Provides an overview of clinical consequences of immunogenicity of biologicals.

1. Immunogenicity of Biologicals:
Clinical Consequences
Dr. S.К. Zyryanov
Professor, Chair of Clinical Pharmacology,
N.I. Pirogov Russian National Research Medical
University (RNRMU)

2. Main Definitions
Biological
A drug, the active substance of which is a
biological substance derived or extracted from
a biological source, including using one or
more of the above mentioned biotechnological
methods: rDNA technology; controlled
expression of genes encoding bioactive protein
production; hybrid and monoclonal antibody
methods

3. What is the difference between biologicals and
low-molecular drugs?
• Molecular weights
• Structure complexity
• Features:
– Structural and physical & chemical properties
– Protein purification degree
– Biological activity
• Stability
• Immunogenicity!
Crommelin DJA, et al. Int J Pharm 2003;266:3-16.

4. Factors Influencing Immunogenicity
Schellekens H. Nat Rev Drug Discov 2002:457–62
Sequence variation Glycosylation
Human Bacterial
Contaminants and
impurities (from
initial production
or downstream
processing)
Formulation
Route of
administration Dose
February
Treatment duration Assay technologies Patient characteristics Unknown factors
Immunogenicity

5. Frequency of Immune Response to Tumor
Necrosis Factor (TNF) Inhibitors
Humira/Adalumimab % of patients with antibodies
Juvenile idiopathic arthritis 5.9% (+MTX) 25.6% (-MTX)
Rheumatoid arthritis 0.6% (+MTS) 12.4% (-MTX)
Remicaid / Infliximab
Adults 38% (3 mg/kg) 12% (6 mg/kg)
Children 2.9%
Enbrel/ Etanerzept
Rheumatoid arthritis 6%
Psoriatic arthritis 7.5%
Ankylozing spondyloatritis 2%
Adult psoriasis 7%
Child psoriasis 9.7%
Juvenile idiopathic artritis 3%
MTX – metatrexat

6. Reported Frequencies of β-1a Interferon
Antibody Formation
Extract from the article by Bertolotto et al J Neurology 2004
Study Determination of positive response to
neutralizing antibodies (Нат)
Analysis
method
Dosage (IM,
weekly)
Share of Нат-positive patients
(%)
Jacobs et al. (1996) [14] Not reported N/A 30 mcg 22*
Herden et al. (1999) [10] Min. 1 out of 20 titres CEA 30 mcg 5
Rudick et al. (1998) [34] Min. 1 out of 20 titres CEA 30 mcg 6
Jacobs et al. (2000) [13] Min. 1 out of 20 titres CEA 30 mcg 2
Clanet et al. (2002) [4] Min. 1 out of 20 titres CEA 30 mcg
60 mcg
2,3
5,8
IM – intramuscular; CEA – cytopathic effect analysis
* Нат frequency was higher than in earlier studies because Jacobs et al. [14] had conducted studies using a non-commercial drug before
the production process was improved.

7. r-protein Indications Immunogenicity
Insulin Diabetes Approx. 44% of patients (5%
IgE)
Growth hormone GH-deficiency 3%-16% of patients
Erythropoietin Anaemia 1:10 000 patients
VIII factor Haemophilia Approx. 35% of patients
IFN- Hepatitis etc. Approx. 25% of patients
IL-2 Certain tumors 20%-100% of patients
Immunogenicity of Recombinant Therapeutic
Proteins
(H.Schellekens, 2002)

8. Antibody Formation Consequences
• Efficacy loss
– Interferon alpha 2
– Interferon beta
– TNF inhibitors
– Beta agalsydase
– And many others
• Cross-neutralization of endogenous factors
– Erythropoietin (EPO)
– Growth and megakaryocyte development factor (GMDF)
• Anaphylaxis reaction; serum reaction
– Monoclonal antibodies

9. Antibody analysis method Interferon drug
Efficacy reduction (relapses, NMR or
bio markers)
PRISMS 4 (2005) CEA Rebif® Yes
IFNβ research group (1996) CEA Betaferon® Yes
IFNβ European Research
Group (1998) and (1999)
CEA Betaferon®
Deisenhammer et al. (1999)
MxA protein interferon alpha
test
Betaferon® Yes
Cook et al. (2001) CEA; neopterin test Avonex®; Betaferon® Yes
Vallittu et al. (2002) CEA; MxA protein test Rebif® 22 Yes
Bertolotto et al. (2005) CEA; MxA protein i-RNA test Avonex®; Rebif® 22; Betaferon® Yes
Pachner et al. (2003)
CEA; MxA protein i-RNA test;
ОАС
Not reported Biomarker reduction
Malucchi et al. (2004) CEA Avonex®; Rebif® 22; Betaferon® Yes
Petkau et al. (2004) CEA; MxA protein test Betaferon® Yes
Perini et al. (2004) CEA Avonex®; Rebif® 22; Betaferon® Yes
Kappos et al. (2005) CEA
Avonex® 30 mcg;
Avonex® 60 mcg
Yes
Sorensen et al. (2003) CEA
Avonex® 30 mcg OW; Rebif® 22
mcg OW; Rebif® 22 mcg TTW;
Betaferon® 8 ММЕ/2days
Interferon neutralization
.Extract from the article by R.A. Farrell and G. Giovannoni, Measuring and management of anti-interferon beta antibodies in subjects with multiple
sclerosis, Mult. Scler. 13 (5) (Jun 2007), pp. 567–577.
ß-interferon neutralizing antibodies reduce the multiple sclerosis treatment efficacy

10. Fig. 5. States of stable disease activity and remissionin patients with and without adalimumab
(ААА) antibodies
Bartelds, G. M. et al. JAMA 2011;305:1460-1468
Copyright restrictions may apply.
Weeks Weeks Weeks
Stable remissionStable minimum activity
Stable minimum activity
Patientshare
Patientshare
Patientshare
Number of persons at risk Number of persons at risk Number of persons at risk
UI/ml
UI/ml

11. Impact of Antibodies on Beta-
Agalsidase Efficacy in Fabry Disease
Benichou et al. Molecular tools and genetics (2008)
Shareofpatients
withnon-zeroscore
Serum negative Low titers Medium titers High titers

12. Biosimilar = generic
‘Biosimilar’ is a biological that is similar to the originator
biological but is not its generic due to differences in the initial
raw materials and manufacturing of the originator biological
and of the biosimilar…
Source: translated from Article 10 (2) b of Directive 2001/83 as amended
Why should we be cautious speaking about
substitutability of biologicals?

13. Regulation of biosimilar manufacturing and application in EC
Amended Directive 2001/83/EC
(2003/63/EC, 2004/27/EC)
Overarching Guidelines
(CHMP/437/04)
Quality Guidelines
(CHMP/49348/05)
Non-Clin & Clin Guidelines
(CHMP/42832/05)
r-Human Insulin Guidance
(CHMP/42832/05)
Somatropin Guidance
(CHMP/94528/05)
r-GCSF Guidance
(CHMP/31329/05)
r-Erythropoeitin Guidance
(CHMP/94526/05)
Immunogenicity Guidance
(CHMP/14327/06)
Low Molecular Weight Heparin
Guidance (CHMP/118264/2007)
r--Interferon Guidance
(CHMP/102046/06)
Guidelines under preparation

14. Findings of the Сomparative Alpha Epoietin
Drug Study
Singh A. K.
Brigham and Women’s hospital & Harvard Medical School,
Boston, USA.
World Congress of Nephrology – Apr 22, 2007
47 samples from the following countries were tested: Argentine,
Brazil, Columbia, India, Indonesia, Iran, Jordan, Korea, Lebanon,
Nigeria, Philippines, Russia, Thailand, Venezuela, Viet Nam and
Yemen
The samples were tested according to the European
Pharmacopoeia quality requirements to alpha epoietin. Eprex®
(alpha epoietin) was used as a reference product.
Singh AK. Poster presented at the World Congress of Nephrology, 21-25 April 2007, Rio de Janeiro, Brazil

15. 15
Singh A. K. Study Findings
 pH
 9 samples deviated from the specification requirements
 Osmosis
 21 samples had greater osmosis
 Protein and erythropoietin content
 1 sample did not meet the specification requirements as concerns the
protein content
 8 samples had excess erythropoietin
 In vitro biological sample
1 sample did not meet the specification requirements
 18 samples deviated from the specification requirements
 In vivo efficacy
48%-163% efficacy differences
 9 samples did not meet the specification requirements
 6 samples deviated from the specification requirements
 Bacterial endotoxin
2 samples contained bacterial endotoxin
Singh AK. Poster presented at the World Congress of Nephrology, 21-25 April 2007, Rio de Janeiro, Brazil

16. 16
 Aggregate content
 29 samples exceeded the specification by the number of aggregates
 7 samples had more than 1% and less than 2% aggregates
 4 samples had 2%-4% aggregates
 18 samples had more than 4% aggregates
 Isoform content
 34 samples had excess isoforms, which may reduce the clinical
effect
 9 out of 34 samples had ≥ 3 additional isoforms
Singh A. K. Study Findings
Singh AK. Poster presented at the World Congress of Nephrology, 21-25 April 2007, Rio de Janeiro, Brazil

17. Alpha Epoietin Drug Comparative Study
Findings (continued)
Epoietin biosimilar
In-vitro bio
sample
In-vivo efficacy on mice,
%
Bacterial endotoxin
(EU/ml)
Aggregates, %
Standard (Eprex®) 80-125 80-125 < 2.5 < 1
Epocrim1 94 73 < 0.5 < 1
Epocrim2 175 76 < 0.5 2.4
Epokin 1 84 91 < 0.5 1.7
Epokrin 1 167 135 < 0.5 1.2
Epokrin 2 155 117 < 0.5 >4
Epokrin 3 175 149 < 0.5 < 1
Eporon 170 163 < 0.5 > 4
Epoet 141 95 < 0.5 2.7
Gerepo 141 48 < 0.5 > 4
Hemax 1 139 97 < 0.5 > 4
Hemax 2 132 101 < 0.5 > 4
Hemax 3 141 90 < 0.5 > 4
Hypercryte 153 113 < 0.5 > 4
Renogen 110 108 < 0.5 > 4
Vepox 177 57 > 2.5 > 4
Zirop 142 Two failures < 0.5 > 4

18. Epokim 1
2
3
Epokrin 1
2
3
Eporon
Epovet 1
2
Espogen 1
2
3
Geerepo
Hemax 1
2
3
Hypercryte 1
2
Vepox
Zirop 1
2
Alkali isoforms
Acid isoforms
0 1 2 3 4 5
Total quantity of additional isoforms
Alpha Epoietin Drug Comparative Study Findings
(continued)

19. Conclusions on Alpha Epoietin drug
Comparative Study Findings:
 Several out of the tested epoietin biosimilars had variable quality and efficacy;
42 out of 47 samples did not meet all European Requirements to alpha
epoietin
 34 samples contained additional alkali isoforms that may reduce clinical drug
efficacy
 2 samples contained bacterial endotoxin that poses risk for the patient’s safety
 22 samples contained more than 2% aggregates that may influence the drug
immunological profile
 Detected differences lead to unpredictable adverse events in clinical practice
 Studied biosimilars had differences in various series of one and the same drug
Specialists applying epoietin generics in their practice should track their
efficacy and safety attentively
Singh AK. Poster presented at the World Congress of Nephrology, 21-25 April 2007, Rio de Janeiro, Brazil

20. 20
July 09ASH
Abstract &
Poster #
Immunogenicity of Low Molecular Weight Heparins and Their
Biosimilars
W.P.Jeske
J.Walenga
HIT Antibodies Synthesis Induction
In Vitro Cross-Reactivity of Branded LMWHs (1 g/mL) with
HIT Antibodies in the Platelet Aggregation Assay
1 2 3 4 5 6 7 8
%Aggregation
0
2 5
5 0
7 5
1. Saline 5. Reviparin
2. Dalteparin 6. Parnaparin
3. Enoxaparin 7. Tinzaparin
4. Nadroparin 8. Heparin
In Vitro Cross-Reactivity of Branded LMWHs (1 g/mL) with
HIT Antibodies in the Platelet Aggregation Assay
1 2 3 4 5 6 7 8
%Aggregation
0
2 5
5 0
7 5
1. Saline 5. Reviparin
2. Dalteparin 6. Parnaparin
3. Enoxaparin 7. Tinzaparin
4. Nadroparin 8. Heparin
In Vitro Cross-Reactivity of Biosimilar Versions of Enoxaparin and
Dalteparin (1 g/mL) with HIT Antibodies in the
Platelet Aggregation Assay
1 2 3 4 5 6 7
%Aggregation
0
2 5
5 0
7 5
1. Saline 5. Dilutol
2. Clenox 6. Lupenox
3. Cutenox 7. Daltehep
4. Dripanina
In Vitro Cross-Reactivity of Biosimilar Versions of Enoxaparin and
Dalteparin (1 g/mL) with HIT Antibodies in the
Platelet Aggregation Assay
1 2 3 4 5 6 7
%Aggregation
0
2 5
5 0
7 5
1. Saline 5. Dilutol
2. Clenox 6. Lupenox
3. Cutenox 7. Daltehep
4. Dripanina

21. Clinical case: Clexan
substitution with a generic
A patient had taken Clexan for 4 years
without complications. Upon transition
to the enoxaparin biosimilar, the
patient experienced 2 life-
threatening haemorrhages within 4
months from the onset of use.
This case suggests that FDA should follow
EMA recommendation to have a more
stringent biosimilar approval procedure:
bioequivalency and the studies that display
safety and efficacy similar to those of the
originator before approval of the
biosimilar.
Biologicals and Biosimilars
Clinical Case

22. М.V. Belov, A.S. Petrochenko, E.M. Pozdnyakova, V.V. Yakusevich. Efficacy of different tromboembolic complication prevention methods in clinical practice
(retrospective study). Clinical Pharmacology and Treatment, 2012, 21 (4)
Biologicals and Biosimilars
Clinical Pre-Surgical Practice Findings
Goal. Assessing the efficacy of tromboembolic
consequences prevention in the pre-surgery
period using unfractionated heparin and
enoxaparin biosimilar.
Findings. Frequency of LLVT did not reliably differ
in the patients receiving unfractionated heparin
and enoxaparin biosimilar.
Opinion. Lack of pronounced advantages of
enoxaparin biosimilar to unfractionated heparin
may be due to heterogeneity of the raw materials
used for manufacturing of the originator and
enoxaparin and biosimilar.
Enoxaparin biosimilar did not differ from
unfractionated heparin in terms of LLVT prevention
efficacy.
Frequency (%) of lower limb venous thrombosis
before surgery (LLVT)
Enoxaparin biosimilarUnfractionated
heparin

23. Immunogenicity Forecasting
 Establishment of physical and chemical
properties
 Epitope analysis (in silico/in vitro)
 Reactions with the patient’s serum
 Animal tests
• Conventionally used animals (relative
immunogenicity?)
• Subprimates
• Immune tolerant transgenic mice

24. Biosimilar Immunogenicity Issues
 The existing analysis methods do not fully predict
biological properties
 The immune system is able to notice changes in a
drug, which are not detected using analytical methods
 Immunogenicity of biologicals may have significant
clinical consequences
24

25. Immunogenicity and Biosimilar
Aspects
 Immunogenicity can only be detected in clinical
studies
 Problems of trackability and substitutability with
similars
 Substitution must not be there the immunogenicity
risk factor is
 Immunogenicity may make a patient immune to a
whole drug class
 Standardization