abstract

Abstract

This technical note demonstrates sensitive detection and accurate mass measurement of intact protein therapeutics and their impurities with high-throughput screening, facilitating rapid drug quality assessment using the Echo MS+ system with the ZenoTOF 7600 system (Figure 1).

AEMS combines precise acoustic droplet ejection for high-speed sampling with electrospray ionization (ESI) MS, eliminating the need for traditional liquid chromatography (LC) separation, leading to significant reduction in analysis time and operation complexity.^1,2 This transformative technology offers high-throughput detection and accurate mass measurement of intact protein therapeutics and their proteoforms to accelerate drug discovery. AEMS is particularly powerful for screening large sample sets in early-stage discovery and clone selection, while maintaining the sensitivity and resolution needed to detect and monitor product quality attributes and low-abundance impurities.

Key Features

Key features of AEMS for intact analysis of protein therapeutics using the Echo MS+ system with the ZenoTOF 7600 system

• Ultra-high throughput: Rapid mass measurement of intact protein therapeutics is achieved at 4 seconds per sample by consuming only 100 nL of sample from a 1536-well plate.
• Enhanced sensitivity: A lower limit of quantitation (LLOQ) of 0.025 µg was obtained for intact mAb analysis.
• High reproducibility: The %CV based on 30 replicate analyses of intact mAbs was <3%.
• Streamlined workflow: The AEMS workflow is streamlined and can be adopted for high-throughput analysis of intact biotherapeutics by scientists with various MS experience.

results1

Figure 1. High-throughput intact mass analysis of mAbs using AEMS.The Echo MS+ system with the ZenoTOF 7600 system provided intact mass measurement of NISTmAb with high-throughput (4 seconds per sample), high reproducibility (%CV<3%) and high sensitivity (LLOQ of 0.025 µg).

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results2

Introduction

Introduction

Protein-based biotherapeutics are highly heterogeneous due to the coexistence of various proteoforms and product-related impurities.³ Intact mass analysis of biotherapeutics is essential to confirm the protein sequence and assess sample impurity to ensure product quality, safety and efficacy.

Traditionally, intact mass analysis is performed with LC-MS using reverse-phase or size-exclusion chromatography. The challenge with LC-MS is the turnaround time for sample analysis, which can take minutes or even hours. AEMS offers an alternative approach similar to ultra-fast direct infusion on a per-well basis. Depending on the analytes and the user's needs, the analysis speed can approach 1 sample per second.^1,2 Intact mass analysis of proteins up to 70 kDa using the AEMS workflow was demonstrated previously on the Echo MS+ system with the ZenoTOF 7600 system.² In this technical note, NISTmAb was analyzed to evaluate the reproducibility, sensitivity and throughput of the AEMS workflow using the Echo MS+ system with the ZenoTOF 7600 system.

results3

Methods

Methods

Sample preparation: The stock solution of NISTmAb (RM 8671, NIST) was diluted to a concentration of 5 mg/mL for the reproducibility study. For the sensitivity evaluation, NISTmAb was buffer exchanged into 100 mM Trizma acetate (Sigma-Aldrich) using the Bio-Spin P-6 Gel Columns (Bio-Rad), followed by serial dilution to a final concentration of 0.25 mg/mL. The samples were then pipetted into an EchoÒ MS+ system-qualified 1536-well low dead volume (LDV) plate (Beckman Coulter) and centrifuged for 2 minutes.

Acoustic ejection method: The key parameters for acoustic ejection of intact NISTmAb using the Echo MS+ system (SCIEX) are shown in Table 1.

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Mass spectrometry: TOF MS data of intact NISTmAb were acquired using the ZenoTOF 7600 system (SCIEX). Table 2 shows the source and TOF MS settings used for the AEMS analysis.

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Data processing: The AEMS data was processed using the SCIEX OS software (SCIEX). The mass reconstruction workflow within SCIEX OS software was used to determine the peak area of intact NISTmAb.

High throughput and high reproducibility

Figure 1 shows 30 replicate AEMS measurements of intact NISTmAb acquired by the Echo MS+ system with the ZenoTOF 7600 system. The data acquisition was completed in less than 2 minutes at a speed of 4 seconds per sample (Figure 1A), demonstrating the system's capability for high-throughput intact analysis of protein therapeutics. The high throughput capabilities of the Echo MS+ system with the ZenoTOF 7600 system did not compromise the quality of the data. Figure 1B shows an example of a high-quality mass spectrum of intact NISTmAb from the last (30^th) AEMS measurement. The deconvolution of this mass spectrum using the protein reconstruction function within SCIEX OS software led to intact mass measurements of various NISTmAb glycoforms (Figure 2C).

The reproducibility of intact mAb analysis is crucial to ensure the comparability of the data acquired across different lots or at different time points. Table 3 shows the peak area or peak area ratio %CV measured for the main glycoforms of NISTmAb from 30 replicate AEMS analyses (Figure 2). The %CV values were less than 3%, indicating the high reproducibility of the AEMS measurement using the Echo MS+ system with the ZenoTOF 7600 system.

intact

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high sensitivity

High sensitivity

Figure 3 shows 3 replicate AEMS measurements of 0.025, 0.05 and 0.1 µg NISTmAb using the Echo MS+ system with the ZenoTOF 7600 system. Similar to the intact data described in Figure 2, high reproducibility was achieved for AEMS analysis of NISTmAb in different quantities (Figure 3A). Excellent TOF MS data were obtained for NISTmAb with as little as 0.025 µg (Figure 3B), demonstrating the high sensitivity of this high-throughput system. The deconvolution result of 0.05 µg NISTmAb confirmed the detection and identification of all the major glycoforms.

Figure 2. Reproducible AEMS analysis of intact NISTmAb (0.025 µg). The Echo MS+ system with ZenoTOF 7600 system produced 30 replicate AEMS data of NISTmAb, with high reproducibility, in less than 2 minutes at a speed of 4 seconds per sample (A). As an example, the mass spectrum from the last acoustic ejection shows an excellent signal and charge state distribution of intact NISTmAb (B). The deconvolution of the mass spectra using SCIEX OS software confirmed the detection of various NISTmAb glycorforms (C).

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Figure 3. Sensitive AEMS analysis of intact NISTmAb. Reproducible AEMS data were obtained for 3 replicate acoustic ejections of 0.025-0.1 µg NISTmAb using the Echo MS+ system with ZenoTOF 7600 system (A). Excellent TOF MS data were obtained for NISTmAb as little as 0.025 µg (B), demonstrating the high sensitivity of this system. The deconvolution result of 0.05 µg NISTmAb confirmed the detection and identification of all the major glycoforms.

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conclusion

Conclusion

• Intact mass analysis of NISTmAb was achieved at a speed of 4 seconds per sample.
• A high-throughput workflow for intact mass analysis of protein therapeutics was demonstrated on the Echo MS+ system with the ZenoTOF 7600 system, with high reproducibility and sensitivity.
• The %CV for 30 replicate AEMS measurements of intact NISTmAb was less than 3%.
• Excellent intact MS data were obtained for NISTmAb as low as 0.025 µg.

references

References

1. Adway O. Zacharias et al. (2023) Ultrahigh-throughput intact protein analysis with acoustic ejection mass spectrometry. J. Am. Soc. Mass Spectrom. 34(1):4-9.

2. High-throughput protein analysis using acoustic ejection mass spectrometry. SCIEX technical note, MKT-31206-A.

3. Anna Robotham and John Kelly. (2020) LC-MS characterization of antibody-based therapeutics: recent highlights and future prospects. Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics. Chapter 1: 1-33.