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 study of Mabs – effect of low nutrient levels – enhancement of glycosylation profiles • Designer-based glycosylation • Effect of glycosylation on a virus • Conclusion

beta - interferon immunoglobulin Protein N-glycan sites erythropoietin 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

Antibody structure - IgG based on X-ray crystallography data heavy chain Fab region V H glycan C H 1 hinge G0F light chain C H 2 domain G1F C H 3 domain Fc region G2F

IgG Fc CH2: galactose binding pocket solvent accessible surface Asn297 Asn297 terminal terminal Gal GlcNAc

Activities of Rituximab

Exoglycosidase digestions Sialidase JBH BTG PNGaseF BKF

Exoglycosidase sequencing of serum IgG on normal phase HPLC ~ 2AB 2AB { 2AB 2AB 2AB 2AB 2AB 2AB ~ * *  1,6  1,3 + sialidase (ABS) 2AB 2AB +  -galactosidase (ABS+BTG) 2AB 2AB +  -fucosidase (ABS+BTG+BKF) +  -hexosaminidase 2AB 2AB (ABS+BTG+BKF+SPH) Gu 6 7 8 9 5

N-Linked Glycosylation Pathway Oligomannose Asn Xaa Ser/Thr Type Dol Endoplasmic Reticulum P  -Glc I  -Glc II  -Glc II  -Man I P NH 2 Oligosaccharide transferase Man Golgi Glc Glc Glc Complex type Man GNAc Tase II Hybrid deficiency type

Type IIa CDG Oligomannose Asn Xaa Ser/Thr Type Dol Endoplasmic Reticulum P  -Glc I  -Glc II  -Glc II  -Man I P NH 2 Oligosaccharide transferase Man Golgi Glc Glc Glc Complex/ single Man antenna GnTase II deficiency

Glycosylation of IgG GlcNAcT-II deficiency (Mgat2) CDG IIa patient • • Dysmorphic features, severe psychomotor retardation • Recurrent infection CDG IIa patient mannose IgG Control IgG Gu 6 7 8 9 M Butler A Critchley HF Hebestreit RA Dwek J Jaeken PM Rudd et al Glycobiology 13: 601-622: 2003 .

Culture environment • G lucose -reduced occupancy at [glc]<0.5 mM • DO dissolved oxygen -reduced galactosylation of Mab at low DO • Ammonia - reduced sialylation - perturbation of antennarity • Degradative enzymes -removes sialic acid

Substrate utilisation and by-product formation in PQXB1/2 hybridomas 10 2 8 ammonia 1.5 lactate 6 glutamine 1 glucose 4 0.5 2 0 0 6 0 1 3 5 2 4 Time (day)

Macroheterogeneity

Production of gamma interferon in extended batch culture N2 60 2-site occupancy N1 1-site occupancy No site occupancy 50 Percentage of glycoform N0 40 30 20 10 0 40 60 80 100 120 140 160 180 200 Time of culture (h) Proportion of N0 increases 5 to 30% during culture Curling et al 1990

Microheterogeneity

The effect of oxygen on Mab production 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 G0F 2 G1F 3 G2F 4 G2FS

60 Change in galactosylation with variable dissolved oxygen G1F DO % GI 50 Relative HPAEC-PAD peak area (%) 1 0.37 2 0.38 GOF G1F 5 0.39 G2F 40 10 0.41 25 0.41 50 0.46 30 G0F 100 0.56 G2 + 0.5*G1 GI = ------------------ 20 (G2 + G1 + G0) G2F Kunkel, J.P., Jan, D.C.H., Butler,M. and Jamieson, J.C. Biotechnol. Progress 16, 462-470: 2000. 10 0 20 40 60 80 100 Kunkel, J.P., Jan, D.C.H., Jamieson, J.C. and Butler, M. Dissolved oxygen (% DO) . J. Biotechnol. 62; 55-71: 1998

Factors that affect galactosylation/ sialylation :- -Accessibility of glycan site -Activity of glycosyl transferases -Activity of transporter + CMP- -Availability of substrates + UDP- -Protein concentration UDP-galactose CMP-sialic acid transporter transporter + CMP- + UDP- Galactosyl transferase Sialyl transferase Sialidase - Degradative enzymes

NSERC strategic Network for the large scale production of single-type glycoform monoclonal antibodies Scientific Director: M. Butler www.mabnet.ca University of Manitoba

Human and llama antibodies 150 kDa 80 kDa Biotechnology Focus 15(5) 11-12: May 2012 www.mabnet.ca

Fig. 2.12 Chinese Hamster Ovary Cells Scanning electron micrograph of non-adherent CHO cells x2,300 (Porter et al. 1973) CHO cells adhering to a plate

www. biogro-technologies.com

Case Study 1 The effect of nutrient concentration on glycosylation 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 cells product Set-point nutrient Fed-batch

Glucose deprivation / starvation 1: Reduced glycosylation site occupancy (Stark & Heath, 1979) 2; High mannose structures • CHO cells • deprive cells of glucose • Pulse label with mannose Asn Man 5 GlcNAc 2 (Rearick et al, 1981)

Consumption of glucose over 24 h Bo Liu 2014

Effect of glucose depletion on:- - Macroheterogeneity - Intracellular glycan precursors - Microheterogeneity

Comparison of Protein A purified Mabs on 4~15% reduced SDS-PAGE gel Initial [Glc] mM 0 5 10 12.5 15 17.5 25 control % lower band 52 40 30 26 0 0 0 0 Bo Liu P34

Mass Spectrometry Analysis (MALDI-MS) A. B. C. Molecular mass kDa 82660 GLYCOPROTEIN 250 150 100 75 50 79340 37 PURIFICATION 50000 60000 70000 80000 90000 100000 110000 m/z MALDI-MS Fig 21. MALDI-MS on protein A purified CHO-EG2 mAb. A. GLYCOPEPTIDE ANALYSIS 33 B. SDS/PAGE mAb produced in media containing 3mM Glc, 4mM Gln Carina Villacres P121 C. MALDI-MS spectra recorded in the linear mode from intact mAb

MALDI-TOF/TOF-MS spectrum of glycans released by acid hydrolysis from a lipid fraction obtained from CHO-EG2 cells 4 x10 2373.80 5 4 Intens. [a.u.] 3 2 Lipid 2211.75 Man 2049.68 1239.44 1563.53 1887.62 1 1725.58 2536.91 1401.48 Glc GlcNAc 0 m/z 1000 1400 1800 2200 2600

Effect of glucose concentration on Lipid- linked oligosaccharides (LLOs) 0 Initial [glucose] 25mM 15mM 5mM Man 0mM Glc GlcNAc GU value 3 4 6 9 5 7 8 10 11 12

Effect of glucose concentration on Mab glycoforms F(6)A2G0 F(6)A2G1 F(6)A2G2 GI Media [Glc] F(6) A2G2S1 0.26 0mM 0.20 5mM 0.27 10mM F(6) A2G2S2 0.43 12.5mM 0.49 15mM 0.57 17.5mM 0.59 25mM GU value 5 6 8 9 10 7 Bo Liu

Galactosylation Index (GI) • Used to calculate overall ratio of galactose residues within glycan profiles • Shift in GI may signal change in culture parameters and metabolism 37

Glucose deprivation affects galactosylation and sialylation on monoclonal antibody

EG2-hFc & DP-12 Glycan Profiles IgG1 EG2-hFc John Doering

Pathway for galactosylation from galactose feeding Galactose Glucose Glc-6P Gal-1P UDP-Glc Glc-6P UDP-Glc UDP-Gal Feeding mixture :- Uridine Golgi Manganese chloride Galactose Gramer et al : Biotech & Bioeng 2011

N-glycan glycosylation profiles EG2 antibody from CHO cells grown in supplemented media. GI=0.83 UMG 4 mM uridine, 8 μM manganese chloride 20 mM galactose. John Doering

Variable galactosylation of antibodies reported in the literature 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

Case Study 2 Methods to produce single glycoform monoclonal antibodies Venkata Tayi and Michael Butler Patent : Serial Number US61/941.172

Fc region Protein A glycans Protein A binding to constant Fc portion of Rituximab Source: Protein Database glycans Protein A / Protein G Fc  Rn Protein A Fc region Protein A binding site is same as the one for FcRn/FcRc receptors Source: Cambridge University,

Schematic view of method(s) for in-vitro glycan remodeling of mAbs to obtain single-glycoforms Reactants mixture in Wash Elution Wash Wash optimized buffer mAb solution buffer buffer buffer buffer Incubation for Incubate at optimum time at RT to Column with optimum capture Abs Modified mAb immobilized temperature with desired affinity ligand glycosylation Wash Wash Wash Loading Modification Glycosyltransferase and/or Monoclonal antibody glycosidase enzymes Affinity protein like Optimized buffer protein A /protein G UDP Solid support Nucleotide sugars