€2,299.00
FabRICATOR-HPLC offers on-column digestion of mAbs for automated subunit generation
FabRICATOR-HPLC contains the FabRICATOR (IdeS) enzyme immobilized on HPLC compatible resin for robust on-column digestion in automated workflows. FabRICATOR-HPLC provides digestion of human IgG and Fc-fusion proteins at one single amino acid position below the hinge generating Fc and F(ab’)2 fragments.
| Application | |
|---|---|
| Product Group | |
| Source/Origin | Cloned from S. pyogenes, Produced in E. coli (His-tagged), 37.7 kDa |
| Product Composition | |
| Digestion Site | |
| Specificity | |
| Substrates | Human IgG1, Human IgG2, Human IgG4, Monkey IgG, Rabbit IgG, Rat IgG, Sheep IgG |
| Reaction Conditions | Operating Conditions: Operating pH 6.5 – 8.0, Operating Temperature 37° C, Maximal Pressure 100 bar, Typical flow rate 0.025-0.05 mL/min., Column Performance: Number of days of continuous operation: >10*, Injections per column: >200*. * Depending on specific application. |
| Digestion Buffers | |
| Buffer Compatibility | 150 mM ammonium acetate, pH 7 (ionic stregth of >150 mM is necessary). Optimization may be required. |
| Inhibitors | |
| Unit Definition | ≥ 95% digestion of 10 μg hIgG1 in less than 5 minutes residence time at 37°C as monitored by RP-LC |
We have tested concentrations as low 0.025 mg/ml without any measurable effect on digestion efficiency. However, low concentration samples require a longer injection time and consequently lead to a broader elution peak from the FabRICATOR HPLC column.
Yes, it is possible to digest the antibody in the first dimension and then separate the subunits in the second dimension. Please see the application note for detailed instructions.
FabRICATOR is a cysteine protease. It should not result in any other modifications than the digestion of the peptide bond. Digestion will be between the two Glycines (…CPAPELLG / GPSVF…) and the C-terminus will regain the carboxyl group – that is addition of one OH-group and the N-terminal amino group will be regained (addition of one H).
In summary, R1-CO-NH-R2 + H2O –> R1-COOH + NH2-R2.
The FabRICATOR HPLC can run >400 samples of 10 ug of IgG.
FabRICATOR HPLC is an HPLC column with the FabRICATOR (IdeS) protease immobilized on a HPLC compatible matrix for automated antibody subunit generation.
We have tested PBS and 150 mM ammonium acetate, pH7 with good results. More buffers were found to be compatible with FabRICATOR digestion in solution (more info) and should be suitable for FabRICATOR HPLC as well. However, the ionic strength of the buffer influences retention of the antibody fragments on the FabRICATOR HPLC column and a minimum of 100-150 mM salt is necessary for IgG1 subunits to elute as a single, narrow peak. Other IgG subclasses or fusion proteins might need some optimization of buffer composition.
