A highly sensitive and robust LC-MS platform for host cell protein characterization in biotherapeutics.

Host cell proteins (HCPs) are a major class of process-related impurities that need to be closely monitored during the production of biotherapeutics. Mass spectrometry (MS) has emerged as a promising tool for HCP analysis due to its specificity for individual HCP's identification and quantitation. However, utilization of MS as a routine characterization tool is still limited due to the time-consuming procedures, non-standardized instrumentation and methodologies, and the limited sensitivity compared to the enzyme-linked immunosorbent assays (ELISA). In this study, we introduced a sensitive (limit of detection (LOD) at 1-2 ppm) and robust HCP profiling platform method with suitable precision and accuracy that can be readily adopted to antibodies and other biotherapeutic modalities without the need for HCP enrichment. The NIST mAb and multiple in-house antibodies were analyzed, and results were benchmarked with other reported studies. In addition, a targeted analysis method with optimized sample preparation for absolute quantitation of lipases was developed and qualified with an LOD of 0.6 ppm and precision of <15%, which can be further improved to an LOD of 5 ppb by using the nano-flow LC.

Awareness of network security and customer value - The company and customer perspective.

The COVID 19 pandemic, by forcing the need to establish and develop relationships via the network, has significantly accelerated the process of digital transformation. For most enterprises, this means the need to change their business model. The basis for each model is subjectively defined customer value. This value is both the input and output of the whole process of building sustainable and profitable relationships with customers. It is believed that in the environment dominated by modern technologies based on the use of the network potential, the value of these relationships reflected in the dually estimated customer value is linked to the awareness of the network potential and the ability to use it. On the basis of the analysis of the purchasing process in the e-commerce industry in Poland and the research, among others, carried out by banks and other scientific centers or institutions dealing with cybersecurity, it is demonstrated that the awareness of the network potential ought to be assessed not only through the prism of benefits that connect both parties with establishing and developing the relationship, but also threats arising from the need to use online mediated relationships. It is believed that the use of the potential of virtual space, in which the customer moves, is determined by the awareness of the network potential, the integral component of which is the awareness of security of establishing, maintaining, and developing relationships. This factor, being directly linked to the risk of the relationship, does and will have a significant impact on the process of creating customer relations in the future and thus the company's value.

Implementation of a High-Resolution Liquid Chromatography-Mass Spectrometry Method in Quality Control Laboratories for Release and Stability Testing of a Commercial Antibody Product.

Liquid chromatography-mass spectrometry (LC-MS) has been widely used throughout biotherapeutic development. However, its implementation in GMP-compliant commercial quality control (QC) laboratories remains a challenge. In this publication, we describe the covalidation and implementation of an automated, high-throughput, and GMP compliant subunit LC-MS method for monitoring antibody oxidation for commercial product release and stability testing. To our knowledge, this is the first report describing the implementation of a high-resolution LC-MS method in commercial QC laboratories for product release and stability testing in the biopharmaceutical industry. This work paves the road for implementing additional LC-MS methods to modernize testing in commercial QC with more targeted control of product quality.

Mass Spectrometry- and Computational Structural Biology-Based Investigation of Proteins and Peptides.

Recent developments of mass spectrometry (MS) allow us to identify, estimate, and characterize proteins and protein complexes. At the same time, structural biology helps to determine the protein structure and its structure-function relationship. Together, they aid to understand the protein structure, property, function, protein-complex assembly, protein-protein interaction, and dynamics. The present chapter is organized with illustrative results to demonstrate how experimental mass spectrometry can be combined with computational structural biology for detailed studies of protein's structures. We have used tumor differentiation factor protein/peptide as ligand and Hsp70/Hsp90 as receptor protein as examples to study ligand-protein interaction. To investigate possible protein conformation, we will describe two proteins-lysozyme and myoglobin. As an application of MS-based assignment of disulfide bridges, the case of the spider venom polypeptide Phα1ß will also be discussed.

Role of Mass Spectrometry in Investigating a Novel Protein: The Example of Tumor Differentiation Factor (TDF).

Better understanding of central nervous system (CNS) molecules can include the identification of new molecules and their receptor systems. Discovery of novel proteins and elucidation of receptor targets can be accomplished using mass spectrometry (MS). We describe a case study of such a molecule, which our lab has studied using MS in combination with other protein identification techniques, such as immunohistochemistry and Western Blotting. This molecule is known as tumor differentiation factor (TDF), a recently-found protein secreted by the pituitary into the blood. TDF mRNA has been detected in brain; not heart, placenta, lung, liver, skeletal muscle, or pancreas. Currently TDF has an unclear function, and prior to our studies, its localization was only minimally understood, with no understanding of receptor targets. We investigated the distribution of TDF in the rat brain using immunohistochemistry (IHC) and immunofluorescence (IF). TDF protein was detected in pituitary and most other brain regions, in specific neurons but not astrocytes. We found TDF immunoreactivity in cultured neuroblastoma, not astrocytoma. These data suggest that TDF is localized to neurons, not to astrocytes. Our group also conducted studies to identify the TDF receptor (TDF-R). Using LC-MS/MS and Western blotting, we identified the members of the Heat Shock 70-kDa family of proteins (HSP70) as potential TDF-R candidates in both MCF7 and BT-549 human breast cancer cells (HBCC) and PC3, DU145, and LNCaP human prostate cancer cells (HPCC), but not in HeLa cells, NG108 neuroblastoma, or HDF-a and BLK CL.4 cells fibroblasts or fibroblast-like cells. These studies have combined directed protein identification techniques with mass spectrometry to increase our understanding of a novel protein that may have distinct actions as a hormone in the body and as a growth factor in the brain.

Proteomics and Non-proteomics Approaches to Study Stable and Transient Protein-Protein Interactions.

Of the 25,000-30,000 human genes, about 2 % code for proteins. However, there are about 1-2 million protein entities. This is primarily due to alternative splicing, post-translational modifications (PTMs) or protein-protein interactions. Proteomics sets out to identify proteins, their sequence and known modifications as well as their quantitation in a biological sample for the purpose of understanding biological processes, protein cellular functions, and their physiological and pathological involvement in diseases.Proteins interact at the molecular level with other proteins, nucleic acids, lipids, carbohydrates and metabolites to perform numerous cellular activities. Protein complexes can consist of sets of more stably (stable PPIs) and less stably (transient PPIs) interacting proteins or combination of both. Here, we discuss the proteomics and non-proteomics approaches to study stable and transient PPIs.

Mass Spectrometry for Proteomics-Based Investigation.

Within the past years, we have witnessed a great improvement is mass spectrometry (MS) and proteomics approaches in terms of instrumentation, protein fractionation, and bioinformatics. With the current technology, protein identification alone is no longer sufficient. Both scientists and clinicians want not only to identify the proteins, but also to identify the protein's post-translational modifications (PTMs), protein isoforms, protein truncation, protein-protein interactions (PPI), and protein quantitation. Here, we describe the principle of MS and proteomics, and strategies to identify proteins, protein's PTMs, protein isoforms, protein truncation, PPIs, and protein quantitation. We also discuss the strengths and weaknesses within this field. Finally, in our concluding remarks we assess the role of mass spectrometry and proteomics in the scientific and clinical settings, in the near future. This chapter provides an introduction and overview for subsequent chapters that will discuss specific MS proteomic methodologies and their application to specific medical conditions. Other chapters will also touch upon areas that expand beyond proteomics, such as lipidomics and metabolomics.

The potential of biomarkers in psychiatry: focus on proteomics.

The etiology and pathogenesis of many psychiatric disorders are unclear with many signaling pathways and complex interactions still unknown. Primary information provided from gene expression or brain activity imaging experiments is useful, but can have limitations. There is a current effort focusing on the discovery of diagnostic and prognostic proteomic potential biomarkers for psychiatric disorders. Despite this work, there is still no biological diagnostic test available for any mental disorder. Biomarkers may advance the care of psychiatric illnesses and have great potential to knowledge of psychiatric disorders but several drawbacks must be considered. Here, we describe the potential of proteomic biomarkers for better understanding and diagnosis of psychiatric disorders and current putative biomarkers for schizophrenia, depression, autism spectrum disorder and attention deficit/hyperactivity disorder.

Protein-protein interactions: switch from classical methods to proteomics and bioinformatics-based approaches.

Following the sequencing of the human genome and many other organisms, research on protein-coding genes and their functions (functional genomics) has intensified. Subsequently, with the observation that proteins are indeed the molecular effectors of most cellular processes, the discipline of proteomics was born. Clearly, proteins do not function as single entities but rather as a dynamic network of team players that have to communicate. Though genetic (yeast two-hybrid Y2H) and biochemical methods (co-immunoprecipitation Co-IP, affinity purification AP) were the methods of choice at the beginning of the study of protein-protein interactions (PPI), in more recent years there has been a shift towards proteomics-based methods and bioinformatics-based approaches. In this review, we first describe in depth PPIs and we make a strong case as to why unraveling the interactome is the next challenge in the field of proteomics. Furthermore, classical methods of investigation of PPIs and structure-based bioinformatics approaches are presented. The greatest emphasis is placed on proteomic methods, especially native techniques that were recently developed and that have been shown to be reliable. Finally, we point out the limitations of these methods and the need to set up a standard for the validation of PPI experiments.

Mass spectrometry investigation of glycosylation on the NXS/T sites in recombinant glycoproteins.

We used a targeted proteomics approach to investigate whether introduction of new N-linked glycosylation sites in a chimeric protein influence the glycosylation of the existing glycosylation sites. To accomplish our goals, we over-expressed and purified a chimeric construct that contained the Fc region of the IgG fused to the exons 7 & 8 of mouse ZP3 (IgG-Fc-ZP3E7 protein). Immunoglobulin heavy chain (IgG-HC protein) was used as control. We then analyzed the IgG-HC and IgG-Fc-ZP3E7 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by Western blotting (WB). We concluded that in control experiments, the glycosylation site was occupied as expected. However, in the IgG-Fc-ZP3E7 protein, we concluded that only one out of three NXS/T glycosylation sites is occupied by N-linked oligosaccharides. We also concluded that in the IgG-Fc-ZP3E7 protein, upon introduction of additional potential NXS/T glycosylation sites within its sequence, the original NST/S glycosylation site from the Fc region of the IgG-Fc-ZP3E7 protein is no longer glycosylated. The biomedical significance of our findings is discussed.

Characterization of tumor differentiation factor (TDF) and its receptor (TDF-R).

Tumor differentiation factor (TDF) is an under-investigated protein produced by the pituitary with no definitive function. TDF is secreted into the bloodstream and targets the breast and prostate, suggesting that it has an endocrine function. Initially, TDF was indirectly discovered based on the differentiation effect of alkaline pituitary extracts of the mammosomatotropic tumor MtTWlO on MTW9/PI rat mammary tumor cells. Years later, the cDNA clone responsible for this differentiation activity was isolated from a human pituitary cDNA library using expression cloning. The cDNA encoded a 108-amino-acid polypeptide that had differentiation activity on MCF7 breast cancer cells and on DU145 prostate cancer cells in vitro and in vivo. Recently, our group focused on identification of the TDF receptor (TDF-R). As potential TDF-R candidates, we identified the members of the Heat Shock 70-kDa family of proteins (HSP70) in both MCF7 and BT-549 human breast cancer cells (HBCC) and PC3, DU145, and LNCaP human prostate cancer cells (HPCC), but not in HeLa cells, NG108 neuroblastoma, or HDF-a and BLK CL.4 cells fibroblasts or fibroblast-like cells. Here we review the current advances on TDF, with particular focus on the structural investigation of its receptor and on its functional effects on breast and prostate cells.

Utility of computational structural biology in mass spectrometry.

Recent developments of mass spectrometry (MS) allow us to identify, estimate, and characterize proteins and protein complexes. At the same time, structural biology helps to determine the protein structure and its structure-function relationship. Together, they aid to understand the protein structure, property, function, protein-complex assembly, protein-protein interaction and dynamics. The present chapter is organized with illustrative results to demonstrate how experimental mass spectrometry can be combined with computational structural biology for detailed studies of protein's structures. We have used tumor differentiation factor protein/peptide as ligand and Hsp70/Hsp90 as receptor protein as examples to study ligand-protein interaction. To investigate possible protein conformation we will describe two proteins, lysozyme and myoglobin.

Using proteomics to unravel the mysterious steps of the HBV-life-cycle.

Infection with Hepatitis B virus (HBV) is the most common cause of liver disease in the world. Infection becomes chronic in up to 10 % of adults, with severe consequences on liver function, including inflammation, fibrosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). HCC is a fast progressing disease causing the death of approximately one million patients annually; current treatment has very limited success, mainly due to late-stage diagnosis and poor screening methodologies. Therefore, unraveling the complex HBV-host cell interactions during progression of the disease is of crucial importance, not only to understand the mechanisms underlying carcinogenesis, but importantly, for the development of new biomarkers for prognostic and early diagnosis. This is an area of research strongly influenced by proteomic studies, which have benefited in the last decade from major technical improvements in accuracy of quantification and sensitivity, large-scale analysis of low-abundant proteins, such as those from clinical samples being now possible and widely applied. This work is a critical review of the impact of the proteomic studies on our current understanding of HBV-associated pathogenesis, diagnostics, and treatment.

Investigating a novel protein using mass spectrometry: the example of tumor differentiation factor (TDF).

Better understanding of central nervous system (CNS) molecules can include the identification of new molecules and their receptor systems. Discovery of novel proteins and elucidation of receptor targets can be accomplished using mass spectrometry (MS). We describe a case study of such a molecule, which our lab has studied using MS in combination with other protein identification techniques, such as immunohistochemistry (IHC) and Western blotting. This molecule is known as tumor differentiation factor (TDF), a recently-found protein secreted by the pituitary into the blood. TDF mRNA has been detected in brain; not heart, placenta, lung, liver, skeletal muscle, or pancreas. Currently TDF has an unclear function, and prior to our studies, its localization was only minimally understood, with no understanding of receptor targets. We investigated the distribution of TDF in the rat brain using IHC and immunofluorescence (IF). TDF protein was detected in pituitary and most other brain regions, in specific neurons but not astrocytes. We found TDF immunoreactivity in cultured neuroblastoma, not astrocytoma. These data suggest that TDF is localized to neurons, not to astrocytes. Our group also conducted studies to identify the TDF receptor (TDF-R). Using LC-MS/MS and Western blotting, we identified the members of the Heat Shock 70-kDa family of proteins (HSP70) as potential TDF-R candidates in both MCF7 and BT-549 human breast cancer cells (HBCC) and PC3, DU145, and LNCaP human prostate cancer cells (HPCC), but not in HeLa cells, NG108 neuroblastoma, or HDF-a and BLK CL.4 cell fibroblasts or fibroblast-like cells. These studies have combined directed protein identification techniques with mass spectrometry to increase our understanding of a novel protein that may have distinct actions as a hormone in the body and as a growth factor in the brain.

Mass spectrometry for proteomics-based investigation.

Within the past years, we have witnessed a great improvement in mass spectrometry (MS) and proteomics approaches in terms of instrumentation, protein fractionation, and bioinformatics. With the current technology, protein identification alone is no longer sufficient. Both scientists and clinicians want not only to identify proteins but also to identify the protein's posttranslational modifications (PTMs), protein isoforms, protein truncation, protein-protein interaction (PPI), and protein quantitation. Here, we describe the principle of MS and proteomics and strategies to identify proteins, protein's PTMs, protein isoforms, protein truncation, PPIs, and protein quantitation. We also discuss the strengths and weaknesses within this field. Finally, in our concluding remarks we assess the role of mass spectrometry and proteomics in scientific and clinical settings in the near future. This chapter provides an introduction and overview for subsequent chapters that will discuss specific MS proteomic methodologies and their application to specific medical conditions. Other chapters will also touch upon areas that expand beyond proteomics, such as lipidomics and metabolomics.

Investigation of stable and transient protein-protein interactions: Past, present, and future.

This article presents an overview of the literature and a review of recent advances in the analysis of stable and transient protein-protein interactions (PPIs) with a focus on their function within cells, organs, and organisms. The significance of PTMs within the PPIs is also discussed. We focus on methods to study PPIs and methods of detecting PPIs, with particular emphasis on electrophoresis-based and MS-based investigation of PPIs, including specific examples. The validation of PPIs is emphasized and the limitations of the current methods for studying stable and transient PPIs are discussed. Perspectives regarding PPIs, with focus on bioinformatics and transient PPIs are also provided.

Identification of potential tumor differentiation factor (TDF) receptor from steroid-responsive and steroid-resistant breast cancer cells.

Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.

Characterization of the anti-HBV activity of HLP1-23, a human lactoferrin-derived peptide.

Lactoferrin (Lf) was shown to exhibit its antiviral activity at an early phase of viral infection and a mechanism whereby the protein interacts with host cell surface molecules has been suggested. In this study, human Lf (HLf) and seven HLf-derived synthetic peptides (HLP) corresponding to the N-terminal domain of the native protein (1-47 amino acids sequence) were assayed for their capacity to prevent hepatitis B virus (HBV) infection and replication using the HepaRG and HepG2.2.2.15 cell lines. Of the series tested, four peptides showed 40-75% inhibition of HBV infection in HepaRG cells, HLP1-23 , containing the GRRRR cationic cluster, being the most potent. Interestingly, this cluster is one of the two glycosaminoglycan binding sites of the native HLf involved in its antiviral activity; however, the mechanism of the HLP1-23 action was different from that of the full-length protein, the peptide inhibiting HBV infection when pre-incubated with the virus, while no effect was observed on the target cells. It is suggested that the cationic cluster is sufficient for the peptide to interact stably with negatively charged residues on the virion envelope, while the absence of the second glycosaminoglycan binding site prevents its efficient attachment to the cells. In conclusion, this peptide may constitute a non-toxic approach for potential clinical applications in inhibiting HBV entry by neutralizing the viral particles.

Impact of Trisulfide on the Structure and Function of Different Antibody Constructs.

Trisulfide is a post-translational modification (PTM) commonly found in recombinant antibodies. It has been demonstrated that trisulfide had no impact on the bioactivity of mono-specific antibodies (MsAbs). However, the impact of trisulfide on multi-specific antibodies has not been evaluated. In this study, two mass spectrometric methods were developed for comprehensive trisulfide characterization. The non-reduced peptide mapping method combined with the unique electron activated dissociation (EAD) provided signature fragments for confident trisulfide identification as well as trisulfide quantitation at individual sites. A higher throughput method using Fab mass analysis was also developed and qualified to support routine monitoring of trisulfide during process development. Fab mass analysis features simpler sample preparation and shorter analysis time but provides comparable results to the non-reduced peptide mapping method. In this study, a bi-specific (BsAb) and a tri-specific antibody (TsAb) were compared side-by-side with a MsAb to evaluate the impact of trisulfide on the structure and function of multi-specific antibodies. Results indicated that trisulfide dominantly formed at similar locations across different antibody constructs and had no impact on the size heterogeneity, charge heterogeneity, or bioactivities of any assessed antibodies. Together with the in vitro stability under heat stress (25 °C and 40 °C for up to four weeks) and rapid conversion from trisulfide to disulfide during in vivo circulation, trisulfide could be categorized as a non-critical quality attribute (non-CQA) for antibody products.

Potential biomarkers in psychiatry: focus on the cholesterol system.

Measuring biomarkers to identify and assess illness is a strategy growing in popularity and relevance. Although already in clinical use for treating and predicting cancer, no biological measurement is used clinically for any psychiatric disorder. Biomarkers could predict the course of a medical problem, and aid in determining how and when to treat. Several studies have indicated that of candidate psychiatric biomarkers detected using proteomic techniques, cholesterol and associated proteins, specifically apolipoproteins (Apos), may be of interest. Cholesterol is necessary for brain development and its synthesis continues at a lower rate in the adult brain. Apos are the protein component of lipoproteins responsible for lipid transport. There is extensive evidence that the levels of cholesterol and Apos may be disturbed in psychiatric disorders, including autistic spectrum disorders (ASD). Here, we describe putative serum biomarkers for psychiatric disorders, and the role of cholesterol and Apos in central nervous system (CNS) disorders.