- SDS-PAGE & Western Blot (WB) — Run reducing and non-reducing gels to reveal heavy- and light-chain integrity, interchain disulfide status, and high-molecular-weight species. WB with isotype- or epitope-specific probes confirms identity of bands and detects low-abundance proteolytic fragments that might be invisible on Coomassie stain. Non-reducing vs reducing comparison pinpoints disulfide disruption or heavy–light chain dissociation.
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Mass Spectrometry (MS) — MS is used at two levels: intact mass analysis and peptide-level mapping.
Intact MS (native or denaturing) provides a mass envelope for the whole antibody (or Fc/ Fab subunits after enzymatic cleavage), resolving glycoform distributions and macro-heterogeneity. Deconvoluted mass spectra highlight clipping, adducts, and predominant glycoforms.
Peptide mapping (LC-MS/MS) following specific protease digestion delivers sequence coverage, localizes modifications (oxidation, deamidation), and confirms cleavage sites. Disulfide mapping workflows establish native disulfide pairing. - Mass Photometry (MP) — Mass photometry measures molecular mass distributions of single particles in solution without labeling. MP detects monomer vs oligomer populations and low-level subpopulations (e.g., dimers, trimers) with excellent sensitivity at native conditions. MP is particularly useful to detect early aggregation species and to quantify the fraction of intact monomer in heterogeneous preparations.
Antibody Integrity Evaluation Service
Introduction
Antibody integrity addresses whether the molecule present in a preparation corresponds to the intended primary sequence, correct chain pairing and disulfide topology, intact post-translational modification (PTM) profile, and preservation of functional paratope structure. Loss of integrity — through clipping, mispaired disulfides, truncation, unexpected PTMs (oxidation, deamidation), or partial denaturation — can reduce binding activity, change pharmacology in model systems, and confound downstream assays. Robust integrity assessment therefore combines orthogonal molecular and functional readouts that detect sequence-level variance, mass heterogeneity, sub-population structure and retained antigen binding.
The Antibody Integrity Evaluation Service provides integrated, multi-platform characterization to identify and quantify molecular variants and to relate those variants to functional competence. The workflow centers on structural (MS, SDS-PAGE/WB, MP) and functional (BLI) assays, supplemented as needed by peptide mapping, intact mass deconvolution, and complementary biophysical methods.
Strategies
Orthogonal structural characterization
Functional correlation
- Biolayer Interferometry (BLI) — BLI provides kinetic and equilibrium binding data (kon, koff, KD) in a label-free format. Comparing BLI sensograms across stress conditions or fractions enables direct correlation between structural variants and loss of antigen binding. BLI can differentiate variants that retain mass/sequence but have impaired paratope conformation.
Integrated interpretation and decision logic
- Combine structural and functional outputs: (a) if intact MS shows clipping and BLI shows reduced binding, clipping likely affects the paratope; (b) if SDS-PAGE shows fragments but MS indicates full sequence for the majority, fragments may reflect minor proteolysis not impacting function; (c) MP quantifies how much monomer remains even when SEC shows broadened peaks. Recommendations include formulation changes, additional polishing steps, or targeted mutagenesis if production-derived degradation is recurrent.
Required Materials
To perform a thorough integrity evaluation, provide the following:
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Antibody material
- Recommended concentration: 0.5–5 mg/mL for full-length IgG (adjust for fragments).
- Minimum volume: 200–500 µL per antibody to permit MS, MP, SDS-PAGE/WB, and BLI replicates. Smaller volumes can be accommodated for focused testing but limit breadth.
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Buffer/excipient information
- Full composition (salts, sugars, surfactants, preservatives), pH, and any prior processing (concentration, diafiltration, lyophilization).
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Intended antigen
- Purified antigen or peptide for BLI binding assays (immobilization strategy will be discussed).
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Reference material (if available)
- A characterized reference batch or sequence file (FASTA) expedites MS database searching and confidence in identifications.
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Project brief
- Expected critical quality attributes, known issues, or prior analytical results.
Samples should be shipped cold with clear labeling. Indicate any biosafety considerations.
FAQs
How is MS used to detect clipping or truncation?
Intact MS yields deconvoluted mass peaks; differences from expected molecular weight indicate truncation or adducts. Peptide mapping localizes the cleavage site by identifying truncated peptides and missing sequence coverage.
What resolution does Mass Photometry offer for aggregates?
MP resolves molecular species by mass at single-molecule sensitivity in native buffer. It can quantify monomer vs small oligomer populations (dimer, trimer) that may be underestimated by ensemble methods.
When is BLI preferable to SPR?
BLI offers rapid, parallel measurements suitable for comparative integrity testing and is tolerant of crude sample matrices. Both platforms are complementary; choice depends on throughput and available antigen immobilization formats.
Can SDS-PAGE & WB detect low-level variants?
Coomassie staining detects major species; WB increases sensitivity for specific epitope-containing fragments. Very low-abundance variants are better identified by MS.
What deliverables are provided?
A comprehensive report including deconvoluted mass spectra, peptide mapping coverage maps, gel/WB images, MP histograms, BLI sensograms with kinetic fits, interpretation, and recommended next steps. Raw data files are included.
Contact Us
For project scoping, sample submission instructions, and customized integrity panels, contact our technical team. Provide antibody identity (sequence or accession if available), target antigen, and any prior characterization data. A technical consultation will define the optimal combination of MS, SDS-PAGE & WB, MP and BLI assays to meet the integrity objectives and to provide clear, actionable conclusions that support purification optimization or downstream use.
For research use only. Not intended for any clinical use.
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