טיהור ואפיון חלבונים ע"י מפלי pH
בכרומטוגרפיה יונית (IEC)


 הרשמה מוקדמת במחירים מוזלים 

הרשמה מוקדמת עד ה-9 באוקטובר 2015

הרשמה החל מה-10 באוקטובר 2015

1,485 ש "ח + מע "מ

1,750 ש "ח + מע "מ


תאריך: 19 אוקטובר 2015
מיקום הקורס: כפר המכביה, רמת גן
מידע כללי
משך הפעילות: יום לימודים מלא
שעות:  09:00-17:00
עלות: 1,750 ש"ח + מע"מ, עד 9 באוקטובר 1,485 ש"ח + מע"מ
נקודות זכות: 1
מק''ט: 15442
שפת הקורס: עברית
על הקורס

Ion exchange chromatography is one of the most common methods used for protein purification both in research and industry both because of its capacity and its economy of scale.  However, it is much less commonly used in protein analysis.  A major reason is the level of resolution of salt gradient elution.  Elution with wide range pH gradients is alternative to salt gradients that can provide both higher resolution and significant information on the electrostatic properties of the eluted proteins.  Unfortunately, wide range pH gradients can be difficult to construct and control. 

This course will cover: 

  1. the basic principles of ion exchange chromatography (IEC) of proteins; 
  2. the elution of proteins from IEC columns by both pH and salt gradients;
  3. the practical aspects of producing robust linear pH gradients that will both improve protein purification results and provide improved tools for the analysis of protein electrostatic properties.

Course Prerequisites
Participants should have some basic familiarity with the principles of chromatography and general biochemical laboratory practice.

קהל היעד
  • R&D Managers;
  • Scientists and technical staff involved with the analysis and characterization of protein & peptide biopharmaceuticals;
  • RA/QA staff and management involved with the analysis and characterization of protein & peptide biopharmaceuticals;
  • Scientists and technical staff involved with the planning, develop, and scale-up of protein purification processes;
  • Academic researchers interested in improving their methods of protein purification and characterization.
התועלת שתופק
  • Improved understanding of the principles of ion exchange chromatography of proteins;
  • Understanding of the potential of pH gradient elution to improve protein purification by ion exchange chromatography;
  • Understanding of the potential pf pH gradient ion exchange chromatography as an analytical tool to provide information on the electrostatic character of a protein (as an adjunct to isoelectric focusing).
השאר פרטים למידע נוסף
  • שם מלא
  • דוא''ל
  • טלפון
  • הערות
  • שלח
תכני הקורס

Major Subdivisions of the Course

  • Section A. pH
    • Some Comments on pH
  • Section B. Proteins
    • Characterization of Proteins
    • Fractionation of Protein Mixtures
  • Section C. Chromatography - Principles
    • General Principles of Chromatography
    • Principles of Ion Exchange Chromatography (IEC)
  • Section D. pH Gradient IEC  (pH-IEC)
    • IEC of Proteins
    • Elution by pH vs Elution by Ionic Strength in IEC
    • Producing Linear Wide Range pH Gradients for IEC
    • Using pH Gradient IEC in Protein Characterization & Analysis
    • Using pH Gradient IEC in Protein Purification
  • Section E. pH-IEC vs IEF, Regulatory Issues, Summary
    • Comparison of pH Gradient IEC with Isoelectric Focusing (IEF)
    • Regulatory Considerations – Biopharmaceutical Purification
    • Regulatory Considerations – Biopharmaceutical Analysis
    • Summary

Outline of the Course

  • Section A. pH
  • A.1:  Some Comments on pH
    • Definition of pH
      • Acidity
      • Measuring Acidity
      • pH Standardization (IUPAC 2002)
    • Measurement of pH
    • Factors Affecting pH
  • Section B. Proteins
  • B.1:  Characterization of Proteins
    • Concepts in Understanding Proteins
      • Amino Acids
      • Peptides and Polypeptides
      • Multiple Polypeptide Complexes
      • Native vs Dissociated;  Native vs Denatured
      • Size and Shape
      • Charge, Polarity, and Hydrophobicity
    • Characteristics of Proteins
      • Biological Characteristics
        • Biological Activity (Activity Units, Specific Activity)
      • Biochemical Characteristics
        • Overall Size (Molecular Weight and Shape)
        • Overall Charge (Isoelectric Point - pI)
        • Subunit Composition
        • Charge Distribution, Polarity, and Hydrophobicity
        • Amino Acid Composition and Sequence
        • Modifications in Amino Acid Sequence
        • Post-Translational Modifications  (Concept of Isoforms)
        • Polymeric Complexes
      • Proteins as Polyelectrolytes
        • pKa vc
        • alues of amino acid side chains
        • Isoelectric point (pI)
        • Protein Charge as Function of pH
        • Surface Electrostatic Properties
  • B.2:  Fractionation of Protein Mixtures
    • Types of Fractionation Techniques
      • Partition
      • Precipitation
      • Chromatography
      • Electrophoresis
    • Techniques Relevant to Protein Analysis
      • Chromatography
      • Electrophoresis
  • Section C. Chromatography - Principles
  • C.1:  General Principles of Chromatography
    • Partition Between Stationary and Mobile Phases
    • Factors Affecting Analyte Separation
    • Forward vs Reverse Phase
    • Isocratic vs Gradient Elution
      • Peak Width and Retention Time in Isocratic Elutions
      • Gradient Elution and Peak Width
  • C.2:    Principles of Ion Exchange Chromatography (IEC)
    • Characteristics of the Stationary Phase
    • Characteristics of the Mobile Phase
    • Interactions between Analyte and Stationary Phase
    • Gradient Elution in IEC
  • Section D. pH Gradient IEC  (pH-IEC)
  • D.1:  Ion Exchange Chromatography of Proteins
    • Proteins as Polyelectrolytes
    • Protein Binding to Ion Exchange Resins
    • Protein Elution from Ion Exchange Resins
  • D.2:    Elution by pH vs Elution by Ionic Strength in IEC
    • Retention of Proteins on IEC Resins
      • Stoichiometric Model
      • Electrostatic Model
      • pH and Protein Retention
    • Producing Elution Gradients
  • D.3:    Producing Linear Wide Range pH Gradients for IEC
    • Principles of Producing Linear pH Gradients
    • Internally Produced pH Gradients
      • Chromatofocusing with Polybuffers (Sluyterman - 1977)
      • Chromatofocusing with Common Buffers (Hutchens – 1986)
      • Chromatofocusing with Simple Mix Common Buffers (Frey – 1998)
    • Externally/Internally Produced pH Gradients
      • Gradient Chromatofocusing  (Anderson – 1997))
    • Externally Produced pH Gradients
      • Slais & Friedl (1992)
      • Ottens Group (2007)
      • CryoBioPhysica (2008)
  • D.4:    Using pH Gradient IEC in Protein Characterization and Analysis
    • Electrostatic Characterization Parameters
    • Electrostatic Determinants of Protein Isoforms
    • Isolation of Protein Isoforms
    • Cross Analysis with IEF
  • D.5:    Using pH Gradient IEC in Protein Purification
    • Bulk & Fine Purification
    • Effect of pH on Protein of Interest
    • Initial Purification by pH Precipitation
    • Peak Resolution – pH Gradient vs Salt Gradient
    • Eluent Removal – pH Adjustment vs Salt Removal
  • Section E. pH-IEC vs IEF, Regulatory Issues, Summary
  • E.1:  Comparison of pH Gradient IEC with Isoelectric Focusing (IEF)
    • Protein characteristics from each method
    • Isoelectric Point (pI) vs pH of Elution
    • IEF band profile vs pH elution profile
  • E.2:   Regulatory Considerations – Biopharmaceutical Purification
    • Regulatory Status of Buffer Systems for pH Gradient Elution
  • E.3:   Regulatory Considerations - Biopharmaceutical Analysis
    • Relevant Regulatory Guidelines
    • Electrostatic Information pH Gradient Chromatography Does Provide
    • Electrostatic Information pH Gradient Chromatography Does Not Provide
  • E.4:  Summary
אודות המרצה

ד"ר מתיו וייסמן

ד"ר ויסמן הוא ביוכימאי חלבונים ואנזימולוג, מומחה בטיהור ואפיון חלבונים (כוללים חלבונים תרופתיים), פיתוח בדיקות אנליטיות לחלבונים, ופיתוח אנזימים ותהליכים מבוססי אנזימים עבור יישומים מסחריים. הוא עבד בתעשיית הביוטכנולוגיה בישראל במשך יותר מעשרים וחמש שנים גם ביצור גם במו"פ.
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