Quantification of therapeutic antibodies and endogenous proteins with LC-MS/MS

Abstract

Therapeutic monoclonal antibodies are widely used for the treatment of various diseases were conventional small molecule based drugs are not effective. For patients with inflammatory autoimmune disease for example, the overproduction of TNF-alpha protein causes painful inflammatory symptoms. The level of TNF-alpha can be reduced through treatment with therapeutic monoclonal antibodies such as infliximab or adalimumab. However, some patients develop anti-drug antibodies that target these therapeutic ‘exogenous’ proteins thus reducing the drug concentration in plasma. This in turn renders the treatment ineffective. In this case quantitative proteomics is used for therapeutic drug monitoring (TDM) of drug concentration in plasma. This can provide the clinician useful insight to what is happening in the patient’s body and can be used to tailor the patient’s care. Quantitative proteomics can also be used for endogenous proteins. Endogenous proteins are synthesized by the DNA within the cell nucleus. Endogenous proteins are the building blocks for all cells that make up the organism, they play a role in food digestion, they offer protection from pathogens and they heal the cells when damaged. The ability to determine whether these proteins are present in the required concentration in plasma can help the clinician to diagnose a disease but also to personalize the treatment plan. Patients with hemophilia A for example, have a mutation in the X chromosome in locus q28. This portion of the X chromosome codes for the synthesis of the 2351 amino acid long FVIII protein. However, if FVIII is not synthesized correctly, this happens when one amino acid is swapped for another, misshaped or misfolded FVIII protein might be formed which could compromise the binding efficiency to its stabilizer protein von Willebrand or to the other factors FIX and FX involved in coagulation, resulting in prolonged bleeding times. Different dosing schemes with recombinant FVIII are thus incorporated dependent on the level of FVIII deficiency. This thesis provides a tutorial for quantitative proteomics with liquid chromatography− tandem mass spectrometry (LC-MS/MS), furthermore various examples of bioanalytical quantification of endogenous and therapeutic proteins in human plasma are presented. Novel sample purification strategies are introduced and optimized though experimental design. Some of these methods such as infliximab and adalimumab are already in use for routine TDM. Others, such as coagulation FVIII, neutralizing anti-drug antibodies, dinutuximab and active anti-thymocyte globulin (ATG) were developed for ongoing pharmacokinetics and pharmacodynamics studies.