Experimental evidence for recovery of mercury-contaminated fish populations.

Anthropogenic releases of mercury (Hg)1-3 are a human health issue4 because the potent toxicant methylmercury (MeHg), formed primarily by microbial methylation of inorganic Hg in aquatic ecosystems, bioaccumulates to high concentrations in fish consumed by humans5,6. Predicting the efficacy of Hg pollution controls on fish MeHg concentrations is complex because many factors influence the production and bioaccumulation of MeHg7-9. Here we conducted a 15-year whole-ecosystem, single-factor experiment to determine the magnitude and timing of reductions in fish MeHg concentrations following reductions in Hg additions to a boreal lake and its watershed. During the seven-year addition phase, we applied enriched Hg isotopes to increase local Hg wet deposition rates fivefold. The Hg isotopes became increasingly incorporated into the food web as MeHg, predominantly from additions to the lake because most of those in the watershed remained there. Thereafter, isotopic additions were stopped, resulting in an approximately 100% reduction in Hg loading to the lake. The concentration of labelled MeHg quickly decreased by up to 91% in lower trophic level organisms, initiating rapid decreases of 38-76% of MeHg concentration in large-bodied fish populations in eight years. Although Hg loading from watersheds may not decline in step with lowering deposition rates, this experiment clearly demonstrates that any reduction in Hg loadings to lakes, whether from direct deposition or runoff, will have immediate benefits to fish consumers.

A 2D LC-MS/MS Strategy for Reliable Detection of 10-ppm Level Residual Host Cell Proteins in Therapeutic Antibodies.

Methodologies employing LC-MS/MS have been increasingly used for characterization and identification of residual host cell proteins (HCPs) in biopharmaceutical products to ensure their consistent product quality and safety for patients. To improve the sensitivity and reliability for HCP detection, we developed a high pH-low pH two-dimensional reversed phase LC-MS/MS approach in conjunction with offline fraction concatenation. Proof-of -concept was established using a model in which seven proteins spanning a size range of 29-78 kDa are spiked into a purified antibody product to simulate the presence of low-level HCPs. By incorporating a tandem column configuration and a shallow gradient through the second-dimension, all seven proteins were consistently identified at 10 ppm with 100% success rate following LC-MS/MS analysis of six concatenated fractions across multiple analysts, column lots and injection loads. Using the more complex Universal Proteomic Standard 1 (UPS-1) as an HCP model, positive identification was consistently achieved for 19 of the 22 proteins in 8-12 ppm range (10 ppm ±20%). For the first time, we demonstrate an effective LC-MS/MS strategy that not only has high sensitivity but also high reliability for HCP detection. The method performance has high impact on pharmaceutical company practices in using advanced LC-MS/MS technology to ensure product quality and patient safety.

A sensitive mutation screening method supporting cell line development for biotherapeutics.

Random genetic mutations, which can occur during cell line development, can lead to sequence variants that comprise pharmaceutical product quality generated by recombinant technology. Mutation screening can minimize the probability of selecting clones harboring sequence variants. Here we report a polymerase chain reaction (PCR)-based mutation screening approach using high-resolution melting (HRM) analysis combined with a mutation enrichment step using limiting dilution to detect low-level mutations at 0.5%. The method allows unknown mutation discovery regardless of its location in a transgene as well as independent of its position in an HRM fragment, ranging from approximately 200 to 300 bp in size.

Determination of critical quality attributes for monoclonal antibodies using quality by design principles.

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes the identification of critical quality attributes (CQAs) as an important first step for QbD development of biopharmaceuticals. A systematic scientific based risk ranking and filtering approach allows a thorough understanding of quality attributes and an assignment of criticality for their impact on drug safety and efficacy. To illustrate the application of the approach and tools, a few examples from monoclonal antibodies are shown. The identification of CQAs is a continuous process and will further drive the structure and function characterization of therapeutic proteins.

Establishing a control system using QbD principles.

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes the elements and tools used to establish acceptance criteria and an attribute testing strategy (ATS) for product variants and process related impurities. The acceptable ranges for CQAs are set based on their potential impact on efficacy and safety/immunogenicity. This approach is focused on the management of patient impacts, rather than simply maintaining a consistent analytical profile. The ATS tools were designed to identify quality attributes that required process and/or testing controls, or that could be captured in a monitoring system to enable lifecycle management of the control strategy.

Engineering upper hinge improves stability and effector function of a human IgG1.

Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2-3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.

Remobilization of legacy arsenic from sediment in a large subarctic waterbody impacted by gold mining.

Arsenic contamination from mining poses an environmental challenge due to the mobility of this redox-sensitive element. This study evaluated arsenic mobility in sediments of Yellowknife Bay (Canada), a large subarctic water body impacted by gold mining during the 20th century. Short-term measurements of arsenic flux from sediment, arsenic profiling of the water column and sediment porewater, and mass balance modelling were conducted to assess the importance of sediment as an arsenic source. Sediment arsenic fluxes were highly variable throughout Yellowknife Bay and ranged from - 65-1520 µg m-2 day-1. Elevated fluxes measured near the mine site were among the highest published for well-oxygenated lakes. Redox boundaries were typically 2-3 cm below the sediment surface as indicated by porewater profiles of iron, manganese, and arsenic, with arsenic maxima of 65-3220 µg L-1 predominately as arsenite. Sediment arsenic flux was positively related to its solid-phase concentration. Modelling indicated sediment was a principal source of arsenic to the water column. Adsorption and precipitation processes in the oxidizing environment of near-surface sediments did not effectively attenuate arsenic remobilized from contaminated sediments. Internal recycling of legacy arsenic between sediment and surface water will impede a return to background conditions in Yellowknife Bay for decades.

Biogeochemical and hydrologic synergy control mercury fate in an arid land river-reservoir system.

Reservoirs in arid landscapes provide critical water storage and hydroelectric power but influence the transport and biogeochemical cycling of mercury (Hg). Improved management of reservoirs to mitigate the supply and uptake of bioavailable methylmercury (MeHg) in aquatic food webs will benefit from a mechanistic understanding of inorganic divalent Hg (Hg(II)) and MeHg fate within and downstream of reservoirs. Here, we quantified Hg(II), MeHg, and other pertinent biogeochemical constituents in water (filtered and associated with particles) at high temporal resolution from 2016-2020. This was done (1) at inflow and outflow locations of three successive hydroelectric reservoirs (Snake River, Idaho, Oregon) and (2) vertically and longitudinally within the first reservoir (Brownlee Reservoir). Under spring high flow, upstream inputs of particulate Hg (Hg(II) and MeHg) and filter-passing Hg(II) to Brownlee Reservoir were governed by total suspended solids and dissolved organic matter, respectively. Under redox stratified conditions in summer, net MeHg formation in the meta- and hypolimnion of Brownlee reservoir yielded elevated filter-passing and particulate MeHg concentrations, the latter exceeding 500 ng g-1 on particles. Simultaneously, the organic matter content of particulates increased longitudinally in the reservoir (from 9-29%) and temporally with stratified duration. In late summer and fall, destratification mobilized MeHg from the upgradient metalimnion and the downgradient hypolimnion of Brownlee Reservoir, respectively, resulting in downstream export of elevated filter-passing MeHg and organic-rich particles enriched in MeHg (up to 43% MeHg). We document coupled biogeochemical and hydrologic processes that yield in-reservoir MeHg accumulation and MeHg export in water and particles, which impacts MeHg uptake in aquatic food webs within and downstream of reservoirs.

Mass spectrometry-based multi-attribute method in protein therapeutics product quality monitoring and quality control.

The multi-attribute method (MAM), a liquid chromatography-mass spectrometry (LC-MS)-based peptide mapping method, has gained increased interest and applications in the biopharmaceutical industry. MAM can, in one method, provide targeted quantitation of multiple site-specific product quality attributes, as well as new peak detection. In this review, we focus on the scientific and regulatory considerations of using MAM in product quality attribute monitoring and quality control (QC) of therapeutic proteins. We highlight MAM implementation challenges and solutions with several case studies, and provide our perspective on the opportunities to use MS in QC for applications other than standard peptide mapping-based MAM.

A screening model analysis of mercury sources, fate and bioaccumulation in the Gulf of Mexico.

A mass balance model of mercury (Hg) cycling and bioaccumulation was applied to the Gulf of Mexico (Gulf), coupled with outputs from hydrodynamic and atmospheric Hg deposition models. The dominant overall source of Hg to the Gulf is the Atlantic Ocean. Gulf waters do not mix fully however, resulting in predicted spatial differences in the relative importance of external Hg sources to Hg levels in water, sediments and biota. Direct atmospheric Hg deposition, riverine inputs, and Atlantic inputs were each predicted to be the most important source of Hg to at least one of the modeled regions in the Gulf. While incomplete, mixing of Gulf waters is predicted to be sufficient that fish Hg levels in any given location are affected by Hg entering other regions of the Gulf. This suggests that a Gulf-wide approach is warranted to reduce Hg loading and elevated Hg concentrations currently observed in some fish species. Basic data to characterize Hg concentrations and cycling in the Gulf are lacking but needed to adequately understand the relationship between Hg sources and fish Hg concentrations.

Mercury in the Gulf of Mexico: sources to receptors.

Gulf of Mexico (Gulf) fisheries account for 41% of the U.S. marine recreational fish catch and 16% of the nation's marine commercial fish landings. Mercury (Hg) concentrations are elevated in some fish species in the Gulf, including king mackerel, sharks, and tilefish. All five Gulf states have fish consumption advisories based on Hg. Per-capita fish consumption in the Gulf region is elevated compared to the U.S. national average, and recreational fishers in the region have a potential for greater MeHg exposure due to higher levels of fish consumption. Atmospheric wet Hg deposition is estimated to be higher in the Gulf region compared to most other areas in the U.S., but the largest source of Hg to the Gulf as a whole is the Atlantic Ocean (>90%) via large flows associated with the Loop Current. Redistribution of atmospheric, Atlantic and terrestrial Hg inputs to the Gulf occurs via large scale water circulation patterns, and further work is needed to refine estimates of the relative importance of these Hg sources in terms of contributing to fish Hg levels in different regions of the Gulf. Measurements are needed to better quantify external loads, in-situ concentrations, and fluxes of total Hg and methylmercury in the water column, sediments, and food web.

Challenges and Strategies for a Thorough Characterization of Antibody Acidic Charge Variants.

Heterogeneity of therapeutic Monoclonal antibody (mAb) drugs are due to protein variants generated during the manufacturing process. These protein variants can be critical quality attributes (CQAs) depending on their potential impact on drug safety and/or efficacy. To identify CQAs and ensure the drug product qualities, a thorough characterization is required but challenging due to the complex structure of biotherapeutics. Past characterization studies for basic and acidic variants revealed that full characterizations were limited to the basic charge variants, while the quantitative measurements of acidic variants left gaps. Consequently, the characterization and quantitation of acidic variants are more challenging. A case study of a therapeutic mAb1 accounted for two-thirds of the enriched acidic variants in the initial characterization study. This led to additional investigations, closing the quantification gaps of mAb1 acidic variants. This work demonstrates that a well-designed study with the right choices of analytical methods can play a key role in characterization studies. Thus, the updated strategies for more complete antibody charge variant characterization are recommended.

Characterization of oligosaccharides in recombinant tissue plasminogen activator produced in Chinese hamster ovary cells: two decades of analytical technology development.

Recombinant tissue plasminogen activator (rt-PA) is a well-characterized glycoprotein with a great deal of published information on its structure, post-translational modifications, and O- and N-glycosylation. Most of the characterization was accomplished in the late 1980s. During the past 2 decades, however, mass spectrometry has made a quantum leap forward offering new capabilities in soft electrospray ionization, speed, resolution, and accuracy of mass measurements. From this point of view, it is worthwhile to revisit the characterization of familiar proteins, such as rt-PA, using the new capabilities of modern analytical technology. In this work, we applied LC-MS with state-of-the-art instrumentation to the characterization of glycoforms of rt-PA. This method takes advantage of accurate mass measurements along with a fast "in-source" voltage switching for the detection of characteristic oxonium ions of saccharides. This method confirmed previously identified glycan structures based on existing knowledge of rt-PA glycans. In addition, we identified two novel glycan structures in rt-PA. A low level of Asn142 N-glycosylation was detected at an atypical Asn-Xaa-Cys consensus motif. It was found to be modified predominantly by biantennary hybrid structures. This N-glycosylation site was confirmed using a recently developed electron-transfer dissociation (ETD) technique. Also using this method, we detected low levels of elongation of fucose-O-Thr61 to di-, tri-, and tetrasaccharides, not previously observed in rt-PA. The results demonstrate that use of state-of-the-art analytical methods can reveal low-level, previously undetected modifications of well-characterized biopharmaceuticals.

Recovery of mercury-contaminated fisheries.

In this paper, we synthesize available information on the links between changes in ecosystem loading of inorganic mercury (Hg) and levels of methylmercury (MeHg) in fish. Although it is widely hypothesized that increased Hg load to aquatic ecosystems leads to increases in MeHg in fish, there is limited quantitative data to test this hypothesis. Here we examine the available evidence from a range of sources: studies of ecosystems contaminated by industrial discharges, observations of fish MeHg responses to changes in atmospheric load, studies over space and environmental gradients, and experimental manipulations. A summary of the current understanding of the main processes involved in the transport and transformation from Hg load to MeHg in fish is provided. The role of Hg loading is discussed in context with other factors affecting Hg cycling and bioaccumulation in relation to timing and magnitude of response in fish MeHg. The main conclusion drawn is that changes in Hg loading (increase or decrease) will yield a response in fish MeHg but that the timing and magnitude of the response will vary depending of ecosystem-specific variables and the form of the Hg loaded.

Percutaneous coronary interventions in a rural hospital without surgical backup: report of one year of experience.

This paper demonstrates that percutaneous coronary intervention (PCI) can be provided in a rural setting with results that are at least as good as the national average without untoward risk to the patient. Percutaneous coronary intervention is the initial treatment of choice for acute myocardial infarction (AMI). Historically, PCI has been available in metropolitan areas, yet 20% of the population lives in rural areas. Rural patients with AMI may not be receiving optimal care, especially if PCI is not readily available. In a rural setting, door-to-balloon time for patients with acute ST-elevation MI was 67.66 +/- 30.80 min. This is significantly better than the 186 min reported in the National Registry Myocardial Infarction (NRMI). These results were achieved with a complication rate that was not significantly different from national averages. This study demonstrates that PCI can be provided safely and with good results in a rural setting.

Evaluation of Heavy-Chain C-Terminal Deletion on Product Quality and Pharmacokinetics of Monoclonal Antibodies.

Due to their potential influence on stability, pharmacokinetics, and product consistency, antibody charge variants have attracted considerable attention in the biotechnology industry. Subtle to significant differences in the level of charge variants and new charge variants under various cell culture conditions are often observed during routine manufacturing or process changes and pose a challenge when demonstrating product comparability. To explore potential solutions to control charge heterogeneity, monoclonal antibodies (mAbs) with native, wild-type C-termini, and mutants with C-terminal deletions of either lysine or lysine and glycine were constructed, expressed, purified, and characterized in vitro and in vivo. Analytical and physiological characterization demonstrated that the mAb mutants had greatly reduced levels of basic variants without decreasing antibody biologic activity, structural stability, pharmacokinetics, or subcutaneous bioavailability in rats. This study provides a possible solution to mitigate mAb heterogeneity in C-terminal processing, improve batch-to-batch consistency, and facilitate the comparability study during process changes.

Effect of copper sulfate on performance of a serum-free CHO cell culture process and the level of free thiol in the recombinant antibody expressed.

A recombinant Chinese hamster ovary (CHO) cell line was used to express a humanized antibody. Product quality analysis of this humanized antibody showed the presence of free thiol, due to unpaired cysteine residues in the Fab region. Decreased potency of this thiol Fab made it critical to minimize the levels of free thiol. In an effort to do this, we evaluated the effect of copper sulfate addition to the cell culture production medium. As a component of the production medium, copper sulfate can act as an oxidizing agent, thereby facilitating disulfide bond formation. Four concentrations of copper sulfate were added at the beginning of 2-L benchtop production cultures of the recombinant CHO cell line: 0, 5, 50, and 100 microM. We found that these copper sulfate additions had no effect on cell growth or antibody production. However, a slight dose-dependent depression in culture viability was observed. Analysis of the purified antibody showed that either the 50 or 100 microM copper sulfate additions reduced the level of free thiol by more than 10-fold.

Non-enzymatic hinge region fragmentation of antibodies in solution.

Liquid formulations of monoclonal antibodies (MAbs) typically undergo fragmentation near the papain cleavage site in the hinge region, resulting in Fab and Fab+Fc forms. The purpose of this study was to investigate whether this fragmentation is due to proteases. Four closely-related MAbs were exchanged into a pH 5.2 acetate buffer with NaCl and stored at -20 degrees C, 5 degrees C, 30 degrees C, or 40 degrees C for 1 month. Fragmentation generated size-exclusion chromatography (SEC) peak fractions that were analyzed by electrospray mass spectrometry to identify the cleavage sites. The effects of protein inhibitors or host cell proteins on fragmentation were also studied. The extent of fragmentation was equivalent for all four antibodies, occurring in the heavy chain hinge region Ser-Cys-Asp-Lys-Thr-His-Thr sequence. The fragment due to cleavage of the Asp-Lys bond showed two forms that differ by 18 Da. A synthetic peptide with the hinge region sequence terminating with Asp did not show fragmentation or the loss of 18 Da after incubation. Protease inhibitors did not affect rates of cleavage or modify sites of fragmentation. Degradation was not affected by host cell protein content. Fragmentation appears to be a kinetic process that is not caused by low levels of host cell proteases.

Charge variants in IgG1: Isolation, characterization, in vitro binding properties and pharmacokinetics in rats.

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.