FcR カラムに直結した質量分析計による
インタクト抗体の糖鎖解析
藤
阿

田
部

洋
志＊ 1
真 由 美＊ 1

Glycoform Analysis of Intact Antibodies Using FcR Column Online
Mass Spectrometer
Youji FUJITA
Mayumi ABE

Immunoglobulin G (IgG) is a widely used antibody that contains a glycosylation site in its Fc region. The type andcombination of glycans vary greatly depending on the host in which the antibody is expressed. It has been reported
that the glycostructure linked to the Fc-region strongly influences antibody-dependent cellular cytotoxicity (ADCC)
activity, which is considered to be a risk factor when producing and developing antibody drugs.
We have created a technique for evaluating the glycan structure and ADCC activity associated with IgGs. It
combines the separation of antibodies by an affinity analysis column (TSKgel® FcR-IIIA-NPR, Tosoh Corporation,
Tokyo, Japan) with detection by mass spectrometry (MS). After analyzing commercially available monoclonal
antibodies (mAbs) using this technique, we confirmed that affinity with the FcR column correlated with a defect in
the core fucose and an increase in the amount of terminal galactose. Furthermore, it was applied to a commerciallyavailable antibody-drug conjugate and the same results as those for the monoclonal antibody were obtained.

抗体医薬品として広く利用されている免疫グロブリン（IgG）は、Fc 領域に糖鎖付加部位を含み、発現宿主によって付加する糖鎖の種類や組み合わせが大きく異なる。Fc 領域に付加した糖鎖構造が抗体依存性細胞傷害（ADCC）活性に大きく影響することが報告されており、抗体医薬品の製造・開発におけるリスク要因と考えられている。
我々は、東ソー株式会社が開発したアフィニティ分析カラム TSKgel FcR − IIIA − NPR による抗体の分離と質量分析計（MS）による検出を組み合わせることで、IgG に付加した状態の糖鎖構造と ADCC 活性を評価する技術を構築した。本技術を市販のモノクローナル抗体に適用し、コアフコースの欠損及び末端ガラクトース付加量の増加に相関した FcR カラムでの吸着の向上が確認された。さらに、抗体薬物複合体への適用を検討し、モノクローナル抗体と同様の結果を得ることができた。

1． Introduction＊1

Analysis and Research, Tosoh Analysis and Research Center
Co., Ltd.

Recently, the research and development of antibody
drugs that use antibodies (Fig.1) as an active ingredient47

TOSOH Research & Technology Review Vol.66（2022）

Fab

FucoseGlycanGlycanMannose
GalactoseN－acetylglucosamine
N－acetylneuraminic acid

Fc

Fig.１

Ganeral structure of antibodies

has been actively promoted. To date, more than 100 ofanalyze antibody samples than the conventional

these types of drugs have been approved worldwide,

methods (LC-MS, MALDI-TOF/MS)4).

approximately 70% of which have been approved in

In this article, we report a glycoform analysis using a

1)

Japan (as of March 2022) . Immunoglobulin G (IgG),

mass spectrometer coupled directly to an FcR column

a widely used antibody drug, is a large glycoprotein(FcR-MS) as an applied technique that can be used towith a bifurcated structure, has a molecular weight

analyze antibody drugs. This technology contributes

of approximately 150,000 Da, and is known to adopt

to quality control and the improvement of antibody

diverse molecular structures as a result of various

drugs. The purpose of this technology is to contribute2)

modification reactions during production . Examples

to the health components (achievement of a healthyof post-translational modifications to IgG include

and long-lived life) within the international targets set

heterogeneity of the C-terminal lysines, deamidation,by the SDGs and to contribute to the goal of creatingoxidation, and glycosylation. Glycans linked to thea foundation for industry and technology innovation in

Fc region (Fc glycans) have been reported to greatly

the form of advances in analytical technology.

affect antibody-dependent cellular cytotoxicity (ADCC)
activity, which is considered a risk factor when
producing and developing antibody drugs3). Antibodies

2．Antibody glycans and ADCC activity

that are produced mainly through cell culturing

Immunoglobulin G contains a glycosylation site

become heterogeneous mixtures with diverse glycan

at residue Asn (residue 297) in the Fc region. It

structures during the manufacturing to administrationconsiderably affects the type and combination of glycans

process, which means that a quality analysis of various

added by the expression host 5). The glycan in thecomponents is required. However, monosaccharide

antibody is mainly composed of five monosaccharides:

monomolecules are fairly small with a molecular weightgalactose (Gal), mannose (Man), fucose (Fuc),of about 180 Da, but antibodies can have a molecularN-acetylglucosamine (GlcNAc), and N-acetylneuraminic

weight of 150,000 Da. This means that advanced

acid (NeuNAc), which form diverse glycan structures

analysis techniques, such as complicated pretreatment

through their complex links. Numerous reports haveand the use of expensive analysis equipment, aresuggested that glycan structures and ADCC activity

required when glycan structure and activity analyses ofhave important effects on antibody drugs. For example,

antibody drugs are carried out.

core Fuc in the glycan structure reduces antibody®

binding to Fc receptor IIIa compared to IgG that

FcR-IIIA-NPR (FcR column) for antibody drugs waslack Fuc, which reduces ADCC activity6). In contrast,

developed by the Tosoh Corporation (Tokyo, Japan).

increasing the amount of bisecting GlcNAc has been

The FcR columns recognize the Fc glycan, and because

reported to enhance ADCC activity 7). Moreover, IgG

the antibody can be separated by the difference in its

with terminal Gal exhibits a higher affinity for Fc-

ADCC activity, it can more rapidly and conveniently

receptor IIIa because the terminal Gal enhances ADCC

Therefore, the affinity analysis column TSKgel48

東ソー研究・技術報告

activity8).

第 66 巻（2022）

analyzing the glycoforms of FcR-separated antibodies

The FcR column can analyze components based on
the ADCC activity of an antibody. The column contains

by performing an intact protein analysis of antibodies
using FcR-MS (Fig.2).

a chromatographic separation agent in which FcRγIIIa
is fixed on the surface of a carrier, which means that theantibody molecules can be separated by differences in
9)［1］Determination of the liquid chromatography
conditions

affinity . When examining the relationship between the

The elution conditions for the antibodies when using

strong and weak ADCC activities derived from antibody

an FcR column were examined. In FcR columns, many

separation by the FcR column and their glycan structure,

antibodies adsorb to FcRγIIIa when the pH is close to

the method used is to separate an antibody using an

neutral (pH 6.0–7.5) and elute under acidic conditions

FcR column, followed by saccharide chain excision(pH 4.0–5.0). Therefore, pH gradient elution with two

using enzymatic digestion or labeling and measurement

solutions of varying pH is usually used. Citrate buffers

by LC-MS. However, it is difficult to sufficiently confirm

with a wide buffering capacity in the above pH range

the association between separation by the FcR column

are suitable eluents, but the use of nonvolatile salts inand the Fc glycan because complicated pretreatment is

LC-MS reduces the volatility of the solvent, resulting

necessary before the glycan is excised and measured

in poor ionization during electrospray ionization (ESI),

using these methods. Therefore, the establishment of adeposition on the ion source, and leads to clogging

new analytical method that can analyze the differencesof the capillary. In this study, ammonium acetatein the glycan structure of the antibody separated by thewas investigated as a volatile buffer in the required

FcR column is desirable.

pH range. After considering the introduction of thesample into the MS equipment and the buffer range ofammonium acetate, gradient conditions based on two

3．FcR-MS analysis

solutions of 20 mM ammonium acetate, pH 6.0 and

LC-MS analysis of intact proteins (intact protein
10)

pH 4.0, were set. The sample was UV-detected (280

analysis) is widely used to characterize proteins . Intactnm) at a flow rate of 1.0 and 0.2 mL/min using 50 μg

protein analysis is a technique that is used to evaluate

trastuzumab (Herceptin, Daiichi Sankyo, Tokyo, Japan)

the molecular weight of a protein by subjecting a protein(Figs.3a, b). In addition, analysis in citrate buffer (pHmolecule to mass spectrometry in its original state

6.5 and pH 4.5) was performed for comparison (Fig.3c).

without protease treatment. Therefore, it is possible to

Similar peaks to those for the citrate buffer wereconfirm the molecular weight in a state where a glycan

obtained when ammonium acetate was used, but there

is bound to an antibody and evaluate a complex where

was a decrease in peak resolution compared to thetwo glycans are bound to one antibody molecule.

citrate buffer.

In this study, we developed a technique capable ofIgG

FcR column

Native MS

Fig.２

Deconvoluted MS

Extracted mass chromatogram

Workflow of the FcR－MS analysis

49

TOSOH Research & Technology Review Vol.66（2022）

Intensity［AU］

0.04

（a）

0.02

0

Intensity［AU］

7.5

0.2

12.5

15

17.5

20

10

12.5

15

17.5

20

12.5

15

17.5

20

（b）

0.1

0

Intensity［AU］

10

0.02

7.5
（c）

0.01

0
7.5

10

Retention time［min］（a）Flow rate 1.0 mL ／ min with ammonium acetate,（b）Flow rate 0.2 mL ／ min with ammonium acetate,（c）Flow rate 1.0 mL ／ min with sodium citrate

Fig.３

Chromatographic profile of trastuzumab using the FcR column［2］Investigation of MS measurement conditions

flow path was connected using a non-metal static mixer

The LC-MS measurement conditions were then(GL Science, Tokyo, Japan). When 50% acetonitrile

examined. Samples were eluted with 50 μg ofcontaining 1.0% formic acid was used as an additional

trastuzumab using an ACQUITY UPLC H-class bio

solvent and added at a volume of 50 v/v% with the(Waters, Milford, MA, USA) at a flow rate of 0.2

LC eluate after separation by the FcR column, themL/min, followed by mass spectrometry. The mass

resolution of the MS spectrum improved and separation

spectrometer was measured in the positive ion modeof the spectrum due to the differences in the Fc glycans

using a Xevo G2-XS QTof (Waters). Mass spectrometry

was confirmed (Fig.4b). This improvement was

performed after ammonium acetate elution revealed

presumed to be due to the fact that the addition of anthat the mass spectra became smooth, indicatingorganic solvent decreases the surface tension of the

low resolution. Therefore, confirming the difference

eluate and improves volatility. Moreover, the addition

between Fc glycans was difficult because they couldof formic acid increases the supply of protons, thereby

not be separated (Fig.4a). This was presumed to be

alleviating the influence of ammonium ions.

due to the decrease in sensitivity caused by the low
volatility of the aqueous solvent and a decrease in［3］Examination of analysis conditions

mass resolution due to competition by the ammonium

We used the mass spectrum acquired in Section 3

addition ion for the proton addition ion to be detected[2] and shown in Fig.4b to perform a deconvolution

during ionization in at the ESI stage. To solve theseanalysis. The protein characterization software UNIFI

problems, we examined whether sensitivity could be(Waters) attached to the device was used for the

improved by adding an organic solvent and formic acidanalysis. The antibody masses calculated from thein a post-column using a makeup pump. To ensure

published amino acid sequence information in Drugthat the solvents were thoroughly mixed together, theBank11) were used, and an analysis was performed to50

東ソー研究・技術報告第 66 巻（2022）

Intensity［Count］

（a）
4e5
3e5
2e5
1000003125

Intensity［Count］

3e6

3150

3175

3200

3225

3250

3275

3300

3325

3350

3150

3175

3200

3225

3250

3275

3300

3325

3350

（b）

2e6

1e6

3125

m／ z

（a）Ammonium acetate only,（b）Addition of post－column solvents（1.0％ FA in 50％ ACN）

Fig.４

Mass spectrum of the intact trastuzumab

G0F ／ G1F
3e6
G0F ／ G0F

Intensity［Count］

2.5e6

G1F ／ G1F

2e6

1.5e6

G0 ／ G0F

G1F ／ G2F

1e6
5e5

1.465e5

G2F ／ G2F

G0 ／ G0

1.47e51.475e5

1.48e51.485e51.49e51.495e5

1.5e5Mass［Da］

Fig.５

Deconvoluted mass spectrum of trastuzumab

search for glycans (Man5, G0, G0F, G1F, and G2F)

MS chromatogram, G0F/G0F containing core Fuc, G0/

present at levels of 1% or more using an excised glycan

G0F without Fuc, and G0/G0 without Fuc were eluted

analysis, which was performed in advance (Fig.5). The

in order and the results indicated an improvement inglycan names and structures used in this study are

ADCC activity due to the lack of core Fuc. Furthermore,listed in Table 1.

the dissolution slowed with each increase in terminal

The mass-to-charge ratio (m /z ) in the polyvalent ion

Gal addition, which also supports a previous report12).

was calculated from the mass of each linked glycan
obtained from the analysis results and a chromatogramfor each extracted ion was generated. These were4．Case Study

overlaid to generate a chromatogram (FcR-MS

To confirm the versatility of the novel FcR-MS

chromatogram) reflecting the glycan conformation ofanalysis technique used in this study, we analyzed cases

the FcR-column-separated antibody (Fig.6). In the FcR-

involving monoclonal antibodies (mAbs) and antibody51

TOSOH Research & Technology Review Vol.66（2022）

Glycostructures commonly found in mAbs

Table１

GlcNAc Man

Name and composition

Classification

Man5［H5N2］High mannose

G0［H3N4］

Complex,nonfucosylated

G0F［H3N4F1］

Complex, fucosylated

G1F［H4N4F1］

Complex, fucosylated

G2F［H5N4F1］

Complex, fucosylated

Gal

Fuc

Structure

G0F ／ G0F

2.5e5
2.25e5Intensity［Count］

2e5
G0F ／ G1F

1.75e51.5e5

G1F ／ G1F

1.25e5
100000G1F ／ G2F

G0 ／ G0F

75000

G2F ／ G2F

G0 ／ G0

50000
25000
0

13

14

15

16

17

18

19

20

21

22

23

24

Retention time［min］

Fig.６

Extracted mass chromatograms of trastuzumab

deficiency and increased terminal Gal levels were

drug conjugates (ADCs).

confirmed, which showed that this method is applicable
to common mAbs.

［1］Monoclonal antibody
The samples contained 50 μg each of rituximab(Rituxan, Zenyaku Kogyo, Tokyo, Japan) and［2］Antibody-drug conjugates

tocilizumab (Actemra, Chugai Pharmaceutical, Tokyo,

An antibody-drug conjugate (ADC) is a monoclonal

Japan). The analyses were performed under theantibody covalently conjugated (complexed) to a small-same conditions described in Section 3. The mAbs

molecule drug using an ADC linker 13). The samples

masses were calculated using the published amino

were measured and analyzed using trastuzumabacid sequence information in the Drug Bank

11)

and

emtansine (Kadcyla, Chugai Pharmaceutical) underthen the analyses were performed (Fig.7). Similar

the same conditions described in Section 3 [1] (Fig.8).

to trastuzumab, enhanced ADCC activity due to Fuc

Trastuzumab emtansine had 1–10 molecules of52東ソー研究・技術報告

2.25e5

G0F ／ G0F
G0F ／ G1F
G1F ／ G1F（a）2e5
Intensity［Count］

第 66 巻（2022）

G1F ／ G2F

1.75e51.5e5
1.25e5

G0 ／ G0F

100000G2F ／ G2F

75000
50000

Man5 ／ G0

25000
0
12

13

14

15

16

17

18

19

20

21

22

23

24

22

23

24

Retention time［min］（b）

Intensity［Count］

2.5e5

G0F ／ G0F
G0F ／ G1F

2e5
1.5e5

G1F ／ G1F

G0 ／ G0F

G1F ／ G2F

100000G2F ／ G2F

50000
0
12

13

14

15

16

17

18

19

20

21

Retention time［min］（a）Rituximab,（b）Tocilizumab

Fig.７

Extracted mass chromatograms

DM-1 bound to the amino groups of lysine residues

were measured in FcR-columns. They also showed that

because trastuzumab adds a mean of 3.5 cytotoxic

drug binding delayed the elution peak14). The ADCC

emtansines (DM-1). The UV chromatogram shows amechanism of action in trastuzumab emtansine issingle smooth peak (Fig.8a) and the mass spectrum

thought to originate from the Fc region of trastuzumab.

shows that neighboring multiply charged ions were

However, our findings suggest that antibody-bounddetected compared to trastuzumab (Figs.8b, c), which

DM-1 may improve the affinity of antibodies for FcRγ

suggests that variations in the additional numbers of

IIIa.

DM-1 molecules can be detected. The deconvolution
analysis detected trastuzumab with 0–5 additional DM-1
molecules (Fig.8d). Extraction chromatograms of the

5．Summarydifferent glycan linkages (G0F/G0F, G0F/G1F, andTo investigate the relationship between the separation

G1F/G1F) in the antibody with two DM-1 additionsof antibodies on the FcR column and the glycan structure

showed that elution slowed with each terminal Gal

to be bound, a new analytical method was created that

addition, even when DM-1 was added. This indicatesused a mass spectrometer coupled directly to an FcR

that the antibody ADCC activity was similar tocolumn.

trastuzumab (Fig.8e). Extraction chromatograms of

A novel FcR-MS analysis technique was created using

antibodies with the same glycan linkage (G0F/G0F)

a volatile buffer and by adding organic solvents andbut with different numbers of DM-1 additions showedformic acid to the post-column. Then determinations

that elution slowed as the number of DM-1 additionsof commercially available antibody drugs wereincreased (Fig.8f). This is consistent with studies

undertaken.

where trastuzumab and its antibody-drug complexesThis method can be used in the quality control of

53

TOSOH Research & Technology Review Vol.66（2022）

Intensity［AU］

0.075

（a）

0.05
0.025

Intensity［Count］

0
12

13

14

15

16

17
18
19
Retention time［min］

20

21

22

23

24

（b）

1.5e6
1e6
5e5
0

2100 22002300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 3500 3600 3700 3800 3900 4000

Intensity［Count］

m／ z

1.5e6

（c）

1.25e61e63000 3025 3050 3075 3100 3125 3150 3175 3200 3225 3250 3275 3300 3325 3350 3375 3400 3425 3450 3475 3500

m／ z

Intensity［Count］

2
1

7.5e5 （d）

3
4

0

5e5

5

2.5e5
0

1.465e51.475e51.485e5（e）1.495e51.505e51.515e5Mass［Da］

1.525e51.535e51.545e5

22

23

24

22

23

24

G0F ／ G0F

50000

Intensity［Count］

G0F ／ G1F
40000

G1F ／ G1F
30000

20000

10000

0
12

70000

13

14

15

16

20

21

Emtansine2（f）

Emtansine1

60000
Intensity［Count］

17
18
19
Retention time［min］

Emtansine3

50000

Emtansine4

40000
30000
20000
10000
0
12

13

14

15

16

17
18
19
Retention time［min］

2021（a）Chromatographic profile using the FcR column,（b）Mass spectrum m ／ z 2000－4000,（c）Mass spectrum m ／ z 3000－3500,（d）Deconvoluted mass spectrum,（e）Extracted mass chromatograms when two DM－1 are bound,（f ）Extracted mass chromatograms for G0F ／G0F

Fig.８

54

FcR－MS analysis of trastuzumab emtansine東ソー研究・技術報告

antibody drugs, to optimize cell culture conditions, andin screening procedures for new drug development. It isalso expected to be utilized in antibody drug production
and development.

第 66 巻（2022）

12）G. P. Subedi, A. W. Barb, mAbs , 8(8), 1512 1524(2016)13）H. A. Burris III, J. Tibbitts, S. N. Holden, M. X.
Sliwkowski, G. D. L. Phillips, Clin. Breast Cancer ,11(5), 275 - 282(2011)

6．Acknowledgements
This research was conducted with technical guidance,14）Y. Matsuda, A. Chakrabarti, K. Takahashi, K.
Yamada, K. Nakata, T. Okuzumi, B. A. Mendelsohn,

J. Chromatogr. B ., 1177, 122753(2021)

antibody drugs, and FcR columns provided by the Life
Science Research Laboratory of the Tosoh Corporation.
We would like to express our gratitude to all those who
cooperated.

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55