Investigating pH Effects on Enzymes Catalyzing Polysorbate Degradation by Activity-Based Protein Profiling.

Host cell proteins (HCPs) are process-related impurities that can negatively impact the quality of biotherapeutics. Some HCPs possess enzymatic activity and can affect the active pharmaceutical ingredient (API) or excipients such as polysorbates (PS). PSs are a class of non-ionic surfactants commonly used as excipients in biotherapeutics to enhance the stability of APIs. The enzyme activity of certain HCPs can result in the degradation of PSs, leading to particle formation and decreased shelf life of biotherapeutics. Identifying and characterizing these HCPs is therefore crucial. This study employed the Activity-Based Protein Profiling (ABPP) technique to investigate the effect of pH on the activity of HCPs that have the potential to degrade polysorbates. Two probes were utilized: the commercially available fluorophosphonate (FP)-Desthiobiotin probe and a probe based on the antiobesity drug, Orlistat. Over 50 HCPs were identified, showing a strong dependence on pH-milieu regarding their enzyme activity. These findings underscore the importance of accounting for pH variations in the ABPP method and other investigations of HCP activity. Notably, the Orlistat-based probe (OBP) enabled us to investigate the enzymatic activity of a wider range of HCPs, emphasizing the advantage of using more than one probe for ABPP. Finally, this study led to the discovery of previously unreported active enzymes, including three HCPs from the carboxylesterase enzyme family.

High level in vivo mucin-type glycosylation in Escherichia coli.

BACKGROUND: Increasing efforts have been made to assess the potential of Escherichia coli strains for the production of complex recombinant proteins. Since a considerable part of therapeutic proteins are glycoproteins, the lack of the post-translational attachment of sugar moieties in standard E. coli expression strains represents a major caveat, thus limiting the use of E. coli based cell factories. The establishment of an E. coli expression system capable of protein glycosylation could potentially facilitate the production of therapeutics with a putative concomitant reduction of production costs. RESULTS: The previously established E. coli strain expressing the soluble form of the functional human-derived glycosyltransferase polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2) was further modified by co-expressing the UDP-GlcNAc 4-epimerase WbgU derived from Plesiomonas shigelloides. This enables the conversion of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) to the sugar donor uridine 5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in the bacterial cytoplasm. Initially, the codon-optimised gene wbgU was inserted into a pET-derived vector and a Tobacco Etch Virus (TEV) protease cleavable polyhistidine-tag was translationally fused to the C- terminus of the amino acid sequence. The 4-epimerase was subsequently expressed and purified. Following the removal of the polyhistidine-tag, WbgU was analysed by circular dichroism spectroscopy to determine folding state and thermal transitions of the protein. The in vitro activity of WbgU was validated by employing a modified glycosyltransferase assay. The conversion of UDP-GlcNAc to UDP-GalNAc was shown by capillary electrophoresis analysis. Using a previously established chaperone pre-/co- expression platform, the in vivo activity of both glycosyltransferase GalNAc-T2 and 4-epimerase WbgU was assessed in E. coli, in combination with a mucin 10-derived target protein. Monitoring glycosylation by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS), the results clearly indicated the in vivo glycosylation of the mucin-derived acceptor peptide. CONCLUSION: In the present work, the previously established E. coli- based expression system was further optimized and the potential for in vivo O-glycosylation was shown by demonstrating the transfer of sugar moieties to a mucin-derived acceptor protein. The results offer the possibility to assess the practical use of the described expression platform for in vivo glycosylations of important biopharmaceutical compounds in E. coli.

A prospective cohort study to assess the relevance of vedolizumab drug level monitoring in IBD patients.

BACKGROUND: Vedolizumab (VDZ) drug monitoring strategies in inflammatory bowel disease (IBD) patients have not been systematically investigated so far. We evaluated the correlation between VDZ trough levels (VTL) and the treatment response in IBD. METHODS: Fifty-one patients with active IBD on or starting a therapy with VDZ were enrolled in this prospective and observational single centre study. Disease activity indices, blood tests, and anthropometric parameters were assessed over a time period of 6 months. One hundred and fifty-five VDZ serum trough levels were measured directly before the next scheduled application using liquid chromatography mass spectrometry (LC-MS/MS). RESULTS: VDZ treatment was found to be clinically effective (Harvey Bradshaw Index (HBI) dropping from 10 to 5.5 points (p < .0005) in Crohn's disease (CD) patients; partial Mayo score (pMS) from 4.4 to 2.1 points (p < .0005) in ulcerative colitis patients (UC). CRP levels tended to decrease and haemoglobin levels to increase under VDZ therapy. CD patients with a serum CRP level lower than 5 mg/l exhibited significantly higher VTL than those with elevated CRP levels (34.9 versus 21.7 µg/ml, p = .00153). UC patients with haemoglobin levels higher 12 g/dl at the time of VTL measurement had significantly higher VTL compared to patients with lower haemoglobin levels (35.4 versus 15.6 µg/ml, p < .0005). CONCLUSIONS: Our data suggest a significant correlation between VTL and response to therapy in IBD patients (higher VTL associated with better response).

Mapping cell envelope and periplasm protein interactions of Escherichia coli respiratory formate dehydrogenases by chemical cross-linking and mass spectrometry.

During anaerobic growth Escherichia coli synthesizes two large, highly homologous respiratory formate dehydrogenases (Fdh's), Fdh-N and Fdh-O, which are associated with the inner membrane but have their respective active site located within the periplasm. The Fdh-N enzyme extends 90 Å into the periplasmic compartment, which in E. coli ranges between 100 and 150 Å from the inner to the outer membrane leaflet. To date, little is known about the interaction partners of Fdh-N and Fdh-O in the periplasmic space that might be involved in stabilizing these enzymes after maturation and translocation across the cytoplasmic membrane has occurred. To address this question, we performed chemical cross-linking in combination with mass spectrometry. We present for the first time the identification of cell envelope interaction partners of Fdh-N and -O from anaerobically grown E. coli using a heterobifunctional amine/photo-reactive cross-linker followed by mass spectrometric analysis of the cross-linked products. We additionally mapped the interface regions within the Fdh/protein complexes for four selected Fdh-binding partners, the chaperone Skp, the l,d-transpeptidase ErfK, OppA, and TolB. Our work yields first structural and functional insights into the mechanisms that support the postmaturation of the multisubunit enzymes Fdh-N and Fdh-O in the periplasm of E. coli.

Pyruvate formate-lyase interacts directly with the formate channel FocA to regulate formate translocation.

The FNT (formate-nitrite transporters) form a superfamily of pentameric membrane channels that translocate monovalent anions across biological membranes. FocA (formate channel A) translocates formate bidirectionally but the mechanism underlying how translocation of formate is controlled and what governs substrate specificity remains unclear. Here we demonstrate that the normally soluble dimeric enzyme pyruvate formate-lyase (PflB), which is responsible for intracellular formate generation in enterobacteria and other microbes, interacts specifically with FocA. Association of PflB with the cytoplasmic membrane was shown to be FocA dependent and purified, Strep-tagged FocA specifically retrieved PflB from Escherichia coli crude extracts. Using a bacterial two-hybrid system, it could be shown that the N-terminus of FocA and the central domain of PflB were involved in the interaction. This finding was confirmed by chemical cross-linking experiments. Using constraints imposed by the amino acid residues identified in the cross-linking study, we provide for the first time a model for the FocA-PflB complex. The model suggests that the N-terminus of FocA is important for interaction with PflB. An in vivo assay developed to monitor changes in formate levels in the cytoplasm revealed the importance of the interaction with PflB for optimal translocation of formate by FocA. This system represents a paradigm for the control of activity of FNT channel proteins.

Selective selC-independent selenocysteine incorporation into formate dehydrogenases.

The formate dehydrogenases (Fdh) Fdh-O, Fdh-N, and Fdh-H, are the only proteins in Escherichia coli that incorporate selenocysteine at a specific position by decoding a UGA codon. However, an excess of selenium can lead to toxicity through misincorporation of selenocysteine into proteins. To determine whether selenocysteine substitutes for cysteine, we grew Escherichia coli in the presence of excess sodium selenite. The respiratory Fdh-N and Fdh-O enzymes, along with nitrate reductase (Nar) were co-purified from wild type strain MC4100 after anaerobic growth with nitrate and either 2 µM or 100 µM selenite. Mass spectrometric analysis of the catalytic subunits of both Fdhs identified the UGA-specified selenocysteine residue and revealed incorporation of additional, 'non-specific' selenocysteinyl residues, which always replaced particular cysteinyl residues. Although variable, their incorporation was not random and was independent of the selenite concentration used. Notably, these cysteines are likely to be non-essential for catalysis and they do not coordinate the iron-sulfur cluster. The remaining cysteinyl residues that could be identified were never substituted by selenocysteine. Selenomethionine was never observed in our analyses. Non-random substitution of particular cysteinyl residues was also noted in the electron-transferring subunit of both Fdhs as well as in the subunits of the Nar enzyme. Nar isolated from an E. coli selC mutant also showed a similar selenocysteine incorporation pattern to the wild-type indicating that non-specific selenocysteine incorporation was independent of the specific selenocysteine pathway. Thus, selenide replaces sulfide in the biosynthesis of cysteine and misacylated selenocysteyl-tRNA(Cys) decodes either UGU or UGC codons, which usually specify cysteine. Nevertheless, not every UGU or UGC codon was decoded as selenocysteine. Together, our results suggest that a degree of misincorporation of selenocysteine into enzymes through replacement of particular, non-essential cysteines, is tolerated and this might act as a buffering system to cope with excessive intracellular selenium.

Well water in karst regions of northeastern Wisconsin contains estrogenic factors, nitrate, and bacteria.

Well water in karst regions is particularly susceptible to contamination by various nonpoint source pollutants such as nitrate, fecal bacteria, and endocrine disrupting chemicals (EDCs). This study analyzed 40 wells in heavily farmed karst areas of northeastern Wisconsin to determine whether these and other pollutants are present, and if so, whether their presence is (1) correlated with other contaminants and (2) exhibits seasonal variation. Nitrate, bacteria, and estrogenicity (indicating the presence of EDCs) were present in at least some of well water samples collected over the course of four time periods between the summers of 2008 and 2009. Although estrogenicity was greatest during the summer months, bacterial contamination was most prevalent during snowmelt. Levels of estrogenicity present in some well water samples approached a threshold concentration that is known to exert endocrine disruption in wildlife. Strong correlations between estrogenicity and other water quality parameters were not found.

Probing single-stranded DNA and its biomolecular interactions through direct catalytic activation of factor XII, a protease of the blood coagulation cascade.

Triggered self-activation of factor XII, a blood coagulation protease, was utilized for the amplified visual detection of ss-DNA targets in a non-sequence specific way. Factor XII holds potential as a low-affinity and therefore non-interfering probe for DNA secondary structure and for the screening of protein binding to ss-DNA. The observation that ss-DNA also accelerates coagulation of human blood plasma is relevant to the emerging field of aptamer therapeutics.

Risk-informed, performance-based inspections at medical facilities.

During the past couple of years, radiation safety professionals have observed a significant change with regard to the inspection philosophy of regulators. The NRC and many Agreement State agencies have implemented a performance-based, risk-informed approach for inspecting medical Radiation Safety Programs. This new, less prescriptive approach originates from the necessity to produce safety benefits commensurate with their cost to the industry and still maintain health and safety performance. While compliance with regulatory requirements is important, regulatory agencies have been focusing on areas that provide greater safety benefit, such as protecting the radiation worker, members of the general public and the environment. This paper discusses simple and practical measures that may assist licensees in preparing for performance-based, risk informed inspections.

Preventing improper disposal of healthcare facility waste containing RAM.

Non-hazardous waste management facilities, which are not authorized to receive licensable radioactive material (RAM), periodically find contaminated waste in shipments from local healthcare facilities. As a consequence, many healthcare facilities are cited each year for losing control and/or improperly disposing of RAM at unauthorized disposal sites. Healthcare radiation safety professionals must ensure that effective measures are in place at their facilities to prevent RAM from inadvertently being included with non-radioactive waste shipments. The objective of this article is to assist in developing and implementing procedures to properly monitor and dispose of waste containing RAM. This article discusses, among other topics, the installation of portal monitors containing both visual and audible alarms to screen medical waste, instruction to individuals handling medical waste and emergency response procedures.

Preventing Improper Disposal of Healthcare Facility Waste Containing RAM.

Non-hazardous waste management facilities, which are not authorized to receive licensable radioactive material (RAM), periodically find contaminated waste in shipments from local healthcare facilities. As a consequence, many healthcare facilities are cited each year for losing control and/or improperly disposing of RAM at unauthorized disposal sites. Healthcare radiation safety professionals must ensure that effective measures are in place at their facilities to prevent RAM from inadvertently being included with non-radioactive waste shipments. The objective of this article is to assist in developing and implementing procedures to properly monitor and dispose of waste containing RAM. This article discusses, among other topics, the installation of portal monitors containing both visual and audible alarms to screen medical waste, instruction to individuals handling medical waste and emergency response procedures.

Characterization of gas-phase adsorption on metal oxide thin films using a magnetoelastic resonance microbalance.

In this study, a magnetoelastic resonance microbalance (MERM) was used to directly measure the gas-phase adsorption behavior of water vapor, isopropyl alcohol, and acetone on a sol-gel-derived titanium dioxide sensor coating. The nature of the MERM platform enables chemical measurements in situations in which wires or physical connections are undesired (or not possible) or in which sensor cost is a major issue. The underlying MERM technique (with an uncoated sensor) showed excellent day-to-day stability, a linear calibration over a 1 kHz change in frequency (or a 1.5-mg change in mass), and the ability to detect a mass change of 15 microg without any efforts at sensitivity optimization. The titanium dioxide coated sensor yielded excellent response to each of the analytes; however, the response did not follow a simple linear calibration function. A more complex calibration model or utilization of the coated sensor in a limited concentration range would be required for quantitative analysis. The process of applying the metal oxide coatings onto the magnetic substrate altered the structure of the thin film layer, resulting in a relatively loose packing of the porous primary titanium dioxide particles to create an open overall honeycomb structure, thereby affecting the adsorption behavior at high relative concentration.

Radiation safety compliance issues at affiliated institutions.

"Affiliation" may be defined as a collaborative interaction between two (or more) organizations in a spirit of mutual benefit through an equitable contribution of resources. Across the United States, hundreds of medical schools and healthcare organizations affiliate with one another for the enhancement of patient care, education, and research. Oftentimes, both parties in the affiliation have active clinical and research programs that involve the use of radioactive material (RAM). The combination of this close affiliation and use of radioactive material presents a number of challenging radiation safety compliance issues. It is important for radiation safety professionals (RSPs) employed by each affiliate to work together to ensure compliance with applicable regulatory requirements.

Radioactive drug research program audit checklist.

Many institutions conduct human research studies that involve the administration of radiopharmaceuticals. The Radiation Safety Officer (RSO) may be the person responsible for ensuring that such studies are performed safely and in compliance with applicable regulations. Sometimes, RSOs assigned this responsibility lack the knowledge and experience necessary to oversee such research activities. In an effort to assist RSOs unfamiliar with radioactive drug research regulations to establish an effective compliance program in this field, the authors developed a comprehensive audit checklist. Periodic use of this checklist may be effective in ensuring compliance with applicable regulations.

Implementation of an X-ray radiation protective equipment inspection program.

In order to comply with the requirements established by healthcare organization accrediting agencies, radiation protective equipment (e.g., lead aprons, gloves, and collars) utilized by x-ray workers must be periodically evaluated for damage. The objective of such evaluations is to identify and remove from service equipment bearing large holes, tears, etc., that could compromise the safety of individuals using this equipment. Many facilities are cited each year for failing to ensure the availability and integrity of such equipment. Unfortunately, written guidance on the proper implementation of inspection programs is not readily available. This paper was developed to assist healthcare organization safety personnel in establishing a radiation protective equipment inspection program at their facilities.