ATS 2021 will feature presentations of original research from accepted abstracts. Mini Symposia and Thematic Poster Sessions are abstract based sessions.
Please use the form below to browse scientific abstracts and case reports accepted for ATS 2021. Abstracts presented at the ATS 2021 will be published in the Online Abstract Issue of the American Journal of Respiratory and Critical Care Medicine, Volume 203, May 3, 2021.
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Method Validation for the Measurement of Alpha-1 Antitrypsin Activity in Clinical Samples
Session Title
TP1 - TP001 MECHANISTIC AND TRANSLATIONAL STUDIES IN COPD
Abstract
A1238 - Method Validation for the Measurement of Alpha-1 Antitrypsin Activity in Clinical Samples
Author Block: A. Weber, S. Riedler, E. Minibeck, A. Engelmaier; Pharmaceutical Science, Baxalta Innovations GmbH, a Takeda company, Vienna, Austria.
RATIONALE: The quality of clinical studies is substantially determined by the analytical methods applied for the testing of the studies’ samples. Rigorous assay validation following the criteria as defined for example by the European Medicines Agency (EMA) guideline for bioanalytical assay validation is a prerequisite for such assays. Alpha-1 antitrypsin (AAT) activity measurement, i.e. measurement of the elastase inhibitory activity, is often integral part of the assay panel for clinical studies addressing AAT deficiency. Here, we present the validation data obtained for a new, sensitive AAT elastase inhibitory activity assay, termed elastase complex formation immunosorbent assay (ECFISA). METHODS: Porcine elastase (Sigma, E7885), coated onto the wells of a microplate at 20 µg/mL, was allowed to bind active AAT. The AAT-elastase complex formed was detected by an anti-AAT peroxidase conjugate. Validation samples with AAT concentrations ranging from 0.2 to 10 µg/mL in a low protein matrix and from 0.01 to 3 mg/mL in human plasma matrix were used. Assay calibration was verified by using the WHO standard 05/612 for AAT. Sample stability was investigated by repeated freezing-thawing and at room temperature. RESULTS: Overall, the assay met all EMA acceptance criteria defined for ligand-binding assays. The six-point calibration curves, ranging from 6 to 192 ng active AAT/mL, showed good accuracy. Errors, expressed as the agreement of the back-fitted assay calibrators with their nominal values, did not exceed ±7%. The AAT concentration determined for the WHO standard represented 100.1% of its assigned value. Validation samples with low protein levels and spiked with low AAT concentrations had mean AAT recoveries from 85.0% to 107.4%. Intra- and inter-run precision (n=6), expressed as relative standard deviations, did not exceed 2.4% and 8.3%, respectively. The assay’s lower limit of quantification (LLOQ) was 0.1 µg/mL. In the plasma matrix, the mean AAT recovery ranged from 94.0% to 106.0%. Intra- and inter-run precision did not exceed 4.2% and 8.2%, respectively. The assay’s LLOQ in plasma matrix was 0.24 µg/mL. Sample stability was shown after repeated freezing thawing for up to three times and at room temperature for up to 4 h. CONCLUSIONS: The assay for the measurement of AAT activity met the acceptance criteria defined by the EMA guideline for bioanalytical assay validation. This qualified the sensitive AAT activity assay ECFISA to be used for the measurement of clinical samples.
Author Block: A. Weber, S. Riedler, E. Minibeck, A. Engelmaier; Pharmaceutical Science, Baxalta Innovations GmbH, a Takeda company, Vienna, Austria.
RATIONALE: The quality of clinical studies is substantially determined by the analytical methods applied for the testing of the studies’ samples. Rigorous assay validation following the criteria as defined for example by the European Medicines Agency (EMA) guideline for bioanalytical assay validation is a prerequisite for such assays. Alpha-1 antitrypsin (AAT) activity measurement, i.e. measurement of the elastase inhibitory activity, is often integral part of the assay panel for clinical studies addressing AAT deficiency. Here, we present the validation data obtained for a new, sensitive AAT elastase inhibitory activity assay, termed elastase complex formation immunosorbent assay (ECFISA). METHODS: Porcine elastase (Sigma, E7885), coated onto the wells of a microplate at 20 µg/mL, was allowed to bind active AAT. The AAT-elastase complex formed was detected by an anti-AAT peroxidase conjugate. Validation samples with AAT concentrations ranging from 0.2 to 10 µg/mL in a low protein matrix and from 0.01 to 3 mg/mL in human plasma matrix were used. Assay calibration was verified by using the WHO standard 05/612 for AAT. Sample stability was investigated by repeated freezing-thawing and at room temperature. RESULTS: Overall, the assay met all EMA acceptance criteria defined for ligand-binding assays. The six-point calibration curves, ranging from 6 to 192 ng active AAT/mL, showed good accuracy. Errors, expressed as the agreement of the back-fitted assay calibrators with their nominal values, did not exceed ±7%. The AAT concentration determined for the WHO standard represented 100.1% of its assigned value. Validation samples with low protein levels and spiked with low AAT concentrations had mean AAT recoveries from 85.0% to 107.4%. Intra- and inter-run precision (n=6), expressed as relative standard deviations, did not exceed 2.4% and 8.3%, respectively. The assay’s lower limit of quantification (LLOQ) was 0.1 µg/mL. In the plasma matrix, the mean AAT recovery ranged from 94.0% to 106.0%. Intra- and inter-run precision did not exceed 4.2% and 8.2%, respectively. The assay’s LLOQ in plasma matrix was 0.24 µg/mL. Sample stability was shown after repeated freezing thawing for up to three times and at room temperature for up to 4 h. CONCLUSIONS: The assay for the measurement of AAT activity met the acceptance criteria defined by the EMA guideline for bioanalytical assay validation. This qualified the sensitive AAT activity assay ECFISA to be used for the measurement of clinical samples.
