Immunogenicity and glycosylation: The key issues for biosimilars Huub Schellekens Utrecht...
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![Page 1: Immunogenicity and glycosylation: The key issues for biosimilars Huub Schellekens Utrecht University.](https://reader033.fdocuments.us/reader033/viewer/2022051215/56649ebd5503460f94bc6c68/html5/thumbnails/1.jpg)
Immunogenicity and Immunogenicity and glycosylation:glycosylation:
The key issues for biosimilarsThe key issues for biosimilars
Huub Schellekens
Utrecht University
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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
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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): ??
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Most biopharmaceuticals induce antibodies
Two mechanisms
Reaction to neo-antigens
Breakdown of immune tolerance
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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
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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
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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
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Pure red cell aplasia associated with EPO
treatment
Data from Nicole Casadevall
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Normal Bone Marrow PRCA Bone Marrow
Bone Marrow SmearBone Marrow Smear
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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
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Why was Eprex implicated?Why was Eprex implicated?
High association between Eprex and PRCA Geographic distributionAssociation with formulation change
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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
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– 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
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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
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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
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What is the role of micelles?What is the role of micelles?
Very unstable No biological data Does not explain
epidemiological data
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Silicon as adjuvantSilicon as adjuvant
Lot of confusion data in the literatureSilicon is inert
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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
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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
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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
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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.
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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
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Reducing immunogenicity
Optimizing production,purification and formulation
Changing sequence (streptokinase, staphylokinase)
Pegylation (ADA)
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GlycosylationGlycosylation
Also an important issue in the biosimilar discussion
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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
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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
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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
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The functions of the The functions of the glycocomponentglycocomponent
Protein folding Protein trafficking Protein targeting Ligand recognition Ligand binding Biological activity Stability Pharmacokinetics Immunogenicity
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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 ?
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Aberrant glycosylation Aberrant glycosylation biosimilar epoetinsbiosimilar epoetins
Retracrit:
> glycoforms without O-glycans.
< N-glycolyl and 0-acetyl neuraminic acid
Epoetin alpha Hexal:
> high mannose
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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?
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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
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What are the unanswered What are the unanswered questions?questions?
– What is biosimilar?– Naming– Label– Safety monitoring
Sensitivity Background data Standardization
– Price Counterfeits