Laboratory Aspects of recombinant porcine FVIII and extended half life concentrates

Transcription

1 Laboratory Aspects of recombinant porcine FVIII and extended half life concentrates Rajiv K. Pruthi, M.B.B.S Co-Director, Special Coagulation Laboratory & Director, Mayo Comprehensive Hemophilia Center Division of Hematology/Internal Medicine Dept of Laboratory Medicine & Pathology Mayo Clinic, Rochester, MN CP MFMER

2 Disclosures Financial None Off label use Selected assays may not be U.S. FDA approved 2011 MFMER

3 Learning Objectives Participants will be able to: Recall the principles of coagulation factor assays available to monitor hemostatic agents Understand the principles of porcine FVIII inhibitor assay List the extended half life coagulation factor concentrates Apply knowledge of assay limitations in the monitoring of the newer hemostatic agents 2011 MFMER

4 FVIII/FIX Assays One-stage Two-stage Chromogenic One paper to read: Peyvandi, F, Oldenburg, J, Friedman, KD JTH. 2016;14: MFMER

5 TT: thrombin time; RT: reptilase time 2011 MFMER

6 Influencing Factor Assays: Contact Activators, Phospholipid & Calibrators Activator Ellagic acid based Ellagic acid Polyphenolic acid (ellagic acid-like) Silica based Colloidal silica Phospholipid (PL) Synthetic Cephalin Soya Vegetable/plant Calibrators Plasma derived standards Chromogenic standards Instrumentation Mechanical methods Micronized silica Silica dioxide Bovine Rabbit brain Concentrate specific standards Optical methods Sulfatides and silica Kaolin Porcine and chicken

7 Measuring FVIII activity: role of the calibrator (native factor vs concentrate) 200 Calibrator Patient plasma Clotting time (s) FVIII Activity Kitchen et al Quality in Lab Hemost & Thromb MFMER

8 Current Status: Variety of FVIII Assays Being Used (ECAT 2013) ECAT 2013 proficiency testing sample 2011 MFMER

9 Discrepancy between OSCA vs chromogenic: Hyate:C and OBI-1 Subject FVIII assay C max (U dl -1 ) Hyate:C 1 OSCA 65 Chromogenic 37 (-28%) 3 OSCA 103 Chromogenic 63 (-40%) 5 OSCA 79 OBI-1 Chromogenic 58 (-21%) 6 OSCA 176 Chromogenic 152 (-24%) 8 OSCA 88 Chromogenic 119 (+31%) 9 OSCA 264 Chromogenic 182 (-82%) Kempton, CL et al Haemophilia 2012; 18: MFMER

10 Assessing for presence/absence of inhibitors: role of substrate 2011 MFMER

11 Patient Plasma Buffered Normal Pooled Plasma FVIII def plasma Porcine FVIII 1:1 mix Incubate 2 hr 37 C FVIII assay FVIII assay

12 Take home message #1: Cross reactivity of human and porcine FVIII inhibitors: congenital hemophilia vs acquired hemophilia A high hfviii inhibitor titer does not consistently imply a high pfviii inhibitor Trend towards a high(er) cross reactivity in congenital hemophilia vs acquired hemophilia Higher cross reactivity of pfviii inhibitor results in a lower post infusion FVIII recovery Kruse-Jarres R et al Haemophilia 2015; 21:162 Kempton, CL et al Haemophilia 2012; 18: MFMER

13 Congenital HA (Kempton et al) Human inhibitor titre r.porcine inhibitor titre Acquired HA (Kruse Jarres et al) MFMER

14 Baseline anti-pfviii inhibitor and post infusion FVIII recovery: acquired HA 800 Immediately after first infusion 800 Highest within 24 h after the first infusion Rise in FVIII activity (%) n.d n.d Kruse-Jarres R et al Haemophilia 2015; 21: MFMER

15 Take home message #2: For rpfviii The FVIII level is typically measured with human FVIII calibrators There is a discrepancy between the one stage and chromogenic FVIII assay For inhibitors assays, rpfviii is required Cross reactivity between human and rpfviii inhibitor is variable and not predictable In acquired hemophilia, presence of porcine FVIII inhibitor does not seem to reduce efficacy of rpfviii 2011 MFMER

16 New Hemostatic Agents: laboratory monitoring Product Characteristics Recombinant porcine FVIII Recombinant porcine Human-cl rhfviii Human cell lines rfviii Single chain Extended Half Life (EHL) Concentrates rfviii; (BAY ) Site specific glycopegylation rfix; (N9-GP) Site specific glycopegylation rfviii;(n8-gp, turoctocog alfa) Site specific glycopegylation rfviii (BAX 855) rfviii (rfviiifc) rfix (rfixfc) rfix (r-fix-fp) Von Willebrand factor rvwf Random peglyation IgG Fc fragment fusion IgG Fc fragment fusion Fusion with albumin Recombinant 2011 MFMER

17 Methodology of field studies Factor (FVIII/FIX) deficient plasma samples Spiked with respective factor at 3 different approximate concentrations 80% (80 IU dl -1 or 0.8 IU ml -1 ) (high) 20% (20 IU dl -1 or 0.2 IU ml -1 ) (intermediate) 5% (5 IU dl -1 or 0.05 IU ml -1 ) (low) Samples tested with available one stage and chromogenic assays Results reported as recovery Generally accepted variation: 80 to 120% of labelled potency 2011 MFMER

18 Fc fusion concentrates: Take Home Message #3 FVIII-Fc fusion protein may be monitored by majority of one-stage FVIII assays Chromogenic assays tend to overestimate FVIII:C 2011 MFMER

19 Fc Fusion protein: FVIII Activator Recovery Ellagic acid based Silica based Kaolin based Acceptable recovery: % recovery of high concentration; % recovery for intermediate/low concentrations Chromogenic Overestimates by 116 to 132% Sommer JM et al Haemophilia 2013 Pruthi RK. Semin Hematol. 2016;53: MFMER

20 Take Home Message #4 FIX-Fc assay reagent dependency was observed Ellagic acid- and (most) silica-based assays were acceptable Kaolin based assays were not recommended (underestimation) (FDA labeling) 2011 MFMER

21 Fc Fusion protein: rfix rfix Ellagic acid based Silica based Acceptable recovery: 88 to 132% Kaolin based Significantly underestimates ~50% of label potency Chromogenic N/A Pruthi RK. Semin Hematol. 2016;53: MFMER

22 rfixfc and reagent effect Sommer ISTH MFMER

23 0.2 IU/mL rfix 0.2 IU/mL rfixfc 0.5 Ellagic acid Silica Kaolin FIX activity, IU/mL

24 rfix albumin fusion Activator Silica based Ellagic acid based Kaolin based Chromogenic Recovery No published comparative field studies * N/A N/A N/A *the only activator used in clinical trial Pruthi RK. Semin Hematol. 2016;53: Santagostino E et al. Blood 2016; 127: MFMER

25 PEGylated products: Take Home Message #5 Most ellagic acid- & (selected silica) based reagents accurately estimate PEGylated FVIII levels Most silica-based reagents underestimate PEGylated FVIII levels PEGylation does not affect chromogenic assays (data not shown) 2011 MFMER

26 One-stage FVIII Assay: Apparent N8-GP Recovery Dependent on Reagent Used Krogh-Meibom et al ISTH 2013 APTT regent Contact activator Phospholipid Result in % of target Actin FSL Ellagic acid Soya & rabbit brain % Actin FS Ellagic acid Soya % SynthAFAX Ellagic acid Synthetic % Cephascreen Polyphenolic acid Cephalin % Phospholin FS Ellagic acid Soya % Dia PTT liquid Ellagic acid Rabbit brain % CK-Prest Kaolin Rabbit brain % Dapptin TC Sulphatides & silica Purified mix % SynthASIL Colloidal silica Synthetic % aptt HS Silica dioxide Porcine & chicken <50% Pathromtin SL Silica dioxide Vegetable <50% APTT-SP liquid Colloidal silica Synthetic <50% STA-PTT-A Colloidal silica Rabbit brain <50% Triniclot Aut PTT Micronized silica Rabbit brain <50% APTT lyoph. Micronized silica Bovine brain <50% Triniclot S Silica Rabbit brain <50% 2011 MFMER

27 Use of N8-GP Product as Calibrator Reduces Reagent-dependent Variability SynthAFAX Actin FSL STA Cephascreen STA CK Prest SynthASIL APTT-SP liquid Pathrombin SL STA PTT automate Ellagic acid Kaolin Silica based 2011 MFMER

28 Increasing Free PEG Affects Silica-based aptt Reagents (BAY ) Ellagic acid Silica Gu J-M, et al Haemophilia. 2014;20: MFMER

29 One stage and chromogenic assays were accurate regardless of activator (manuscript submitted) ISTH Congress, MFMER

30 Take Home Message #6 Most ellagic acid-, silica- and kaolinbased reagents resulted in under/over estimation of PEGylated FIX Chromogenic assays resulted in expected FVIII and FIX recovery (data not shown) 2011 MFMER

31 PEGylated FVIII N8-GP BAY BAX 855 Ellagic acid based Acceptable recovery (100+25%) Silica based Kaolin based Majority underestimate (<50% recovery) Acceptable recovery (100+25%) [n=1] Acceptable Significantly underestimates (~10% recovery) N/A Recovery approx 75 to 130% (0.8 IU/ ml 60 to 160% (0.2 IU/ ml) 70 to 175% 0.05 IU/ ml) Chromogenic Acceptable recovery 96 to 110% Ellagic acid based Acceptable recovery FIX 100 to 150% 50 to 200% (0.05 IU/ ml) Silica based Overestimates

32 Conclusions: what did we learn? Variability between various one stage assays Variability between one stage and chromogenic assays For both porcine and human FVIII Porcine FVIII inhibitor tend to be lower in congenital hemophilia than acquired hemophilia Presence of low to moderate titer porcine inhibitor titers appeared not to influence clinical response to rpfviii Optimal reagent for monitoring of long-acting factor concentrates varies with molecular modification of protein 2011 MFMER

33 Conclusions: what do we need to do? Hemophilia treatment centers Be aware of assay limitations Communicate with your coagulation laboratory (know your assay) Coagulation laboratories Communicate with hemophilia centers Validate performance characteristics of in-house reagents with newer concentrates Participate in quality assurance surveys Pharmaceutical and diagnostics companies Make available spiked plasma samples for in-house optimization of assays More granularity in performance characteristics of reagents for assaying EHL concentrates (publications). Organizations (NASCOLA, CLSI, CAP,ECAT others) Educational/quality assurance efforts 2011 MFMER