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.