2
Structure Characterization and Differentiation of Biosimilar and Reference Products Using Unique Combination of Complementary Fragmentation Mechanisms
Overview
R lt
Purpose:
To analyze difference of protein structure in biosimilar and reference products
using Orbitrap LC-MS/MS
Methods:
A unique data-dependent instrument method that utilizes two different
fragmentation mechanisms was applied for peptide sequence and PTM identification and
esu s
1. Peptide identification and
The data was analyzed and t
100% sequence coverage for
quantification using a Thermo Scientific
TM
Orbitrap Fusion
TM
Tribrid
TM
mass spectrometer.
While generating HCD MS/MS spectra on peptides in a data-dependent experiment, the
method identifies glycopeptides on-the-fly using the diagnostic ions from glycan
fragmentation. A subsequent ETD fragmentation is then triggered on the same peptide to
produce information of amino acid sequence and site of glycosylation The new Thermo
peptide forms was calculated
range for identified peptide
modified peptides with less th
shown). Figure 1 shows an ex
.
Scientific
TM
PepFinder
TM
1.0 software for peptide imaging, was used for data analysis.
Results:
A LC-MS/MS workflow was developed for differentiating minor difference of protein
structure in biosimilar and reference products using an Orbitrap Fusion LC-MS/MS and
PepFinder 1.0 software. This new approach offers efficient, confident and comprehensive
l i
t
l f bi i il
bilit
t d b t l
f l t t l t
i
f
Figure 1. 100% sequence c
ana ys s, no on y or os m ar compara y s u y u a so or o - o- o compar son o a
same compound.
Introduction
Bi i il
b
i
f i
bi
h
i l
d
d f
os m ars are su sequent vers ons o nnovator op armaceut ca pro ucts create a ter
the expiration of the patent on the innovator product. The approval of a biosimilar product by
a regulatory agency requires thorough characterization that demonstrates comparability with
a reference product in quality, safety and efficacy. High resolution mass spectrometry
provides accurate characterization of various protein properties including primary structure,
type and location of post translational modifications (PTMs) and low abundant sequence
-
,
variants or impurities. In this study, we developed a robust approach for comparability study
of biosimilar and reference product. Minor differences in products including glycosylation
were systematically compared using high resolution LC/MS/MS with complementary
fragmentation methods and a new peptide mapping software package.
Table 1 Identified glycosyl
Methods
Samples
An original drug, a recombinant variant and its biosimilar product, TPA, I-TNK and G-TNK,
.
number of glycoforms ide
Site of glycosylation
N 103
were digested using trypsin after reduction and alkylation. Tenectelplase (TNK) is a
recombinant TPA with the following minor sequence changes:
T103->N (Becomes N-glycosylation site)
N117->Q (Removes N-glycosylation site)
KHRR (296-299) -> AAAA
N 103
N117
N 184
Liquid chromatography
Peptides were separated using with a Thermo Scientific
TM
EasySpray
TM
source setup
containing 50-cm C
18
column (2 µ particle size) and a high-pressure easy nanoLC (U-
HPLC). The LC solvents were 0.1% formic acid in H
2
O (Solvent A) and 0.1% formic acid in
acetonitrile (Solvent B). Flow rate was 250 µL/min. A 70 min gradient was used to elute
N 448
N 448
N 448
peptides from the column.
Mass spectrometry
Samples were analyzed using an Fusion mass spectrometer with a Thermo Scientific
TM
EASY-ETD™ ion source. An instrument method designed for glycopeptide analysis was
Figure 2. Characterization
C441-R449 with glycosylat
showing peptide backbo
used for this study. This method primarily acquires HCD MS/MS spectra on peptides in a
data-dependent top-ten experiment. However, if diagnostic sugar oxonium ions from glycan
fragmentation are detected in the HCD MS/MS spectrum, a subsequent ETD fragmentation
is then triggered on the same precursor peptide to produce amino acid backbone sequence
information to identify the site of glycosylation. Therefore, for each glycopeptide, this HCD
d t d d t ETD th d (HCD dETD)
t
i
f HCD d ETD t
preserved) and fragmentati
C441-R449 , N448
pro uc - epen en
me o
p
genera es a pa r o
an
spec ra,
producing information for the peptide sequence and the site of glycosylation as well as
confirming glycan structure. Orbitrap MS spectra were acquired at 120,000 resolution (at
m/z
200) with an AGC target of 4x10
5
. MS/MS spectra were acquired at 30,000 resolution
(at
m/z
200) with an AGC target of 5x10
4
. Capillary temperature was set to 275 °C and the
S lens level was set at 60 The priority for precursor selection for data dependent MS/MS
366.1
c3
-
.
-
was for the highest charge state followed by the lowest
m/z
. HCD collision energy was 30
and ETD activation time was charge dependent based on the standard calibration.
Data analysis
Data was analyzed using PepFinder 1 0 software This software provides automated
200
300
400
500
178.1
c1
279.1
c2
494.2
c4
138.1
204.1
(Gn)
(Gn)
274.1
366.1
(GGn)
.
.
analyses of liquid chromatography/tandem mass spectrometry data for large-scale
identification and quantification of known and unknown modifications. Peptide identification
is achieved by comparing the experimental fragmentation spectrum to the predicted
spectrum of each native or modified peptide. Peak areas of related peptide ions under their
selected-ion chromatograms (SIC) are used for relative quantification of modified peptides A
200
300
400
500
600
168.1
186.1
292.1
(S)
528.2
(GGnM)
.
mass tolerance of 5 ppm was used to ensure accurate identification.
