Supporting Information
CHARACTERIZATION OF ACIDIC AND BASIC VARIANTS OF IgG1 THERAPEUTIC MONOCLONAL ANTIBODIES
BASED ON NON-DENATURING IEF FRACTIONATION
Oluwatosin O. Dada, Nomalie Jaya, John Valliere-Douglass, and Oscar Salas-Solano.
Department of Analytical Sciences, Seattle Genetics, Inc., 21823 30th Drive SE, Bothell, WA 98021
S-1.0. IPG-IEF Recovery and Reproducibility
Protein amount in fractions 12 through 22 was calculated by measuring absorbance at 280 nm using
nanodrop 8000 (Thermo scientific, Watham, MA)
Figure S-1: mAb1 amount calculated from absorbance at 280 nm for six replicates. All variants focused
within wells 12 through 22. The average recovery is 60%. Wells 12, 13, 14 were combined into Fraction
14 while wells 20, 21, 22 were combined into fraction 20.
S-2.0
CE-SDS Analysis of Fractions 14 – 20
CE-SDS analyses were performed under reducing and non-reducing conditions. Higher fragments
observed in acidic variants.
Figure S-2A: Non-reduced CE-SDS of fractions 14-20. Higher fragmentation observed in acidic variants
compared to the main (F19) and the basic (F20). HL-Heavy Light, HH – Heavy Heavy, HHL Heavy Heavy
Light, LHHL – Whole antibody.
B
Figure S-2B: Reduced CE-SDS, of fractions 14-20. Higher level of backbone clips and Non-reducible
species in acidic variants. MMW - Medium Molecular Weight, HMW – High Molecular Weight
L
HL
HH
100
90
80
70
60
50
40
30
20
10
0
LHHL % TCA
%Time Corrected Area
10
9
8
7
6
5
4
3
2
1
0
F14 F15 F16 F17 F18 F19 F20
IPG-IEF Fractions
Figure S-3A: The level of LC (L),HC-LC (HL), HC-HC (HH), HC-HC-LC (HHL) species as measured using Nonreduced CE-SDS,
3.00
2.50
MMW
%TCA
2.00
Non-Reducible
1.50
1.00
0.50
0.00
F14
F15
F16
F17
F18
IPG-IEF Fractions
F19
F20
Figure S-3B: Backbone clips (MMW) and Non-reducible species as measured using Reduced CE-SDS.
Fragments are preferentially segregated in fraction 14-17.
S-3.0. HC Mass spectra of mAb1 fractions
Figure S-4: Reduced mass analysis of Fractions 14 through 19 and the control (starting material) showing
higher level of sialylated species in the acidic varaints in addition to the typically observed fucosylated
biantennary glycans with 0 (G0F), 1 (G1F), or 2 (G2F) galactose species.
FigureS-5A: Light chain mass analysis of Fractions 14 through 19 and the control (starting material).
+162 Da in the acidic fractions indicative of glycation.
Figure S-5B: Fab subunit of mAb1 variants analyzed by LC-MS. +162 mass shift indicates glycation in the
acidic fractions. Fractions 14, 16, and 18 not analyzed due to limited material.
S-4.0. Reduced Mass Analysis of Deglycosylated Samples
Purified Off-Gel fractions were buffer exchange into 10 mM phosphate. Prior to reduction, the samples
were treated with PNGase F enzyme in 37oC water bath for 30 min. After deglycosylation, the samples
were denatured and reduced in the presence of guanidine-HCL and dithiothreitol (DTT). RP-HPLC
separation was performed on an Agilent 1200 series LC system using an injection of approximately 30 μg
on a 2.1 × 150 mm Waters 1.7 m particle BEH C4 column and the antibody subdomains were eluted
with an acetonitrile/trifluoroacetic acid (TFA) gradient. MS analysis was carried out on an Agilent 6510
QTOF mass spectrometer (Agilent, Santa Clara CA) in positive ion mode and the raw data was converted
to zero charge mass spectra using a maximum entropy deconvolution algorithm embedded in Agilent
MassHunter software, version B.04.00.
Figure S-6: Mass spectra of deglycosylated heavy chain. +72 Da is due to ethylcarboxyl arginine
modification.
S-5.0. ASP-N Peptide Mapping
The reduced Asp-N digestion procedure is similar to that of Lys-C. Reductive alkylation was carried out
under denaturing conditions by heating the samples with DTT in the presence of guanidine-HCl and
subsequently alkylating the reduced cysteine residues with sodium iodoacetate (IAA). The reduced and
alkylated samples were buffer exchanged into Tris-HCl at pH 7.5 using NAP-5 gel filtration columns and
separately digested with Roche Asp-N at a 1:10 w:w ratio. The samples were digested for 4 hours at
37oC at which time the reaction was quenched by addition of acetic acid. Approximately 20 g of
digested sample was injected onto a 2.1 x 150 mm Waters 1.7 m particle BEH CSH-C18 HPLC column
The peptides were eluted with an acetonitrile/Tetrafluoro acetic acid (TFA) gradient on a Thermo Accela
1250 equiped with LEAP PAL HTX-xl robotics. MS analysis was carried out on a Thermo Q-Exactive mass
spectrometer (Thermo, Waltham MA) in positive mode at a resolution of 17,500 for MS and MS/MS
scans. The Q-Exactive RAW data files were processed with Thermo Xcalibur version 2.2. The data were
also processed with Mascot Distiller version 2.4.3.3 (Matrix Science, Boston MA) and Search against
product specific protein databases in Mascot server version 2.4.
Figure S-7A: MS spectra from Lys-C peptide mapping result for fraction 20 showing the glycine (G),
amidated proline (P), and unprocessed C-terminal lysine (K) isoforms of the C-terminal peptide on the
heavy chain subunit.
A
B
Figure S-7B: MS/MS spectra of C-terminal peptide of the heavy chain in fraction 20 (BV) analyzed using
Asp-N peptide mapping. Top is the unmodified peptide with y1 = 173.09 and the bottom is the modified
peptide showing y1 = 115.09.
S-6.0. Representative Chromatogram and MS spectrum of detected modifications in fraction 20 (BV)
Figure S-8: Representative peptidemap elution order of heterogeneity species of C-terminal peptide
(SLSLSPGK) on one of the heavy chain arm of mAb1.