Comprehensive N-Glycan Profiling of Cetuximab Biosimilar Candidate by NP-HPLC and MALDI-MS
Comprehensive N-Glycan Profiling of Cetuximab Biosimilar Candidate by NP-HPLC and MALDI-MS
Sheng Liu 0 1
Wenjie Gao 0 1
Yao Wang 1
Zhenyu He 1
Xiaojun Feng 0 1
Bi-Feng Liu 0 1
Xin Liu 0 1
0 Britton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics & Molecular Imaging Key Laboratory, Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology , Wuhan , China , 2 Wuhan Centers for Disease Prevention and Control , Wuhan , China
1 Editor: Roger Chammas, Universidade de Sao Paulo , BRAZIL
Monitoring glycosylation of the mAbs have been emphasized and routinely characterized in biopharmaceutical industries because the carbohydrate components are closely related to the safety, efficacy, and consistency of the antibodies. In this study, the comprehensive glycan profiling of a biosimilar candidate of cetuximab was successfully characterized using Normal phase high-performance liquid chromatography (NP-HPLC) in combination with Matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The presence of minor N-linked glycans containing sialic acid lactone residues (NeuAcLac) was observed in the biosimilar for the first time, which could influence the quantitative analysis of sialylated glycans and interfere with quantification of neutral glycans when it was analyzed by high performance liquid chromatography fluorescence (HPLC-FL). To overcome this issue, mild alkali treatment was used to hydrolyze lactone of the sialic acid to their neutral formation, which had no impact on the analysis of other glycans before and after the treatment. As a result, the mild alkali treatment might be helpful to obtain quantitative glycan profiling of the mAbs drugs with enhanced accuracy and robustness.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The authors gratefully acknowledge the
financial support from the National Natural Science
Foundation of China (20905027 and 81402198).
W.J.G. also thanks the financial support from the
Independent Innovation Fund of HUST
Competing Interests: The authors have declared
that no competing interests exist.
Therapeutic recombinant monoclonal antibody (mAbs) drugs have emerged as a clinically
important drug class, and more than 30 therapeutic antibodies have been approved for clinical
]. However, development of biosimilars is becoming a trend due to the coming off-patent
of approximate 50% alternative of the existing mAbs and the expensiveness of the production
and characterization of mAbs. All currently approved mAbs are based on IgGs and are most
usually produced with the use of mammalian expression systems, such as mouse myeloma
NS0, Chinese hamster ovary (CHO), and mouse myeloma Sp 2/0 cell lines [2±4]. Typical
mAbs are comprised of two identical light chains and two identical heavy chains subunits
interconnected by intramolecular disulfide bonds (Fig 1A). A conserved N-glycosylation site
was contained in the CH2 domain at Asn297 and about 30% of polyclonal human IgG
molecules bear N-linked oligosaccharides in the Fab region [5±7].
It has been well documented that the attached N-glycans on mAbs play crucial roles in many
biological and physicochemical processes such as enhancement of the structural integrity,
resistance against protease, effectiveness of serum half-life in vivo, and antibody-dependent cellular
cytotoxicity [8±12]. For example, the sialic acids, a family of 9-carbon monosaccharides found
as terminal residues on many glycan structures attached to glycoproteins, were closely related to
some biological process such as cell±cell adhesion, cell surface receptor recognition, and
progression of human malignancies [13±15]. In addition, non-human N-glycans could be
presented in the non-human expression systems which will be a considerable risk in relation to
immunogenicity and possible factor for incidence of some disease, and possible masking of
existing antigenic sites on the peptide backbone causes a crucial side effect or insufficient
]. Therefore, N-glycan profiling analysis of the mAbs is growing in importance and
becoming a critical process in antibody characterization and regulatory evaluation [
High-performance liquid chromatography (HPLC) has been a conventional method for
routine monitoring of N-glycosylation during process development and quality control of
]. However, it suffers from unstable identification of structurally diverse glycans
and could not provide direct information on glycan structure. Modern biological mass
spectrometry (bio-MS) techniques based on soft ionization methods, such as matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS) has provided a powerful platform for
the qualitative analysis of glycans from mAbs, thanks to its unique features of enhanced
sensitivity, high throughput and high-molecular-weight detection abilities [
inaccurate glycan quantification could occur because the ionization efficiency was different for
neutral and acid glycans [
]. Thus, several orthogonal techniques must be used to identify
and quantify glycoforms, glycosylation profiling and carbohydrate contents of the mAbs.
In this study, we determined the glycan profiling of a biosimilar of cetuximab, which was
produced in CHO cell lines in our lab, in a detailed structure and quantitative manner using
NP-HPLC and in combination with MALDI-MS. However, several abnormal N-linked glycans
containing NeuAcLac residues were observed in the biosimilar by MALDI-MS which was not
reported previously. NanoLC-ESI-MS/MS was employed to elucidate the detailed structural
information of minor aberrant glycans. It should be noted that even the amount of the unusual
glycans were limited, their existence directly impacted on the accuracy of the quality control.
In addition, the aberrant glycans were relatively stable after an overnight digestion in slight
alkali buffer. Therefore, ammonium hydroxide solution (pH 10.0) was employed to transform
the NeuAcLac containing glycans to their natural forms, which could be removed by rotary
concentrator (as shown the flowchart of our method in Fig 1B). More importantly, it could not
influence the quantitative analysis of total glycan pattern. With this simple treatment, a more
accurate and robust quantification of glycans was obtained for the biosimilar. From the results
obtained in this study, we strongly believe that the mild alkali treatment might be a
prerequisite step for not only the accurate quantitative analysis but also the qualitative analysis of the
glycans in therapeutic antibodies.
2. Materials and Methods
2.1. Chemicals and reagents
The biosimilar of cetuximab (IgG1 type) used in this paper was expressed in transfected CHO
cells lines by our lab. The biosimilar of bevacizumab was given by Wuhan Xinshengye Co.
Limited (Wuhan, China), which was also expressed by CHO cell lines. Peptide-N-glycosidase
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Fig 1. a) A representative schematic structure of monoclonal antibody and N-glycosylation sites on it. The main
glycan moieties of the Fab and Fc fragment were shown in the frame. Structures and the monosaccharides are
depicted following the CFG notation; b) flowchart of our method in this study.
F (PNGase F) and endoglycosidase buffer kit were obtained from LCP Biomed (Lianyungang,
China). Dimethyl sulfoxide (DMSO), 2-aminobenzoic acid (2-AA), 2-Picoline-borane (2-PB),
trifluoroacetic acid (TFA), porous graphitic carbon (PGC) cartridges, microcrystalline
cellulose (MCC) and ammonium hydroxide were purchased from Sigma-Aldrich (St. Louis, MO,
U.S.A). Glacial acetic acid, ethanol, 1-butanol were obtained from Aladdin (Shanghai, China).
LC-MS grade Formic acid (FA), pure water for NanoLC ESI-MS and PierceTM Fab
Preparation Kit were attained from Thermo Fisher Scientific (Waltham, MA, U.S.A). Cetuximab,
LC-MS grade Acetonitrile (ACN) and methanol were purchased from Merck KGaA
(Darmstadt, Germany). All reagents were HPLC grade and all the reaction solutions were prepared
with water purified by the Direct-Q system (Millipore, Bedford, MA).
2.2. Fragmentation of the cetuximab biosimilar
Fab and Fc fragments of the mAb biosimilar were prepared with PierceTM Fab Preparation Kit
(Thermo Scientific) according to the manufacturer's instruction. Briefly, 200 μL immobilized
papain resin was equilibrated with 200 μL digestion buffer through centrifugation with spin
columns. The equilibration step was repeated for three times. An amount of 200 μg (10mg/
mL) mAbs diluted in 200 μL digestion buffer was added to the equilibrated immobilized
papain and the sample was incubated for 8h with an end-over-end mixer at 37ÊC. After
incubation, the digestion buffer with Fab and Fc fragments was collected by centrifugation at 2000
g and the resin was washed with 100 μL PBS for two times for optimal recovery. The Fab and
Fc fragments was then applied to an equilibrated NAb Protein A Plus Spin Column and
incubated with end-over-end mixing for 10 min. The flow through fraction containing Fab
fragments was collected with a new 1.5 mL collection tube by centrifugation at 2000 g and wash
column with 100 μL PBS for two more times. The Fc fragments was collected by washing the
Protein A Plus Spin Column with IgG elution buffer and also repeated for two more times.
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2.3. N-glycan release and purification
N-glycans of the intact cetuximab, biosimilar, Fab and Fc fragments of the biosimilar were
enzymatically cleaved with N-glycosidase F according to previously published procedure with
little modification [
]. 100 μg of the mAb and cetuximab were dissolved in 90 μL of sodium
phosphate (50 mM, pH 7.5, LCP Biomed, China) containing 0.2% SDS and 10 mM
dithiothreitol. The sample was incubated at 100ÊC for 10 min prior to adding 10 μL of 10% Nonidet P-40.
The reaction mixture was incubated with PNGase F (10 units) for 18 h at 37ÊC. Following
digestion, sample was then boiled for 5min to deactivate the enzyme. The released glycans
were purified using PGC cartridges as previously reported [
]. Briefly, the sample was diluted
with 0.5 mL water and subsequently purified using PGC cartridge. The cartridge was initially
washed with 3 mL of ACN and 3 mL of 80% (v/v) ACN containing 0.1% (v/v) TFA, followed
by 3 mL of water. The sample was then loaded on the PGC cartridge and washed with 3 mL of
water to remove impurity and salts. Finally, sample was eluted with 1.0 mL of 40% (v/v) ACN
containing 0.1% (v/v) TFA. The eluent was collected and the fraction was dried by a rotary
concentrator (Hamburg, Germany) for further analysis. The dried glycans from intact mAb
were also treated with 50 μL mild ammonium hydroxide (pH 10) at room temperature for 1h,
which was then dried by rotary concentrator for further analysis.
2.4. Fluorescence labeling and purification of 2-AA derivatized oligosaccharides
2-AA labeling of glycans from intact biosimilar, Fab and Fc and mild ammonium hydroxide
treated were conducted as previously reported with minor modifications [
]. Briefly, the
dried glycans were mixed with 25 μL freshly prepared labeling solution (4.8 mg/mL 2-AA in
DMSO containing 30% glacial acetic acid) and 25 μL freshly prepared reducing agent (10.7 mg
2-picoline-borane in DMSO). The mixture was shaked for 30 s and incubated at 65ÊC for 2 h.
After incubation, the glycan derivatives were diluted with 0.5 mL of equilibration solution
(1-butanol/H2O/ethanol (4:1:1, v/v/v)) and then purified using a self-packed MCC SPE [
Briefly, the self-packed MCC cartridges were first washed with 3.0 mL of water to prevent
contamination by cellulose-derived materials into the eluent and then equilibrated with 3.0 mL
binding solution of 1-butanol/H2O/ethanol (4:1:1, v/v/v). The reaction mixture was diluted in
500 μL binding solution and then applied to the cartridge. The cartridge was washed with 1
mL binding solution for three times to remove the excessive derivative reagents and other
impurities. Finally, the Asn-glycan derivatives were eluted with 1 mL of ethanol/H2O (1:1, v/v)
and dried by a rotary concentrator.
2.5. NP-HPLC analysis of 2-AA labeled oligosaccharides
The 2-AA labeled glycans were reconstituted in 40 μL of solution consisting of ACN/50 mM
HCOONH4 (1:4, v/v) for NP-HPLC analysis. Samples were separated by TSK-Gel Amide-80
5 μm 4.6×250 mm column (Tosoh, Bioscience Shanghai Co, LTD) on a Shimadzu LC-20 AD
separation module (Shimadzu, Milford, MA) equipped with a Shimadzu temperature control
module and a Shimadzu RF-10AXL fluorescence detector. The column temperature was set at
40ÊC. Solvent A was 50 mM ammonium formate adjusted to pH 4.4 with formic acid solution
and solvent B was acetonitrile. A 65-min run was used as keeping solvent B at 70% for 4 min at
a flow rate of 1 mL/min and then the linear gradient of 70±50% solvent B over 59 min,
followed by 1 min at 50±0% B and 3 min at 0% B, returning to 70% B over 1 min. Fluorescence
detector was set with excitation and emission wavelengths of 360 and 419 nm, respectively
]. Finally, all the glycan fractions were automatically collected based on the elution time of
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the peaks using an automatic fraction collector, dried in Speedvac concentrator and analyzed
2.6. MALDI-TOF MS analysis
Matrix-assisted laser desorption ionization time-of flight mass spectrometry
(MALDI-TOFMS) analysis was performed using an Applied Biosystems 4800 Proteomics analyzer (AB
SCIEX, Concord, Canada) that was equipped with a 355 nm Nd:YAG laser. The spectrometer
was operated in the positive reflectron mode. DHB matrix was prepared in 50% ACN aqueous
solution with a final concentration of 10 mg/mL. To suppress potassium adduct formation in
the mass spectra, 10 mM sodium acetate aqueous in 1:1 methanol: water was applied to
dissolve the dried sample. 0.5 μL samples mixed with 0.5 μL of freshly prepared DHB matrix were
directly loaded onto the stainless steel MALDI plate and allowed to dry in a gentle stream of
warm air. Samples were ablated with a power of 4000 while the laser rastered over the target
surface. A total of 1,000 laser shots were employed in each sample spot. The MS data
processing was further performed by DataExplorer 4.0 (AB SCIEX, Concord, Canada).
2.7. NanoLC-ESI-MS and MS/MS analysis
The 2-AA labeled glycans and native glycans were analyzed using Triple TOF 5600 mass
spectrometer (AB SCIEX, USA) equipped with a NanoLC system (NanoLC Ultra, Eksigent,
Dublin, CA). The 2-AA labeled glycans were enriched on-line using a trap column (150 μm i.d. ×
10 mm long; C18, 5 μm; Eksigent, Dublin, CA) and then separated using a 25min gradient on
the analytical column (75 μm i.d. × 100 mm long; 5 μm; Eksigent, Dublin, CA) at a flow rate of
300 nL/min. A gradient condition was employed, i.e., 95±70% A for 10 min, 70±40% A for 5
min, 40±20% for 5 min and 95% A for 10 min for the 2-AA labeled glycans. Native glycans
were enriched on-line using a trap column (150 μm i.d. × 1cm long; PGC, 5 μm; Proteomics
Front, China) and then analyzed by a separation column (75 μm i.d. × 10 cm long; PGC, 5 μm;
Proteomics Front, China.). A 50 min gradient was used for the native glycan analysis: 0 min
5% solvent B; 5 min5% solvent B; 30 min 30% solvent B; 40 min 80% solvent B; 41 min 95%
solvent B; 45 min 95% solvent B; 46 min 5% solvent B; 50 min 5% solvent B. The solvent A
consisted of 5% ACN solution containing 0.1% FA (v/v), and the solvent B consisted of 95%
ACN solution containing 0.1% FA (v/v). For the two columns, the MS was all manipulated in
positive-ion mode with a nano ion spray voltage typically maintained at 2.3 kV, and the source
temperature was set at 150ÊC. The MS scanning range was acquired from 500 to 2,000 (m/z)
with up to 20 precursors selected for MS/MS from m/z 100±2,000. MS scans were performed
for 0.25 s, and the following 20 MS/MS scans were performed for 0.1 s each, which resulted in
the full cycle for 2.8 s. Data were processed with PeakView 1.2 software (AB SCIEX, USA), and
the elucidation of the glycans were edited by GlycoWorkbench 2.1 software [
3. Results and discussion
3.1. Glycans profiling analysis by MALDI-TOF MS
Thanks to the advantages of ease of operation and accuracy in composition assignment of
glycans, MALDI-MS is often used as a first step for glycan profiling analysis to generate
information about the nature and diversity of glycans released from native, recombinant glycoproteins
or even more complex biological samples. In this study, the native glycans released by PNGase
F were enriched by PGC SPE prior to the direct MALDI analysis. The sample was handled by
mass spectrometer in the positive reflectron mode. As shown in Fig 2A, the main glycans in
the biosimilar of cetuximab were G0F, G1F, and G2F, which was in agreement with the glycan
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Fig 2. MALDI-TOF MS spectrum of N-glycans enzymatically released from the biosimilar of cetuximab
and cetuximab. a) native N-glycans before mild alkali treatment (pH 10 ammonium hydroxide); b) native
Nglycans of the biosimilar after mild alkali treatment; c) native N-glycans from the cetuximab. The cartoons of
possible structures of glycans were adapted from Glycoworkbench and structure is depicted following the CFG
profile of cetuximab that shown in Fig 2C. However, there were still differences between the
cetuximab and the biosimilar. Although two kinds of structures that could cause severe
hypersensitivity reactions was not observed in the biosimilar, three sialylated glycans, proposed
chemical composition of GlcNAc4Man3Gal1Fuc1NeuNAc1, GlcNAc4Man3Gal2Fuc1NeuNAc1
and GlcNAc4Man3Gal2 Fuc1NeuNAc2 with 18 or 36 Da loss were detected, which was
corresponding to 1920.71, 2082.77 and 2355.87 with only one sodium ions adduct, respectively. The
detailed structural information about the aberrant glycans was verified by nanoLC-ESI-MS/
MS as mentioned below. Up to now, only a recent article described the appearance of
dehydrated sialic acids in the serum of mouse [
]. We supposed that the sialic acid in the glycans
were dehydrated and a structure of lactone (NeuAcLac) was formed. It is well known that
lactone was unstable under alkali condition [
]. However, the NeuAcLac residues containing
glycans were relatively stable in the biosimilar even after an overnight incubation (16±18 h) in
the PNGase F digestion buffer (pH 7.5). Previously, we demonstrated that man-made lactone
of sialic acids could be decomposed with pH 10 solution, which has minor influence to the
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glycan profiling [
]. Therefore, a slightly high pH ammonium hydroxide solution (pH 10)
was used to accelerate hydrolysis of the lactone in the glycans, which could be later conveniently
removed by evaporating in speed vacuum. The glycan profiling from the biosimilar after alkali
treatment was shown in Fig 2B. However, due to the low ionization response and in or out
postsource of sialylated glycans, only two sialylated glycans in free form was observed at m/z 1960.82
and 2122.88, corresponding to GlcNAc4Man3Gal1Fuc1NeuAc1 and GlcNAc4Man3Gal2
Fuc1NeuAc1 with two sodium ions adduct. Clearly, the above results verified our hypothesis. And
the detailed glycan structures in the biosimilar were shown in S1 Table.
Other mAbs, a biosimilar of Bevacizumab, without dehydrated glycans was also used to
evaluate the impact of mild alkali treatment to the glycan profiling. As shown in the S1 Fig, the
chromatogram that obtained with and without mild alkali treatment are almost overlapped. In
addition, the peaks from two of the chromatograms were collected and analyzed by
nanoLCESI-MS, which showed the same glycan structures in the corresponding peaks (Data not
shown). Furthermore, technical replicates were conducted (n = 3) to analyze the
reproducibility of this method. Average CV for the N-glycans peaks was 2.39% before and after mild alkali
treatment. These results indicated that the mild alkali treatment could not impact the glycan
profiling with or without the esterified glycans.
3.2. Glycan profiling of 2-AA labeled from the biosimilar by NP-HPLC
HPLC with fluorescence detection is one of the most widely used detection method in the
quality control of biopharmaceuticals due to the high sensitivity and possibility to estimate the
relative amounts of glycans in glycoprotein. Since fluorescence detection does not allow for
the direct structure elucidation, the identity of glycans must be confirmed either by MS or
chromatography of labeled standards. However, the latter approach is limited by the number
of commercially available standards. As the reasons presented above, NP-HPLC was applied in
this study to quantitative analysis the 2-AA labeled glycans from the biosimilar and nanoLC
combined with MS was used to determine the structure of glycans.
The representative chromatogram of glycans from the biosimilar was shown in Fig 3A and
the relative intensity of each peak was calculated and displayed in S2 Table. Four major peaks
were observed in the chromatogram of the biosimilar, which represented the glycans
GlcNAc4Man3Fuc1 (peak 2), GlcNAc4Man3Gal1Fuc1 (peak 4), GlcNAc4Man3Gal2Fuc1NeuAc1 (peak 7)
and GlcNAc4Man3Gal2Fuc1NeuAc2 (peak 8), respectively. GlcNAc4Man3Gal1Fuc1 at peak 4
consists of two isomeric glycans with the same molecular weight but different oligosaccharide
structural distribution, displayed in the chromatogram at different retention time. Relative
peak area was utilized to quantitative analysis of the glycans in the biosimilar. Although there
were no significant difference before and after mild alkali treatment in the chromatogram, the
relative peak area of several peaks were still changed (Fig 3A and 3B). As shown in S2 Table,
the percentage of peak 6 (including GlcNAc4Man3Gal2Fuc1 and
GlcNAc4Man3Gal1Fuc1NeuAcLac1), changed from 7.12±0.70% to 5.66±0.23%, was strongly decreased. We also observed
that the lactonization of the sialic acid in glycans could decrease the interaction with the solid
phase of the column which shorten the retention time. There is no doubt that this could
interfere with the quantitative analysis of the glycans. After the mild alkali treatment, the glycans
with free sialic acid returned to their due position. Consequently, accurate quantitative analysis
could be obtained using our simple mild alkali treatment method. In addition, each peak was
collected by an automatic fraction collector and the structure information was further
identified with nanoLC-ESI-MS/MS.
Although we initially used MALDI-TOF MS to confirm the molecular structure by mass
assignment, we found that relative peak intensities detected in the glycan spectra were not well
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Fig 3. Typical NP-HPLC spectrum of 2-AA labeled glycans from the biosimilar of cetuximab. a) 2-AA
labeled mAbs glycans before mild alkali treatment; b) 2-AA labeled mAbs glycans after mild alkali treatment.
correlated with the relative abundance obtained by NP-HPLC (Figs 2A and 3A). It is general
accepted that MALDI-MS does not allow real quantitative for oligosaccharides unless stable
isotope-labeled analogs are incorporated as internal standards [
]. As a result, HPLC-FL
might be the golden standard for the quantitative analysis of the glycans in the
3.3. NanoLC-ESI-MS and MS/MS analysis
Mass-only compositional assignments could not obtain the detailed structural information of
the glycans. In order to further elucidate minor glycans in the biosimilar, the structural
information of the abnormal glycans were confirmed by the nanoLC-ESI-MS/MS. Although the
CID-based tandem MS/MS often failed to produce sufficient number of cross-ring fragments,
it could provide abundant B/Y ions. As shown in Fig 4, several sets of diagnostic ions between
the normal and abnormal structures were shown in a typical MS/MS spectrometry. For
example, m/z 436, 639, 801, 1549 and 1695, corresponded to fragment ions from the aberrant glycan
moiety of GlcNAc4Man3Gal2NeuAcLac1, and m/z 454, 657, 819, 1567 and 1713, corresponded
to fragment ions from the normal glycan moiety of GlcNAc4Man3Gal2NeuAc1. Clearly, the 18
Da mass shift of CID MS/MS fragment spectra demonstrated the different chemical
component of aberrant and normal glycan. Spectrum was also screened for the presence of typical
NeuAc-associated fragments in the normal glycans, including m/z 292 (NeuAc), 274
(NeuAcH2O), and 657 (Hex1HexNAc1NeuAc1) [
]. In contrast, there was no typical
NeuAc-associated diagnostic fragment in the abnormal N-glycans. These results indicated that minor
glycans in the biosimilar were NeuAcLac-contained glycans. Structural assignment in the glycan
structure and tandem MS/MS analysis was performed using the GlycoWorkbench suite [
3.4. Fab and Fc N-glycan analysis
Approximately 15±20% of IgGs bear N-linked oligosaccharides in the IgG Fab region, in
addition to those attached at the conserved glycosylation site at Asn297 in the IgG Fc [
Fig 4. NanoLC-ESI-MS/MS spectrum of native glycans. a) MS/MS spectra of m/z 2060 with chemical
composition of GlcNAc4Man3Gal2NeuAcLac1; b) MS/MS spectra of m/z 2078 with chemical composition of
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Fig 5. Typical NP-HPLC spectrum of 2-AA labeled glycans from the Fab and Fc fragment of the
biosimilar of cetuximab. a) N-glycans on the Fab fragment; b) N-glycans on the Fc fragment. The
compositions and structural schemes of glycans in each chromatographic peak are shown in S2 Table of the
Electronic Supplementary Material.
results demonstrated that the biosimilar of cetuximab also had two N-linked glycosylation
sites. The Fab and Fc fragments of the mAb biosimilar were obtained by papain digestion and
the glycans on the Fab and Fc fragments were released by PNGase F and labeled by 2-AA. The
2-AA labeled glycans in the Fab and Fc was then analyzed by NP-HPLC. The representative
chromatogram of Fab and Fc fragment was shown in Fig 5. In addition, NanoLC-ESI-MS/MS
was employed to identify 2-AA glycan structures in Fab and Fc fragments, which was shown
in S3 Table. The major of the Fc glycans were core fucosylated complex biantennary
oligosaccharides with zero, one, or two Gal residues and less than 5% of Fc glycans in the biosimilar
are sialylated. From previously reported literature, Fc glycosylation was required for the
induction of antibody-mediated effector functions including ADCC and CDC by altered the
threedimensional structure of the protein, and the terminal Gal content of the IgG affect the CDC
]. The glycans in the Fab have been dominated by biantennary complex-type structures, in
contrast to Fc glycans, which were highly sialylated. The Fab N-glycans could be involved in
immunomodulation, because they influence the affinity and avidity of antibodies for antigens
as well as antibody half-life [
]. Since there were minor sialylated glycans on the Fc
fragment, most of the sialylated glycans resulted from the Fab fragment bearing the
NeuAcLaccontained glycans. The biological effects about the minor abnormal glycans from the
biosimilar were not reported, and we focused on the accurate glycan profiling analysis of the
biosimilar in this study. Potential risk of the NeuAcLac-contained glycans in the therapeutic antibody
will be studied in the future.
In this study, we identified the glycan profiling of one mAbs tested as biosimilar candidate of
cetuximab which produced in CHO cell lines. NP-HPLC coupled to fluorescence detection in
combined with MALDI-TOF MS analysis have allowed us to comprehensively identify and
confirm the presence of glycans in the biosimilar. The major glycan moieties in the biosimilar
were in agreement with the innovator of cetuximab. However, several minor aberrant
Nlinked glycans containing NeuAcLac residues were observed in the biosimilar which was not
reported previously. In addition, NanoLC-ESI-MS/MS was employed to elucidate the altered
minor glycans in the biosimilar. Glycans of the fragments (Fab and Fc) about the biosimilar
were also investigated. Results showed that the sialylated glycans in the biosimilar were mainly
in the Fab fragment. In order to get more accurate quantification of the biosimilar, a slightly
alkali treatment using ammonium hydroxide was conducted to transform NeuAcLac residues
containing glycans to their neutral forms. As the treatment could not impact the glycan
profiling, we strongly suggested mild alkali treatment as a prerequisite step for accurate glycan
quantification of the therapeutic recombinant.
S1 Fig. HPLC spectrum of 2-AA labeled glycans from a mAb without lactone containing
glycans before and after mild alkali treatment (a) and the relative percentage area of the peaks (b).
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S1 Table. Detected N-glycans from the biosimilar of cetuximab by MALDI-MS.
S2 Table. The major peaks of the biosimilar and their abundance (%) determined by
NP-HPLC with 2-AA labeling before and after ammonium hydroxide treatment.
S3 Table. Major glycans detected in the Fab and Fc of the biosimilar.
The authors gratefully acknowledge the financial support from the National Natural Science
Foundation of China (20905027 and 81402198). W.J.G. also thanks the financial support from
the Independent Innovation Fund of HUST (2015TS092).
Conceptualization: ZH XL.
Data curation: ZH XL.
Formal analysis: SL WJG YW ZH XJF BFL XL.
Funding acquisition: XL.
Investigation: SL WJG YW ZH XJF BFL XL.
Methodology: ZH XJF BFL XL.
Project administration: XL.
Resources: ZH XL.
Supervision: ZH XL.
Validation: ZH XL.
Visualization: SL WJG XL.
Writing ± original draft: SL WJG YW XL.
Writing ± review & editing: SL WJG YW ZH XJF BFL XL.
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