Characterization of IGIV Products by Tests. Indicative for the Thromboembolic Potential

Transcription

1 Characterization of IGIV Products by Tests Indicative for the Thromboembolic Potential Plasma Product Biotechnology Meeting, Cyprus Wolfgang Teschner, Ursula Mais-Paul, Brigitte Talir and Hans-Peter Schwarz Baxter Innovations GmbH 1

2 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

3 Thrombin Generation Assay (TGA) Test Principle 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 CaCl 2 and the generated thrombin is continuously detected by a thrombinspecific 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 Specificity 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 *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 Sep-Dec;32(5-6):

4 Thrombin Generation Assay (TGA) The coagulation cascade TGA test principle 4

5 FXIa Assay - Natural Substrate, FIX Based Assay Test Principle Serial dilutions of IgG are incubated with physiological amounts of FIX, FX and FVIII in the presence of phospholipid vesicles and CaCl 2 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 Specificity The amount of FXa depends on the FXIa concentration in the sample. Purified FXIa in the range of ~ pm ( 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/cacl 2 If both controls are negative, the assay is specific for FXIa 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,

6 NAPTT Assay Test Principle Serial dilutions of IgG product are added to FXI deficient plasma, and clotting is initiated by phospholipid and CaCl 2 Results are expressed either as the amount of protein (in mg) in the product which is necessary to shorten the NAPTT of factor XIdeficient plasma to a specified time or as the clotting time obtained at the highest protein concentration applied in the test system Specificity Global assay, assessing all activated coagulation factors in the samples except FXIIa Serial dilutions of IgG FXI deficient plasma Addition of phospholipids, CaCl 2 Measurement of clotting time 6

7 Anticomplementary Activity Assay (ACA) Test Principle 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 2.5x10 8 red blood cells out of a total of 5x10 8 red blood cells) After incubation the remaining amount of complement is titrated using hemolysin sensitised sheep red blood cells 7

8 Comparison of TGA, FXIa, NAPTT and ACA Test Results Test TGA (% of normal plasma control at 5 mg/ml protein) FXIa (mu/ml) NAPTT (mg at 180 sec) Gammagard Liquid 106 <0.04 >10 42 C 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 C <0.04 >5 30 C <0.04 >5 39 ACA (%) Conclusion: Only competitor product C1 showed high TGA and FXIa values, shortened NAPTT and failed to fulfill the Pharm. Eur. ACA criterion of <50% 8

9 Chromogenic Substrate Description Name Formula Selectivity S-2222 Bz-Ile-pyroGlu-Gly-Arg-pNA FXa S-2251 H-D-Val-Leu-Lys-pNA Plasmin S-2302 H-D-Pro-Phe-Arg-PNA Kallikrein, FXIIa S-2266 H-D-Val-Leu-Arg-pNA FXIa, Glandular kallikrein S-2288 H-D-Ile-Pro-Arg-pNA Data and sketches are taken from CoaChrom; Bz: benzoyl; pna: p-nitroanilid; pyroglu: pyroglutaminic acid Broad spectrum, tpa 9

10 Overview of Activities Detectable with Chromogenic Substrates Substrate S-2222 S-2251 S-2266 S-2288 S-2302 Specificity FXa Plasmin FXIa, glandular kallikrein nmol/ml min Broad spectrum Kallikrein, FXIIa Gammagard Liquid <5 <5 <5 <5 <5 C1 <5 < 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 Conclusion: The amidolytic activity profile generated with different chromogenic showed elevated values for competitor product C1 substrates 10

11 The Wessler Test Quantitative evaluation of thrombogenicity Score no clot 0 few macroscopic strands of fibrin 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 11

12 Wessler Test Results Product Wessler Score Average male male female female Gammagard Liquid C 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 12

13 Conclusions 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 13

14 Acknowledgements TGA: FXIa: NAPTT: ACA: Chromogenic substrate tests: Wessler test: Coagulation expertise: Michael Dockal Sabine Knappe Klara Michalkova Katalin Varadi Michaela Schädler Robert Weiss Martina Simon Andrea Wanjura Geoffrey Pot Alfred Weber Eva-Maria Muchitsch Peter Turecek 14