3
Thermo Scientific Poster Note
•
PN-64136-ASMS-EN-0614S
unoassay (MSIA) workflows for
b di
f
h
l
Th
Analytical Affinity Purification
MSIA Streptavidin D.A.R.T.’S were treated with a solution of biotinylated TNF-α to
immobilize the affinity ligand on the microcolumn surface Plasma samples spiked
Intact Analysis of Reduce
Figure 1
shows the elution profile o
µg/mL) purified from human plasma
o es rom uman p asma. e
lasma by using MSIA™
biotinylated TNF-α.
sma by binding to an
.
with adalimumab were incubated with the TNF-α-derivatized MSIA Streptavidin
D.A.R.T.’S. Analytical affinity purification of adalimumab was achieved by repetitive
pipetting (aspirating and dispensing) of the sample solution though the functionalized
MSIA Streptavidin D.A.R.T.’S. Following purification, the MSIA Streptavidin
D A R T ’S t i i
b d d li
b
h d d th t
t d ith l ti
light chain (LC). Deconvolution of th
masses, 23409.33 and 23413.68. T
reduction of the two disulfide linkag
of the theoretical LC average mass
spectrum gave a mass at 50644 22
b was then either (1) reduced
ed/ trypsinized for bottom-up
etry (HRAM MS) detection with
eter.
FIGURE 1: Intact analysis of reduce
reduced adalimumab showing the e
. . . . con a n ng oun a a muma were was e an en rea e w e u on
buffer to release purified adalimumab. Affinity purification steps were automated by
using the Thermo Scientific™ Versette™ automated liquid handler.
In these experiments the purified adalimumab underwent two types of treatment just
prior to MS analysis Following elution the adalimumab either subjected to (1)
. ,
C-terminal lysine and the addition o
represents the addition of a hexose
mab at concentrations as low as
(HC). B) and C) Raw MS spectra for
average mass (M+H) of LC. E) The
.
,
reduction or (2) reduction, alkylation and digestion (120 ng of trypsin) for bottom up
analyses. Digested samples were internally calibrated through the addition of 5.95
fmol/µL of the Pierce Retention Time Calibration (PRTC) peptide mixture to each
sample.
60
70
80
90
100
undance
7.49
L
A
ntibody Drug Conjugates, Single
apidly becoming the preferred
This preference stems from their
fects, relative to conventional
MS Detection and Analysis
Liquid Chromatography
For bottom-up peptide analysis, purified adalimumab (ranging from 5-500 ng/mL in
0
10
20
30
40
50
RelativeAb
s is rapidly evolving and
ing recognized that new
consistent analytical
at determine quantity, character
nctionality, are all paramount to
plasma) was prepared by Analytical Affinity Purification (described above) and then
reduced/alkylated/digested. The resulting peptide fragments were injected onto a
Thermo Scientific Hypersil™ Gold aQ 2.1 x 100 mm column heated to 70 °C.
Peptides were eluted at 150 µL/min using a gradient of 2-35% formic acid (0.1%) in
acetonitrile in 45 minutes on an Ultimate 3000 RSLC.
6.0
6.5
7.0
7.5
he MSIA Streptavidin Workflow
umab.
For intact analysis of the reduced adalimumab, 1.8
μ
g/mL was recovered from
plasma and injected into a Thermo Scientific™ ProSwift™ column (RP-4H 0.5 mm x
100 mm) heated to 60 °C. The heavy chain and light chain were eluted at 200 µL/min
using a gradient of 15-35% formic acid (0.1%) in acetonitrile in 8.2 minutes on an
Ulti
t 3000 RSLC
80
90
100
2129.2008
B
ing concentrations. The sample
ith 250
μ
L PBS.
ma e
.
Mass Spectrometry
All samples were analyzed on a Q Exactive mass spectrometer.
Ad li
b di
t
l
d i
t
10 d t d d t
th d F ll
30
40
50
60
70
Relative Abundance
1378.0564
1233.0984
1464.2771
1801.7598
2342.0249
a muma ges s were ana yze us ng a op a a- epen en me o . u
scans were acquired at a resolving power of 70,000 (FWHM) at
m/z
200 and an AGC
target value of 1E6. HCD spectra were acquired at a resolving power of 17,500
(FWHM) at
m/z
200 and an AGC target value of 1E5 with a normalized collision
energy of 27 and a 20 second dynamic exclusion duration.
tic Antibodies
1000
1500
2000
25
m/z
0
10
20
2
D
Intact heavy chain and light chain were analyzed with a full scan taken from m/z 900-
4500 at a resolving power of 17,500 (FWHM) at
m/z
200 and an AGC target value of
3E6.
Data Analysis
Bottom-up data was searched using SEQUEST® HT in Thermo Scientific
TM
Proteome Discoverer
TM
Software (1.4 SP1) against a database of adalimumab
heavy chain and light chain appended with 115 common contaminant proteins.
Spectra were searched with a 15 ppm precursor tolerance and a 0.02 Da fragment
ion tolerance with dynamic pyroglutamylation of N-termini, oxidation of methionines,
carbamidomethylation of cysteines and deamidation of asparagines and glutamines.
Results were filtered to 1% FDR using Percolator.
Reduced, intact data was deconvolved using Thermo Scientific
TM
Protein
Deconvolution
TM
Software (3 0) with the ReSpect™ algorithm
Bottom-Up Analysis of Adali
Plasma samples were spiked with
500 ng/mL and 5000 ng/mL. Adali
.
.
Results
The MSIA Streptavidin Workflow combines traditional ligand binding with MS
α-derivatized MSIA Streptavidin D.
to reduction, alkylation, and trypsin
coverage determination and PTM
detection to provide a highly sensitive, robust and reproducible method to therapeutic
mAb analytics. By using the innate affinity of the therapeutic mAb for its target
antigen, a metric of function is also provided as a part the general analysis, while
HRAM MS detection provides additional analytical flexibility over other developing
QQQ methods.
