1. Immunogenicity and glycosylation: The key issues for biosimilars
Huub Schellekens
Utrecht University

2. 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

3. 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): ??

4. Most biopharmaceuticals induce antibodies
Two mechanisms
Reaction to neo-antigens
Breakdown of immune tolerance

5. 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

6. 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

7. Consequences of antibodies
Loss 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

9. Pure red cell aplasia associated with EPO treatment
Data from Nicole Casadevall

10. Bone Marrow Smear
Normal Bone Marrow PRCA Bone Marrow

11. Pure red cell aplasia associated with anti-EPO antibodies
Nicole Casadevall
PRCA case with natural antibodies
cases with antibodies associated with
epoetin treatment

12. Why was Eprex implicated?
High association between Eprex and PRCA
Geographic distribution
Association with formulation change

13. Time course of individual PRCA cases
Since Dec 93 t
Epo-refractory anemia (diagnosis) l
Pure Red Cell Aplasia (diagnosis)
Since Feb 93
Epoetin alfa SC Eprex
Epoetin alfa IV Eprex
Epoetin beta SC NeoRecormon
Darbepoetin
Since
Dec 95

14. 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

15. Main stabilizers used in the epoetin formulations
Epogen®/Procrit® (US)
Eprex® (pre 1998)
Eprex® (post 1998)
NeoRecormon® (1990 launch)
HSA
HSA
Polysorbate 80
Polysorbate 20
Glycine
Glycine
Complex of 5 other amino acids
Calcium chloride
Urea

16. 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

17. What is the role of micelles?
Very unstable
No biological data
Does not explain epidemiological data

18. Silicon as adjuvant Lot of confusion data in the literature
Silicon is inert

19. The evidence for this leachates hypothesis is based on high-pressure liquid chromatography of formulated EPREX®/ERYPO®.
Comparison of the elution profiles of solutions from syringes with uncoated or coated rubber plungers shows the presence of many additional peaks when the plunger is not coated.
The compounds indicated by these peaks are low-molecular-weight elastomers, curing agents, antioxidants, and plasticizers, all of which are normal constituents of synthetic rubber.
Several of these types of molecule, particularly phthalates (used as plasticizers), have been shown in animal experiments to act as adjuvants for immunogenic responses.1–3
References
1. Larsen ST, et al. Toxicology 2001;169:37-51.
2. Larsen ST, et al. Pharmacol Toxicol 2002;91:
3. Larsen ST, et al. Food Chem Toxicol 2003;41:

20. 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

21. 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

22. 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

23. Daily i.p.

25. Reducing immunogenicity
Optimizing production,purification and formulation
Changing sequence (streptokinase, staphylokinase)
Pegylation (ADA)

26. Also an important issue in the biosimilar discussion
Glycosylation
Also an important issue in the biosimilar discussion

27. Glycosylation of biosimilar epoetins can be expected to be different
What types of glycosylation are there?
What is the biological significance of glycosylation

28. 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 N-acetylgalactoseamine
Then extended by glycosyltransferases

29. 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: GlcNAc, 9 mannose and 3 glucose molecules.
Trimming by removing glucoses and mannoses and possible adding of GlcNAc

30. The functions of the glycocomponent
Protein folding
Protein trafficking
Protein targeting
Ligand recognition
Ligand binding
Biological activity
Stability
Pharmacokinetics
Immunogenicity

31. 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 ?

32. Aberrant glycosylation biosimilar epoetins
Retracrit:
> glycoforms without O-glycans.
< N-glycolyl and 0-acetyl neuraminic acid
Epoetin alpha Hexal:
> high mannose

33. Epoetin lacking O-linked glycosylation
About 20% lesser activity
Delorme at al. Biochemistry 1992
Explanation for the lower activity of Retracrit?

35. 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

36. What are the unanswered questions?
What is biosimilar?
Naming
Label
Safety monitoring
Sensitivity
Background data
Standardization
Price
Counterfeits