Antibodies represent an important class of proteins due to their central role in the immune response. Moreover, there is an increasing interest in the use of recombinant antibodies as novel drug therapies.

1. Characterization of Intact Antibodies by Pre-Fractionation Using Gel Electrophoresis and ESI-MS
James B Harkins IV, Charles E. Witkowski II, Jeremy L. Norris
Protein Discovery, Inc., Knoxville, TN
• Increased therapeutic applications of antibodies necessitates
analytical methods for the rapid, sensitive characterization of
antibody structure.
• Ideally, antibody characterization by mass spectrometry
would involve a combination of both top-down and bottom-up
analysis to confirm protein primary structure and characterize
PTMs.
• Few high resolution separation/fractionation tools are
available for intact proteins. Coupling 1D gels and LC/MS
allow one to analyze antibodies using a bottom up approach.
Intact mass measurements using LC/MS allow one to validate
the bottom-up result. These two strategies require different
approaches to sample preparation.
• This presentation introduces a single sample preparation
strategy for monoclonal antibodies, enabling isolation of the
light and heavy chain in a manner fully compatible with both
top-down and bottom-up methods of analysis.
OVERVIEW
Antibodies represent an important class of proteins due to
their central role in the immune response. Moreover, there is
an increasing interest in the use of recombinant antibodies as
novel drug therapies. The structural analysis of antibodies is
important for a variety of reasons ranging from understanding
the structure of biologically active antibodies to monitoring
antibody production to ensure a quality product. Post
translational modifications play an important role in the
biological activity of antibodies. Of the many modifications,
glycosylation is highly variable depending on the method of
antibody productions and the variations are highly correlated
with variations in biological activity. This project presents a
strategy for the isolation and characterization of antibodies
that combines gel electrophoresis and LC/MS.
INTRODUCTION
Reagents
Monoclonal IgG antibody was purchased from Abcam.
All other reagents were purchased from Sigma.
Sample preparation
A 1 mg/mL solution of antibody was prepared in 50
mM ammonium bicarbonate. Reduction was
performed by adding 10uL of antibody solution to
88uL of 50mM ammonium bicarbonate and 2 µL of 0.5
M DTT. The solution was heated to 50°C for 30
minutes. 4.8 µL of IAA was added to the solution
along with 15.2uL of 50mM ammonium bicarbonate
and the reaction proceeded for 1 hr at room
temperature in the dark. A volume of 30 µL of 5x
Gelfree Sample Buffer was added to the sample,
yielding 150 µL of volume containing 10 µg of total
reduced antibody.
Gelfree 8100 fractionation
The reduced IgG was fractioned using the Gelfree
8100 as directed by the manufacturer’s suggested
method. The collection time for the antibody were
between 53-62 minutes for the light chain and 65-92
minutes for the heavy chain.
SDS Removal
SDS was removed from the sample using Detergent
Removal Spin Columns (Pierce).
Digestion Protocol (bottom-up only)
The Gelfree fractions containing the reduced and
alkylated antibody fragments were digested by adding
trypsin (1:100 enzyme/substrate) and heating for 1
hour at 37°C.
Mass Spectrometry
Intact Analysis
A volume of 30 µl of each acidified sample were
loaded on a 2.1 x 50 mm C4 XBridge column with 3.0
µM particle size, 300 Å (Waters). LC-MS data were
acquired on a LTQ Orbitrap (Thermo) coupled to a
Agilent 1100 capillary LC system. A 25 minute step
gradient was used for elution (A: 1% FA in H2O; B:
ACN).
Bottom-up
A volume of 8 µl of each peptide digest was loaded on
a 0.075 x 150 mm C18 Chip LC column (Agilent). LC-
MS data were acquired on an Agilent 6340 Ion Trap
coupled to a Agilent 1200 nano LC system. A 45
minute gradient was used for elution (A: 1% FA in
H2O; B: 90% ACN).
ANTIBODY CHARACTERIZATION WORKFLOW
Figure 2: Schematic of the Gelfree device. The
Gelfree 8100 uses SDS-PAGE using specialty pre-
cast gels to separate analytes based on molecular
weight. As molecular weight fractions elute from
the end of the gel, they are entrapped in a 150 µL
liquid layer defined by the end of the gel and a
molecular weight cut-off membrane.
Figure 1. The Gelfree 8100 instrument (left) is an
eight channel electrophoretic controller that
supplies voltage independently to each of the
eight channels in the pre-cast cartridge (right). To
use the system, the user programs the sequence (or
chooses from a pre-programmed sequence) for each of
the eight electrophoretic channels and starts the
experiment. The device automatically pauses the
experiment when each time interval has expired,
allowing the user to extract the molecular weight
fraction of interest. Measurement information for the
eight channels is displayed to the user in tabular and
graphical format during the course of an experiment.
GELFREE 8100 FRACTIONATION SYSTEM
• Mass spectrometric characterization of antibodies requires
multiple approaches, including both top-down and bottom-
up approach.
• Gelfree 8100 Fractionation System provides a simplified,
universally applicable, method for isolation of antibodies and
antibody fragments.
• Gelfree 8100 fractionation can be optimized by the user to
ensure high recovery of the protein of interest.
• Gelfree fractionation is compatible with the intact analysis by
ESI; likewise, the sample can be analyzed using bottom-up
techniques.
The authors acknowledge Michael Ford and Ravi Amunugama from NextGen
Sciences for performing the intact mass measurements.
SUMMARY
Sample
Preparation
•Reduction or enzymatic cleavage of light and
heavy chain
Fraction by
Gelfree 8100
•Light chain and heavy chain are separated into
independent fraction
Obtain intact
molecular
weight
•Top-down analysis is performed to obtain
molecular weight of intact protein
Structural
confirmation
•Digestion of protein
•Analysis of peptides using bottom-up analysis
OPTIMIZATION OF FRACTIONATION
Figure 5. Analysis of intact
mass of light chain and
heavy chain mAb.
Gelfree collection times were
optimized for collection of the
light and heavy chain mAb in
distinct fractions. The optimized
recovery of the light and heavy
chain are demonstrated using
1D gel analysis (a). Analysis of
these fractions using ESI-MS is
shown for the light chain (b-c)
and the heavy chain (d-e).
Heavy chain mass indicates
presence of the G0F form of the
mAb.
0
10
20
30
40
50
60
70
25.0 27.0 29.0 31.0 33.0 35.0 37.0
PercentIntensity
Elapsed Time (min)
Resolution of Bovine Serum Albumin
1 µg
5 µg
10 µg
25 µg
92%
97%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1ug 25 ug
Recovery
Protein Recovery
1 µg load 25 µg load
GF C GF C
Figure 4: Optimization of recovery. Simple adjustment
of the collection times allows for a protein to be collected in
a single fraction at near quantitative recovery. Using the
elution time data in Figure 3, collection times were
optimized for maximum recovery. Gel image shows the
Gelfree fraction (GF) containing albumin, 1 µg and 25 µg,
compared to control.
Optimization of Protein
Fractionation: A Model Study
Gelfree 8100 can be programmed by
the user to isolate the proteins of
interest into a single fraction at high
recovery.
Figure 3: Resolution of separation
as a function of protein load.
Bovine serum albumin peak width as
a function of protein load. Peak width
varies between 2 and 7 minutes,
FWHM. Width increases at higher
loading. Fractions were collected at
90 second intervals to define the
elution window.
1 µg
5 µg
10 µg
25 µg
200kDa
116kDa
97kDa
66kDa
55kDa
37kDa
31kDa
22kDa
14kDa
6kDa
Heavy
Chain
Light
Chain
F1 F2 F3 F4 F5
b) c)
d) e)
ESI Mass Spectra Deconvoluted Mass Spectra
a)
GN
M
M
M
GN
M
Figure 6. Further analysis
of mAb heavy chain. Tryptic
digestion of the heavy chain
mAb (Fraction 4, Figure 5)
allows for further
characterization of the heavy
chain. A glycopeptide found in
the chromatogram (a) was
selected for fragmentation (b).
A selected range is highlighted
and annotated to show
fragmentation of the glycan
(c).
IGG_HEAVY_2_NL_3.D: BPC100.0-2200.0 +AllMS
0.0
0.2
0.4
0.6
0.8
1.0
1.2
7x10
Intens.
10 20 30 40 50 Time [min]
a)
974.9
989.2
1028.6
1044.1
1073.8
1120.3
1160.8
1188.8
1201.1
1237.6
1252.2
1278.0
1316.9
1371.6
1425.6
1445.0
1479.8
1493.6
1547.2
1602.5
1660.6
+MS2(1063.7), 27.8min #1815
0.0
0.2
0.4
0.6
0.8
1.0
4x10
Intens.
1000 1100 1200 1300 1400 1500 1600 m/z
366.1
437.2
512.3
673.2
974.9
1044.1
1120.3
1160.8
1201.1
1252.2
1371.6
1425.6
1479.8
1547.2
1602.5
1660.6
1749.91800.5 1874.7
+MS2(1063.7), 27.8min #1815
0.0
0.2
0.4
0.6
0.8
1.0
4x10
Intens.
200 400 600 800 1000 1200 1400 1600 1800 2000 m/z
b) c)
GN = N-acetyl
glucosamine
M = Mannose