Biophysical characterization is essential decoding the complexity of biopharmaceuticals. In this post, from Venture Center’s Only One Analytical Technique series, we explore how these tools provide insights into the structure, stability, and function of biologics such as monoclonal antibodies, fusion proteins, and ADCs.
From the "Only One Analytical Technique" talk series by Venture Center. Watch the full video here -
https://www.youtube.com/watch?v=9CBhB-VdHyc
Why Biophysical Characterization Matters
Biological drugs, particularly monoclonal antibodies, are structurally large and complex. Unlike small molecules (simple and stable), biologics can be structurally diverse, heterogeneous, and sensitive to manufacturing conditions. To ensure product quality, safety, and efficacy, comprehensive characterization is critical. This is where biophysical tools come in.
Classes of Therapeutics and Growing Complexity
- Small Molecules : Simple chemical structures, like aspirin.
- Complex Small Molecules : Includes peptides and nucleotides.
- Biologics : mAbs, ADCs, fusion proteins, bispecific/multispecific antibodies, and now
even cell therapies.
With advancements in antibody engineering and payload conjugation, the complexity of biologics has grown exponentially. Multi-payload, multi-specific formats demand equally robust analytical approaches.
Regulatory Expectations and CQAs
Regulatory guidelines (e.g., ICH Q6B) demand detailed analysis of Critical Quality Attributes (CQAs), such as :
- Aggregation
- Glycosylation and glycan variants
- Charge heterogeneity
- Truncations and fragmentation
- Post-translational modifications (PTMs)
Failure to assess these can affect immunogenicity, potency, and pharmacokinetics.
Key Analytical Objectives for Biologics
- Primary structure : Sequence confirmation and PTM detection
- Higher order structure : Secondary and tertiary structural integrity
- Aggregation profile : Detection of dimers, trimers, and high molecular weight species
- Charge variants : Due to deamidation, glycation, etc.
- Glycan profiling: Intact, subunit, peptide, and released levels
Techniques and Workflows Used
Spectroscopic Methods
- CD (Circular Dichroism)
- FTIR
- UV-Vis
- Fluorescence spectroscopy
Used to study secondary and tertiary structure, thermal unfolding, and binding interactions.
Chromatographic Methods
- Size exclusion chromatography (SEC)
- Ion exchange (IEX)
- Hydrophobic interaction chromatography (HIC)
Ideal for size variants, charge variants, and hydrophobicity mapping.
Mass Spectrometry (MS)
- Intact mass analysis
- Subunit and peptide mapping
- Glycan and PTM identification
Mass spectrometry enables multi-attribute monitoring (MAM), allowing simultaneous tracking
of multiple CQAs.
Light Scattering and Aggregation Tools
- Dynamic light scattering (DLS)
- Multi-angle light scattering (MALS)
- Analytical ultracentrifugation
Used to detect and quantify aggregation states.
Capillary Electrophoresis (CE)
- Particularly useful for high-resolution separation of charge variants.
- CBA has recently added CE to its toolkit, expanding its advanced capabilities.
Differential Scanning Calorimetry (DSC)
- Measures thermal stability by tracking melting temperature.
Native vs Denaturing MS
- Native MS : Retains non-covalent structures; ideal for detecting aggregates and complexes.
- Denaturing MS : Breaks down into subunits or peptides for detailed sequencing and PTM analysis.
Automation and 2D-LC-MS Workflows
2D liquid chromatography setups automate fraction collection, desalting, and injection into MS. This eliminates manual desalt steps, improves throughput, and enhances reproducibility.
Multi-Attribute Methods (MAM)
Mass spectrometry is increasingly replacing conventional HPLC assays by monitoring multiple attributes in a single run. Advantages:
- Reduces equipment footprint
- Consolidates workflows
- Enhances specificity and data richness
Glycan Analysis Approaches
- Intact and subunit level : Detected via MS
- Peptide level (glycopeptides) : Localizes glycosylation sites
- Released glycan analysis : Fluorescence-based and MS-based
Kits with pre-optimized workflows accelerate sample prep, labeling, and detection.
Aggregation Profiling Techniques
- SEC with MALS: Quantifies aggregates with absolute molecular weight.
- Fluorescence-based aggregation assays: High-throughput, microplate compatible, label-based.
Real-Time Monitoring and PAT Integration
Emerging Process Analytical Technology (PAT) tools integrate analytical methods into manufacturing.
- Real-time sample analysis from upstream or downstream processes
- Enables immediate decision-making during production
CBA’s Role in Biophysical Characterization
As part of Venture Center’s offerings, the Center for BioPharma Analysis (CBA) provides:
- GLP-compliant, NABL-accredited facility
- Integrated structural and functional analysis under one roof
- High-end tools like HRMS, DSC, CE, SEC-MALS, FTIR, fluorescence spectroscopy,
and more - End-to-end advisory support for bio/pharma innovators
CBA empowers industry, startups and researchers in India with advanced tools and expertise to accelerate biologics development and regulatory readiness.
Contact us at cba@venturecenter.co.in to discuss your biophysical characterization
needs.
Learn more at - https://bioanalysis.in/