Analyses development in plasma fractionation for in ... · Analyses development in plasma...
Transcript of Analyses development in plasma fractionation for in ... · Analyses development in plasma...
Analyses development in plasma fractionation for in process and final control
using BiacoreTM and 2-Dimensional gel electrophoresis
2 / Camilla Estmer Nilsson /
5/16/2011
Content
1. Introduction
Plasma process
Analyses in hIgG process
2. Biacore™: specific concentration
IgG concentration assay
IgG subclass distribution assay
3. Impurity profile
1-Dimensional (1-D) gel electrophoresis with western blotting
2-D gel electrophoresis analysis
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Analysis flow in process
<10 min per sample
Thank God! It´s pure!! Hmm….
What have I done?
WOW! 23.4 mg/ml!
Specific!!
Samples to concentration
analysis
Samples to impurity profile
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Plasma
Sepharose™ 4 FF
Q sepharose HP
Virus inactivation
SP Sepharose HP
Superose™ 12 pg
Ultrafiltration
Sterile filtration
DEAE Sepharose FF1
Virus inactivation
Heparin Sepharose FF
Q Sepharose FF
Ultra-diafiltration
Sterile filtration
Ultrafiltration
Sephadex™ G-25 C
Euglobulin precipitation
DEAE Sepharose FF2
CM Sepharose FF
Ultrafiltration
Heat treatment
Sephacryl™ S-200
Ultra-diafiltration
Sterile filtration
Ultrafiltration
Q Sepharose FF
Ultrafiltration
Virus inactivation
CM Sepharose FF
Ultra-diafiltration
Sterile filtration
Factor VIII
Albumin
Factor IX IgG
Plasma process
IgG process samples
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Assay IVIG
Total protein (A280, Biuret , Kjeldahl) F P
Protein composition (PAGE, Zone EF) F P
Consistency of subclasses of IgG F
Impurity profile (WB) F P
Molecular size distribution (gel filtration) F P
pH F P
Conductivity P
Osmolality F
Prekallikrein activator (PKA) F
Anticomplement activity (ACA) F
Fc function F
Heparin F
Water F
Anti-A and anti-B haemagglutinins F
Anti-D antibodies, HBV, HBA antigens F
Sterility F
Pyrogens F
Viral markers F P
Endotoxins (LAL) F P
S/D Tween,TNBP F P
Analyses in process and final control IgG for intravenous administration (IVIG)
Biacore™;
Surface plasmon resonance
Specific protein concentration
2-Dimensional gel electrophoresis
(2-DE)
P = In process
F = Final QC
(According to European
Pharmacopoiea)
WB= western blot PAGE = polyacrylamid gel electrophoresis EF= electrophoresis
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Content
1. Introduction
Plasma process
Analyses in hIgG process
2. Biacore™: specific concentration
IgG concentration assay
IgG subclass distribution assay
3. Impurity profile
1-Dimensional (1-D) gel electrophoresis with western blotting
2-D gel electrophoresis analysis
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BiacoreTM
Surface plasmon resonance
Sensor chip
Sensor chip
Specific
antibody
Sample
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0
1000
2000
3000
0 20 40 60 80 100 120 140 160
Time (s)
Re
lati
ve
Re
spo
nse
(R
U)
[standard]
(µg/ml)
50
20
3.2
8
1.3 0.5 x
20 sec
Injection
30 sec
Regeneration
x
x
x
x
x x x
x
x
x
x x 0
1000
2000
3000
0 10 20 30 40 50 60
Re
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ve
Re
spo
nse
RU
Concentration µg/ml
Standard curve
x
15
Injection & regeneration of standards
160 seconds cycle time
Construct standard curve Plot response vs concentration
Curve fitting
Sample injection
Interpolate sample response
Aquire sample concentration
Concentration analysis In real time
Biacore™
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Ligand: α-hIgG (~10000 RU immobilisation)
Excellent assay stability
Possible to use master standard curve
Result in <10 minutes per sample
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1000
2000
3000
0 10 20 30 40 50
Concentration (µg/ml)
Re
lati
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esp
on
se (
RU
)
IgG standard* curves during > 1 week
1000 cycles run
Std curve 0.5 50 μg/ml
IgG concentration analysis
Biacore™
Re
lati
ve R
esp
on
se (R
U)
0
500
1000
1500
2000
2500
3000
3500
0 200 400 600 800 1000
Cycle number
CV =1.3%
standard control sample
Sensor
chip
a-hIgG Fc
Plasma
sample
* Calibrated against international reference material (DAKO)
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Biacore™ IgG concentration analysis of 20 process samples
Immediate
specific IgG
Calculated
specific IgG
Sensor
chip
Anti-IgG Fc
Plasma
sample
Biuret total protein SDS-PAGE
Evaluation in ImageQuant™ Calculation in Excel
Biacore IgG conc assay
Sample dilutions
T200 Evaluation
Sample dilutions
Sample dilutions
Hands on time: 1.5h Total time: 4.5h
Hands on time: 10h Total time: 16h
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Step Fraction Biacore assay IgG conc
mg/ml (CV)
Biuret total protein mg/ml (CV)
SDS-PAGE IgG amount
(%)
Biuret+SDS-PAGE IgG conc.
mg/ml
Plasma Start 7.8 (4.7%) 76 (2.7%) 15 11
Sepharose™ 4 FF FIX, Alb, IgG FVIII
3.4 (2.4%) 0.01 (7.6%)
27 (2.1%) 2.4 (4%)
15 0.0
4.0 0.0
DEAE Sepharose FF1 Alb, IgG
FIX 2.8 (1.7%)
0.1 (3.7%) 22 (2.2%)
4.1 (5.1%) 20
0.0 4.4
0.0
Euglob. Prec. Supernatant 3.6 (2.9%) 25 (7.5%) 11 2.7
DEAE Sepharose FF2 IgG Alb
2.3 (1.3%) 0.1 (4.3%)
3.3 (2.8%) 22 (2.2%)
80 0.0
2.6 0.0
Q Sepharose FF IgG 2.0 (1.7%) 2.5 (1.3%) 97 2.4
CM Sepharose FF IgG 4.6 (2.7%) 4.6 (1.5%) 100 4.6
Sterile filtration IgG 25 (1.3%) 28 (0.7%) 100 28
IgG concentration analysis of process samples
Biacore™
y = 1,0371x
R 2 = 0,9785
0
5
10
15
20
25
30
35
40
0 5 10 15 20 25 30 35 40
Biacore assay (mg/ml)
Biuret * SDS-PAGE (mg/ml)
Immediate specific
concentration
Calculated specific
concentration
1 In FIX trail 2 In Alb trail
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4 flow cells immobilised with specific anti-hIgG Ab: ~8000-10000 RU Contact time: 120s
IgG subclass distribution Biacore™
Standard dilution
hIgG1 (µg/ml)
hIgG2
(µg/ml)
hIgG3
(µg/ml)
hIgG4
(µg/ml)
1280 2,8 1,8 0,4 0,2
640 5,7 3,7 0,8 0,3
320 11 7,3 1,7 0,6
160 23 15 3,3 1,2
80 45 30 6,6 2,5
40 90 60 13 5,0
Original concentrations
mg/ml*
3.62 (54.1%)
2.34 (35%)
0.53 (7.9%)
0.20 (3.0%)
* Concentrations according to international reference material (DAKO)
Fc 1
a-IgG1
flow
Sample
Sensor chip Fc 2
a-IgG2
Fc 3
a-IgG3
Fc 4
a-IgG4
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s s s
Time (s) 50 150 250 350 0 50 150 250 350 0 50 150 250 350 0 50 150 250 350 0
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Fc1: IgG1 (54%) Fc2: IgG2 (35%) Fc3: IgG3 (8%) Fc4: IgG4 (3%)
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Concentration (μg/ml)
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0 12 0
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0 16 0
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Concentration (μg/ml) Concentration (μg/ml) Concentration (μg/ml)
Re
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(RU
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IgG subclass distribution Biacore™
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IgG subclass distribution of process samples
Biacore™
Step Fraction IgG1
(mg/ml)
IgG2
(mg/ml)
IgG3
(mg/ml)
IgG4
(mg/ml)
Sum of IgG1-4
(mg/ml)
Total IgG Biacore (mg/ml)
Plasma Start 4.5 53%
2.9 35%
0.6 7.4%
0.3 4.2%
8.4 7.7
Sepharose™ 4 FF FIX, Alb, IgG 1.9 54%
1.2 35%
0.2 7.1%
0.1 3.8%
3.5 3.4
DEAE Sepharose FF1 Alb, IgG 1.6 54%
1.0 35%
0.2 7.0%
0.1 3.8%
2.9 2.8
Euglob. Prec. Supernatant 1.9 54%
1.3 37%
0.2 6.8%
0.1 2.9%
3.5 3.6
DEAE Sepharose FF2 IgG 1.2 53%
0.8 37%
0.2 6.8%
0.05 2.3%
2.2 2.3
Q Sepharose FF IgG 1.1 57%
0.8 41%
0.03 1.5%
0.01 0.3%
1.9 2.0
IgG UF3 IgG 17 64%
9.3 35%
0.3 1.3%
0.05 0.2%
27 29
0 1 2 3 4 5 6 7 8 9
10
0 1 2 3 4 5 6 7 8 9 10
Sum of IgG1-4 (mg/ml)
Total IgG Biacore (mg/ml) y=0.9947x
R2=0.9873
1 In FIX trail 2 In Alb trail
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Biacore™
IgG subclass distribution ELISA vs Biacore
Control sample
IgG1
(mg/ml)
IgG2
(mg/ml)
IgG3
(mg/ml)
IgG4
(mg/ml)
Sum of IgG1-4
(mg/ml)
Hands on time 1/5
samples**
Total time 1/5
samples**
Biacore 10.7 63%
6.1 36%
0.2 1.2%
0.03 0.18%
17 40min/1h 2h/6h
ELISA* 7.4 54%
5.9 44%
0.1 0.9%
0.2 1.2%
14 2.5h/4h 5h/6.5h
*commercial ELISA for IgG subclasses
** 2 replicates, 4 dilutions
One IgG subclass ELISA has only space for 5 samples
(2 replicates, 4 dilutions = 40 wells)
Standards
Control samples
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Analysis flow in process
<10 min per sample
Hmm…. What have I
done?
WOW! That was fast!
Specific!!
Samples to concentration analysis Subclass
distribution
17 / Camilla Estmer Nilsson /
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Content
1. Introduction
Plasma process
Analyses in hIgG process
2. Biacore™: specific concentration
IgG concentration assay
IgG subclass distribution assay
3. Impurity profile
1-Dimensional (1-D) gel electrophoresis with western blotting
2-D gel electrophoresis analysis
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Impurities in IgG product
Detection limit:
In European Pharmacopoeia: ”…not more than 5% of protein has a different mobility shift from that of the principal band.” (Protein composition by Zone electrophoresis)
Plasma protein
MW
(kDa)
Plasma concentration
(mg/L)
At 3 log reduction
(mg/L)
Transferrin 88 3500 0,877
Fibrinogen 330 3000 0,751
a2-Macroglobulin 725 2000 0,501
Ceruloplasmin 150 350 0,088
Fibronectin 450 300 0,075
Plasminogen 92 200 0,050
Prothrombin (II) 72 150 0,038
Antithrombin 58 145 0,036
Heparin cofactor II 66 80 0,020
HMW kininogen 120 70 0,018
Plasmin inhibitor 63 60 0,015
Prekallikrein 86 50 0,013
Factor XII 80 30 0,008
Factor V 330 20 0,005
Factor X 59 8 0,002
Factor XI 160 5 0,001
Protein C 62 4 0,001
Protein S 70 10 0,003
WB/2-DE~ 0.01mg/L
Silver staining ~0.05mg/L
Coomassie™ ~ 0.5mg/L
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Western Blotting
Why do gel based protein analysis?
Confirm presence, molecular weight, pI or identity with Western blotting.
Proteins separated on a gel
Proteins transferred to a membrane
Blocking and binding of specific Ab to target protein
Labeled secondary Ab binding to primary Ab
Detection of the target protein Chemiluminescence ECL Prime™
0
30
60
90
120
150
180
Time (min) 2.5 0.0049 0.31
Amount transferrin (ng)
Signal stability and quantitation limit of ECL Prime
Detection limit:
~0.05ng (in 5µl loaded) => 0.01mg/L
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ID Step Fraction
1 Plasma Start
2 Sepharose™ 4 FF FIX, Alb, IgG
3 4
DEAE Sepharose FF1 Alb, IgG FIX
5 6
Euglob. Prec. Precipitate Supernatant
7 8 9
DEAE Sepharose FF2 IgG Alb Discard
10 11
Q Sepharose FF IgG Discard
12 13
CM Sepharose FF IgG Flow though
14 Final formulation IgG
a-Fibrinogen
a-IgA
a-Transferrin
a-IgG
1 2 3 4 5 6 7 8 9 10 11 12 13 14 M
Western Blotting a-mouse-HRP (IgG, IgA) a-rabbit-HRP (Tr, Fr)
ECL prime
SDS-PAGE 4-12% Gradient
Post staining
14
250
150
100
75
50
37
25
1 2 3 4 5 7 8 9 10 11 12 13 M M TR* FR*
250
150
100
75
50
37
25
1-D PAGE with western blotting Purity profile analysis of IgG
6
<0.01mg/L in final IgG?
1 In FIX trail 2 In Alb trail
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Benefits of 2-D DIGE vs conventional 2-DE
Sensitive
• Detects very low abundant proteins
• Detects the smallest possible real differences in protein abundance
Wide Dynamic Range
• Detects high and low abundant proteins simultaneously ( >5 orders of magnitude)
Efficiency and convenience
• Pre-labeling of samples
• Multiplexing of 2 samples simultaneously significantly reduces analysis time
Biological variation
• Reduces experimental gel-to-gel-variation by the use of an internal standard
CyDyes => increased sensitivity and dynamic range Detection limit as in WB ~ 0.01mg/L However, detection not dependent on antibody recognition!
Staining Detection limit
Dynamic range
Silver ~ 1 ng ~101
Coomassie™ Blue ~ 10 ng ~102
Deep Purple™
Sypro™ Ruby
~ 1–2 ng ~103.5
CyDyes™
Multiplexing possible!
~ 0.25 ng ~104
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2-D DIGE workflow
Protein sample 1 label with Cy3
Pooled internal standard label with Cy™2
Protein sample 2 label with Cy5
Mix labeled samples
IEF + 2-D separation
Imager Evaluation Software
Cy2
Cy3
Cy5
Buffer exchange Vivaspin™
+ -
pI
IEF= iso electric focusing
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2-D DIGE Experimental set up
Step Fraction CyDye™ 2-D gel
Start Final
Plasma IgG
Cy3 Cy5
1
DEAE Sepharose™ FF1 Alb, IgG FIX
Cy3 Cy5
2
Euglob. Prec. Precipitate Supernatant
Cy3 Cy5
3
DEAE Sepharose FF2 IgG Alb pooled with discard
Cy3 Cy5
4
Q Sepharose FF IgG Discard
Cy3 Cy5
5
CM Sepharose FF IgG Flow through
Cy3 Cy5
6
Internal control Mix of all samples Cy2 on all gels
1 In FIX trail 2 In Alb trail
Gel 1
plasma IgG final
Internal control overlay
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2-D DIGE Evaluation in DeCyder™
Start plasma
IgG DEAE Sepharose™ FF2
Discard Q Sepharose FF
Haptoglobin
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DEAE Sepharose FF2 Q Sepharose FF
2-D DIGE Results
CM Sepharose FF
start/final DEAE Sepharose FF1
Plasma IgG
Alb, IgG FIX
IgG Alb pooled with discard
IgG Discard
IgG Flow through
Euglobin precipitation
Precipitate Supernatant
Cy3 Cy5
IgG
Albumin Transferrin IgA
Antitrypsin
Fibrinogen b
Apolipoprotein
Fibrinogen a
Fibrinogen g
1 In FIX trail 2 In Alb trail
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Acknowledgements
Bioprocess group
Anna Mattsson
Helena Skoglar
Karolina Busson
Lena Sandberg
Martin Hall
Mikael Berg
Biacore™ group
Johan Kärnhall
Åsa Frostell
Electrophoresis group
Anneli Jorsback
Karin Söderquist
Maria Winkvist
http://www.gehealthcare.com
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General Electric Company. Biacore, CyDyes, Vivaspin, ECL Prime, ImageQuant, Sepharose,
Superose , Sephadex and DeCyder are trademarks of GE
Healthcare. © 2011 General Electric Company – All rights reserved.
First published May 2011
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