Glycosylation analyses of recombinant proteins by LC-ESI mass spectrometry

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1 Glycosylation analyses of recombinant proteins by LC-ESI mass spectrometry Dr Malin Bäckström Mammalian Protein Expression Core Facility P4EU meeting Porto Nov 11-12, 2013

2 MPE - A tissue culture facility for protein expression in mammalian cells Culture of cell lines in different formats T flasks, roller bottles, spinner bottles Bioreactors with controlled ph and oxygen for optimal production

3 What MPE core facility offers: Recombinant proteins: Generation of expression vectors Mutagenesis Generation of stable expressing clones in different cell lines Production of larger amounts of protein ( 100 mg) by perfusion culture in bioreactors Transient protein expression in 1L scale in bioreactors Concentration of product down to easy-to-handle volumes Simple purification (His-tag, Protein G) Other: Culture of cell lines Culture of hybridoma cells for monoclonal antibody production Mycoplasma testing of cell lines

Transient transfection of CHO-S, Lec3.2.8.

1 (adapted to suspension) are transfected using NOVACHOice

4 Transient transfection of CHO-S, Lec S and 293F cells CHO-S and Lec (adapted to suspension) are transfected using NOVACHOice (Merck Millipore) in Freestyle medium 293F cells are transfected using PEI in Freestyle medium

Production of MUC2 D3 proteins in Lec3.2.8.

5 Production of MUC2 D3 proteins in Lec cells glycosylation deficient CHO cells Lec ; a CHO mutant producing high-mannose N- glycans and GalNAc O- glycans (deficient in α-glucosidase I responsible for N-glycan trimming in the ER) Asn Ser/Thr N-glycans (removable by EndoH) O-glycans Lec cells were adapted to suspension culture at MPE, for the production of MUC2 D3 domain for structural determination His-purified proteins from transient transfection of Lec cells in 1L bioreactor (dec 2012)

6 Production of glycosylated recombinant proteins N-glycans (in many proteins) O-glycans (less common, but very extensive in some proteins) Mammalian cells (CHO, HEK etc) Yeast Insect cells

7 MUC1 mucin; a densely O-glycosylated membrane protein on epithelial surfaces Endogenous membranebound form: n tandem repeats TM MUC1 signal peptide Recombinantly expressed as an Ig fusion protein: ** * ** HGVTSAPDTRPAPGSTAPPA CT Proteolytic cleavage site MUC1 signal peptide Extra-cellular part of MUC1 with 16 tandem repeats Murine IgG2a Fc (exon1-3) Extracellular domain made up by a variable number of multiple tandem repeats of 20 amino acids 5 potential O-glycosylation sites per tandem repeat

8 Aberrant O-glycosylation of MUC1 in breast carcinoma cells Normal GalNAc-Thr/Ser (Breast) carcinoma Galb1-3GalNAc (core 1=T) SAa2-6GalNac-Thr/Ser (sialyl-tn) GlcNAcb1,6 Galb1-3GalNAc (core 2) SAa2-3Galb1-3GalNac (sialyl-t) Chain extension Brockhausen et al., 1995, Eur J Biochem Lloyd et al., 1996, J Biol Chem Hanisch et al., 1996, Eur J Biochem Muller and Hanisch, 2002, J Biol Chem Chain termination (further sialylation possible)

9 Release of O-glycans from blotted protein on PVDF membrane MS 2 MS 3 MS 4 etc. O-glycans are released chemically using NaBH 4 /KOH

10 O-glycans on MUC1 in the prostate cancer cell line DU-145 (Bäckström et al., J Proteome Res, 2009)

11 Modification of O-glycans by co-transfection with specific glycosyltranserases (Sewell et al., J Biol Chem, 2006)

12 Structure of O-glycans and O-glycan occupany in MUC1-Ig from CHO and (Sewell et al., J Biol Chem, 2006) CHO/ST6GalNAcI cells (Sewell et al., J Biol Chem, 2006)

13 Cleavage of protein into glycopeptides to analyze the glycan site occupancy Here: clostripain Other common enzymes: trypsin, Asp-N etc (Sewell et al., J Biol Chem, 2006)

14 LC-MS with ECD fragmentation to analyze which sites are glycosylated Now: ETD dissociation more commonly used for this purpose Sihlbom et al., 2009 Glycobiology

15 N-glycans can have high-mannose, hybrid or complex structures

Signal peptide N-79 N-96 N-117 TM N-glycosylation of overexpressed dendritic cell glycoprotein CD83 Full-length CD83 1 19 V Ig domain

16 Signal peptide N-79 N-96 N-117 TM N-glycosylation of overexpressed dendritic cell glycoprotein CD83 Full-length CD V Ig domain CD83EX-Ig 144myc murine IgG2a Fc His EK CHO/CD83 FL CD83EX-Ig 293/CD83 FL hcd83ext (E.coli) (Western blot anti-cd83)

17 Relative Abundance N-glycans on CD83EX-Ig from CHO K1 released by PNGAseF RT: NeuAcGalGalNAc-ol (O-glycan) Time (min)

18 Relative Abundance N-glycans on CD83EX-Ig from Lec RT: NL: 1.27E4 Base Peak F: ITMS - p ESI Full ms [ ] MS mb_090323_ Time (min)

19 N-glycans on CD83EX-Ig; summary CHO K1 Lec High-mannose none Hybrid none Complex none

20 Potential oligosaccharide structures of N-glycans on CD83 FL

21 Summary N-glycans CD83 CD83 N-glycans 6000 Mw (Da) Sialic acids (n) CD83EX-Ig Lec CD83-Ig CHO K1 CD83FL HEK293

22 Methodology for glycosylation analysis N-glycans: Released by PNGAse F O-glycans: Chemical release After purification the oligosaccharides are analysed using graphite columns and negative ion mode by LC-ESI MS and MS n Glycopeptides are analysed in positive ion mode with milder fragmentation techniques (ECD, ETD etc) to localize glycan substitutions

23 O-glycosylation in insect cells PSGL-1-Ig fusion protein with many O- glycans (mucin-like) Expressed in Trichoplusia ni (Hi-5) and Spodoptera frugiperda (Sf9) cell lines O-glycans were released and analyzed by LC-ESI MS Gaunitz et al., Glycobiology, 2013

24 Characterisation of O-linked glycosylation in different expression systems Insect (Hi-5) Yeast (Pichia) CHO

25 O-glycosylation in insect cells Monosaccharide Sf9 (mole/mole protein) Hi-5 (mole/mole protein) Monosaccharide analysis with HPAEC-PAD Fucose Galactosamine Glucosamine Galacatose Glucose 11 3 Mannose Galacturonic acid 2 2 Glucoronic acid Iduronic acid - - Sialic acid analysis with DMB HPLC Neu5Gc Neu5Ac

26 a) Reducing end GalNAcol is included as HexNAc. Identification of Hex-HeNAcol as Gal β3galnacol Composition Tentative structure RT Novel termination and elongation in insect cell lines Hex-HexNAc-dHex-HexA (M - n H) n - (Min) (M + Na) + (M + Reducing end GalNAcol cores: M/z LC-MS M/z ESI-MS a) Galβ3GalNAcol O n β HexA-GalNAcol O n HexA-(Fuc-)GalNAcol c) O n Extensions: Δ d) HexNAc-HexA-GalNol Δ n.f n HexA-Galβ3GalNAcol n Hex-(HexA-)GalNAcol n HexNAc-(Hex-)GalNAcol n.f n HexNAc-Hex-GalNAcol n.f n HexNAc-HexA-GalNAcol n HexNAc-HexA-(Fuc-)GalNAcol n Hex-HexNAc-(Hex-)GalNAcol n.f n Δ HexNAc-4HexNAc-HexA-GalNol Δ n.f n Galβ3(HexNAc-HexA)GalNAcol n HexNAc-HexA-Galβ3GalNAcol n HexNAc-4HexNac-HexA-GalNAcol e) n HexNAc-4HexNac-HexA-(Fuc-)GalNAcol n Extensions + Decorations: + + S + PC S f) 1 S{HexNAc-HexA-GalNAcol n.f n g) PC 1 PC{HexNAc-HexA-GalNAcol n.f PC 1 PC{HexNAc-4HexNAc-HexA-GalNAcol n.f PC 1 PC{HexNAc-HexNAc-HexA-(Fuc-)GalNAcol n.f 111 h) PC 1 n.d n

27 Thank you! Mammalian Protein Expression core facility Dr Elisabeth Thomsson Richard Lymer Mucin Biology Group, University of Gothenburg Prof Gunnar C Hansson Glycoinflammatory Group, University of Gothenburg Dr Niclas G Karlsson

Supplementary Figure 1. PD-L1 is glycosylated in cancer cells. (a) Western blot analysis of PD-L1 in breast cancer cells. (b) Western blot analysis

Supplementary Figure 1. PD-L1 is glycosylated in cancer cells. (a) Western blot analysis of PD-L1 in breast cancer cells. (b) Western blot analysis of PD-L1 in ovarian cancer cells. (c) Western blot analysis