Improved HPLC method with automated pre-column sample derivatisation for serum pegylated L-asparaginase activity measurement in paediatric acute lymphoblastic leukaemia patients.

Therapeutic drug monitoring of pegylated L-asparaginase (ASNase) ensures the drug effectiveness in childhood acute lymphoblastic leukaemia (ALL) patients. The biological drug property with variable immunogenic host clearance, and the prescription of its generic formulation urge the need for a reliable assay to ensure an optimal treatment and improve outcome. This study aimed to optimise an existing isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method with an automated pre-column sample derivatisation and injection program, and a computational algorithm for measuring serum pegylated ASNase activity in children with ALL. Nath et al.'s method in 2009 was adopted and modified using a pegylated ASNase. A set of Microsoft Excel macros was developed for the serum drug activity computation. An Agilent InfinityLab LC Series 1260 Infinity II Quaternary System with fluorescence detection was employed with an Agilent Poroshell 120 EC-C18 4.6×100 mm, 2.7 µm analytical column. System flow rate was optimised to 2.0 mL/min with 40×10-6/bar pump compressibility. The O-phthaldialdehyde (OPA) solution composition was optimised to 1 % o-phthaldialdehyde, 0.8 % 2-mercaptoethanol, 7.13 % methanol, and 1.81 % sodium tetraborate. The pre-column derivatisation program mixed 0.1 µL sample with 25 µL OPA solution before the automated injection. Method validation was according to the ICH guidelines. Total analysis time was 15 min, with L-aspartic acid eluted at 0.96 min and internal standard at 4.7 min. The calibration curves showed excellent linearity (R ≥0.9999). Interday precision for the drug activity at 0.1 IU/mL, 0.5 IU/mL, and 1 IU/mL were 4.15 %, 3.05 %, and 3.09 % (n = 6). Mean %error for the drug activity at 0.1 IU/mL, 0.5 IU/mL, and 1 IU/mL were 0.90±4.41 %, -1.37±3.04 %, and -3.03±3.02 % (n = 6). Limit of quantitation was 0.03 IU/mL. Majority of the patients' serum drug activity fell within the assay calibration range. Our improved method is automated, having shorter analysis time with a well-maintained separation resolution that enables a high-throughput analysis for application.

A Review of L-Asparaginase Hypersensitivity in Paediatric Acute Lymphoblastic Leukaemia Patients with Regard to the Measurement of Anti-Asparaginase Antibodies and Their Genetic Predisposition.

L-asparaginase is effective as part of the first line childhood acute lymphoblastic leukaemia (ALL) treatment regimen but suffers the risk of antibody production causing immune-mediated sequelae. This article aimed to describe the clinical implication of L-asparaginase hypersensitivity and review the types of antibodies and genetic polymorphisms contributing to it. Clinical or subclinical L-asparaginase hypersensitivity may lead to suboptimum therapeutic effect and jeopardise the clinical outcome in ALL children. Anti-asparaginase antibodies immunoglobulin (Ig)G, IgM and IgE were identified in the L-asparaginase hypersensitivities. Enzyme-linked immunosorbent assay (ELISA) is commonly used to quantify the IgG and IgM levels. The role of IgE in mediating L-asparaginase hypersensitivity is contradictory. Moreover, the presence of antibodies may not necessarily correlate inversely with the L-asparaginase efficacies in some studies. Patients with specific genetic variants have been shown to be more susceptible to clinical hypersensitivity of L-asparaginase. With the advance of technology, gene polymorphisms have been identified among Caucasians using whole-genome or exon sequencing, but the evidence is scanty among Asians. There is lack of pre-clinical study models that could help in understanding the pathophysiological pathway co-relating the gene expression and anti-asparaginase antibody formation. In conclusion, future research studies are required to fill the current gap in understanding the immune mediated reactions towards L-asparaginase upon its administration and its potential impact to the disease outcome.