22 May / June 2017
containing various numbers of emtansine molecules. This results in various drug to antibody ratios (DAR) from 1 to 8 in the final product. In order to accurately quantify all species of the drug product, several bioanalytical assays are required.
The high capacity BioBA sample enrichment kit used in this study utilises magnetic beads to facilitate capture and recovery of samples from complex matrices such as plasma. The BioBA magnetic beads are cellulose based particles with a macroporous structure and are on average 30-50 µm in size. The hydrophilic cellulose surface and macroporous structure yield a particle with low nonspecific binding and higher surface area than polymeric particles. Magnetic beads offer several advantages including: ease of handling, scalability, improved sample recovery, parallel processing of samples using a variety of magnetic stands, and use in high-throughput formats with robotics. A study on the binding capacity of the BioBA beads using biotinylated anti-human IgG and monitoring its disappearance from the supernatant over time using a bicinchoninic assay showed that binding was complete (96%) after 60 minutes (Figure 1).
Figure 4. Signal Intensity Improvement using M3 MicroLC. A 4 fold improvement in S/N ratio was achieved using microflow LC. XIC data for microflow and traditional flow LC for 10 µg/ml (Top) and 5 ng/ml (Bottom)
FTISADTSK is shown here with improved S/N.
column 50 x 0.3 mm HALO Peptide ES-C18 2.7 µm 160 Å column was used (Sciex). Mobile phase A in the analytical gradient was water with 0.1% formic acid, mobile phase B was acetonitrile with 0.1% formic acid with flowrate of 10 µL/min. The column temperature was set to 40°C. Injection volume was 25 µL, and the autosampler needle and valve wash consisted of two cycles using mobile phase B, followed by one cycle using mobile phase A. The gradient method was as follows: 0 min, 3% 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; 10.0 min, 95% B; 10.1 min, 3% B; 15.0 min, 3% B. For trapping conditions, mobile phase A in the loading gradient was water with 0.1% formic acid, mobile phase B was acetonitrile with 0.1% formic acid. Sample was loaded from the injection loop onto the trap column using 100% A for 2.5 min at 50 µL/min flow rate. The trap was then washed with 95% B followed by 100% A each at 50 µL/min for 5 min after every injection.
Mass Spectrometry and Data Processing: A Sciex QTRAP® 6500+ with IonDrive™ Turbo V source was used. For the microflow
LC experiments, the standard electrode was replaced with a 25 µm ID electrode (Sciex). The transitions and MS parameters were optimised using DiscoveryQuant™ software (Sciex) and kept constant for both the traditional flow and microflow LC experiments. MultiQuant™ 3.0.2 software (Sciex) was used for data analysis. Sample for both microflow and traditional flow LC-MS/ MS analysis was prepared on the same day to exclude variations in response due to sample preparation. Three replicate LC-MS/ MS injections were acquired for both the traditional flow and trap-and elute microflow LC analysis.
Results and Discussion
Ado-trastuzumab emtansine is the first HER2-targeted treatment for metastatic breast cancer. It combines the mAb trastuzumab with the cytotoxic agent emtansine bound through a non-cleavable chemical linker to lysine groups on the mAb. Due to the availability of multiple lysine sites for emtansine conjugation, the final drug product is a heterogeneous mixture
The beads have high binding capacity with 1 mg of beads capable of binding 77 µg of biotinylated anti-human IgG, allowing for capture of more target analyte proteins than other polymer based commercially available magnetic beads (Figure 2).
The magnetic beads in the BioBA kit
are coated with streptavidin which has an extremely high affinity for biotin. This enables an easily customisable immunocapture strategy. Various LBA LC-MS/MS assays can be developed by first binding the appropriate biotin conjugated immunocapture reagent to the streptavidin coated beads. For example, for heterogeneous ADC drug products such as ado-trastuzumab emtansine, the amount of conjugated ADC species can be determined using an anti-payload antibody for immunocapture. To assay the total antibody (free + conjugated), a generic anti-human Fc antibody can be employed for immunocapture, or a target specific immunocapture strategy can be employed with recombinant target protein or an anti-idiotype antibody. For the current study, an assay was created to determine the total antibody (DAR 0 to 8) in the drug product by binding biotinylated goat anti- human IgG antibody to the beads as the immunocapture reagent. This antibody is
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68
