MSACL 2023 Abstract

Development and Evaluation of an LC-MS/MS Method for Therapeutic Drug Monitoring of Infliximab for Use in the Routine Clinical Laboratory Melissa Sam (1,2,3), Catherine Toong (1,2,3,5), David Harman (4), William Alex Donald (2) (1). NSW Health Pathology (2.) University of New South Wales (3.) The Ingham Institute of Applied Medical Research (4.) Western Sydney University (5.) Liverpool Hospital, South-Western Sydney Local Health District Melissa Sam, Bachelor of Science (Biomedical) Master of Science (Research) in Medicine (Presenter) NSW Health Pathology, Australia UNSW South-Western Sydney Clinical School UNSW School of Chemistry Ingham Institute for Applied Medical Research, Sydney, Australia >> POSTER (PDF)

Presenter Bio: Mrs Melissa Sam is a Senior Immunology Research Scientist (Senior Scientific Officer) with NSW Health Pathology at Liverpool Hospital in Sydney, NSW, Australia. She also holds conjoint positions at the University of New South Wales and the Ingham Institute for Applied Medical Research. She received her BSc (Biomedical Science) from University of Technology Sydney, and her MSc (Medicine) by Research from University of New South Wales. She has recently commenced her PhD in Chemistry at UNSW, focusing on Mass Spectrometry techniques for therapeutic drug measurement of monoclonal antibodies. She is currently working collaboratively on several clinical trials throughout Australia and is working to develop LCMS testing of infliximab for the Immunology laboratory at Liverpool Hospital.

Introduction:
Therapeutic drug monitoring (TDM) of anti- tumour necrosis factor-alpha drug, infliximab (IFX) is increasingly used in patient management protocols in several autoimmune diseases. Traditionally both drug and anti-drug antibody (ADA) levels are measured using immunoassays, most commonly, ELISA. Measurement of serum drug levels of IFX and ADM and their anti-drug antibodies has been shown to improve patient outcomes over clinical assessment alone. Yet, as discussed by Papamichael et al (2022) in their review, there are significant ‘un-met needs’ in the current modes of testing. Reasons hypothesised by the authors include lack of harmonisation between different assay methods, cost efficiency and long turn-around times between sample collection and result. In addition, most immunoassay methods are not drug tolerant. The presence of the mAb prevents detection of the ADA, and in the same way, the presence of ADAs prevent measurement of the mAb levels. This is due to the formation of complexes between drug (mAb) and ADAs. This produces a gap in information for the clinician in cases of primary or secondary non-response to mAb therapy. Currently, there are several pre-treatment protocols for dissociating drug-antibody complexes in serum and measuring the ADAs on ELISA. However, these are not standardised, nor approved by the therapeutic goods administration, Australia (TGA) and so are not used routinely.

There is limited data on the utility of total drug levels, and along with that, limited ability to detect early development of ADAs that would otherwise be masked by drug interference. The questions is, can patient outcomes be improved if ADAs could be detected earlier? To answer this question, there needs to be further development in assays that can quantify ADAs in the presence of free drug. Recently, assays for the measurement of IFX drug levels have been developed using LC-MS/MS techniques. The benefit of LC-MS/MS assays are that their sensitivity and specificity provide information on drug levels and potential ADA development that is otherwise unavailable. The aims of this study are to develop and validate LC-MS/MS assays for measuring IFX drug levels (both total and free) for routine use and to use these assays to investigate the development of ADAs in patients with undetectable levels on traditional immunoassay.

Methods:
We spiked IFX into buffer at concentrations of 0.5-100 mg/L to produce a calibration curve. Samples were digested using an optimised trypsin digestion protocol. Replicates were analysed to determine intra- and inter-assay precision (%CV). For determination of total IFX spiked into serum, IFX was isolated using ZEBA© and Melon Gel© columns. Free IFX in serum was isolated using an immunoassay capture sample pre-treatment and subsequent on plate tryptic digestion. Samples containing spiked IFX and commercial anti-IFX (ATI) were measured using total IFX method. IFX and ATI ELISAs were performed using a Triturus autoanalyser and commercial ELISA kits.

Results:
We optimised an LC-MS/MS assay for measurement of IFX on the Shimadzu 8050 that has been evaluated for recovery rate, precision, sample stability and carryover. We developed standard curves for the measurement of IFX in serum across the range of 0.5- 100 mg/ml (for total IFX) and 0.5-50mg/ml (for free IFX). Results show good precision for both total IFX assay (R2 = 0.99, %CV < 7) and free IFX assay (R2 = 0.99, %CV < 12). Sample pre- treatment method to isolate total IFX showed good recovery compared to no sample pre-treatment when measured by ELISA. Samples spiked with IFX and commercial ATI were measured on total IFX LC-MS/MS method and compared to ELISA (mean =11.1 mg/L vs <0.49 on ELISA) demonstrating the interference of ATIs in traditional methods. We found that samples spiked with IFX and commercial ATIs performed similarly to samples spiked with IFX alone (Mean =11.1 mg/L vs 12.5 mg/L) on our total IFX, LC-MS/MS assay.

Conclusions:
We have developed an LC-MS/MS assay for the measurement of total IFX and free IFX in serum, which has a good recovery rate, precision and a broader measurement range compared to ELISA. Samples containing IFX-ATI complexes produced similar results to samples containing IFX alone using total IFX assay, showing proof of principle that total IFX can be measured in the presence of ATI. We are currently testing IFX-ATI spiked samples on free IFX assay, and patient samples with suspected ADA development on both total and free IFX assays.