Development and Validation of an Efficient and Highly Sensitive Enzyme-Linked Immunosorbent Assay for Alemtuzumab Quantification in Human Serum and Plasma

Therapeutic Drug Monitoring, vol. 45, pp. 79–86

Abstract

BACKGROUND: Alemtuzumab is a humanized monoclonal antibody that targets the CD52 glycoprotein expressed on most lymphocytes, subsequently inducing complement-mediated and antibody-mediated cytotoxicity. Owing to its ability to induce profound immune depletion, alemtuzumab is frequently used in patients before allogeneic hematopoietic stem cell transplantation to prevent graft rejection and acute graft-versus-host disease. In this clinical context, a stable immunoassay with high sensitivity and specificity to determine alemtuzumab levels is essential for performing pharmacokinetic and pharmacodynamic analyses; however, the available methods have several limitations. Here, we report the successful development and validation of an efficient and highly sensitive enzyme-linked immunosorbent assay technique based on commercially available reagents to quantify alemtuzumab in human serum or plasma. METHODS: This enzyme-linked immunosorbent assay technique was developed and validated in accordance with the European Medicines Agency guidelines on bioanalytical method validation. RESULTS: The assay sensitivity (lower limit of quantification) is 0.5 ng·mL -1 , and the dynamic range is 0.78-25 ng·mL -1 . To accommodate quantification of peak concentration and concentrations below the lympholytic level (<0.1 mcg·mL -1 ), patients' serum samples were prediluted 20-400 times according to the expected alemtuzumab concentration. The overall within-run accuracy was between 96% and 105%, whereas overall within-run precision (coefficient of variation) was between 3% and 9%. The between-run assessment provided an overall accuracy between 86% and 95% and an overall coefficient of variation between 5% and 14%. CONCLUSIONS: The developed assay provides accurate insight into alemtuzumab exposure and its effects on the clinical response to treatment, which is key to optimizing treatment strategies.

Authors 8

1. Federica Achini Aachen Laboratory for Paediatric Immunology

   Leiden University · University of Zurich · Leiden University Medical Center · University Children's Hospital Zurich · Willem-Alexander Kinderziekenhuis

   Affiliation as printed

   Division of Paediatric Stem Cell Transplantation and Haematology, Children's Research Centre (CRC), University Children's Hospital of Zurich, Zurich, Switzerland;

   Laboratory for Paediatric Immunology, Willem Alexander Children’s Hospital, Leiden University Medical Centre, Leiden, the Netherlands;

   Division of Paediatric Stem Cell Transplantation and Haematology, Children's Research Centre (CRC), University Children's Hospital of Zurich, Zurich, Switzerland

   Laboratory for Paediatric Immunology, Willem Alexander Children's Hospital, Leiden University Medical Centre, Leiden, The Netherlands

2. Cornelia M. Jol‐van der Zijde Aachen Laboratory for Paediatric Immunology

   Leiden University · Leiden University Medical Center · Willem-Alexander Kinderziekenhuis

   Affiliation as printed

   Laboratory for Paediatric Immunology, Willem Alexander Children’s Hospital, Leiden University Medical Centre, Leiden, the Netherlands;

   Laboratory for Paediatric Immunology, Willem Alexander Children's Hospital, Leiden University Medical Centre, Leiden, The Netherlands

3. Anja M. Jansen-Hoogendijk Aachen Laboratory for Paediatric Immunology

   Leiden University · Leiden University Medical Center · Willem-Alexander Kinderziekenhuis

   Affiliation as printed

   Laboratory for Paediatric Immunology, Willem Alexander Children’s Hospital, Leiden University Medical Centre, Leiden, the Netherlands;

   Laboratory for Paediatric Immunology, Willem Alexander Children's Hospital, Leiden University Medical Centre, Leiden, The Netherlands

4. Arjan C. Lankester Aachen

   Leiden University · Leiden University Medical Center · Willem-Alexander Kinderziekenhuis

   Affiliation as printed

   Department of Paediatrics, Willem Alexander Children’s Hospital, Leiden University Medical Centre, Leiden, the Netherlands; and

5. Robbert G. M. Bredius Aachen

   Leiden University · Leiden University Medical Center · Willem-Alexander Kinderziekenhuis

   Affiliation as printed

   Department of Paediatrics, Willem Alexander Children’s Hospital, Leiden University Medical Centre, Leiden, the Netherlands; and

6. Maarten J. D. van Tol Aachen Laboratory for Paediatric Immunology

   Leiden University · Leiden University Medical Center · Willem-Alexander Kinderziekenhuis

   Affiliation as printed

   Laboratory for Paediatric Immunology, Willem Alexander Children’s Hospital, Leiden University Medical Centre, Leiden, the Netherlands;

   Laboratory for Paediatric Immunology, Willem Alexander Children's Hospital, Leiden University Medical Centre, Leiden, The Netherlands

7. Dirk Jan A. R. Moes corresponding Aachen Department of Clinical Pharmacy and Toxicology

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Department of Clinical Pharmacy and Toxicology, Leiden University Medical Centre, Leiden, the Netherlands

   Department of Clinical Pharmacy and Toxicology, Leiden University Medical Centre, Leiden, The Netherlands

8. Marco W. Schilham corresponding Aachen Laboratory for Paediatric Immunology

   Leiden University · Leiden University Medical Center · Willem-Alexander Kinderziekenhuis

   Affiliation as printed

   Laboratory for Paediatric Immunology, Willem Alexander Children’s Hospital, Leiden University Medical Centre, Leiden, the Netherlands;

   Laboratory for Paediatric Immunology, Willem Alexander Children's Hospital, Leiden University Medical Centre, Leiden, The Netherlands