2
Middle-down Analysis of Monoclonal Antibody Middle using Nano-flow Liquid Chromatography and a Novel Tribrid Orbitrap Mass Spectrometer
Overview
Purpose:
Sensitive analysis of monoclonal antibody using middle-down approach on a
novel Thermo Scientific™ Orbitrap Fusion
TM
Tribrid
TM
mass spectrometer
Results
Full Mass Spectrum of Inta
Intact mAb (candidate NIST
Methods:
A method combining nano-flow liquid chromatography and complementary
higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD)
fragmentation on an Orbitrap Fusion Mass Spectrometer was developed and
implemented
Results:
A monoclonal antibody was enzymatically and chemically cleaved into Fd’
shown below was acquired at
CID at 70. The application of
help remove the adducts and
protein dependent.
,
Fc/2 and light chain respectively. Intact masses of the monoclonal antibody and its
proteolytic fragments were measured. The proteolytic fragments (Fd’, Fc/2 and light
chains) were directly fragmented using HCD and ETD. An average 50% amino acid
coverage was achieved and glycosylation site was unambiguously identified
FIGURE 1. Full MS spectru
lot #3F1b) obtained from L
T:
FTMS + p ESI sid=70.00 Full ms [1000.00-6000.
90
100
Introduction
Monoclonal antibodies (mAbs) are an increasingly important line of therapeutics for the
30
40
50
60
70
80
RelativeAbundance
28
2797.
2695.5589
2647.4794
2601 0195
biopharmaceutical industry. The demand to better understand the biochemical and
biophysical properties of mAbs has become critical. Recent developments in high
resolution mass spectrometry (MS) with multiple dissociation techniques have clearly
shown its distinctive power for characterization of intact proteins and in particular its
subunits. The mass spectrometry–based study of mAbs in middle-down approach
f f
f
2400
2600
2
0
10
20
.
2558.9576
2430.4122
FIGURE 2. Expanded view
glycoforms at charge +51
provides a wealth o in ormation to interpret its structural eatures. Here, we describe
the use of an Orbitrap Fusion mass analyzer in combination with nano liquid
chromatography (LC) for characterizing a mAb protein using different dissociation
techniques.
T:
FTMS + p ESI sid=70.00 Full ms [1000.00-6000.00
70
80
90
100
nce
2906
Methods
Sample Preparation
Th i t t Ab t i
ti ll
l
d b l
th hi
i
i t
0
10
20
30
40
50
60
RelativeAbunda
2903.7472
2899.8132
2895.7333
e n ac m pro e n was enzyma ca y c eave e ow e nge reg on n o a
F(ab')2 fragment and two Fc fragments. Aliquot of candidate NIST RM 8670 mAb lot
#3F1b was treated with FabRICATOR
®
(Genovis, Sweden) at 37
o
C for 1 hour. The
resulting F(ab’)2 and Fc fragments were further denatured and reduced in 50mM
dithiothreitol (Sigma, Saint Louis, MO) at 56
o
C for 1 hour. The proteolytically
fragmented mAb was diluted to 1 5 µg/µL using 0 1% formic acid in water
Intact LC-MS Analysis of Pro
A mixture including approximat
2895
2900
2905
.
.
.
Liquid Chromatography
Fd' and Fc/2 fragments after reduction were chromatographically eluted from a Thermo
Scientific™ PepSwift™ column, Monolithic easy spray column (200 µm x 25 cm,
Thermo Fisher Scientific Amsterdam the Netherlands) One µL of the stock was
was eluted at 800 nl/min and d
Fusion MS. The mAb fragment
800 nl/min gradient of 5-60% i
eluted at 20.67minute, followe
chain at 24.76min.
,
,
.
loaded per injection. Nano flow reverse phase chromatography was performed with a
800 nl/minute gradient of 5-60% in 32minutes using the Thermo Scientific™ EASY-
nLC™ 1000 system. The proteins were directly detected by a standard Orbitrap fusion
mass spectrometer. Liquid chromatography solvents used include the aqueous as
0.1% formic acid in water (Fisher Scientific, Fair Lawn, New Jersey) and the organic as
Full MS spectra acquired at 24
isotope distribution of the ~25,
spectra were deconvoluted for
deconvoluted monoisotopic m
mass accuracy of 2 2ppm com
RT:
18.71 - 27.44
SM:
7G
20 67
0.1% formic acid (Fisher Scientific, Fair Lawn, New Jersey) in acetronitirile (Fisher
Scientific, Fair Lawn, New Jersey).
Mass Spectrometry
The Fd’, Fc/2 and light chain eluted were directly detected by a standard Orbitrap
.
23113.3041Da.
FIGURE 3. Total Ion Current
PepSwift Monolithic Nano C
Fc/2
70
80
90
100
nce
.
Fusion MS under both full scan mode and tandem scan mode using higher-energy
collisional dissociation (HCD) and electron transfer dissociation (ETD). Full mass
spectra of mAb fragments were acquired at 240,000 resolution at
m/z
200 with mass
range
m/z
400-2000. AGC setting for full MS spectrum was at 1e5 with 100ms
maximum injection. Ion transferring temperature was set at 300
o
C. Full mass spectra
30
40
50
60
Relative Abunda
of intact mAb were acquired at 15,000 resolution at
m/z
200 with mass range
m/z
1000-6000.
Data Analysis
The full mass spectra were analyzed with Thermo Scientific
TM
Protein Deconvolution
TM
3 0 ft
f
l
l
d t
i
ti
Th t d
t
f Fd’ F /2
19
20
21
0
10
20
. so ware or mo ecu ar mass e erm na on . e an em mass spec ra o
, c
and light chain were deconvoluted by Xtract. The deconvoluted spectra were
processed by ProSightPC 3.0 software for middle-down data interpretation.
