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mixed. The plate was plated on a magnetic stand for 10 m then the supernatant was transferred to an injection plate.
Trypsin Digestion Protocol
For samples requiring a trypsin digestion, the supernatant of the wash/elution step was placed into a 96-well plate. The eluted samples were diluted with ammonium bicarbonate, ensuring pH >7.0. Samples were then heat denatured to 95°C. After cooling to <50°C, trypsin was added, and the mixture was shaken at 300 RPM for 1 h at 50°C. After centrifugation, the sample was used for LC-MS/MS analysis.
Results and Discussion
The uniformity of the bioZen MagBeads was assessed by scanning electron microscopy (SEM). The particle size of the streptavidin- coated beads was 1 µm and displayed excellent uniformity (Figure 2). Along with a patented streptavidin coating process, the consistent particle size lends to more efficient binding, thus lower background.
Mag Bead
bioZen MagBeads Lot 1 bioZen MagBeads Lot 2
Correlation Coefficient 0.9914 0.9941
Figure 3: Correlation Coefficient Assessment of Multiple Bead Lots.
correlation coefficients greater than 0.99 for both lots, a robust LBA protocol was confirmed. This lot-to-lot reproducibility assessment is important to ensure consistency in biotinylated capture antibody, which could lead to potential variation in the linear dynamic range of the assay.
Figure 2: Uniform Particle Size of Magnetic Beads Determined by SEM.
To assess consistent immunocapture, two different lots of the activated magnetic beads were evaluated (Figure 3). With
Insulin analogues are growing in the biotherapeutic industry [10] and thus, the bioanalytical immunocapture workflow is significant. The experiment commenced with a calibration curve to ensure good linearity with the external standard (Figure 4). A correlation coefficient of 0.99814 was determined for a LDR from 50-10,000 pg/mL. Proceeding to analyse insulin, we assessed samples at 50 pg/mL and 500 pg/mL to qualify the binding capacity of the magnetic beads (Figures 5 and 6, respectively).
Excellent recovery of each sample was observed even at 50 pg/mL, although some inherent background noise was also observed at this concentration.
Hybrid LBA/LC-MS/MS
Rituximab is a classic example of a commercial therapeutic monoclonal antibody in the biopharmaceutical industry. Utilising the magnetic bead LBA protocol in tandem with the trypsin digestion protocol described above, six rituximab signature peptides were analysed on a bioZen Peptide XB-C18 LC column (Figure 7). Good chromatographic separation of the peptides was observed, and each peptide demonstrated appropriate ionisation for the LC-MS/MS analysis. Notably, the integrity
Figure 4: Calibration Curve from 50 pg/mL-10,000 pg/mL.
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