Significance of the Topic
Proteolytic clipping of therapeutic antibodies can compromise drug efficacy and safety and constitutes a critical quality attribute in biopharmaceutical development.
Dedicated analytical workflows are required to detect and characterize low-abundance truncation variants that may escape conventional peptide mapping.
Objectives and Study Overview
This work aimed to evaluate a streamlined approach for identifying and confirming clipping variants of Vedolizumab, a recombinant humanized IgG1, by combining high-resolution intact mass screening with Middle-Down sequencing.
SpeB protease cleavage in the hinge region was used as a model for generating antibody truncations, and BioPharma Compass 2021 workflows were assessed for rapid variant detection and validation.
Methodology
Vedolizumab biosimilar samples were digested with SpeB (FabULOUS kit) and deglycosylated (IgGZERO kit), followed by reduction with TCEP to yield Fd, Fc/2 and LC subunits.
Intact mass profiling was performed by LC-MS to screen for expected and unexpected clipping variants.
Candidate masses were subjected to MS/MS via both ETD and CID in a Middle-Down approach to confirm sequence identities and map cleavage sites.
Data processing employed the MAM Protein Screening workflow for intact mass analysis and the Top-Down Sequencing workflow for fragment validation in BioPharma Compass 2021.
Instrumentation Used
- LC system: Bruker Elute UPLC with Waters Protein BEH C4 column (300 Å, 1.7 µm, 2.1×100 mm) and 0.1% formic acid in water and acetonitrile solvents with a stepped gradient.
- Mass spectrometer: Bruker maXis II ETD ultrahigh-resolution QTOF operating at 150–2800 m/z for MS and 100–2400 m/z for ETD/CID fragmentation.
- MS/MS settings: ETD with controlled ion accumulation (ICC target 1×10^9), injection times 16–22 ms, reaction times 4–6 ms; CID stepping at 25/30 eV.
- Software: BioPharma Compass 2021 using SNAP deconvolution, MAM screening and Top-Down sequencing workflows.
Results and Discussion
Intact mass analysis confirmed the main hinge cleavage products Fd [1–229], Fc/2 [230–450] and LC [1–219] as major peaks, while four additional, low-abundance peaks were detected.
Middle-Down ETD delivered higher average sequence validation percentages (~50%) compared to CID (~37%), enabling reliable confirmation of three minor cleavage sites by fragment ion coverage.
Two additional sites were validated by complementary mass pairs and mass accuracy (<1 ppm) matching, and one site by isotopic pattern fidelity alone.
The combined workflow mapped six non-canonical hinge-region cleavages in addition to the expected site, illustrating ragged Fc/2 and Fd termini.
Benefits and Practical Applications
The integrated high-resolution intact mass screening and Middle-Down sequencing approach allows rapid, unambiguous identification of antibody clipping variants.
This method supports biopharmaceutical comparability, stability and aging studies by monitoring proteoform integrity as part of multi-attribute monitoring strategies.
Future Trends and Opportunities
Advances in automation and workflow integration will facilitate routine incorporation of clipping analysis into quality control pipelines.
Improvements in fragmentation techniques, data analytics and AI-driven pattern recognition are expected to enhance sensitivity and throughput for comprehensive proteoform profiling.
Expansion to other classes of biotherapeutics and novel enzymes could further broaden applicability.
Conclusion
BioPharma Compass 2021 workflows, combined with ultrahigh-resolution QTOF and Middle-Down sequencing, enable efficient screening and confirmation of antibody clipping variants with sub-2 ppm mass accuracy.
This approach streamlines proteoform analysis and bolsters the monitoring of critical quality attributes for therapeutic antibodies.
References
- Resemann A., et al. Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing. MAbs. 2016;8(2):318–30.
- DrugBank. Vedolizumab (DB09033).
Content was automatically generated from an orignal PDF document using AI and may contain inaccuracies.
Protein Clipping Variant Analysis of Vedolizumab
using dedicated workflows in BioPharma Compass
Proteolytic degradation of biopharmaceuticals during their life cycle can pose health
risks and require dedicated analysis.
Authors: Christian Albers 1, Detlev Suckau 1, Tomasz Gozdziewicz 2;
1
Bruker Daltonics, Bremen, Germany; 2 Polpharma Biologics S.A., ul. Trzy Lipy 3, 80-034 Gdansk, Poland.
Keywords:
Proteoform analysis,
protein degradation,
enzyme specificity,
Middle-Up, Middle-Down,
sequence analysis,
Elute_UPLC, maXis II
ETD, BioPharma
Compass 2021
Abstract
In this work we used enzy-
matically introduced antibody
truncation variants as a case
study. These clipping variants
were detected based on accurate
mass measurements using a
maXis II ETD and confirmed by
LC-ETD and LC-CID Middle-
Down protein sequencing.
Typically, ETD yielded higher
sequence coverages of the
proposed clipping candidates.
However, CID Middle-Down
spectra provided sufficiently
long terminal sequence tags to
confirm candidates as well.
Dedicated clipping variant ana-
lysis workflows in BioPharma
Compass
2021 enabled the
detection and confirmation of
these variants rapidly and easily.
Introduction
For Biopharmaceuticals, degra-
dation of the active drug
substance during production,
formulation or storage poses a
threat to drug efficacy and safety
and is a critical quality attribute
(CQA). The detection of such trun-
cated protein species – clipping
variants – can be difficult to
achieve through classic peptide
mapping with trypsin.
Vedolizumab is a recombinant,
humanized IgG1 monoclonal antibody
directed against the human lympho-
cyte α4β7 integrin, a key mediator
of gastrointestinal inflammation. It is
used in the treatment of moderate
to severe active ulcerative colitis and
Crohn's disease.
In this case study, a Vedolizumab
biosimilar was characterized after
SpeB treatment, which cleaves IgG1
predominantly in the hinge region at
...KTHT | CPPCPAPE... corresponding
to T229|C230 on Vedolizumab’s
heavy chain. Minor cleavage products
with uncharacterized specificity have
also been observed.
We analyzed the reduced SpeB
digest products with LC-MS using
an ultrahigh resolution maXis II ETD
QTOF instrument to characterize
the enzyme specificity and evaluate
the performance of the BioPharma
Compass
2021 clipping variants
detection workflow. Middle-Down
ETD and CID sequence validation
measurements were subsequently
performed to confirm the findings.
Experimental
Samples
A Vedolizumab biosimilar candidate
drug substance (Polpharma Biologics)
was measured after treatment with
FabULOUS and IgGZERO enzyme
kits (Genovis) followed by reduction
using TCEP. Concentration after di-
lution was approximately 0.5 mg/mL.
The resulting major subunits are
named following the usual IgG sub-
unit nomenclature: Fd, Fc/2 and LC.
Figure 1: Experimental design for the Vedolizumab protein clipping analysis. SpeB digestion produced Fab and Fc fragments. After reduction Fd, Fc, and LC
subunits were analyzed by LC-MS, yielding the major expected subunits plus some low abundant fragments. Protein clipping analysis in BioPharma Compass
provided candidates based on accurate mass and isotope pattern. They were confirmed by direct sequence analysis of the clipping product candidates by
ETD and CID.
F
ab
F
d
... PKSCDKTHT
1 FabULOUS (SpeB) digest
in the hinge region
2 Reduction of disulfide
cross links
3 LC-MS
Intact mass based
clipping variant screening
4 CID + ETD Middle-Down
verification of clipping
variant candidates
CPPCPAPELAGAPSV ...
F
c
F
c/2
Figure 2: Top: Total Ion Chromatogram of the SpeB-digested, reduced and deglycosylated Vedolizumab
with the annotated subunits peaks 5 - 7 . Peaks 1 - 4 were further characterized as putative protein
clipping variants, which elute earlier in the chromatogram.
1 2
3
4
5
6
7
S
ig
na
l in
te
nsi
ty
x107
13.5
14.5
15.5
18.5
16.5
19.5
17.5
13
14
15
18
16
19
17
20
Time [min]
2.0
1.0
3.0
4.0
5.0
6.0
0
Fc/2 NF_15.24
LC_16.85
Fd pQ_18.78
LC-MS and MS/MS
A Bruker Elute UPLC with Waters
Protein BEH C4, 300Å 1.7 µm,
2.1 x 100 mm column was used for
the protein separation.
Solvents: A: 0.1% Formic Acid in Water;
B: 0.1% Formic Acid in ACN
Gradient: 0 min 10% B
1.5 min 10% B
2.5 min 15% B
34 min 45% B
35 min 95% B
40 min 95% B
40.2 min 10% B
45 min 10% B
A Bruker maXis II ETD Ultrahigh
Resolution QTOF was used for
MS spectra acquisition in the
150-2800 m/z range. The samples
were also subjected to ETD and CID
fragmentation for Middle-Down
protein sequence analysis using a
mass range from 100-2400 m/z.
The precursor ion intensity for ETD
experiments was controlled in all
experiments using these parameters:
ICC Target Intensity Controlling:
1000 Mio
ICC Time Accumulation Analyte
min: 1 ms
ICC Time Accumulation Analyte
max: 600 ms
Injection Time: 16 - 22 ms
Extended Reaction time: 4 - 6 ms
For CID, a 50/50 Stepping with 25 eV
and 30
eV collision energy was
applied to all precursor ions.
Data Analysis
All datasets were transferred to
BioPharma Compass 2021 for
data processing and analysis. The
Vedolizumab LC and HC sequences
were imported from [1] as FASTA
file. N-terminal pyro Q was defined
as variable modification together
with G0F, G1F and G2F as major
glycoforms. The MAM Protein
Screening workflow was used
for intact mass analysis of the
SpeB proteolysis products and the
Top-Down Sequencing ESI work-
flow for the sequence analysis of the
CID and ETD spectra. Both work-
flows allowed to specifically analyze
protein clipping variants. All molecular
weights calculated were based
on the monoisotopic masses
determined using the SNAP
algorithm in BioPharma Compass.
Results
The regular SpeB cleavage products
The cleavage products resulting
from Vedolizumab IgGZERO
deglycosylation, SpeB treatment and
reduction predominantly yielded the
expected Fd [1-229], Fc/2 [230-450]
and LC [1-219] subunits (peaks 5-7).
In addition 4 additional peaks (1-4)
were observed by LC-MS analysis
(Figure 2).
Chromatographic peaks 5-7 contained
predominantly – but not exclusively,
see Figure 9 – the expected subunits
as confirmed by the maximum
entropy-deconvoluted mass spectra
(Figure 3).
Figure 3: MaxEnt deconvoluted MS spectra of chromatogram peaks 5 - 7 , containing the IgGZERO deglycosylated Fc/2, the LC and pyro-glutamylated Fd
subunit. All spectra were isotopically resolved and the calculated isotope patterns (red, LC in peak 6) show perfect alignment with the mass spectra. Minor
additional peaks are indicative of, e.g., potential protein clipping products of the drug sample (see Figure 9). Monoisotopic molecular weights are annotated
to the peaks.
5
6
7
In
te
nsi
ty
In
te
nsi
ty
x106
x105
23,900
25,000
23,910
25,100
23,920
25,200 25,300 25,400
Da
Da
0.2
0.2
0.1
0.1
0.3
0.3
0.4
0.4
0.5
0.5
0.6
0.6
0.8
0.8
0.7
0.7
0.9
0.9
1.0
1.0
0
0
In
te
nsi
ty
x105
24,300 24,400 24,500 24,600 24,700
Da
1.00
0.50
0.75
1.00
1.25
1.50
1.75
0
25142.528
Fc/2 NF
23891.777
LC
24576.039
Fd pQ
Figure 4: Top: ETD Middle-Down sequencing spectrum of the Fd subunit in peak 7; c-, y- and z+1 fragment ions were annotated. Center: Sequence coverage
map of the ETD fragments (blue); top row of bricks represents observed c-ions, bottom row represents matching y- and z+1-ions. Bottom: Sequence
coverage map of the CID fragments (red) from the CID spectrum (not shown); top row of bricks represents observed a- and b-ions, bottom row represents
matching y-ions. The yellow bricks indicate tolerated gaps in the observed fragment ion ladders for the calculation of the SVP [2].
In
te
nsi
ty
x103
1000
G
D
y
z+1
z+1 (13)
z+1 (21)
z+1 (34)
z+1 (45)
z+1 (49)
z+1 (2)
D
D
D
T
T
Y
L
N
G
S
S
S S
E V
V
V
V
K
K
K
K
K
K
K
c (27)
c (31)
c
A S V K V S
S G Y
T
S
Y
R
L
I G
W
2000
3000
4000
5000
500
1500
2500
3500
4500
5500
m/z
0.50
0.25
0.75
1.00
1.25
0
1
2
3
Figure 5: MaxEnt deconvoluted MS spectra of chromatogram peaks 1 - 3 . Some clipping candidates are annotated, some aren’t even visible here such as
HC [246-450] in peak 3 at 23605.831 Da (∆Mr -0.86 ppm).
In
te
nsi
ty
x105
12,800
12,400
12,600
Da
Da
1.00
0.50
0.75
1.00
1.25
1.50
1.75
0
In
te
nsi
ty
x104
12,200
12,600
13,000
12,400
12,800
Da
3.0
1.0
2.0
4.0
5.0
6.0
7.0
0
In
te
nsi
ty
x105
11,000
13,000
12,000
14,000
3.0
1.0
2.0
4.0
5.0
0
12397.121
LC [68-181]
12397.127
LC [68-181]
12698.287
HC [338-450]
12698.292
HC [338-450]
12269.029
HC [342-450]
Subsequent LC-ETD and LC-CID ana-
lyses of each of the chromatographic
peaks provided sequence information
of the fragmented protein subunits,
which were automatically calculated
based on the Vedolizumab protein
sequence. Sequence Coverage (SC)
and Sequence Validation Percentage
(SVP) [2] were determined; the SVP
calculation tolerated a terminal gap
size of 15 and internal gap size of 3
missing fragments in the ion series.
The observed SVP and SC values
obtained for ETD were Fc/2:41/27%,
LC: 60/43% and Fd:48/36%, and for
CID were Fc/2:36/29%, LC:47/38%
and Fd:28/21%, respectively. In
average, 50% SVP were obtained from
LC-ETD analysis for the 3 subunits
and 37% SVP from LC-CID. The
results confirmed the Vedolizumab
subunit sequence even in case of the
weakest spectra of the Fd (Figure 4).
Clipping variant analysis
The initial list of proposed clipping
candidates based on intact mass
can be quite extensive if somewhat
relaxed mass tolerances, e.g., 10 ppm
in this case, are applied to match
sequence candidates (Figure 6).
However, the applied calibration
provided for a mass accuracy better
than 2 ppm, which coincided with
the only sequence identified by ETD
analysis in peak 2 (Figure 7). LC
[68-181] in Figure 5, amongst other
candidates, was ruled out based on
poor mass accuracy (Figure 6) and
the absence of a match with the
respective ETD spectrum.
Figure 6: Proposed clipping variants in peak 2 after application of a 10 ppm mass tolerance. The 2
entries at 12698.3 Da. with <2 ppm errors are the best candidates and were further analyzed by ETD
and CID.
Figure 7: ETD spectrum of the 12698.3 Da protein in peak 2 matched the sequence HC [338-450]. None of the other candidate sequences at 12698.3 Da
were identified by Middle-Down sequencing.
In
te
nsi
ty
x103
250
750
1250
1750
2250
2750
3250
3750
500
1000
1500
2000
2500
3000
3500
m/z
0.50
0.25
0.75
1.00
1.25
0
K
K
A
G
c (8)
c (11)
c (17)
c (22)
z+2 (32)
z+1 (30)
z+1 (27)
z+1 (25)
z+1
z+2
c (15)
R
V
T
R
Q
Y
S
D
E
G
S
N
Q
L
y
K
H
Q
Q
L
T
K
N
V
Q
S
I
c
The HC [342-450] clipping product
at 12269.029 Da in peak 3 was
confirmed by ETD and CID, the CID
spectrum is shown in Figure 8.
The Mr 24348.231 base peak in the
MaxEnt spectrum of Peak 4 was
confirmed by ETD as HC [238-450]
with a 29% SVP (data not shown).
In addition, peaks were proposed as
clipping candidates, which weren’t
directly confirmed by Middle-Down
sequencing but represented the
N-terminal and C-terminal fragments
of the same cleavage sites:
HC [1-227]|HC [228-450] and
HC [1-228]|HC [229-450], adjacent
to the theoretical SpeB cleavage site
HC [1-229]|HC [230-450] (Figure 10).
All 4 peaks were matched with an
average mass accuracy of 0.8 ppm
and well matching isotope patterns.
Figure 8: CID spectrum of the 12269.029 Da protein in peak 3 matching the sequence HC [342-450]. None of the other candidate sequences at 12698.3 Da
were identified by Middle-Down sequencing.
In
te
nsi
ty
m/z
1.0
500
1500
3000
4000
5000
6000
7000
8000
1000
2500
3500
4500
5500
6500
7500
2000
2.0
x104
0
Figure 9: Intact mass spectra from chromatographic peaks 5 (top) and 7 (bottom). The annotated peaks show cleavage behind positions HC 227 and HC 228.
All peaks show a perfect overlap with the calculated isotope pattern (red) with a mass error between 0.4 and 1.5 ppm. The HC [228-450] was matched by
the SNAP algorithm even against a significant background of chemical noise.
In
te
nsi
ty
x105
24,800
24,900
25,000
25,100
25,200
25,300
25,400
25,500
Da
Da
1.0
2.0
0
15.24
25243.572
HC [229-450]
25380.648
HC [228-450]
In
te
nsi
ty
x105
24,100
24,200
24,300
24,400
24,500
24,600
1.0
2.0
0
18.78
24337.957
[1-227] Gln->pyro-Glu
24475.019
HC [1-228] Gln->pyro-Glu
G
V Y
A V
a
b
G Q
Q V
V
Y
S
A
F
P
D
W
E
E
I
V
Y T
T
L
b (31)
L
L
C
P
S
b (11)
P
H
F
D
Y L
L
F F
D
P
E
E
Q G N
V
S
P
D
G
S
S
y (47)
y (52)
y (67)
H
Y
T
N
y
y (3)
y (18)
y (23)
S
Q
Summary
Three LC-MS runs of SpeB-digested
and reduced Vedolizumab (LC-MS,
LC-ETD, LC-CID) confirmed the
expected cleavage site in the hinge
region. In addition, the combined
approach to qualify candidates by
intact mass measurements for
Middle-Down sequencing yielded
6 more, minor, cleavage sites, 3 of
which were confirmed by direct ETD
and CID analysis (Figures 7, 8), 2 were
validated by their complementary
nature and mass accuracy (Figure 9,)
and one by mass accuracy and
isotopic fidelity alone, in the absence of
alternative possible matches (Figure
caption 5). The results are summarized
in Figure 10. In case of Vedolizumab,
ragged ends of the Fc/2 and the Fd
were obtained in the hinge region;
and an overall of 6 unspecific
cleavage sites were observed with
the implemented new workflows in
BioPharma Compass.
The identity and amount of the
observed cleavage products
were comparable between the
Vedolizumab originator and bio-
similar drug substances (data not
shown), which indicates that clipping
analysis might become a useful
protein-based MAM approach
in comparability, stability and aging
studies.
Conclusions
• A workflow was described to identify antibody
clipping variants based on subunit mass
measurements and Middle-Down sequencing.
• A mass accuracy of well below 2 ppm was
important to provide sufficient specificity to define
candidate sequences. This was achieved with
monoisotopic mass assignments using the SNAP
algorithm.
• ETD and CID on the maXis II ETD UHR-QTOF
were suitable to verify all clipping variants
subjected to MS/MS analysis, though ETD typically
provided 20-35% more sequence information.
• BioPharma Compass 2021 Clipping workflows,
both for the intact mass-based prediction as well
as for the Middle-Down sequencing facilitated the
prediction and confirmation of clipping variants
significantly to speed up potential drug substance
clipping analysis.
Figure 10: Vedolizumab HC sequence from [1] with the confirmed main cleavage site (green), CID/ETD-confirmed sites (orange) and sites where the
identification was based on accurate mass and isotopic pattern matching (red).
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For Research Use Only. Not for Use in Clinical Diagnostic Procedures.
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References
[1] www.drugbank.ca/drugs/DB09033
[2] Resemann A, et al. (2016). Full validation of therapeutic antibody sequences by middle-up mass
measurements and middle-down protein sequencing. MAbs. 8(2):318-30. www.ncbi.nlm.nih.gov/
