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
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: 100-2000 mg / kg of body weight per every 3 to 4 weeks hundreds of grams per year! 30.000 kg per year! Imbach, P. et al. (1981) Lancet 1 Tha-In, T. et al. (2008) Trends Immunol. 29
2006 Science 313
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
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. 2006 IVIG 1 g/kg desial. IVIG 1 g/kg IVIG 0.1 g/kg SNA-IVIG 0.1 g/kg non-sna-ivig??
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 0.033 g/kg SNA-Fc 0.033 g/kg from Kaneko et al. 2006
Questions to be answered IVIG in Vienna: Baxter (Kiovig) Octapharma (Octagam) +/- Yield of SNA-fractionation our part: Fc-sialylation Fc C H 2
SNA chromatography of IVIG % 100 80 60 40 20 0 nonbinding n binding b Samples: Octagam or Kiovig 20 mg each, same result
Analysis of glycopeptides Glycopeptides glycan Glycans protein
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
LC-ESI-MS of tryptic IgG peptides: Glycan analysis 100 BPI (TIC) DTLMISR 835.43 LTVDK 575.37 20.63 26.05 26.74 SLSLSPG 660.36 VVSVLTVLHQDWLNGK 904.71 37.50 Capillary RP-LC with ESI-Q-TOF detection 0 100 10 15 20 25 30 35 40 45 50 EIC 879 0 10 15 20 25 30 35 40 45 50 Equal sensitivity for neutral and sialylated glycans EEQYNSTYR Stadlmann J, Pabst M. et al., 2008 Proteomics 8 3+ 932.8 3+ 986.9 3+ 878.8 3+ 1083.9 1000.86 1054.89 2+ 2+ 1398.7 1317.7 2+ 1479.7 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1216.64 1135.61 1297.68
Quantitation of sialylated glycans 35 < G1F > NP RP-LC-ESI < G0F > GnGnF 6 4 6 A GnF GnA 4 F 6 < G2F > 4 4 6 A A F < G2FS > Na 3-4 4 6 A F iso < G2FS2 > Na 3-4 3-4 6 Na F 3 0 2 5 2 0 1 5 Abundance (%) 1 0???? 5 45 50 55 60 65 70 75 80 85 90 95 100 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
Glycopeptides from IgG1 and IgG2 differ gp1a EEQYNSTYR 100 gp1b 21.06 21.41 gp1a gp1b m/z (3+) = 1093.6 m/z (3+) = 932.7 TKPREEQYNSTYR 0 gp2a EEQFNSTFR 100 Relative intensity gp2b m/z (4+) = 812.4 23.47 25.02 gp2a m/z (3+) = 922.0 gp2b TKPREEQFNSTFR 0 18 20 22 24 26 28 Stadlmann J, Pabst M. et al., 2008 Proteomics 8
Sialylation of Fc glycopeptides of SNA fractions neutral glycans sialylated glycans Stadlmann J. et al., 2009 Proteomics 9
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
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
Selected ion intensity Analysis of oligosaccharides from unbound and bound fraction 35.3 37.9 39.7 42.0 733.3 814.3 895.4 35.2 A 4 GnF 6 37.8 GnGnF 6 GnA 4 F 6 39.7 AAF 4 4 6 42.1 46.5 959.9 1040.9 1186.5 + 44.5 46.6 + a) c) b) d) 44.4 48.9 34 36 38 40 42 44 46 48 50 34 36 38 40 42 44 46 48 50 Elution time (min) LC-ESI-MS of reduced glycans on porous graphitic carbon (PGC) at ph 3
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.)
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 40 45 50 55 60 65 Time (min) Pabst et al. 2007, Anal. Chem. 79 Mass + Retention time = Structure...
SIM 895.9 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
The idiots approach to glycan structure analysis MS/MS of diantennary N-glycan isomers b2 H1N1 366.2 528.2 913.4 1055.4 822.4 1116.5 1217.5 1278.5 y H4N4 1420.5 1481.6 1643.7 366.2 b3 H2N1 528.2 y H3N2 913.4 690.3 751.3 893.4 954.4 1116.5 1440.6 1278.5 1420.6 1217.5 1481.6 1643.7 Spectra acquired with known structures. Interpretation of fragment masses only academic game
Back to IVIG Conclusion: SNA-fractionation due to Fab-sialylation
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 % 100 80 60 40 20 Thus, the above conclusion is not quite correct 0 n b 4E10-hum
pep pep pep pep Analysis of a sialylated mab (4E10-hum) 2958.1 3249.3 starting material S0 40 % S1 54 % t 2796.2 3540.3 S2 6 % n 2796.1 2958.1 3249.3 SNA unbound fraction S0 63 % S1 37 % S2 0 % 2958.2 3249.2 3540.3 SNA binding fraction S0 16 % S1 75 % S2 9 % 2600 2900 3200 3500
Holistic view of Ab-glycosylation S0-S0 S1-S1 (0 sialic acids)
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 = 0.16 0.43 0.29 0.05 0.07 < 0.01 16 % 84 % 88 % 12 % 59 (55) % 41 (45) %
Combinations of glycoforms of IVIG p (S0) = 0.8971 p (S1) = 0.1024 p (S2) = 0.00052 S0-S0 S0-S1 S1-S0 S1-S1 S0-S2 S2-S0 S1-S2 S2-S1 S2-S2 fraction in mixture: 0.805 0184 0.01 0.001 -- -- 99 % 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. 1 % of total 3249.3 2958.1 2796.2 3087.2 3540.3 S1-S1 S2-S0 2600 2900 3200 Mass 3500 SNA-binding glycoforms of IgG
Absolute EIC intensity SNA-binding of a normal glycoprotein IVIG Fab as the model S0 S1 S2 Fab - neutral 4.5 41.3 37.7 39.2 35.4 0 26.5 28.8 31.3 46.3 Analysis of free Fab glycans by carbon LC-ESI-MS in pos. mode 0 Fab - sialylated 36.7 24 28 32 36 40 44 48 52 Retention time (min) 41.0 44.6 49.5
Quantitation of sialylated glycans by ESI 60 ES- 50 Is it allowed to compare 40 30 peak intensities of glycans with or without sialic acid? 20 10 0 AGnF AAF ANaF NaNaF 40 ES+ In positive mode, YES! 30 20 10 0 AGnF AAF ANaF NaNaF Fluorescence ESI-MS ph 3 ESI-MS ph 9
SNA-binding requirements with Fab domain AGn 4677 GnGnFbi 13865 AGnFbi 18470 AAFbi 8045 neutral MGnF 20064 MAF 11224 GnGnF 34922 AGnF 101333 AAF 72085 72.9 % 100 80 60 Fab-glycosylation non-glycosylated mono-sia NaAF 46552 NaAFbi 22471 di-sia NaNa 7557 NaNaF 29210 17.7 % 9.4 % 27.1 % 40 20 0 neutral sialylated SNAfractionation % 87.7 % non-glyc + neutral 100 80 60 40 20 0 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
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?
Analysis of Fc fractions IgG1 + IgG3 ALPAPIEK IgG2 GLPAPIEK IGHG2 824.47 A IGHG4 830.47 838.48 IGHG 1+3 polyclonal human IgG (IVIG) IgG4 GLPSSIEK and other peptides B 838.49 Fc-fragment from papain digestion 824.47 C Undigested material 830.47 822 824 826 828 830 832 834 836 838 840 842 844 Mass Stadlmann J, et al., 2009 Proteomics 9
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
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
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