In Partnership with:

Biopharmaceuticals and GlycosylationBiopharmaceuticals and Glycosylation

ABIC, Cork, Ireland

August 27 2008

Lokesh JoshiStokes Professor of Glycosciences

Associate Director, Center for BioAnalytical SciencesNational University of Ireland Galway

The Market For Glycoprotein DrugsThe Market For Glycoprotein Drugs

Estimated Annual World Wide Sales of Selected

Recombinant Proteins

Figure: Pavlou and Reichert, Nature Biotechnology 22, 1513 - 1519 (2004)

Total Biopharmaceutical Sales(proteins, mAbs and nucleic acids)

2004 US $ ~ 33 billion

2010 est US $ ~70 billion

Walsh, Nat. Biotech., 2006, 24:769

2010 Expected Contributions

Non-Ab Proteins US $ > 30 billion

Pavlou and Reichert, Nat. Biotech., 2004

mAbs US $ ~ 20 billion

Hiatt and Pauly, PNAS, 2006

Why do drugs fail?Why do drugs fail?

Toxicity and immunogenicity (49%)

long term safety is still totally unpredictable

Bioavailability and half life (15%)

half life cannot be predicted, only guessed

Metabolism (3%)

drug/drug interactions; parent or metabolite

Human Error (33%)

Faulty understanding of pathophysiology

Structure and Function(Catalytic activity, stability, association, half-life, localization etc.)

PHOSPHORYLATION, AMIDATION, SULFATION, ACETYLATION,

CARBOXYMETHYLATION, ADP- RIBOSYLATION, N-TERMINAL CLEAVAGE,

C-TERMINAL CLEAVAGE, ASPARAGINE LINKED GLYCOSYALTION,

SERINE/THREONINE LINKED GLYCOSYLATION, HYDROXYLYSINE

GLYCOSYLATION, HYDROXYPROLINE GLYCOSYLATION, UBIQUITINATION,

PROTEOLYTIC CLEAVAGE, CROSS LINKING, NON-ENZYMATIC GLYCATION,

HALOGENATION, ADENYLATION, ASPARTYL ISOMERIZATION, DISULFIDE

BOND FORMATION, PRENYLATION, METHYLATION, LIPOYLATION,

ARGINYLATION, PROLINE HYDROXYLATION, LYSINE HYDROXYLATION,

ISOMERIZATION, MYRISTOYLATION, ACYLATION, PALMITOYLATIN,

FARNESYLATION, GERANYLGERANYLATION, DEPHOSPHORYLATION,

DEAMINATION, DEIMINATION, DEGLYCOSYLATION, DEACYLATION,

DESULFATION, DEUBIQUITINATION, ISOPRENYLATION, GPI-ANCHOR,

PROTEOGLYCANS

Representative PostRepresentative Post--Translational ModificationsTranslational Modifications

HumansHumansE. coliE. coli FungiFungi PlantsPlants FishFishInsectsInsects MammalsMammals

Glycoconjugatecomplexity

Glycoconjugatecomplexity

GlycosylationGlycosylationThe Most Challenging Aspect ofThe Most Challenging Aspect of PharmaproteinPharmaprotein EngineeringEngineering

--Where the Variation LiesWhere the Variation Lies

Helenius & Aebi. Science, 291, 2001

NN--Glycosylation PathwayGlycosylation Pathway

Glycoforms

OO--Glycosylation BiosynthesisGlycosylation Biosynthesis

Man Fuc

GDP-Man

Man-l-P

GTP

NADP

Dol-P-Man

GDP-Fuc

Man-6-P

Dol-P

ATP

Fuc-1-P

GTP

ATP

ManNAc-6-P

PEP

Neu5AcCMP-Neu5Ac Neu5Ac-9-PCTP

ManNAc

UDP- GlcNAcUDP-GalNAcGalNAcATP

GalNAc-1-PUTP

GlcNAc-6-P

GlcNAc-l-P

UTP

Dol-P-Glc

GlcNAc

UDP-Xyl

-CO2

UDP-GlcA

UDP-Glc

Glycogen

NAD+

UDP-Gal

Gal

Gal-1-P

UTP UDP-Glc

Dol-P

ATP

Glc-1-P

Glc

Glc-6-P Fru-6-P

ATP

Glutamine

GlcN-6-P

-NH3

Pi

ATP

AcCoANAD

Glycolysis

CMP-Neu5Ac

NADP

Overview of Sugar Metabolism in CellsOverview of Sugar Metabolism in Cells

- Synthesis

- Activation

- Transport

- Transfer

- Removal

* Asialoglycoprotein Receptors* Asialoglycoprotein Receptors* Hepatic Gal/GalNAc Receptors* Hepatic Gal/GalNAc Receptors* Mannose Receptors* Mannose Receptors

Protein of Interest

Host & expressionssystem

Extracellular pH & buffersystem

NH4 accumulation

CO2 accumulation

Temperature

Nutrient feeds

Operating mode (batchvs. perfusion)

Harvest times

Agitator shear

Bioreactor type

Manufacturing site

Factors that can InfluenceFactors that can InfluenceGlycosylation of Biopharma DrugsGlycosylation of Biopharma Drugs

Sialic acid

Galactose

N-acetylglucosamine

Sialic acid

Galactose

N-acetylglucosamine

Mannose

Fucose

Asn

Recombinant Protein GlycosylationRecombinant Protein Glycosylation

• Sources of heterogeneity• Site-occupancy

• Antennarity

• Sialylation / Fucosylation

• Pharmaceutical Relevence• Bioactivity

• Pharmacokinetics

• Immunogenicity

• Stability

Serum HalfSerum Half--Life & ClearanceLife & Clearance

The increase of in vivo potency of glycosylated and

hyperglycosylated proteins appears to be due to an increase in

serum half-life or circulating residence time.

Darbepoetin alfa (DA) (hyperglycosylated rhEPO) exhibits approx 3-fold

longer half-life in humans.

FSH isoforms with high sialic acid content were found to have reduced

renal clearance and increased in vivo bioactivity.

Hyperglycosylated FSH had 3-4 fold increased half-life, enhancing its in

vivo bioactivity.

Serum HalfSerum Half--Life & ClearanceLife & Clearance

rEPO – serum half-life

Non-silaylated – 2 min

Sialylated - 3 hr

rFactor VIII – serum half-life

Non-silaylated – 5 min

Sialylated - 4 hr

Ngantung et al., 2006

Most Biopharmaproteins Induce AntibodiesMost Biopharmaproteins Induce Antibodies

Current analytical methods cannot fully predictbiological properties.

The immune system can detect alterations inproducts missed by analytical methdos.

Immunogenicity to the biopharmaceuticals mayhave serious clinical conseuqences.

Immunogenicity to BImmunogenicity to Biopharmaceuticalsiopharmaceuticals

Limits efficacy for many biological therapeutics IgG antibodies can neutralize a therapeutic protein,

IgG antibodies can block action of endogenous homolog

IgE antibodies can cause anaphylaxis

Poses ongoing concern for licensed productsfollowing changes in manufacture, packaging, andclinical indication

CHARLOTTESVILLE, Va., March 12 2008 -- The unusual but severe allergicreactions to the cancer agent cetuximab (Erbitux), a monoclonal antibody,appear to be caused by preexisting immunoglobulin E (IgE) antibodies,researchers here found.

TheThe IgEIgE antibody was specific for the sugar galactoseantibody was specific for the sugar galactose--αα--1,31,3--galactose expressedgalactose expressedin the cell line used to producein the cell line used to produce cetuximabcetuximab.. The etiology may be tick bites andhypersensitive patients may be identifiable.

The researchers tested blood samples from cetuximab-treated patients from threelocations across the country as well as several control groups. The ImmunoCAPtest captured antibodies that had bound to an antigen, which allowed theresearchers to determine what kind of reaction was occurring.

The assay did detect IgE antibodies as suspected, and more than 95% of thesewere bound to the FabFab portionportion of the cetuximab heavy chain, where galactose-α-1,3-galactose is found.

ErbituxErbitux

Expression System AffectsExpression System AffectsGlycosylationGlycosylation

Mammalian Cells CHO

NS0

C127

Transgenic Animals

Bacterial

Fungi

Yeast

Insect

Plant

Expression System AffectsExpression System AffectsGlycosylationGlycosylation

Bioequivalency ofrecombinantlyengineeredproteins is themost criticalissue

Gomord and Faye 2004

Plant Glycosylation Challenges and Solutions

MALDI-TOF mass spectra of glycans from Arabidopsisthaliana endogenous proteins

R. Strasser et al., FEBS Letters 561 (2004) 132

Plant Glycosylation Challenges and Solutions

C: FucTA/FucTB double knockout(fuct).D: FucTA/FucTB/XylTtriple knockout plants (xylt/fuct).

A: Wild-type A. thaliana.B: XylT knockout (xylt).

Down-regulated expression of plant-specific glycoepitopes inalfalfa through:

Transformation with Human β-(1→4)Gal Transferase Gene

Natural form and fusion with plant Golgi targeting domain

Also, siRNA silencing of XylT and FucT

Overall result:Less β-(1→2)Xyl and α-(1→3)Fuc on glycoproteins whichimproves utility of alfalfa for recombinant use.

Sourrouille et al, 2008, Plant Biotech. J., 6:702-721,

Plant Glycosylation Challenges and Solutions

Physcomitrella patens engineered to reduce β-(1→2)Xyl andα-(1→3)Fuc (Koprivova 2004/Heuther 2005).

Transient protoplast transformation with gene for hEPO

Weise et al, 2007, Plant Biotech. J., 5:389-401

Plant Glycosylation Challenges and Solutions

Authors demonstrated a maximum production ofrhEPO in XylT/FucT knockout plants at 144hours

Yield ~ 14 ng/mL

Maximum yield was over twice that of sameconstruct(s) in WT moss

Oil body targeting of recombinant human insulin in seeds.

Oleosin-human pre-insulin fusion protein expressed in seedsof Arabidopsis

Nykiforuk et al, 2006, Plant Biotech. J., 4:77-85

Biopharmaceutical Production in Plants

Authors recovered insulin up to0.13% w/w of transgenic seedprotein

Oil-body prepared protein wasshown to be biologically active

Culture Conditions Affect GlycosylationCulture Conditions Affect Glycosylation

In vivo Activity of rhErythropoieitin

0

20000

40000

60000

80000

100000

120000

140000

160000

0 1 2 3 4 5 6 7 8 9

Sample

In vivo Activity of rhErythropoeitinObtained from different sources

0

20000

40000

60000

80000

100000

120000

140000

160000

0 1 2 3 4 5 6 7 8 9

Sample

inviv

o

500%

Yuen, C.T. et al., 2003

Roller bottleFed-batch reactorHollow fiber

reactor

Culture Conditions Affect GlycosylationCulture Conditions Affect Glycosylation

BatchBatch--toto--Batch Variation in glycosylationBatch Variation in glycosylation

Gervai et al. (2003), Glycosylation of human recombinant gonadotrophins:characterization and batch-to-batch consistency. Glycobiology 13(3): 179-189.

Antigenicity can differ for productAntigenicity can differ for productfrom different manufacturingfrom different manufacturing sitessites

Schellekens – ppt presentation “Immunogenicity: The key issue for biosimilars”

Antigenicity ofidentical rhIFN-produced at differentsites

CAMBRIDGE, Mass., April 21 /PRNewswire-FirstCall/ -- Genzyme Corporation

(Nasdaq: GENZ) announced today that the FDA has informed the company of itsFDA has informed the company of its

opinion thatopinion that Myozyme(RMyozyme(R) () (alglucosidasealglucosidase alfaalfa) produced at the 160L bioreactor scale) produced at the 160L bioreactor scale

andand MyozymeMyozyme produced at the 2000L scale should be classified as two differeproduced at the 2000L scale should be classified as two differentnt

products because of differences in the carbohydrate structures oproducts because of differences in the carbohydrate structures of the molecules.f the molecules.

Currently, Genzyme has U.S. approval to sell Myozyme manufactured at the 160L

scale, and the company has been seeking clearance from the FDA for Myozyme

produced at the 2000L scale. Production at this larger scale has already been

approved in more than 40 countries.

Myozyme is the only treatment for Pompe disease -- a severe, progressively

debilitating and life-threatening inherited disorder affecting a very small number of

people throughout the world.

Host & expressionssystem

Extracellular pH & buffersystem

NH4 accumulation

CO2 accumulation

Temperature

Nutrient feeds

Operating mode (batchvs. perfusion)

Harvest times

Agitator shear

Bioreactor type

Manufacturing Site

Glycosylation of Biopharma DrugsGlycosylation of Biopharma DrugsCan be affected by several factors:Can be affected by several factors:

SummarySummary

GFl 1.0

Library of Humanized Yeast Strains

GFl 2.0 GFl 3.0 GFl 4.0 GFl 5.0 GFl 6.0

GFl 1.1 GFl 2.1 GFl 3.1 GFl 4.1 GFl 5.1 GFl 6.1

GFl 3.2 GFl 4.2 GFl 5.2GFl 6.2

GFl 3.3 GFl 4.3 GFl 5.3 GFl 6.3

GFl 4.4 GFl 5.4 GFl 6.4

GFl 1.2GFl 2.2

GFl 2.3

GFl 6.x

Choi et al., PNAS, 2003Hamilton et al. Science, 2003Bobrowicz et al., Glycobiology, 2004Gerngross T.U.., Nature Biotechnology, 2004Wildt and Gerngross, Nature Microbiology Reviews, 2005

NeoseNeose--GlycoAdvanceGlycoAdvance improves Sialylationimproves Sialylation

Need to overcome glycosylation challenges

Better biochemical and molecular understanding of glycosylation process

regulation

This knowledge will help in engineering glycosylation pathways

Better analytical tools for RAPID carbohydrate analysis

Genome microarray and Glycan microarray analytical tools

Better mathematical models to understand the complex interactions

involved in controlling glycosylation

Synthetic Glycobiology

SummarySummary