Genovis / SmartEnzymes / Antibody Digestion / FabRICATOR / FabRICATOR HPLC
SmartEnzymes™


Immobilized enzyme for on-column digestion of IgG below the hinge. FabRICATOR (IdeS) is an IgG-specific cysteine protease that digests antibodies at a single amino acid site below the hinge, generating homogenous F(ab’)2 and Fc fragments.

After digestion, the fragments can be analyzed using reversed-phase HPLC, mass spectrometry or other analytical method. Automation of antibody digestion and subunit generation enables applications in online monitoring of bioreactors, for example as part of a 2D-LC-MS setup, coupling the reactor directly to the MS. This automated approach of antibody subunit analysis reduces operator time, sample handling and increases the throughput.

 

  • Automated and consistent middle-level workflows
  • Robust on-column enzyme performance
  • Easy to integrate into multidimensional LC setups

Available Products

FabRICATOR HPLC 2.1 x 50 mmImmobilized enzyme for on-column digestion of IgG below the hingeA0-FRC-050€2,299.00Buy / Request a Quote

For information on how to order, visit Place an Order or contact us directly at order@genovis.com.

Storage

When not in use, store the column at +4-8°C in 10 mM sodium phosphate, 140 mM NaCl, pH 7.4. Store the column with the endcaps in place, carefully sealed to prevent drying. For long-term storage of the column, 0.02% (w/v) sodium azide can be added as a preservative.

Product Overview

Column dimensions: 2.1 mmD x 50 mmL
Support resin: POROS®*
Typical flow rate: 0.025-0.05 mL/min
Maximum Pressure: 100 bar
Operating pH: 6.5 - 8.0
Operating temperature: 37°C
Storage conditions: +4-8°C
Number of days of continuous operation: >10 **
Injections per column: >200 **
Start material: Human IgG1-4, Fc-fusion proteins
 
** Depending on specific application

Performance

The performance of the FabRICATOR HPLC column at 37°C is typically >200 injections with a digestion efficiency of >95% for 10 µg human IgG1 (1 mg/ml) sample in PBS, pH 7.4.

In-house performance testing* of the FabRICATOR HPLC column highlights the stability and reproducibility of this product. The column delivered consistent digestion for 14 days at 37°C. More than 450 injections of mAb were made during testing and no carry-over was observed. Reproducible Fc glycan profile analysis was obtained showing the column to be suitable for online process monitoring. Modified Fc glycans on a therapeutic mAb were also detected using FabRICATOR HPLC for digestion followed by LC-MS.

* Tested with trastuzumab in PBS, injections every 40 minutes, at a flow rate of 25 µl/min, samples collected, reduced and analyzed by RP-HPLC.

Instructions

Product Specifications

Safety Data Sheet

Certificate of Analysis

FAQ and Support

Popular FAQ

b

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.

b

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.

b

How many samples can I run through a column of FabRICATOR HPLC?

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

b

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.

b

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.

 

Find all FAQ here

Support Enzymes for Biopharmaceuticals

Support

Contact our enzyme experts for support and technical questions.

* POROS® included in FabRICATOR® HPLC.
This product is provided under an intellectual property license from Life Technologies Corporation. The transfer of this product is conditioned on the buyer using the purchased product solely in research or for analytical testing during manufacturing, all conducted by the buyer, excluding contract research or any fee for service research, and the buyer must not (1) use this product or its components for (a) diagnostic, therapeutic or prophylactic purposes; (b) testing, analysis or screening services, or information in return for compensation on a per-test basis; and/or (c) sell or transfer this product or its components for resale, whether or not resold for use in research. For information on purchasing a license to this product for purposes other than as described above, contact Thermo Fisher Scientific, 5826 Newton Drive, Carlsbad, CA 92008 USA or outlicensing@thermofisher.com. The trademark POROS® trademark is the property of Life Technologies Corporation.

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