Characterization of CRM197 for conjugate vaccine using icIEF (Maurice) and Capillary Western (SimpleWestern)

John Loughney

Associate Principle Scientist

Vaccine Analytical Development

CE Pharm 2017, Boston

1

• Introduction – CRM197

• icIEF (Maurice)

−Assays Titer

Identity Test

Stability

• Capillary Western (Wes, NanoPro)

• Conclusions

Presentation Outline

2

Polysaccharide Conjugate Vaccines

+

Polysaccharide protein

Polysaccharide Conjugated protein

kDa kDa MDa

“The Most Complex Biological Product Ever Made” Emilio Emini (Oct 1, 2007) Newsweek

OMPC, DT, TT, HiD

3

CRM197 CRM197

Cross-Reacting Molecule 197 (CRM197)

► Non-toxic (no enzymatic activity)

► Mutant Form of Diphtheria Toxin

► G52E (Glycine to glutamic acid) – Causes the ADP-ribosyltransferase activity to

be lost

► Expressed Pseudomonas Fluorescens

► MW ~ 58.4 kDa

► pI ~ 5.85

4

► Maurice technology (iCE280 / iCE3)

► Samples were pre focused for 1 min at 1500V, then

focused 8 mins at 3000V.

► Protein is mixed with 3-10; 4-6.5 (1:2) ampholyte

carries.

► 20% glycerol final was added as a stabilizer.

► CRM197 is detected by absorbance at 280nm and

fluorescence at 330 nm (ex = 280 nm).

CRM197 icIEF Assay

Rustandi et al Electrophoresis 2014, 35, 1065–1071

5

15ug/mL

4.5 5 5.5 6 6.5 7 7.5

pI

0

50

100

150

200 A

bsorb

ance (

mA

U)

-200

-100

0

100

200

300

400

500

600

700

800

900

1,000

1,100

1,200

1,300

1,400

1,500

1,600

1,700

1,800

1,900

2,000

2,100

2,200

2,300

2,400

2,500

2,600

2,700

2,800

2,900

3,000

3,100

3,200

3,300

3,400

3,500

3,600

Flu

ore

scence

Mkr 4.65 Mkr 7.65

6

17

S/N 38

3,530,

S/N 159

CRM197 pI = 5.90

pI

4.5 5.0 5.5 6.0 6.5 7.0 7.5

OD

(2

80 n

m)

mA

U

0

100

200

300A

7.6

4.65

250 mg/mL

125 mg/mL 62.5 mg/mL 31 mg/mL

15 mg/mL

pI

4.5 5.0 5.5 6.0 6.5 7.0 7.5

Flu

ore

sce

nce

0

10000

20000

30000

40000

50000

250 mg/mL

125 mg/mL

62.5 mg/mL

31 mg/mL

B

15 mg/mL

y = 14.93x - 14.181 R² = 1

0.0

500.0

1000.0

1500.0

2000.0

2500.0

3000.0

3500.0

4000.0

0.0 100.0 200.0 300.0

Pe

ak A

rea

µg/mL

cIEF CRM Std (UV) cIEF CRM Std (FLD)

FLD UV

Loughney et al Anal Biochem 2017, 534, 19–23

y = 2150.8x + 15380 R² = 0.9978

0.0

100000.0

200000.0

300000.0

400000.0

500000.0

600000.0

0.0 100.0 200.0 300.0

Peak A

rea

ug/mL

7

CRM197 CRM197

Typical cIEF profiles for Process Samples (iCE280)

pI

4.5 5.0 5.5 6.0 6.5 7.0 7.5

Ab

so

rba

nce

(2

80

nm

)

0.0

0.2

0.4

0.6

pI 4.22

pI 7.6

Sample 1

Sample 2

Sample 3

Sample 4

Final product

8

Incre

asin

g p

urity

CRM197

Value of FLD observed for Upsteam samples.

9

pI

4.5 5.0 5.5 6.0 6.5 7.0 7.5

OD

(280nm

) m

AU

0

100

200

300

400

pI

4.5 5.0 5.5 6.0 6.5 7.0 7.5

Flu

ore

scence

0

5000

10000

15000

20000

25000

30000C D 7.6

4.65 CRM197

CRM197

FLD UV

pI

4.5 5.0 5.5 6.0 6.5 7.0 7.5

OD

(2

80 n

m)

mA

U

0

200

400

600

pI

4.5 5.0 5.5 6.0 6.5 7.0 7.5

Flu

ore

scence

0

10000

20000

30000

40000

50000

60000

pI

4.5 5.0 5.5 6.0 6.5 7.0 7.5

Flu

ore

scence

0

10000

20000

30000

pI

4.5 5.0 5.5 6.0 6.5 7.0 7.5

OD

(280 n

m)

mA

U

0

500

1000

1500

2000

A B

C D

S1

S2

S3

S4

S5

S6

S7

S8

S1

S2

S3

S4

S5

S6

S7

S8

CRM197 7.6

7.6

4.65

10

CRM197 ???

Capillary Western Technology (Simple Western)

• Charge-based Separation (cIEF) Western (Blotless Western, FireFly, CB1000, NanoPro, Peggy-Sue)

• Size-based Separation (CE SDS) Western (Simon, Sally, Peggy, Sally Sue, Peggy Sue, Wes)

Peggy Sue Sally Sue Wes

11

Capillary Western method Overview

Rustandi et al Electrophoresis 2012, 33, 2790–2797 12

CRM197 (ng/mL)

0 10 20 30 40 50

Peak A

rea

0.0

4.0e+4

8.0e+4

1.2e+5

230

180

116

66

40

12

kDa MW 1 2 3 4 5 S5 S6 S7 S8 B

A

C CRM197

S5

S6

S7

S8

CRM197 Quantitation with Wes

13

Std Curve

Published in: Rustandi et al Electrophoresis 2014, 35, 1065–1071

CRM197 Identity test (icIEF)

14

Published in: Rustandi et al Electrophoresis 2014, 35, 1065–1071

CRM197 Identity test (icIEF)

15

Published in: Rustandi et al CE of Proteins and Peptides 2015

CRM197 Identity test (Capillary Western)

CRM197

CRM197

16

Automated Pseudo-Dot Blot Assay

Hamm et al Anal. Biochem. 478 (2015) 33–39

CRM197 ID test in conjugate vaccine (Capillary Western)

17

pI

4.5 5.0 5.5 6.0 6.5 7.0 7.5

OD

(280)

0.0

0.2

0.4

0.6

0.8

1.0

4.65

7.6

0M

1M

3M

2M

CRM197 Stability Assay (icIEF)

Rustandi et al Electrophoresis 2014, 35, 1065–1071 18

CRM197 Stability Kinetic Curves

-70 C

-20 C

4 C

22 C

19

Rustandi et al CE of Proteins and Peptides 2015

CRM197 Stability Compared with IEF Capillary Western

20

Summary

• Maurice with fluorescence detection provides useful tools in quantifying CRM197 in lysate samples.

• Simple Western provides critical tool to quantify CRM197 in low yield and complex “dirty” samples through immuno-specific detection.

• Maurice and Wes can be used for identity test and relatively easy to operation.

• Maurice and NanoPro can perform CRM197 stability analysis.

• Great potential for release and stability in QC.

21

► Richard Rustandi

► Richard Peluso

► Van Hoang

► Sha Ha

► Kevin Minster

► Melissa Hamm

► Carrie Anderson

► Brian Peklansky

► Vaccine Process Development

► Vaccine Analytical Development

► Vaccine Basic Research

► MMD

Acknowledments

22