SWATH-MS as a strategy for CHO host cell protein identification and quantification supporting the characterization of mAb purification platforms.

Host cell proteins (HCPs) are process-related impurities expressed by the host cells during biotherapeutics' manufacturing, such as monoclonal antibodies (mAbs). Some challenging HCPs evade clearance during the downstream processing and can be co-purified with the molecule of interest, which may impact product stability, efficacy, and safety. Therefore, HCP content is a critical quality attribute to monitor and quantify across the bioprocess.

Combination of IM-Based Approaches to Unravel the Coexistence of Two Conformers on a Therapeutic Multispecific mAb.

Multispecific antibodies, which target multiple antigens at once, are emerging as promising therapeutic entities to offer more effective treatment than conventional monoclonal antibodies (mAbs). However, these highly complex mAb formats pose significant analytical challenges. We report here on the characterization of a trispecific antibody (tsAb), which presents two isomeric forms clearly separated and identified with size exclusion chromatography coupled to native mass spectrometry (SEC-nMS).

Mechanistic understanding of metal-catalyzed oxidation of polysorbate 80 and monoclonal antibody in biotherapeutic formulations.

Surfactants are commonly used in biotherapeutic formulations to prevent the formation of aggregates and protect proteins from denaturation. Among them polysorbates are the most widely used. However, they are known to be prone to degradation, mainly via enzymatic hydrolysis and oxidation.

"High-risk" host cell proteins (HCPs): A multi-company collaborative view.

Host cell proteins (HCPs) are process-related impurities that may copurify with biopharmaceutical drug products. Within this class of impurities there are some that are more problematic. These problematic HCPs can be considered high-risk and can include those that are immunogenic, biologically active, or enzymatically active with the potential to degrade either product molecules or excipients used in formulation.

Automated mass spectrometry multi-attribute method analyses for process development and characterization of mAbs.

More than 370 biotherapeutics drug products have been approved by regulatory agencies on the US and EU markets and this industry continues to expand. Process change and optimization is necessary to develop new effective biologics in a cost effective and productive way. Consequently, improvement of analytical techniques is required for better product characterization according to Quality by Design (QbD) approach recommended by regulatory agencies.

Improving the analytical toolbox to investigate copurifying host cell proteins presence: N-(4)-(β-acetylglucosaminyl)- l-asparaginase case study.

Levels of host cell proteins (HCPs) in purification intermediates and drug substances (DS) of monoclonal antibodies (mAbs) must be carefully monitored for the production of safe and efficacious biotherapeutics. During the development of mAb1, an immunoglobulin G1 product, unexpected results generated with HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit triggered an investigation which led to the identification of a copurifying HCP called N-(4)-(β-acetylglucosaminyl)-l-asparaginase (AGA, EC3.5.

Multi-OMIC profiling of survival and metabolic signaling networks in cells subjected to photodynamic therapy.

Photodynamic therapy (PDT) is an established palliative treatment for perihilar cholangiocarcinoma that is clinically promising. However, tumors tend to regrow after PDT, which may result from the PDT-induced activation of survival pathways in sublethally afflicted tumor cells. In this study, tumor-comprising cells (i.

Proteomic comparison of the EWS-FLI1 expressing cells EF with NIH-3T3 and actin remodeling effect of (R/W) cell-penetrating peptide.

Unlabelled: EWS-FLI1 expression in NIH-3T3 fibroblasts has a profound impact on the phenotype, resulting in the cytoskeleton and adhesive capacity disorganization (EF cells). Besides this, (R/W), a cell-penetrating peptide (CPP), has an intrinsic actin remodeling activity in EF cells. To evaluate the impact of the oncogenic protein EWS-FLI1 on proteins expression levels, a quantitative comparison of tumoral EF and non-tumoral 3T3 proteomes was performed.

Photocross-Linked Peptide-Protein Complexes Analysis: A Comparative Study of CID and ETD Fragmentation Modes.

Protein-protein interactions are among the keys to organizing cellular processes in space and time. One of the only direct ways to identify such interactions in their cellular environment is to covalently bond the interacting partners to fix the interaction. Photocross-linking in living cells is thus a very promising technique.

Synthesis of an activated phosphonated bifunctional chelate with potential for PET imaging and radiotherapy.

The synthesis of a phosphonated acyclic bifunctional chelate L* for the labeling of biomaterial is described. L* is based on a pyridine backbone, functionalized in ortho positions by aminomethyl-bis-methylphosphonic acids, and, in the para position, by a side chain containing a reactive NHS carbamate function. The stability of L* in aqueous solutions at different pH values was studied by mass spectrometry, showing the activated function to be sensitive to hydrolysis above neutral pH.

Improving the selectivity of the phosphoric acid β-elimination on a biotinylated phosphopeptide.

This study aims at improving the MALDI-TOF detection of a phosphorylated peptide containing a cysteine residue by β-elimination of H(3)PO(4) hardly enriched by classical methods. The experimental conditions were optimized on this phosphopeptide (biot-pAdd) and its nonphosphorylated counterpart (biot-Add). The major side-reactions were H(2)S elimination on the cysteine residues and H(2)O elimination on the non phosphorylated serine residue of biot-Add.