A comprehensive strategy for the identification of biologics by liquid-chromatography-mass spectrometry for release testing in a regulated environment.

Identification (ID) testing is a regulatory requirement for biopharmaceutical manufacturing, requiring robust, GMP-qualified assays that can distinguish the therapeutic from any other in the facility. Liquid Chromatography-Mass Spectrometry (LC-MS) is a powerful analytical tool used to identify and characterize biologics. While routinely leveraged for characterization, LC-MS is relatively rare in Quality Control (QC) settings due to its perceived complexity and scarcity of MS-trained personnel. However, employing LC-MS for identification of drug products has many advantages versus conventional ID techniques, including but not limited to its high specificity, rapid turn-around time, and ease of method execution. In this work, we outline the development and implementation of a comprehensive LC-MS based ID strategy for biologics release testing. Two main workflows (WFs) were developed: i) WF1, a subunit-based assay measuring the molecular weight of the light chain (LC) and heavy chain (HC) of an antibody upon reduction, and ii) WF2, intact mass measurement of the biologic upon N-deglycosylation by PNGase F. The proposed strategy is shown to be applicable for over 40 diverse model biologics including monoclonal antibodies (mAbs), biobetters such as antibody prodrugs/afucosylated mAbs, fusion proteins, multi-specific antibodies, Fabs, and large peptides, all with excellent mass accuracy (error typically < 20 ppm) and precision. It requires a single-step sample preparation and a single click to run and process the data upon method setup. This strategy has been successfully implemented for release testing in GMP labs. Challenges and considerations for the establishment of QC-friendly methods are discussed. It is also shown that these methods can be applied to the ID of more analytically complex biotherapeutics, such as fixed-dose combination (FDC) and drug products co-formulated with trace-level additives.

A Detailed Protocol for Generation of Therapeutic Antibodies with Galactosylated Glycovariants at Laboratory Scale Using In-Vitro Glycoengineering Technology.

N-linked glycosylation is an important post translational modification that occurs on Asparagine 297 residue or a homologous position on the Fc portion of monoclonal antibodies (mAbs). mAb Fc glycans play important roles in antibody structure, stability, and function including effector function and pharmacokinetics. The Fc glycans are made up of a wide variety of sugars including galactose, mannose, and sialic acid. The role of galactose in mediating antibody effector functions is not well understood. Hence, there is widespread interest in the antibody research community to understand the role of galactose in antibody effector functions as galactose is a major constituent of antibody glycans. This requires generation of highly enriched galactosylated variants that has been very challenging via cell culture process. To tackle this challenge, we developed a laboratory scale biochemical process to produce highly enriched galactosylated variants. In this article, we report optimized lab-scale workflows and detailed protocols for generation of deglycosylated, hypo-galactosylated and hyper-galactosylated variants of IgG therapeutic antibodies using the in-vitro glycoengineering technology. The optimized workflows offer short turnaround time and produce highly enriched deglycosylated/hypo-galactosylated/hyper-galactosylated IgG glycovariants, with high purity & molecular integrity as demonstrated by data from an example IgG.

Proteomic Profiling of Hsp90 Inhibitors.

Mass spectrometry assays demonstrate that Hsp90 inhibitors alter the expression of approximately one-quarter of the assayable proteome in mammalian cells. These changes are extraordinarily robust and reproducible, making "proteomics profiling" the gold standard for validating the effects of new Hsp90 inhibitors on cultured cells. Proteomics assays can also suggest novel hypotheses regarding drug mechanisms. To assist investigators in adopting this approach, this Chapter provides detailed protocols for conducting simple proteomics assays of Hsp90 inhibition. The protocols present a robust label-free approach that utilizes pre-fractionation of protein samples by SDS-PAGE, thereby providing reasonably good penetration into the proteome while addressing common issues with sample quality. The actual programming and operation of liquid chromatography-tandem mass spectrometers is not covered, but expectations for achievable performance are discussed, as are alternative approaches, common challenges, and software for data analysis.

The anticancer drug AUY922 generates a proteomics fingerprint that is highly conserved among structurally diverse Hsp90 inhibitors.

AUY922 is a potent synthetic Hsp90 antagonist that is moving steadily through clinical trials against a small range of cancers. To identify protein markers that might measure the drug's effects, and to gain understanding of mechanisms by which AUY922 might inhibit the proliferation of leukemia cells, we characterized AUY922's impacts on the proteomes of cultured Jurkat cells. We describe a robust and readily assayed proteomics fingerprint that AUY922 shares with the flagship Hsp90 inhibitors 17-DMAG and radicicol. We also extend our proteomics findings, demonstrating that an unrelated antagonist of protein folding potentiates the antiproliferative effects of AUY922. Results provide a set of candidate biomarkers for responses to AUY922 in leukemia cells and suggest new modalities for enhancing AUY922's anticancer activities.

Elucidation of the Hsp90 C-terminal inhibitor binding site.

The Hsp90 chaperone machine is required for the folding, activation, and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains; however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein.