Antibody Characterization Tools
Antibody Characterization Tools
Antibodies are structurally complex, highly heterogeneous macromolecules that are sensitive to external conditions and inherently less stable. Because of this complexity, therapeutic antibody R&D requires a comprehensive, precise, and highly robust set of bioanalytical tools to support:
♦︎ Antibody characterization
♦︎ Monoclonal antibody (mAb) purification
♦︎ Peptide mapping analysis
♦︎ Glycosylation analysis
♦︎ Post-translational modification (PTM) analysis
♦︎ Assessment of monoclonal antibody internalization
Across all stages of large-molecule drug development, protein analysis and identification are critical. Aladdin provides a validated portfolio of antibody characterization tools covering antibody purification, peptide mapping, glycosylation, and PTM analysis. These tools can also be used to detect process residuals during manufacturing, helping accelerate end-to-end progress from research to development.
Antibody Purification Magnetic Beads — Protein G Beads, Protein A Beads
Protein G and Protein A magnetic beads are highly specific, high-capacity magnetic affinity media that enable convenient purification of antibodies from samples such as cell culture supernatants, ascites, and serum. Whether for single-sample handling, parallel multi-sample workflows, or high-throughput automation, these beads deliver stable and efficient antibody purification.
Product Features:
♦︎ High-density covalent coupling: Dense surface immobilization of Protein A/G for strong binding capacity and excellent stability.
♦︎ Lower usage: More binding sites mean fewer beads are needed to achieve the same results.
♦︎ Low non-specific adsorption: Low background for cleaner purification and higher recovery.
Specification | Protein G Magnetic Beads | UltraBio™ Protein G Magnetic Agarose Beads | UltraBio™ Protein G Plus Magnetic Beads | rProtein G Magnetic Beads |
Catalog No. | ||||
Matrix | Silica-based magnetic beads | Agarose magnetic beads | Nanoscale amino-functional magnetic beads | Magnetic agarose microspheres |
Ligand | Recombinant Protein G | Protein G | Recombinant Protein G | Recombinant Protein G |
Particle size | 200 nm | 50 μm (20–80 μm) | 200 nm | 20–80 μm |
Bead concentration | 10 mg/mL | 10% v/v | 10 mg/mL | 380–580 units/mg |
Binding capacity | ≥0.85 mg hIgG/mL | 30–40 mg hIgG/mL | ≥0.9 mg hIgG/mL | ≥50 mg hIgG/mL |
Applications | IP, Co-IP, ChIP, RIP | |||
Storage / Shelf life | Stable for 2 years at 2–8 °C | 2–8 °C (20% ethanol) | Store at 4 °C; 2-year shelf life | 2–8 °C (20% ethanol) |
Specification | Protein A Magnetic Beads | UltraBio™ Alkali-Tolerant Protein A Magnetic Agarose Beads | Protein A/G Magnetic Beads | UltraBio™ Protein A/G Magrose Beads |
Catalog No. | ||||
Matrix | Silica-based magnetic beads | Agarose magnetic beads | Silica-based magnetic beads | Agarose magnetic beads |
Ligand | Recombinant Protein A | Recombinant Protein A | Recombinant Protein A/G | Protein A/G |
Particle size | 200 nm | 50 μm (20–80 μm) | 200 nm | 10–37 μm |
Bead concentration | 10 mg/mL | 10% v/v | 10 mg/mL | 20% v/v |
Binding capacity | ≥0.9 mg hIgG/mL | 30–40 mg hIgG/mL | ≥0.7 mg hIgG/mL | 35–45 mg hIgG/mL |
Applications | IP, Co-IP, ChIP, RIP | |||
Storage / Shelf life | Stable for 2 years at 2–8 °C | 2–8 °C (20% ethanol) | Stable for 2 years at 2–8 °C | 2–8 °C (20% ethanol) |
Applications:
♦︎ Antibody purification: Capture IgG from serum/supernatant, wash, and elute.
♦︎ Immunoprecipitation (IP/Co-IP/ChIP/RIP): Use an antibody to pull down the target protein/complex/chromatin/RNA for downstream analyses.
Brief Workflow:
Immunoglobulin IgG-Degrading Enzymes — IdeS and IdeZ
Recombinant Immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS) and Immunoglobulin-degrading enzyme Z (IdeZ) are expressed in E. coli and purified. They specifically recognize IgG and cleave at a defined site below the IgG hinge region, yielding intact F(ab’)₂ fragments and Fc fragments.
Product Features:
♦︎ Strict substrate specificity: Recognizes IgG only.
♦︎ Broad species compatibility: Recognizes IgG from mouse, rabbit, monkey, sheep, and chimeric human–animal antibodies.
Product Name | Recombinant IdeS Protease (MS Grade) | Recombinant IdeZ Protease (MS Grade) |
Catalog No. | ||
Form | Liquid | Liquid |
Origin | Streptococcus pyogenes | Streptococcus equi |
Cleavage site | Specific site below the IgG hinge region | Specific site below the IgG hinge region |
Digestion time | Incubate at 37 °C for 0.5–1 h | Incubate at 37 °C for 0.5–2 h |
Applications:
♦︎ LC/MS-based characterization of the physicochemical and structural attributes of therapeutic antibodies.
♦︎ Preclinical AAV studies: Literature and practice indicate IdeS can be administered in animal models prior to gene therapy dosing to remove pre-existing anti-AAV neutralizing antibodies, thereby reducing interference from pre-existing antibodies.
Cleavage Sites:
Differences in IdeS and IdeZ protease activity across human and mouse IgG subclasses. Cleavage sites are indicated in bold.
Human
Subclass | Hinge/CH2 Sequence | IdeS Activity | IdeZ Activity |
IgG1 | CPPCPAPELLGGPSVF | +++ | +++ |
IgG2 | CPPCPAPP_VAGPSVF | +++ | +++ |
IgG3 | CPRCPAPELLGGPSVF | +++ | +++ |
IgG4 | AHHAQAPEFLGGPSVF | +++ | +++ |
Mouse
Subclass | Hinge/CH2 Sequence | IdeS Activity | IdeZ Activity |
IgG1 | PCICTVPEV_SSVF | – | – |
IgG2a | CPPCAAPNLLGGPSVF | + | +++ |
IgG2b | CHKCPAPNLEGGPSVF | – | – |
IgG3 | GSSCPAGNILGGPSVF | + | +++ |
Mass Spectrometry–Grade Recombinant Trypsin
Compared with routine protein analysis, peptide mapping of antibody proteins imposes higher requirements on the protease’s purity, specificity, and cleavage efficiency. Aladdin’s mass spectrometry–grade recombinant trypsin offers higher site specificity and cleavage efficiency, reduced self-digestion, and improved digestion at hard-to-cleave sites—delivering more precise and reliable support for the identification and characterization of biotherapeutic proteins.
Key Features:
♦︎ High specificity: Specifically cleaves peptide bonds at the C-terminus of Lys (K) and Arg (R). The resulting peptides are of suitable size and readily ionize in positive mode, enabling MS and proteomics characterization.
♦︎ High purity & stability: Recombinant expression in Pichia pastoris with high-efficiency chromatographic purification; methylation suppresses autolysis, yielding low background and more stable activity.
♦︎ Animal-component–free: Recombinant production with no animal-derived materials used during manufacturing; free from adventitious viral contamination.
Item | Description |
Product Name | Recombinant Trypsin (MS Grade) |
Catalog No. | |
Physical Form | Lyophilized protein |
Molecular Weight | 23 kDa |
Storage | Store powder at −20 °C |
Reconstitution | 1 mM HCl or 50 mM acetic acid |
Digestion Buffer | 50 mM ammonium bicarbonate, or 50 mM Tris-HCl at pH 8.5 |
Activity | ≥ 13,000 U/mg protein |
pH Range | 8–9 |
Stability / Reagent Tolerance (max.) | 1% SDC, 0.1% SDS, 2 M urea, 20% acetonitrile |
Cleavage Specificity | Specific hydrolysis at the C-terminal peptide bond of Lys (K) and Arg (R) |
Item | Description |
Product Name | Rapid-Trypsin (MS) |
Catalog No. | |
Physical Form | Lyophilized |
Molecular Weight | 23.5 kDa |
Storage | Long-term storage at −20 °C (up to 2 years). After reconstitution, aliquot to avoid repeated freeze–thaw cycles. |
Reconstitution | 50 mM acetic acid |
Activity | ≥ 3,800 USP U/mg protein |
Cleavage Specificity | Specific cleavage at the C-terminus of Lys (K) and Arg (R) |
Figure 1. Results indicate that Rapid Trypsin completes digestion within 3 h.
Applications:
♦︎ Suitable for MS-based identification and characterization of antibodies and routine proteins.
♦︎Compatible with LC/MS and RP-HPLC-UV platforms.
Alternative Protease — rAsp-N, Mass Spec Grade
rAsp-N (Mass Spec Grade) is a high-activity recombinant protease well suited for proteomics studies of complex mixtures and for peptide mapping of purified proteins (e.g., therapeutic monoclonal antibodies). The enzyme excels at resolving protein composition and structure in complex samples, efficiently and reproducibly hydrolyzing proteins into peptides of appropriate length, thereby significantly improving the accuracy and reliability of downstream mass spectrometric analysis.
Key Features:
♦︎ Higher cost-effectiveness vs. native Asp-N.
♦︎ Larger size than native Asp-N, providing higher consistency during preparation/manufacturing.
♦︎ Use cases: Proteomics analysis of complex mixtures and peptide mapping of purified proteins (such as antibodies).
Item | Description |
Product Name | Endoproteinase Asp-N |
Catalog No. | |
Form | Lyophilized powder |
Source | Pseudomonas fragi |
Specificity / Principle | Specific cleavage at the N-terminus of Aspartic acid (D) and Cysteine (C) residues |
Activity | ≥ 1,800 U/mg protein |
Predicted Molecular Weight | 22.4 kDa |
Enzyme-to-Protein Ratio (w/w) | 1:10–1:50 |
Digestion pH | 7.5–8.5 |
Digestion Buffers | 20 mM Tris; or 50 mM ammonium bicarbonate (ABC); or HEPES buffer |
Alternative Protease — rLys-C, Mass Spec Grade
rLys-C (Mass Spec Grade) is a recombinant Lys-C expressed in E. coli, with performance equivalent to the native enzyme. It specifically cleaves at the C-terminus of lysine residues. The enzyme retains hydrolytic activity even under strongly denaturing conditions such as 6 M urea, markedly improving digestion efficiency for difficult or resistant proteins. rLys-C shows optimal activity at pH 8–9.
Key Features:
♦︎ Equivalent activity: Recombinant lysyl endopeptidase (Lys-C) expressed in E. coli delivers activity comparable to the native enzyme from Achromobacter.
♦︎ High specificity, no contaminating proteases: Free of Lys-N, Arg-C, and other adventitious proteases; highly specific for the post-Lys site, with complete cleavage even at DK/PK motifs.
♦︎ High purity & MS-ready: Methylation suppresses autolysis. High purity and high activity meet mass-spec–grade requirements for protein characterization and sample preparation.
Item | Description |
Product Name | Lysyl Endopeptidase (MS) |
Catalog No. | |
Physical Form | Lyophilized protein, with 3 mg trehalose per vial |
Molecular Weight | 27 kDa |
Storage | Store powder at −20 °C. For long-term storage, reconstitute, aliquot, and freeze at −20 °C; avoid repeated freeze–thaw cycles. |
Reconstitution | 1 mM HCl or 50 mM acetic acid |
Digestion Buffer | 50 mM ammonium bicarbonate (ABC), or 50 mM Tris-HCl at pH 8.5 |
Optimal pH Range | pH 8–9 (maximal Lys-C activity) |
Stability (tolerance, max.) | 0.2% SDS; 1.0% Triton; 6 M urea; 20% acetonitrile |
Cleavage Specificity | Lys-C specifically hydrolyzes at Lys (K) sites (C-terminal to Lys); difficult sites after denaturation can also be cleaved. |
Figure 2. LC–MS molecular-weight spectrum.
Glycosylation Analysis — PNGase F & Endo H
Glycosylation broadly affects protein folding, stability, secretion/trafficking, activity, and immune recognition, and is a key post-translational modification regulating protein function and biological processes. Two N-glycosidases are commonly used in experiments:PNGase F cleaves at the Asn–GlcNAc linkage, removing virtually all classes of N-glycans (except those with core α1,3-fucose) and releasing the intact glycan.Endo H endoglycosidase specific for high-mannose and immature hybrid-type N-glycans; it cleaves within the chitobiose core, leaving one GlcNAc on the peptide, and is ineffective on mature complex-type N-glycans.Used together, the enzymes rapidly reveal glycan maturation state and localize N-glycosylation sites.
Key Features:
♦︎ High purity: No contaminating proteases or other glycosidases; purity ≥ 95%.
♦︎ Lot-to-lot consistency: Strict QC; every lot verified for activity and function.
♦︎ Broad workflow compatibility: Glycerol-free; digestion systems are compatible with HPLC and mass spectrometry workflows.
Item | PNGase F (Glycerol-free) (MS) | Recombinant Endo H (MS Grade) |
Catalog No. | ||
Form | Liquid | Lyophilized |
Source | Cloned from Elizabethkingia miricola | Cloned from Streptomyces plicatus |
Type | Recombinant glycosidase | Recombinant glycosidase |
Cleavage | Cleaves between the innermost N-acetylglucosamine (GlcNAc) and the asparagine residue of nearly all N-linked glycans (high-mannose, hybrid, complex), except those bearing core α1,3-fucose. | Cleaves within the chitobiose core of high-mannose and certain hybrid N-glycans from N-linked glycoproteins; cuts between the two GlcNAc residues of the core, yielding a glycoprotein that retains one GlcNAc. Ineffective on mature complex-type glycans. |
Applications | In-vitro removal of N-glycans from antibodies, Ig-fusion proteins, and other glycoproteins; structural characterization of released glycans and deglycosylated proteins. | Assess glycosylation state, characterize glycan structures, and selectively remove high-mannose/early hybrid N-glycans from proteins. |
Figure 3. Schematic of PNGase F cleavage mechanism
Figure 4. Schematic of Endo H cleavage mechanism
Aladdin：https://www.aladdinsci.com/