Comprehensive characterization of three IgG forms using CESI-MS

Transcription

1 Comprehensive characterization of three IgG forms using CESI-MS Bryan Fonslow 1, Olga V. Friese 2, and K. Steven Cook 2 1 SCIEX, Brea, CA and 2 Pfizer, Chesterfield, MO

2 mabs, ADCs, biosimilars, and biobetters are generated from IgG molecules Beck et al., Nature Reviews Immunology, 2010, 10,

3 Capillary electrophoresis with mass spectrometry is ideally-suited for IgG characterization High separation efficiency of biomolecules Maltotetroase Intensity Neurotensin Intensity High ionization efficiency of biomolecules with reduced ion suppression CESI The integration of Capillary Electrophoresis (CE) with Electrospray Ionization (ESI) into a single dynamic process within the same device

4 Evaluation of CESI-MS for the characterization of different IgG forms IgG1, IgG2, & IgG4 Peptide mapping Reduce, alkylate, & digest IgG1 Disulfide mapping Alkylate & digest IgG2 IgG4 Intact IgG Charge Heterogeneity & Reduced Analysis Intact IgG1 Reduced IgG4 Non-reduced IgG4

5 Evaluation of CESI-MS for the characterization of different IgG forms IgG1, IgG2, & IgG4 Peptide mapping Reduce, alkylate, & digest ~25 ng of digest analyzed

6 Separation and identification of small and large peptides TISK ( Da ; +1) VDK ( Da ; +1) QAPGK ( Da ; +2) DYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHK ( Da ; +5) SLSLSPG ( Da; +1)

7 CESI separation and sensitivity contribute to comprehensive peptide mapping coverage of IgGs Trypsin digestion only ~25 ng of digest analyzed Identified Potential Critical Quality Attributes: Glycosylation Deamidation (Asp and IsoAsp) Pyroglutamate formation Methionine oxidation C-terminal lysine heterogeneity Lysine glycation

8 Separation and relative quantification of glycopeptides G0F Man5 G1 G2 IgG1 G1F G0 G2F G1F-linked peptide MS/MS spectra MS/MS identification of glycan and peptide sequence

9 Separation and relative quantification of glycopeptides G0F Man5 G1 G2 IgG1 Man5 G0 G1F G0 G2F Man6 G1 Man7 IgG2 G0F G0 A2G1 A2G1F A2G2 G0F G1F IgG4 G1F Man5 G2F G2F A2G2 Man5 Naturally peptide- labeled glycans as ionization and separation tags

10 Evaluation of CESI-MS for the characterization of different IgG forms IgG1, IgG2, & IgG4 Disulfide mapping Alkylate & digest ~25 ng of digest analyzed

11 HC203-HC321 HC134-LC194 HC134-HC147 HC203-HC147 Disulfide linkages identified on IgG4 HC134-LC194 LC88-LC23 LC134-LC194 HC261-HC321 HC226-HC229 Additional potential usages positional disulfide isomers and more highly-linked peptides

12 Evaluation of CESI-MS for the characterization of different IgG forms IgG1, IgG2, & IgG4 Intact IgG Charge Heterogeneity & Reduced Analysis Intact IgG1 Reduced IgG1

13 AU AU AU AU AU AU AU AU cief charge heterogeneity analysis of IgGs with UV detection UV - 280nm 1 mg/ml Pfizer IgG1 w/ markers Quality IgG1 with pi markers UV - 280nm 1 mg/ml Pfizer IgG2 Quality pi 7.11 IgG pi range Minutes Minutes UV - 280nm 1 mg/ml Pfizer IgG1 w/ markers Quality pi 8.65 IgG UV - 280nm 1 mg/ml Pfizer IgG4 w/ markers Quality pi 7.22 IgG4 pi range pi range Minutes Minutes 0.04

14 AU AU Charge heterogeneity analysis of IgG1 by CESI-MS CESI-TripleTOF 6600 MS 0.10 UV - 280nm 1 mg/ml Pfizer IgG1 w/ markers pi 8.65 cief-uv pi range Minutes ~ 10 mg/ml sample desalted into 50 mm ammonium acetate, ph 4 BGE 3% acetic acid (titp-cze mode) ~3.5 nl injection ~ 35 ng injected

15 Charge heterogeneity analysis of IgG1 by CESI-MS ~ 10 mg/ml sample desalted into 50 mm ammonium acetate, ph 4 BGE 3% acetic acid (titp-cze mode) ~3.5 nl injection ~ 35 ng injected

16 Charge heterogeneity analysis of IgG1 by CESI-MS ~ 10 mg/ml sample desalted into 50 mm ammonium acetate, ph 4 BGE 3% acetic acid (titp-cze mode) ~3.5 nl injection ~ 35 ng injected

17 Charge heterogeneity analysis of IgG1 by CESI-MS Extracted ion electropherograms of single charge states

18 Charge heterogeneity analysis of IgG1 by CESI-MS Extracted ion electropherograms of single charge states

19 Reduced IgG1 CESI-MS analysis verifies heavy and light chain sequences G0F HC LC G1F Unknown higher MW species (impurities?)

20 Identification of main IgG1 charge isoform peaks -2 deamidations +1 oxidation Higher MW impurity? Da

21 Identification of IgG1 heavy chain dimer peak

22 AU AU Charge heterogeneity analysis of IgG4 by CESI-MS UV - 280nm 1 mg/ml Pfizer IgG4 w/ markers Quality pi 7.22 cief-uv pi range CESI-TripleTOF 6600 MS CESI-TripleTOF 6600 MS Minutes 0.04 ~ 10 mg/ml sample desalted into 50 mm ammonium acetate ph 4 BGE 3% acetic acid (titp-cze mode) ~3.5 nl injection ~ 35 ng injected

23 AU AU Charge heterogeneity analysis of IgG2 by CESI-MS 0.16 UV - 280nm 1 mg/ml Pfizer IgG2 pi pi range G0F-GlcNAc G1F Deamidated Oxidized Lys loss (2) Minutes

24 Charge heterogeneity analysis of IgG2 by CESI-MS

25 Charge heterogeneity analysis of IgG2 by CESI-MS Different glycosylation and modification profiles

26 Charge heterogeneity analysis of IgG2 by CESI-MS Deamidations Different glycosylation and modification profiles

27 Charge heterogeneity analysis of IgG2 by CESI-MS Deamidations Different glycosylation and modification profiles

28 Charge heterogeneity analysis of IgG2 by CESI-MS Deamidations and oxidation Different glycosylation and modification profiles

29 Evaluation of CESI-MS for the characterization of different IgG forms IgG1, IgG2, & IgG4 Peptide mapping Reduce, alkylate, & digest IgG1 Disulfide mapping Alkylate & digest IgG2 IgG4 Intact IgG Charge Heterogeneity & Reduced Analysis Intact IgG1 Reduced IgG4 Non-reduced IgG4

30 Combining CE & ESI-Mass Spectrometry CESI 8000 High Performance Separation-ESI Module High resolution, ultra-low flow rate CE separations coupled with high resolution, high sensitivity MS TripleTOF 6600 System

31 Software analysis of CESI-TripleTOF MS data Peptide mapping experiments BioPharmaView TM software and ProteinMetrics Byonic Disulfide mapping experiments BioPharmaView TM software and ProteinMetrics Byonic Intact charge heterogeneity experiments BioPharmaView TM software

32 Conclusions CESI-TripleTOF MS analyses provides new and orthogonal information about IgG molecules Peptide and disulfide mapping achieve: 100% sequence coverage from ~25 ng and single digest Characterization of potential critical quality attributes including glycosylation and disulfide bonds CZE-based charge heterogeneity analysis provides: Separation resolution of major charge variants Charge variant-specific identification and correlation from deconvoluted MS spectra

33 Acknowledgements SCIEX Separations Chitra Ratnayake Rajeswari Lakshmanan Marcia Santos Clarence Lew Andras Guttman BioPharma Eric Johansen St John Skilton Pfizer Michael Jones James Carroll ProteinMetrics Eric Carlson Chris Becker Wilfred Tang