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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