2
Differentiate Minor Difference of Protein Structure in Biosimilar and Reference Products Using High-Resolution Orbitrap LC-MS/MSS
Overview
Purpose:
To develop a high-resolution LC/MS workflow for the analysis of the protein
structure differences between biosimilar and reference products using a Thermo
Scientific™ Orbitrap™ bench-top hybrid quadrupole-Orbitrap mass spectrometer
Results
Nine LC-MS/MS data files, t
and G-TANK, were analyzed
.
Methods:
A top-ten data-dependent high-energy collision dissociation (HCD) method
was performed to analyze the samples using a bench-top mass spectrometer. Data
was analyzed using a new software that is under development.
Results:
An LC-MS/MS workflow was developed for differentiating minor differences of
1. Peptide identification an
The top ten data-dependent
high-resolution and accurate
identification. For each raw fi
confidence peptide identifica
protein structure between biosimilar and reference products using a benchtop Orbitrap
LC-MS/MS and a new software that is under development. This workflow provides
qualitative and quantitative biosimilar to reference product comparison.
Introduction
was achieved for each of the
map and sequence coverag
Figure 1. Peptide map (top
Biosimilar products are required by regulatory authorities to have appropriate and
comparable quality, safety and efficacy with a reference biologic product. Mass
spectrometry can offer in-depth characterization to explore the similarity and difference
between a candidate biosimilar and a reference biologic. In this study, we developed a
robust approach for comparability study of biosimilar and reference products. Any
minor difference in sequence modification and glycosylation can be well characterized
and compared by using combination of high resolution Orbitrap LC-MS/MS with a
powerful software to systematically interpret the results.
In this study tissue plasminogen activator (TPA) and a generic variant of TPA (TNK)
,
are well characterized by the robust approach. In addition, two TNK forms (G-TNK as
a reference product and I-TNK as a biosimilar form) are also compared to explore the
similarity and difference.
Methods
Samples
Three samples, TPA, I-TNK, G-TNK, were digested using trypsin after reduction and
alkylation. Tenectelplase (TNK) is a recombinant TPA with the following minor
The Q Exactive MS provides
of magnitude of abundances
shown) which ensures confi
sequence changes:
T103 -> N (Becomes N-glycosylation site)
N117 -> Q (Removes N-glycosylation site)
KHRR (296-299) -> AAAA
Liquid chromatography
,
specific cleavage versions a
quality MS/MS spectra of a p
(bottom) which is of 0.1% in
Figure 2. MS/MS spectra
Peptides were separated using Thermo Scientific™ EASY-Spray™ technology
containing a 50-cm C
18
column (2 µ particle size) and a Thermo Scientific™ EASY-
nLC™ LC. LC solvents are 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 60 min gradient was used to
elute peptides from the column
(bottom) which is of 0.13%
.
Mass spectrometry
A top-ten data-dependent high-energy collision dissociation (HCD) method was
performed using a Thermo Scientific™ Q Exactive™ MS system to analyze the
samples The following MS and MS/MS settings were used: MS scan range 100-2000
204.1
y2
5
190-GTH
.
m/z
. FT-MS was acquired at 70,000 resolution at
m/z
200 with AGC target of 1x10
6
.
MS2 was acquired at 17,500 resolution at
m/z
200 with AGC target of 2x10
5
. The spray
voltage was 1.8kV. Capillary temperature was 275 °C. S-lens level was set at 55.
Data analysis
150
200
250
300
350
400
450
500
147.1
y1
y4++
256.1
284.1
317.2
y3
b4
398.2
b8-H2O++
430.3
y4
b9++
471.2
b10++
y12[3+]b5
514.7
b11++
y14[3
204.1
y2
282.1
190-GTHS
Data was analyzed using a new software that is under development. This software
provides automated analyses of liquid chromatography/tandem mass spectrometry
(LC-MS/MS) 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
317.2
y3
3962
430.3
y4
471.2
b10++
W double ox
modified peptide. Peak areas of related peptide ions under their selected-ion
chromatograms (SIC) are used for relative quantification of modified peptides.
2. Glycosylation of TPA, I-TN
A total of four glycosylation s
99% glycosylated They are
150
200
250
300
350
400
450
500
147.1
y1
186.1
y2-H2O
231.1
b7-2H2O[3+]
355.1
b7-H2O++
.
b5
b11++
523
y14-3H2
.
TNK, and N117 in TPA. The f
and only 19% of this site is g
site (N184) compared to G-T
acid sequence, suggesting d
MS/MS spectra of three iden
