Immunogenicity and
glycosylation:
The key issues for biosimilars
Huub Schellekens
Utrecht University
Immunogenicity and biotech
comparability

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
proteins
 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
immunogenicity
Structural properties
Sequence variation
Glycosylation
Other factors
Assays
Contaminants and impurities
Formulation
Downstream processing
Route of application
Dose and length of treatment
Patient characteristics
Unknown factors
Consequences of antibodies
Loss of efficay
Insulin
Enhancement of efficacy
Streptokinase
Staphylokinase
Growth hormone
ADA
Salmon calcitonin
Factor VIII
Neutralization of native protein
Interferon alpha 2
MDGF
Interferon beta
EPO
IL-2
GnRH
TNFR55/IgG1
General immune effects
Denileukin diftitox
Allergy
HCG
Anaphylaxis
GM-CSF/IL3
Serum sickness, etc
Pure red cell aplasia
associated with EPO
treatment
Data from Nicole Casadevall
Bone Marrow Smear
Normal Bone Marrow
PRCA Bone Marrow
Pure red cell aplasia associated with
anti-EPO antibodies
Nicole Casadevall
- 1996 PRCA case with natural antibodies
- 2002 13 cases with antibodies associated with
epoetin treatment
Why was Eprex implicated?

High association between Eprex and PRCA
 Geographic distribution
 Association with formulation change
Time course of individual PRCA cases
1997
1998
1999
2000
2001
2002
2003
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 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
Since Dec 93
Since Feb 93
t Epo-refractory anemia (diagnosis)
 Pure Red Cell Aplasia (diagnosis)
Epoetin alfa SC Eprex
Epoetin alfa IV Eprex
Epoetin beta SC NeoRecormon
Darbepoetin
Since Dec 95
Product formulation
– 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
Main stabilizers used in the
epoetin formulations
Epogen®/Procrit® Eprex®
(US)
(pre 1998)
Eprex®
(post 1998)
NeoRecormon®
(1990 launch)
HSA
Polysorbate 80
Polysorbate 20
Glycine
Glycine
HSA
Complex of
5 other
amino acids
Calcium chloride
Urea
Factors potentially contributing
to the immunogenicity of Eprex®




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?

Very unstable
 No biological data
 Does not explain
epidemiological data
Silicon as adjuvant

Lot of confusion data in the literature
 Silicon is inert
The 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
Mishandling

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 immunogenicity

Purity of the product
 Epitope analysis
 Reaction with patient sera
 Animal experiments
• Convential animals (relative
immunogenicity)
• Non-human primates
• Immune tolerant transgenic mice
Daily i.p.
5 ug Avonex
Transgenic immune-tolerant
5 ug Avonex
Wildtype (C57Bl/6)
t=0
t=7
t=14
t=21
0.50
0.25
0.75
A 415nm - A 490nm
A 415nm - A 490nm
0.75
t=0
t=7
t=14
t=21
0.50
0.25
0.00
0.00
1
2
3
Mouse
4
5
1
2
3
Mouse
4
5
40 ug Betaseron s.c. 2x/week
Wildtype (C57Bl/6)
t=0
t=7
t=14
t=21
0.75
0.50
0.25
0.00
1
-0.25
2
3
4
5
Mouse
5 ug Betaseron daily i.p.
Transgenic immune-tolerant
1.00
A 415nm - A 490nm
A 415nm - A 490nm
1.00
t=0
t=7
t=14
t=21
0.75
0.50
0.25
0.00
1
-0.25
2
3
Mouse
4
Reducing immunogenicity

Optimizing production,purification and formulation

Changing sequence (streptokinase, staphylokinase)

Pegylation (ADA)
Glycosylation
Also an important issue in the
biosimilar discussion
Glycosylation of biosimilar
epoetins can be expected to
be different

What types of glycosylation are there?
 What is the biological significance of
glycosylation
O-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 Nacetylgalactoseamine
 Then extended by glycosyltransferases
N-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
glycocomponent









Protein folding
Protein trafficking
Protein targeting
Ligand recognition
Ligand binding
Biological activity
Stability
Pharmacokinetics
Immunogenicity
Glycosylation 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
biosimilar epoetins

Retracrit:
> glycoforms without O-glycans.
< N-glycolyl and 0-acetyl neuraminic acid

Epoetin alpha Hexal:
> high mannose
Epoetin lacking O-linked
glycosylation

About 20% lesser activity
 Delorme at al. Biochemistry 1992

Explanation for the lower activity of
Retracrit?
Conclusion

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
questions?
–
–
–
–
What is biosimilar?
Naming
Label
Safety monitoring



Sensitivity
Background data
Standardization
– Price

Counterfeits