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INTRODUCTION

COMPREHENSIVE PTM CHARACTERIZATION OF THE NIST MAB REFERENCE STANDARD USING HRMS

Nilini Ranbaduge, Jing Fang, William Alley, Henry Shion, Ying Qing Yu Waters Corporation, Milford, MA 01757

CONCLUSIONS

Figure 7: The fluorescence chromatogram reporting the identity of all glycan structures discovered in the NIST mAb released N-glycan sample. The structures were assigned by UNIFI following its mass confirmation using RFMS Glycan GU Scientific Library.

Figure 6: Monitoring the deamidation of Asn at peptide level. The MS/MS spectra presents the fragmentation observed for N136 on the light chain.

Figure 4. The charge deconvoluted mass spectra for scFc, LC and Fd’ subunits of the NIST mAb. The PTMs identified at mAb subunit level are; N-terminal pyroglutamic acid, C-terminal lysine truncation, N-glycans present, glycation (LC and Fd’). Subunit level %abundance measurements correlate with that of peptide level and released glycan. PQ - N-term. pyroglutamic acid modification

Table 5: Summary table of the deaminated Asn and the %abundance at peptide level. N437 presents a newly identified site for NIST mAb.

The manufacturing process of mAb products is tightly controlled to reduce the inherent heterogeneity that may raise efficacy and safety concerns and uses thorough characterization methods to ensure their quality.

LC-HRMS is an ideal analytical tool that can be used to determine the level of heterogeneity of mAbs.

Here, we demonstrate the capability of a new benchtop Vion IMS QTof MS controlled by a compliant, workflow driven software, UNIFI, for mAb characterization using NIST RM 8671 as a test case.

The Vion/UNIFI system provides a system solution for ob-taining molecular mass, PTM assignment and relative abundance measurements of mAbs at intact protein, sub-unit, peptide, or released glycan levels.

METHODS

Table 1. Summary table of global C-terminal lysine truncation and glycation levels

Figure 3. Charge deconvoluted intact mass spectrum of the NIST mono-clonal antibody. UNIFI Intact RT-MS workflow with MaxEnt 1-based data processing results in less than 10 ppm mass accuracy for the most abun-dant glycoforms. The PTM assignment identified and mass confirmed dif-ferent combinations of glycoforms with confidence.

Table 2. Summary table of PTMs identified at subunit level

Figure 1. A workflow outlining the analytical methods used in charac-terization of NIST mAb1 (RM 8671). The main PTMs were mass con-firmed at both intact mAb and its subunits level and quantified. Peptide mapping was used to identify the location of the major modifications and the structure of the glycans were confirmed and quantified via a re-leased glycan assay utilizing RapiFluor-MS.

The NIST mAb RM was useful for LC/MS method development for PTM characterizations.

Vion/UNIFI system with the build-in analytical workflows are ideal for comprehensive PTM analyses.

Comprehensive PTM analyses were performed at intact, subunit, peptide, and released glycan levels with fully automated work-flows.

A few glycations and deamidations presented here are being re-ported for the first time.

Figure 5: The most abundant charge state of the truncated peptides is +1 and the full-length peptides, is +2. The total MS response for both groups of peptides was calculated based on all charge states detected in the analysis. About 5% of the total peptides contain its C-term. lysine. The %truncation at peptide level (~5%) is comparable to that determined at the intact protein level (~ 10%) given in Table 1.

Figure 8: The %relative abundance of different glycan attributes identified in the NIST mAb sample by released N-glycan analysis

RESULTS

Table 4: Summary table providing the site specificity and the relative site occupancy for glycation of the NIST mAb.

Table 3: Summary table providing the amino acid identity and the relative site occupancy for oxidation of the NIST mAb.

Peptide mapping: C-term. lysine truncation Released glycans: N-glycan structures

Intact MS: accurate mass

Peptide mapping: Deamidation

Peptide mapping: Oxidation

Peptide mapping: Glycation

Deglycosylated intact MS: %PTM global

Subunit MS: PTMs

Subunit MS: %PTMs

Released glycans: %N-glycans

References

1. Schiel et al.; State-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization Volume 1-3. ACS Symposium Series; American Chemical Society: Washington, DC, 2014.

2. Lauber et al: Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection, Anal. Chem., 2015, 87 (10), 5401–9.

ThP 597

Figure 2. The UNIFI workflows consist of an acquisition method, processing method, and a report.

Sample Preparation:

The NIST mAb RM 8671 sample preparation was as follows:

Intact mass analysis: diluted in 25 mM ammonium acetate

Subunit mass analysis: digested with endopeptidase IdeS enzyme by disulfide bond reduction using DTT

Peptide mapping: denatured and digested with trypsin followed by reduction and alkylation.

Released glycan: prepared using the Glycoworks RapiFlour-MS Glycan kit2

Intact and subunit: ACQUITY UPLC BEH C4, 1.7 µm, 2.1 x 50 mm

Peptide mapping: ACQUITY UPLC BEH 300 C18, 2.1 x 100 mm

Released glycan with FLR labeling: ACQUITY UPLC BEH Glycan Amide column 2.1 x 150 mm

ACQUITY UPLC TUV ACQUITY UPLC FLR Vion IMS QTOF MS acquisition modes (MS, DDA, DIA) Data acquisition, processing and reporting: UNIFI Scientific Informa-

tion System v1.8.2 Workflows: Intact protein, peptide mapping, accurate mass

screening, glycan UNIFI scientific library RFMS glycan library

LC/MS: LC System: ACQUTIY UPLC H-Class Bio System