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OVERVIEW

In this study, we have obtained additional structural information for mAb standards by the use of Impactor, ESI/Impactor and G-ESI source geometries without the need for complex sample preparation methods.

Impactor Spray sources produce mass spectra that exhibit mild charge reduction and additional Light Chain (LC) ion fragments.

ESI/Impactor Spray sources exhibit strong charge reduction, LC fragment ions and previously unobserved Intact minus Light Chain (I-LC) fragment ions.

Experiments with a gap electrospray (G-ESI) source reveal that electrical discharges are likely to play an important role in the formation of the LC and I-LC ions that are unobserved with conventional ESI.

INTRODUCTION Monoclonal antibodies (mAbs) are complex recombinant proteins that are increasingly used as therapeutics for many diseases. LC/MS with electrospray ionisation (ESI) is routinely employed to monitor the quality and safety of mAbs during both the development and manufacturing cycles. In addition to obtaining molecular weight information on the intact biomolecules, LC/ESI/MS can be used in association with other processes, such as enzymatic IdeS, electron transfer dissociation (ETD) and chemical reduction by DTT to obtain structural information from mAb fragment ions. Whilst these methods increase our ability to characterize mAbs, they do however increase the complexity and time of analysis. More recently, Impactor Spray sources have been developed as an alternative to ESI sources and can offer sensitivity enhancements and other benefits under certain analytical conditions. Here, we have used a UPLC/MS method with a standard Impactor source, an ESI/Impactor variant and a gap electrospray (G-ESI) source to reveal additional structural information for mAb analytes that cannot be obtained from conventional ESI sources.

CHARGE REDUCTION AND NOVEL FRAGMENTATION OF mAbs WITH AN ESI/IMPACTOR API SOURCE

Steve Bajic and Jeff Brown Waters Corporation, Altrincham Rd, Wilmslow SK9 4AX, UK.

METHODS

Mass Spectrometry

All data were obtained on a Waters Synapt G2-Si orthogonal Q-TOF MS. Mass spectra were acquired in TOF-MS mode (100-4000Da, scan time = 1.0s). Spectral quality was enhanced by the use of a Waters WREnS program to optimise the flow of argon gas into the trap and transfer regions between the quadrupole and the TOF analyser. Figure 1 is a schematic of the three source types used in this study. These were formed by modifications to a Waters Xevo ESI source. All three sources utilised a pneumatic N2 nebuliser and a ø1.6mm cylindrical, stainless steel target. The ESI/Impactor source (Figure 1(b)) differs from a standard Impactor source (Figure 1(a)) in that the high voltage is applied to the sprayer and the target is grounded. In the G-ESI source (Figure 1(c)), the electrosprayed plume traverses the gap that exists between the grounded target and the ion inlet cone. In this latter source type, the plume does not impact on the target.

RESULTS Figure 2(a) shows the resulting mass spectrum for Trastuzumab obtained using the UPLC/MS method described above with a conventional ESI source. This shows the multiply charged ion series that corresponds to intact mAb ions where the zoomed spectrum for the 50+ charge states confirms that the mAb is, in fact, composed of at least 5 main glycoforms that differ in nominal mass by 162Da. This type of UPLC-ESI-MS experiment is considered to be routine for current mAb analyses and, as such, represents one example of quality control monitoring of commercial mAbs. In comparison, Figure 2(b) shows the resulting Trastuzumab mass spectrum that is obtained by replacing the ESI source with a standard Impactor source. Here, the Impactor source produces the same multiply-charged intact mAb ion series and with a comparable ion intensity (sensitivity) to that obtained with an ESI source. In contrast, however, the impactor source data reveals a small degree of charge reduction (spectrum shifted to the higher m/z) and importantly, the appearance of a second multiply-charged ion series that corresponds to the ionized light chain (LC) fragment of Trastuzumab (Figure 3). The intensity of the light chain ion series is found to increase

CONCLUSION

An Impactor Spray source can be used to obtain intact mAb mass spectra whilst also providing light chain (LC) fragment ion information.

An ESI/Impactor source can produce intact, LC and I-LC ions that are characteristic of the mAb structure and are obtained in the absence of complex sample preparation methods.

An ESI/Impactor source can significantly charge-reduce intact and I-LC ions which may be advantageous for the analysis complex samples in biological matrices.

The ESI/Impactor method may be useful as a rapid, critical quality attribute for mAb QC.

Whilst the generation of intact, LC and I-LC fragment ions is possible with discharging ESI sources, charge reduction is only observed by impacting the spray plume on a surface.

spectrum for the Waters mAb data on a true mass scale (charge state = 0) which confirms that the nominal loss of 24198Da from the intact mAb is due to the loss of the light chain. In fact, it is observed that the I-LC ions have a typical +10Da mass excess which may be due to a systematic mass accuracy error or possibly reduction of one or more disulphide bridges in the fragment ion. These data also suggest that it may be possible to lose both light chains from the Waters mAb by this process, although this was not observed with the other mAb samples. The spectrum of Figure 2(a) shows that both LC and I-LC fragment ions are not observed under conventional ESI conditions where the capillary voltage is 3kV and the capillary/ion inlet distance is typically 10mm. However, it can be shown that a modified ESI arrangement (G-ESI in Figure 1(c)) can result in the formation of both these ions fragment ion types for mAbs. In the case of G-ESI, the capillary voltage is increased to typically 4-5kV and the ESI probe is positioned such that the spray plume traverses the gap between the impactor target and the ion inlet cone. As far as plume impact is concerned, the target is now completely passive, but importantly, serves the purpose of influencing the shape and magnitude of the gap field in this region. This high voltage gap arrangement results in visible discharges between the capillary and the impactor target which are arrested by the use of a 1MΩ current-limiting resistor. It can be demonstrated that a visibly discharging ESI probe can also produce LC and I-LC fragment ions for mAbs, such as is shown in Figure 7(b). Figure 7 compares the Trastuzumab mass spectra obtained by (a) an ESI/Impactor source and (b) a G-ESI source. The G-ESI source is shown to produce I-LC ions () over a limited m/z range (<2600Da) whilst the ESI/Impactor source produces a clear I-LC ion series () that extends beyond 4000Da (data not shown). The ability to produce multiply charged ion series over a wide m/z range will greatly improve the mass accuracy of algorithms that transform mass spectra from the m/z scale to a true mass scale. Furthermore, the ability to charge reduce the ion series to higher m/z values is important for real biological samples since the higher m/z regions tend to contain significantly reduced background ion contamination which further increases spectral quality and mass accuracy of transformed data. Thus, whilst a discharging ESI source may reproduce some of the features of the ESI/Impactor source, the latter would seem to offer enhanced performance for mAb analysis in real samples. Since the above data suggest a causal link between the appearance of LC and I-LC ions and the onset of electrical discharge, it is interesting to compare the current-voltage (I-V) characteristics of some of the sources described in this study. Figure 8 highlights the differences in the I-V plots for the standard ESI, G-ESI and ESI/Impactor sources. In this figure, the total current is the sum of the gap current and the parasitic current that is conducted to ground via the liquid flow in the capillary tube. Here, it is observed that the standard ESI capillary produces no visible discharge in the capillary voltage range 0-5kV. The addition of a

Figure 1. A schematic of (a) the impactor spray, (b) the ESI/Impactor and (c) the gap electrospray (G-ESI) sources used in this study

(a) (b) (c)

Target

Ion Inlet

HV

HV HV Heater

Figure 2. Trastuzumab mass spectra obtained by UPLC/MS using (a) a conventional ESI and (b) an Impactor Spray source.

Light Chain Series *

50+

50+

20%

11%

15+

Intact mAb

LC

HC

GlycanGlycan

GlycanGlycan

ESI 3kV

Impactor 3.5kV*

(a)

(b)

Figure 4. Trastuzumab mass spectrum obtained by UPLC/MS using an ESI/Impactor source. (a) Charge reduced mass spectrum showing the I ()

and LC (*) ion series and (b) a zoomed view of the I-LC ion series ().

50+

48+

LCIntactmAb (I)

IntactmAb – LC(I‐LC)

*GG

GG

*

40+

(a)

(b)

with increasing target voltage in the range 3-5kV. Thus the standard Impactor source can reveal additional structural information for mAbs that is not obtained with conventional ESI sources.

A unique feature of the ESI/Impactor source is its ability to vary the degree of charge reduction in multiply charged spectra by tuning the position of the impact point of the spray on the target. This is demonstrated in Figure 3 for Horse Heart Myoglobin (0.6mL/min, 20/80 water/acetonitrile) where the charge reduction increases as the impact point moves to the left of the point of maximum ion intensity. This tuning procedure leads to an order of magnitude increase in the intensity of the 9+ ion. To highlight the critical nature of the tuning, it should be appreciated that the difference between the probe positions in Figure 3 (a) and (b) is only ~150µm. Figure 4 shows a charge reduced Trastuzumab mass spectrum that was obtained with a ESI/Impactor

Samples and UPLC

Commercially available mAb standards were diluted in HPLC grade water to a concentration of 1mg/mL. 2µL of sample was injected onto a UPLC column (Waters Acquity, 2.1mm x 50mm, UPLC Protein BEH C4, 1.7µm) under gradient elution at a flow rate of 0.2mL/min. Mobile phases were water and acetonitrile with 0.1% formic acid.

Figure 3. Charge reduction of Horse Heart Myoglobin on an ESI/Impactor source. (a) Tuned for maximum ion intensity and (b) tuned for charge re-duction.

9+

9+

20+

IncreasedChargeReduction

3kV

(a)

(b)

(c)Horse Heart Myoglobin

Quadrupole MS

Figure 5. Characteristic I-LC fragment ions () obtained on other com-mercially available mAbs

53+

45+

45+

51+

43+

53+

NIST mAb

Waters mAb

Denosumab

GGG G

Figure 6. Transformed mass spectrum for the Waters mAb obtained on an ESI/Impactor source

Waters mAb

Intact mAb(I)

Loss of 1 light chain

Loss of 2 light chains

source. The ESI/Impactor source was initially tuned for charge reduction using a cytochrome C solution as previously described. Repeat injections of Trastuzumab were then made on-column where the sprayer position was progressively moved in 25µm steps between

injections until the intensity of the light chain ion series (* in Figure 4(a))

was equal to the intensity of the main intact mAb ion series ( in Figure 4(a)). Referring to Figure 4(a), it will be seen that the ESI/Impactor source produces the light chain and intact mAb ions where the centre of the intact distribution is shifted by typically 7 charge states when compared to the ESI spectrum of Figure 2(a). However, if we zoom into the m/z region highlighted by the circle in Figure 4(a), we observe another, hitherto unobserved ion series shown in Figure 4(b) and labeled with markers. These novel fragmentation ions correspond to the loss of a light chain from an intact Trastuzumab ion, presumably by the cleavage of the disulphide bridges. The inset of Figure 4(a) summarizes the ion schemes produced by the ESI/Impactor source: viz. light chain ions (LC), intact mAb ions (I) and intact mAb minus light chain ions (I-LC). To our knowledge, the I-LC ions cannot be produced by CID, ETD, post column addition of charge reduction agents and are not observed under normal ESI conditions.

In order to determine whether the novel I-LC fragmentation pathway is generic to all mAbs, the current method was repeated with a number of commercially available mAb standards. Figure 5 shows the mass spectra obtained for NIST mAb, Waters mAb and Denosumab standards with an ESI/Impactor ionisation source. Here, it is seen that the same characteristic I-LC ions are obtained for each mAb sample (labelled with markers in the figure). Figure 6 is a transformed mass

close proximity target (~3mm) in the case of G-ESI results in a small increase in gap current and the onset of visible discharge at 4kV. In contrast, the ESI/Impactor source, that sprays directly at the target, gives rise to greater pre-breakdown currents and produces LC and I-LC ions well in advance of visible discharges at 4kV. Furthermore, there is no evidence to suggest that atmospheric pressure discharges in the absence of a surface (ESI and G-ESI) can lead to significant charge reduction of mass spectra. In this respect, it is likely that both the stable gap current and the critical impact conditions at the target surface play an important role in the ionisation/fragmentation and charge reduction mechanisms pertaining to the ESI/Impactor source.

Figure 8. A comparison of the discharge characteristics of the ESI, G-ESI and ESI/Impactor sources used in this study. Here, the total current is the sum of the gap current and the parasitic current to ground.

NoVisibleDischarge

10-30µA

6-12µA

ESI/Impactor

G‐ESI 4kV

ESI/Impactor 3kV

2250‐3000 Th

(a)

(b)

Figure 7. Trastuzumab mass spectra obtained by (a) an ESI/Impactor source and (b) a modified electrospray arrangement (G-ESI).

(a)

(b)