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INTRODUCTION

LC/MS peptide mapping data is commonly used for assessing biosimilarity of biotherapeutics. The comparison based on identified components is a time-consuming process and requires a detailed examination of the data. Often, the biosimilarity conclusions are made based only on the identified components, and any unknown

modifications, sequence variants or impurities that are not documented are seldom considered in the assessment. This can result in erroneous conclusions. However, components (features) in the samples, known and unknown, are all recorded at the ion level but the volumes of data generated are large, and the complexity is high. This work presents an integrated workflow to assess the biosimilarity between an innovator Etanercept and two biosimilar drugs at the ion level based on LC/MSE peptide mapping data.

COMPREHENSIVE ASSESSMENT OF THE BIOSIMILARITY OF PROTEIN BIOTHERAPEUTICS BASED ON ION SIGNAL STATISTICS

IN LC/MS PEPTIDE MAPPING DATA

Stephane Houel1; Mark Bennett2; Ying Qing Yu1; Weibin Chen1 1Waters Corp., Milford, MA 01757, USA; 2Nonlinear dynamics, Newcastle, UK

METHODS

Sample preparation:

Etanercept (innovator and biosimilars) samples were first

denatured in urea and Tris-HCl buffer followed by reduction and alkylation with DTT and IAM respectively. PNGase F was used to

deglycosylate N-glycans while neuraminidase (2-3,-6,-8,-9) was used to desialylate O-glycans. Enzymatic reactions were

incubated at 37oC for 5 hours. Trypsinization was performed at 37 oC overnight. Each sample was prepared in duplicate.

Data acquisition:

Each sample was injected three times. LC: Waters ACQUTIY UPLC I-Class

Column: Acquity UPLC PST 2.1x150mm BEH C18 300Å, 1.7 µm MS: Waters SynaptTM G2-Si HDMS

Instrument Waters SynaptTM G2-Si HDMS Mode: ESI positive mode

Capillary Voltage: 3.0 kV

Cone Voltage: 10 V Source Temperature: 100 °C

Desolvation Temperature: 350 °C

Data Processing: Progenesis QI Only features with charge states between 2 and 13 were

considered in the analysis.

Figure 1. Data were collected using a Synapt G2-Si HDMS system

CONCLUSION

The results show there is a higher degree of similarity between Biosimilar 1 and Innovator (with more

common features and comparable intensities) than that for Biosimilar 2 and Innovator.

In comparison with Biosimilar 1, Biosimilar 2 displays more and higher intensity unique features, suggesting

there is major differences between Biosimilar 2 and Innovator.

Both Biosimilar 1 and Biosimilar 2 show different distributions in common O-glycopeptides (with O-Core 1)

than Innovator.

Feature detection and quantification with Progenesis QI can be used to assess biosimilarity.

RESULTS

Dig

est

A

Dig

est

B

Figure 2. Venn diagram comparing features between Innovator and Biosimilar 1 (left panel) and Innovator and Biosimilar 2 (right panel) for digest A and B.

Figure 4. Relative frequency and cumulative relative frequency for the fold change between Inno-vator and Biosimilar 1 (a, c) and the Innovator and Biosimilar 2 (b, d).

0

250

500

750

1000

1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06

Fre

qu

en

cy (

cou

nts

)

Normalized Abundance

Innovator

Biosimilar 1

0

250

500

750

1000

1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06

Fre

qu

en

cy (

cou

nts

)

Normalized Abundance

Innovator

Biosimilar 2

Figure 3. Frequency of feature intensity in log10 for Innovator and Biosimilar 1 (left panel) and Innovator and Biosimilar 2 (right panel). Solid Lines: Digest A - Dotted Line: Digest B

Innovator vs. Biosimilar 1 Innovator vs. Biosimilar 2

Dig

est

A

Dig

est

B

Dig

est

A

Dig

est

B

0

10

20

30

40

50

60

70

80

0

1

2

3

4

5

6

7

8

9

10

1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06

Fre

qu

en

cy f

or

Bio

sim

ilar

2

(co

un

ts)

Fre

qu

en

cy f

or

Inn

ova

tor

and

B

iosi

mila

r 1

(co

un

ts)

Normalized Abundance

Innovator

Biosimilar 1

Biosimilar 2

Figure 5. Venn diagram comparing features between the Innovator, Biosimilar 1 and Biosimilar 2 for digests A and B.

Figure 6. Frequency of feature intensity in log10 for features unique to the Innovator, Biosimilar 1 and Biosimilar 2 for digests A and B.

Innovator vs. Biosimilars 1 and 2

Figure 8. Principal component analysis of features corresponding to O-core1 glycopeptides (previously identified) for digest A (a) and digest B (b).

Core1-glycopeptides: Innovator vs. Biosimilars 1and 2

Figure 7. MS/MS of one unique feature of Biosimilar 2

Figure 9. Ratio of feature average intensities between Innovator and Biosimilar 1 (a) and between Innovator and Biosimi-lar 2 (b) for previously identified O-core1-glycopeptides.

Figure 10. Expression profile for tryptic peptides T14 containing 1 O-core1 (a) and T15 containing 6 O-core1 modifications.

a) Digest A b) Digest B

0

5

10

15

20

25

30

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

Rel

ativ

e Fr

equ

ency

(%

)

Fold Change

Digest A

Digest B

a)

0

5

10

15

20

25

30

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5

Rel

ativ

e Fr

equ

ency

(%

)

Fold Change

Digest A

Digest B

b)

0

20

40

60

80

100

1.1

1.2 1.3

1.4

1.5

1.6

1.7

1.8

1.9 2

2.1

2.2

2.3

2.4

2.5

>2.5

Cu

mu

lati

ve R

elat

ive

Fre

qu

en

cy (

%)

Fold Change

Digest A

Digest B

c)

0

20

40

60

80

100

1.1

1.2 1.3

1.4

1.5

1.6

1.7

1.8

1.9 2

2.1

2.2

2.3

2.4

2.5

>2.5

Cu

mu

lati

ve R

elat

ive

Fre

qu

en

cy (

%)

Fold Change

Digest A

Digest B

d)

Innovator BIO 1 BIO 2

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

T01

_0-O

Co

re1

T01

_1-O

Co

re1

T14

_3-O

Co

re1

T14

_2-O

Co

re1

T14

_1-O

Co

re1

T14

_1-O

Co

re1

T15

_7-O

Co

re1

T15

_6-O

Co

re1

T15

_6-O

Co

re1

T15

_5-O

Co

re1

T15

_2-O

Co

re1

T17

_1-O

Co

re1

T17

_0-O

Co

re1

Rat

io (I

nn

ova

tor

/ B

iosi

mila

r 1)

digest A

digest B

a)

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

T01

_0-O

Co

re1

T01

_1-O

Co

re1

T14

_3-O

Co

re1

T14

_2-O

Co

re1

T14

_1-O

Co

re1

T14

_1-O

Co

re1

T15

_7-O

Co

re1

T15

_6-O

Co

re1

T15

_6-O

Co

re1

T15

_5-O

Co

re1

T15

_2-O

Co

re1

T17

_1-O

Co

re1

T17

_0-O

Co

re1

Rat

io (I

nn

ova

tor

/ B

iosi

mila

r 2)

digest A

digest B

b)

0

10

20

30

40

50

60

70

80

0

1

2

3

4

5

6

7

8

9

10

1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06

Fre

qu

en

cy f

or

Bio

sim

ilar

2

(co

un

ts)

Fre

qu

en

cy f

or

Inn

ova

tor

and

B

iosi

mila

r 1

(co

un

ts)

Normalized Abundance

Innovator

Biosimilar 1

Biosimilar 2

Replicate 1

Replicate 2

Replicate 3

a)

A B

C

D

A

B

C D

A B

C

D

A B

C

D

A

B

C

D

b)

A

B

C

D