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Host cell proteins (HCPs) must be sufficiently cleared from recombinant biopharmaceuticals during the downstream process (DSP) to ensure product quality, purity, and patient safety. For monitoring of HCP clearance, the typical method chosen is an enzyme-linked immunosorbent assay (ELISA) using polyclonal anti-HCP antibodies obtained from an immunization campaign. This polyclonal reagent is a critical factor for functionality and confidence of the ELISA. Therefore, it is important to ensure that the pool of ELISA antibodies covers a broad spectrum of the HCPs that potentially could persist in the final drug substance. Typically, coverage is determined by gel-based approaches. Here, we present a quantitative proteomics approach combined with purification of HCPs by immunoaffinity chromatography (qIAC-MS) for assessment of ELISA coverage. The cell culture fluid (CCF) of a mock fermentation and a recombinant monoclonal antibody product were characterized in detail to investigate whether the HCPs used for immunization of animals accurately represent HCPs that are relevant to the process. Using the qIAC-MS approach, the ELISA antibody coverage was determined for mock fermentation and product CCF, as well as several different DSP intermediates. Here, the use of different controls facilitated the identification and quantification of HCPs present in the polyclonal reagent and those that nonspecifically bound to IAC material. This study successfully demonstrates that the described qIAC-MS approach is not only a suitable orthogonal method to commonly used 2D SDS-PAGE-based analysis for evaluating ELISA antibody coverage, but that it further identifies HCPs covered as well as missed by the ELISA, enabling an improved risk assessment of HCP ELISA.

A novel mechanism of monolayer poration leading to the rapid exfoliation and lysis of microbial membranes is reported., The spread of bacterial resistance to traditional antibiotics continues to stimulate the search for alternative antimicrobial strategies. All forms of life, from bacteria to humans, are postulated to rely on a fundamental host defense mechanism, which exploits the formation of open pores in microbial phospholipid bilayers. Here we predict that transmembrane poration is not necessary for antimicrobial activity and reveal a distinct poration mechanism that targets the outer leaflet of phospholipid bilayers. Using a combination of molecular-scale and real-time imaging, spectroscopy and spectrometry approaches, we introduce a structural motif with a universal insertion mode in reconstituted membranes and live bacteria. We demonstrate that this motif rapidly assembles into monolayer pits that coalesce during progressive membrane exfoliation, leading to bacterial cell death within minutes. The findings offer a new physical basis for designing effective antibiotics.

International audience; : Due to the lack of macromolecular fossils, the enzymatic repertoire of extinct species has remained largely unknown to date. In an attempt to solve this problem, we have characterized a cyclase subunit (HisF) of the imidazole glycerol phosphate synthase (ImGP-S), which was reconstructed from the era of the last universal common ancestor of cellular organisms (LUCA). As observed for contemporary HisF proteins, the crystal structure of LUCA-HisF adopts the (βα)8-barrel architecture, one of the most ancient folds. Moreover, LUCA-HisF (i) resembles extant HisF proteins with regard to internal 2-fold symmetry, active site residues, and a stabilizing salt bridge cluster, (ii) is thermostable and shows a folding mechanism similar to that of contemporary (βα)8-barrel enzymes, (iii) displays high catalytic activity, and (iv) forms a stable and functional complex with the glutaminase subunit (HisH) of an extant ImGP-S. Furthermore, we show that LUCA-HisF binds to a reconstructed LUCA-HisH protein with high affinity. Our findings suggest that the evolution of highly efficient enzymes and enzyme complexes has already been completed in the LUCA era, which means that sophisticated catalytic concepts such as substrate channeling and allosteric communication existed already 3.5 billion years ago.

In the course of tryptophan biosynthesis, the isomerization of phosphoribosylanthranilate (PRA) is catalyzed by the (βα)₈-barrel enzyme TrpF. The reaction occurs via a general acid-base mechanism with an aspartate and a cysteine residue acting as acid and base, respectively. PRA isomerase activity could be established on two (βα)₈-barrel enzymes involved in histidine biosynthesis, namely HisA and HisF, and on a HisAF chimera, by introducing two aspartate-to-valine substitutions. We have analyzed the reaction mechanism underlying this engineered activity by measuring its pH dependence, solving the crystal structure of a HisF variant with bound product analogue, and applying molecular dynamics simulations and mixed quantum and molecular mechanics calculations. The results suggest that PRA is anchored by the C-terminal phosphate-binding sites of HisA, HisF and HisAF. As a consequence, a conserved aspartate residue, which is equivalent to Cys7 from TrpF, is properly positioned to act as catalytic base. However, no obvious catalytic acid corresponding to Asp126 from TrpF could be identified in the three proteins. Instead, this role appears to be carried out by the carboxylate group of the anthranilate moiety of PRA. Thus, the engineered PRA isomerization activity is based on a reaction mechanism including substrate-assisted catalysis and thus differs substantially from the naturally evolved reaction mechanism used by TrpF.