Influence of temporary emigration on wood turtle (Glyptemys insculpta) detectability, with implications for abundance estimation.

Reliable population estimates are important for making informed management decisions about wildlife species. Standardized survey protocols have been developed for monitoring population trends of the wood turtle (Glyptemys insculpta), a semi-aquatic freshwater turtle species of conservation concern throughout its distribution in east-central North America. The protocols use repeated active search surveys of defined areas, allowing for estimation of survey-specific detection probability (p) and site-specific abundance. These protocols assume population closure within the survey area during the survey period, which is unlikely to be met as wood turtles are a highly mobile species. Additionally, current protocols use a single-pass design that does not allow for separation of availability (pa) and detectability (pd). If there are systematic influences on pa or pd that are not accounted for in the survey design or data analysis, then resulting abundance estimates could be biased. The objectives of this study were to determine if pa is a random process and if pa and pd are influenced by demographic characteristics. We modified the wood turtle survey protocol used in the upper Midwest to include a double-pass design, allowing us to estimate pa and pd using a robust design capture-recapture model. The modified protocol was implemented at 14 wood turtle monitoring sites in Minnesota and Wisconsin between 2017 and 2022. Our results indicated that pa was non-random and that pd increased with turtle carapace length. Our study suggests that model assumptions for current wood turtle population models may be violated, likely resulting in an overestimation of abundance. We discuss possible protocol and modeling modifications that could result in more accurate wood turtle abundance estimates.

Sensitive and rapid analysis of plasmid DNA topology isoforms by capillary gel electrophoresis with laser induced fluorescence in uncoated capillary.

The work describes the use of SYBR Gold to improve the detection sensitivity of plasmid DNA topoisomers by capillary gel electrophoresis with laser induced fluorescence in an uncoated capillary. The impact of different dyes, including ethidium bromide, SYBR Green and SYBR Gold, was compared based on detection and separation of DNA plasmid topoisomers. Use of SYBR Gold enabled improvement of detection sensitivity by 15-fold while maintaining good separation resolution of the different topoisomers. The baseline dropped with the use SYBR Gold but was overcome by the employment of a capillary with longer ineffective length (40 vs. 20 cm). Separation resolution and reproducibility were impacted by the concentration of SYBR Gold and hydroxypropyl methylcellulose. With the use of a short capillary (10 cm effective length and 50 cm total length), fast separations of supercoiled, linear, open circular, and other isoforms were accomplished within 8 min. Appropriate capillary cleaning with 0.1 M sodium hydroxide/0.1 M hydrochloric acid and capillary storage with 0.1 M hydrochloric acid ensured good separation reproducibility of 217 runs during an extended period of use.

Atomic Engineering of Molecular Qubits for High-Speed, High-Fidelity Single Qubit Gates.

Universal quantum computing requires fast single- and two-qubit gates with individual qubit addressability to minimize decoherence errors during processor operation. Electron spin qubits using individual phosphorus donor atoms in silicon have demonstrated long coherence times with high fidelities, providing an attractive platform for scalable quantum computing. While individual qubit addressability has been demonstrated by controlling the hyperfine interaction between the electron and nuclear wave function in a global magnetic field, the small hyperfine Stark coefficient of 0.34 MHz/MV m-1 achieved to date has limited the speed of single quantum gates to ∼42 µs to avoid rotating neighboring qubits due to power broadening from the antenna. The use of molecular 2P qubits with more than one donor atom has not only demonstrated fast (0.8 ns) two-qubit SWAP gates and long spin relaxation times of ∼30 s but provides an alternate way to achieve high selectivity of the qubit resonance frequency. Here, we show in two different devices that by placing the donors with comparable interatomic spacings (∼0.8 nm) but along different crystallographic axes, either the [110] or [310] orientations using STM lithography, we can engineer the hyperfine Stark shift from 1 MHz/MV m-1 to 11.2 MHz/MV m-1, respectively, a factor of 10 difference. NEMO atomistic calculations show that larger hyperfine Stark coefficients of up to ∼70 MHz/MV m-1 can be achieved within 2P molecules by placing the donors ≥5 nm apart. When combined with Gaussian pulse shaping, we show that fast single qubit gates with 2π rotation times of 10 ns and ∼99% fidelity single qubit operations are feasible without affecting neighboring qubits. By increasing the single qubit gate time to ∼550 ns, two orders of magnitude faster than previously measured, our simulations confirm that >99.99% single qubit control fidelities are achievable.

Is the future female for turtles? Climate change and wetland configuration predict sex ratios of a freshwater species.

Climate change and land-use change are leading drivers of biodiversity decline, affecting demographic parameters that are important for population persistence. For example, scientists have speculated for decades that climate change may skew adult sex ratios in taxa that express temperature-dependent sex determination (TSD), but limited evidence exists that this phenomenon is occurring in natural settings. For species that are vulnerable to anthropogenic land-use practices, differential mortality among sexes may also skew sex ratios. We sampled the spotted turtle (Clemmys guttata), a freshwater species with TSD, across a large portion of its geographic range (Florida to Maine), to assess the environmental factors influencing adult sex ratios. We present evidence that suggests recent climate change has potentially skewed the adult sex ratio of spotted turtles, with samples following a pattern of increased proportions of females concomitant with warming trends, but only within the warmer areas sampled. At intermediate temperatures, there was no relationship with climate, while in the cooler areas we found the opposite pattern, with samples becoming more male biased with increasing temperatures. These patterns might be explained in part by variation in relative adaptive capacity via phenotypic plasticity in nest site selection. Our findings also suggest that spotted turtles have a context-dependent and multi-scale relationship with land use. We observed a negative relationship between male proportion and the amount of crop cover (within 300 m) when wetlands were less spatially aggregated. However, when wetlands were aggregated, sex ratios remained consistent. This pattern may reflect sex-specific patterns in movement that render males more vulnerable to mortality from agricultural machinery and other threats. Our findings highlight the complexity of species' responses to both climate change and land use, and emphasize the role that landscape structure can play in shaping wildlife population demographics.

Assessing Antigen-Adjuvant Complex Stability Against Physical Stresses By wNMR.

This study applies an emerging analytical technology, wNMR (water proton nuclear magnetic resonance), to assess the stability of aluminum adjuvants and antigen-adjuvant complexes against physical stresses, including gravitation, flow and freeze/thaw. Results from wNMR are verified by conventional analytical technologies, including static light scattering and microfluidic imaging. The results show that wNMR can quickly and noninvasively determine whether an aluminum adjuvant or antigen-adjuvant complex sample has been altered by physical stresses.

Solution Oligonucleotide APIs: Regulatory Considerations.

Manufacture of oligonucleotide active pharmaceutical ingredients (APIs) typically consists of solid-phase synthesis, deprotection and cleavage, purification and filtration, and isolation from aqueous solutions through lyophilization. In the first step of drug product manufacture, the API is dissolved in water again and excipients are added. While isolation of oligonucleotide APIs can be meaningful in many cases, there may be cases where keeping the API in solution provides benefit, and multiple technical aspects must be taken into account and balanced when determining the appropriate API form. A significant factor is whether an API in solution will contain additional components. While APIs in solution containing additional components (so-called formulated APIs) are well established for biological products, there are regulatory guidelines in place that represent hurdles for industry to using a formulated API approach for oligonucleotide drugs. The present communication outlines conditions where a formulated API approach can be chosen in compliance with existing guidelines. Relevant aspects pertaining to risk management, GMP standards, facility design, control strategies, and regulatory submission content are discussed. In addition, the authors propose that existing guidelines be modernized to enable the use of a formulated API approach for additional reasons than the ones described in the existing regulatory framework. The manuscript aims to promote a dialog with regulators in this field.

Evaluating the effect of expert elicitation techniques on population status assessment in the face of large uncertainty.

Population projection models are important tools for conservation and management. They are often used for population status assessments, for threat analyses, and to predict the consequences of conservation actions. Although conservation decisions should be informed by science, critical decisions are often made with very little information to support decision-making. Conversely, postponing decisions until better information is available may reduce the benefit of a conservation decision. When empirical data are limited or lacking, expert elicitation can be used to supplement existing data and inform model parameter estimates. The use of rigorous techniques for expert elicitation that account for uncertainty can improve the quality of the expert elicited values and therefore the accuracy of the projection models. One recurring challenge for summarizing expert elicited values is how to aggregate them. Here, we illustrate a process for population status assessment using a combination of expert elicitation and data from the ecological literature. We discuss the importance of considering various aggregation techniques, and illustrate this process using matrix population models for the wood turtle (Glyptemys insculpta) to assist U.S. Fish and Wildlife Service decision-makers with their Species Status Assessment. We compare estimates of population growth using data from the ecological literature and four alternative aggregation techniques for the expert-elicited values. The estimate of population growth rate based on estimates from the literature (λmean = 0.952, 95% CI: 0.87-1.01) could not be used to unequivocally reject the hypotheses of a rapidly declining population nor the hypothesis of a stable, or even slightly growing population, whereas our results for the expert-elicited estimates supported the hypothesis that the wood turtle population will decline over time. Our results showed that the aggregation techniques used had an impact on model estimates, suggesting that the choice of techniques should be carefully considered. We discuss the benefits and limitations associated with each method and their relevance to the population status assessment. We note a difference in the temporal scope or inference between the literature-based estimates that provided insights about historical changes, whereas the expert-based estimates were forward looking. Therefore, conducting an expert-elicitation in addition to using parameter estimates from the literature improved our understanding of our species of interest.

Rapid and Noninvasive Quantification of Capsid Gene Filling Level Using Water Proton Nuclear Magnetic Resonance.

The present work reports an enabling novel technology for quantifying the gene content in adeno-associated viral capsids. The method is based on the water proton nuclear magnetic resonance (wNMR) technique. Instead of analyzing the capsid directly, it utilizes water molecules to distinguish empty and full capsids, as water interacts with them differently. The transverse relaxation rate of water protons, R2(1H2O), readily distinguishes empty and full capsids and is capable of quantifying the fraction of full capsids in a mixture of full and empty ones. It involves no sample preparation and no reagents. Measurement is rapid (data collection takes 1-2 min), noninvasive (the capsid sample can stay inside the original sealed and labeled container to be used in other studies or administered to a patient), and performed using a wide-bore benchtop NMR instrument. The method can be readily implemented at a production plant for product release as part of product quality control.

Enzymatic depolymerization of streptococcus pneumoniae type 8 polysaccharide.

Although there have been decades of research on streptococcus pneumoniae, it is still among the leading cause of infectious disease in the world. As a type of capsular polysaccharide (CPS) of streptococcus pneumoniae, pneumococcal polysaccharides are essential components for colonization and virulence in mammalian hosts. This study aimed to characterize the CPS structure of type 8 streptococcus pneumoniae, which is one of the most fatal serotypes. In this work, heparinase I&III was used to successfully digest pneumococcal type 8 polysaccharide (Pn8P). We characterized the oligosaccharide generated from the enzymatic depolymerization of Pn8P by size exclusion chromatography, mass spectrometry and nuclear magnetic resonance. This is the first study to enzymatically depolymerize and characterize Pn8P.

Sensitive quantitation of low level free polysaccharide in conjugate vaccines by size exclusion chromatography-reverse phase liquid chromatography with UV detection.

The level of free polysaccharide is a critical quality attribute of polysaccharide-protein conjugate vaccines. The work presented describes a simple and sensitive method for the determination of low level free polysaccharides in multiple polysaccharide-protein conjugates. The method utilizes a reverse phase (RP) column to perform a size exclusion chromatography (SEC) separation of free polysaccharide and a reverse phase liquid chromatography (RPLC) separation of free protein and protein-polysaccharide conjugate. The use of phosphate buffer in the mobile phase enables the universal and sensitive detection of low level free polysaccharides at UV 200 nm. The method has been validated to monitor low level free polysaccharide (<1 %) in multiple polysaccharide-protein conjugates. The limit of quantitation is 2 µg/ml or 0.3 % free polysaccharide in 0.6 mg/ml polysaccharide-protein conjugate. The accuracy is in the range of 94.1.0-108.5 %.

On-line coupling of hydrophobic interaction column with reverse phase column -charged aerosol detector/mass spectrometer to characterize polysorbates in therapeutic protein formulations.

Polysorbates are complex mixtures of over a thousand components with a wide range of hydrophobicity. This paper describes a methodology for characterization of heterogeneity and stability of polysorbates in therapeutic protein formulation. The method utilizes on-line coupling of a hydrophobic interaction chromatography (HIC) column with reverse phase liquid chromatography (RPLC) and charged aerosol detection (CAD)/mass spectrometer (MS). The addition of a low concentration of formic acid and organic solvent (e.g. 0.05% formic acid and 3% acetonitrile) in the mobile phase enables the use of the HIC column to separate small molecule excipients (including major components of polysorbates) and the large protein molecules by a mixed mechanism of size exclusion chromatography (SEC) and RPLC. The protein and the charged excipients, which elute early from the HIC column, are directed by a switching valve to waste. The polysorbates and other neutral excipients, which elute later from the HIC column, are directed to the RPLC column for further separation. The separated polysorbate components are detected by CAD, and characterized by MS. This method has been used to characterize the degradation of polysorbate 80 (PS80) in placebo and protein formulations. Our studies have revealed different degradation behavior of PS80 in placebo vs. protein formulation.

Probing Conformational Diversity of Fc Domains in Aggregation-Prone Monoclonal Antibodies.

PURPOSE: Fc domains are an integral component of monoclonal antibodies (mAbs) and Fc-based fusion proteins. Engineering mutations in the Fc domain is a common approach to achieve desired effector function and clinical efficacy of therapeutic mAbs. It remains debatable, however, whether molecular engineering either by changing glycosylation patterns or by amino acid mutation in Fc domain could impact the higher order structure of Fc domain potentially leading to increased aggregation propensities in mAbs. METHODS: Here, we use NMR fingerprinting analysis of Fc domains, generated from selected Pfizer mAbs with similar glycosylation patterns, to address this question. Specifically, we use high resolution 2D [13C-1H] NMR spectra of Fc fragments, which fingerprints methyl sidechain bearing residues, to probe the correlation of higher order structure with the storage stability of mAbs. Thermal calorimetric studies were also performed to assess the stability of mAb fragments. RESULTS: Unlike NMR fingerprinting, thermal melting temperature as obtained from calorimetric studies for the intact mAbs and fragments (Fc and Fab), did not reveal any correlation with the aggregation propensities of mAbs. Despite >97% sequence homology, NMR data suggests that higher order structure of Fc domains could be dynamic and may result in unique conformation(s) in solution. CONCLUSION: The overall glycosylation pattern of these mAbs being similar, these conformation(s) could be linked to the inherent plasticity of the Fc domain, and may act as early transients to the overall aggregation of mAbs.

Considerations for the Use of Polysorbates in Biopharmaceuticals.

PURPOSE: Polysorbates are commonly added to protein formulations and serve an important function as stabilizers. This paper reviews recent literature detailing some of the issues seen with the use of polysorbate 80 and polysorbate 20 in protein formulations. Based on this knowledge, a development strategy is proposed that leads to a control strategy for polysorbates in protein formulations. METHODS: A consortium of Biopharmaceutical scientists working in the area of protein formulations, shared experiences with polysorbates as stabilizers in their formulations. RESULTS: Based on the authors experiences and recent published literature, a recommendation is put forth for a development strategy which will lead into the appropriate control strategy for these excipients. CONCLUSIONS: An appropriate control strategy may comprise one or more elements of raw material, in-process and manufacturing controls. Additionally, understanding the role, if any, polysorbates play during stability will require knowledge of the criticality of the excipient, based upon its impact on CQAs due to variations in concentration and degradation level.

Control Strategy for Small Molecule Impurities in Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceuticals. As such, there are no specific guidelines addressing impurity limits and qualification requirements. The current ICH guidelines on impurities, Q3A (Impurities in New Drug Substances), Q3B (Impurities in New Drug Products), and Q6B (Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) do not adequately address how to assess small molecule impurities in ADCs. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) formed an impurities working group (IWG) to discuss this issue. This white paper presents a strategy for evaluating the impact of small molecule impurities in ADCs. This strategy suggests a science-based approach that can be applied to the design of control systems for ADC therapeutics. The key principles that form the basis for this strategy include the significant difference in molecular weights between small molecule impurities and the ADC, the conjugation potential of the small molecule impurities, and the typical dosing concentrations and dosing schedule. The result is that exposure to small impurities in ADCs is so low as to often pose little or no significant safety risk.

Spin Diffusion Editing for Structural Fingerprints of Therapeutic Antibodies.

The growing importance of biologics and biosimilars as therapeutic and diagnostic agents is giving rise to new demands for analytical methodology that can quickly and accurately assess the chemical and physical state of protein-based products. A particular challenge exists in physical characterization where the proper fold and extent of disorder of a protein is a major concern. The ability of NMR to reflect structural and dynamic properties of proteins is well recognized, but sensitivity limitations and high levels of interference from excipients in typical biologic formulations have prevented widespread applications to quality assessment. Here we demonstrate applicability of a simple one-dimensional proton NMR method that exploits enhanced spin diffusion among protons in well-structured areas of a protein. We show that it is possible to reduce excipient signals and allow focus on structural characteristics of the protein. Additional decomposition of the resulting spectra based on rotating frame spin relaxation allows separate examination of components from aggregates and disordered regions. Application to a comparison of two different monoclonal antibodies and to detection of partial pH denaturation of a monoclonal antibody illustrates the procedure.

Ion-pair reversed phase liquid chromatography with ultraviolet detection for analysis of ultraviolet transparent cations.

This paper describes the use of an anionic ion-pair reagent (IPR) to impove the ultraviolet (UV) detection and hydrophobic retention of polar and UV transparent cations. Anionic IPR added to the mobile phase forms an ion-pair with cations. Formation of the ion-pair causes a redshift in the absorption wavength, making it possible for direct UV detection of UV-inactive cations. The ion-pairs with increased hydrophobicity were separated by reversed phase liquid chromatography (RPLC). Different perfluorinated caboxylic acids (trifluoroacetic acid, heptafluorobutyric acid, nonafluoropentanoic acid) were evaluted as IPR in the separation and detection of the common cations sodium, ammonium and Tris(hydroxymethyl)aminomethane (Tris). The effects of the IPR type and concentration on separation and detection have been investigated to understand the separation and detection mechanisms. The optimal separation and detection condtions were attained with mobile phase containing 0.1% nonafluoropentanoic acid and with the UV detection at 210nm. UV detection and charged aerosol detection (CAD) were compared in the quantitation of the cations. The limit of quantitation (LOQ) of sodium and Tris with UV detection is comparable to that by CAD. The LOQ of ammonium with UV detection (1ppm or 3ng) is about 20-fold lower than that (20ppm or 60ng) by CAD. The RPLC-UV method was used to monitor ammonium clearance during ultrafiltration and diafiltration in the manfucaturing of biopharmceutical drug substance.

Size exclusion chromatography of polysaccharides with reverse phase liquid chromatography.

This work describes the use of a reverse phase (RP) column for size-based separation of hydrophilic polysaccharides. The entire separation window (before and after void volume) is used to provide unique separation of polysaccharides from other components in a glycol-conjugate vaccine or complex media of fermentation broth. The technique has also been applied to the separation of polysaccharides of different sizes. The effect of chromatographic parameters including type of packing material (CN, C8 and C18), pore size (80 and 300Å) of stationary phase, concentration of organic solvent on the separation was investigated. In addition, characterization of size-based separation was evaluated by coupling of RP column with multi-angle laser light scattering (MALLs) detector.

On-line coupling of size exclusion chromatography with mixed-mode liquid chromatography for comprehensive profiling of biopharmaceutical drug product.

A methodology based on on-line coupling of size exclusion chromatography (SEC) with mixed-mode liquid chromatography (LC) has been developed. The method allows for simultaneous measurement of a wide range of components in biopharmaceutical drug products. These components include the active pharmaceutical ingredient (protein) and various kinds of excipients such as cations, anions, nonionic hydrophobic surfactant and hydrophilic sugars. Dual short SEC columns are used to separate small molecule excipients from large protein molecules. The separated protein is quantified using a UV detector at 280 nm. The isolated excipients are switched, online, to the Trinity P1 mixed-mode column for separation, and detected by an evaporative light scattering detector (ELSD). Using a stationary phase with 1.7 µm particles in SEC allows for the use of volatile buffers for both SEC and mix-mode separation. This facilitates the detection of different excipients by ELSD and provides potential for online characterization of the protein with mass spectrometry (MS). The method has been applied to quantitate protein and excipients in different biopharmaceutical drug products including monoclonal antibodies (mAb), antibody drug conjugates (ADC) and vaccines.