Detailed Analysis of Polyclonal IgG with Emphasis on Sialylation and Sub-Classes After Fractionation on a Sialic-Acid Binding Lectin

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Universität für Bodenkultur Wien Institute of Biochemistry Detailed Analysis of Polyclonal IgG with Emphasis on Sialylation and Sub-Classes After

1 Universität für Bodenkultur Wien Institute of Biochemistry Detailed Analysis of Polyclonal IgG with Emphasis on Sialylation and Sub-Classes After Fractionation on a Sialic-Acid Binding Lectin by Friedrich Altmann University of Natural Resources and Applied Life Sciences, Vienna Department of Chemistry

2 Indications for Intravenous IgG (IVIG) Allogeneic bone marrow transplant Chronic lymphocytic leukemia Idiopathic thrombocytopenic purpura Pediatric HIV Primary immunodeficiencies Kawasaki disease Chronic inflammatory demyelinating polyneuropathy (CIDP) Kidney transplant with a high antibody recipient or with an ABO incompatible donor Dosage of IVIG: mg / kg of body weight per every 3 to 4 weeks hundreds of grams per year! kg per year! Imbach, P. et al. (1981) Lancet 1 Tha-In, T. et al. (2008) Trends Immunol. 29

3 2006 Science 313

5 Sambucus nigra agglutinin I SNA Scheme of SNA I RIP RIP Ric Ric Ric Ric Neu5Ac-(a2,6)-Gal-(b1- or Neu5Ac-(a2,6)-GalNAc-(b1- RIP ribosome inactivating protein = N-glycosidase Ric ricin B like = CHO-binding domain

6 Conclusions of Kaneko, Ravetch et al.: > Fractionation on SNA of 6A6 Ab reduced cytotoxicity if sialylated > Sialylation lower in IgG after immunization > Fractionation on SNA of IVIG role of sialylation PBS control foot path swelling from Kaneko et al IVIG 1 g/kg desial. IVIG 1 g/kg IVIG 0.1 g/kg SNA-IVIG 0.1 g/kg non-sna-ivig??

of Fc on SNA Fc sialylation is the trigger mol = mol PBS control IVIG 1 g/kg

7 Conclusions of Kaneko et al.: > Fractionation on SNA of IVIG role of sialylation > Frac. of Fc on SNA Fc sialylation is the trigger mol = mol PBS control IVIG 1 g/kg desial. IVIG 1 g/kg IVIG 0.1 g/kg SNA-IVIG 0.1 g/kg PBS control Fc 0.33 g/kg desial. Fc 0.33 g/kg Fc g/kg SNA-Fc g/kg from Kaneko et al. 2006

8 Questions to be answered IVIG in Vienna: Baxter (Kiovig) Octapharma (Octagam) +/- Yield of SNA-fractionation our part: Fc-sialylation Fc C H 2

9 SNA chromatography of IVIG % nonbinding n binding b Samples: Octagam or Kiovig 20 mg each, same result

10 Analysis of glycopeptides Glycopeptides glycan Glycans protein

11 Analysis of glycopeptides Denaturation S-Alkylation Trypsin digestion Essentially a proteomic workflow...yet, the Scope of the Data- Mining is different! RP-LC-ESI-MS

12 LC-ESI-MS of tryptic IgG peptides: Glycan analysis 100 BPI (TIC) DTLMISR LTVDK SLSLSPG VVSVLTVLHQDWLNGK Capillary RP-LC with ESI-Q-TOF detection EIC Equal sensitivity for neutral and sialylated glycans EEQYNSTYR Stadlmann J, Pabst M. et al., 2008 Proteomics

13 Quantitation of sialylated glycans 35 < G1F > NP RP-LC-ESI < G0F > GnGnF A GnF GnA 4 F 6 < G2F > A A F < G2FS > Na A F iso < G2FS2 > Na Na F Abundance (%) 1 0???? Retention time (min) NP-HPLC of 2-AB labeled hu-igg N-glycans 0 G n G n F A G n F / G n A F A A F N a A F N a N a F Comparison with LC-ESI of glycopeptides AB AB

14 Glycopeptides from IgG1 and IgG2 differ gp1a EEQYNSTYR 100 gp1b gp1a gp1b m/z (3+) = m/z (3+) = TKPREEQYNSTYR 0 gp2a EEQFNSTFR 100 Relative intensity gp2b m/z (4+) = gp2a m/z (3+) = gp2b TKPREEQFNSTFR Stadlmann J, Pabst M. et al., 2008 Proteomics 8

$glycopeptides of SNA fractions$ neutral glycans sialylated

15 Sialylation of Fc glycopeptides of SNA fractions neutral glycans sialylated glycans Stadlmann J. et al., 2009 Proteomics 9

16 What was the basis for SNA-fractionation? FACTS: RP-LC-ESI-MS of only reveals glycopeptides with defined amino acid sequence is blind for polyclonal Fab-glycosylation PLAN: o Analysis of total glycan pool

17 Analysis of oligosaccharides variable constant PNGase F Pool of glycans from Fab and Fc Available methods: Normal-phase HPLC (HILIC) CZE HPAEC HPLC MALDI carbon-lc-esi-ms

18 Selected ion intensity Analysis of oligosaccharides from unbound and bound fraction A 4 GnF GnGnF 6 GnA 4 F AAF a) c) b) d) Elution time (min) LC-ESI-MS of reduced glycans on porous graphitic carbon (PGC) at ph 3

19 The idiots approach to glycan structure analysis Separation of glycans on porous graphitic carbon (PGC) Detection by ESI-MS, maybe with MS/MS Assignment based on reference compounds of known structure Features: - sensitive Pabst et al. 2007, Anal. Chem. 79 Mass + Retention time = Structure... - robust - isomer specific - no blind spots (neutral, sialylated, sulfated etc.)

20 SIM intensity The idiots approach to glycan structure analysis Diantennary N-glycans on PGC Conditions: PGC column (100 x 0.32 mm, 5 µm) with ammonium formate, ph 3.0 AcCN gradient with Waters CapLC EIC bovine fibrin Cow Time (min) Pabst et al. 2007, Anal. Chem. 79 Mass + Retention time = Structure...

21 SIM The idiots approach to glycan structure analysis Gala1-3Galb1-4GlcNAcb1- Straight chain B alpha-gal or Galili epitope Zenapax, made in murine cell Stadlmann J, Pabst M. et al., 2008 Proteomics 8

22 The idiots approach to glycan structure analysis MS/MS of diantennary N-glycan isomers b2 H1N y H4N b3 H2N y H3N Spectra acquired with known structures. Interpretation of fragment masses only academic game

23 Back to IVIG Conclusion: SNA-fractionation due to Fab-sialylation

24 Analysis of a sialylated mab 4E10 is an anti-hiv mab (Katinger, Polymun Vienna) 4E10 has no glycosylation site other than in the Fc 4E10-hum contains a2,6-sialic acid 4E10-hum contains 60 % sialylated N-glycans SNA fractions % Thus, the above conclusion is not quite correct 0 n b 4E10-hum

$1 2958.1 3249.3 SNA unbound fraction S0 63 % S1 37 % S2 0 % 2958.2 3249.2 3540.$

25 pep pep pep pep Analysis of a sialylated mab (4E10-hum) starting material S0 40 % S1 54 % t S2 6 % n SNA unbound fraction S0 63 % S1 37 % S2 0 % SNA binding fraction S0 16 % S1 75 % S2 9 %

26 Holistic view of Ab-glycosylation S0-S0 S1-S1 (0 sialic acids)

27 Combinations of glycoforms of 4E10 p (S0) = 0.4 p (S1) = 0.54 p (S2) = 0.06 S0-S0 S0-S1 S1-S0 S1-S1 S0-S2 S2-S0 S1-S2 S2-S1 S2-S2 fraction in mixture: 0.4 x 0.4 = 2 x 0.4 x0.54 = 0.54 x 0.54 = 2 x 0.4 x 0.06 = 2 x 0.54 x 0.06 = 0.06 x 0.06 = < % 84 % 88 % 12 % 59 (55) % 41 (45) %

28 Combinations of glycoforms of IVIG p (S0) = p (S1) = p (S2) = S0-S0 S0-S1 S1-S0 S1-S1 S0-S2 S2-S0 S1-S2 S2-S1 S2-S2 fraction in mixture: % 1 % Hypothesis: 1 % of IVIG binds to SNA by virtue of Fc-sialylation

$pep pep pep pep SNA fractionation of Fc 4 mg of Fc Eluted protein only detectable by ELISA (in 0.5 M lactose)! ca.$

29 pep pep pep pep SNA fractionation of Fc 4 mg of Fc Eluted protein only detectable by ELISA (in 0.5 M lactose)! ca. 1 % of total S1-S1 S2-S Mass 3500 SNA-binding glycoforms of IgG

30 Absolute EIC intensity SNA-binding of a normal glycoprotein IVIG Fab as the model S0 S1 S2 Fab - neutral Analysis of free Fab glycans by carbon LC-ESI-MS in pos. mode 0 Fab - sialylated Retention time (min)

31 Quantitation of sialylated glycans by ESI 60 ES- 50 Is it allowed to compare peak intensities of glycans with or without sialic acid? AGnF AAF ANaF NaNaF 40 ES+ In positive mode, YES! AGnF AAF ANaF NaNaF Fluorescence ESI-MS ph 3 ESI-MS ph 9

32 SNA-binding requirements with Fab domain AGn 4677 GnGnFbi AGnFbi AAFbi 8045 neutral MGnF MAF GnGnF AGnF AAF % Fab-glycosylation non-glycosylated mono-sia NaAF NaAFbi di-sia NaNa 7557 NaNaF % 9.4 % 27.1 % neutral sialylated SNAfractionation % 87.7 % non-glyc + neutral nonbinding n binding b 12.3 % of Fabs bind to SNA mono-sialylated? One sialic acid is enough to promote binding of Fab fragments to SNA

33 SNA binding of Fc Possible explanation: - First sialic acid buried between Fc-peptide chains - Second sialic acid cannot be accomodated and is hence accessible to SNA C H 2 domain S0-S0 S1-S0 S1-S1 S2-S0 cannot bind to SNA binds to SNA Human receptors = SNA?

34 Analysis of Fc fractions IgG1 + IgG3 ALPAPIEK IgG2 GLPAPIEK IGHG A IGHG IGHG 1+3 polyclonal human IgG (IVIG) IgG4 GLPSSIEK and other peptides B Fc-fragment from papain digestion C Undigested material Mass Stadlmann J, et al., 2009 Proteomics 9

35 Conclusions technical part - Glycopeptides by RP-LC-ESI-MS glycan composition site-specific glycosylation quantitation of neutral and sialylated glycans subclass profile. - Oligosaccharides by PGC-LC-ESI-MS: glycan structure quantitation of neutral and sialylated glycans sulphated glycans N- and O-glycans

36 Conclusions IVIG part - Sambucus nigra agglutinin binds to glycoprotein with 1 sialic acid - Two sialic acids are required for SNA-binding of glycans in Fc-region - This reflects the special steric situatio of Fc-glycans - Only 1 % of IVIG fulfills this requirement

37 Acknowledgements Johannes Stadlmann Martin Pabst Josephine Grass the protein-glycosylation analysis ( proglycan ) group of the Vienna Institute of Biotechnology at BOKU University Renate Kunert Dietmar Katinger VIBT / Polymun Alfed Weber Heinz Anderle Hans-Peter Schwarz Baxter BioScience Vienna

Electron transfer dissociation (ETD)

Electron transfer dissociation (ETD) Electron transfer dissociation (ETD) Originally developed for FT-ICR MS as ECD ( C for capture) Now often used on other trapping instruments, even Q-TOFs Trick: Bringing