Easy assay by LC is suitable for ADCs

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  • Published: Apr 1, 2018
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Easy assay by LC is suitable for ADCs

Simpler analytical methods needed for ADCs

Antibody–drug conjugates (ADCs) selectively deliver drugs to medical targets such as tumours, thus minimising side effects. These pharmaceuticals are increasingly used in cancer treatment, but accurate assays are challenging. One critical parameter in the analysis of ADCs is the drug–antibody ratio (DAR), which is the ratio of antibody–drug conjugate to total antibody. Analysis of ADCs requires measuring (typically with different platforms) the amount of conjugates, total antibodies and unbound drug.

The Chungnam University researchers developed a single-platform method for such measurements. An LC-TOF-MS/MS (liquid chromatography time-of-flight MS/MS) method was used to detect the conjugate brentuximab vedotin, which contains the active (but toxic) drug monomethyl auristatin E (MMAE) linked to a monoclonal antibody.

LC-MS quantifies ADC and total antibody

The sample preparation for total antibody determination involved addition of an internal standard (a deuterated peptide), mixing with magnetic beads and digestion using modified trypsin, giving a peptide mixture that includes the non-deuterated analogue (the ‘signature peptide’) of the internal standard. The sample preparation for the determination of antibody–drug conjugate involved addition of the internal standard (verapamil), mixing with magnetic beads and freeing the MMAE from the antibody using the enzyme cathepsin B, which cleaves the linker.

In order to find the DAR of commercial brentuximab vedotin, hydrophobic interaction chromatography (HIC) was employed, with a Shimadzu Nexera XR instrument fitted with a TSKgel butyl-NPR column and UV detection at 280 nm. Gradient elution was used, with mobile phase A being aqueous 1.5 M ammonium sulphate and 25 mMol phosphate buffer (pH 7) and mobile phase B being a 4:1 mixture of 25 mMol phosphate buffer and acetonitrile (pH 7.2). The amount of B was taken from 0 to 100% over ca. 20 min. The DAR, calculated from the drug load and peak area, was similar to previous figures for brentuximab vedotin.

A 7-day stability test was carried out in vitro using rat plasma, while a 14-day in vivo pharmacokinetic test was carried out with rats that had been intravenously dosed with brentuximab vedotin. Samples were taken at intervals, worked up as above and examined by LC-MS.

The HPLC for the LC-MS system was made up of two LC-AD20 pumps linked to a CBM-20A system controller (both Shimadzu), a LEAP Technologies CTC HTS PAL autosampler and a Phenomenex Kinetex phenyl-hexyl column. The aqueous mobile phase (A) was 0.1% aqueous formic acid, while the organic phase (B) was acetonitrile containing 0.1% formic acid. For analysis of the conjugated drug and free MMAE, the solvent gradient was: 15% B for 0.5 min, 15–95% B over 0.9 min, constant 95% B for 0.2 min, 95–15% over 0.1 min and 15% for 1.3 min (total time 3.0 min). For analysis of total antibody (peptide assay post digestion), the gradient was: 15% B for 0.5 min, 15–70% B over 0.9 min, 70–95% B over 0.1 min, constant 95% B for 0.2 min, 95–15% over 0.1 min and 15% for 1.5 min (total time 3.3 min).

Mass spectrometry employed a Sciex 5600 quadrupole time-of-flight/triple time-of-flight instrument with electrospray ionisation in positive ion mode. Suitable signals were found to quantify the analytes with respect to the internal standards.

The method was shown to give good calibration curves and a high signal-to-noise ratio. The rat serum studies showed a significant loss of conjugated material after 1 day. However, a species-dependent matrix effect was seen, with human serum not giving as high a signal as rat serum. The in vivo rat studies showed the expected decrease in conjugate concentration with time, but little free MMAE.

New Method Simplifies ADC analysis

The short LC-MS run times and the use of only one analytical system are definite advantages to the new method. This protocol simplifies the analysis of conjugate drugs and should be adaptable to other ADCs with cathepsin-cleavable linkers.

Related Links

Biomedical Chromatography, 2018, Early View paper. Byeon et al.. A single liquid chromatography-quadrupole time-of-flight mass spectrometric method for the quantification of total antibody, antibody-conjugated drug and free payload of Antibody–drug conjugates.

Bioanalysis, 2016, 8, 1663-1678. Grafmuller et al.. Unconjugated payload quantification and DAR characterization of antibody–drug conjugates using high-resolution MS.

Wikipedia, Brentuximab vedotin

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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