Nanoparticle-based Sample Preparation and High- Resolution Mass Spectrometry for Bioprocess and Quality Control in Biopharmaceutical Production

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 XII 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.