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May 10, 2011 1
Characterization of IGIV Products by Tests
Indicative for the Thromboembolic Potential
Plasma Product Biotechnology Meeting, Cyprus
May 10, 2011
Wolfgang Teschner, Ursula Mais-Paul, Brigitte Talir and Hans-Peter Schwarz
Baxter Innovations GmbH
PPB 2011 Cyprus
Overview of the Characterization Tests
• Assays to determine procoagulant activity
– Global in vitro assays
• Thrombin generation assay (TGA)
• Non-activated partial thromboplastin time (NAPTT) assay in FXI deficient plasma
– FXIa specific in vitro assay
• Factor XIa determination with a FIX based assay
– In vivo assay
• Wessler test
• Amidolytic activity assays
– Chromogenic substrates S-2288, S-2266, S-2222, S-2251 and S-2302
• Anticomlementary activity assay
– According to the European Pharmacopoeia monograph (§ 2.6.17)
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Thrombin Generation Assay (TGA)
3
*Hemker HC, Giesen P, AlDieri R, Regnault V, de Smed E, Wagenvoord R, Lecompte T, Béguin S. The calibrated automated thrombogram (CAT): a
universal routine test for hyper- and hypocoagulability. Pathophysiol Haemost Thromb. 2002 Sep-Dec;32(5-6):249-53.
• A variation of the Calibrated Automated Thrombograph (CAT) as described by Hemker* is used for analysis of IgG samples
• Coagulation in normal plasma is initiated in the absence and presence of IgG (5 mg/ mL plasma) by low concentration of Tissue Factor, Phospholipids and CaCl2 and the generated thrombin is continuously detected by a thrombin-specific fluorescence substrate
• Readouts are four major parameters: lag time, thrombin peak, time to peak and endogen Thrombin potential (ETP). Thrombin peak was selected for evaluation of IgG samples
• Thrombin peak is calculated and expressed as % of the thrombin peak measured in the normal plasma in the absence of IgG
Test Principle
• Global test, assessing the function of all hemostatically active factors in the sample. Outcome of the assay dependent on certain determinants in plasma, i.e., prothrombin and antithrombin concentration
Specificity
May 10, 2011 PPB 2011 Cyprus
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Thrombin Generation Assay (TGA)
The coagulation
cascade
TGA test principle
PPB 2011 Cyprus
FXIa Assay -
“Natural Substrate, FIX Based Assay”
5
• Serial dilutions of IgG are incubated with physiological amounts of FIX, FX and FVIII in the presence of phospholipid vesicles and CaCl2 at 37°C. During that time FXIa activates FIX, which in turn forms a complex with FVIII on the phospholipid surface and this complex activates FX
• FXa activity is measured in a subsample added to a chromogenic substrate specific for FXa in the presence of EDTA to stop any further activation
Test Principle
• The amount of FXa depends on the FXIa concentration in the sample. Purified FXIa in the range of ~1.25 – 10 pM (0.04 - 0.32 mU/mL) is used to establish a reference curve
• To assure the specificity of the assay two controls are measured: • Control for FIXa- and/or FXa-like activity – FIX is omitted • Control for FXa-like activity of the IgG - only FXa substrate and
phospholipid/CaCl2
If both controls are negative, the assay is specific for FXIa
Specificity
Based on: von dem Borne PAK, Koppelman SJ, Bouma BN,Meijers JCM: Surface independent factor XI activation by thrombin in the presence
of high molecular weight kininogen. Thromb. Haem.72; 374, 1994.
May 10, 2011 PPB 2011 Cyprus
NAPTT Assay
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• Serial dilutions of IgG product are added to FXI deficient plasma, and clotting is initiated by phospholipid and CaCl2
• Results are expressed either as the amount of protein (in mg) in the product which is necessary to shorten the NAPTT of factor XI-deficient plasma to a specified time or as the clotting time obtained at the highest protein concentration applied in the test system
Test Principle
• Global assay, assessing all activated coagulation factors in the samples except FXIIa
Specificity
May 10, 2011 PPB 2011 Cyprus
Serial dilutions
of IgG FXI
deficient
plasma
Addition of
phospholipids,
CaCl2
Measurement of
clotting time
Anticomplementary Activity Assay (ACA)
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• Test is part of the Pharm. Eur. monograph for IGIV preparations
• A defined amount of immunoglobulin (10mg) is incubated with a defined amount of guinea-pig complement (20 CH50) (1 CH50 = amount of complement that, in the given reaction conditions will produce the lysis of 2.5x108 red blood cells out of a total of 5x108 red blood cells)
• After incubation the remaining amount of complement is titrated using hemolysin sensitised sheep red blood cells
Test Principle
May 10, 2011 PPB 2011 Cyprus
Test TGA
(% of normal plasma control
at 5 mg/mL protein)
FXIa
(mU/mL)
NAPTT (mg at 180 sec)
ACA (%)
Gammagard Liquid 106 <0.04 >10 42
C1 451 4.06 1.44 51
C2 107 <0.04 >10 36
C3 97 <0.04 >10 39
C4 109 <0.04 >10 46
C5 269 <0.04 >5 34
C6 125 <0.04 >5 37
C7 125 <0.04 >5 47
C8 121 <0.04 >5 35
C9 125 <0.04 >5 45
C10 116 <0.04 >5 30
C11 110 <0.04 >5 39
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Comparison of TGA, FXIa, NAPTT and
ACA Test Results
May 10, 2011
Conclusion: Only competitor product C1 showed high TGA and FXIa values,
shortened NAPTT and failed to fulfill the Pharm. Eur. ACA
criterion of <50% PPB 2011 Cyprus
Chromogenic Substrate Description
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S-2222
S-2251
S-2266
S-2302
S-2288
Bz-Ile-pyroGlu-Gly-Arg-pNA
H-D-Val-Leu-Lys-pNA
H-D-Pro-Phe-Arg-PNA
H-D-Val-Leu-Arg-pNA
H-D-Ile-Pro-Arg-pNA
Data and sketches are taken from CoaChrom; Bz: benzoyl; pNA: p-nitroanilid; pyroGlu: pyroglutaminic acid
Name Formula Selectivity
FXa
Plasmin
Kallikrein, FXIIa
FXIa,
Glandular kallikrein
Broad spectrum,
tPA
May 10, 2011 PPB 2011 Cyprus
Substrate S-2222 S-2251 S-2266 S-2288 S-2302
Specificity FXa Plasmin FXIa, glandular
kallikrein
Broad
spectrum
Kallikrein,
FXIIa
nmol/mL min
Gammagard Liquid <5 <5 <5 <5 <5
C1 <5 <5 27.1 29 70.1
C2 <5 <5 <5 <5 <5
C3 <5 <5 <5 <5 <5
C4 <5 <5 <5 <5 <5
C5 <5 <5 <5 <5 <5
C6 <5 <5 <5 <5 <5
C7 <5 <5 <5 <5 <5
C8 <5 <5 <5 <5 <5
C9 <5 <5 <5 <5 <5
C10 <5 <5 <5 <5 <5
C11 <5 <5 <5 <5 <5
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Overview of Activities Detectable with
Chromogenic Substrates
May 10, 2011
Conclusion: The amidolytic activity profile generated with different chromogenic substrates
showed elevated values for competitor product C1
PPB 2011 Cyprus
The Wessler Test
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Quantitative evaluation of
thrombogenicity Score
no clot 0
few macroscopic strands of fibrin 0.5 - 1
several small thrombi 2
two or more large thrombi 3
several large thrombi 3.5
single thrombus forming a
complete clot in vein segment 4
May 10, 2011 PPB 2011 Cyprus
Product Wessler Score Average
male male female female
Gammagard Liquid 2 1 2 0.5 1.38
C1 4 3.5 4 4 3.88
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Wessler Test Results
May 10, 2011
Conclusion: In the Wessler in vivo test Gammagard Liquid showed a very low
thromboembolic potential. In contrast competitor product C1 showed a
high test score, single thrombus forming clots indicative for a high
thromboembolic potential
The test results of the in vivo Wessler test are in good agreement with the
test results obtained by the in vitro tests for Gammagard Liquid and
competitor product C1
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Conclusions
May 10, 2011
• In the thrombin generation assay Gammagard Liquid showed no
effect on normal plasma in a concentration of 5 mg/mL
• FXIa values for Gammagard Liquid were below the detection limit
of the specific FXIa assay ( <0.04 mU/mL). Baxter uses a FXIa
test, which mimics the physiological situation and measures FXIa
activity on its natural protein substrate FIX
• Gammagard Liquid does not shorten the non-activated partial
thromboplastin time measured in FXI deficient plasma
• Amidolytic activity for Gammagard Liquid is below the detection
limit of the chromogenic assays applied
• Gammagard Liquid shows a very low Wessler score in vivo. The
in vivo results are in good agreement with the results in vitro
PPB 2011 Cyprus
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Acknowledgements
May 10, 2011
TGA: Michael Dockal
Sabine Knappe
Klara Michalkova
FXIa: Katalin Varadi
Michaela Schädler
NAPTT: Robert Weiss
Martina Simon
Andrea Wanjura
ACA: Geoffrey Pot
Chromogenic substrate tests: Alfred Weber
Wessler test: Eva-Maria Muchitsch
Coagulation expertise: Peter Turecek
PPB 2011 Cyprus
