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 with 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
Which enzymes are available for antibody characterization?
The collection includes Endo H and Endo S for glycan analysis, plus SpeB and IdeS for generating defined antibody subunits.
How do Endo H and Endo S differ in glycan specificity?
Endo H cleaves high-mannose and hybrid N-glycans but not complex N-glycans, while Endo S preferentially cleaves complex glycans on IgG and has limited activity on high-mannose or hybrid forms.
What are the key differences between SpeB and IdeS for antibody fragmentation?
SpeB and IdeS generate different antibody fragments due to their distinct cleavage sites. For human IgG1, SpeB cleaves above the hinge to produce Fab and Fc fragments, enabling analysis of monovalent antigen binding without Fc-mediated interactions. IdeS cleaves below the hinge to generate F(ab')2 and Fc fragments, supporting subunit-level structural characterization. ACROBiosystems offers both enzymes with demonstrated IgG digestion activity.
How long are the listed antibody-digestion workflows?
The displayed examples use about 60 minutes for SpeB and about 30 minutes for IdeS. Follow the enzyme-specific protocol for antibody amount and reaction conditions.
How can antibody fragmentation help distinguish antigen-binding properties from Fc-mediated effects?
Intact IgG antibodies can exhibit avidity effects and Fc-dependent interactions that complicate binding analysis. Generating Fab fragments allows monovalent antigen recognition to be evaluated separately from these contributions. ACROBiosystems offers SpeB for producing Fab and Fc fragments from IgG, supporting binding characterization where removal of Fc-related interactions is required.
What enzyme activity and quality information should be considered when selecting reagents for antibody characterization?
Enzyme selection should consider substrate specificity, defined activity, digestion efficiency, and compatibility with the intended antibody format. ACROBiosystems provides product-specific activity definitions and validated reaction examples for Endo H, Endo S, SpeB, and IdeS. These data help researchers assess reagent suitability and establish consistent glycan analysis or antibody fragmentation workflows.
- Background
- Product List
- Tools for Glycan Analysis
- Antibody Subunit Fragmentation
- FAQ