Improved characterization of trastuzumab deruxtecan with PTCR and internal fragments implemented in middle-down MS workflows.

Antibody-drug conjugates (ADCs) are highly complex proteins mainly due to the structural microvariability of the mAb, along with the additional heterogeneity afforded by the bioconjugation process. Top-down (TD) and middle-down (MD) strategies allow the straightforward fragmentation of proteins to elucidate the conjugated amino acid residues. Nevertheless, these spectra are very crowded with multiple overlapping and unassigned ion fragments. Here we report on the use of dedicated software (ClipsMS) and application of proton transfer charge reduction (PTCR), to respectively expand the fragment ion search space to internal fragments and improve the separation of overlapping fragment ions for a more comprehensive characterization of a recently approved ADC, trastuzumab deruxtecan (T-DXd). Subunit fragmentation allowed between 70 and 90% of sequence coverage to be obtained. Upon addition of internal fragment assignment, the three subunits were fully sequenced, although internal fragments did not contribute significantly to the localization of the payloads. Finally, the use of PTCR after subunit fragmentation provided a moderate sequence coverage increase between 2 and 13%. The reaction efficiently decluttered the fragmentation spectra allowing increasing the number of fragment ions characteristic of the conjugation site by 1.5- to 2.5-fold. Altogether, these results show the interest in the implementation of internal fragment ion searches and more particularly the use of PTCR reactions to increase the number of signature ions to elucidate the conjugation sites and enhance the overall sequence coverage of ADCs, making this approach particularly appealing for its implementation in R&D laboratories.

Advanced mass spectrometry workflows for accurate quantification of trace-level host cell proteins in drug products: Benefits of FAIMS separation and gas-phase fractionation DIA.

Therapeutic monoclonal antibodies (mAb) production relies on multiple purification steps before release as a drug product (DP). A few host cell proteins (HCPs) may co-purify with the mAb. Their monitoring is crucial due to the considerable risk they represent for mAb stability, integrity, and efficacy and their potential immunogenicity. Enzyme-linked immunosorbent assays (ELISA) commonly used for global HCP monitoring present limitations in terms of identification and quantification of individual HCPs. Therefore, liquid chromatography tandem mass spectrometry (LC-MS/MS) has emerged as a promising alternative. Challenging DP samples show an extreme dynamic range requiring high performing methods to detect and reliably quantify trace-level HCPs. Here, we investigated the benefits of adding high-field asymmetric ion mobility spectrometry (FAIMS) separation and gas phase fractionation (GPF) prior to data independent acquisition (DIA). FAIMS LC-MS/MS analysis allowed the identification of 221 HCPs among which 158 were reliably quantified for a global amount of 880 ng/mg of NIST mAb Reference Material. Our methods have also been successfully applied to two FDA/EMA approved DPs and allowed digging deeper into the HCP landscape with the identification and quantification of a few tens of HCPs with sensitivity down to the sub-ng/mg of mAb level.

Effect of Dietary Seaweed Supplementation in Cows on Milk Macrominerals, Trace Elements and Heavy Metal Concentrations.

This study investigated the effect of seaweed supplementation in dairy cow diets on milk yield, basic composition, and mineral concentrations. Thirty-seven Icelandic cows were split into three diet treatments: control (CON, no seaweed), low seaweed (LSW, 0.75% concentrate dry matter (DM), 13-40 g/cow/day), and high seaweed (HSW, 1.5% concentrate DM, 26-158 g/cow/day). Cows were fed the same basal diet of grass silage and concentrate for a week, and then were introduced to the assigned experimental diets for 6 weeks. The seaweed mix of 91% Ascophyllum nodosum: 9% Laminaria digitata (DM basis), feed, and milk samples were collected weekly. Data were analyzed using a linear mixed effects model, with diet, week, and their interaction as fixed factors, cow ID as random factor, and the pre-treatment week data as a covariate. When compared with CON milk, LSW and HSW milk had, respectively, less Se (-1.4 and -3.1 µg/kg milk) and more I (+744 and +1649 µg/kg milk), while HSW milk also had less Cu (-11.6 µg/kg milk) and more As (+0.17 µg/kg milk) than CON milk. The minimal changes or concentrations in milk for Se, Cu, and As cannot be associated with any effects on consumer nutrition, but care should be taken when I-rich seaweed is fed to cows to avoid excessive animal I supply and milk I concentrations.