Aim of the study: Quantification of circulating biomarkers is affected by drug administration. A method was developed to measure both free and bound biomarkers in the serum of drug-treated patients.
Analyte: Circulating protein biomarkers.
Methodology: ELISA methods applied for the measurement of biomarkers in clinical trials should be fit for purpose. It is strongly recommended that sources from relevant disease population are included in the validation of the method to cover disease induced Interferences on measured biomarkers. Moreover, it is important to consider possible drug-induced effects on circulating proteins, including binding of the drug to the measured biomarkers.
System: Human serum.
Therapeutic area: Autoimmune and inflammatory disease.
Development stage: Clinical trial.
Customer: A multinational pharmaceutical company commercializing biological and antibody based drugs.
Results: Quantification of two specific circulating biomarkers after drug administration to patients was consistently low. Mimicking samples, quantification of the biomarkers was shown to be inversely correlated with drug concentration in serum. Therefore, a method was set up to quantify both the free fraction of biomarker, as the total (free + bound) fraction of biomarker, in order to correctly assess correlation with disease progression after treatment. Samples, prior to direct sandwich ELISA, were subjected to acid dissociation varying time, concentration and type of acid, but biomarker levels were not reliably quantified since biomarker proteins were no more able to bind the antibodies of the ELISA. Therefore, biomarker and drug structure were studied and reduction of disulfide bridges of the antibody-based drug was tried to dissociate biomarkers from the drug. Although biomarkers were not destroyed with this method, quantification of total biomarker concentration was shown to be dependent on the concentration of drug in the samples. Thus, an excess of drug substance was added to each sample prior to analysis, a winning approach. Optimizing also reduction agent and concentration, total biomarker concentration was quantified, as assessed through quality controls of spiked biomarker in human serum in the presence of antibody-based drug.
Advantage of the methodology: Relevance of biomarker concentration can be undermined if drugs have direct effects on their binding to drug proteins or other proteins induced in the drug response. Measurement of total biomarker concentration can be relevant for clinical correlation. Dissociation of bound circulating biomarkers for quantification of total biomarker in human serum can be necessary to obtain relevant information about disease state in patients.