Technical Note
Utilisation of AEX-MS for charge variant analysis ofIgG4-based mAbs!
ation exchange chromatography (CEX) is an excellent
method of characterising the charge heterogeneity ofbiomolecules. This is also true for most commercially available monoclonal antibodies (mAbs). They are often IgG1based and possess a high isoelectric point (pI) of usually ≥8.
Therefore, CEX coupled with mass spectrometry (MS) isC

the traditional approach [1]. In contrast, anion exchange
chromatography (AEX) has only been used for relatively
acidic proteins.
However, for IgG4-based mAbs AEX may be an alternativeapproach. They possess a pI<8 and therefore CEX is lesssuitable.

This technical note describes the successful application ofan AEX method for charge heterogeneity analysis of IgG4based mAbs coupled to a mass spectrometry (MS) detec-tor. Several IgG4-based mAbs with different pIs (between6.1–7.3) as well as NISTmAb (pI=9.2) were analysed using
a strong anion exchange column (SAX), BioPro IEX QF [2].

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Technical Note
Native AEX-MS method development
The combination of AEX and MS requires a special setup(see Figure 1) in which a stainless-steel T-piece after thecolumn divides the flow. Most of the flow is directed to theUV detector, while the remaining sub-microlitre per minuteflow is directed to the nanoelectrospray ionisation mass

spectrometer (NSI-MS). NSI is used because it can toleratehigh salt concentrations of up to 600 mM ammonium acetate. To further improve the spray stability, isopropanol isused as a dopant, modified desolvation gas.

Figure 1: Native AEX-MS analysis before (blue) and after PNGase F-treatment (black) [2].
Table 1: Chromatographic conditions of the AEX-MS method [2].

Column:Part No.:
Eluent:

BioPro IEX QF (5µm) 100 x 4.6 mm ID
QF00S05-1046WPA) 10 mM ammonium acetate, pH 6.7
B) 300 mM ammonium acetate, pH 6.8
Gradient:
0%B (0–2 min), 0–100%B (2–18 min), 100%B (18–22 min)Flow rate:
0.4 mL/min
Temperature:
45 °C intact mAb
25 °C subunit analysis
Injection:
5 or 10 µg mAb sample
Detection:
NSI-MS (nanoelectrospray ionisation)
UVSample:In-house IgG4-based mAbs (Regeneron)
NISTmAb
Post column setup: Post column stainless-steel T-piece to direct the majority to the UV detector
Remaining sub-microlitre per minute flow directed to the NSI-MS

A salt gradient from 10 mM ammonium acetate (A) to 300 mM ammonium acetate (B) (pH unadjusted) is used to analyse an IgG4 based mAb (mAb-B) with a pI of 6.8. Different temperatures of 25, 35 and 45 °C are tested (see Figure 2).
At 45 °C, variant separation is significantly improved and sharper peaks are obtained. Four basic and two acidic peaks ofmAb-B can be detected.

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Technical NoteFigure 2: Native AEX-MS analysis of mAb-B (pI=6.8) at column temperatures of 25 °C (blue),
35 °C (orange), and 45 °C (red) [2].

These conditions were applied to various IgG4-based mAbs (pI 6.1–7.3) as well as to the NISTmAb, which is IgG1-based
and has a pI of 9.2. Figure 3 shows that the AEX-MS method is suitable for IgG4-based mAbs with moderate pIs, but notfor IgG1-based mAbs with higher pIs. The separation improves as the pI gets lower.

Figure 3: Native AEX-MS charge variant analysis of three different IgG4-based mAbs and NISTmAb, shown basic (B) and acidic (A) variant peaks as wellas the main species (M), 5 µg injection [2].

mAb-6 has a pI of 7.3, which is higher than the mobilephase pH, but sufficient separation still occurs. This suggests that it is the surface charge rather than the intrinsic charge that causes the AEX-based separation. The
acidic variants separated correlate with those commonlyobserved by CEX-MS such as deamidation, glycation andsialic acid (Neu5Ac)-containing species.
Deamidated variants were found in several peaks. The additional abundant peak A1 of mAb-1 contains deamidationthat correlates with a known deamidation site in its complementary determining regions (CDR). However, the othermAbs tested have no deamidation sites in their CDRs, soPage 3/6

these two acidic peaks are probably due to site-specificdeamidation in the Fc region.
The basic variants observed can be identified as unprocessed C-terminal Lys (C-term K) and mAb species withdifferent numbers of Fc N-glycans. In addition, the glycoforms Man5/Man5 with unprocessed C-terminal K andG0F/G0F-GlcNac are observed for mAb-1. mAb-4 demonstrates that this AEX-MS method is very sensitive to themacroheterogeneity of Fc N-glycosylation. The main fully
glycosylated (FG) species is separated from the partiallyglycosylated (PG) peak B1 and the non-glycosylated (NG)species B2, which elute earlier.

Technical Note
Comparison of the AEX-MS method with CEX-MS
The IgG4-based mAb-B with a pI of 6.8 is used to compare the two methods (Figure 4). From the CEX-MS method, twoacidic peaks can be observed and identified as deamidated and glycated variants. There are no basic peaks.
Table 2: Chromatographic conditions of the CEX-MS method [2].

Column:Part No.:
Eluent:

BioPro IEX SF (5 µm) 100 x 4.6 mm ID
SF00S05-1046WPA) 20 mM ammonium acetate, pH adjusted to 5.6 with 20 mM acetic acidB) 150 mM ammonium acetate, pH 6.8Gradient:
0%B (0–2 min), 0–100%B (2–18 min), 100%B (18–22 min)Flow rate:
0.4 mL/min
Temperature:
45 °C
Injection:
10 µg mAb sample
Detection:
NSI-MS (nanoelectrospray ionisation)
UVSample:
In-house IgG4-based mAb, pI=6.8 (Regeneron)
Post column setup: Post column stainless-steel T-piece to direct the majority to the UV detector
Remaining sub-microlitre per minute flow directed to the NSI-MS

In comparison, the AEX-MS analysis shows four basic peaks
including partially and non-glycosylated mAb species and1 or 2 unprocessed C-terminal K in addition to two acidic
peaks. However, A1 consists of a deamidated and glycated variant and A2 contains another deamidated species.

The use of AEX-MS probably provides the possibility toseparate site-specific deamidation variants. The overall
separation using AEX-MS is better as the method provides
sharper peaks and additional information about the basevariants.

Figure 4: Comparison of (a) CEX-TIC and (b) AEX-TIC of an IgG4 mAb (pI=6.8) [2].

Further improvement of the basic variant separation
Since mAbs with a lower pI are better separated, it was tested to see whether the separation can be improved by lowering the pI through PNGase F (peptide:N-glycosidase F)-mediated deglycosylation. This reaction removes N-glycans and simultaneously converts the glycan-bearing as-paragine (Asn) residue to aspartic acid (Asp). Since all IgG4mAbs contain an Asn residue in the Fc region of each of thetwo heavy chains, up to two Asn to Asp conversions can beexpected.

Figure 5: Native AEX-MS analysis of mAb-4 (right) and mAb-8 (left) before (blue) and after PNGase F-treatment (black) [2].

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Technical Note
After the PNGase F-treatment of mAb-4, the retention timeof the FG main species increases by about 1 min due to theelevated acidity induced by the now two Asp residues. The
retention time of the PG species B1 shifts by only about
0.5 min, while the retention time of the NG species B2
remains unchanged. In the case of mAb-8, an additional
minor variant (B1a) can be detected and identified as apartially glycosylated species.

Figure 5 shows the differences between the untreated andthe PNGase F-treated mAbs mAb-4 (pI=6.6) and mAb-8(pI=6.9). The PNGase F-treatment decreased the pI to 6.4
for mAb-4 and 6.6 for mAb-8. Due to the reduced pI theoverall retention as well as the variant separation and peak
sharpness are improved for both mAbs.

Monitoring critical Fc quality attributes
tion and to further investigate the glycosylated species. In
contrast to previous results, a lower temperature of 25 °C
showed improved peak shape and charge variant separation for the Fc fragment analysis compared to analysis at
higher temperatures, as shown in Figure 6 for mAb-4.

This AEX-MS method can also be used for subunit analysisof mAbs after digestion with IdeS protease. Since the pIof F(ab’)2 fragments is relatively high, these fragments arepoorly retained and are not taken into account further.
The Fc fragments were treated with PNGase F-mediatedglycosylation to take advantage of the improved resolu-

B4: PG Fc + 1 x C-term Lys
B3: NG Fc
B2: FG Fc + 1 x C-term LysB1: PG Fc
B1*: PG Fc

M: FG Fc
M*: FG Fc
A1: Deamidated FG Fc
A2: Deamidated FG Fc

25 °C

35 °C

45 °CFigure 6: Native AEX-MS analysis of the PNGase F-treated and IdeS digested mAb at different temperatures: 25 °C (blue), 35 °C (orange) and 45 °C (red) [2].

Four basic and two acidic variants can be identified. The
main peak as well as B1 show tailing shoulder peaks, withidentical mass to the corresponding peak, which could beconformational isomers. In addition to the NG B3 and thePG B1 peak, B2 is identified as a fully glycosylated species
with one unprocessed C-term Lys while B4 is identified as apartially glycosylated species with one unprocessed C-term
Lys. These findings were confirmed by peptide mapping.

Figure 7: AEX-MS analysis of the PNGase F and IdeS-treated mAb-8 at (a) T=0, and (b) T = 6M @ 25 °C [2].

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Technical Note
Possible deamidation sites are NG at VVSVLTVLHQDWLNGK; NK at VSNK; NG and NN at GFYPSDIAVEWESNGQPENNYK. Therefore, the fractions of the deamidated variants were identified by peptide mapping. The resultsof the peptide mapping of the main and acidic fractions areshown in Table 3. It is noticeable that A1b and A1c both contain the deamidated VSNK peptide, but A1b mainly contains
the isoAsp form, whereas A1c mainly contains the Asp form.
It is striking that this AEX-MS method is capable of separating site-specific deamidation products at the Fc level, evenat isoform resolution, so that these attributes can be monitored without the need for peptide mapping. This saves
time and reduces sources of error such as deamidation artefacts that can occur in peptide mapping.

mAb-8 was thermally stressed to achieve higher levels ofdeamidation and to facilitate fractionation after IdeS digestion and deglycosylation. Therefore, mAb-8 was incubatedat 25 °C for 6 months. The basic Fc variants of mAb-8 arealso assigned to the unprocessed C-term K and Fc N-glycosylation macroheterogeneity. These basic variants did
not change after thermal stress. While an increase in peaks
for the acidic variants was observed, which can be completely attributed to deamidation.
Analysis of Fc fragments improved the resolution of separated deamidated variants. Four acidic peaks are resolved from the analysis of the thermally stressed mAb.
Since there are only a few deamidation sites present
in the IgG4 Fc region, it is likely that this AEX-MS method can separate site-specific deamidations.

Table 3: Relative abundance of deamidated tryptic peptides from peptide mapping Analysis of the main and acidic (A1a, A1b, A1c, and A2) fractions fromthermally stressed PNGase F treated and IdeS digested mAb-8 [2].

Relative Abundance (%)

Deamidated Peptides
Main Fraction

A1a Fraction

A1b Fraction

A1c Fraction

A2 Fraction

GFYPSDIAVEWESNGQPEDNYK

0.1

7.3

4.1

1.8

2.1

VS(isoD)K

0.3

0.0

28.5

15.1

0.0

VSDK

0.2

0.0

2.3

17.7

0.1

GFYPSDIAVEWESDGQPENNYK

0.2

0.1

0.1

0.1

2.0

GFYPSDIAVEWES(isoD)GQPENNYK

1.0

0.1

0.7

0.9

23.9

Summary
AEX-MS analysis is very suitable for characterising charge
heterogeneity in IgG4-based mAbs. Compared to CEX, theAEX method shows overall better separation of the IgG4
based mAb with moderate pI and provides additional information.
The resolution of the glycosylated variants can be further
improved by PNGase F-mediated deglycosylation.

AEX-MS methods are suitable for the Fc critical quality attribute monitoring of IgG4-based mAbs, while CEX remains
the better option for F(ab’)2 subunit analysis.
With this AEX-MS method the Fc critical quality attributes
can be performed without peptide mapping, saving timeand reducing errors/and even more important reproducibleresults.

Literature[1] Y. Yan, A. P. Liu, S. Wang, T. J. Daly, N. Li, Ultrasensitive
Characterization of Charge Heterogeneity of Therapeutic
Monoclonal Antibodies Using Strong Cation Exchange
Chromatography Coupled to Native Mass Spectrometry,Anal. Chem. 2018, 90, 13013-20.

for Charge Heterogeneity Characterization of Monoclonal
Antibodies, Anal. Chem. 2022, 94, 6355−62.

[2] A. P. Liu, Y. Yan, S. Wang, N. Li, Coupling Anion Exchange Chromatography with Native Mass Spectrometry

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