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Relevance of Stability Studies on Demonstrating Biosimilarity
Laura Giribaldi, PhD
Amega Biotech, Argentina
CASSS, CMC Strategy Forum Latin America 2019March 13, Brasilia
Development, Production and Commercialization of Development, Production and Commercialization of BIOSIMILARSBIOSIMILARS
4414 years producing Biosimilars14 years producing Biosimilars
442 Production Plants2 Production Plants
442 R&D Labs2 R&D Labs
441 Preclinical site1 Preclinical site
AMEGA BIOTECH: AMEGA BIOTECH:
CONFIDENTIAL
441 Preclinical site1 Preclinical site
44R&D scientific staff 50 peopleR&D scientific staff 50 people
4415 Biosimilars developed15 Biosimilars developed
44Exports to over 40 countriesExports to over 40 countries
TECHNOLOGICAL PLATFORMS
BACTERIAL EXPRESSION (E. Coli, Inclusion bodies)BACTERIA (E. coli, soluble)
PEPTIDES (E. coli)CHO, PerfusionCHO, fed-batch
PEGYLATIONPEGYLATION
AMEGA BIOTECH R&D SITES
Gemabiotech R&D Lab
Staff : Total 22
(4 PhD, 7 MS, 11 Tech)
R&D platforms
- Molecular Biology
- Cell lines & Bioprocesses
Zelltek R&D Lab
Staff : Total 20
(4 PhD, 5 MS, 11 Tech)
R&D platforms
- Bioprocess
- Purification
Gemabiotech preclinical Site
Staff : Total 10
(1 PhD, 3 MS, 6 Tech)
-Medical Direction
-Histopathology laboratory- Cell lines & Bioprocesses
- Purification
- Analytical dev
(MS: ESI-MS/MS, MALDI-TOF MS)
- Cell Banks QC
- Purification
- Pilot Upstream Facility
- Analytical dev
- Hybridoma development
- Post-transfer process validation
-Animal facilities
-Preclinical studies
-In vivo biological activity
Assays
DP
Protein content
pH
Osmolarity
ANALYTICAL CAPABILITIES
Glycosylation
N-glycans, charged
N-glycans, structure
N-glycan occupation
O-glycans
Monosaccharides
Sialic acidIdentity and structure
Aminoacid sequence (MS/MS)
Biologic activity
Binding assays
Cell proliferation assays
Viral Intereference assays
TNF Interference assay
In vivo EPO assay
In vivo FSH assay
Enzymatic assays
Preclinical tests (rats)Toxicity Local toleranceImmunogenicity PK
Impurities
SEC Aggregation
Aggregation DLS
Hidrophobic variants
Nonreducing SDS-PAGE
Charged variants
Oxidation and deamination (MS)
Aminoacid sequence (MS/MS)
Intact mass (MS)
Immunoidentification
Isoelectric point
Peptide mapping
Disulfide bonds (MS)
Free cystein
Thermodynamic structural stability
Fluorescence spectroscopy
Enzymatic assays
ADCC
CDC
– To establish degradation pathways.
– To differentiate and elucidate the structure of degradation products.
– To determine the intrinsic stability of a drug substance informulation.
– To reveal the degradation mechanisms.
Forced degradation studies
– To reveal the degradation mechanisms.
– To understand the chemical properties of drug molecules.
– To generate more stable formulations.
– To produce a degradation profile similar to that of what would beobserved in a formal natural stability study.
– To solve stability-related problems.
–To be included in comparability exercise –Biosimilars and Manufacturing process
Forced degradation studies
Biosimilars and Manufacturing processchanges
Forced Degradation Studies for Biosimilar Development
Howde, Berkowitz. BIOPHYSICAL CHARACTERIZATION OF PROTEINS IN DEVELOPING BIOPHARMACEUTICALS, 2015
“A stress is applied in a well-controlled repeatable way to two biopharmaceuticalsamples that only slightly differ in the native state, but the difference is too small todetect. On applying and then removing the stress, the resulting states may now moreeasily reveal the subtle underlining difference that initially existed”
Aim: To adjust the stress condition to achieve a measurable degradation
Temperature (60°C) hours 2 8 24 1 3 7
Acid hydrolisis (pH 3) hours 2 8 24 1 3 7
Selected expositionCondition Exposition
Sample: DP Product A – 40,000 IU/ml
1- Exploratory studies
Exposure Exposure
Acid hydrolisis (pH 3) hours 2 8 24 1 3 7
Basic hydrolisis (pH 8) hours 2 8 24 1 3 7
Oxidation (H2O2 0.2% v/v) hours 6 16 24 48 66 6 16 24
Oxidation (H2O2 2% v/v) hours 6 16 24
Agitataion (200 rpm) days 1 3 7 30 45
Freeze/Thawing cycles 16 30 60 60
After studying several exposition times, experimental conditions were selectedin order to achieve the adequate degradation (not too harsh that most of drugproduct has degraded and not too light that degradation level is betweenmethod variability)
Agitation (
C4-HPLC
D e
g r
a d
a t
i o
n
p r
o f
i l e
s
SEC-HPLC In vitro biological activity
2-To establish stability indicating nature of a developed method
D e
g r
a d
a t
i o
n
p r
o f
i l e
s
2- To establish stability indicating nature of a developed method
D e
g r
a d
a t
i o
n
p r
o f
i l e
s
C4-HPLC SEC-HPLC In vitro biological activity
D e
g r
a d
a t
i o
n
p r
o f
i l e
s
Stability indicating methods analysis
Analytical method
SEC
C4 HPLC
In vitro BA
ELISA
Sialic acid
Western blot
SDS-PAGE
Freeze
Thawing1 3 7 1 3 7 1 3 7
Stress conditionTemperature (h) Acid hydrolisis (h) Basic hydrolisis (h) Oxidation (h)
Agitation6 16 24
Stability indicating methods and the conditions that promote degradation for this product were identified.
SDS-PAGE
N-glycans WAX
CZE
Fluorescence spec
Thermal Shift
CD
RMN
No degradation detected
Degradation detected
3- Comparability of the type and extent of degradation profile between biosimilar and reference product
Temperature
3 h 7 h 3 h 7h
Temperature
3 h 7 h 3 h 7h
1- Non stressed Product A2, 3- AmegaBiotech DP4, 5- Reference product
Similar degradation profile was obtained for AmegaBiotech DP and Reference product bothin SDS-PAGE and Western blot
Comparability analysis
30000
40000
50000
60000
70000
80000
In v
itro
Bio
logi
cal A
ctiv
ity
(IU
/ml)
Biological Activity
30
40
50
60
70
80
90
100
Agg
rega
tes
and
dim
mer
s co
nte
nt
(%)
HMW Impurities
Similar behavior was observed for two batches of AmegaBiotech Product A (blue) and two batches of reference product (red)
0
10000
20000
Control 60°C pH 3 + 60°C
In v
itro
Bio
logi
cal A
ctiv
ity
(IU
/ml)
0
10
20
30
Control 60°C pH 3 + 60°C
Agg
rega
tes
and
dim
mer
s co
nte
nt
(%)
Etanercept: FDS and comparability
Conditions Duration Analysis performed
25°C ± 3 °C 8 and 15 days SEC-HPLC HIC-HPLC TNF-α neutralization cell based assay
Sample: DP Product B – 50 mg/ml
50°C ± 3 °C 8 and 15 days SEC-HPLC HIC-HPLC TNF-α neutralization cell based assay
Low pH
(pH 3- 25 ± 3 °C) 8 and 15 days
SEC-HPLC HIC-HPLC TNF-α neutralization cell based assay
High pH
(pH 9, 25 ± 3 °C) 8 and 15 days
SEC-HPLC HIC-HPLC TNF-α neutralization cell based assay
Oxidation
(0.5% v/v H2O2) 3 days
SEC-HPLC HIC-HPLC TNF-α neutralization cell based assay
HMWs variants
Slope ratios were used to compare degradation rates
Truncated variants
Degradation kinetics were similar for AmegaBiotech Product B (green) and Reference product (blue)
Size exclusion chromatography - High performance liquid chromatography (SEC-HPLC)Stress condition: Oxidation
Refe
rence P
roduct
The degradation profile of all samples was highly similar since they all show an increase of Product B fragments (LMWs), denoted by peaks with less retention time than the peak of monomeric form
Am
egaB
iote
chP
roduct
Size exclusion chromatography - High performance liquid chromatography (SEC-HPLC)Stress condition: Oxidation
Refe
rence P
roduct
The degradation profile of all samples was highly similar since they all show an increase of Product B fragments (LMWs), denoted by peaks with less retention time than the peak of monomeric form
Am
egaB
iote
chP
roduct
Potency (TNF-a neutralization) In vitro cell based assay
No significant differences were found between degradation rates of both products
CD near UV- Tertiary structuree
llip
tic
ity
(de
g/m
ol)
Ref product pH 3 15 days
Ref product pH 3 8 days
Ref product pH 3 8 days
Ref product pH 3 15 days
AB Product B pH 3 15 days
AB Product B pH 3 8 days
AB Product B control
Control Condition
Mo
lar
ell
ipti
cit
y
Wavelength (nm)
AB Product B pH 3 8 days
AB Product B pH 3 15 days
AB Product B pH 3 8 days
CD near UV- Tertiary structure
Acid Hydrolisis- 8 days
ell
ipti
cit
y(d
eg
/mo
l)
days
days
Ref product pH 3 15 days
Ref product pH 3 8 days
Ref product pH 3 8 days
Ref product pH 3 15 days
AB Product B pH 3 15 days
AB Product B pH 3 8 days
AB Product B control
Unlike erythropoietin, CD is able to detect changes in etanerceptstructure.Similar spectra are obtained for AmegaBiotech and Reference products after acid hydrolisis (7 and 15 days)
Mo
lar
ell
ipti
cit
y
Wavelength (nm)
days
days
AB Product B pH 3 8 days
AB Product B pH 3 15 days
AB Product B pH 3 8 days
CD near UV- Tertiary structure
Acid Hydrolisis- 15 days
ell
ipti
cit
y(d
eg
/mo
l)
days
days
Ref product pH 3 15 days
Ref product pH 3 8 days
Ref product pH 3 8 days
Ref product pH 3 15 days
AB Product B pH 3 15 days
AB Product B pH 3 8 days
AB Product B control
Unlike Product A, CD is able to detect changes in Product B structure.Similar spectra are obtained for AmegaBiotech and Reference products after acid hydrolisis (8 and 15 days)
Mo
lar
ell
ipti
cit
y
Wavelength (nm)
days
days
AB Product B pH 3 8 days
AB Product B pH 3 15 days
AB Product B pH 3 8 days
Conclusions
• Both for Product A and Product B, forced degradation conditions wereselected, which allowed sufficient degradation to occur over a reasonabletime period
• Stability indicating methods were product-specific
• Degradation was studied not only at the final state but also at intermediatestates (kinetics)states (kinetics)
• FDS showed similarity between compared samples
• FDS allowed to increase product knowledge
• Forced degradation studies constitute a powerful tool for comparabilitystudies in biosimilar development, since they leverage opportunities toidentify structural differences between them
Thank you!Thank you!
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