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The multi-attribute method (MAM) was conceived as a single assay to potentially replace multiple single-attribute assays that have long been used in process development and quality control (QC) for protein therapeutics. MAM is rooted in traditional peptide mapping methods; it leverages mass spectrometry (MS) detection for confident identification and quantitation of many types of protein attributes that may be targeted for monitoring. While MAM has been widely explored across the industry, it has yet to gain a strong foothold within QC laboratories as a replacement method for established orthogonal platforms. Members of the MAM consortium recently undertook an interlaboratory study to evaluate the industry-wide status of MAM. Here we present the results of this study as they pertain to the targeted attribute analytics component of MAM, including investigation into the sources of variability between laboratories and comparison of MAM data to orthogonal methods. These results are made available with an eye toward aiding the community in further optimizing the method to enable its more frequent use in the QC environment.

The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography-mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671. Results from 28 participating laboratories show that the NPD parameters being utilized across the industry are representative of high-resolution MS performance capabilities. Certain elements of NPD, including common sources of variability in the number of new peaks detected, that are critical to the performance of the purity function of MAM were identified in this study and are reported here as a means to further refine the methodology and accelerate adoption into manufacturer-specific protein therapeutic product life cycles.

We report a modular approach to synthesize maleimido group containing hydrophilic dolastatin 10 (Dol10) derivatives as drug-linkers for the syntheses of antibody-drug conjugates (ADCs). Discrete polyethylene glycol (PEG) moieties of different chain lengths were introduced as part of the linker to impart hydrophilicity to these drug linkers. The synthesis process involved construction of PEG maleimido derivatives of the tetrapeptide intermediate (N-methylvaline-valine-dolaisoleucine-dolaproine), which were subsequently coupled with dolaphenine to generate the desired drug linkers. The synthetic method reported in this manuscript circumvents the use of highly cytotoxic Dol10 in its native form. By using trastuzumab (Herceptin®) as the antibody we have synthesized Dol10 containing ADCs. The presence of a discrete PEG chain in the drug linkers resulted in ADCs free from aggregation. The effect of PEG chain length on the biological activities of these Dol10 containing ADCs was investigated by in vitro cytotoxicity assays. ADCs containing PEG6 and PEG8 spacers exhibited the highest level of in vitro anti-proliferative activity against HER2-positive (SK-BR-3) human tumor cells. ADCs derived from Herceptin® and PEG8-Dol10, at a dose of 10 mg kg-1, effectively delayed the tumor growth and prolonged the survival time in mice bearing human ovarian SKOV-3 xenografts.

Up to 15% of the population older than 30 years suffers from symptomatic thumb carpometacarpal (CMC) osteoarthritis (OA), with the incidence increasing to ~33% in postmenopausal women. The thumb CMC joint has been reported as the most painful joint when compared to other hand joints affected by OA. It is therefore no surprise that this is a common chief complaint of patients and has a significant effect on work and life satisfaction. The purpose of this article was to review currently available literature to discuss nonoperative and operative techniques utilized to treat the various stages of thumb CMC arthritis. A variety of nonoperative and operative techniques have been described in the literature, each with its own benefits and pitfalls. This review concludes that while many treatment options exist, there remains no perfect treatment, but the goal of improving quality of life and patient satisfaction remains the same.

In recent years, researchers have begun to adopt a perspective evaluating “winners and losers” regarding the consumption and value of ecosystem services. “Winners” tend to benefit from the ecosystem service and “losers” absorb most associated costs. Our study focuses on water use in Oklahoma (USA) and a plan to divert water from the Kiamichi River in southeastern Oklahoma for consumption at residences in the Oklahoma City metropolitan area. Our study is, in part, a follow-up from an initial 2013 survey of Oklahoma City residents and residents of the Kiamichi. For this paper, a survey was distributed within the state of Oklahoma to evaluate changes to ecosystem service willingness to pay and valuation. This survey also included an experimental element assessing if exposure to additional information about ecosystem services influenced respondents on ecosystem service valuation, or willingness to pay. Our results generally aligned with those found in the 2013 survey. Oklahoma City residents are not aware of where their water is coming from and are not willing to pay to protect ecosystem services, despite an overall increase in activism. Our results indicate that a smaller number of significant factors determining willingness to pay for ecosystem service maintenance were identified than the study in 2013. Exposure to additional information had no effect on peoples’ preferences. We found that public opinion surrounding environmental support is context-specific, political conservatism may not always impede valuation of environmental protections. We conclude that cultural, moral, and political values interact in their influence on expressions of valuation and willingness to pay for ecosystem services.

Switchgrass (Panicum virgatum L.) is a native, warm season perennial that may be suitable for advanced renewable bioenergy feedstock production in semi-arid regions, where year-round soil cover is desirable as the primary environmental concern related to vegetation harvest is wind erosion. One objective of this project was to explore potential effects of two switchgrass varieties, Blackwell and Kanlow, on both abiotic and biotic soil quality indicators. No significant differences were detected in soil erodibility parameters, but numerically the cultivar with the lesser biomass and fewer plants per unit area had soil properties that had greater potential for wind erosion. Regardless of cultivar, soil P was enriched in the wind-erodible fraction compared with the bulk soil samples. Biological properties did not differ for the two switchgrass varieties, but microbial biomass and dehydrogenase enzyme activity was greater for samples collected 15 cm from the ne...

Obwohl es bereits an zahlreichen Hochschulen interessante Lehrangebote zu Nachhaltigkeit gibt, besteht nach wie vor ein unzureichender Einbezug der in den letzten Jahren in der Praxis erarbeiteten Erfahrungen. Zahlreiche Behorden und Ingenieurburos fuhren bereits praktische Anwendungen zur Implementierung nachhaltiger Planungslosungen durch, deren Ergebnisse und Erfahrungen in der akademischen Lehre multipliziert werden sollten. Insbesondere betrifft dies die Bereiche Integriertes Wasser- und Abfallmanagement, Okosystemrenaturierung und Okosystemleistungen, aber auch nachhaltiges Landmanagement und den Water-Energy-Food Nexus. Den Rahmen fur die inhaltliche Planung bildet die Mitteilung der EU-Kommission vom 06.05.2013 „Grune Infrastruktur (GI) – Aufwertung des europaischen Naturkapitals“. Dabei spielt im Zusammenhang mit der Vermittlung des Wissens zu Nachhaltigkeit nicht nur der bekannte Teaching-Research Nexus (TRN) eine masgebliche Rolle, sondern auch die Erganzung durch die Praxis, weil in der Wirtschaft insbesondere in den letzten Jahren oftmals ein System selbstlernender Wissenserweiterung praktiziert wird. Der TRN ist im Hinblick auf den dreipoligen Nachhaltigkeitsansatz erweiterbar, um den Bezug zur Praxis hinreichend zu berucksichtigen. Daher schlagen wir als Rahmenansatz fur die Umsetzung von Nachhaltigkeit in der angewandten Lehre in Anlehnung an das „research-teaching-practice triangle“ nach Kaplan (1989) die Begrifflichkeit Teaching-Research-Practice Nexus (TRPN) vor. Fur die praktische Umsetzung wurde an der Hochschule Magdeburg-Stendal das Projekt „Umweltmanagement und Umweltzertifizierung in Theorie und Praxis“ realisiert, an dem die Studierenden mit grosem Interesse teilnahmen, da es darum geht, den eigenen lebensweltlichen Kontext des Studiums mit Bezug auf das Umweltmanagement in der eigenen Institution in die wissenschaftlich fundierte fachliche Ausbildung zu integrieren.

In the frame of higher education, the Teaching-Research-Practice Nexus (TRPN) considers an equal linking of the subjects to achieve sustainability in applied teaching through a holistic framework, which generally refers to the “research-teaching-practice triangle” according to Kaplan (Account Horiz: 129–132, 1989). The methodology underlines the challenges associated with a Nexus approach: the topics to be linked, their linking mechanisms and the respective communication mechanisms. The present investigation results are based on a data collection through an international questionnaire at institutions of Higher Education. Although there are already courses on sustainability theories at several universities, there is still room for development in terms of the transfer of experiences from projects on sustainable development outside the academic sector gained over the last few years. Governmental agencies, NGO’s and the private sector started already implementing practical solutions for sustainable urban planning and development, whose results and experiences should be multiplied in academic teaching. This concerns, for instance, subjects like the ecological footprint, integrated resource management, circular economy and ecosystem services. This contribution discusses the options for the implementation of the TRPN in an international context, and opportunities for universities to become more sustainable by using a balanced proportion of teaching, research, and practice.

A series of novel multivalent drug linkers (MDLs) containing cytotoxic agents were synthesized and conjugated to antibodies to yield highly potent antibody-drug conjugates (ADCs) with drug/antibody ratios (DARs) higher than those typically reported in the literature (10 vs. ≈4). These MDLs contain two copies of a cytotoxic agent attached to biocompatible scaffolds composed of a branched peptide core and discrete polyethylene glycol (PEG) chains to enhance solubility and decrease aggregation. These drug linkers produced well-defined ADCs, whose DARs could be accurately determined by LC-MS. Using this approach, ADCs with significantly lower aggregation and higher DAR than those of conventional drug linker design were obtained with highly hydrophobic cytotoxic agents such as monomethyldolastatin 10 (MMAD). The in vitro potencies of the MDL-derived conjugates matched that of ADCs of similar DAR with conventional linkers, and the potency increased proportionally with drug loading. This approach may provide a means to prepare highly potent ADCs from a broader range of drugs, including those with lower cytotoxicity or poor solubility, which otherwise limits their use for antibody-drug conjugates. This may also provide a means to further improve the potency achievable with cytotoxins currently used in ADCs.

Syntheses and characterization of aminooxy terminated polymers of N-(2-hydroxyproyl) methacrylamide (HPMA) of controlled molecular weight and narrow molecular weight distribution are presented here. Design of a chain transfer agent (CTA) containing N-tert-butoxycarbonyl (t-Boc) protected aminooxy group enabled us to use reversible addition-fragmentation (RAFT) polymerization technique to polymerize the HPMA monomer. An amide bond was utilized to link the aminooxy group and the CTA through a triethylene glycol spacer. As a result, the aminooxy group is linked to the poly(HPMA) backbone through a hydrolytically stable amide bond. By varying the monomer to initiator ratios, polymers with targeted molecular weights were obtained. The molecular weights of the polymers were determined by gel permeation chromatography (GPC) and mass spectrometry (ESI and MALDI-TOF). The t-Boc protecting group was quantitatively removed to generate aminooxy terminated poly(HPMA) macromers. These macromers were converted to rhodamine B terminated poly(HPMA) by reacting N-hydroxysuccinimide (NHS) ester of the dye with the terminal aminooxy group to form a stable alkoxyamide bond. Utility of these dye-labeled polymers as molecular probes was evaluated by fluorescence microscopy by studying their intracellular uptake by renal epithelial cells. These aminooxy terminated poly(HPMA) were also tested as biocompatible carriers to prepare chemoselective bioconjugates of proteins using transferrin (Tf) as the protein. Oxidation of the sialic acid side chains of Tf generated aldehyde functionalized protein that was reacted with aminooxy terminated poly(HPMA), which resulted in protein-polymer bioconjugates carrying oxime linkages. These bioconjugates were characterized by gel electrophoresis and MALDI-TOF mass spectrometry.

Objective: Paliperidone palmitate once-monthly injectable (PP1M) is approved in Japan and other countries for the treatment of schizophrenia. During the 6 month Japanese early postmarketing phase vigilance (EPPV) period, 32 deaths were reported. This report reviews potential contributing factors to the fatal outcomes in the PP1M-treated population. Research design and methods: All spontaneously reported adverse events following PP1M use received during EPPV from 19 November 2013 to 18 May 2014 were entered into the global safety database and these events were analyzed. Results: During the EPPV period, 10,962 patients were estimated to have been treated with PP1M in Japan. The mortality reporting rate during this EPPV period was higher than that observed in the US or globally after PP1M launch (5.84, 0.43, and 0.38 per 1000 patient-years, respectively), but was consistent with the mortality incidence rates (10.2 per 1000 person-years) observed during interventional clinical studies in Japan and in observational patient cohorts. Of the 32 deaths reported during the Japanese PP1M EPPV period, 19/32 (59.4%) were in patients over 50 years of age, 23/32 (71.9%) reported cardiovascular risk factors and 25/32 (78.1%) received antipsychotic polypharmacy. Conclusions: Based on this review of the 32 fatal cases in the PP1M EPPV period, the observed death rate does not necessarily result from a risk with PP1M treatment in Japanese patients. The higher mortality reporting rates in Japan may be attributed to a variety of factors: the effectiveness of mortality reporting in the unique Japanese EPPV program, the advanced age of the fatal cases, high cardiovascular risk factors, multiple underlying diseases and high antipsychotic polypharmacy among the cases with fatal outcomes.

The prevention of adventitious agent contamination is a top priority throughout the entire biopharmaceutical production process. For example, although viral contamination of cell banks or cell cultures is rare, it can result in serious consequences (e.g., shutdown and decontamination of manufacturing facilities). To ensure virus free production, numerous in vivo and in vitro adventitious agent assays and biophysical characterizations such as electron microscopy are conducted on cell banks, raw materials, process materials, and drug substances throughout the manufacturing process. Molecular assays such as PCR and other nucleotide-based techniques are also routinely used for screening and identification of any viral agents. However, modern techniques in protein identification of complex protein mixtures have not yet been effectively integrated throughout the industry into current viral testing strategies. Here, we report the identification and quantitation of Vesivirus 2117 particles in bioreactor fluid from infected Chinese hamster ovary cell cultures by global protein sequencing using mass spectrometry in combination with multi-dimensional liquid-chromatography. Following mass spectrometric data acquisition and rigorous data analysis, six virus specific peptides were identified. These peptides were fragments of two structural proteins, capsid protein pre-cursor (four unique peptides) and small structural protein (two unique peptides), from the same species: Vesivirus 2117. Using stable heavy isotope-labeled peptides as internal standards, we also determined the absolute concentration of Vesivirus particles in the bioreactor fluid and the ratio of two capsid proteins (VP1:VP2) in the particles as approximately 9:1. The positive identification of Vesivirus 2117 was subsequently confirmed by RT-PCR.

Targeting tubulin binders to cancer cells using antibody–drug conjugates (ADCs) has great potential to become an effective cancer treatment with low normal tissue toxicity. The nature of the linker used to tether the tubulin binder to the antibody and the conjugation sites on the antibody and the small molecule are important factors in the ADC stability and effectiveness. We explored the use of tubulin-targeting dolastatin 15 derivatives (Dol15) tethered covalently to a representative antibody, trastuzumab, via cleavable and non-cleavable linkers at varying antibody reactive sites (i.e., lysine residues, partially reduced hinge region disulfide bonds) and drug coupling sites (i.e., C-terminus, N-terminus), to investigate which constructs were more effective in killing HER2-positive cells in vitro and in vivo. We found that Dol15 conjugated to trastuzumab via lysine residues at the drug C-terminus using a non-cleavable linker (trastuzumab–amide–C-term–Dol15) produced target-dependent growth inhibition of cells endogenously expressing high HER2 levels (i.e., SK-BR-3, SK-OV-3) in vitro. This ADC was effective at varying doses (i.e., 10 and 20 mg/kg) in the SK-OV-3 human ovarian cancer xenograft. Tethering Dol15 via partially reduced disulfide bonds at the drug C-terminus via a non-cleavable linker (trastuzumab–MC–C-term–Dol15) resulted in an equally effective ADC in vitro, showing that site of antibody conjugation did not influence ADC activity. However, tethering Dol15 at the drug N-terminus using non-cleavable and cleavable linkers (trastuzumab–MC–N-term–Dol15 and trastuzumab–MC–VC–PABC–N-term–Dol15, respectively) resulted in ineffective ADCs. Thus, Dol15 tethered at the C-terminus may be a useful tubulin-targeting agent for conjugation at various antibody reactive sites.

Antibody-drug conjugates (ADCs) have been proven clinically to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prepare ADCs by targeting primary amines or hinge disulfides have a number of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymatically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidation of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatography and LC-MS analyses revealed the incorporation of ~1.6 cytotoxic agents per antibody molecule, approximating the number of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-positive tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-positive tumor xenograft model. These findings suggest that enzymatic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chemical process to ensure reproducible drug loading.

Currently, the principal chemistries for the preparation of antibody-drug conjugates (ADC) target either lysines or cysteines for coupling cytotoxic drugs for delivery to target cells expressing tumor-specific antigens. All of these chemistries generate populations of molecules which differ in critical properties which are known to affect efficacy, pharmacokinetics, and the therapeutic window. Of key interest are methods to minimize this heterogeneity to achieve reproducible product profiles and efficacy. A current trend in the development of ADC is the evaluation of suitable targets, antibodies, and payloads, occurring well before process development to produce conjugates of clinical quality. This creates a need for an ability to generate comparably high-quality products early in development and at sufficient scale for evaluating in vitro potency and in vivo efficacy, as well as the early identification of any deficiencies in critical quality attributes including solubility and stability. Here we elaborate detailed protocols using maleimide-based chemistry for the conjugation to reduce hinge disulfides in antibodies by several cytotoxic drugs. We present a method for the initial characterization of the reduction/alkylation reaction using polyethylene-glycol (PEG) as a drug surrogate, a 5 mg scale drug conjugation to provide material for initial characterization including cell proliferation assays and a 150 mg scale process for performing efficacy studies in small animals. These methods yield well-defined predictable product profiles at high yield and with low impurities. These procedures include details relevant to the execution of these methods in a safe and contained manner within a typical laboratory environment.

Translocation of gastrointestinal bacteria in HIV-infected individuals is associated with systemic inflammation, HIV progression, mortality, and comorbidities. HIV-infected individuals are also susceptible to fungal infection and colonization, but whether fungal translocation occurs and influences HIV progression or comorbidities is unknown.Serum (1→3)-β-D-glucan (BG) was measured by a Limulus Amebocyte Lysate assay (Fungitell) in 132 HIV-infected outpatients. Selected plasma cytokines and markers of peripheral T-cell activation were measured. Pulmonary function testing and Doppler echocardiography were performed. Relationship of high (≥40 pg/mL) and low (40 pg/mL) levels of BG with HIV-associated variables, inflammation markers, and pulmonary function and pulmonary hypertension measures were determined.Forty-eight percent of patients had detectable BG, and 16.7% had high levels. Individuals with high BG were more likely to have CD4 counts less than 200 cells/μL (31.8% vs. 8.4%, P = 0.002), had higher log10 HIV viral levels (2.85 vs. 2.13 log copies/mL, P = 0.004), and were less likely to use antiretroviral therapy (68.2% vs. 90.0%, P = 0.006). Plasma IL-8 (P = 0.033), TNF-α (P = 0.029), and CD8CD38 (P = 0.046) and CD8HLA-DR (P = 0.029) were also increased with high levels. Abnormalities in diffusing capacity (P = 0.041) and in pulmonary artery pressures (P = 0.006 for pulmonary artery systolic pressure and 0.013 for tricuspid regurgitant velocity) were more common in those with high BG.We found evidence of peripheral fungal cell wall polysaccharides in an HIV-infected cohort. We also demonstrated an association between high serum BG, HIV-associated immunosuppression, inflammation, and cardiopulmonary comorbidity. These results implicate a new class of pathogen in HIV-associated microbial translocation and suggest a role in HIV progression and comorbidities.

Background Despite the high prevalence of respiratory symptoms and obstructive lung disease in HIV-infected subjects, the prevalence of bronchodilator reversibility (BDR) and asthma has not been systematically studied during the era of combination antiretroviral therapy (ART). Objective We sought to determine the prevalence of asthma diagnosis and related pulmonary function abnormalities in an HIV-infected cohort and to identify potential mechanisms. Methods We performed a cross-sectional analysis of 223 HIV-infected subjects with data on respiratory symptoms and diagnoses, pulmonary function, sputum cell counts, and asthma-related cytokines and chemokines in serum/sputum. Results Doctor-diagnosed asthma was present in 46 (20.6%), and BDR (≥200 mL and ≥12% increase in FEV 1 or forced vital capacity) was present in 20 (9.0%) participants. Pulmonary symptoms and function were worse in those with doctor-diagnosed asthma. Doctor-diagnosed asthma was independently associated with female sex ( P = .04), body mass index of greater than 29.6 kg/m 2 (vs 2 , P = .03), history of bacterial or Pneumocystis pneumonia ( P = .01), and not currently taking ART ( P = .04) and in univariate analysis with parental history of asthma (n = 180, P = .004). High sputum eosinophil percentages (>2.3% based on the highest decile) were more likely in those with doctor-diagnosed asthma ( P = .02) or BDR ( P = .02). Doctor-diagnosed asthma tended to be more common with high sputum IL-4 ( P = .02) and RANTES ( P = .02) levels, whereas BDR was associated with high plasma macrophage inflammatory protein 1α ( P = .002) and sputum macrophage inflammatory protein 1β ( P = .001) levels. Conclusion Asthma diagnosis and BDR are prevalent in an HIV-infected outpatient cohort, and associations with family history, obesity, allergic inflammation, prior infection, absence of ART, and increased HIV-stimulated cytokines suggest possible mechanisms of HIV-associated asthma.