Mike Butler University of Manitoba CANADA Seminar Plan Structure - - PowerPoint PPT Presentation
July 31st 2014: PEWS Control of glycosylation in cell bioprocesses Mike Butler University of Manitoba CANADA Seminar Plan Structure of Mabs Glycosylation of Antibodies effect of dissolved oxygen The secret of camelids Case
– effect of dissolved oxygen
– effect of low nutrient levels – enhancement of glycosylation profiles
beta-interferon immunoglobulin erythropoietin Protein N-glycan sites gp120 25 huCD36 9 huICAM-1 (CD54) 8 hu-tPA 3 hu-Epo 3 hu-IFN gamma 3 rhu ant-IL-8 (IgG) 2 hu-CSF 2 hu-IFN beta 1
Fc region
hinge
Fab region
CH 2 domain CH 3 domain CH 1 VH glycan light chain heavy chain
Antibody structure - IgG based on X-ray crystallography data G2F G0F G1F
terminal GlcNAc terminal Gal
solvent accessible surface Asn297 Asn297
BKF Sialidase BTG JBH
PNGaseF
+ -galactosidase (ABS+BTG) + sialidase (ABS) + -fucosidase (ABS+BTG+BKF) +-hexosaminidase (ABS+BTG+BKF+SPH)
{
2AB 2AB 2AB 2AB
~ ~
2AB 2AB
5 6 8 9 7 Gu
1,6 1,3 * *
Asn Xaa Ser/Thr
Dol P P
NH2
Golgi Hybrid type Man Complex type Oligosaccharide transferase Glc Glc Glc
Endoplasmic Reticulum -Glc I -Glc II -Glc II -Man I
Man
Oligomannose Type
GNAc Tase II deficiency
Asn Xaa Ser/Thr
Dol P P
NH2
Golgi Man Oligosaccharide transferase Glc Glc Glc
Endoplasmic Reticulum -Glc I -Glc II -Glc II -Man I
Man
Oligomannose Type Complex/ single antenna
GnTase II deficiency
Control IgG
6 8 9 7 Gu
CDG IIa patient IgG
M Butler A Critchley HF Hebestreit RA Dwek J Jaeken PM Rudd et al Glycobiology 13: 601-622: 2003.
mannose
2 1.5 1 0.5 1 2 3 4 5 6 10 8 6 4 2 ammonia lactate glutamine glucose Time (day)
Substrate utilisation and by-product formation in PQXB1/2 hybridomas
Time of culture (h)
40 60 80 100 120 140 160 180 200
Percentage of glycoform
10 20 30 40 50 60 2-site occupancy 1-site occupancy No site occupancy
Curling et al 1990 N2 N1 N0
Proportion of N0 increases 5 to 30% during culture
Butler, M. Optimisation of the cellular metabolism of glycosylation for recombinant
proteins produced by mammalian cell systems. Cytotechnology 50: 57-76 2006
HPAEC-PAD chromatograms of PNGase released glycans from Mab
1 2 3 4 G0F G1F G2F G2FS
Change in galactosylation with variable dissolved oxygen
Dissolved oxygen (% DO)
20 40 60 80 100
Relative HPAEC-PAD peak area (%)
10 20 30 40 50 60
GOF G1F G2F
G2F G0F G1F
Kunkel, J.P., Jan, D.C.H., Butler,M. and Jamieson, J.C.
Kunkel, J.P., Jan, D.C.H., Jamieson, J.C. and Butler, M. . J. Biotechnol. 62; 55-71: 1998
DO % GI 1 0.37 2 0.38 5 0.39 10 0.41 25 0.41 50 0.46 100 0.56
G2 + 0.5*G1 GI = ------------------ (G2 + G1 + G0)
Factors that affect galactosylation/ sialylation :-
Galactosyl transferase
+ UDP- + CMP-
Sialyl transferase
+ UDP- + CMP- UDP-galactose transporter CMP-sialic acid transporter
Sialidase
Scientific Director: M. Butler University of Manitoba
150 kDa 80 kDa www.mabnet.ca Biotechnology Focus 15(5) 11-12: May 2012
Scanning electron micrograph of non-adherent CHO cells x2,300 (Porter et al. 1973)
CHO cells adhering to a plate
Liu, B., Spearman,M., Doering,J., Lattova,E., Perreault,H. and Butler,M.
“The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody.” Journal of Biotechnol ogy 170: 17-27 2014
Kinetics of nutrient consumption and product accumulation in the fed-batch mode of culture
nutrient Fed-batch cells product
Set-point
(Rearick et al, 1981)
Asn Man5GlcNAc2
1: Reduced glycosylation site occupancy (Stark & Heath, 1979) 2; High mannose structures
Bo Liu 2014
Comparison of Protein A purified Mabs on 4~15% reduced SDS-PAGE gel
Bo Liu P34 Initial [Glc] mM 0 5 10 12.5 15 17.5 25 control % lower band 52 40 30 26 0 0 0 0
Mass Spectrometry Analysis (MALDI-MS)
PURIFICATION MALDI-MS GLYCOPROTEIN
79340 82660 50000 60000 70000 80000 90000 100000 110000 m/z
B. A.
Fig 21. MALDI-MS on protein A purified CHO-EG2 mAb. A. GLYCOPEPTIDE ANALYSIS B. SDS/PAGE mAb produced in media containing 3mM Glc, 4mM Gln C. MALDI-MS spectra recorded in the linear mode from intact mAb
Molecular mass kDa 50 75 37 250 150 100
33
C.
Carina Villacres P121
2373.80 1887.62 2211.75 1725.58 1563.53 2049.68 1 2 3 4 5 4 x10
1000 1400 1800 2200 2600 m/z 1401.48 1239.44 2536.91
GlcNAc Man Glc Lipid
MALDI-TOF/TOF-MS spectrum of glycans released by acid hydrolysis from a lipid fraction obtained from CHO-EG2 cells
3 4 5 6 7 8 9 10 11 12 25mM 15mM 5mM 0mM GU value
Initial [glucose]
Effect of glucose concentration on Lipid- linked oligosaccharides (LLOs)
GlcNAc Man Glc
Effect of glucose concentration on Mab glycoforms
5 6 7 8 9 10 GU value 0mM Media [Glc] 5mM 10mM 12.5mM 15mM 17.5mM 25mM GI 0.26 0.20 0.27 0.43 0.49 0.57 0.59
F(6)A2G0 F(6)A2G1 F(6)A2G2 F(6) A2G2S2 F(6) A2G2S1 Bo Liu
37
Glucose deprivation affects galactosylation and sialylation
IgG1 EG2-hFc
John Doering
Galactose UDP-Gal Gal-1P
Golgi
Glucose Glc-6P UDP-Glc
UDP-Glc Glc-6P
Pathway for galactosylation from galactose feeding Feeding mixture :- Uridine Manganese chloride Galactose Gramer et al : Biotech & Bioeng 2011
N-glycan glycosylation profiles EG2 antibody from CHO cells grown in supplemented media.
UMG 4 mM uridine, 8 μM manganese chloride 20 mM galactose.
GI=0.83 John Doering
Source/ Cell type Antibody Variable parameter GI range Publication Source
Murine (Sp2/0) Chimeric / human IgG1 3 commercial Mabs 0.32-0.39 Raju & Jordan, 2012 Hamster (CHO) Human/ humanized IgG1 3 commercial Mabs 0.09-0.23 Raju & Jordan, 2012 Hamster (CHO K1) Human-camelid EG2 Uridine/Mn2+/gal 0.73-0.83 Liu et al, 2014*
*Liu, B., Spearman,M., Doering,J., Lattova,E., Perreault,H. and Butler,M. J. Biotechnol , 170: 17-27 2014
Venkata Tayi and Michael Butler Patent : Serial Number US61/941.172
Protein A binding site is same as the one for FcRn/FcRc receptors Source: Cambridge University, Protein A binding to constant Fc portion of Rituximab Source: Protein Database Protein A / Protein G Protein A Fc region glycans Protein A glycans Fc region FcRn
Wash Loading Wash Modification Incubation for
temperature Wash buffer Reactants mixture in
buffer Wash buffer Wash Column with immobilized affinity ligand Incubate at RT to capture Abs Wash buffer mAb solution Elution buffer Modified mAb with desired glycosylation
UDP
Monoclonal antibody Affinity protein like protein A /protein G Glycosyltransferase and/or glycosidase enzymes Nucleotide sugars Solid support Optimized buffer
Schematic view of method(s) for in-vitro glycan remodeling of mAbs to obtain single-glycoforms
28 30 32 34 36 38 40 42 Retention time (min)
Modification of a chimeric heavy chain monoclonal antibody (EG2-hFc)
28 30 32 34 36 38 40 42
Retention time (min)
Glycan profile of EG2-hFc from culture harvest
chain antibody
EGFR on tumor cells
28 30 32 34 36 38 40 42 Retention time (min)
28 30 32 34 36 38 40 42
Retention time (min)
GlcNAc Mannose Galactose Fucose Sialic acid
Modification of Cetuximab monoclonal antibody
26 28 30 32 34 36 38 40 42
Retention time (min)
Glycan profile of Cetuximab from culture harvest
GlcNAc Mannose Galactose Fucose Sialic acid
26 28 30 32 34 36 38 40 42
Retention time (min)
IgG1-type antibody
26 28 30 32 34 36 38 40 42
Retention time (min)
26 28 30 32 34 36 38 40 42
Retention time (min)
Modification of antibodies to α(2,6) Sialylated antibodies
30 32 34 36 38 40 42 44
Retention time (min)
Glycan profile of aIL8-hFc from culture harvest
30 32 34 36 38 40 42 44 Retention time 28 30 32 34 36 38 40 42 44 Retention time
α(2,6) α(2,6) α(2,6) α(2,6) α(2,6) α(2,6) 28 30 32 34 36 38 40 42
Retention time (min)
Glycan profile of EG2-hFc from culture harvest
α(2,3) α(2,3) α(2,3)
265,000x
HA N-glycan profiles of (A) Sf9-rHA, (B) Mimic-rHA, and (C) CHO-rHA
Shih-Chang Lin, Jia-Tsrong Jan, Ben Dionne, Michael Butler, Ming-Hsi Huang, Chung-Yi Wu, Chi-Huey Wong, Suh-Chin Wu. “Different immunity elicited by recombinant H5N1 hemagglutinin proteins containing pauci- mannose, high-mannose, or complex type N-glycans.” Plos One 14;8(6): 10.1371 : 2013
3D structure model. (A) Insect cell expressed HA attached with pauci-mannose N-glycans and (B) mammalian cell expressed HA attached with complex-type N-glycans were created by the crystal structure of HA (A/Vietnam/1194/04, PDB ID: 2IBX) and Glyprot. H5N1 Immunogenicity of HA Glycoproteins
Neutralizing antibodies against homologous and heterologous clades of H5N1 viruses.
Lin et al Plos One 14; 8(6): 10.1371 : 2013
The biantennary glycan structure on the conserved site of IgG has reduced galactosylation at low DO or under reducing conditions
<[Glucose] or < [Glutamine] may cause deficient glycosylation
Strategic feeding of galactose, mannose, fructose or N-acetylmannosamine may increase terminal glycosyltransferase
Glycosylation profile depends upon cell host and the profile affects functional properties of the virus
Strategic to produce single glycoform Mabs during downstream processing
Maureen Spearman Bo Liu Carina Villacres John Dearing Ben Dionne Institute of Biology, Taiwan Suh-Chin Wu