Immunogenicity and Immunogenicity and glycosylation:glycosylation:

The key issues for biosimilarsThe key issues for biosimilars

Huub Schellekens

Utrecht University

Immunogenicity and biotech Immunogenicity and biotech comparabilitycomparability

Current analytical methods cannot fully predict biological properties

The immune system can detect alterations in products missed by analytical methods

Immunogenicity of biopharmaceuticals may have serious clinical consequences

History of the medical use History of the medical use proteinsproteins

Proteins of animal origin (e.g. equine antisera, porcine/bovine insulin): foreign proteins

Human derived proteins (e.g.growth hormone, factor VIII): no immune tolerance

Recombinant human proteins(e.g.insulin, interferons, GM-CSF): ??

Most biopharmaceuticals induce antibodies

Two mechanisms

Reaction to neo-antigens

Breakdown of immune tolerance

Types of immune reaction against biopharmaceuticals

Breaking of self-tolerance

Type of product Human homologues

Characteristics of antibody production

Slow, after long treatment, binding antibodies, disappear after treatment

Cause Mainly impurities and aggregates

Factors influencing Factors influencing immunogenicityimmunogenicity

Structural propertiesSequence variation

Glycosylation

Other factorsAssays

Contaminants and impurities

Formulation

Downstream processing

Route of application

Dose and length of treatment

Patient characteristics

Unknown factors

Consequences of antibodiesConsequences of antibodiesLoss of efficay 

Insulin

Streptokinase

Staphylokinase

ADA

Salmon calcitonin

Factor VIII

Interferon alpha 2

Interferon beta

IL-2

GnRH

TNFR55/IgG1

Denileukin diftitox

HCG

GM-CSF/IL3

Enhancement of efficacy

Growth hormone

Neutralization of native protein

MDGF

EPO

General immune effects

Allergy

Anaphylaxis

Serum sickness, etc

Pure red cell aplasia associated with EPO

treatment

Data from Nicole Casadevall

Normal Bone Marrow PRCA Bone Marrow

Bone Marrow SmearBone Marrow Smear

Pure red cell aplasia associated with Pure red cell aplasia associated with anti-EPO antibodiesanti-EPO antibodies

Nicole Casadevall

- 1996 PRCA case with natural antibodies- 2002 13 cases with antibodies associated with epoetin treatment

Why was Eprex implicated?Why was Eprex implicated?

High association between Eprex and PRCA Geographic distributionAssociation with formulation change

Time course of individual PRCA cases1999 2000 2001 2002 2003J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D J

1997 1998J F M A M J J A S O N D J F M A M J J A S O N D

Epo-refractory anemia (diagnosis)

Pure Red Cell Aplasia (diagnosis)

Epoetin alfa SC Eprex

Epoetin alfa IV Eprex

Darbepoetin

Epoetin beta SC NeoRecormon

Since Dec 93

Since Feb 93

Since Dec 95

– Recent concern over use of HSA in Europe because of potential transmission of infectious viruses or BSE prions

– In 1998, HSA was replaced with polysorbate 80 in prefilled syringes of Eprex® distributed ex-US

Product formulationProduct formulation

Main stabilizers used in the Main stabilizers used in the epoetin formulationsepoetin formulations

Eprex® (post 1998)

Polysorbate 80

Glycine

NeoRecormon®

(1990 launch)

Polysorbate 20

Glycine

Complex of5 other amino acids

Calcium chloride

Urea

Epogen®/Procrit® (US)

HSA

Eprex® (pre 1998)

HSA

Factors potentially contributing Factors potentially contributing to the immunogenicity of to the immunogenicity of EprexEprex®® Formation of micelles associated with Epo

(Hermeling et al, 2003) Silicon droplets in the prefilled syringes Leachates from rubber stoppers Mishandling

What is the role of micelles?What is the role of micelles?

Very unstable No biological data Does not explain

epidemiological data

Silicon as adjuvantSilicon as adjuvant

Lot of confusion data in the literatureSilicon is inert

The leachate theoryThe leachate theory

No biological rationale– Adjuvants do not break B cell tolerance

No experimental data showing breaking tolerance

Does not explain epidemiological data and pathogenesis

MishandlingMishandling

Mishandling with a slightly less stable product may explain all features of PRCA– Biological rationale– Fits with data concerning other product– Fits the pathogenesis– Fits with the epidemiological data

Prediction of immunogenicityPrediction of immunogenicity

Purity of the product Epitope analysis Reaction with patient sera Animal experiments

• Convential animals (relative immunogenicity)

• Non-human primates • Immune tolerant transgenic mice

5 ug AvonexWildtype (C57Bl/6)

1 2 3 4 50.00

0.25

0.50

0.75t=0t=7t=14t=21

Mouse

A 415nm - A 490nm

5 ug AvonexTransgenic immune-tolerant

1 2 3 4 50.00

0.25

0.50

0.75t=0t=7t=14t=21

Mouse

A 415nm - A 490nm

Daily i.p.Daily i.p.

40 ug Betaseron s.c. 2x/weekWildtype (C57Bl/6)

1 2 3 4 5

-0.25

0.00

0.25

0.50

0.75

1.00t=0t=7t=14t=21

MouseA 415nm - A 490nm

5 ug Betaseron daily i.p.Transgenic immune-tolerant

1 2 3 4

-0.25

0.00

0.25

0.50

0.75

1.00t=0t=7t=14t=21

MouseA 415nm - A 490nm

Reducing immunogenicity

Optimizing production,purification and formulation

Changing sequence (streptokinase, staphylokinase)

Pegylation (ADA)

GlycosylationGlycosylation

Also an important issue in the biosimilar discussion

Glycosylation of biosimilar Glycosylation of biosimilar epoetins can be expected to epoetins can be expected to

be differentbe differentWhat types of glycosylation are there?What is the biological significance of

glycosylation

O-linked glycosylationO-linked glycosylation

O-linked to specific serine or threonine but consensus sequence not identified

Apparently defined by secondary structural elements like β turn.

Start with the attachment of a single monosaccharide normally N-acetylgalactoseamine

Then extended by glycosyltransferases

N-linked glycosylationN-linked glycosylation

N-linked to Asn-X-Ser/Thr Most consensus sequences non-glycosylated.

Depends on secondary structures. Glycosylation before folding Starts with binding of DTP-oligosaccharide: 2

GlcNAc, 9 mannose and 3 glucose molecules. Trimming by removing glucoses and mannoses

and possible adding of GlcNAc

The functions of the The functions of the glycocomponentglycocomponent

Protein folding Protein trafficking Protein targeting Ligand recognition Ligand binding Biological activity Stability Pharmacokinetics Immunogenicity

Glycosylation of epoetinGlycosylation of epoetin

40% sugar Three linked N-glycosylation sites at Asn 24, 38 and 83 One O-linked site at serine 126 Heterogeneity caused by variation in core structures and

sialic acid Removal of N-glycosylation sites has no effect on in vitro

activity, but greatly reduces the in vivo activity Half life in rodents IV 5-6h but < 2 min if desialylated Adding N-glycosylation sites increases half-life. Single O-linked side chain removal has little effect ?

Aberrant glycosylation Aberrant glycosylation biosimilar epoetinsbiosimilar epoetins

Retracrit:

> glycoforms without O-glycans.

< N-glycolyl and 0-acetyl neuraminic acid

Epoetin alpha Hexal:

> high mannose

Epoetin lacking O-linked Epoetin lacking O-linked glycosylationglycosylation

About 20% lesser activity Delorme at al. Biochemistry 1992

Explanation for the lower activity of Retracrit?

ConclusionConclusion

The clinical consequences of immunogenicity may be severe

Only clinical trials decisive to reveal immunogenicity

The main difference between biosimilars is glycosylation

Clinical consequences of differences in glycosylation unknown

What are the unanswered What are the unanswered questions?questions?

– What is biosimilar?– Naming– Label– Safety monitoring

Sensitivity Background data Standardization

– Price Counterfeits