20 February / March 2020
Uniform and Reliable Magnetic Beads for Protein Immunocapture Workflows
by M. Christina Malinao, Matt Brusius, Brian Rivera, Chad Eichman Affiliation: Phenomenex, Inc, 411 Madrid Ave, Torrance, CA, 90501 Email Address:
ChadE@Phenomenex.com
Magnetic beads are ubiquitous in the field of genomics but are also a critical component of protein therapeutic analysis [1,2]. Typically utilised in the pharmacokinetic (PK) and pharmacodynamic (PD) laboratories, magnetic beads are used to extract and quantitate proteins from biologic matrices, which is predominantly considered a ligand binding assay (LBA) or immunocapture. The sensitivity, accuracy, and reliability of these quantitation methods are essential during safety and efficacy testing. Enzyme-linked immunosorbent assays (ELISA) are the most established LBA protocol, but magnetic beads are emerging as the tool of choice to streamline the immunocapture process. A common magnetic bead immunocapture approach starts with streptavidin-coated beads that are activated with a biotinylated anti-idiotypic reagent. This strategy allows specific binding to the protein of choice and takes steps toward platforming the protocol.
More modern strategies are geared toward combining LBA with LC-MS/MS which creates a more sensitive process with increased linear dynamic range (LDR) [3,4]. While magnetic beads are a mature technology, advances in the grafting process can lead to increased binding capacity and more dependable data. This article demonstrates a hybrid LBA/LC-MS/MS approach [5-7], utilising immunocapture of large molecule therapeutics with streptavidin-coated magnetic beads, followed by quantitation of signature peptides using LC-MS/MS. This strategy necessitates a consistent and reliable sample preparation procedure that can be platformed to different large molecule therapeutic modalities, which offers a considerable benefit over traditional methods [8,9].
Materials and Methods
Rituximab and insulin aspart (Novolog) were purchased from Myoderm®
(Norristown,
PA). Trypsin was purchased from Promega® Corporation (Madison, WI). SiLuMab and Dulbecco’s Phosphate Buffered Saline (DPBS) were purchased from Sigma-Aldrich® (St Louis, MO). bioZen™ MagBeads, bioZen Peptide XB-C18 LC column, and bioZen Peptide PS-C18 LC column are from Phenomenex®
(Torrance, CA). LC-MS/MS
methods were performed on an Agilent® 1290 equipped with a SCIEX® SCIEX X500B QTOF.
6500+ or a
Magnetic Bead Activation – Representative Protocol (Figure 1)
A 25 µL aliquot of bioZen MagBeads (20 mg/mL) was washed with 500 µL PBS buffer. Excess liquid was discarded using a magnetic stand (3x). The beads were reconstituted to original volume with PBS. 5 µg of anti-insulin and proinsulin antibodies were added and incubated at room temperature for 1 h with shaking speed of 1200 RPM using a deep well plate thermoshaker. The excess liquid was discarded using a magnetic stand. The beads were washed with 500 µL PBS buffer and the excess liquid was discarded using a magnetic stand (3x). The beads were reconstituted to original volume with PBS.
Immunocapture – Representative Protocol (Figure 1)
Figure 1: Sample Preparation Procecure Visual.
250 µL plasma samples were added to a 96-well plate. The activated beads were vortexed to mix thoroughly and 25 µL was added to each well containing plasma. The plate was covered and spun down at 800 RPM for 3 s before incubating at 1200 RPM for 2 h on a thermoshaker. The excess liquid was discarded using a magnetic stand.
Washing and Elution – Representative Protocol
200 µL of 0.5 % CHAPS in PBS buffer was added to the beads and the solution was mixed then centrifuged at 800 RPM for 3 s. The liquid was
then discarded using a magnetic stand. The beads were washed with 200 µL PBS, mixed and shaken for 10 m at 1200 RPM using a deep well plate thermoshaker. The resultant mixture was centrifuged at 800 RPM for 3 s and the excess liquid was discarded using a magnetic stand for 2 m. 70 µL methanol/ water/acetic acid (50:48:2) was added, mixed, and shaken at 1200 RPM for 10 min using a deep well plate thermoshaker. The mixture was centrifuged at 800 RPM for 3 s then placed on a magnetic stand for 10 m. The supernatant was transferred to a different 96-well plate and 45 µL water was added and
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