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Physicochemical Characterization of Remicade® and its Biosimilar RemsimaTM
Anna Schwendeman
5 October 2015
PQRI Conference, Bethesda, MD
Biologics, mostly mAbs, are top sellers
Best selling prescription drugs 2014. Quartz qz.com2
*
*
*
*
*
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* mAbs
“Patent cliff” presents opportunities biosimilars
3
Rickwood, Di Biase, IMS Health (Dec 2012)
Biologics Price Comparison Act = Hatch Waxman Act
Remicade® and RemsimaTM
• Remicade® (Janssen Biotech Inc) is approved for arthritis (RA),
spondylitis (AS), psoriasis (PS), Crohn’s (CD) and colitis (UC)
• World wide sales in 2014 total of $9.2B
• Biosimilar InflectraTM/RemsimaTM/CT-P13 (Celltrion)
approved in EU, Canada and South Korea
• In EU RemsimaTM was approved for all indications based on AS
(PLANETAS, 250 pt) and RA (PLANETRAS, 606 pt) clinical
studies and in vitro characterization
• In Canada RemisimaTM was not approved for UC and CD
• RemsimaTM is currently under review by FDA
• Difference in surgery rate for UC and CD patients on
Remicade® and RemsimaTM (Single center study in Ireland)
• Phase 3 (214 pt) and observational study (500 pt) in UC and
CD is ongoing
Weiner LM, et al. Nat Rev Immunol. 2010 May;10(5).
Domain Specificity of mAb Effector Functions
5
Fab: antigen
neutralization
Fc: receptor mediated
interactions (e.g. ADCC)
Rheumatoid Arthritis
Inflammatory Bowel Disease
Remicade® and RemsimaTM Comparison in vitro
Infliximab
chimeric IgG1
C6428H9912N1694O1987S46 = 1441930 Da
Major N-Glycoforms
Variable region-murine binding
site against TNF-α
N-Glycosylation
-Asn300
0 or 1 K450
G0F
G1FG2F
Man 5
0
0
214
449/450
Property Remicade Remsima
Sequence Identical Identical
TNF-α binding Identical Identical
FcγRIIIα
binding
Strong Lower
ADCC assay Strong Lower
N-Glycan (Asn
300)
G0F, G1F,
G2F, Man5
G0F, G1F,
G2F
C-terminal
lysine
More
prevalent
FC-region Affects Clearance, Immunogenicity and Receptor Binding
7Wendy S. Putnam, et al. Trends in Biotechnology 2010; 28(10).
Hmiel LK, et al. Anal Bioanal Chem. 2015 Jan;407(1).
Goals of Study
• Assess structural differences and similarities
• Investigate how differences/similarities affect
mAb aggregation in stressed conditions
Remicade® and RemsimaTM Samples
Sample Lot no. Exp Date
Remicade 14AO52P1 2017.01
Remicade 14GO43P1 2017.09
Remicade EIM74016P1 2017.08
Remicade EKL97011P1 2017.10
Remsima 12B1C006BA1 2014.12.19
Remsima 12B1C014BA1 2015.05.04
Remsima 12B1C021BA1 2015.12.03
Remsima 12B1C015BA6 2017.05.08
Identical freeze-dried formulation:
Sucrose, Tween 80
Similarities
11
eV eV
Collision Induced Unfolding (CIU) – IM-MS
Trypsin Digest – Compete Proteomic Analysis
Primary sequence coverage consistent with Remicade®
Similar Oxidation and Deamidation
0
1
2
3
4
5
6
137 138 152 158 31 57 318 364 387 392
N N
LC HC
% X
IC R
ati
o
% Deamidation, N=3
Remicade
Remsima
0
5
10
15
20
25
55 35 18 34 85 255 431 110 316 35 47 280 384
M W M W W W
LC HC LC HC
% X
IC R
ati
o
% Oxidation, N=3
Remicade
Remsima
Oxidation Di-Oxidation
Similar Sequence Variance – Mutations
0
0.05
0.1
0.15
0.2
0.25
200 32 176 202 29 191 205 125 140 284 319 405 307 411 48 95 128 308 311
Gly->Asp Ser->Asn Val->Leu Gly->Asp Ser->Asn Val->Leu
Light Chain Heavy Chain
Remicade Remsima
Re
lati
ve a
bu
nd
an
ce,
%
Disulfide Bonds: Expected and Shuffled
C229
C232
C229
C232
C214
C134
C22
C98
C147
C203
C223
C264
C324
C370
C428
C22
C98
C147
C203
C223
C264
C324
C370
C428
C23
C88
C194
C23
C88
C134
C194
C214
Light
Chain
Heavy
ChainLight
Chain
Heavy
Chain
Cys-1 Cys-2 Remicade Remsima Cys-1 Cys-2 Remicade Remsima
LC-023 LC-088 3.6E+01 3.0E+02 HC-147 LC-134 4.5E+02 5.1E+02
LC-134 1.6E+02 1.5E+02 HC-022 7.4E+01
LC-194 3.7E+01 3.8E+01 HC-264 9.4E+01 1.2E+02
HC-223 1.8E+02 HC-223 LC-134 1.5E+01 5.6E+01
HC-264 1.1E+02 5.9E+01 HC-022 2.2E+01
HC-428 5.5E+01 2.6E+01 HC-264 1.4E+02 6.6E+01
LC-134 LC-088 6.7E+01 HC-428 7.4E+01
LC-134 8.8E+01 1.5E+02 HC-264 LC-134 1.2E+02 1.5E+02
HC-223 1.0E+02 9.5E+01 HC-022 8.3E+01 1.2E+02
HC-264 1.2E+02 1.5E+02 HC-264 3.1E+02 3.0E+02
HC-428 3.8E+01 HC-324 HC-147 2.3E+01 1.7E+01
LC-194 LC-088 4.5E+01 1.0E+02 LC-134 8.3E+01 4.2E+01
LC-194 6.8E+01 4.3E+01 LC-194 3.1E+01 3.3E+01
HC-022 5.2E+01 8.7E+01 HC-022 2.1E+01 3.3E+01
HC-232 2.2E+01 6.0E+00 HC-098 2.7E+00
HC-264 1.3E+02 7.8E+01 HC-428 3.9E+01 4.1E+01
HC-428 5.5E+01 7.7E+01 HC-370 LC-134 2.2E+02 3.1E+02
LC-214 LC-134 3.8E+02 4.4E+02 HC-022 3.0E+01
LC-194 1.5E+02 1.2E+02 HC-264 3.1E+01
HC-022 1.7E+01 HC-428 LC-134 1.5E+02 1.9E+02
HC-264 2.4E+02 1.6E+02 HC-229 3.7E+01
HC-428 1.4E+01 HC-022 1.6E+01 4.9E+01
HC-022 HC-264 4.9E+01 6.4E+01 HC-264 2.2E+02 2.0E+02
HC-098 LC-134 1.1E+02 1.1E+02 HC-428 1.7E+01 4.5E+01
HC-264 5.9E+01 5.1E+01
(blank) HC-229, 232 2.8E+02 5.2E+02
• Shuffled S-S bonds detected at <1% of expected S-S bonds
• Most prevalent shuffled disulfide bonds
Remicade: LC134—LC214, LC134-HC147
Remsima: LC134—LC214, LC134-HC147, HC229-HC232
• Tri-sulfide detected at <0.01% of expected S-S bonds
Differences
Major Glycoforms Differences
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
XIC
Ra
tio
%
Remicade Remsima
G0FG1F G2FG0F-GlcNacMan 5
Remicade Remsima0
5
10
15
20
25
% a
-fu
co
sy
late
d
Remicade
Remsima
Total levels of non-fucosylated mAb
Remicade: 19.7 + 1.6% Remsima: 13.2 + 0.7%
Higher Glycation Levels for Remsima (Trypsin)
0.00E+00
1.00E+08
2.00E+08
3.00E+08
4.00E+08
5.00E+08
6.00E+08
Co
un
ts
Remicade Remsima Remicade Remsima0
1
2
3
4
% Glycation
% G
lycati
on
Remicade
Remsima
FcγIIIa Binding is Weaker for Remsima
Run 1 Run 2 Run 3 Run 4
3.2 µM 1.6 µM 0.8µM 0.4 µM
20
KD
(n
M) Sample kd (1/Ms) x 10 -2 KD (nM)
Remicade 3.08 ± 0.39 162 ± 18
Remsima 6.15 ± 0.96 351 ± 48
Aggregation Studies
Humidity Stressed Samples
28 days
14 days
7 days
0 days
0.0E+00
1.0E+07
2.0E+07
3.0E+07
4.0E+07
5.0E+07
5
35
65
95
12
5
15
5
18
5
21
5
24
5
27
5
30
5
33
5
36
5
39
5
Co
nce
ntr
ati
on
(pa
rtic
les/
mL)
Particle size (nm)
Remicade
Remsima
84
89
94
99
104
0 10 20 30
% m
on
om
er
Time (days)
RC-0 RC-97 RS-0 RS-97
0% RH
97% RH
Remicade
28 days/ 97% RH
Monomer
Dimer
Trimer
454 kDa
Remsima
28 days/ 97% RH
Monomer
Dimer
Trimer
483 kDa
Remicade
0 50 75 97 0 50 75 97MW
Remicade Remsima
250 --
150 --
100 --
75 --
50 --
37 --
Aggregation and Hydrolysis Under Stress
24
Stress Increases Oxidation and Deamidation
M-5
5W
-35
W-9
4M
-18
M-3
4M
-85
M-2
55M
-431
W-4
7W
-110
% X
IC R
atio
M-5
5W
-35
M-1
8M
-34
M-8
5M
-255
M-4
31W
-110
W-3
16
% X
IC R
ati
oDeamidation
N-1
37
N-1
38
N-1
52
N-1
58
N-3
1
N-5
7
N-3
18
N-3
64
N-3
87
N-3
92
0
2
4
6
8
Remicade
Remsima
LC HC
N-1
37N
-138
N-1
52N
-158
N-3
1N
-57
N-2
79N
-289
N-3
18N
-364
N-3
87N
-392
N-4
24N
-437
0
2
4
6
8
Deamidation: Humidity stressed
% X
IC R
atio
Remicade Humid
Remsima Humid
25
Increased Glycation Level in Stressed Samples
- Glycation levels comparable between the two products after stress
- Examination of FCγIIIa binding for stressed samples is ongoing
K-4
9K
-126
K-1
49K
-169
K-1
83K
-190
K-2
07 K-3
K-1
9K
-43
K-5
4K
-78
K-1
36K
-208
K-2
25K
-249
K-2
51K
-291
K-2
93K
-320
K-3
29K
-337
Inte
nsity
K-4
9K
-126
K-1
49K
-169
K-1
83K
-190
K-2
07 K-3
K-1
9K
-43
K-7
8K
-136
K-2
25K
-249
K-2
51K
-291
K-2
93K
-329
Inte
nsity
Summary and Future Work
• Remicade® and Remsima™ are highly similar in term of primary
structure, oxidation, deamidation, mutations, aggregation kinetics
• Measurable differences observed in glycosylation, glycation,
FcγIIIa binding and aggregation pattern
• Relative impacts of glycosylation and glycation differences on
activity, immunogenicity and pharmacokinetics needs to me
further examined
• Similarities/differences with respect to mechanisms of mAbs
aggregation needs to be further examined
• Forced degradation, IM-MS and complete proteomic analysis
appear to be useful tools to characterize mAb products that
require further development
Acknowledgements
Michael Ford Sergei Saveliev
Chris Becker Eric Carlson
S. Schwendeman B. Ruotolo Yuwei Tian
Mi Hee Lim
David Keire Michael Boyne
Rose Ackermann Karthik Pisupati Alex Benet
Tom Tolbert