HPLC OPERATOR

COURSE OUTLINE

Introduction to Biomolecule Analysis

Overview of Biomolecules

• Importance of biomolecule characterization in research and industry
• Challenges in HPLC-based biomolecule separation

Types of Biomolecules Analyzed by HPLC

• Amino Acids and Peptides
• Proteins and Their Structural Complexity
• Primary, secondary, tertiary, and quaternary structures
• Post-Translational Modifications (PTMs)
• Phosphorylation, glycosylation, disulfide bonds
• Monoclonal Antibodies (mAbs)
• Antibody-Drug Conjugates (ADC)
• Oligonucleotides and RNA-Based Therapeutics

Fundamentals of Biotechnology in HPLC

Nucleotides and Nucleic Acids

• DNA, mRNA, and tRNA structures
• Transcription and translation processes

ICH Q6(B) Guidelines for Biopharmaceuticals

• Regulatory requirements for quality control and characterization

HPLC Techniques for Biomolecule Analysis

Size Exclusion Chromatography (SEC) for Proteins

• Understanding Protein Aggregation and Its Impact
• SEC Column Selection and Mobile Phase Optimization
• Basic SEC Method Development for Biomolecule Purity Assessment

Ion Exchange Chromatography (IEX) for Charge Variants

• Charge Variability in Biomolecules
• Modes of IEX: Cation vs. Anion Exchange
• Optimizing pH and Salt Gradients for Selective Separation

Reversed-Phase Chromatography (RPC) for Protein and Peptide Analysis

• Selection of Columns and Mobile Phases
• Separation of Proteins Based on Primary Structure
• Peptide Mapping and Characterization
• Derivatized Amino Acid Analysis Techniques

Hydrophilic Interaction Chromatography (HILIC) for Polar Biomolecules

• Overview of HILIC and Its Applications
• Glycan Analysis and Carbohydrate Profiling
• Un-derivatized Amino Acid Analysis Using HILIC

Hydrophobic Interaction Chromatography (HIC) for Hydrophobic Proteins

• Fundamentals of HIC and Retention Mechanisms
• Common Applications in Protein and Antibody Analysis