Suitability of analytical methods for detection of thrombogenic factor XIa in Privigen

Transcription

1 Suitability of analytical methods for detection of thrombogenic factor XIa in Privigen Absence of thrombogenic factors in Privigen M. Moses, K. Ruhwedel, H.-A. Stöhr, L. Duse, A. Feussner, W. Wormsbächer, and U. Kalina Preclinical Research and Development, CSL Behring GmbH, 8 th Plasma Product Biotechnology meeting, May 14 th, 2013, Lanzarote, Spain Marburg, Germany

2 Introduction Proteolytic contaminants in intravenous immunoglobulin (IVIG) products led to thromboembolic events Since then, IVIG drug products have been under intensive investigation FXIa was rated to be the major contributor to thrombogenicity of some immunoglobulin preparations 1,2 1 Wolberg et al. Am J Hematol 2000;65:30 2 Etscheid et al. Vox Sang 2012;102:40 2

3 Ensuring high quality and safety of IVIG 3 For more than 65 years, CSL Behring has been fractionating human plasma for use in lifesaving biotherapies such as Privigen IVIG treatment Rigorous plasma and final product testing in place Screen for antibodies markers of infection Nucleic acid testing for viral genomes Robust purification process and virus inactivation/elimination 1 Activated factors potentially present during manufacturing are depleted to minimize the risk of thromboembolic events Please attend Presentation 403: Ibrahim El Menyawi, May 15 th CSL Behring Stucki et al. Biologicals 2008;36:239e247

4 Privigen is a state-of-the-art IVIG product A ready-to-use 10% liquid IVIG First and only IVIG stabilized with proline 1 Sugar-free Proline reduces IgG aggregation and minimizes fragmentation Room-temperature storage for 36 months 2 IgA d25 µg/ml 1 Bolli et al. Biologicals 2010;38:150 2 Cramer et al. Vox Sanguinis 2009;96:219 4

5 Potential factors influencing the presence of proteolytic contaminants in IVIG products Manufacturing steps could cause the activation of coagulation factors Ion exchange resin can trigger contact activation of clotting factors such as FXIIa that generates kallikrein and FXIa Manufacturing steps can remove activated coagulation factors during processing Heparin resins 1 can bind and deplete coagulation factors Octanoic acid step removes many impurities from plasma such as FXIa 2 1 Patent published: WO 2012/152953A1 2 Presentation 403: El Menyawi et al. May 15 th 5

6 Study objective Ensure the absence of activated coagulation factors in Privigen drug product: Characterize suitability and sensitivity of test methods Compare different methods Evaluate the potential presence of procoagulant activity in various Privigen lots 6

7 Privigen contains no thrombogenic activity Commercial lots of Privigen were analyzed and the absence of procoagulant activity was confirmed by all test methods applied: TGA: below detection limit NaPTT: below detection limit FXIa-like activity: below detection limit 7 May-13

8 Experimental design Samples of Privigen and buffer control solution (1% [v/v] human albumin in imidazole buffer) spiked with FXIa Commercially available FXIa material (human FXIa, Haematologic Technologies Inc., Vermont, USA) used Concentration range ng/ml 8

9 Study design Test methods for suitability to detect FXIa TGA (thrombin generation assay) NaPTT (non-activated partial thromboplastin time) aptt (activated partial thromboplastin time) FXIa amidolytic activity Intrinsic und extrinsic activation in FXI-depleted plasma Ph. Eur. requirements for intravenous FIXproducts 1 Determination of clotting time in standard human plasma without activation FXIa-like activity in FXI-depleted plasma FXIa-specific chromogenic substrate 1 European Pharmacopeia ,

10 10 Thrombin generation assay

11 TGA: FXIa-triggered coagulation Developed to monitor coagulation function by the temporal measurement of thrombin generation Based on a physiological coagulation model triggered by FXIa Slow-acting fluorogenic substrates used to detect thrombin generation over time FXIa Throm bin Fluorogenic substrate Activated product A thrombogram is used to measure thrombin activity, including velocity and time course of thrombin generation 11

12 Measureable phases in TGA 12 Initiation phase Lag time: clotting time defined as the interval from the start of substrate/calcium addition until formation of 10 nm of thrombin Propagation phase (thrombin generation) Peak height: indicates the proportion of thrombin generated Area under the curve or endogenous thrombin potential Time to peak: time required for maximum thrombin generation Detection depends on the levels of 400 coagulation factors and inhibitors, 200 and experimental conditions 0 Hemker et al. Pathophysiol Haemost Thromb 2003;33:4 Chandler & Roshal Am J Clin Pathol 2009;132:169 Thrombin generation (nm) Time to peak Peak height Time (min) Area under the curve

13 Two modified TGA approaches investigated Activator reagent Extrinsic activation TGA Technothrombin TGA RB Assay (Technoclone Co.) Intrinsic activation TGA Pathromtin SL (Siemens Co.) diluted with phospholipids In physiological conditions, thrombin is generated by the extrinsic pathway 1 (e.g. hemophilia A and B patients) FXI can be activated by surfaces (such as ion-exchange matrices) triggering the activation of the intrinsic pathway Evaluated TGA quantification methods: Peak height (nm thrombin) Time to peak (min), within 60 min of reaction 1 Woodle S et al. J Thromb Haemost 2011; 9 (Suppl 2):P-WE

14 Proportional decrease of time to peak with increased spiked FXIa levels Thrombin generation (nm) ng/ml 30 ng/ml 10 ng/ml 3 ng/ml 1 ng/ml Duplicate measurements of Privigen spiked with FXIa (extrinsic activation TGA in undiluted samples) showed good reproducibility for FXIa levels ng/ml ng/ml 0 ng/ml Time (min) 14

15 Comparable time to peak by intrinsic or extrinsic activation in Privigen spiked with FXIa Intrinsic activation TGA Extrinsic activation TGA Time to peak (min) ,01 0, Spiked FXIa (ng/ml after dilution) Spiked FXIa in Privigen before dilution (ng/ml) Time to peak (min) ,01 0, Spiked FXIa (ng/ml after dilution) 0 µg/ml FXIa samples were e20 min 0 µg/ml FXIa samples were e40 min Assays performed with undiluted and diluted (1:3, 1:9) samples 15

16 Comparable thrombin peak by intrinsic or extrinsic activation in Privigen spiked with FXIa Intrinsic activation TGA Extrinsic activation TGA 600 Spiked FXIa in Privigen before dilution (ng/ml) 600 Thrombin peak (nm) Thrombin peak (nm) ,01 0, Spiked FXIa (ng/ml after dilution) ,01 0, Spiked FXIa (ng/ml after dilution) Assays performed with undiluted and diluted (1:3, 1:9) samples 16

17 Summary of TGA results Similar results were obtained with extrinsic and intrinsic activation TGA High sensitivity and reproducibility for FXIa e1 ng/ml Reagents and test settings influenced assay variability substantially Further improvements and modifications of the test methods are under investigation for IVIG product testing 17

18 18 Non-activated partial thromboplastin time

19 NaPTT: activated coagulation factor assay Included in the Ph. Eur related to analyzing prothrombin complex and purified Factor IX products The presence of activated coagulation factors in the sample leads to a decrease in the clotting time A sample is considered activated when clotting time is significantly lower than the control value (e.g. buffer sample) A long clotting time indicates a low potential for thrombogenicity Test samples Platelet - poor plasma * Phospholipids + CaCl 2 Monitor coagulation time (e.g. BCS analyzer) * Defined as <10 4 platelets/µl 19

20 NaPTT can detect low amounts of spiked FXIa 350,0 300,0 Spiked FXIa in Privigen Spiked FXIa in buffer Mean clotting time (s) 250,0 200,0 150,0 100,0 50,0 Detection limit Official threshold,0 0, Spiked Factor XIa (ng/ml) Clotting time in Privigen without spiked FXIa and control samples were approx sec 20

21 Summary of NaPTT results NaPTT has a good sensitivity for FXIa >1 ng/ml High reproducibility for samples with clotting times below 200 sec 1:3 dilution appears to be sufficient as minimal dilution factor to compensate any effects of the sample matrix; however, this needs further validation Further improvements and modifications of the test method are under investigation 21

22 22 Activated partial thromboplastin time

23 aptt: procoagulant potential assessment Coagulation test that encompasses all steps of the intrinsic coagulation pathway from activation of the contact phase system to fibrin formation Intrinsic activation is triggered by a surface activator Test samples FXI-deficient plasma Phospholipids Surface Phospholipids activator + CaCl 2 Monitor coagulation time (e.g. BCS analyzer) Both FXI and FXIa are detected and thus aptt assesses any procoagulant potential of a sample, i.e. FXIa-like activity 23

24 Summary of aptt results The detection of both FXI and FXIa is a strength of the assay Spiking experiments with FXIa in both buffer solution and Privigen showed: aptt is suitable only for general characterization of the procoagulant activity of a sample Moderate recovery rate of H70% Detection limit of >10 ng/ml 24

25 25 FXIa chromogenic assay

26 FXIa chromogenic assay FX activation, mediated by FXIa, is detected using a FXaspecific chromogenic substrate (SXa-11) FXIa FXa Chromogenic substrate (SXa-11) Free p-nitroalanine A

27 Summary of FXIa chromogenic assay results The dynamic range of the assay required sample dilution FXIa detection using the chromogenic substrate was highly sensitive (e0.14 ng/ml) The recovery was high, especially for samples containing a low amount of FXIa Allows specific quantification of FXIa (e.g. for impurity profiling) Can be used as a complementary assay to broad assessment of activated coagulation factors (e.g. NaPTT, TGA) 27

28 Assessment of FXIa detection methods Assay NaPTT Intrinsic and extrinsic TGA FXIa chromogenic assay aptt Suitability High sensitivity for FXIa (>1 ng/ml) Good reproducibility for clotting time <200 s Detection of activated coagulation factors High sensitivity for FXIa (e1 ng/ml) Detects FXIa-like activities Specific and highly sensitive quantification of FXIa (e0.14 ng/ml) Detects both FXI and FXIa, but low sensitivity 28

29 Conclusions FXIa spiking experiments provided strong evidence that the analytical methods used for testing can detect trace amounts of procoagulant activity in immunoglobulin samples FXIa was absent in all Privigen lots analyzed Therefore, the absence of procoagulant activity in Privigen was demonstrated 29

30 30 Back-up slides

31 Coagulation cascade a, activated factor Ca, calcium PL, phospholipid 31 (adapted from Davie et al. Biochemistry 1991;30:10363) Cell-based coagulation model Initiation phase: complex of FVIIa and tissue factor activates FX (and FIX) and generates trace amount of thrombin Propagation phase: generated thrombin activates FVIII, FV, and platelets, which leads to the formation of tenase and prothrombinase complexes, resulting in a thrombin burst

32 TGA: materials & methods Materials Automated Ceveron Alpha test system (Technoclone Co.) FXI-depleted human plasma (Siemens Co.) Reaction buffer, substrate, control low/high (Technoclone Co.) 25 mm CaCl 2 solution (Technoclone Co.) Phospholipids (Rossix Co.) Several tests commercially available 32

33 NaPTT: materials & methods Materials Automated coagulation analyzer (BCS, Siemens Co.) Standard human plasma (Siemens Co.) 25 mm CaCl 2 solution (Siemens Co.) Cephalin reagent (Stago Co.) as phospholipid Tris-chloride buffer ph

34 aptt: materials & methods Materials Automated coagulation analyzer (BCS, Siemens Co.) FXI-depleted human plasma (Siemens Co.) Pathromtin SL, silica particles as surface activator (Siemens Co.) 25 mm CaCl 2 solution (Siemens Co.) Imidazole buffer (Siemens Co.) with 1% (v/v) human albumin (CSL Behring) Assay performed with undiluted and diluted (1:3, 1:9) samples FXI and FXIa used as reference/standard material Presence of FXIa in FXI-deficient plasma leads to a decrease in the coagulation time 34

35 FXIa chromogenic assay: methods Commercial kit available (Biophen Factor XIa, Hyphen- Biomed Co.) including FXIa calibrator for FXIa concentration determination Test adapted for automated coagulation analyzer (BCS, Siemens Co.) Absorbance of free p-nitroalanine generated by amidolysis of the chromogenic substrate is monitored 35

36 References Bolli R et al. L-Proline reduces IgG dimer content and enhances the stability of intravenous immunoglobulin (IVIG) solutions. Biologicals 2010;38: Cramer M et al. Stability over 36 months of a new liquid 10% polyclonal immunoglobulin product (IgPro10, Privigen) stabilized with L-proline. Vox Sanguinis 2009;96: CSL Behring Immune Globin Therapies: Excellence and innovation in manufacturing. 2013; Etscheid, M. et al. Identification of kallikrein and FXIa as impurities in therapeutic immunoglobulins: implications for the safety and control of intravenous blood products. Vox Sang. 2012;102:40 46 European Pharmacopeia 7.6, , Activated Coagulation Factors. Jan 2013 Stucki M et al. Investigations of prion and virus safety of a new liquid IVIG product. Biologicals 2008;36:239e247 Woodle S et al. J Thromb Haemost 2011; 9 (Suppl 2):P-WE-233 Wolberg AS. et al. Coagulation factor XI is a contaminant in intravenous immunoglobulin preparations. Am J Hematol 2000;65: