Alexandru C. Lazar

Synthesis and Evaluation of Hydrophilic Linkers for Antibody–Maytansinoid Conjugates

The synthesis and biological evaluation of hydrophilic heterobifunctional cross-linkers for conjugation of antibodies with highly cytotoxic agents are described. These linkers contain either a negatively charged sulfonate group or a hydrophilic, noncharged PEG group in addition to an amine-reactive N-hydroxysuccinimide (NHS) ester and sulfhydryl reactive termini. These hydrophilic linkers enable conjugation of hydrophobic organic molecule drugs, such as a maytansinoid, at a higher drug/antibody ratio (DAR) than hydrophobic SPDB and SMCC linkers used earlier without triggering aggregation or loss of affinity of the resulting conjugate. Antibody-maytansinoid conjugates (AMCs) bearing these sulfonate- or PEG-containing hydrophilic linkers were, depending on the nature of the targeted cells, equally to more cytotoxic to antigen-positive cells and equally to less cytotoxic to antigen-negative cells than conjugates made with SPDB or SMCC linkers and thus typically displayed a wider selectivity window, particularly against multidrug resistant (MDR) cancer cell lines in vitro and tumor xenograft models in vivo.

Recent advances in the MS analysis of glycoproteins: Theoretical considerations.

Protein glycosylation is involved in a broad range of biological processes that regulate protein function and control cell fate. As aberrant glycosylation has been found to be implicated in numerous diseases, the study and large‐scale characterization of protein glycosylation is of great interest not only to the biological and biomedical research community, but also to the pharmaceutical and biotechnology industry. Due to the complex chemical structure and differing chemical properties of the protein/peptide and glycan moieties, the analysis and structural characterization of glycoproteins has been proven to be a difficult task. Large‐scale endeavors have been further limited by the dynamic outcome of the glycosylation process itself, and, occasionally, by the low abundance of glycoproteins in biological samples. Recent advances in MS instrumentation and progress in miniaturized technologies for sample handling, enrichment and separation, have resulted in robust and compelling analysis strategies that effectively address the challenges of the glycoproteome. This review summarizes the key steps that are involved in the development of efficient glycoproteomic analysis methods, and the latest innovations that led to successful strategies for the characterization of glycoproteins and their corresponding glycans. As a follow‐up to this work, we review innovative capillary and microfluidic‐MS workflows for the identification, sequencing and characterization of glycoconjugates.

Recent advances in the MS analysis of glycoproteins: Capillary and microfluidic workflows

Recent developments in bioanalytical instrumentation, MS detection, and computational data analysis approaches have provided researchers with capabilities for interrogating the complex cellular glycoproteome, to help gain a better insight into the cellular and physiological processes that are associated with a disease and to facilitate the efforts centered on identifying disease‐specific biomarkers. This review describes the progress achieved in the characterization of protein glycosylation by using advanced capillary and microfluidic MS technologies. The major steps involved in large‐scale glycoproteomic analysis approaches are discussed, with special emphasis given to workflows that have evolved around complex MS detection functions. In addition, quantitative analysis strategies are assessed, and the bioinformatics aspects of glycoproteomic data processing are summarized. The developments in commercial and custom fabricated microfluidic front‐end platforms to ESI‐ and MALDI‐MS instrumentation, for addressing major challenges in carbohydrate analysis such as sensitivity, throughput, and ability to perform structural characterization, are further evaluated and illustrated with relevant examples.

Glycoproteomics on the rise: Established methods, advanced techniques, sophisticated biological applications

Glycosylation is the most complex form of protein PTMs. Affected proteins may carry dozens of glycosylation sites with tens to hundreds of glycan residues attached to every site. Glycosylated proteins have many important functions in biology, from cellular to organismal levels, being involved in cell–cell signaling, cell adhesion, immune response, host–pathogen interactions, and development and growth. Glycosylation, however, expands the biological functional diversity of proteins at the expense of a tremendous increase in structural heterogeneity. Aberrant glycosylation of cell surface proteins, as well as their detectable fingerprint in plasma samples, has been associated with cancer, inflammatory and degenerative diseases, and congenital disorders of glycosylation. Therefore, there are on‐going efforts directed toward developing new technologies and approaches for glycan sequencing and high‐throughput analysis of glycosylated proteins in complex samples with simultaneous characterization of both the protein and glycan moieties. This work is aimed primarily at pinpointing the challenges associated with the large‐scale analysis of glycoproteins and the latest developments in glycoproteomic research, with focus on recent advancements (2011–2012) in microcolumn separations and MS detection.

Exploring the glycoproteomics landscape with advanced MS technologies

The advance of glycoproteomic technologies has offered unique insights into the importance of glycosylation in determining the functional roles of a protein within a cell. Biologically active glycoproteins include the categories of enzymes, hormones, proteins involved in cell proliferation, cell membrane proteins involved in cell–cell recognition, and communication events or secreted proteins, just to name a few. The recent progress in analytical instrumentation, methodologies, and computational approaches has enabled a detailed exploration of glycan structure, connectivity, and heterogeneity, underscoring the staggering complexity of the glycome repertoire in a cell. A variety of approaches involving the use of spectroscopy, MS, separation, microfluidic, and microarray technologies have been used alone or in combination to tackle the glycoproteome challenge, the research results of these efforts being captured in an overwhelming number of annual publications. This work is aimed at reviewing the major developments and accomplishments in the field of glycoproteomics, with focus on the most recent advancements (2012–2014) that involve the use of capillary separations and MS detection.

Determination of Charge Heterogeneity and Level of Unconjugated Antibody by Imaged cIEF

Imaged capillary isoelectric focusing (icIEF) is capable of monitoring the charge heterogeneity profile of conjugated antibodies. The electropherogram from icIEF can be integrated to quantitate the amount of unconjugated antibody present in a conjugate sample. This chapter describes an icIEF method where a conjugate sample was first prepared by mixing with appropriate ampholytes, pI markers, and additives. Then, the sample was focused in a fluorocarbon-coated fused silica capillary, where absorbance images were taken. Quantitation of the unconjugated antibody was achieved by using a calibration curve.

Proteomics for Drug Discovery

The combination of photoaffinity labeling (PAL) and quantitative chemoproteomics enables the comprehensive, unbiased determination of protein interaction profiles to support target identification of bioactive small molecules. This approach is amenable to cells in culture and compatible with pharmacologically relevant transmembrane target classes like G-protein coupled receptors and ions channels which have been notoriously hard to access by conventional chemoproteomics approaches. Here, we describe a strategy that combines PAL probe titration and competition with excess parental compounds with the goal of enabling the identification of specific interactors as well as assessing the functional relevance of a binding event for the phenotype under investigation.