Protein Aggregation and Immunogenicity

Protein Aggregation and Immunogenicity
Critical Role of the Formulation
Wim Jiskoot
Division of Drug Delivery Technology
Leiden/Amsterdam Center for Drug Research (LACDR)
PSWC-AAPS
New Orleans
17 November 2010

Practically all proteins are immunogenic
• Proteins of animal origin (> 1920s)
e.g. porcine/bovine insulin
• Human derived proteins (> 1950s)
growth hormone, factor VIII
• Recombinant human proteins (> 1980s)
insulin, interferons, GM-CSF, epo
• Monoclonal antibodies (> 1980s)
mouse, chimeric, humanized, human
• Follow-on biologics / biosimilars (> 2000s)
growth hormone, epoetin, filgrastim ...
Impurities,
contaminants
Product quality

Immunogenicity - definition
T cell
activation
Innate immunity
CDC
(complement
dependent
cytotoxicity)
Cell mediated
cytotoxicity
The ability of a
substance (e.g. antigen
or vaccine) to elicit an
immune response
Hypersensitivity
ADCC
(antibody-dependent cellmediated
cytotoxicity )
Cytokine
storm
Anti-drug antibodies

Immunogenicity - definition
T cell
activation
Innate immunity
CDC
(complement
dependent
cytotoxicity)
Cell mediated
cytotoxicity
The ability of a
substance (e.g. antigen
or vaccine) to elicit an
immune response
Hypersensitivity
ADCC
(antibody-dependent cellmediated
cytotoxicity )
Anti-Drug Antibodies
Cytokine
storm
Anti-drug antibodies

Clinical consequences of immunogenicity vary and are
highly unpredictable
Loss of therapeutic efficacy (common)
IFN-beta, IFN-alfa, anti-TNF proteins, insulin, monoclonals…
Increase of therapeutic efficacy (rare)
growth hormone
Neutralization of endogenous protein (rare)
epoetin, MDGF
General immune effects
allergy, anaphylaxis, serum sickness, etc.
None (common)

Formation of anti-drug antibodies in patient receiving
an anti-TNFα MAb
MAb conc. (ng/ml)
10000
1000
100
30
20
10
Anti-MAb (% binding)
10
0
0 10 20 30 40 50 60
Week
Courtesy of Lucien Aarden

Factors influencing immunogenicity of proteins

Factors influencing immunogenicity of proteins
So, THE immunogenicity of a protein does not exist
Product Characteristics Indication Immunogenicity (%)
Rituxan/rituximab chimeric/ CD20 NHL 0
Rituxan/rituximab chimeric / CD20 SLE 65
Rituxan/rituximab chimeric / CD20 PSS 27

Factors influencing immunogenicity
Product Quality
&
Formulation

Simplified summary:
protein aggregates are immunogenic

Aggregates and immunogenicity: clinical evidence
• Intravenous immunoglobulins (1950s, 1960s)
– Aggregate removal resulted in less immunogenic products
• Human growth hormone purified from formalin-fixed
pituitary glands (1950s)
– The higher the aggregate levels (up to 70%!) the more
immunogenic
• Betaseron
– Contains substantial aggregate levels and is more immunogenic
than other interferon-beta products
• Interleukin 2
– Aggregated formulation induced antibodies in most patients
• Interferon-alfa
– Formulation with oxidized and aggregated products was more
immunogenic than other formulations
Hermeling et al., Pharm Res, 2004; Rosenberg, AAPS J, 2006

Antibody response to human growth hormone: aggregate
level determines antibody response and persistence
(Moore and Leppert 1980)
% 125 I-hGH Bound
50
40
30
Highly aggregated HGH
Minimally aggregated HGH
0.28
0.24
0.20
0.16
0.12
0.08
0.04
0
40
60
Monomer
80
100
20
10
0.28
0.24
0.20
0.16
0.12
0.08
0.04
Monomer
0
3
6 9 12
Months of therapy
15
28
0
40
60 80 100
Courtesy of Amy Rosenberg

Immunogenicity: the formulation matters
Neutralizing antibodies
Immunogenicity differences between IFN-a2 formulations
2000
1800
1600
1400
1200
1000
800
600
400
200
0
0 1 2 3 4 5 6 7 8
Duration of treatment (months)
B (n = 86)
C (n = 110)
D (n = 81)
E (n = 74)
A (n = 190)
Ryff, J Interferon Cytokine Res, 1997

Immunogenicity: the formulation matters
Reformulated REBIF (interferon beta) shows reduced
immunogenicity in phase IIIb clinical trial
260 patients - persistent neutralizing antibodies:
Old = 14%, high titers, 58% relapse free
RNF2 = 2.5%, low titers, 67% relapse free
Courtesy of Matthew Baker

Immunogenicity: the formulation matters
Number of Epoetin Alfa
PRCA Cases
Anti-epoetin antibody-related pure red cell aplasia
Formulation change
Replacement of albumin by Tween 80/glycine in epoetin alfa (outside USA)
80
70
60
50
40
30
20
10
0

Why are recombinant human proteins immunogenic
Simpliflied scheme of the immune system
M. Salmon, 1994
Courtesy of Matthew Baker

Fate of auto-reactive B cells after encountering
self-protein arrays on virus like particles (VLPs)
Monomeric B-cell receptor /
self-Ag complexes
Oligomerization of B-cell receptor /
self-Ag signaling complexes
Toleragenic Signals
Proliferative Signals

Any substance can be rendered immunogenic!
Soluble molecules:
B-cell tolerance
Repetitive epitopes on polymeric or
particulate drug carriers: B-cell stimulation
Prerequisites:
• At least 10-20 repetitive epitopes ()
• Inter-epitope distance between 5-10 nm
Jiskoot et al., Pharm Res 26: 1303-1314 (2009)

B-cell stimulation by epitope arrays
Wouldn’t protein aggregates act similarly
= protein molecule
And particles with adsorbed proteins
= protein molecule
Poliovirus
FMD Virus

Protein immunogenicity – our current knowledge
• Practically all rh therapeutic proteins and antibodies are
immunogenic
• We do not exactly know why
• Besides patient- and treatment-related factors,
formulation and product quality are important
• Aggregates are suspicious as risk factors
• We do not know which aggregates
• We do not know the critical aggregate dose

Protein immunogenicity – our current knowledge
Product quality
Formulation and
characterization tools,
PAT, QbD, …
Clinical immunogenicity
Immunogenicity assays
THE GAP:
Predictive models to link the two;
understanding immune mechanisms

Tools to predict immunogenicity
• Process / formulation characterization
(including aggregates, particles…)
• Animal studies - transgenics
• In silico B-cell prediction
• In silico T-cell prediction
• T-cell epitope screening assays
uncapable of
predicting
formulationrelated
factors
• T-cell activation assays
• Clinical studies
• Postmarketing surveillance / pharmacovigilance

Tools to predict immunogenicity
• Process / formulation characterization
(including aggregates, particles…)
• Animal studies - transgenics
• In silico B-cell prediction
• In silico T-cell prediction
• T-cell epitope screening assays
• T-cell activation assays
THE MAIN CHALLENGE NOW:
• Clinical How studies to bridge the gap
• Postmarketing surveillance / pharmacovigilance

Tools to predict immunogenicity
Transgenic immune tolerant mouse
models to correlate
Product Quality & Formulation
to
Immunogenicity

Human interferon-a2 immune tolerant mice
IgG Titer
100000
rhIFN a2b
ovalbumin
10000
1000
Titers on
day 22
100
10
Wildtypes
Transgenics
Week 1 Week 2 Week 3
Day 1 Day 8
Day 15
Day 22
5 μg / injection
Blood sampling
(s.c. or i.p.)

Preparation of aggregated rhIFNa2
optical density
Fluorescence intensity
OD 214 nm
SDS-PAGE (non-reducing)
HPLC-SEC
0.20 Native
Hydrogenperoxide
0.15
Metal-catalyzed
Glutaraldehyde
0.10
Boiled
0.05
0.00
10 20 30 40
Time (min)
0.30
0.25
0.20
0.15
0.10
0.05
UV/Vis spectroscopy
Native
Hydrogen peroxide
Metal-catalyzed
Glutaraldehyde
Boiled
Trp fluorescence spectroscopy
3.0×10 7 Native
Hydrogenperoxide
2.0×10 7
Metal-catalyzed
Glutaraldehyde
1.0×10 7
Boiled
0.00
200 250 300 350 400 450
Wavelength (nm)
0
300 325 350 375 400 425 450
Emission wavelength (nm)
Hermeling et al., Pharm Res 22, 1997-2006 (2005)

Only “native like” aggregates induce antibodies against native
recombinant human IFNa2a in transgenic mice
anti-rhIFNa2b IgG titer
1000
t=14 days t=21 days
100
10
5/5
4/5
N M G H B
N= untreated
M= metal-catalyzed oxidized
G= glutaraldehyde treated
H= oxidized by H 2 O 2
B= boiled
Hermeling et al., Pharm Res 22, 1997-2006 (2005)

Immunogenicity of “native like” aggregates of recombinant
human IFNa2a in transgenic mice is dose dependent
IgG titer
b
Wildtype
Transgenic
10000
1000
**
***
***
5/5
4/5
5/5
100
2/5
10
ND
A B C D E
Increasing aggregate content
Hermeling et al., J Pharm Sci 95, 1084-1096 (2006)

Memory study - injection and blood sampling scheme
Week 1 Week 2 Week 3
t = 0 t = 7 t = 14 t = 21 days
… Week 9
Week 4
Wash out
Week 10 Week 11
Boost
Blood sampling
Injection (i.p.) with
5 μg protein

Transgenic immune tolerant mice do not show immunological
memory against aggregated rh IFNa2a
Anti-rhIFNa2a antibody titers before and after a
6-week wash-out period
Sauerborn et al. (2010) Manuscript in preparation

Antibody response against aggregated rhIFNa2a is T cell
dependent – in both wildtype and transgenic mice
Anti-rhIFNa2a antibody titers after a 3-week
injection protocol
– CD4 + T cells
depleted
+ undepleted
Sauerborn et al. (2010) Manuscript in preparation

Antibody response against aggregated rhIFNa2a is T cell
dependent – in both wildtype and transgenic mice
Anti-rhIFNa2a antibody titers after a 3-week
injection protocol
Pneumovax
(TI antigen)
– CD4 + T cells
blocked
+ not blocked
Sauerborn et al. (2010) Manuscript in preparation

Aggregated rhIFNb does not act as an adjuvant for native
rhIFNa2a in transgenic mice
Anti-rhIFNa2a antibody titers after a 3-week
injection protocol
rhIFNb
Sauerborn et al. (2010) Manuscript in preparation

IgG titer
IgG titer
Adsorption of rhIFNb to metal (but not glass or polystyrene)
beads enhances its immunogenicity in transgenic mice
Anti-rhIFNb antibody titers after a 3-week injection
protocol
10000
10000
p

Conclusions from studies with transgenic mice
Transgenic immune tolerant mice are useful models to
study product-related factors of immunogenicity:
• Some aggregates were shown to be immunogenic, others not
• Immunogenicity of aggregates is dose dependent
• Protein adsorption to non-proteinaceous subvisible particles can
promote antibody formation
• Immune mechanism differs from a classical vaccination reaction:
- No memory
- CD4 + T-cell dependent

Proposed mechanisms of antibody induction by
protein aggregates
V. Filipe et al. In: Aggregation of Therapeutic Proteins (W. Wang and C.J. Roberts, Eds.),
John Wiley & Sons, Hoboken, NJ, pp. 403-433 (2010)

Protein immunogenicity – wrap up
Still work to do!

Acknowledgments
The immunogenicity team
Miranda van Beers
Melody Sauerborn
Suzanne Hermeling
Vasco Filipe
Riccardo Torosantucci
Andrea Hawe
Ruediporn (Sun) Tantipolphan
Vera Brinks
Basak Kukrer
UIPS
Utrecht Institute for
Pharmaceutical Sciences
Ted Randolph
Jared Bee
Daan Crommelin
Huub Schellekens
And may others – students, technicians, collaborators…

Thank you !
Hope to see you again at the
Third Open Scientific EIP Symposium
Immunogenicity of Biopharmaceuticals
8-9 December 2010, Gent, Belgium
Info & registration: http://e-i-p.eu

Epitope presentation in protein aggregates
Native
protein molecules
V. Filipe et al. In: Aggregation of Therapeutic Proteins (W. Wang and C.J. Roberts, Eds.),
John Wiley & Sons, Hoboken, NJ, pp. 403-433 (2010)

IgG titer
Transgenic immune tolerant mice do not show immunological
memory against aggregated rhIFNb
Anti-rhIFNb antibody titers before and after a 6-week
wash-out period
100000
10000
1000
100
10
Before After Before After
Wildtype
Transgenic
Cf clinical data: Perini et al., (2001)
Millonig et al. (2009)
Van Beers et al.
Pharm Res 27: 1812–1824 (2010)

Antibody response against aggregated rhIFNb is T cell
dependent – in both wildtype and transgenic mice
Anti-rhIFNb antibody titers after a 3-week injection
protocol
– CD4 + T cells
depleted
+ undepleted
Sauerborn et al. (2010) Manuscript in preparation

Switching from IFNβ-1b to IFNβ-1a
• Decrease in BAB levels after switch to Avonex®
• No immunological memory
Total
IgG
3 months wash-out
(Betaferon®)
(Avonex®)
Patient A B C D
Perini et al. 2001