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Figure 5. Disulfide bond mapping workflow. The protein was digested without disulfide bond reduction. Trypsin was used for proteolysis.

Figure 2. HDX workflow. IgG2 was digested online by pepsin. Sequence coverage was 88% and 93% for light and heavy chain, respectively. Deuterium incorporation was determined at each time point for peptides consistently observed in all three batches.

OVERVIEWThis study demonstrates a comprehensive analytical workflow combining peptide mapping for disulfide bond-linked isoform analysis and Hydrogen Deuterium Exchange mass spectrometry (HDX-MS) analyses for batch-to-batch protein confirmation comparison for a IgG2 monoclonal antibody.

INTRODUCTIONRecombinant human monoclonal antibodies (mAbs) are widely used in biotherapeutics research and development as potential therapies for various diseases(1). After antibody production several analytical techniques focused on primary and higher-order structural analyses are conducted to adequately characterize these complex therapeutics. Recent reports detailing the characterization of IgG2 mAbs revealed the presence of disulfide-linked structural isoforms, termed IgG2-A, IgG2-B, and IgG2-A/B.

In this study, we present an analytical workflow to automatically map out the disulfide -linked isomers using data independent LC/MS acquisition and disulfide bond mapping informatics tools. To compliment the peptide mapping data, HDX-MS using on-line pepsin digestion and peptic peptide mapping was used to investigate the consistency of multiple IgG2 batches.

HIGHER-ORDER STRUCTURAL CHARACTERIZATION OF AN IgG2 MONOCLONAL ANTIBODY BY DISULFIDE MAPPING AND HYDROGEN DEUTERIUM EXCHANGE MASS SPECTROMETRY

Taiji Kawase1, Kenji Hirose1, Joomi Ahn2, Stephane Houel2, Asish Chakraborty2, Ying Qing Yu2, and Justin B. Sperry3

1Nihon Waters K.K., Tokyo, Japan, 2Waters Corporation, Milford, MA, 3Analytical R&D, Biotherapeutics Pharm. Sci., Pfizer Inc., Chesterfield, MO

Figure 1. (A) Disulfide mapping data were collected using Synapt G2-S. (B) nanoACQUITY UPLC HDX system with Xevo G2 MS was used for HDX MS analysis.

METHODSHDX MS System:LC: nanoACQUTIY UPLC with HDX technology. The analytical column was an ACQUITY UPLC® BEH C18 1.7 μm 1.0 x 100 mm. The trap column was an ACQUITY VanGuard® Pre-column, BEH C18, 1.7 μm 2.1 x 5 mm MS: Waters XevoTM G2 QTof MS ESI positive mode was used. Capillary/Cone: 3.0 kV/37 V, Source/Desolvation temperature: 80 °C / 175 °C, Desolvation gas: 800 L/h Software: PLGS v. 2.5 and DynamX 1.0 Peptide Mapping System: LC: Waters ACQUTIY UPLC H-Class with BEH C18 300Å column (2.1x100mm, 1.7 μm)MS: Waters SynaptTM G2-S QTof MS ESI positive mode was used. Capillary/Cone: 3.0 kV/60 V, Source/Desolvation temperature: 150 °C / 350 °C, Desolvation gas: 800 L/h Software: BiopharmaLynx 1.3.2

RESULTS AND DISCUSSION

(B)(A)

Figure 3. IgG2 structure illustration with disulfide bond linkages in isoform A, B, and A/B. The bottom panel highlights the peptides affected by the isomer species. Peptides highlighted in purple are present in all isoforms. Peptides highlighted in blue are specific to the A and A/B isoforms. Peptides highlighted in red are unique to each individual isoform. For example, cysteine #5 is involved with two different disulfide linkages, forming two different isoforms.

Protein Denaturation &

Tryptic Digestion

UPLC MSE

BiopharmaLynx : Peptide Map

Disulfide BondMap

DisulfideWorkflow

IgG2 A IgG2 B IgG2 A/B

Cys#1

Cys #2

Cys #3

Cys #4

Cys #5

Cys#3 : LC, 132-143AA Cys#4: LC, 180-195AA

Figure 4. (A), (B) and (C) are respectively HDX results of peptides containing cysteine #3 (132-143AA, light chain), #4 (180-195AA, light chain) and #5 (196-214AA, light chain). Peptides containing cysteine #3 and #4 are not affected by the disulfide connectivity differences in the IgG2 isoforms (see Figure 3 highlighted in purple). The deuterium exchange profile of these peptides exhibit a typical EX2 profile, consistently appeared throughout the time-course as well as lot #1, #2 and 3.

(A) (B)

Figure 7. The left panel (A,C,E) and the right panel (B, D, F) represent the normalized intensity and the % deviation of peptides from multiple batches of IgG2 (lot #1, 2 and 3) respectively.(A) and (B) represent peptides that DO NOT have disulfides. (C) and (D) represent disulfide bond peptides that are common to allconformations, except 1:T20-2:T11 (marked Blue in Figure 3), which is unique to conformation A and A/B. (E) and (F) represent disulfide bond peptides that are unique to each conformation (marked red in Figure 3). Independently of the value of the normalized ratio, peptides that are common to all conformation show a % deviation of less than10%. This result indicates that the proportion of those peptides is constant between samples as expected. Peptides that are unique to a specific conformation show the highest % deviation and can be used to compare the proportion of each conformation in different samples.

Cys#5 : LC, 196-214AA, involved in two different disulfide bonds

(C)

Reference : (1) Nature Reviews Drug Discovery 9, 767-774 (October 2010)

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Figure 4. (C) represents HDX result of a peptide containing cysteine #5 (196-214AA, light chain); this pep-tide contains an inter-chain disulfide between the light chain at residue 214 and the heavy chain. The deu-terium exchange profile of this particular peptide exhibits an EX1-like behavior, as observed as having multiple deuterium distributions at long deuterium exchange times; not from carry-over from the pepsin column (data not shown). Although there are subtle differences in the isoform abundances for lot #1, there were no observed differences in the deuterium uptake profile for this peptide across the three lots.

Three lots of an IgG2 were analyzed by disulfide mapping and HDX-MS. The disulfide mapping analysis identified subtle differences in the abundance of each IgG2 isoform in lot 1 as compared to lots 2 and 3. The peptide contains cysteine #5 (196-214AA in light chain) is involved with two different disulfide bond linkages in IgG2. HDX MS data at long exchange times reveals multiple exchange species indicative of EX1-like behavior, which suggests at least two major higher-order conformations are present in solution. The HDX MS data for this particular peptide did not discern the subtle differences in abundance of the isoforms. Disulfides bond peptides that are unique to a conformation show the highest variability and can be used to evaluate the proportion of each conformation between samples. HDX MS and disulfide bond mapping together are powerful tools to assess the higher-order structure and the abundance of IgG2 disulfide-mediated isoforms from batch-to-batch. Future work includes increasing sequence coverage to identify all peptides uniquely involved in the IgG2 isoforms, specifically those peptides involved in the hinge region of the antibody. Obtaining this information will determine if subtle changes in IgG2 isoform abundances affect the higher-order structure of these antibodies.

CONCLUSION

Figure 6. Peptide mapping including disulfide bond linked peptides are performed using BiopharmaLynx v. 1.3.2. The above screen capture is used as an example to show that all tryptic peptides containing disulfide bonds present in the IgG2-A isoform were successfully identified via BiopharmaLynx 1.3.2 . The unique peptides present in the IgG2-A/B and B were respectively identified using BiopharmaLynx and manual analysis. Nomenclature: Label 1:T20-2:T11 = tryptic peptide number 20 of chain 1 (light chain in this case) linked to tryptic peptide 11 of chain 2 (heavy chain in this case). This peptide is shown in blue on figure 3.

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