Cathepsin D: Removal strategy on protein A chromatography, near real time monitoring and characterisation during monoclonal antibody production.

Monoclonal antibody (mAb) fragmentation is a well-known degradation pathway that results in product loss and can significantly impact product quality, efficacy, or even cause immunogenic reactions, thus potentially endangering patients' health. It is recognised that residual proteases present among host cell proteins (HCPs) such as those expressed by Chinese Hamster Ovary (CHO) can induce fragmentation, and failure of their complete removal during downstream processing could cause fragmentation during mAb production and in the final drug product. We identified, using a protease inhibitor screen, an aspartic protease that contributes to proteolytic fragmentation of partially purified mAbs in multiple projects.

Investigation of cathepsin D-mAb interactions using a combined experimental and computational tool set.

Cathepsin D has been identified as a challenge to remove in downstream bioprocessing of monoclonal antibodies (mAbs) due to interactions with some mAbs. This study focused on investigating the mechanisms of interaction between cathepsin D and two industrial mAbs using a combined experimental and computational approach. Surface plasmon resonance was used to study the impact of pH and salt concentration on these protein-protein interactions.

The Hybrid Search: A Mass Spectral Library Search Method for Discovery of Modifications in Proteomics.

We present a mass spectral library-based method to identify tandem mass spectra of peptides that contain unanticipated modifications and amino acid variants. We describe this as a "hybrid" method because it combines matching both ion m/z and mass losses. The mass loss is the difference between the mass of an ion peak and the mass of its precursor.

Identification of an IgG CDR sequence contributing to co-purification of the host cell protease cathepsin D.

Recombinant therapeutic monoclonal antibodies (mAbs) must be purified from host cell proteins (HCPs), DNA, and other impurities present in Chinese hamster ovary (CHO) cell culture media. HCPs can potentially result in adverse clinical responses in patients and, in specific cases, have caused degradation of the final mAb product. As reported previously, residual traces of cathepsin D caused particle formation in the final product of mAb-1.

Target-Agnostic Identification of Functional Monoclonal Antibodies Against Klebsiella pneumoniae Multimeric MrkA Fimbrial Subunit.

The increasing incidence of Klebsiella pneumoniae infections refractory to treatment with current broad-spectrum antibiotic classes warrants the exploration of alternative approaches, such as antibody therapy and/or vaccines, for prevention and treatment. However, the lack of validated targets shared by spectrums of clinical strains poses a significant challenge. We adopted a target-agnostic approach to identify protective antibodies against K.

A Monoclonal Antibody to ADAM17 Inhibits Tumor Growth by Inhibiting EGFR and Non-EGFR-Mediated Pathways.

ADAM17 is the primary sheddase for HER pathway ligands. We report the discovery of a potent and specific ADAM17 inhibitory antibody, MEDI3622, which induces tumor regression or stasis in many EGFR-dependent tumor models. The inhibitory activity of MEDI3622 correlated with EGFR activity both in a series of tumor models across several indications as well in as a focused set of head and neck patient-derived xenograft models.

A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies.

Co-purification of a subset of host cell proteins (HCPs) with monoclonal antibodies (mAbs) during the capture of mAbs on Protein A affinity chromatography is primarily caused by interactions of HCPs with the mAbs. To date, there is limited information about the identity of those HCPs due to the difficulty in detecting low abundance HCPs in the presence of a large amount of the mAb. Here, an approach is presented that allows identification of HCPs that specifically associate with the mAb, while avoiding interference from the mAb itself.

Improved detection of host cell proteins (HCPs) in a mammalian cell-derived antibody drug using liquid chromatography/mass spectrometry in conjunction with an HCP-enrichment strategy.

Rationale: Host cell proteins (HCPs), which are process-related impurities typically present at low levels in recombinant biopharmaceutical products, are often measured using an immunological technique, such as an enzyme-linked immunosorbent assay (ELISA). In contrast to ELISA which only provides the total amount of HCP, liquid chromatography/mass spectrometry (LC/MS) can provide both qualitative and quantitative information about the major HCP species. In this study, an HCP-enrichment step was optimized and combined with LC/MS to identify and determine the relative abundance of HCPs present in a monoclonal antibody (mAb) drug product.