Drug discovery and development

Charge Variant Assessment of Nanobodies
at the Intact Level by CESI-MS
Sensitive workﬂow for the detection of charge variants of nanobodies from small sample volumes
Stephen Lock,1 Rob Haselberg,2 Raimond Heukers,3 Martine J. Smit,3 Govert W. Somsen, and Elena Domínguez-Vega2,4
1 SCIEX, Warrington, United Kingdom
2 Division of BioAnalytical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands
3 Division of Medicinal Chemistry, Vrije Universiteit Amsterdam, The Netherlands
4 Leiden University Medical Center, Leiden, The Netherlands

Introduction
Traditional LC-MS methods struggle to separate proteins thatdiffer in mass by <10 Da. When proteins larger than 10 kDa ofnearly identical mass co-elute, their spectra overlap, making
it nearly impossible to deconvolute the data and identify theindividual species. Therefore, identification of charge variants
such as deamidation, which causes a mass charge of just
1 Da, is very difficult using traditional LC-MS approaches. CESI
provides a separation mechanism orthogonal to LC-MS-based
approaches for separation. The inherent separation efficiencyof capillary electrophoresis (CE) provides sharp peaks forbetter separation and identification. The goal of this work was
to separate and detect challenging charge variants, such as
deamidation of nanobody proteins at the intact level, without
the need for digestion of the sample using CESI-MS.

The CESI 8000 Plus System

Key Features• Direct combination of capillary electrophoresis with mass
spectrometer, enabling the charge-based separation of proteinsat very low flow rates, including separation of deamidated
forms of the proteins• Minute (<10 nL) injection volumes from sample volumes of5 μL possible, which helps in sample limited analysis

Methods
Sample preparation: Nanobodies were C-terminally tagged with
Myc-(EQKLISEEDLNG) and 6x-histidine tags, and produced inBL21 E.coli.1 Periplasmic extracts were prepared by a freezethaw cycle of E.coli in phosphate buffered saline (PBS) and theFigure 1. Separation of a nanobody (peak 1) from its deamidation form
(peak 2) using CESI-MS.

nanobodies isolated by ion-metal affinity chromatography usingp1

Drug discovery and development
Ni-NTA agarose resin. Elution of the nanobodies from the Ni-NTA

analysis of intact protein pharmaceuticals as well as peptides

resin was performed with 250 mM imidazole in PBS. Finally,

obtained after their proteolytic digestion. Intact protein (andimidazole was removed via multiple dialysis steps against PBS

top-down) analysis is beneficial for biopharmaceutical analysis

using a 3.5 kDa MWCO SnakeSkin dialysis membrane.2 Theseas it offers a general picture of the heterogeneity of the protein

final nanobody samples were diluted into 10 mM ammonium

population while inducing minimal changes caused by sample

acetate (pH 3.0) to a final concentration of approximately

treatment. Reverse-phase LC separates species based on their

1 μM before CESI-MS analysis.

hydrophobicity and often struggles to separate proteins of very

CESI-MS methods. For analysis, the CESI 8000 Plus High
Performance Separation-ESI Module was used in conjunction
with a neutrally coated separation capillary and coupled
to a Bruker maXis HD Ultra-High Resolution QTOF mass
spectrometer (Bruker Daltonics, Bremen, Germany) equippedwith a nano-electrospray source.1 Prior to each analysis, thecapillary was rinsed with water (3 min, 100 psi) and a backgroundelectrolyte (BGE) of 50 mM ammonium acetate at pH 3.0

similar size, such as charge variants of the same protein. CESIMS, on the other hand, separates proteins based on their charge
to hydrodynamic volume ratio.3 While deamidation corresponds
to a gain of 1 Da only, it also adds an excess negative charge on
the protein, allowing CESI to separate these variants. Currently
available MS systems are accurate enough to distinguish a mass
shift of 1 Da on proteins less than 30 kDa, enabling deamidation
charge variant identification.

(10 min at 50 psi), and the conductive line was filled with 50 mM

The nanobody preparations Nb1 and Nb2, both with peptide

acetic acid (3 min, 75 psi). The sample was pressure injected

chains of approximately 15 kDa, were tested. The bivalent

by applying 2.5 psi for 15 s (which was equivalent to 6.3 nL)

construct Nb1-35GS-Nb1, generated via a fusion of 2 Nb1

followed by an injection plug of BGE (0.5 psi for 20 s). The

domains attached through a 35-mer peptide linker ((- GGGGS-)7),1separations were performed at +30 kV (0.5 psi forward pressure,

was also analyzed. During method development, the biggest20 min) with the voltage ramped down at the end of the run. The

increase in peak resolution was obtained by minimizing themass spectrometer was operated in positive ionization modepressure applied to the inlet capillary during the separation.

using a capillary voltage of 1,200 V and drying gas temperature

A pressure of 0.5 psi, providing an overall flow rate of 5 nL/min,of 180 °C (flow rate 2 L min−1). Mass spectra were collected in

produced stable spray and good resolution, and it was therefore

profile mode using an m/z range of 500 to 3,000 with molecular

selected for protein analysis.

mass determinations performed using the “maximum entropy”
algorithm of the data analysis software.

Important

The analysis of the monovalent Nb1 sample produced 2 partiallyseparated peaks migrating at 12.0 and 12.5 min (Figure 2A) of
14590.97 and 14591.90 Da, respectively (Table 1). The massobserved for the first migrating peak agreed with the theoretical• A separation current above 5 μA might cause permanent
damage to the separation capillary and its coating.

mass of Nb1. The second peak, which had an area of about• In general, please do not apply >2,000 V to generate
electrospray as it may result in capillary damage.

Nb1 as this modification caused a reduction in pI and a change

Results and discussion

Figure 2B), which could be assigned to the intact conjugate

There is a growing interest in the potential therapeutic use(5) migrating closely after the main peak had a mass differenceof nanobodies as possible alternatives to mAbs. Nanobodies

of +0.93 Da from the native Nb1-35GS-Nb1 and highlighted theare antibody fragments derived from the variable domains ofpresence of considerable deamidation in the sample. In Figure

heavy- chain-only antibodies and are relatively small proteins

2B, 3 minor peaks were also observed (Table 1) and these could(12-15 kDa) exhibiting a binding capacity and specificity likebe assigned to the monovalent Nb1 and its deamidated form,mAbs while exhibiting higher chemical stability.1

and possibly to the Nb1 molecule carrying a fragment of theMass spectrometry (MS) has become a common techniquefor antibody characterization and can provide data on protein
structure, purity and heterogeneity. MS has been used for the52% relative to peak 1, was assigned to a deamidated form ofof molecular mass (+0.984 Da). The bivalent Nb1 construct(Nb1-35GS-Nb1) yielded a main peak at 13.3 min (peak 4 in(deconvoluted mass, 28835.74 Da; Table 1). The significant peak

35GS linker (15527.70 Da). The analysis of the Nb2 nanobody is
depicted in Figure 2C and highlighted significant heterogeneity.
Up to 22 peaks were observed between 12 and 17 min. Apartp2

Drug discovery and developmentfrom the native sample peak 2, other peaks appeared to originate
from products of truncation, a result of non-specific digestion ofthe Myc tag originated by residual proteases in the sample(Table 2). Next to truncated species, several deamidated formsof Nb2 were observed (Table 2). Nb2 has multiple asparagine
residues and, in this example, CESI-MS was capable ofseparating several isomeric species resulting from deamidation
at different asparagine sites of Nb2 (e.g., peaks 3, 5 and 6 andpeak 7 correspond to singly and doubly deamidated forms ofNb2, respectively).

Figure 2. Base peak electropherograms (CESI-MS) for nanobodies Nb1(panel A), Nb1-35GS-Nb1 fusion (panel B) and Nb2 (panel C).

Peaka

Observed mass (Da)Theoretical mass (Da)Assignmentb

Δ Mass (Da)114590.97

14591.18

Nb1

0.21

2

14591.90

14592.17

deam Nb10.27

Peaka

Observed mass (Da)Theoretical mass (Da)Assignmentb

Δ Mass (Da)114590.97

14591.18

Nb1

0.21

214591.90

14592.17

deam Nb10.27

Nb1

Nb1

a Peak numbering according to Figures 2A (Nb1) and 2B (Nb1-35GS-Nb1).
b deam: deamidated.
Table 1. Assignment of species observed during CESI-MS of Nb1 and Nb1-35GS-Nb1 as shown in Figures 2A and Figure 2B(MS spectra can be found in Haselberg et al.4
p3

Drug discovery and development

PeakaObserved mass (Da)Theoretical mass (Da)Assignmentb

Δ Mass (Da)11639.79

1640.61

E129-H142

0.82

215498.58

15499.16

Nb20.58315499.57

15500.15

1-deam Nb2

0.58

4

1953.47

1954.05

L126-H142

0.58

515499.55

15500.15

1-deam Nb2

0.60

1640.79

1641.58

1-deam E129-H142

0.79615500.54

15501.16

2-deam Nb2

0.62715500.54

15501.16

2-deam Nb2

0.6281640.78

1641.61

1-deam E129-H142

0.83

91954.53

1955.04

1-deam 126-H142

0.511015087.41

15087.76

Nb2 − (H140-H142)

0.35

11

1953.98

1955.04

1-deam L126-H142

0.82

1215088.30

15088.75

1-deam Nb2 −(H140-H142)

0.451313562.69

13563.15

Nb2 − (L126-H142)

0.451413875.88

13876.54

Nb2 − (E129-H142)

0.66

1514004.94

14005.66

Nb2 − (E130-H142)

0.72

1613305.97

13306.84

Nb2 − (Q124-H142)

0.87

1713434.50

13434.97

Nb2 − (K125-H142)

0.47

1814604.39

14605.26

Nb2 − (A136-H142)

0.87

1914605.38

14606.25

1-deam Nb2 − (A136-H142)

0.87

20

14605.39

14606.25

1-deam Nb2 − (A136-H142)

0.86

2114606.38

14607.24

2-deam Nb2 − (A136-H142)

0.86

2214606.38

14607.24

2-deam Nb2 − (A136-H142)

0.86

a Peak numbering according to Figure 2B.
b 1-deam, singly deamidated; 2-deam, doubly deamidated.
Table 2. Assignment of species observed during CESI-MS of Nb2 as shown in Figure 2C.

p4

Drug discovery and development
Conclusions

References

In this study we have demonstrated that CESI-MS can separate

1. R.H. de Wit et al. CXCR4-Specific Nanobodies as Potential
Therapeutics for WHIM syndrome. Journal of Pharmacology
and Experimental Therapeutics, 363 (2017) 35–44.

charge variants of nanobodies and nanobody fusion proteins
including deamidated forms as well as products of truncation of
the antibody which happened during preparation of the protein
samples.

4

Further information
For further information on this topic we would like to refer readers
to the full scientific publication on which this application note isbased.4

2. R.H. de Wit, R. Heukers, H.J. Brink, A. Arsova, D. Maussang,
P. Cutolo, B. Strubbe, H.F. Vischer, F. Bachelerie, M.J. Smit.
CXCR4-Specific nanobodies as potential therapeutics for
WHIM syndrome. J. Pharmacol. Exp. Therapeut. 363(2017) 35–44.
3. S. Lock. The separation of intact phosphorylated proteinsby CESI-MS. SCIEX Application Note (2018), Document No.
RUO-MKT-02-7110-A.
4. R. Haselberg, T. De Vijlder, R. Heukers, M.J. Smit, E.P.
Romijn, G.W. Somsen, E. Domínguez-Vega. Heterogeneity
assessment of antibody-derived therapeutics at the intact
and middle-up level by low-flow sheathless capillary electrophoresis-mass spectrometry. Analytica Chimica Acta (2018).
https://doi.org/10.1016/j.aca.2018.08.024.

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