Automated parallel analysis of antibodies

FabRICATOR® MagIC enables the popular middle-level LC-MS workflows in a magnetic format for automated parallel analysis of antibodies and other IgG-based biopharmaceuticals. FabRICATOR MagIC contains the IgG-specific FabRICATOR (IdeS) enzyme immobilized on magnetic agarose beads for digestion of antibodies into F(ab’)2 and Fc/2 subunits in under 20 minutes with minimal hands-on time. The workflow using FabRICATOR MagIC can be used to study oxidation, glycosylation and other critical quality attributes of therapeutic antibodies in development and manufacturing.

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FabRICATOR immobilized on magnetic agarose beads

  • Automated workflow for middle level analysis
  • Compatible with automation platforms
  • High throughput parallel analysis of antibodies
  • Complete digestion in 10-20 minutes

Fast parallel sample preparation with minimal hands-on time

Therapeutic antibodies and other IgG-based biopharmaceuticals require careful characterization and monitoring of critical quality attributes such as oxidation, glycosylation and other post-translational modifications. The numerous samples associated with clone selection, process development and stability studies of antibodies are typically analyzed by liquid chromatography mass spectrometry (LC-MS). Sample preparation on a sufficient scale prior to LC-MS is however often challenging. To address the sample preparation challenge, we immobilized our favorite cysteine protease FabRICATOR (IdeS) on magnetic agarose beads, now called FabRICATOR MagIC. Immobilized on magnetic beads, FabRICATOR provides fast automated digestion of multiple antibodies in parallel, facilitating middle-level characterization workflows. Figure 1 highlights how FabRICATOR MagIC can reduce the experimental and operational time with minimal user interactions, reducing the risk of sample handling errors, while increasing the throughput.

Figure 1: Comparison of middle-level and bottom-up sample preparation approaches. Here we compare the time savings and reduced user interaction for parallel automated subunit generation using FabRICATOR MagIC to that of manual subunit generation using lyophilized FabRICATOR and peptide mapping using trypsin.

Simple one-pot reaction to generate reduced subunits

FabRICATOR MagIC rapidly produces F(ab’)2 and Fc/2 subunits without any risk of overdigestion. Subsequent reduction of the disulfide bonds leads to the separation of the F(ab’)2 into the smaller LC and Fd’ subunits, allowing for a more detailed analysis of the resulting subunits. This can be achieved in a one-pot reaction using FabRICATOR MagIC and 5 mM TCEP. Reverse phase LC-MS analysis of the resulting fragments demonstrates both complete digestion and complete reduction of the disulfide bonds, Figure 2.

Figure 2: Subunit generation using FabRICATOR MagIC. A mAb sample was analyzed by reverse phase LC-MS at the intact level (upper panel), after digestion with FabRICATOR MagIC (middle panel) or after digestion with FabRICATOR MagIC in the presence of 5 mM TCEP (lower panel).

Flexible and adaptable for automation platforms

To make rapid automated digestion of multiple antibodies in parallel available to all labs, FabRICATOR MagIC is designed to be flexible and adaptable to a range of automation platforms. We have optimized a workflow for FabRICATOR MagIC on the Thermo Scientific™ KingFisher™ Purification System but it can be transferred to other automation platforms and will perform equally well on a shaking table if automation is not available. After digestion, the deep well plate containing the processed samples can be directly transferred to the autosampler of the LC-MS for fast chromatography or to other analytical methods for analysis.

FabRICATOR MagIC is compatible with the addition of 0.05% Polysorbate 20 to the digestion buffer to minimize the sheer stress that may occur in automated systems.

When processing samples using a KingFisher instrument, the sample preparation is very straightforward. The samples, resin and buffer are pipetted into the designated wells or plates and the plate(s) are placed into the instrument. The BindIt™ protocol designed for use with FabRICATOR MagIC will control the incubation temperature and times for each step. Figure 3 shows a schematic view of the workflow using FabRICATOR MagIC on a KingFisher instrument.

FabRICATOR MagIC workflow

Figure 3: FabRICATOR MagIC workflow on KingFisher. IgG, FabRICATOR MagIC and PBS are dispensed into a 96-deepwell plate according to the instructions for the KingFisher workflow. FabRICATOR MagIC beads are transferred (1) to wells containing PBS for equilibration (2). The beads are then collected (3), added to the antibody samples (4) and incubated with mixing for 10-20 min at 37°C (5). Thereafter the beads are collected (6) and transferred to waste wells and the pure F(ab’)2 and Fc are left in the sample wells (7).

Fast monitoring of oxidation, glycosylation and other CQAs

FabRICATOR MagIC automated sample preparation can be used to analyzed and monitor different CQAs. Oxidation of mAbs during production or storage is one such CQA that may impact the quality of the final therapeutic product. To show how FabRICATOR MagIC can address this, a mixture of six mAbs was subjected to forced degradation (3 months at room temperature) and analyzed by reverse phase LC-MS. When the mAbs were analyzed at the intact level (Fig. 4, upper panel), a marked difference in the abundance of mAb5 could be detected while a number of new peaks appeared. However, no reliable mass data could be obtained. Using FabRICATOR MagIC, the source of the change in mAb5 was revealed to be within the Fab region (Fig. 4, middle panel). Subsequent reduction to Fc/2, LC and Fd’ fragments made it possible to identify the difference was due to tryptophan oxidation on the mAb5 Fd’ fragment, revealed by formation of an oxolactone (+14 Da) and dioxolactone (+30 Da), as well as succinimidation of the mAb5 LC (-18 Da). Furthermore, an isomerization of the mAb3 light chain was detectable (Fig. 4, lower panel). This example illustrates the power of FabRICATOR MagIC in middle-level analysis of mAbs by creating fragments small enough to pin-point specific modifications without the need for time and resource consuming peptide mapping, all achieved quickly with relatively little hands-on time.

Figure 4: Analysis of tryptophan oxidation during forced degradation studies. A mix of 6 mAbs were analyzed by reverse phase LC-MS at the intact level (upper panel) after digestion with FabRICATOR MagIC (middle panel) and after FabRICATOR MagIC with reduction using 5 mM TCEP (lower panel). Differences between the control sample (blue) and the forced degradation sample (orange) are highlighted.

Glycan profiling in under 30 minutes

The antibody Fc glycosylation profile is an important CQA since it affects efficacy, serum clearance and immunogenicity. Therefore, the Fc glycan profile needs to be monitored from early development, in process development to final quality control and batch release. Traditional analytical approaches based on fluorescent labelling of released glycans with subsequent analysis by HILIC-HPLC or CE entail multi-step sample preparation protocols that are time and resource intensive. Digestion of a mAb with FabRICATOR MagIC followed by middle-level analysis using LC-MS offers a fast and straightforward alternative to Fc glycan analysis. It is easily automated, as shown above, for higher throughput and reduced risk of handling errors.

Figure 5: Analysis of Fc glycosylation by middle-level LC-MS.  The glycan profile of trastuzumab was determined by quantifying the different glycoforms from a deconvoluted mass spectrum of the Fc/2 fragment.

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