Automated Antibody Subunit Analysis using FabRICATOR-HPLC

FabRICATOR-HPLC is an HPLC column for fast on-column digestion of monoclonal antibodies. The cysteine protease FabRICATOR (IdeS) immobilized on the column delivers rapid and consistent subunit generation, perfect for automated online analysis in a middle-level LC/MS workflow.



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”Rapid On-column Monoclonal Antibody Subunit Generation for Automated Glycan Analysis”


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Automated Antibody Subunit Analysis

Middle-level LC-MS workflows facilitate the understanding of multiple critical quality attributes (CQAs) such as glycosylation, oxidation, and C-terminal lysine clipping. Such detailed knowledge of post-translational modifications (PTM) is required for the development and manufacturing of biopharmaceuticals. FabRICATOR-HPLC provides rapid on-column digestion of monoclonal antibodies with consistent subunit generation. The column with immobilized FabRICATOR (IdeS) enzyme digests IgG rapidly and specifically just below the hinge, generating F(ab’)2 and Fc fragments without any risk of over-digestion (Fig.1).

Figure 1. FabRICATOR digests IgGs to F(ab´)2 and Fc. After reduction, the size of the fragments Fd’, Fc/2 and LC are 23–25 kDa, which is well suited for high resolution MS analysis.

Monitoring Ongoing Reactions using FabRICATOR-HPLC

FabRICATOR-HPLC can be used in a standard LC-MS setup for routine analysis. But more advanced configurations are possible, for example with a 2D-LC. Ultimately a bioreactor can be connected directly to the MS in an automated online middle-level workflow (Fig. 2). This would significantly reduce operator time, sample handling errors and increase throughput.

Fabricator-HPLC middle level flow
Figure 2. Potential set-up for online monitoring of a bioreactor in a middle-level workflow.

To demonstrate the potential, we added β1-4-Galactosidase (β-gal) to trastuzumab and determined the changes in the Fc glycan profile at different time points using middle-level analysis with FabRICATOR-HPLC. The β-gal hydrolyzes the terminal galactoses converting the G1(±F) and G2(±F) to G0(±F). The resulting Fc glycan spectra (Fig. 3a) show the expected transformation as mAb galactosylation decreased from 51 to 22% over a period of 18h (Fig. 3b). This indicates that the FabRICATOR-HPLC column is suitable for online monitoring of changing antibody quality attributes over time.

Figure 3. Middle-level assessment of trastuzumab Fc glycosylation after addition of β-galactosidase. Trastuzumab was treated with β-Galactosidase at 37°C. 10 µg modified trastuzumab was injected onto the FabRICATOR-HPLC column at different time points. The resulting fragmented samples were analyzed by RP-HPLC and ESI-Q-TOF mass spectrometry. From the deconvoluted mass spectra the glycosylation profiles were determined.

Robust On-column Digestion Performance

Digestion efficiency is maintained by FabRICATOR-HPLC during continuous operation at 37°C for 14 days. This aligns with the production of monoclonal IgG cell lines which are typically grown for 10-14 days to reach maximum antibody titers. In our tests, 10 µg trastuzumab samples were injected every 40 minutes under native conditions in PBS at a flow rate of 25 µl/min. The resulting antibody fragments were collected, reduced and analyzed by RP-HPLC (Fig. 4a). Digestion of the antibody was complete until around day 10, where some residual intact heavy chain became detectable. However, even in the samples from day 14, digestion exceeded 90% (Fig. 4b), producing enough antibody fragments to perform a middle-level analysis by mass spectrometry (see next section).

Fabricator-HPLC protocolFigure 4. Column performance under continuous operation. 20 samples taken on 10 different days over a period of 14 days were analyzed by RP-HPLC on a BEH C4 column (Waters) followed by detection by UV absorption at 280 nm. a) Comparison between digestion of trastuzumab using the standard in-solution FabRICATOR protocol (top panel) and digestion using FabRICATOR-HPLC (bottom panel). UV traces from 20 samples are overlaid. b) Quantification of digestion performance of FabRICATOR-HPLC over a period or 14 days. The amount of digested vs. intact trastuzumab heavy chain was quantified from the UV traces depicted in Figure 4a.

Reproducible Glycan Analysis

The performance and operational stability of the FabRICATOR-HPLC column is demonstrated here with trastuzumab Fc glycosylation. Fc glycosylation is a critical quality attribute for therapeutic monoclonal antibodies and one of the PTMs that are easily monitored by middle-level analysis. Ten samples taken on different days over a 14-day period of continuous operation were analyzed by LC-MS (Fig. 5a). The resulting glycosylation profiles were very reproducible with standard deviations of less than one percent. Comparison between samples digested by FabRICATOR-HPLC with samples prepared with the standard in-solution FabRICATOR protocol showed no significant differences in the glycosylation profiles (Fig. 5b).

middle-level analysis fabricator-HPLCFigure 5. Middle-level analysis of Fc glycosylation using FabRICATOR-HPLC. 10 µg samples of trastuzumab were digested either by FabRICATOR in solution or using FabRICATOR-HPLC. Antibody subunits were separated by RP-HPLC on a BEH C4 column (Waters) and analyzed by ESI-Q-TOF mass spectrometry (Bruker Impact II). a) Deconvoluted mass spectra of the trastuzumab Fc/2 fragment generated by in-solution FabRICATOR digestion (black), or FabRICATOR-HPLC on days 1, 7 and 14 (red). The results correspond to previously published data. b) Glycosylation profiles of trastuzumab were determined from LC-MS analysis of Fc/2 fragments generated by in-solution FabRICATOR digestion (black, n=6) or FabRICATOR-HPLC (orange, n=20). The bar represents the mean of all measurements, the error bars show the standard deviation.

Substrate Versatility of FabRICATOR-HPLC

FabRICATOR-HPLC is versatile and gives consistent subunit generation for three other well-known therapeutic antibodies, adalimumab, rituximab and cetuximab. All three are completely digested as seen by the lack intact HC detected in UV chromatograms (Fig. 6a). Their respective Fc glycan profiles show glycosylation patterns in accordance to previously published results. The column can be applied to human IgG1-4, to fusion proteins, ADCs, bispecific antibodies and other molecules for which FabRICATOR is active.

IgG1 analysed fabricator-HPLC
Figure 6. Digestion of IgG1 analysed by RP-HPLC followed by Middle-level analysis of Fc glycans. 10 µg of each therapeutic mAb was fragmented by FabRICATOR-HPLC and subsequently reduced. Antibody subunits were separated and analysed by LC-MS. a) Top panel: RP-HPLC analysis of the fragmented IgG1s demonstrating complete fragmentation into Fc/s, LC and Fd´ subnunits. b) Bottom panel: Deconvoluted mass spectra of the Fc/2 subunits for each of the mAbs. Glycoforms marked with an asterisk have not undergone lysine clipping.

Complete Digestion in under 5 min

At 25 µl/min the anti­body resides on the column for approximately 4 min. Under these conditions the FabRICATOR digestion reaction reaches completion on a new column and upwards of 90% of antibody is digested after 2 weeks of continuous operation (Fig. 7). Increasing the flow rate to 50 µl/min or 100 µl/min reduces the contact time to 2 and 1 min respectively and a moderate decrease in digestion efficiency is observed on a new column.

Fabricator-HPLC different flow rates
Figure 7. FabRICATOR-HPLC digestion of trastuzumab at different flow rates. Trastuzumab (10 µg) was passed through the FabRICATOR-HPLC column on day 1 and 14 at flow rates of 25 (black), 50 (blue) or 100 (red) µl/min, corresponding to a contact time 4, 2 and 1 minute respectively. Digestion performance was assessed by RP-HPLC. The percentage of intact heavy chain is stated for each sample.

No Carry Over

FabRICATOR-HPLC shows no carry-over between injections. An injection of 10 µg of adalimumab after 500 previous injections of trastuzumab were analysed by RP-HPLC/MS and evaluated for signs of trastuzumab carry-over to the adalimumab sample (Fig. 8). Neither the trastuzumab LC, Fd´ or HC fragments were detectable in the UV chromatogram or MS data of the adalimumab sample.

Sample Carry-over fabricator-HPLC
Figure 8. Assessment of sample carry-over. Trastuzumab (black) and Adalimumab (green) were digested through two consecutive injections on FabRICATOR-HPLC. a) RP-HPLC analysis of the last trastuzumab (black) and the first adalimumab (green) sample. Antibody subunit fragments are labelled. The arrows indicate the retention time for the trastuzumab heavy chain and Fd´ fragment which are clearly absent from the adalimumab sample. b) Overlay of the deconvoluted mass spectra of the light chains of trastuzumab (black) and adalimumab (green). Note the absence of a peak corresponding to the trastuzumab light chain in the adalimumab sample.



Automated Subunit Generation

FabRICATOR-HPLC is an HPLC column for fast on-column digestion of monoclonal antibodies.

  • Human IgG1-4, IgG from monkey, rat, rabbit and sheep
  • Over 95% digestion of 10 µg IgG in less than 5 minutes
  • FabRICATOR-HPLC Column for automated subunit analysis

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