Analyses development in plasma fractionation for in ... · Analyses development in plasma fractionation for in process and final control using BiacoreTM and 2-Dimensional ... CV =1.3%

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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


5/16/2011

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


5/16/2011

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


5/16/2011

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


5/16/2011

Content

1. Introduction

Plasma process





3. Impurity profile




5/16/2011

BiacoreTM

Surface plasmon resonance

Sensor chip

Sensor chip

Specific

antibody

Sample


5/16/2011

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

lati

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™


5/16/2011

Ligand: α-hIgG (~10000 RU immobilisation)

Excellent assay stability

Possible to use master standard curve

Result in <10 minutes per sample

0

1000

2000

3000

0 10 20 30 40 50

Concentration (µg/ml)

Re

lati

ve R

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)


5/16/2011

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


5/16/2011

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


5/16/2011

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


5/16/2011

0

500

1000

1500

2000

2500

3000

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

Re

lati

ve

Re

spo

nse

(RU

)

Fc1: IgG1 (54%) Fc2: IgG2 (35%) Fc3: IgG3 (8%) Fc4: IgG4 (3%)

0

200

400

600

800

0 180

Concentration (μg/ml)

0

1000

2000

3000

4000

0 12 0

200

400

600

800

1000

0 16 0

400

800

1200

1600

0 100

Concentration (μg/ml) Concentration (μg/ml) Concentration (μg/ml)

Re

lati

ve

Re

spo

nse

(RU

)

IgG subclass distribution Biacore™


5/16/2011

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



5/16/2011

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


5/16/2011

Analysis flow in process

<10 min per sample

Hmm…. What have I

done?

WOW! That was fast!

Specific!!

Samples to concentration analysis Subclass

distribution


5/16/2011

Content

1. Introduction

Plasma process





3. Impurity profile




5/16/2011

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


5/16/2011

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


5/16/2011

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?



5/16/2011

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


5/16/2011

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


5/16/2011

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


Gel 1

plasma IgG final

Internal control overlay


5/16/2011

2-D DIGE Evaluation in DeCyder™

Start plasma

IgG DEAE Sepharose™ FF2

Discard Q Sepharose FF

Haptoglobin


5/16/2011

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



5/16/2011

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

GE, imagination at work and GE monogram are trademarks of

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

All goods and services are sold subject to the terms and

conditions of sale of the company within GE Healthcare which

supplies them.

A copy of these terms and conditions is available on request.

Contact your local GE Healthcare representative for the most

current information.

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Bjorkgatan 30

SE-751 84 Uppsala

Sweden

http://www.gehealthcare.com/

Documents

Analyses development in plasma fractionation for in ... · Analyses development in plasma fractionation for in process and final control using BiacoreTM and 2-Dimensional ... CV =1.3%