(W1230-03-15) Development of a Plate-Based Assay for the Quantitation of Tenecteplase Activity in Plasma Samples

Purpose: Thromboelastography (TEG) is a useful diagnostic test that utilizes whole blood to measure hemostasis and fibrinolysis. However, the use of TEG is a challenge in a clinical setting because of the time constraints of using whole blood and the relatively low throughput of the assay. This study endeavored to develop an equivalent test to measure clot lysis of a plasma sample in response to a fibrinolytic drug in a high throughput manner.

Methods: TEG assays were carried out on citrated whole blood activated with kaolin and calcium on TEG 5000 instruments. The plasma assay was 96-well plate-based and activated with kaolin and calcium and turbidity was measured at 340 nm. Tenecteplase (TNKase) was added to both assays exogenously over a range of 1-15 nM (in-assay final concentration) and clot lysis was measured as percent lysis at 30 minutes after maximum amplitude in the TEG and 50% lysis after maximum turbidity in the plasma assay. Once equivalency was established, the plate based assay was further modified to expand the quantitative range of Tenecteplase activity by incorporating unfractionated heparin or using a thrombin activator.

Results: Tenecteplase was titrated over a functional range (1-15 nM) in the TEG and turbidimetric assays with identical kaolin/calcium activators. The TEG assay (Figure 1) displayed linearity over a very limited range (4-8 nM) whereas the turbidimetric method showed linearity over a significantly wider range (5-15 nM). For the purposes of testing Tenecteplase levels in clinical plasma samples, the assay required a larger dynamic range. The incorporation of heparin into the plasma system (Figure 2A and 2B), which yields a weaker clot structure that is more susceptible to lysis through hinderance of thrombin generation, expands the range of the assay significantly (7.5 to 37.5 nM, in plasma concentration). In an attempt to further improve the low end range of the assay, the kaolin/calcium trigger was replaced with a simple thrombin trigger (Figure 2C and 2D). This change created an environment with significantly faster, yet sufficiently weaker clot formation, which greatly improved the range of the curve on the lower end (LLOQ of 1.25 nM). However, there was a trade off with a loss of accuracy at higher levels likely due to the weaker clot structure which is quickly lysed with increasing dosages of Tenecteplase. Both assays display good precision and accuracy of plasma Tenecteplase activity across their entire range (Figure 3).

Conclusion: A turbidimetric, plate-based assay for plasma samples was developed and is shown to be superior to the TEG assay as it encompasses a larger linear range of Tenecteplase sensitivity. Depending on the activator, (kaolin/calcium vs thrombin alone) the assay can be used to measure plasma Tenecteplase levels in two different ranges (1.25-25 nM for thrombin activated and 7.5-37.5 nM for kaolin activated). For the purposes of measuring plasma Tenecteplase activity in clinical samples, the thrombin activated assay will be of particular utility as it will be able to measure low levels of Tenecteplase present at subclinical doses as used in phase I trials. The assay also displays dilutional linearity when samples above this range are diluted with normal pool plasma which will make it a valuable tool for therapeutic doses seen in phase II/III trials. The kaolin/calcium activated assay in the presence of heparin is a more useful tool for CMC purposes and the measurement of relative potency of potentially new or biosimilar fibrinolytic drug products. Furthermore, both assays can be potentially used to quantitatively measure antifibrinolytic products in plasma samples. The establishment of a plate-based plasma assay to measure the activity of fibrinolytics/antifibrinolytics in storable clinical samples is a significant improvement on the methodology. The assays described here vastly improve upon the sensitivity of the current methodology and allow for quantitation of fibrinolytic drug products in plasma samples in a high throughput manner.

Figure 1. A comparison of the percent clot lysis in the TEG and the plate based assay at relevant time points for varying levels of TNKase. The TEG LY30 parameter is percent clot lysis 30 minutes after the maximum amplitude is measured. The plate based assay values are the percent clot lysis at a time that is 2x the time to peak.

Figure 2. Standard curve data for the kaolin and calcium triggered assay (A, B) and the thrombin triggered assay (C, D). (A, C) Absorbance at 340 nm for each level of TNKase in the curve over the length of the assay. (B, D) Concentration of TNKase (nM) vs Time to 50% lysis (sec) as measured by absorbance with a 5-parameter fit curve.

Figure 3. The spike recovery data for both the (A) kaolin and calcium triggered assay and (B) the thrombin triggered assay.