21 Experimental
Magnetic Bead Preparation: Magnetic immunocapture beads from the BioBA sample prep kit (Sciex) were prepared with biotinylated goat anti-human IgG antibody (included in the kit) according to the kit protocol.
Figure 2. Binding Capacity of Magnetic Beads. Comparison of magnetic bead binding capacities for several commercially available magnetic beads using biotinylated anti-human IgG and following manufacturers directions.
Sample Preparation: 10x spiking solutions of ado-trastuzumab emtansine were first prepared in 1X BioBA bind/wash buffer containing 0.01% BSA (bovine serum albumin), then spiked into Sprague-Dawley rat plasma, K2EDTA (BioreclamationIVT) at the final concentrations of 0.5-100,000 ng/ml. SILuMab (Sigma Aldrich), was used as internal standard (IS) and was added to the plasma samples prior to BioBA immunocapture processing. A blank and double blank sample were also prepared. The double blank only had 1X BioBA bind/ wash buffer containing 0.01% BSA and the blank sample had rat plasma with additional internal standard. Spiked plasma samples (50 µL) were mixed with internal standard and processed based on the BioBA generic method with some modified steps to reduce background signal and improve signal to noise (S/N) ratio in order to utilise the advantage of higher sensitivity provided by microflow LC. The modified steps include: an extra hour of incubation of conjugated beads with sample followed with two wash steps with 500 µL of BioBA bind/wash buffer and 500 µL of 50 mM ammonium bicarbonate. Each immunopurified sample was digested with 1 µg total Trypsin/Lys-C.
Figure 3. BioBA Immunoenrichment and Sample Processing Workflow. The workflow includes the enrich- ment of mAbs from plasma using magnetic streptavidin beads conjugated with an anti-Human IgG for enrichment and quantification of the total antibody with a generic or specific signature peptide.
the bioanalysis of mAbs and other proteins in biological fluids. Because of their large size, proteins are typically not quantified intact but instead are digested into peptides, with one or more peptides chosen as a proxy for quantification of the protein. However, quantification of mAbs and other proteins from plasma often requires greater sensitivity than can be achieved with LC-MS/ MS alone using traditional LC flow rates. Additionally, as potency increases, dosage is decreased, thereby resulting in less biotherapeutic in circulation to measure.
Here we describe a universal immunocapture enrichment strategy
coupled with sample preparation and microflow LC-MS/MS analysis and apply it to the total antibody analysis of the ADC ado-trastuzumab emtansine in plasma. Streptavidin coated magnetic beads bound with a universal capture reagent accelerate method development for total antibody enrichment. The use of microflow LC provides greater sensitivity than traditional flow LC. The hybrid LBA microflow LC-MS/ MS workflow provides a customisable immunocapture strategy that enables the rapid development of high sensitivity pharmacokinetic assays of biotherapeutics during pre-clinical or phase I-IV studies.
Traditional Flow Liquid Chromatography: A Shimadzu Prominence HPLC system with two LC-20AD pumps, CTO-20A column oven, and a SIL-20AC autosampler was used. The column was a 100 x 2.1 mm Kinetex C18 2.6 µm 100 Å column (Phenomenex). Mobile phase A, water with 0.1% formic acid, and mobile phase B, acetonitrile with 0.1% formic acid, was used at a flow rate of 0.5 ml/min. Wash solvent for the autosampler was 20/20/60 methanol/ acetonitrile/IPA. Injection volume was 25 µL, and the column was kept at 40°C. The gradient method was as follows: 0 min, 5% B; 0.7 min, 5% B; 0.8 min, 10% B; 3.5 min, 25% B; 5.0 min, 40% B; 5.1 min, 95% B; 5.9 min, 95% B; 6.0 min, 3% B; 7.0 min, 3% B.
Microflow Liquid Chromatography: A Sciex M3 MicroLC-TE system, with two microLC gradient pumps and an integrated autosampler was used in combination with a source mounted column oven (Sciex). A 10 x 0.3 mm trap column packed with 5 µm 120 Å ChromXP C18 CL and an analytical
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