Mass Spectrometry-Based Glycoprofiling of Biopharmaceuticals by Using An Automated Data Processing Tool - SimGlycan®

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Chromatography · Electroseparation
3 2023
Biomedicine · Foods · Environment

SEPARATION
Applications
Methods
ISSN 1615-9306 · JSSCCJ 46 (3) 2023 · Vol. 46 · No. 3 · February 2023
SCIENCE
JO U R N A L O F
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16159314, 2023, 3, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/jssc.202200521 by University of Queensland Library, Wiley Online Library on [20/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Received: 29 June 2022 Revised: 15 November 2022 Accepted: 22 November 2022
DOI: 10.1002/jssc.202200521
RESEARCH ARTICLE
Mass spectrometry-based glycoprofiling of

biopharmaceuticals by using an automated data processing
tool: SimGlycan
R
Amita Puranik1 Rupanjan Goswami2 Purushottam Sutar3 Devika Tupe3

Pratap Rasam3 Prajakta Dandekar4 Ratnesh Jain1
1 Department of Chemical Engineering,

Institute of Chemical Technology, The therapeutic and immunological properties of biopharmaceuticals are gov-
Mumbai, India erned by the glycoforms contained in them. Thus, bioinformatics tools capable
2 PREMIER Biosoft, San Francisco,
of performing comprehensive characterization of glycans are significantly impor-
California, USA
3 Shimadzu
tant to the biopharma industry. The primary structural elucidation of glycans
Analytical (India) Private
Limited, Mumbai, India using mass spectrometry is tricky and tedious in terms of spectral interpreta-
4 Departmentof Pharmaceutical Sciences tion. In this study, the biosimilars of a therapeutic monoclonal antibody and
and Technology, Institute of Chemical an Fc-fusion protein with moderate and heavy glycosylation, respectively, were
Technology, Mumbai, India
employed as representative biopharmaceuticals for released glycan analysis
Correspondence using liquid chromatography–tandem mass spectrometry instead of conven-
Prajakta Dandekar, Department of tional mass spectrometry-based analysis. SimGlycan R is a software with proven
Pharmaceutical Sciences and Technology,
Institute of Chemical Technology, ability to process tandem MS data for released glycans could identify eight
Matunga, Mumbai 400019, India. additional glycoforms in Fc-fusion protein biosimilar, which were not detected
Email: pd.jain@ictmumbai.edu.in
during mass spectrometry analysis of released glycans or glyco-peptide mapping
Ratnesh Jain, Department of Chemical of the same molecule. Thus, liquid chromatography–tandem mass spectrom-
Engineering, Institute of Chemical etry analysis of released glycans not only complements conventional liquid
Technology, Matunga, Mumbai 400019,
chromatography–mass spectrometry-based glycan profiling but can also identify
India.
Email: rd.jain@ictmumbai.edu.in additional glycan structures that may otherwise be omitted during conven-
tional liquid chromatography–tandem mass spectrometry based analysis of
mAbs. The mass spectrometry data processing tools, such as PMI Byos™,
SimGlycanR
, etc., can display pivotal analytical capabilities in automated liquid
chromatography–mass spectrometry and liquid chromatography–tandem mass
spectrometry-based glycan analysis workflows, especially for high-throughput
structural characterization of glycoforms in biopharmaceuticals.
KEYWORDS
Fc-fusion protein, glycoprofiling, monoclonal antibodies, SimGlycan
R
List of abbreviations: ADCC, antibody dependent cell cytotoxicity; Asn, asparagine; CDC, cell dependent cytotoxicity; CDR, complementarity
Determining Region; CQA, critical-quality-attribute; DTT, dithiothreitol; HIC, hydrophobic interaction chromatography; IAA, iodoacetamide; mAb,
monoclonal antibody; MAM, multi-attribute-monitoring; PGC, porous graphitic column; PTM, posttranslational modification; TIC, total ion
chromatogram; VEGFR, vascular endothelial growth factor receptors.
J Sep Sci 2023;46:2200521. www.jss-journal.com © 2022 Wiley-VCH GmbH. 1 of 9

https://doi.org/10.1002/jssc.202200521
2 of 9 PURANIK et al.
1 INTRODUCTION glycoforms detached from their peptide or protein back-

bone. MS-based primary structural elucidation of glycans
Glycosylation is an important post-translational modifica- is tricky and tedious in terms of spectral interpretation.
tion (PTM) that influences mechanism of action, stability, Moreover, the analysis also requires awareness about the
safety, and efficacy of the therapeutic proteins [1]. The anomeric linkages, carbohydrate sequences, and the frag-
structure and expression of glycans is widely influenced mentation pattern of monosaccharides under different
by the cell culture process parameters [2, 3]. Thus, it experimental conditions, such as permethylation, reduc-
is considered as a critical quality attribute (CQA) that ing end modifications, adducts used, etc. [6] A software
needs a regular monitoring at different stages of develop- compliant with the use of customized glycan database gen-
ment and manufacturing of biopharmaceuticals [4]. Thus, erated based on in-silico MS/MS fragmentation as well as
to ensure process and product consistency, an in-depth retention time information can support the recognition
and comprehensive characterization of glycosylation in of glycans beyond restricted entries in reference database.
therapeutic proteins is indispensable [1, 3]. Glycans in The precision in glycan characterization is thus, subjected
biopharmaceuticals can broadly be divided into two cat- to multiple factors such as structural properties of the ana-
egories based on the site of attachment, viz. (a) N-glycans lyte, mode of separation, sample preparation strategies,
or oligosaccharides attached to asparagine residue through algorithm used for the acquired data processing, etc. [6, 14]
a nitrogen atom and (b) O-glycans or oligosaccharides In this study, we have revealed how the mode of data
attached to threonine or serine residue through an oxy- acquisition (i.e., MS-alone vs. LC–MS/MS) during released
gen atom. N-glycans are further sub-categorized into three glycan analysis and data processing approaches can play
type’s viz. oligomannose, complex glycans, and hybrid a pivotal role in determining the range of glycoforms that
glycans [1, 2]. may be detected via mass spectrometric analysis. Two rep-
Characterization of glycosylation can be performed at resentative biopharmaceuticals, a mAb and an Fc-fusion
various levels viz. (a) at intact glycoprotein, (b) glycoprotein, with 2–3% and 15–20% of glycosylation, respec-
peptide, and (c) glycosylated protein subunit, and (d) tively were analyzed by released glycan analysis with
released glycan levels. Each of these analytical approach LC–MS and LC–MS/MS. SimGlycan R
, a commercial soft-
can offer solutions for specific questions such as iden- ware for released glycan level analysis, was used to confirm
tification of glycan pairing, micro-heterogeneity, glycan MS1 profile and process MS/MS level data for released
composition, identification of glycosidic linkages, glycan glycan analysis. Glyco-peptide level analysis was also car-
quantitation, etc. [5] However, released glycan analysis is ried out in LC–MS/MS mode to confirm glycosylation
the direct, simplest, and most preferred strategy for glyco- sites and complement the released glycan analysis. The
profiling of majority of therapeutic glyco-proteins, at both glyco-peptide and MS1 level analysis for released glycans
research and commercial levels [6]. Recent advances in gly- demonstrated a good correlation. However, LC-MS/MS
can separation techniques (such as capillary electrophore- level characterization of released glycans from Fc fusion
sis, porous graphitic column chromatography, etc.) and protein supported the identification of eight additional
overall glycoprofiling workflows have been reviewed by glycoforms that were missed during LC–MS-based pro-
Yang and Bartlett [7] and Li et. al. [8]. filing. The study demonstrated critical role of MS level
Informatics-based tools play a critical role in systematic fragmentation and suitable data processing software to
glycan characterization. Thus, numerous databases and obtain comprehensive information, with enhanced ana-
software have emerged over the last couple of decades to lytical confidence that is an under-explored area in the
facilitate glycan data analysis. Some of them have been biopharmaceutical characterization domain.
reviewed and discussed elaborately by Zhang et al. [5]
Walsh et al. emphasize that the software-based annotation
of peaks with specific glycan structure for an LC–MS data 2 MATERIALS AND METHODS
is typically facilitated by two factors viz. (i) the retention
time (RT) on an LC column and (ii) the mass of individ- 2.1 Materials
ual eluted moieties [9]. The software aims at matching
these factors with the reference values in glycan reposi- Commercially available trastuzumab biosimilar - Cann-
tories. The currently preferred softwares for glyco-peptide mAb™, (Biocon Limited, Bengaluru, India) was purchased
and/or released glycan analysis, such as Byonic™ [10], Sug- from a local healthcare supplier. Fc-fusion protein biosimi-
arQb [11], and pGlyco [12], typically rely on built-in glycan lar was provided as a gift sample for research purpose by an
database dependent search algorithms [13]. Indian biopharmaceuticals manufacturing organization.
The structural information acquired from LC–MS/MS LC–MS grade acetonitrile, water, and formic acid were pur-
is enormous for the individual glycan species, that is, chased from Honeywell research chemicals, Germany for
PURANIK et al. 3 of 9
mobile phase preparation. Milli-Q Plus system (electrical The technical replicates of both the analytes were
resistivity 18.2 M cm, Millipore, Bedford, USA was used for imported in the software in Shimadzu Corporation native
getting Milli-Q water that was used for all solutions and file format (.lcd) and subjected to LC–MS peak processing
reagents preparation apart from mobile phase preparation. using an optimized set of peak picking parameters. A com-
Analytical grade urea used during peptide mapping sample plete coverage of the LC compounds was achieved with
preparation, was purchased from Avra Synthesis Pvt. Ltd., three main strategies viz. (i) low threshold for peak pick-
Ahmedabad, India. Iodoacetamide and dl-Dithiothreitol ing, (ii) stringent validations of the LC compounds, and
were procured from Sigma–Aldrich, Missouri, USA. MS- (iii) align compounds across the technical replicates.
grade trypsin was obtained from Promega Corporation,
USA. Amicon R
Ultracel centrifugal ultrafilters (MWCO Low threshold for peak picking
10,000) were obtained from Merck Millipore Ltd., Darm- MS1 threshold intensity of 0.1% with respect to the base
stadt, Germany. ThermoMixer-C from eppendorf (Eppen- peak was permitted to ensure the qualification of wide
dorf, Westbury, NY) was used for continuous mixing range of LC compounds during the generation of poten-
at desired temperature. LC–MS/MS analysis was carried tial candidate’s peak list. In order to avoid the possibility of
out on LCMS-9030 QTOF connected with Nexera-X2 liq- missing any low abundant candidate LC compound, chro-
uid chromatography system from Shimadzu Corporation, matographic peak width in the range of 0.1–2 min and the
Japan. N-glycans release and labeling was carried out by minimum XIC (extracted ion chromatogram) peak height
using AdvanceBio Gly-X InstantPC kit from Agilent Tech- of 1000 (absolute intensity) was permitted. The software
nologies (Agilent Technologies, Santa Clara, CA, USA). was also configured to collate the intensity values of a tar-
Chromatographic separation of glycopeptides and released get compound from majority of the MS1 scans within its
glycans was carried out by using Shim-pack Arata pep- XIC. Collating the intensity values helped the low abun-
tide C18 column (2.2 μm, 100 × 2.0 mm) and AdvanceBio dant compounds to qualify as a candidate compound for
Glycan Map column (2.1 × 150 mm; 1.8 μm), respectively. further validation.
Stringent LC compound validation

2.2 Methods The huge pool of candidate LC compounds generated due
to lower peak picking threshold needed further validation.
2.2.1 Sample preparation and LC–MS In SimGlycan R
, an intensity filter was set in between the
profiling of released glycans isotopologues that ensured picking only the compounds
with acceptable isotopic cluster patterns. Also, the software
Sample preparation and LC–MS-based analysis of released was instructed to detect only those compounds for which
glycans was carried out as reported in our previous at least three peaks [M+0], [M+1], and [M+2] within the
manuscript [15]. “Released glycans IgG” workflow in PMI isotopic envelope were observed. In addition to these, the
Byos™ was used for processing the MS profiling data facility of specifying the charge state range (1 to 3) of the
of released glycans with “N-Glycan 309 mammalian no compounds provided a controlled validation in picking
sodium” reference library. the authentic LC compounds. In Supporting Information
S2, Figure S1.11 represents the optimized set of peak pick-
ing parameters used during SimGlycan R
based released
2.2.2 Released glycan analysis for MS and glycan analysis of the representative biopharmaceuticals.
MS/MS level data by using SimGlycan R
Align compounds across the technical replicates

LC–MS/MS data processing and released glycan analysis The peaklists of the technical replicates of both analytes
were carried out using SimGlycan R
v.5.94 software. Cus- were aligned using m/z and retention time tolerance to
tom databases were created in SimGlycan R
with 14 and 103 discover the unique and universal LC compounds. The
unique compositional glycan structures for trastuzumab MS/MS scans were later clustered with the appropriate
and Fc-fusion protein biosimilars, respectively. A propri- compounds by using a proprietary algorithm.
etary algorithm in the software automatically generated The MS/MS scans clustered under these compounds
the structure-specific in-silico fragments for all the unique were subjected to MS/MS database search using a pre-
constituent glycoforms based on Domon and Costello cursor and product ion error tolerance of 10 and 20 ppm,
nomenclature [15]. In Supporting Information S2, Figures respectively. The experimental MS/MS fragment ions were
S1.9 and S1.10 represent a typical SimGlycan R
user inter- matched against in-silico fragment ions from the target
face, displaying the theoretical/in-silico fragments gener- database. Based on the nature of the fragmentation tech-
ated for A2 and SA2A2 glycan structures, respectively. nique used, only the protonated glycosidic ion species
T A B L E 1 Description of individual protein specific tic glycoproteins has been provided in FASTA format in
N-glycosylation sites confirmed during glyco-peptide mapping level supplementary file-1 [17, 18].
analysis using LC-MS/MS mode
N-Glycosylation Corresponding
Glycoprotein position glyco-peptide 3.1 Trastuzumab biosimilar
Trastuzumab Biosimilar N-300 EEQYNSTYR
VEGFR targeted Fc-fusion N-36 VTSPNITVTLK Both software, that is, PMI Byos™ and SimGlycan R
con-
protein biosimilar N-68 GFIISNATYK firmed the presence of twelve glycoforms in trastuzumab
N-123 LVLNCTAR biosimilar, as depicted in Figure 1. However, MS/MS
N-196 NSTFVR level analysis of the released glycans from trastuzumab
N-282 EEQYNSTYR biosimilar could not detect three glycoforms, namely
HexNAc(2)Hex(3), HexNAc(4)NeuAc(1)Fuc(1)Hex(5)
and HexNAc(4)NeuAc(2)Fuc(1)Hex(5). The failure
(single glycosidic and glycosidic-glycosidic) were consid- to detect sialylated, that is, neuraminic acid contain-
ered for fragment matching. The software was tuned ing glycans [HexNAc(4)NeuAc(1)Fuc(1)Hex(5) and
to report only those candidate glycan structures, which HexNAc(4)NeuAc(2)Fuc(1)Hex(5)] was attributed to
had 80% of their monosaccharide residues explained by the inherent sensitivity of sialylated glycans towards
structure specific characteristic ions in the MS/MS spectra. fragmentation and ionization [19]. Sialylated glycans
Typical SimGlycan R
graphical user interface (GUI) dis- are typically present at a lower extent as compared to
playing the MS/MS Database Search Parameters have been other fucosylated or mannosylated glycans, in case of
provided in Figures S1.12, S1.13, and S1.14 of the Supporting trastuzumab biosimilar [20]. This was also confirmed by
Information S2. observing the lower intensity of these glycans unlike the
The software also facilitated generation of annotated major contributors. Thus, these low contributing glycans
mass spectra for the visual confirmation on accuracy of the may not have given detectable level of fragmentation and
identification. The portable Microsoft Excel report gener- thus were not observed during MS/MS analysis.
ated by the software included detailed information for the The MS/MS profile for two representative glycan struc-
identified glycans and their corresponding structures. tures viz. (Fuc)1 (GlcNAc)3 (Man)3 (i.e., G0F-N acetyl
glucosamine) and (GlcNAc)3 (Man)3(Fuc)1 (Gal)1 (i.e.,
G1F – N acetyl glucosamine) has been provided in sup-
2.2.3 Glyco-peptide level analysis plementary information-2 (figure 1.3 and 1.4, respectively).
Total ion chromatogram (TIC) for the released glycans of
Tryptic digestion based sample preparation, LC–MS data trastuzumab biosimilar has been provided in Supporting
acquisition and data processing by using Byonic v 2.7.2 Information S1 as Figure S1. Of the 12 glycoforms observed
(Protein Metrics Inc., San Carlos, CA) for both the repre- during released glycan analysis, MS profiling confirmed all
sentative biopharmaceuticals was carried out as reported 12 glycans whereas MS/MS analysis confirmed nine gly-
in our earlier manuscript [15]. cans. The identified glycoforms were fairly similar to those
reported in previous studies related to glyco-profiling of
trastuzumab [20].
3 RESULTS AND DISCUSSION The glycopeptide mapping performed for trastuzumab
biosimilar resulted in 94.4% and 96% of amino acid
The released glycan analysis was carried out in both, sequence coverage in the light chain and heavy chain,
LC–MS as well as LC–MS/MS mode. The data analysis respectively [17]. N-glycosylation was observed at N-300
for LC–MS mode was carried out using PMI Byos™ position in EEQYNSTYR peptide of heavy chain, as ver-
and also confirmed by using SimGlycan R
software. ified using previous reports on characterization of this
Byos™ files displayed glycan composition as unresolved molecule [20]. Total ion chromatogram (TIC) with appro-
stereoisomers, such as “Hex” or “HexNAc”. N-glycan priate peak annotation for glyco-peptide mapping of
biosynthesis being highly conserved in humans, the struc- trastuzumab biosimilar has been depicted in Figure S2.
tural identity of these monosaccharides was completed Four type of glycoforms were observed through glyco-
by assigning them to either mannose (Man), galactose peptide level analysis of trastuzumab biosimilar. All the
(Gal), or N-acetylglucosamine (GlcNAc) [16]. Table 1 identified glycans were well within 5% relative standard
presents the individual protein specific N-glycosylation deviation, thus demonstrating repeatability and precision
sites, confirmed by glyco-peptide mapping analysis, using in analysis. One more glycan, HexNAc(2) Hex(5), was
LC–MS/MS. Amino acid sequences for both the therapeu- observed in TKPREEQYNSTYR peptide, with two missed
F I G U R E 1 Glycoforms identified by released glycan analysis of trastuzumab biosimilar at MS and MS/MS levels, using Byos
R and
SimGlycan R
softwares, respectively.
F I G U R E 2 Major glycoforms observed during

glyco-peptide level analysis of trastuzumab biosimilar (Data
presented here represents the average results for the
acquisition carried out for at least two sample replicates).
cleavages. Though the relative percent of this glycoform eters such as cell line properties, media, feed composition,
was >5% in all three replicates, there was significant etc. [16, 17].
inconsistency in terms relative percentage. Released glycans analyzed by MS-profiling and by
Comparison of Figure 1 and Figure 2 demonstrated LC–MS/MS were compared with those identified during
that the released glycan analysis approach, as anticipated, LC–MS/MS based glyco-peptide analysis of trastuzumab
could identify a better number of glycoforms as com- biosimilar. The results demonstrated a good correlation
pared to the four major glycans (i.e., G0F, G0F-N, G1F, between LC–MS/MS analysis of the released glycans, with
and G1F-N) observed during the glyco-peptide approach. glyco-peptide level analysis.
Figure 2 represents the average percent relative abun-
dance of four major glycoforms identified by glyco-peptide
analysis using LC–MS/MS. Majority of these glycoforms 3.2 Fc-fusion protein biosimilar
matched with the studies related to released glycan anal-
ysis of trastuzumab [15]. The minor differences observed In case of Fc-fusion protein biosimilar, totally 44 glycans
in the previously reported glycoforms and those observed were identified through released glycan and glyco-peptide
in this study were attributed to the origin of therapeutic analysis. Out of these, 22 glycans were confirmed through
biosimilar and the obvious variations in bioprocess param- released glycan analysis carried out with MS profiling
F I G U R E 3 Additional glycan structures identified through LC–MS/MS analysis with data processing carried out by using SimGlycan
R
in VEGFR targeted Fc-fusion protein.
mode and confirmed by both software, that is, PMI Byos™ quantification of N-glycans in biopharmaceuticals. Perme-
as well as SimGlycan R
. Glyco-peptide mapping (in LC– thylation of N-glycans has been reported as a promising
MS/MS mode) and released glycan analysis (in LC–MS strategy to stabilize glycans prior to MS-based analysis.
mode) of Fc-fusion protein, using PMI Byos™ software for It is reported to offer added benefits such as improved
data processing, has been previously reported for devel- MS signals for such sensitive, low abundant glycoforms
opment and optimization of a multi-attribute-monitoring [21]. Table 2 provides a summary of glycoforms observed
(MAM) workflow for characterization of CQAs in Fc- in Fc-fusion protein biosimilar during glyco-peptide and
fusion protein [15]. Thus, in order to avoid a data overlap released glycan analysis conducted by LC–MS/MS.
with our earlier investigation, only the additional data, The discrepancy in terms of number of glycans observed
that is, the data processed with SimGlycan R
software was attributed to the variation in the mode of analysis
has been reported in this manuscript. LC–MS/MS data i.e. glycopeptide vs. released glycan analysis, and differ-
of released glycans processed with SimGlycan R
software ent modes of separation, that is, RP vs. HILIC, etc. Thus,
could identify eight additional glycoforms, which were not both, the analytical approach as well as an appropriate
detected during MS-profiling of released glycans or glyco- data processing software are critical especially in case
peptide mapping. These additional glycans have been of heavily glycosylated therapeutic glyco-proteins, such
demonstrated in Figure 3. as Fc-fusion protein, described in this study. Past few
Majority of the additional unique glycoforms identified years have witnessed enormous development in glycan
in Fc-fusion protein biosimilar with LC–MS/MS analy- analysis technologies and overall knowledge about pro-
sis of released glycans, followed by data processing using tein glycosylation. Thus, in glycosylation analysis, it has
SimGlycan R
software, were either fucosylated or con- become a consensus that the question dictates the exact
tained neuraminic acid. Fucosylated glycans have critical approach that needs to be followed. [5] However, a com-
role in determining the stability and functional proper- prehensive understanding about glycan diversity at early
ties of biopharmaceuticals. Sialylation also plays a critical product development stage is a key factor to successful
role in ADCC (antibody dependent cell cytotoxicity) activ- development of safe and efficient biopharmaceuticals [22].
ity as well as half-life of the biotherapeutics. [21] Some of As highlighted by Sun et.al., in glycomic analysis, it is
the sialylated glycans demonstrated similar properties as convenient to rely on a comprehensive database contain-
explained in case of trastuzumab biosimilar i.e. there were ing accurate molecular weights (MWs) and compositional
3 sialylated glycoforms observed during MS profiling and information about all probable glycans for mass search-
confirmed by both, PMI Byos™ as well as SimGlycan R
ing. However, an authentic spectral database can only
MS profiling, but were not detected during MS/MS frag- be established based on a large number of experiments,
mentation. [19] The loss of information about sialylated using a variety of samples, making this approach both
glycans during LC–MS/MS could lead to bias in precise time and labor intensive. A review article by Rojas-Macias
T A B L E 2 Summary of glycoforms observed in VEGFR targeted Fc-fusion protein during glyco-peptide and released glycan level analysis
(Data presented here represents the average results for the acquisition carried out for at least two sample replicates)
Observed during released glycan analysis
Observed during Data processed using Data processed using
Glycan Moiety Glyco-peptide analysis PMI (MS) SimGlycan (MS/MS)
N2H3 (HexNAc)2 (Hex)3 No Yes Yes
N4H3 (HexNAc)4 (Hex)3 No Yes Yes
N4H3F1 (HexNAc)4 (Hex)3 (Fuc)1 Yes Yes Yes
N2H4 (HexNAc)2 (Hex)4 Yes Yes Yes
N4H4F1 (HexNAc)4 (Hex)4 (Fuc)1 No Yes Yes
N3H4NA1 (HexNAc)3 (Hex)4 (NeuAc)1 No Yes Yes
N4H5F1 (HexNAc)4 (Hex)5 (Fuc)1 Yes Yes Yes
N4H6F1 (HexNAc)4 (Hex)6 (Fuc)1 No Yes Yes
N4H5F1NA1 (HexNAc)4 (Hex)5 (Fuc)1 (NeuAc)1 No Yes Yes
N4H5F1NA2 (HexNAc)4 (Hex)5(Fuc)1 (NeuAc)2 No Yes Yes
N5H6F1NA1 (HexNAc)5 (Hex)6 (fuc)1 (NeuAc)1 No Yes Yes
HexNAc(3)Hex(6) Yes No Yes
(Fuc)1 (Gal)1 (GlcNAc)3 (Man)3 (NeuAc)1 No No Yes
(Gal)1 (GlcNAc)3 (Man)5 (NeuAc)1 No No Yes
(Fuc)1 (Gal)1 (GlcNAc)3 (Man)3 No Yes Yes
(Gal)1 (GlcNAc)3 (Man)5 No No Yes
(Gal)1 (GlcNAc)3 (Man)4 (NeuAc)1 No No Yes
(Fuc)1 (GlcNAc)3 (Man)3 No No Yes
Man6 (H6N2) No No Yes
HexNAc(3)Hex(4)Fuc(1) Yes No Yes
HexNAc(4)Hex(5)Fuc(1) Yes No No
HexNAc(3)Hex(3)Fuc(1) Yes No Yes
N4H5NA1 (HexNAc)4 (Hex)5 (NeuAc)1 Yes Yes No
N5H6NA2 (HexNAc)5 (Hex)6 (NeuAc)2 No Yes No
N6H7F1NA3 (HexNAc)6 (Hex)7 (fuc)1 (NeuAc)3 No Yes No
et al. sheds light on limitations of the existing analyti- A.P. has performed data analysis and written first draft
cal approaches for glycan characterization and the current of the manuscript. R.G. processed the data and assisted
landscape of bioinformatics infrastructure for N and O in drafting the manuscript. P.R. and R.J. supervised and
linked glycomics [23]. Thus, the use of customized in- reviewed the manuscript. R.J. and P.D. supported with
silico fragmentation-based database can complement in necessary funding for resources, reviewed and edited the
cases where information about mass-alone (i.e., MS1) is manuscript.
insufficient for accurate annotation of glycan structure.
Tandem MS level analysis can offer detailed information, AC K N OW L E D G M E N T S
with masses of fragments, when some of the isobaric struc- The authors express gratitude to the analytical scientists
tures have nearly same precursor masses and thus create from Indian biopharmaceuticals manufacturing organiza-
ambiguity during structural annotation. In our study, there tions for providing the insightful suggestions. The authors
was no presence of isobaric glycan structures. However, are thankful to Premier Biosoft Ltd. for critical support in
in an application note by Agilent technologies, tandem SimGlycan R
software.
MS based identification of isobaric structures (NeuGc +
fucose isobaric with NeuAc + galactose) has been sys- CONFLICT OF INTEREST
tematically described [19]. Reyes et al. have reviewed the The authors declare that they have no conflict of interest.
mass spectrometric strategies for separation and charac-
terization of isomeric forms of glycopeptides and glycans FUNDING
[24]. Thus, software tools such as SimGlycan R
, which A.P. is grateful to the Department of Science and Tech-
supports the analysis of tandem MS data for glycans, are nology for the Prime Minister’s Fellowship Scheme from
anticipated to complement the glycan characterization of SERB-CII. A.P., R.J., and P.D. are thankful to the Depart-
biopharmaceuticals with a new dimension. ment of Science and Technology, Govt. of India, for the
DST FIST grant. A.P., R.J., and P.D. are thankful to Shi-
madzu Analytical (India) Private Limited and Spinco
4 CONCLUDING REMARKS Biotech Pvt. Ltd. for supporting this work with QTOF
instrumental collaboration.
Comprehensive analysis of glycan diversity is very impor-
tant at almost every stage of biopharmaceuticals devel- D A T A AVA I L A B I L I T Y S T A T E M E N T
opment. Stages such as clone selection, optimization of The data that supports the findings of this study are
media, feed, and bioprocess parameters are more prone available in the supplementary material of this article.
to result in variation in glycosylation. MS-based analysis
of released glycans largely relies on software and database ORCID
for complex data interpretation. While progress has been Prajakta Dandekar https://orcid.org/0000-0001-5968-
made regarding development of analytical reagents and 1072
methods for characterization of glycans, informatics-based
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Mass Spectrometry-Based Glycoprofiling of Biopharmaceuticals by Using An Automated Data Processing Tool - SimGlycan®

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16159314, 2023, 3, Downloaded from https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/jssc.202200521 by University of Queensland Library, Wiley Online Library on [20/03/2023].

Biomedicine · Foods · Environment

Mass spectrometry-based glycoprofiling of

Amita Puranik1 Rupanjan Goswami2 Purushottam Sutar3 Devika Tupe3

1 Department of Chemical Engineering,

J Sep Sci 2023;46:2200521. www.jss-journal.com © 2022 Wiley-VCH GmbH. 1 of 9

1 INTRODUCTION glycoforms detached from their peptide or protein back-

Stringent LC compound validation

Align compounds across the technical replicates

F I G U R E 2 Major glycoforms observed during

in VEGFR targeted Fc-fusion protein.

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