Biopharmaceutical Characterization Application Compendium - page 186

Degradation Profiling of a Monoclonal Antibody Using Multiple Fragmentation Techniques and a Novel Peptide Mapping Software
The goal of these analyses is to develop an effective workflow for
degradation profiling of a monoclonal antibodies utilizing the sensitive liquid
chromatography-mass spectrometry technique in combination with a software tool for
automatic identification and relative quantification of protein modifications.
Enzymatically digested monoclonal antibody was analyzed by liquid
chromatography and mass spectrometry with complementary fragmentation
techniques. The HCD and ETD data were interpreted by Thermo Scientific
Chemical degradation of amino acids was profiled. Both expected and
unexpected modifications were identified.
Monoclonal antibodies (mAbs) are the fastest growing classes of therapeutic agents.
Like other proteins, mAbs are subject to various degradation pathways. MAb
degradation can occur during manufacturing process and upon storage in liquid,
frozen, or solid state at different pH and temperature conditions. These degradations
are heavily characterized in the biopharmaceutical industry throughout the
development, manufacturing and storage stages. The characterization of chemical
degradation includes amino acid verification, N-terminal and C-terminal processing,
deamidation, oxidation, etc. Recent development and optimization of mass
spectrometry with multiple fragmentation techniques is expected to significantly
improve efficiency and sensitivity of the conventional characterization methods.
Furthermore, the use of a novel peptide mapping software yields detailed and ultimate
qualitative insight into mAb degradation profiling.
Sample Preparation
Commercially available monoclonal antibody rituximab was purchased and used in all
experiments. Rituximab was supplied at a concentration of 10 mg/mL, formulated in 9.0
mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate
80,and water. For degradation profiling of the mAb, different conditions for stressing
rituximab include pH extremes (pH 10), oxygen (exposure to hydrogen peroxide), light
(exposure to natural day light) and temperature (40
For pH extremes, buffer pH 10 was made by 5% NH
OH. To stress the mAb protein, 5
µL rituximab was mixed with 45 µL buffer and stored in dark at 40
For oxidative stress, 5 µL rituximab was mixed with 45 µL 5% H
and stored at room
temperature for 45 minutes prior to digestion.
For light stress, 20 µL protein was stored in an Eppendorf tube and exposed to natural
day light for two days.
For additional temperature stress conditions, 20 µL stock was stored at 40
C in dark
for 48 hours (annotated as “native, 40
All mAbs were mixed with 1:1 ratio (v:v) to 2,2,2-Trifluoroethanol (Sigma, St. Louis,
Missouri) and reduced in 200 mM dithiothreitol for 20 min at 90
C in dark. The reduced
proteins were then alkylated with 100mM iodoacetamide (Sigma, St. Louis, Missouri) in
the dark for 1 hour. The proteins were diluted by 1:10 10 mM ammonium bicarbonate
and incubated with 1:20 trypsin to protein (w:w) for 6 hours. Digestion was quenched
by acidifying the sample to pH 3 using formic acid.
Liquid Chromatography
An approximate 2.5 µg digest was loaded per injection. Peptides were separated by
reverse phase liquid chromatography using an Accucore C18 column (100 x 2.1 mm,
2.6 µm particle size, Thermo Fisher Scientific). For all experiments, the solvents used
were water with 0.1 % FA (A) and acetonitrile with 0.1% FA (B). Gradient was
performed at 300 µL/min, 5-35 % in 50 minutes.
Mass Spectrometry
Peptides, eluted from reverse phase liquid chromatography, were directly analyzed
using a Thermo Scientific
Orbitrap Fusion
Mas Spectrometer
. Full MS
was acquired at 120,000 resolution with mass range of
350-2,000 followed by data
dependent MS/MS spectra . Tandem spectra acquired include alternating HCD and
ETD spectra. HCD
spectra were collected with pr
spectra were only collected w
Thermo Scientific™
was utilized with source para
1, vaporizer temperature at 3
spray voltage at 3,500 v. The
dissociation (ETD) and high e
Data Analysis
The LC-MS data were proces
analyzed with this novel pepti
and identification of modificati
unexpected modifications. Th
signal threshold at 2e4, maxi
within CHO N-glycan, maxim
for unspecified modifications
Protein Sequence Coverag
Rituximab is an IgG1 class ch
chains with 213 amino acids
residue Asn301 of each heav
length. The variety and relativ
antibody characterization. Ad
environmental factors such a
shear. Thus, besides complet
characterizations, which accu
are in great need.
In this study, LC-MS data was
1.0. As shown in Figure 1, 10
from day light stressed rituxi
were utilized for matching of t
sequences shows the signal i
color coded block represent t
100 % sequence coverage w
stressed rituximab as shown i
foundation for surveying degr
Protein Modifications
A list of modification sites of
match and listed in Table 1. T
peptide is theoretically modifi
the experimental spectrum as
FIGURE 1. Light chain sequ
code is for the signal inten
represent retention time of
1...,176,177,178,179,180,181,182,183,184,185 187,188,189,190,191,192,193,194,195,196,...223
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