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INTRODUCTION

Biopharmaceutical products provide a complex analytical

challenge to the modern pharmaceutical industry. Due to the

expansion of the biopharmaceutical industry and the

broadening range of drugs being investigated,

instrumentation is required to carry out advanced experiments

in a routine manner.

Over the past decade native ion mobility spectrometry-MS

(IMS-MS) has become commonplace in academic and

industrial research labs investigating protein folding1, protein-

ligand2 and protein-protein interactions, and protein complex

architecture3. However, more recently it is starting to gain

traction as an everyday tool.

Traditionally, native-IMS-MS is carried out using static

infusion from glass nanoflow capillaries following extensive

sample clean-up. However, with UPLC size-exclusion

chromatography (SEC) in a volatile, neutral pH mobile phase,

the benefits of native IMS-MS can be realized in a more

automated setting.

We describe a method to carry out native IMS-MS of antibody

therapeutics on a high resolution LC-IMS-MS platform. We

also describe investigations into the applicability of high

resolution-IMS-MS (HR-IMS-MS) for biopharmaceutical

analysis using a cyclic IMS-enabled research platform.

NATIVE ION MOBILITY MASS SPECTROMETRY FOR THE CHARACTERIZATION OF BIOTHERAPEUTICS

Dale A. Cooper-Shepherd, Jakub Ujma, Kevin Giles, Nick Tomczyk, Laetitia Denbigh Waters Corporation, Wilmslow, Cheshire, UK, SK9 4 AX

Figure 2. Schematic of the Vion IMS QTof.

METHODS

Size exclusion chromatography (SEC)-native IMS-MS was

conducted on trastuzumab (TmAb) and the antibody-drug

conjugate trastuzumab-emtansine (T-DM1, without

deglycosylation). Tmab and T-DM1 were reduced in the

presence of 5 mM dithiothreitol (DTT). Fragments were

released by reduction and incubation with IdeS enzyme

(Figure 1). SEC was conducted on an ACQUITY I-class UPLC

system with UPLC BEH200 SEC 2.1 x 150 mm, 1.7 µm

column. 100 mM ammonium acetate was used as the mobile

phase. SEC was conducted online with a Vion IMS QTof

(Waters Corp., Wilmslow, UK)(Figure 2). Source temperatures

and voltages were optimized for the transmission of intact

natively-folded protein ions. The acquisition mass range was

set to 8,000 m/z. Data were processed using UNIFI software.

The Vion IMS QTof applies the declustering (cone) voltage

after the IMS cell, which has the advantage of obtaining well-

desolvated protein ions whilst not disrupting native structure

and losing structural information.

References

1. T. W. Knapman, et al. Curr Anal Chem 2013, 9(2), 181-191.

2. T. M. Allison, et al. Nat Commun 2015; 6:8551

3. C. V. Robinson. Biochem Soc Trans 2017, 45(1):251-260.

RESULTS

SEC-Native MS spectra obtained from the Vion IMS QTof

were of high quality (Figure 4). TmAb showed excellent

separation of glycoforms, with peak widths in line with the

theoretical value. UNIFI data processing allows visualisation

of critical attributes (e.g. Glycoform ratio).

CONCLUSION

Native IMS-MS using SEC UPLC and the Vion IMS QTof yields high quality data for mAb and ADC characterization, including single-experiment determination of LC, HC, and intact mAb masses.

Workflows in UNIFI make repeat analyses streamlined for ease of comparison and data interpretation, e.g. DAR calculation.

IMS provides a means to compare 3D structure of ions in the gas phase.

A cyclic IMS-enabled research platform shows promise for the analysis of high MW proteins.

OVERVIEW

Here we present methods to characterize biotherapeutics

using native-ion mobility spectrometry-mass

spectrometry.

Size-exclusion chromatography coupled to the Vion

IMS QTof is a high performance solution for native-IMS

-MS analysis of biotherapeutics.

SEC-native IMS-MS has the potential to compare 3D

structures of antibody species.

Native MS under reducing conditions allows

determination of intact antibody and subunits in one

experiment.

A cyclic IMS-enabled research platform shows

promise for high molecular weight protein ions.

A)

B)

C)

Figure 5. TmAb structural differ-

ences under native conditions. A)

The ATD of non-reduced TmAb

shows two species, 1 and 2. Spe-

cies 1 is the major species and is

more compact than the minor spe-

cies 2 B) Under reducing condi-

tions species 2 contributes equally

to the intensity, indicating a greater

extent of unfolding in the structure.

A)

B)

Native IMS information is also obtained using the Vion IMS

QTof, allowing the determination of drift times for all ions.

SEC-Native-IMS was conducted on TmAb under standard

non-reducing and reducing conditions. Comparison of the drift

times of the intact native mAb under both conditions showed

significant differences, suggesting differences in their higher-

order structure (Figure 5). The reducing conditions have likely

affected the structure of TmAb by breaking a number of

disulphide bonds, which may cause the mAb to become less

rigid and exhibit a larger collision cross-section.

In addition to the intact mAb, native half-mAb and native light

chain (LC) were observed (Figure 6) under reducing

conditions, illustrating how the covalent structure of the

antibody is disrupted. Also, when elevated energies are used

both LC and HC product ions can be produced from the

native mAb. The light chain and heavy chain signals overlap

in m/z with the native light chain, but are well separated in

mobility meaning they do not interfere. These data show that

under reducing conditions it is possible to obtain the LC, HC,

and intact mAb masses in a single experiment.

A)

B)

Figure 6. Trastuzumab under partially-reducing conditions. A) Native

half-mAb, and light chain are observed as well as native intact mAb.

Elevated energies yield heavy chain and light chain as CID products. B)

UNIFI component plot showing masses of light chain, heavy chain, half-

mAb and intact mAb obtained from a single injection of trastuzumab un-

der partially-reducing conditions.

Figure 7. Calculating DAR of T-DM1 using native MS and UNIFI. A)

High quality native MS data were obtained without the need for deglyco-

sylation. B) MS data acquisition, deconvolution, mass identification and

DAR calculation are included in one method for swift results.

A)

B)

Enhanced resolution ion mobility experiments were performed

on a modified SYNAPT HDMS (ESI-Q-IMS-Tof) system fitted

with a prototype cyclic IMS device and dual gain ADC. Data

were processed in MassLynx and DriftScope software. The

cyclic IMS device and array (Figure 3) provide a 100cm,

single pass, mobility path length at 90° to the instrument axis

offering IMS resolution of 60-70 Ω/ΔΩ. By controlling the array

region ions can be made to do further passes increasing the

IMS path length by 1 meter per pass. In this way IMS

resolution in excess of 200 is possible. For more information

on this research platform please see poster TP385.

SEC-Native-MS was also conducted on an antibody-drug

conjugate trastuzumab-emtansine (T-DM1). The high quality

native MS data were acquired and automatically processed

using UNIFI software, including the calculation of drug-

antibody ratio (DAR) (Figure 7). As for TmAb, native IMS can

be conducted on T-DM1 under reducing conditions to obtain

masses of LC, HC and intact ADC (Figure 8).

Figure 8. T-DM1 under partially reducing conditions. Observed species

are analogous to those in Figure 6, with the addition of conjugations

originating from the ADC.

To investigate the high mass capability of our cyclic IMS-

enabled research platform, both intact deglycosylated T-DM1

and IdeS/reduced TmAb were prepared for static infusion

native IMS-MS.

Figure 3. Schematic of the cyclic IMS region of the research platform

Figure 9. Investigating the cyclic IMS-enabled research platform. A)

DAR 0 to DAR 4 ATDs from the 27+ charge state of T-DM1 after 2

passes. B) Mobility selection of regions of the ATD of the 13+ charge

state of the native Fc fragment of TmAb. After 4 passes around the de-

vice, mobility regions are selected and subjected to an additional pass.

Mobility-selected regions show different glycoform profiles due to mobil-

ity separation. Ion losses in the cell are minimal with additional passes.

A)

B)

Figure 1. Monoclonal antibody structure. The intact mAb can be decom-

posed into fragments. Using the IdeS enzyme and DTT reduction, the

light chain (LC), Fd’ and Fc fragments are produced. Using DTT alone,

the LC and heavy chain (HC) are produced.

Cyclic IMS of biotherapeutics - Preliminary studies

A)

B)

Figure 4. SEC-Native MS of TmAb. A) The SEC TIC trace is integrated

and combined to give the native mass spectrum at ~5500 m/z. B) Zoom

of the 27+ charge state of TmAb showing well separated glycoform

peaks. C) UNIFI component plot showing identified species from the de-

convoluted native mass spectrum.