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

*

*

*

*

*

*

* 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