What is the FabRICATOR-HPLC?

FabRICATOR-HPLC is an HPLC column with the FabRICATOR (IdeS) protease immobilized on a HPLC compatible matrix for automated antibody subunit generation.

Which digestion buffers are compatible with FabRICATOR-HPLC?

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.

Can I use FabRICATOR-HPLC in a 2D-LC setting?

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.

Can I process diluted IgG samples using FabRICATOR-HPLC?

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.

How many samples can I run through a column?

The FabRICATOR-HPLC can run >400 samples of 10 ug of IgG.

Does FabRICATOR introduce modifications?

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.