Enzymes for In-Depth Antibody Characterization
Therapeutic antibodies used as biopharmaceuticals are highly complex molecules requiring strict monitoring and control during manufacturing. Due to its nature as a biological drug, drug safety and innate variation in production methods is always a cause for concern. Thus, a series of workflows for comprehensive, in-depth analytical characterization should be applied to analyze and characterize product quality.
With the approval of monoclonal antibody products dramatically increasing each year such as mAbs, Fc-fusions, Fab, antibody-drug conjugates, bispecific mAbs (bsAbs) and bispecific T cell engagers (BiTE), understanding individual post-translational modifications (PTMs) can give a better insight and prediction as to the in vivo efficacy, pharmacokinetics, and mechanism of action of manufactured Abs.
Characterization of Abs is usually performed in several perspectives, starting from top-down analysis (overall structure) to bottom-up analysis (peptide sequencing), and finally, middle-level analysis (Fc, Fv, PTMs).
To enable antibody characterization methods, ACROBiosystems has developed a series of enzymes to assist with the characterization of antibodies and their related PTMs.
Product List
| Product Details | Cat. No. | Unit Definition |
|---|---|---|
| Endo H (500U/μl) | ENH-S5116 | One unit is defined as the amount of enzyme required to remove >95% of the carbohydrate from 10µg of denatured RNase B in 1 hour at 37°C in a total reaction volume of 10µl. |
| Endo S (200U/μl) | ENS-S5143 | One unit deglycosylates ≥ 95% of 1 μg human IgG, when incubated in 10 mM sodium phosphate, 150 mM NaCl, pH7.4 at 37°C for 30 min. |
| SpeB (40U/μl) | SPB-S5115 | One unit digests ≥ 95% of 1 µg human IgG1 when incubated in PBS ith 5 mM DTT or TCEP, pH 7.4 at 37°C for 1 hour. |
| IdeS (20U/μl) | IDS-S5143 | One unit digests ≥ 95% of 1 μg human IgG when incubated in 10 mM sodium phosphate, 137 mM NaCl, 2.7 mM KCl, pH7.4 at 7°C for 30min. |
Tools for Glycan Analysis
The structure and type of N-linked glycans can affect the immunogenicity, pharmacokinetics, and pharmacodynamics of therapeutic proteins such as monoclonal antibodies (mAbs) and Fc fusion proteins. Enzymes such as Endo H and Endo S provide type-specific cleavages of N-linked glycans for subsequent analysis.
Endo H (500U/μl) (Cat .No. ENH-S5116)
Application:
Endo H cleaves asparagine-linked (N-linked) hybrid or high mannose oligosaccharides but not complex oligosaccharides.
Cleavage of high mannose or hybrid N-glycans
Product Features:
Selective action: Does not cleave complex glycans
≥95% purity, as determined by SDS-PAGE
Optimal pH：5-6
Reaction time: 1h
≥95% purity, as determined by SDS-PAGE
Optimal pH：5-6
Reaction time: 1h
Endo S (200U/μl) (Cat .No. ENS-S5143)
Application:
Endo S preferentially cleaves complex oligosaccharides and avoids the cleavage of asparagine-linked (N-linked) hybrid or high mannose oligosaccharides.
Cleaves complex glycans, limited activity on high-mannose and hybrid-type glycans.
Product Features:
Highly efficient IgG-specific endoglycosidase
≥95% purity as determined by SDS-PAGE
Reaction time: 30 min
≥95% purity as determined by SDS-PAGE
Reaction time: 30 min
Antibody Subunit Fragmentation
SpeB (40U/μl) (Cat. No. SPB-S5115)
Application:
SpeB enzyme digests IgG to generate Fab and Fc fragments. Purified and homogenous Fab fragments can be used in many applications, including:
Studying the monovalent binding of Fab fragments to the antigen.
Elimination of Fc-mediated effector functions.
Reduced unspecific binding from Fc interactions.
Elimination of Fc-mediated effector functions.
Reduced unspecific binding from Fc interactions.
Product Features:
Cleavage site (Human IgG1): KTHT / CPPCPAP (above the hinge)
Reaction time: 60min
Reaction time: 60min
IdeS (20U/μl) (Cat. No. IDS-S5143)
Application:
IdeS protease specifically cleaves IgGs below the hinge region into F(ab´)2 and Fc fragments.
Product Features:
Cleavage site (Human IgG1): CPAPELLG / GPSVF (below the hinge)
Reaction time: 30 min
Reaction time: 30 min
FAQ
What types of tool enzymes are used in antibody characterization and biopharmaceutical research?
Tool enzymes are commonly used as analytical reagents to support antibody characterization, protein engineering, and molecular analysis. Different enzyme classes serve different research purposes, such as IgG fragmentation, glycan analysis, protein processing, or nucleic acid manipulation. Selecting the appropriate enzyme depends on the target molecule, analytical objective, and assay format.
How are IdeS and other proteases used for IgG antibody characterization?
Proteases such as IdeS and SpeB are used to generate antibody fragments for structural and functional analysis. IdeS specifically cleaves IgG at the hinge region, producing defined antibody fragments that support Fc/Fab characterization. SpeB provides broader IgG digestion capability and can be used when different fragmentation strategies are required.
What is the difference between Endo H and Endo S for antibody glycan analysis?
Endo H and Endo S are glycosidases with different substrate preferences for N-linked glycans. Endo H is commonly used for analysis of high-mannose and hybrid glycans, while Endo S shows activity toward IgG-associated complex glycans. Selecting between these enzymes depends on the glycan structure of interest and the intended characterization approach.
How can IgG fragmentation help reduce Fc-related interference in antibody assays?
IgG fragmentation can help separate antigen-binding regions from Fc-mediated effects during antibody analysis. Enzymes such as IdeS or SpeB can generate antibody fragments that support evaluation of Fab-related binding properties or structural characteristics. The appropriate fragmentation strategy depends on the assay objective and required antibody format.
What should be considered when selecting recombinant tool enzymes for antibody analysis?
Selection of recombinant tool enzymes depends on enzyme specificity, target molecule, required activity, and downstream detection method. For antibody characterization, factors such as cleavage preference, glycan specificity, and compatibility with the analytical workflow are important considerations. ACROBiosystems provides recombinant tool enzymes with product-specific characterization to support research applications.
What quality information is available for recombinant tool enzymes?
Quality evaluation of recombinant tool enzymes typically includes protein purity, molecular characterization, and functional activity assessment under reported assay conditions. Enzymatic activity data help researchers determine whether a product is suitable for their intended analytical application. ACROBiosystems provides characterization information for selected tool enzymes to support assay development and antibody analysis studies.
- Background
- Product List
- Tools for Glycan Analysis
- Antibody Subunit Fragmentation
- FAQ