Abstract Urban riverscapes are integral to the livability and sustainability of cities and suburbs. Conserving and restoring these riverscapes requires assessing their condition over a broad range of spatial scales, from watershed to planning segment to reach scale. Any riverscape assessment must examine the hydrogeomorphic and ecological characteristics and behaviors in order to provide decision-making information to resource managers. The novel assessment method introduced in this article, Urban Riverscape conditions-Based Assessment for management Needs (URBAN), provides a framework to characterize the physical condition of and the social-ecological values within a watershed, as well as the anthropogenic impacts affecting those functions and values, that can inform restoration planning and riverscape management. URBAN is founded upon the third tier of the Urban Riverscapes Assessment Framework – “How should we assess the values and functions of an urban riverscape?” Assessment of physical conditions and characteristics in urban riverscapes using the URBAN indicators and metrics provides critical information about the underlying watershed system status and stream evolution trajectory. URBAN includes both data collection and the evaluation of desktop and field data to determine stream characteristics. Data collection emphasizes reach-typing (or stream classification) and related or relevant aspects of stream physical condition. URBAN is unique in its emphasis upon stream “values,” which links the physical processes of a stream and its social-ecological values to anthropogenic stressors that may influence the long-term recovery of degraded streams. These linkages also provide critical information for planning instruments, regulatory requirements, and community conversations. Coupled with GIS tools, urban riverscape studies at multiple scales, like URBAN, provide objective methods to understand and interpret multi-causal mechanisms and trends that influence the physical condition and potential recovery of degraded urban riverscapes. This article provides a summary of those methods and the tools applied by way of remote and rapid field assessments, through the lens of an urban watershed assessment conducted in the Colorado Front Range. The outcome of this work advances the scientific basis underlying applied riverscape management and broad-scale preservation and renovation planning.

Restoring the health of urban streams has many of the characteristics of a wicked problem. Addressing a wicked problem requires managers, academics, practitioners, and community members to make negotiated tradeoffs and compromises to satisfy the values and perspectives of diverse stakeholders involved in setting restoration project goals and objectives. We conducted a gap analysis on 11 urban stream restoration projects to identify disconnections, underperformance issues, and missing processes in the project structures used to develop restoration project goals and objectives. We examined the gap analysis results to investigate whether managers appropriately identified problem statements and met stated objectives. Projects that aimed to restore overall stream health commonly fell short for various reasons, including limited stakeholder and community input and buy-in, revealing potential limitations in the breadth of objectives, values, and stakeholder perspectives and knowledge types. Projects that emphasized integrating community values and diverse knowledge types tended to meet the expected outcomes of restoring stream processes through incremental solutions. Managers implementing more holistic solutions and values-driven approaches are more likely to consider diverse viewpoints from a variety of community local institutions. Based on these and other results, we propose a conceptual framework that integrates diverse perspectives and knowledge to enhance social and ecological outcomes of urban stream restoration. The framework also emphasizes the importance of setting objectives that support incremental solutions to foster more realistic expectations amongst stakeholders.

The services that rivers provide and how they affect the landscape plays a dominate role in urban planning and development. Urban riverscapes, which consist of stream channels, their floodplains, biotic communities, and manmade features, are complex social-ecological and hydrogeomorphic systems. Yet, despite recognition of their place and value, rivers are often degraded in urban settings. Successfully managing urban riverscapes requires improved methods to assess them and to more effectively link stressors to values, and to incorporate these considerations in planning. Assessment of urban riverscapes' physical condition and function-a hydrogeomorphic assessment-is necessary to make these links, and inform more appropriate management strategies for sustainable and valued riverscape systems. The framework and methods used for such an assessment should be appropriate to the urban context, insofar as they are applicable to a range of streams from lightly degraded to highly utilized or constructed. Above all, the framework must prioritize the connection of human communities to riverscapes. In this article, we outline a framework for urban riverscape assessment which considers four facets of urban riverscapes: human values, hydrology, geomorphology, and ecology. The four facets, assessed across multiple nested scales, provide a flexible basis for context-driven hydrogeomorphic assessment, which is vital to informing better planning and management of urban riverscapes. The framework can be integrated with other facets (e.g. geochemical, aquatic ecology) depending on the scope of the assessment. By linking intrinsic, relational, and use-based values to physical conditions, watershed managers can select relevant and measurable indicators that directly inform interventions in the riverscape, catchment, or urban zones to improve riverscape function and urban vitality through planning mechanisms. This assessment framework facilitates dialogue between managers, practitioners, scientists, and the community; enabling technical and non-technical inputs to the development of assessment criteria, and a shared vision to inform targets and goals.

To evaluate the different degrees of residual structure in the unfolded state of interferon-τ using chemical denaturation as a function of temperature by both urea and guanidinium hydrochloride.

Deamidation of asparagine (Asn) residues is one of the most common chemical degradation pathways observed in proteins. This reaction must be understood and controlled in therapeutic drug candidates, as chemical changes can affect their efficacy and safety. The analytical tools available for detection of deamidation reaction products, such as isoaspartic acid residues, are either chromatographic or electrophoretic, and require MS detection for absolute identification of peaks. High-throughput measurement of protein degradation has typically been limited to probing the target's physical state using spectroscopic techniques. Here, we describe a high throughput assay for isoaspartate residues using fluorescent detection in a microtiter plate format. The method allows for fast detection of protein deamidation in a cost-efficient manner. The method can be employed even if the target peptide or protein contains free Cys residues. The technique appears to be selective, linear, and accurate.

The peptide teriparatide, also known as parathyroid hormone (1-34), PTH(1-34), was developed for intranasal delivery, requiring extended stability of the reconstituted product for up to four weeks at room temperature. Lyophilized formulations of PTH(1-34), containing glycine and trehalose and using lactate as the buffer, are stable for months upon storage. However, the physical stability of the peptide after reconstitution unexpectedly varied considerably, depending on peptide concentration and storage temperature, with precipitation seen within two to four weeks in some samples. By comparison, equivalent samples that did not undergo lyophilization did not display any precipitation upon storage in the liquid state for as long as twelve weeks. PTH(1-34) appears to adopt a higher order structure that is perturbed by the combined stresses of freezing and drying, leading to greater propensity to aggregate, which is accentuated at higher peptide concentrations and at higher temperatures. The precipitation seems to be correlated with increased amounts of subvisible particles. This study shows the importance of peptide conformation in long-term stability and illustrates the ability of lyophilization to cause increased propensity to aggregate, even in a peptide.

Buffers comprise an integral component of protein formulations. Not only do they function to regulate shifts in pH, they also can stabilize proteins by a variety of mechanisms. The ability of buffers to stabilize therapeutic proteins whether in liquid formulations, frozen solutions, or the solid state is highlighted in this review. Addition of buffers can result in increased conformational stability of proteins, whether by ligand binding or by an excluded solute mechanism. In addition, they can alter the colloidal stability of proteins and modulate interfacial damage. Buffers can also lead to destabilization of proteins, and the stability of buffers themselves is presented. Furthermore, the potential safety and toxicity issues of buffers are discussed, with a special emphasis on the influence of buffers on the perceived pain upon injection. Finally, the interaction of buffers with other excipients is examined.

Comparing higher order structure (HOS) in therapeutic proteins is a significant challenge. Previously, we showed that changes in solution conditions produced detectable changes in the second-derivative amide I Fourier transform infrared (FTIR) spectra for a variety of model proteins. Those comparisons utilized vector-based approaches, such as spectral overlap and spectral correlation coefficients to quantify differences between spectra. In this study, chemometric analyses of the same data were performed, to classify samples into different groups based on the solution conditions received. The solution conditions were composed of various combinations of temperature, pH, and salt types. At first, principal component analysis (PCA) was used to visually demonstrate that FTIR spectra respond to changes in solution conditions, which, presumably indicates variations in HOS. This observed when samples from the same solution condition form clusters within a PCA score plot. The second approach, called soft independent modeling of class analogy (SIMCA), was conducted to account for the within-class experimental error for the lysozyme spectra. The DModX values, indicative of the distance of each spectra to their respective class models, was found to be a more sensitive quantitative indicator of changes in HOS, when compared with the modified area of overlap algorithm. The SIMCA approach provides a metric to determine whether new observations do, or do not belong to a particular class or group. Thus, SIMCA is the recommended approach when multiple samples from each condition are available.

Demonstrating comparability of secondary structure composition as part of higher order structure (HOS) in therapeutic proteins is a significant challenge. Previously, we showed that the variability of second derivative amide I Fourier transform infrared (FTIR) spectra were small enough that significant differences in secondary structures could be seen for a variety of model proteins. Those comparisons used spectral overlap and spectral correlation coefficients to quantify spectral differences. However, many of the excipients used in downstream purification process, drug substance, and drug product formulation, such as free amino acids and sugars, can interfere with the absorbance in the amide I region. In this study, analysis of amide II FTIR spectra is shown as an alternative to using spectral data from the amide I region to analyze protein secondary structure to assess their HOS. This research provided spectral overlap and spectral correlation coefficient mathematical approaches for analysis of amide II FTIR spectra to demonstrate comparability of protein secondary structure. Spectral overlap and spectral correlation coefficients results show strong correlations between changes in the second derivative of amide II and amide I FTIR spectra for various model proteins under different conditions, which demonstrate the applicability of using amide II FTIR spectra for the comparability of protein secondary structure. These results indicate that the analysis of the second derivative of amide II FTIR spectra may be used to monitor and demonstrate comparability of protein secondary structure during downstream process and formulation development of protein therapeutics.

Better understanding of protein higher order structures (HOS) is of major interest to researchers in the field of biotechnology and biopharmaceutics. Monitoring a protein's HOS is crucial towards understanding the impact of molecular conformation on the biotechnological application. In addition, maintaining the HOS is critical for achieving robust processes and developing stable formulations of therapeutic proteins. Loss of HOS contributes to increased aggregation, enhanced immunogenicity and loss of function. Selecting the proper biophysical methods to monitor the secondary and tertiary structures of therapeutic proteins remains the central question in this field. In this study, both Fourier Transform Infrared (FTIR) and vibrational circular dichroism (VCD) spectroscopy are employed to characterize the secondary structures of various proteins as a function of temperature and pH. Three proteins with different secondary structures were examined, human serum albumin (HSA), myoglobin, and the monoclonal antibody, ofatumumab. This work demonstrates that VCD is useful technique for monitoring subtle secondary structure changes of protein therapeutics that may occur during processing or handling.

Eight lyophilized formulations of a IgG1 monoclonal antibody (MAb) were prepared containing increasing levels of sucrose. In addition, three of the formulations had sorbitol added at a level of 5% w/w relative to sucrose. The samples were stored for up to 4 weeks at 40°C, which is well below the Tg. Upon reconstitution, the levels of subvisible particles were measured using microflow imaging (MFI). The formulation containing no sucrose contained exceedingly high levels of subvisible particles, accounting for as much as 25% of the weight of the protein. Addition of sucrose markedly decreased the number of subvisible particles, with the maximal sucrose:protein weight ratio being 2:1 (the highest level tested). Addition of sorbitol further decreased subvisible particle levels, even for formulations where the sucrose:protein ratio was relatively high. This suggests that even small amounts of a plasticizer like sorbitol can improve the storage stability of a lyophilized antibody formulation, probably by dampening β-relaxations within the amorphous glass.