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.

Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug molecules per antibody molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid molecules generated charge envelope profiles that differ from those of the non-conjugated antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug molecules per antibody molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species.

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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 mass spectrometry (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.

Typically, in real-time aerosol mass spectrometry (RTAMS), individual airborne particles are ablated and ionized with a single focused laser pulse. This technique yields information that permits bulk characterization of the particle, but information about the particle's surface is often masked or diluted by the particle bulk. Here we show that it is possible to probe the surface composition of individual airborne particles by separating the desorption and ionization steps using a two-laser real-time aerosol mass spectrometry technique (L2RTAMS). First, a weak excimer laser pulse was used to desorb the semivolatile components of the particle surface when the particle was in the center of the ion trap. After a short delay, another excimer laser pulse was used to ionize the semivolatile surface components in the gas phase and subsequently mass analyzed. The results from the one- and two-laser techniques were compared and found to be complementary. The L2RTAMS technique was found very sensitive to polycyclic aromatic hydrocarbons (PAHs). PAHs, of the type emitted from diesel engines, were found on particle surfaces of National Institute of Standards and Technology (NIST) standard reference materials (SRMs) from Indiana Harbor Canal (1645) and urban particulate matter (1648). PAH partitioning on the environmental particles is discussed.