chromatography to enhance sensitivity
    • Working in the highest sensitivity conditions...is it possible?
    • Knowing ESI Theory to improve source Optimization
      • A message from Prof. Fenn (Nobel prize for Chemistry in 2002)
      • The 4 major processes in ESI
      • Ion evaporation: surface tension and droplet production
      • D&D: Desorption & Desolvation
      • Rayleigh Limit
      • Practical considerations from ESI Theory
        • ESI mechanism for Large Molecules and Proteins
          • Charged residue mechanism (CRM)
        • Droplet vs. protein
      • ESI as Electrochemical Cell and impact on Protein oxidation
        • Platinum ESI capillary vs. stainless steel ESI capillary
        • The electrochemical effect
  •  

  • Lesson 4
    Developing the Right Chromatography for Biotherapeutics
    • LC parameters
      • HPLC strategies for ESI
      • Drawbacks of developing LC-MS/MS methods without good chromatographic separations
      • Isocratic and gradient LC methods
    • Chromatographic parameters to consider when developing a method in LC-MS
      • Column Geometry and size
      • Flow rate and LC Columns
      • Speed & Sensitivity
      • Importance of pore size (300A)
      • Mobile Phase composition
      • Importance of Temperature effect in protein analysis
        • Problems with protein fronting/tailing
        • Problems with protein recovery from stationary phases
      • LC column selection & decision tree
        • What column is best suited for a particular application?
        • Criteria for optimal column chemistry for LC-MS/MS protein applications
    • Stationary phases for Large Molecules
      • Reverse phase (RP)
      • Polar embedded phase
      • Silica based columns vs. polymeric columns
      • UHPLC - Ultra High Performance Liquid Chromatography
        • Compatibility of UHPLC with MS, are we more productive?
        • Efficiency vs. Selectivity
      • Fuse-core columns
        • LC-MS/MS efficiency with Fused-Core columns
        • Matrix interferences on Fuse-core columns
    • Pre-column vs. filter frit: Dead volume issues in high throughput LC-MS/MS
      • The importance of the Connecting tubes
      • Effect Of Dead Volume On System Performance
      • Particulate material in the sample flow stream
      • Pre-column filter: troubleshooting
    • Dwell time & fast chromatography in LC-MS
    • Specific applications in micro/nano flow/spray
      • Nanospray
        • Tips positioning
        • Chromatography in the tip
        • Tandem LC - Tandem MS (LC/LC-MS/MS)
      • Microflow and microESI Optimization for Protein analysis
        • Routine capillary analysis
        • Main advantages of using microESI
          • High sensitivity and low injection volume
          • Sharp chromatographic peaks
          • Extremely low flow rate and chemical noise
        • Columns for microESI
          • Flow rate & column optimization
        • Why does microESI work so well?
  •  

  • Lesson 5
    Matrix effects, Ion Suppression and Carryover
    • Matrix effects and Ionization effects in the source
      • Matrix Effect, Ion suppression and S/N on Different API sources
      • General criteria to fight matrix effect
    • Ion suppression
      • Instrument set-up
      • How to minimize ion-suppression
      • Differential suppression
    • Carryover
      • Definition
      • Guidelines
      • Fighting carryover
      • Special washing solutions
      • Accumulation
      • Carryover & contamination
  •  

  • Lesson 6
    LCMS vs. LBA in Quantification of Biotherapeutics
    • Compare LCMS vs.LBA for:
      • Reagents vs. Equipment
      • Detector System
      • Reference Standard
      • Calibration Curve & Prozone
      • Assay Validation
      • Parameters
      • Matrix interference
    • LCMS advantages:
      • No-reagent bases
      • Faster method development
      • Better linearity
      • No impact from ADAs (immunogenicity)
      • Better selectivity
      • Ability to measure post-translational modifications
  •  

  • Lesson 7
    Principles of Protein analysis by Mass Spectrometry
    • Functional and structural proteins
    • Basic understanding of multiple charging of proteins
    • Transcription and translation
    • Primary sequence and amino acids that produce positive and negative charges
    • Protein digestion
      • Trypsin and pepsin digestion
      • Method development strategies
      • MS/MS nomenclature for digested peptides
    • Post-translation modifications
      • Case study: Identification of possible phosphorylation sites
    • Multiply changed ions
      • Calculations for the charge state
      • Case study: Angiotensin
      • Case study: Interleukin
      • Large Molecule case studies:
        • Fibrinopepide
          • Charge state (MS)
          • Fragmentation: Tag in MS/MS
        • Myoglobin
          • Sample Digestion
          • Charge state (MS)
          • Fragmentation: Tag in MS/MS
  •  

  • Lesson 8
    Sample Preparation and Enrichment for LM by LCMS
    • Solid Phase Extraction (SPE)
      • Principles
      • Method development strategies
      • Applications
    • Immunoaffinity (IA)
      • Capturing Reagents
      • Protein a and g
      • Method development strategies
      • Applications
    • Signature peptide
      • The concept of proteotypic peptides
      • Strategies to choose the signature peptide
      • Using multiple signature peptides
    • Internal standard for LM by LCMS methods
      • Labeled protein
      • Labeled signature peptide (flanked peptide)
      • Analog peptide
  •  

  • Lesson 9
    Method Development Flow Chart and Problem Solving
    • Decision tree on Sample Preparation/enrichment
    • Decision tree for Mass spectrometry type
      • Protein size and type (fusion, PEGylated, biomarkers protein and monoclonal antibodies)
      • Sensitivity & Selectivity (S&S) requested for each project
      • Intact vs. digested
      • Immunoaffinity (IA) vs. SPE
      • Protein precipitation (PPT) followed by pellets digestions
      • Triple Quad vs. HRMS
      • Other method development strategies
    • Practical Case Studies: Step-by-Step method development of biotherapeutics and interactive group exercises on method development followed by troubleshooting discussions

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