HPLC-SEC
High-Performance Liquid Chromatography Size-Exclusion Chromatography for protein purity and aggregation analysis
HPLC-SEC (High-Performance Liquid Chromatography Size-Exclusion Chromatography) is a gold-standard analytical technique for protein characterization. Our platform uses HPLC-SEC to assess protein purity, aggregation state, and molecular weight distribution with high precision and reproducibility.
Technology Overview
Size-exclusion chromatography separates proteins based on their hydrodynamic size as they pass through a porous stationary phase. Larger molecules elute first, followed by smaller molecules in order of decreasing size.
Size-Based Separation
Hydrodynamic radius determines elution time
- Larger proteins elute earlier
- Smaller proteins elute later
- Aggregates separated from monomers
- Fragments distinguished from intact protein
High-Resolution Analysis
Precise molecular characterization
- Native protein conditions
- Quantitative purity assessment
- Aggregation state analysis
- Molecular weight estimation
Key Applications
Quantitative assessment of protein purity
Measurements:
- Main peak purity percentage
- Impurity identification and quantification
- Related substance analysis
- Degradation product detection
Applications:
- Quality control testing
- Lot release criteria
- Process development
- Stability studies
Sensitivity: Can detect impurities at 0.1% levels
HPLC-SEC provides orthogonal purity measurements to complement other analytical techniques.
Quantitative assessment of protein purity
Measurements:
- Main peak purity percentage
- Impurity identification and quantification
- Related substance analysis
- Degradation product detection
Applications:
- Quality control testing
- Lot release criteria
- Process development
- Stability studies
Sensitivity: Can detect impurities at 0.1% levels
HPLC-SEC provides orthogonal purity measurements to complement other analytical techniques.
Detection and quantification of protein aggregates
Measurements:
- High molecular weight species (HMWS)
- Dimer and multimer quantification
- Soluble aggregate analysis
- Aggregation kinetics monitoring
Applications:
- Formulation development
- Storage stability studies
- Process optimization
- Comparability assessments
Detection range: 0.1% to 50% aggregation levels
Some aggregates may be too large to enter the column and will be excluded from analysis.
Detection of protein degradation and fragmentation
Measurements:
- Low molecular weight species (LMWS)
- Clip products identification
- Degradation pathway analysis
- Fragmentation kinetics
Applications:
- Stability indicating methods
- Forced degradation studies
- Process-related impurity monitoring
- Method validation
Resolution: Can separate fragments differing by >5% in molecular weight
Fragmentation analysis helps identify degradation pathways and optimize storage conditions.
Analytical Specifications
Column Technology
Detection Methods
UV Detection
280 nm absorbance for aromatic amino acids
- Universal protein detection
- Quantitative measurements
- High sensitivity (μg/mL range)
- Interference-free detection
Multi-Wavelength Detection
Spectral analysis across UV range
- Peak purity assessment
- Coelution detection
- Spectral confirmation
- Method robustness
Fluorescence Detection
Enhanced sensitivity for specific applications
- Intrinsic tryptophan fluorescence
- Labeled protein detection
- Low-level impurity detection
- Specialized applications
Light Scattering
Absolute molecular weight determination
- Model-independent MW calculation
- Aggregation state confirmation
- Polydispersity measurements
- Advanced characterization
Method Development and Optimization
Column Selection
Choose appropriate column based on separation requirements and sample characteristics.
Selection criteria:
- Molecular weight range of analytes
- Required resolution
- Sample complexity
- Analysis time requirements
Our method development team can recommend optimal column selection based on your specific protein characteristics.
Mobile Phase Optimization
Develop buffer conditions that maintain protein stability while achieving optimal separation.
Key parameters:
- Buffer composition (phosphate, Tris, HEPES)
- pH optimization (typically 6.5-7.5)
- Salt concentration (50-300 mM)
- Additive selection (glycerol, sucrose)
Mobile phase conditions should match or closely approximate protein storage conditions.
Method Validation
Establish method performance characteristics according to regulatory guidelines.
Validation parameters:
- Accuracy and precision
- Linearity and range
- Limit of detection/quantification
- Robustness and ruggedness
- System suitability criteria
Validated methods ensure consistent, reliable results across analysts and instruments.
System Suitability
Establish criteria to ensure method performance for each analysis.
Typical criteria:
- Retention time precision (RSD <2%)
- Peak area precision (RSD <5%)
- Resolution requirements (Rs >1.5)
- Theoretical plate count (N >2000)
- Peak symmetry (0.8-1.5)
Data Analysis and Interpretation
Chromatogram Analysis
Statistical Analysis
Precision assessment:
- Repeatability (same analyst, same day)
- Intermediate precision (different analysts/days)
- Reproducibility (different laboratories)
- Statistical acceptance criteria
Trend analysis:
- Control chart monitoring
- Process capability assessment
- Out-of-specification investigations
- Continuous improvement initiatives
Quality Control and Compliance
ICH guidelines for analytical method validation
Key requirements:
- Method validation protocols
- Stability indicating capability
- Forced degradation studies
- Robustness evaluation
- Transfer to QC laboratories
Documentation:
- Analytical method validation reports
- Standard operating procedures
- Method transfer protocols
- Change control procedures
ICH guidelines for analytical method validation
Key requirements:
- Method validation protocols
- Stability indicating capability
- Forced degradation studies
- Robustness evaluation
- Transfer to QC laboratories
Documentation:
- Analytical method validation reports
- Standard operating procedures
- Method transfer protocols
- Change control procedures
Equipment qualification and performance verification
Qualification elements:
- Installation qualification (IQ)
- Operational qualification (OQ)
- Performance qualification (PQ)
- Periodic requalification
Performance monitoring:
- System suitability testing
- Calibration verification
- Preventive maintenance
- Performance trending
21 CFR Part 11 compliance for electronic records
Requirements:
- Audit trail maintenance
- Electronic signature controls
- Data backup and archival
- Access control measures
Best practices:
- Data review procedures
- Error correction protocols
- Investigation procedures
- Training documentation
Advanced Applications
Method Hyphenation
SEC-MALS
Size-exclusion chromatography with multi-angle light scattering
- Absolute molecular weight determination
- Polydispersity measurements
- Aggregation state confirmation
- Model-independent analysis
SEC-DLS
Size-exclusion chromatography with dynamic light scattering
- Hydrodynamic radius measurements
- Polydispersity assessment
- Real-time size distribution
- Aggregation monitoring
SEC-MS
Size-exclusion chromatography with mass spectrometry
- Exact molecular weight determination
- Modification identification
- Sequence confirmation
- Impurity characterization
2D-LC
Two-dimensional liquid chromatography
- Enhanced resolution
- Complex sample analysis
- Orthogonal separation mechanisms
- Comprehensive characterization
Specialized Applications
Integration with Other Technologies
HPLC-SEC data complements other analytical techniques:
- nanoDSF: Correlate thermal stability with aggregation propensity
- DLS: Confirm size measurements and polydispersity
- Binding assays: Assess impact of aggregation on binding activity
- Cell-based assays: Correlate purity with biological activity
HPLC-SEC may not detect all forms of aggregation, particularly large, insoluble aggregates that cannot enter the chromatographic system.
Troubleshooting Common Issues
Method Development Guidelines
Column Selection Strategy
Assess Protein Size Range
Determine the molecular weight range of your protein and expected impurities.
Size considerations:
- Main protein molecular weight
- Expected aggregate sizes (dimers, trimers, etc.)
- Potential fragment sizes
- Required resolution between species
Choose columns with appropriate separation range covering your protein size ±50% for optimal resolution.
Select Column Type
Choose column chemistry and particle size based on separation requirements.
Column options:
- TSKgel SuperSW3000: 10-500 kDa proteins
- TSKgel SuperSW4000: 20-1000 kDa proteins
- Superdex 200: 10-600 kDa proteins
- BioSuite UltraSEC: 5-1250 kDa proteins
For challenging separations, consider high-resolution or ultra-high performance columns.
Optimize Mobile Phase
Develop buffer conditions that maintain protein stability and achieve good separation.
Buffer optimization:
- pH: Match protein stability range (usually 6.5-7.5)
- Ionic strength: 50-300 mM salt
- Buffer system: Phosphate, Tris, or HEPES
- Additives: Consider glycerol, sucrose, or stabilizers
Test multiple buffer conditions and select based on peak shape, resolution, and protein stability.
System Suitability Requirements
Critical parameters:
- Resolution: Rs ≥ 1.5 between main peak and closest impurity
- Theoretical plates: N ≥ 2000 for main peak
- Peak symmetry: 0.8-1.5 for main peak
- Baseline stability: ±2% over run time
- Retention time precision: RSD ≤ 2% (n=6)
Validation Considerations
Specificity
Demonstrate method specificity
- Forced degradation studies
- Spiking studies with impurities
- Stress testing conditions
- Peak purity assessment
Linearity
Establish quantitative range
- Concentration vs. area response
- Impurity detection limits
- Working range definition
- Correlation coefficient >0.99
Precision
Assess method precision
- Repeatability (same day)
- Intermediate precision (different days)
- Acceptance criteria (RSD <5%)
- Statistical evaluation
Robustness
Test method robustness
- Flow rate variations (±10%)
- Temperature changes (±5°C)
- pH variations (±0.2 units)
- Mobile phase composition (±5%)
Best Practices
Sample Preparation
- Filtration: 0.22 μm filter to remove particulates
- Concentration: Optimize for good signal without overloading
- Storage: Minimize time between preparation and analysis
- Standards: Include molecular weight standards for calibration
Data Analysis
- Integration: Consistent integration parameters across samples
- Calibration: Regular molecular weight calibration verification
- Controls: Include system suitability and reference standards
- Documentation: Thorough record keeping of all parameters
Quality Control
- System suitability: Test before each analytical sequence
- Standard curves: Verify calibration regularly
- Blank injections: Monitor for carryover between samples
- Performance trending: Track key performance indicators over time
HPLC-SEC may not detect all forms of aggregation, particularly large, insoluble aggregates that cannot enter the chromatographic system. Consider complementary techniques for comprehensive aggregation assessment.
Regular method performance monitoring and system suitability testing ensure consistent, reliable results over time. Our analytical team provides comprehensive method development and validation support.