Biomarker discovery for cervical cancer

Proteomics of human boy fluids is still in its early stage of development with major methodological challenges ahead. This implies that much attention is given to improving the methods and strategies. One major challenge is that many samples that have been acquired in the past may not fulfill the stringent requirements of storage and sample preparation to allow comparable proteomics analyses. It is therefore important to assess the factors that may affect the final proteomics result through systematic and reproducible analyses. Therefore accuracy and sensitivity of the analytical instrumentation is not the only critical factor in this research. Blood (plasma or serum) and urine can be easily sampled from patients or healthy volunteers and are therefore often the first choice when trying to discover novel biomarkers or biomarker patterns to diagnose cancer and other diseases. There are, however, drawbacks such as the masking of low-abundance by high abundance proteins and the possible effect of sampling and sample handling procedures (e.g. different times for blood clotting). A number of different approaches to deplete highly abundant proteins from human serum were studied throughout this thesis. Further, different analytical techniques were applied, such as a miniaturized, microfluidics-based LC-MS system (chip-LC-MS) to enhance overall sensitivity. It is shown that chip-LC-MS has at least twice the resolution of the previously used standard capillary LC-MS method. Since blood composition will change under the influence of external factors, the influence of clotting time on proteome of serum was studied. It was found that most proteins were not affected by clotting time except for those directly involved in this process, such as the fibrinopeptides. Next, we describe a more comprehensive approach for evaluating the influence of various pre-analytical parameters on the serum proteome. A factorial design strategy was applied to assess the importance of seven factors considered to be of relevance, including the level of hemolysis, the digestion conditions, and the storage conditions. Finally, we analyzed serum samples from cervical cancer patients at various stages of disease before and after treatment followed by data processing and statistical data analysis. While we did not discover major changes in the serum proteome using this method, subtle changes in the protein composition were observed in relation to treatment, the significance of which are being further investigated. It is thus demonstrated that the described methods are applicable to highly complex body fluids such as serum and that further studies into the relevance of the discovered changes of the serum proteome are warranted.

Analytical tools for the characterization of deamidation in monoclonal antibodies

This review focuses on the deamidation of asparagine in monoclonal antibodies, one of the well-known degradation pathways. The importance of close monitoring of deamidation from early developability assessment to in vivo studies is discussed consequentially. The application of chromatographic and electrophoretic instruments for the separation of deamidated variants is described. Current mass spectrometric approaches for characterization of deamidated antibody variants and localization of modification sites are discussed accordingly. The review also covers the challenges in bioanalysis of deamidated forms in complex biological matrices during in vivo studies.</p

Non-Antibody-Based Binders for the Enrichment of Proteins for Analysis by Mass Spectrometry

There is often a need to isolate proteins from body fluids, such as plasma or serum, prior to further analysis with (targeted) mass spectrometry. Although immunoglobulin or antibody-based binders have been successful in this regard, they possess certain disadvantages, which stimulated the development and validation of alternative, non-antibody-based binders. These binders are based on different protein scaffolds and are often selected and optimized using phage or other display technologies. This review focuses on several non-antibody-based binders in the context of enriching proteins for subsequent liquid chromatography-mass spectrometry (LC-MS) analysis and compares them to antibodies. In addition, we give a brief introduction to approaches for the immobilization of binders. The combination of non-antibody-based binders and targeted mass spectrometry is promising in areas, like regulated bioanalysis of therapeutic proteins or the quantification of biomarkers. However, the rather limited commercial availability of these binders presents a bottleneck that needs to be addressed

Meta-Inflammation and Metabolic Reprogramming of Macrophages in Diabetes and Obesity:The Importance of Metabolites

Diabetes mellitus type II and obesity are two important causes of death in modern society. They are characterized by low-grade chronic inflammation and metabolic dysfunction (meta-inflammation), which is observed in all tissues involved in energy homeostasis. A substantial body of evidence has established an important role for macrophages in these tissues during the development of diabetes mellitus type II and obesity. Macrophages can activate into specialized subsets by cues from their microenvironment to handle a variety of tasks. Many different subsets have been described and in diabetes/obesity literature two main classifications are widely used that are also defined by differential metabolic reprogramming taking place to fuel their main functions. Classically activated, pro-inflammatory macrophages (often referred to as M1) favor glycolysis, produce lactate instead of metabolizing pyruvate to acetyl-CoA, and have a tricarboxylic acid cycle that is interrupted at two points. Alternatively activated macrophages (often referred to as M2) mainly use beta-oxidation of fatty acids and oxidative phosphorylation to create energy-rich molecules such as ATP and are involved in tissue repair and downregulation of inflammation. Since diabetes type II and obesity are characterized by metabolic alterations at the organism level, these alterations may also induce changes in macrophage metabolism resulting in unique macrophage activation patterns in diabetes and obesity. This review describes the interactions between metabolic reprogramming of macrophages and conditions of metabolic dysfunction like diabetes and obesity. We also focus on different possibilities of measuring a range of metabolites intra-and extracellularly in a precise and comprehensive manner to better identify the subsets of polarized macrophages that are unique to diabetes and obesity. Advantages and disadvantages of the currently most widely used metabolite analysis approaches are highlighted. We further describe how their combined use may serve to provide a comprehensive overview of the metabolic changes that take place intracellularly during macrophage activation in conditions like diabetes and obesity

Enrichment and Liquid Chromatography-Mass Spectrometry Analysis of Trastuzumab and Pertuzumab Using Affimer Reagents

[Image: see text] Trastuzumab and pertuzumab are monoclonal antibodies used in the treatment of human epidermal growth factor receptor-2 (HER2)-positive breast cancer. Therapeutic proteins may undergo chemical modifications that may affect the results of bioanalytical assays, as well as their therapeutic efficacy. Modifications may arise during production and storage, as well as after administration to patients. Studying in vivo biotransformation of monoclonal, therapeutic antibodies requires their enrichment from plasma to discriminate them from endogenous antibodies, as well as from other plasma proteins. To this end, we screened Affimer reagents for selectivity toward trastuzumab or pertuzumab. Affimer reagents are alternative binding proteins possessing two variable binding loops that are based on the human protease inhibitor stefin A or phytocystatin protein scaffolds. Affimer reagents were selected from an extensive library by phage display. The four best-performing binders for each therapeutic antibody were prioritized using a microtiter plate-based approach combined with liquid chromatography–mass spectrometry (LC–MS) in the selected reaction monitoring (SRM) mode. These Affimer reagents were immobilized via engineered 6-His or Cys tags to Ni(2+)- or maleimide beads, respectively. Recovery values of 70% and higher were obtained for both trastuzumab and pertuzumab when spiked at 100, 150, and 200 μg/mL concentrations in human plasma followed by trypsin digestion in the presence of 0.5% sodium deoxycholate and 10 mM dithiothreitol (DTT). Notably, the maleimide beads showed undetectable unspecific binding to endogenous immunoglobulin G (IgGs) or other plasma proteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enrichment method was applied to samples from stress tests of the antibodies at 37 °C to mimic in vivo conditions

Effect of Trastuzumab-HER2 Complex Formation on Stress-Induced Modifications in the CDRs of Trastuzumab

Asparagine deamidation and aspartic acid isomerization in the complementarity determining regions (CDRs) of monoclonal antibodies may alter their affinity to the target antigen. Trastuzumab has two hot spots for deamidation and one position for isomerization in the CDRs. Little is known how complex formation with its target antigen HER2 affects these modifications. Modifications in the CDRs of trastuzumab were thus compared between the free antibody and the trastuzumab-HER2 complex when stressed under physiological conditions at 37°C. Complex formation and stability of the complex upon stressing were assessed by size-exclusion chromatography. Deamidation of light-chain Asn-30 (Lc-Asn-30) was extensive when trastuzumab was stressed free but reduced about 10-fold when the antibody was stressed in complex with HER2. Almost no deamidation of heavy-chain (Hc-Asn-55) was detected in the trastuzumab-HER2 complex, while deamidation was observed when the antibody was stressed alone. Hc-Asp-102 isomerization, a modification that critically affects biological activity, was observed to a moderate degree when the free antibody was stressed but was not detected at all in the trastuzumab-HER2 complex. This shows that complex formation has a major influence on critical modifications in the CDRs of trastuzumab.</p

Determination of Binding Sites on Trastuzumab and Pertuzumab to Selective Affimers Using Hydrogen-Deuterium Exchange Mass Spectrometry

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a method to probe the solvent accessibility and conformational dynamics of a protein or a protein-ligand complex with respect to exchangeable amide hydrogens. Here, we present the application of HDX-MS to determine the binding sites of Affimer reagents to the monoclonal antibodies trastuzumab and pertuzumab, respectively. Intact and subunit level HDX-MS analysis of antibody-affimer complexes showed significant protection from HDX in the antibody Fab region upon affimer binding. Bottom-up HDX-MS experiments including online pepsin digestion revealed that the binding sites of the affimer reagents were mainly located in the complementarity-determining region (CDR) 2 of the heavy chain of the respective antibodies. Three-dimensional models of the binding interaction between the affimer reagents and the antibodies were built by homology modeling and molecular docking based on the HDX data.</p

Pertuzumab Charge Variant Analysis and Complementarity-Determining Region Stability Assessment to Deamidation

Pertuzumab is a monoclonal antibody used for the treatment of HER2-positive breast cancer in combination with trastuzumab. Charge variants of trastuzumab have been extensively described in the literature; however, little is known about the charge heterogeneity of pertuzumab. Here, changes in the ion-exchange profile of pertuzumab were evaluated by pH gradient cation-exchange chromatography after stressing it for up to 3 weeks at physiological and elevated pH and 37 °C. Isolated charge variants arising under stress conditions were characterized by peptide mapping. The results of peptide mapping showed that deamidation in the Fc domain and N-terminal pyroglutamate formation in the heavy chain are the main contributors to charge heterogeneity. The heavy chain CDR2, which is the only CDR containing asparagine residues, was quite resistant to deamidation under stress conditions according to peptide mapping results. Using surface plasmon resonance, it was shown that the affinity of pertuzumab for the HER2 target receptor does not change under stress conditions. Peptide mapping analysis of clinical samples showed an average of 2-3% deamidation in the heavy chain CDR2, 20-25% deamidation in the Fc domain, and 10-15% N-terminal pyroglutamate formation in the heavy chain. These findings suggest that in vitro stress studies are able to predict in vivo modifications. </p

Pertuzumab Charge Variant Analysis and Complementarity-Determining Region Stability Assessment to Deamidation

Pertuzumab is a monoclonal antibody used for the treatment of HER2-positive breast cancer in combination with trastuzumab. Charge variants of trastuzumab have been extensively described in the literature; however, little is known about the charge heterogeneity of pertuzumab. Here, changes in the ion-exchange profile of pertuzumab were evaluated by pH gradient cation-exchange chromatography after stressing it for up to 3 weeks at physiological and elevated pH and 37 °C. Isolated charge variants arising under stress conditions were characterized by peptide mapping. The results of peptide mapping showed that deamidation in the Fc domain and N-terminal pyroglutamate formation in the heavy chain are the main contributors to charge heterogeneity. The heavy chain CDR2, which is the only CDR containing asparagine residues, was quite resistant to deamidation under stress conditions according to peptide mapping results. Using surface plasmon resonance, it was shown that the affinity of pertuzumab for the HER2 target receptor does not change under stress conditions. Peptide mapping analysis of clinical samples showed an average of 2-3% deamidation in the heavy chain CDR2, 20-25% deamidation in the Fc domain, and 10-15% N-terminal pyroglutamate formation in the heavy chain. These findings suggest that in vitro stress studies are able to predict in vivo modifications. </p