2
Highly Sensitive, Robust MS-Based Workflow for Therapeutic Monoclonal Antibody Analysis from Complex Matrices
Overview
Purpose:
To demonstrate Mass Spectrometric Immunoassay (MSIA) workflows for
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Analytical Affinity P
MSIA Streptavidin D.A.R.T.’
immobilize the affinity ligan
e ana ys s o ow eve erapeu c monoc ona an o es rom uman p asma. e
model system demonstrated targeted adalimumab.
Methods:
Adalimumab was purified from human plasma by using MSIA™
Streptavidin D.A.R.T.’S.™ that were pre-treated with biotinylated TNF-α.
Adalimumab was selectively purified from human plasma by binding to an
with adalimumab were incu
D.A.R.T.’S. Analytical affini
pipetting (aspirating and dis
MSIA Streptavidin D.A.R.T.’
D A R T ’S t i i
b
immobilized TNF-α antigen. The purified adalimumab was then either (1) reduced
for downstream intact analysis or (2) reduced/alkylated/ trypsinized for bottom-up
analysis by High Resolution Accurate Mass Spectrometry (HRAM MS) detection with
the Thermo Scientific™ Q Exactive™ mass spectrometer.
. . . . con a n ng oun
buffer to release purified ad
using the Thermo Scientifi
In these experiments the p
prior to MS analysis Follo
Results:
The ability to reproducibly detect adalimumab at concentrations as low as
5 ng/mL directly from human plasma.
Introduction
.
reduction or (2) reduction, a
analyses. Digested sample
fmol/µL of the Pierce Reten
sample.
Monoclonal antibodies, and their derivative forms (Antibody Drug Conjugates, Single
Domain Antibodies, Fragment Antibodies, etc), are rapidly becoming the preferred
drug choice in the treatment of numerous diseases. This preference stems from their
improved efficacy, selectivity, and decreased side effects, relative to conventional
MS Detection and A
Liquid Chromatography
For bottom-up peptide anal
small molecule therapeutics. As this therapeutic class is rapidly evolving and
expanding utility into other indications, it is quickly being recognized that new
analytical tools are required for for the accurate and consistent analytical
measurement of these drugs. Improved methods that determine quantity, character
(Drug Antibody Ratio, Biotransformation, etc) and functionality, are all paramount to
plasma) was prepared by A
reduced/alkylated/digested.
Thermo Scientific Hypersil
Peptides were eluted at 15
acetonitrile in 45 minutes o
any drug sponsor. Described here is application of the MSIA Streptavidin Workflow
for the affinity purification and MS detection of adalimumab.
Methods
For intact analysis of the re
plasma and injected into a
100 mm) heated to 60 °C.
using a gradient of 15-35%
Ulti
t 3000 RSLC
Sample Preparation
Human plasma was spiked with adalimumab in varying concentrations. The sample
size used was 500
μ
L of plasma and it was diluted with 250
μ
L PBS.
ma e
.
Mass Spectrometry
All samples were analyzed
Ad li
b di
t
a muma ges s were a
scans were acquired at a re
target value of 1E6. HCD s
(FWHM) at
m/z
200 and an
energy of 27 and a 20 seco
Figure 1. MSIA Streptavidin Workflow for Therapeutic Antibodies
Intact heavy chain and light
4500 at a resolving power o
3E6.
Data Analysis
Bottom-up data was search
Proteome Discoverer
TM
So
heavy chain and light chain
Spectra were searched wit
ion tolerance with dynamic
carbamidomethylation of cy
Results were filtered to 1%
Reduced, intact data was d
Deconvolution
TM
Software
Results
The MSIA Streptavidin Wor
detection to provide a highl
mAb analytics. By using the
antigen, a metric of function
HRAM MS detection provid
QQQ methods.
