Singlicate well assay for the analysis of Enoxaparin Pharmacodynamic Biomarkers (Anti-Factor Xa, Anti-Factor IIa, and TFPI) for the Bioequivalence study.

Singlicate well assay for the analysis of Enoxaparin Pharmacodynamic Biomarkers (Anti-Factor Xa, Anti-Factor IIa, and TFPI) for the Bioequivalence study. - Poster presented at 18th WRIB

Explore our poster presented at the 18th WRIB, highlighting our innovative ‘Singlicate Well Assay’ for analyzing Enoxaparin Pharmacodynamic Biomarkers, including Anti-Factor Xa, Anti-Factor IIa, and TFPI, pivotal for Bioequivalence studies.

Poster Authors: Ravi Maurya, Pravin Patel, Bhavesh Prajapati, Komal Patel, Bhoomika Bhatt, Astha Yadav, Rishabh Patel, Hitendra Rana, Pragnesh Parmar, Megha Barot, Prashant Kale, M S Ramakrishnan


INTRODUCTION

Enoxaparin is a low molecular weight heparin (LMWH) used to treat and prevent venous thrombosis. LMWH acts as an anticoagulant by inactivating factor Xa and factor IIa and promoting the release of tissue factor pathway inhibitor (TFPI), preventing blood coagulation. Enoxaparin is a complex mixture of oligosaccharides with variations in chemical structure and size, making direct conventional pharmacokinetic measurement unfeasible. Instead, regulatory requirements accept measuring pharmacodynamic markers such as anti-factor Xa, anti-factor IIa, and TFPI for comparison between the biosimilar and the reference LMWH.

We have developed the single well assay, and microtiter plates were read using Softmax Pro GXP software. All three methods are optimized to improve assay performance, reproducibility, and high throughput using the single well.

CHALLENGE

The challenge lies in meeting regulatory expectations, particularly regarding the analytical validation criteria for biomarker assays. While pharmacokinetic (PK) assays and in vitro diagnostic devices (IVD) have well-defined acceptance criteria, these standards may not seamlessly apply to biomarker assays due to their unique requirements. Biomarker assays demand distinct expectations, necessitating a refinement of analytical validation to suit the specific context of drug development.

Novel Aspects

  • Methods utilize the principles of regulated pharmacokinetics (PK) and ligand binding assay (LBA) design to repurpose diagnostic instrument commercial kit assays. This involves incorporating features such as a minimum of six-point calibration curves (CC), multiple layers of quality controls (QCs), and comprehensive validation parameters including accuracy, precision, selectivity, specificity, and stability.
  • Implements singlicate analysis by utilizing single wells instead of duplicate wells, streamlining the assay process for efficiency and resource optimization.
  • Incorporates ISR for assessing reproducibility, a practice not typically performed for biomarkers. This ensures robustness and reliability by evaluating the consistency of results across independent sample analyses.


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