Abstract:
Biopharmaceuticals, such as monoclonal antibodies (mAb), have recently become
increasingly important in the treatment of many different diseases. Usually these molecules
have complex molecular structures which poses great challenges for their characterization.
However, full characterization is essential for FDA and EMA drug approval. Nowadays,
antibodies are usually analyzed by high-resolution mass spectrometry (HR-MS). One
approach is the middle-down analysis, where enzymes such as pepsin are used to digest the
antibodies into specific fragments which are in a more suitable size-range and can be more
easily analyzed. However, mAb characterization usually starts with top-down analysis of
intact antibodies using HR-MS or liquid chromatography (LC) hyphenated to HR-MS (LC-MS)
for determining the molecular mass to charge (m/z) of the protein.
In this work new methods for sample preparation in protein analytics of biopharmaceuticals
have been developed. In particular, the main approach discussed herein describes the
sample preparation of therapeutic proteins with heterogeneous nanobiocatalysts based on
gold nanoparticles (GNPs) with coated or immobilized enzymes such as pepsin. The different
synthesis steps of the nanoparticulate carriers were investigated and compared in size and
function using classical methods, such as Vis-spectroscopy, dynamic light scattering (DLS),
Lowry assay and Michaelis-Menten kinetic analysis. Newer methods such as Resonant Mass
Measurement and Taylor Dispersion Analysis were also used for this purpose. In order to
extend the toolbox of methods in this regard, the results of these modern characterization
methods were compared with those of the classical ones. For functional studies of the gold
nanoparticle-conjugated enzyme, the comparison of enzyme activities with free, unbound
enzymes (e.g. pepsin) is an important aspect for the performance evaluation of the new
nanobiocatalysts. The immobilization of enzymes to nanoparticulate carriers has some
advantageous. Since the gold nanoparticles have a high density, separation of the nano
biocatalysts from the sample is easily possible with simple benchtop centrifuges, as they are
present in almost every modern laboratory. Thus, the enzyme can be easily removed after
reaction and does not contaminate the sample with another protein (enzyme) which might
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give interferences in subsequent MS analysis. However, it has to be ascertained that
immobilization does not reduce the enzyme activity. In this work it is documented that pepsin
immobilized on gold nanoparticles has even higher enzymatic efficiencies than pepsin in free
solution.
The aim of the present work is to provide an overview of the synthesis and characterization
of GNPs as a nano framework for enzymes used for protein and antibody analysis. Also, the
current state of technology in methods for the use of GNPs should be pointed out here.
