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Thrombin generation test based on a 96-channel pipettor for evaluation of FXIa procoagulant activity in pharmaceuticals

Nature Protocols volume 16, pages 3981–4003 (2021)Cite this article

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Abstract

Thrombin generation (TG) assays are used widely to investigate both diseases and drugs that impact thrombosis and bleeding. TG assays were also instrumental in the identification of thrombogenic impurities in immune globulin products, which were associated with thrombotic adverse events in patients. TG assays are therefore now used by quality control laboratories of plasma derivative drug manufacturers and regulatory agencies responsible for the safety testing and release of immune globulin products. In this protocol, we describe a robust and sensitive version of the TG assay for quantitative measurement of thrombogenic activity in immune globulin products. Compared with the version of the assay commonly used in clinical laboratories that compares individual patient plasma samples with normal donor samples, our TG assay is suitable for quick (170–260 min) semiautomated analysis of multiple drug samples against the World Health Organization international standard for factor XIa. Commercially available reagents can be used for the assay, and it does not require specialized equipment. The protocol can be easily adapted for the measurement of the procoagulant activity of other biopharmaceuticals, e.g., coagulation factors.

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Fig. 1: Overview of TG and TG curve parameters.
Fig. 2: Experimental workflow.
Fig. 3: Typical symmetrical strip plot plate layout.
Fig. 4: AutoTGT worksheet.
Fig. 5: aPotency worksheet.
Fig. 6: Visualizing standard curves on _StdCurvesALL graph.
Fig. 7: Results of in-house TG assay in FXI depleted plasma.
Fig. 8: Results of in-house TG assay in congenitally FXI-deficient plasma (an example of poor experiment performance when the tissue factor concentration was not optimized for this particular lot of plasma).
Fig. 9: Results of Technothrombin kit-based TG assay in FXI-depleted plasma.
Fig. 10: Results of CAT kit-based TG assay in FXI depleted plasma.

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Data availability

An example data set for analysis is provided in Supplementary Data 1. Source data are provided with this paper.

Code availability

The analysis script is included in Supplementary Data 1.

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Acknowledgements

This study was supported in part by a Postgraduate and Postbaccalaureate Research Fellowship Award to M.S. as a recipient of the Oak Ridge Institute for Science and Education (ORISE) Fellowship, administered through an interagency agreement between the US Department of Energy and the FDA. This is an informal communication and represents the authors’ own best judgment. These comments do not bind or obligate FDA. We thank J. Jackson for careful review of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Leonid A. Parunov, Maria E. Shea, Yideng Liang.

Authors and Affiliations

  1. Center for Biologics Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, USA

    Leonid A. Parunov, Maria E. Shea, Yideng Liang, Stepan S. Surov, Maitreyi Chattopadhyay, Timothy K. Lee, Dorothy E. Scott & Mikhail V. Ovanesov

Authors
  1. Leonid A. Parunov
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  2. Maria E. Shea
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Contributions

L.A.P., M.V.O., M.C., Y.L. and S.S.S. developed and optimized the protocol. M.V.O. and L.A.P. wrote the Origin code for analyzing thrombin generation data. M.S., M.V.O. and L.A.P. prepared the manuscript. S.S.S., Y.L., M.C., T.K.L. and D.E.S. assisted in manuscript text preparation.

Corresponding author

Correspondence to Mikhail V. Ovanesov.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Bas de Laat and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Liang, Y. L. et al. Res. Pract. Thromb. Haemost. 5, 211–222 (2020): https://doi.org/10.1002/rth2.12467

Sridhar, G. et al. Transfusion 54, 2553–2565 (2018): https://doi.org/10.1111/trf.12663

Ekezue, B. F. et al. J. Thromb. Haemost. 13, 2168–2179 (2015): https://doi.org/10.1111/jth.13155

Supplementary information

Supplementary Information

Supplementary Tables 1–7.

Reporting Summary

Supplementary Software

Zipped folder containing software used to run TG analysis and a short demo with instructions.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 6

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Fig. 8

Statistical source data.

Source Data Fig. 9

Statistical source data.

Source Data Fig. 10

Statistical source data.

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Parunov, L.A., Shea, M.E., Liang, Y. et al. Thrombin generation test based on a 96-channel pipettor for evaluation of FXIa procoagulant activity in pharmaceuticals. Nat Protoc 16, 3981–4003 (2021). https://doi.org/10.1038/s41596-021-00568-4

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