2
Comprehensive Sequence and Post-translational Modifications Analysis of Monoclonal Antibody by Flash Digest and LC-High Resolution MS
FIGURE1. Complete Workfl
Results
Tryptic Digestion Time Opti
Flash Digest is a very active,
with heating technology for fa
use of a high concentration of
order to push the non-comple
concentration near the compl
Using the Flash Digest kit (be
incubating native, non-reduce
120 min. The filtered samples
Overview
Purpose:
To develop a workflow for fast and comprehensive characterization of
peptide sequence, identification and relative quantitation of post-translational
modifications (PTMs) of monoclonal antibody (mAb).
Methods:
Native and oxidatively stressed IgG mAb were enzymatically digested by
Flash Digest
TM
kit with trypsin. Peptide samples were analyzed by online LC-MS on a
Thermo Scientific™ Q
Exactive
™ mass spectrometer.
Peptide sequence mapping,
identification and quantification of PTMs were performed
by Thermo Scientific™
PepFinder
TM
software (version 1.0).
Results:
Comprehensive and simultaneous sequence and PTM analysis with a
particular focus on oxidation of IgG mAb was realized by combining rapid digestion,
high resolution, accurate mass (HRAM) data and PepFinder software. This workflow
greatly shortens the sample preparation and data analysis time while providing great
sensitivity to detect low level PTMs.
Introduction
As well established and fast growing biotherapeutics, mAbs have been approved for
the treatment of diseases such as cancer, inflammatory, infectious and autoimmune
diseases etc.
1,2
To ensure product efficacy and safety, the quality of biotherapeutics
needs to be closely monitored. Various analytical methods have been used to study
quality attributes such as structural integrity, aggregation, glycosylation pattern or
amino acid degradation. Here, we report a fast and sensitive approach by combing fast
enzymatic digestion, high resolution mass spectrometry and user friendly new data
processing software for sequence and post translational modifications analysis. This
approach provides an effective way to characterize protein therapeutics in bioprocess
development.
Methods
Sample Preparation
Differential oxidative stress was induced by 5 mM hydrogen peroxide and quenched by
the addition of 1mM sodium thiosulfate at various time points. The native and
oxidatively stressed IgG samples were trypsin digested using a Flash Digest kit
(Perfinity Biosciences Inc). Digestion time was optimized by incubating native, non-
reduced IgG mAb at 70
°
C for various durations from 15 to 120 minutes. One portion of
the digest was analyzed by online UHPLC-ESI-MS/MS. The other portion was reduced
and alkylated before similar analysis. All chemicals were purchased from Sigma Aldrich
unless it is specified.
Liquid Chromatography
Native and Oxidatively stressed tryptic peptide samples were analyzed on Thermo
Scientific™
Dionex
™
UltiMate
™ 3000 XRS system and OAS
autosampler coupled to
the Thermo Scientific
TM
Q Exactive
TM
MS. Peptides were separated on an ACQUITY®
BEH 130 C18 column (2.1x100mm, 1.7µm, Waters) with column temperature set as 40
°
C at a flow rate of 300 µL/min with solvent A (0.05% trifluoroacetic acid in H
2
O) and
solvent B (0.045% trifluoroacetic acid in acetonitrile).
Injection amount: 8.0 µg digested protein on column
Time [min] Flow [µL/min] Mixture [%B]
0 300 0.1
5 300 0.1
94 300 35
94.5 300 95
99.5 300 95
100 300 0.1
110 300 0.1
Mass Spectrometry
The Q Exactive MS interface
Acquisition method was set w
top 5 data dependent MS/MS
Data Analysis
The mapping of mAb sequen
performed in PepFinder softw
characterization of biotherape
identification of disulfide bond
deamidation etc by mono-isot
fragments indicated with a co
with color code for signal inte
summary report with relative
friendly interface. For unknow
indicated with accurate mass
=== HESI Source: ===
Spray Voltage (+)
Capillary Temperature (+)
Sheath Gas (+)
Aux Gas(+)
Sweep Gas(+)
Heater Temperature (+)
S-lens
Full MS Scan in positive mo
Scan range=
m/z
300-1800;
Top 5 data dependent MS/M
NCE=27; Isolation window=
