Buffer Components Research Articles

Stabilization of Dry Sucrose Glasses by Four LEA_4 Proteins from Arabidopsis thaliana.

Cells of many organisms and organs can withstand an (almost) total water loss (anhydrobiosis). Sugars play an essential role in desiccation tolerance due to their glass formation ability during dehydration. In addition, intrinsically disordered LEA proteins contribute to cellular survival under such conditions. One possible mechanism of LEA protein function is the stabilization of sugar glasses. However, little is known about the underlying mechanisms. Here we used FTIR spectroscopy to investigate sucrose (Suc) glass stability dried from water or from two buffer components in the presence of four recombinant LEA and globular reference proteins. Buffer ions influenced the strength of the Suc glass in the order Suc < Suc/Tris < Suc/NaP. LEA proteins strengthened the sugar H-bonded network and the molecular structure in the glassy state. The position of νOH peak and the wavenumber–temperature coefficient (WTCg) provided similar information about the H-bonded network. Protein aggregation of LEA proteins was reduced in the desiccation-induced Suc glassy state. Detailed knowledge about the role of LEA proteins in the stabilization of dry sugar glasses yields information about their role in anhydrobiosis. This may open the possibility to use such proteins in biotechnical applications requiring dry storage of biologicals such as proteins, cells or tissues.

Analysis of biopharmaceutical formulations by Time Domain Nuclear Magnetic Resonance (TD-NMR) spectroscopy: A potential method for detection of counterfeit biologic pharmaceuticals.

1H Time-Domain Nuclear Magnetic Resonance (TD-NMR) is used to characterize solutions of antibodies that simulate biologic pharmaceutical formulations. The results from these measurements are compared with those from solutions in which the concentration or identity of the antibody has been altered. TD-NMR is shown to be very sensitive to differences in the amount of antibody in solution, with the ability to detect variations in as low as 2mg/mL. It is therefore capable, by comparison with data from known formulations, of determining whether a particular sample is likely to be of an authentic biologic formulation. This method expands on the previous use of HPLC, UV/VIS, Near-IR and High-Resolution NMR to detect adulterated pharmaceutical materials. While the sensitivity of the method is high, it is a fingerprinting methodology, illustrating differences but not elucidating their origin. The extracted relaxation times reflect the combined effect of all solutes (antibody, buffer components, etc.) on the solvent (water).

Impurity profiling of siRNA by two-dimensional liquid chromatography-mass spectrometry with quinine carbamate anion-exchanger and ion-pair reversed-phase chromatography

A short RNA with the sequence of the antisense strand of Patisiran has been selected as test material for the investigation of its common impurities using three different two-dimensional liquid chromatography (2D-LC) platforms. On the one hand, a quinine (QN) carbamate-based weak anion-exchange (AX) stationary phase (QN-AX) and a classical C18 reversed phase (RP) stationary phase in ion-pair (IP) mode with tripropylammonium acetate, respectively, have been used in the first dimension (1D) to provide the selectivity for impurities formed during the synthesis of the RNA. In the next step, certain peaks of interest from 1D have been transferred by multiple-heart-cutting (MHC) into a 2D in which an ESI-MS-compatible non-ionpairing RP method has been used for desalting via a diverter valve to remove non-volatile phosphate buffer components and ion-pair agents, respectively. Thus, a sensitive electrospray-ionization quadrupole time of flight mass spectrometry (ESI-TOF-MS) analysis of resolved impurity peaks of the siRNA has become possible under MS-friendly conditions. With both 2D-LC setups, peak purity of the ON has been evaluated by selective comprehensive (high resolution) sampling of the main peak. In a third MHC 2D-LC approach, the QN-AX LC mode was online coupled with the IP-RPLC in the 2D using UV detection. It allows the separation of additional impurities which coeluted in the first dimension. The potential of these methods for comprehensive impurity profiling of ON therapeutics is illustrated and discussed.

Online hyphenation of size-exclusion chromatography and gas-phase electrophoresis facilitates the characterization of protein aggregates.

Gas‐phase electrophoresis yields size distributions of polydisperse, aerosolized analytes based on electrophoretic principles. Nanometer‐sized, surface‐dry, single‐charged particles are separated in a high laminar sheath flow of particle‐free air and an orthogonal tunable electric field. Additionally, nano Electrospray Gas‐Phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) data are particle‐number based. Therefore, small particles can be detected next to larger ones without a bias, for example, native proteins next to their aggregates. Analyte transition from the liquid to the gas phase is a method inherent prerequisite. In this context, nonvolatile sample buffers influence results. In the worst case, the (bio‐)nanoparticle signal is lost due to an increased baseline and unspecific clustering of nonvolatile components. We present a novel online hyphenation of liquid chromatography and gas‐phase electrophoresis, coupling a size‐exclusion chromatography (SEC) column to an advanced nES GEMMA. Via this novel approach, it is possible to (i) separate analyte multimers already present in liquid phase from aggregates formed during the nES process, (ii) differentiate liquid phase and spray‐induced multimers, and (iii) to remove nonvolatile buffer components online before SEC–nES GEMMA analysis. Due to these findings, SEC–nES GEMMA has the high potential to help to understand aggregation processes in biological buffers adding the benefit of actual size determination for noncovalent assemblies formed in solution. As detection and characterization of protein aggregation in large‐scale pharmaceutical production or sizing of noncovalently bound proteins are findings directly related to technologically and biologically relevant situations, we proposed the presented method to be a valuable addition to LC‐MS approaches.

Crystal structure of human CRM1, covalently modified by 2-mercaptoethanol on Cys528, in complex with RanGTP.

CRM1 is a nuclear export receptor that has been intensively targeted over the last decade for the development of antitumor and antiviral drugs. Structural analysis of several inhibitor compounds bound to CRM1 revealed that their mechanism of action relies on the covalent modification of a critical cysteine residue (Cys528 in the human receptor) located in the nuclear export signal-binding cleft. This study presents the crystal structure of human CRM1, covalently modified by 2-mercaptoethanol on Cys528, in complex with RanGTP at 2.58 Å resolution. The results demonstrate that buffer components can interfere with the characterization of cysteine-dependent inhibitor compounds.

Light-Induced Histidine Adducts to an IgG1 Molecule Via Oxidized Histidine Residue and the Potential Impact of Polysorbate-20 Concentration.

Histidine (His) undergoes light-induced reactions such as oxidation, crosslinking and addition. These reactions are initiated by singlet oxygen (1O2) to generate His photo-oxidation products, which are subject to nucleophilic attack by a non-oxidized His residue from another protein or by nucleophilic buffer components such as Tris and His. This report aims to identify light-induced His-adducts to a monoclonal antibody (mAb-1) due to the reaction of His molecules in the buffer with the photooxidized His residues under ICH light conditions. Since polysorbate-20 (PS-20) is a commonly used excipient in biotherapeutics formulation, it is also important to study the impact of PS-20 concentration on protein photostability. We identified and characterized light-induced His-adducts of mAb-1 by LC-MS/MS. We showed that the levels of light-induced His-adducts generally correlate with the solvent accessibility of His residues in the protein. In addition, the presence of PS-20 at concentrations commonly used in protein drug formulations can significantly increase the levels of light-induced His-adducts. Since His residues are present in a conserved region in the Fc domain, and may be present in the complementarity-determining region (CDR), the impact on the biological functions of the His-adducts observed here should be further studied to evaluate the risk of their presence.

Protein formulation through automated screening of pH and buffer conditions, using the Robotein® high throughput facility.

Among various factors, the direct environment (e.g. pH, buffer components, salts, additives, etc.…) is known to have a crucial effect on both the stability and activity of proteins. In particular, proper buffer and pH conditions can improve their stability and function significantly during purification, storage and handling, which is highly relevant for both academic and industrial applications. It can also promote data reproducibility, support the interpretation of experimental results and, finally, contribute to our general understanding of the biophysical properties of proteins. In this study, we have developed a high throughput screen of 158 different buffers/pH conditions in which we evaluated: (i) the protein stability, using differential scanning fluorimetry and (ii) the protein function, using either enzymatic assays or binding activity measurements, both in an automated manner. The modular setup of the screen allows for easy implementation of other characterization methods and parameters, as well as additional test conditions. The buffer/pH screen was validated with five different proteins used as models, i.e. two active-site serine β-lactamases, two metallo-β-lactamases (one of which is only active as a tetramer) and a single-domain dromedary antibody fragment (VHH or nanobody). The formulation screen allowed automated and fast determination of optimum buffer and pH profiles for the tested proteins. Besides the determination of the optimum buffer and pH, the collection of pH profiles of many different proteins may also allow to delineate general concepts to understand and predict the relationship between pH and protein properties.

Cryo-EM grid optimization for membrane proteins

SummaryCryo-EM grid preparation is an important bottleneck in protein structure determination, especially for membrane proteins, typically requiring screening of a large number of conditions. We systematically investigated the effects of buffer components, blotting conditions and grid types on the outcome of grid preparation of five different membrane protein samples. Aggregation was the most common type of problem which was addressed by changing detergents, salt concentration or reconstitution of proteins into nanodiscs or amphipols. We show that the optimal concentration of detergent is between 0.05 and 0.4% and that the presence of a low concentration of detergent with a high critical micellar concentration protects the proteins from denaturation at the air-water interface. Furthermore, we discuss the strategies for achieving an adequate ice thickness, particle coverage and orientation distribution on free ice and on support films. Our findings provide a clear roadmap for comprehensive screening of conditions for cryo-EM grid preparation of membrane proteins.

FPGA friendly NoC simulation acceleration framework employing the hard blocks

A major role is played by Modeling and Simulation platforms in development of a new Network-on-Chip (NoC) architecture. The cycle accurate software simulators tend to become slow when simulating thousands of cores on a single chip. FPGAs have become the vehicle for simulation acceleration due to the properties of parallelism. Most of the state-of-the-art FPGA based NoC simulators utilize soft logic only for modeling the NoCs, leaving out the hard blocks to be unutilized. In this work, the FIFO Buffer and Crossbar switch functionalities of the NoC router have been embedded in the Block RAM (BRAMs) and the DSP48E1 slices with large multiplexer respectively. Employing the proposed techniques of mapping the NoC router components on the FPGA hard blocks, an NoC simulation acceleration framework based on the FPGA is presented in this work. A huge reduction in the use of the Configurable Logic Blocks (CLBs) has been observed when the FIFO buffer and Crossbar components of the NoC topology’s router micro-architecture are embedded in FPGA hard blocks. Our experimental results show that the topologies implemented employing the proposed FPGA friendly mapping of the NoC router components on the hard blocks consume 43.47% fewer LUTs and 41.66% fewer FFs than the topologies with CLB implementation. To optimize the latency of the NoC under consideration, a control unit called “buf_empty_checker” has been employed. A reduction in average latency has been observed compared to the CLB based topology implementation employing the proposed mapping. The proposed work consumes 10.88% fewer LUTs than the CONNECT NoC generation tool. Compared to DART, a reduction of 73.38% and 66.55% in LUTs and FFs has been observed with respect to the proposed work. The average packet latency of the proposed NoC architecture is 24.8% and 19.1% lesser than the CONNECT and DART architectures.

Investigation of the structure and dynamics of gallium binding to high-affinity peptides elucidated by multi-scale simulation, quantum chemistry, NMR and ITC.

Gallium (as Ga3+) is a Group IIIa metal and its recovery from wastewaters has become increasingly important for its reuse. The use of peptides for recycling offers a low-cost and environmentally-friendly option but the structural characteristics of peptides likely to bind Ga3+ are largely unknown. Multiple computational methods, coupled with experimental verification via NMR and Isothermal Calorimetry (ITC), were used to establish that Ga3+ binds with high affinity to peptide sequences and to elucidate the structural characteristics that contributed. It was demonstrated that peptide pre-organisation is key to Ga3+ binding and that a favourable binding position is necessarily governed by the size and shape of the electrostatic environment as much as individual electrostatic interactions with peptide residues themselves. Given favourable conditions, Ga3+ retrieved plausible binding positions involving both charged and uncharged residues that greatly increases the range of bonding possibilities with other peptide sequences and offers insights for binding other metals. The addition of pH buffer substantially improved the affinity of Ga3+ and a structural role for a buffer component was demonstrated.

Carbon Dioxide Expiration and Performance Fatigability Following Aerobic Exercise Training: A Longitudinal, Observational, Pilot Study.

This study examined the influence of aerobic exercise training (AET) on components of carbon dioxide expiration (VCO2), cardiorespiratory function, and fatigability. Twenty healthy adults completed peak cardiopulmonary exercise (CPX) and submaximal tests before and after a vigorous, 4-week AET regimen. Each test was followed by a 10-min recovery and endurance test at 70% of peak wattage attained during CPX. Fatigability was assessed using testing durations and power output. Respiratory buffering (excess VCO2) and non-buffering (metabolic VCO2) were calculated. Data were analyzed for significance (p<0.05) using regressions and paired t-tests. Significant improvements in all measures of fatigability were observed after AET. A significant increase in excess VCO2 was observed, though not in metabolic VCO2. Excess VCO2 was strongly predictive of fatigability measures. Significant decreases in fatigability are often observed in clinical populations such as obstructive or restrictive lung disease or pulmonary hypertension following AET, even when peak cardiorespiratory function does not appear to adapt. Decreases in fatigability appear to predict longevity with no yet identified mechanism. These results suggest that respiratory buffering and metabolic components of VCO2 may adapt independently to AET, introducing foundational plausibility for an influence of respiratory buffering adaptation to AET on fatigability status.

Studying Protein-DNA Interactions by Hydrogen/Deuterium Exchange Mass Spectrometry.

Protein hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) can be used to study interactions of proteins with various ligands, to describe the effects of mutations, or to reveal structural responses of proteins to different experimental conditions. It is often described as a method with virtually no limitations in terms of protein size or sample composition. While this is generally true, there are, however, ligands or buffer components that can significantly complicate the analysis. One such compound, that can make HDX-MS troublesome, is DNA. In this chapter, we will focus on the analysis of protein-DNA interactions, describe the detailed protocol, and point out ways to overcome the complications arising from the presence of DNA.

DYNAMIC SIMULATION ANALYSIS OF CAPTURE AND BUFFER SYSTEM BASED ON CLAW-TYPE DOCKING MECHANISM

The existing docking mechanism for on-orbit service cannot provide strong support for our country's follow-up lunar exploration project due to its large size, complex structure and single docking target. Lightweight docking mechanism is also an essential link due to the limit of carrying capacity. In order to study the docking mechanism that can serve the high-orbit missions such as future Moon Space Station and manned lunar landing, a new claw-type docking mechanism was designed, which adopted androgynous configuration and can realize the interchange between active vehicle and passive vehicle, the V-shaped slot and claw hook and other structural components are used to realize the capture and energy consumption functions in the docking process of aircraft, so as to realize the stable connection between two vehicles. The docking mechanism has the advantages of small size, light weight, simple structure and easy realization of functions. The dynamic analysis of the capture buffer system was carried out, and the influence of the parameters of buffer components on its capture performance was calculated, the establishment of the digital virtual prototype was completed in ADAMS software, and the simulation research was carried out in combination with the actual two typical initial docking conditions. The results show that the energy consumption in the docking process under the two working conditions meets the design requirements, and the capture can be completed with a smaller impact force of the V-shaped slot. The results prove the feasibility of the capture buffer system, and verify the capability of the docking mechanism with this configuration has better ability to complete tasks.

Probing Microenvironmental Acidity in Lyophilized Protein and Vaccine Formulations Using Solid-state NMR Spectroscopy.

Biophysical and biochemical instability of therapeutic proteins in the solution state may necessitate the development of products in the solid form, due to their enhanced stability. Lyophilization is a widely used method to ensure dry state stabilization of biological products. A commonly encountered issue is the pH shifts that can occur due to undesired crystallization of a buffer component, resulting in loss of protein activities. However, it is technically challenging to noninvasively investigate the physicochemical environment in the lyophile matrix. In this work, we demonstrate an approach based on solid-state NMR to investigate the microenvironmental acidity in lyophilized protein formulations, using histidine, a commonly used buffer agent, as a molecular probe. The solid-state acidity in the lyophilized matrix can be assessed by monitoring the chemical shift changes of histidine. The protonation and tautomeric states of histidine lyophilized at a range of pH values from 4.5 to 11.0 were identified from full 13C and 15N resonance assignments in one-dimensional and two-dimensional NMR experiments. The results demonstrated a pH-dependence of histidine chemical shift in the amorphous state. Moreover, we successfully applied this protocol to investigate the microenvironmental pH in lyophilized formulations of the HPV vaccine and lactate dehydrogenase protein.

Mass spectrometric and kinetics characterization of modified species of Growth Hormone Releasing Hexapeptide generated under thermal stress in different pH and buffers

Growth Hormone Releasing Peptide-6 (GHRP-6) is a promising molecule (H-His1-d-Trp- Ala-Trp-d-Phe-Lys6-NH2) for the treatment of several diseases. Studies on the degradation pathways of this molecule under stressed conditions are needed to develop appropriate formulations. Degradation products (DPs) of GHRP-6, generated by heating in the dark at 60 °C with pH ranging from 3.0 to 8.0 and in presence of common buffers, were isolated by RP-HPLC and characterized by ESI-MS/MS. C-terminal deamidation of GHRP-6 was generated preferentially at pH 3.0 and 8.0. Hydrolysis and head-to-tail cyclization were favored at pH ranging from 6.0 to 7.0 in phosphate containing buffers. A DP with +12 Da molecular mass was presumably originated by the reaction with formaldehyde derived from some of the additives and/or elastomeric closures. Certain DPs derived from the acylation reaction of the tri- and di-carboxylic buffering species were favored at pH 3.0–6.0 and indicate that buffer components, including those “Generally Recognized as Safe”, may potentially introduce chemical modifications and product heterogeneity. Nano LC–MS/MS analysis revealed GHRP-6 was also detected as a low-abundance species with Trp oxidized to 5-hydroxy, kynurenine, and N-formylkynurenine. The kinetics for the formation of the major degradation products was also studied by RP-HPLC.

In-depth interrogation of protein thermal unfolding data with MoltenProt.

Protein stability is a key factor in successful structural and biochemical research. However, the approaches for systematic comparison of protein stability are limited by sample consumption or compatibility with sample buffer components. Here we describe how miniaturized measurement of intrinsic tryptophan fluorescence (NanoDSF assay) in combination with a simplified description of protein unfolding can be used to interrogate the stability of a protein sample. We demonstrate that improved protein stability measures, such as apparent Gibbs free energy of unfolding, rather than melting temperature Tm, should be used to rank the results of thermostability screens. The assay is compatible with protein samples of any composition, including protein complexes and membrane proteins. Our data analysis software, MoltenProt, provides an easy and robust way to perform characterization of multiple samples. Potential applications of MoltenProt and NanoDSF include buffer and construct optimization for X‐ray crystallography and cryo‐electron microscopy, screening for small‐molecule binding partners and comparison of effects of point mutations.

Soil Organic Carbon Attenuates the Influence of Plants on Root-Associated Bacterial Community.

Plant-derived carbon (PDC) released by roots has a strong effect on root-associated bacterial community, which is critical for plant fitness in natural environments. However, the freshly exuded PDC can be diluted by the ancient soil-derived carbon (SDC) at a short distance from root apices. Thus, the rhizosphere C pools are normally dominated by SDC rather than PDC. Yet, how PDC and SDC interact to regulate root-associated bacterial community is largely unknown. In this study, a grass species and a legume species were planted in two contrasting matrixes, quartz sand and soil, to assess the role of PDC and SDC in regulating root-associated bacterial community, and to explore whether SDC affects the influence of PDC on bacterial community in soil. Our results indicated that the legume plant showed significantly positive priming effect on soil organic matter decomposition but the grass plant did not. PDC significantly shaped bacterial community in sand culture as indicated by PCR-DGGE and high-throughput sequencing of bacterial 16S rRNA gene. Intriguingly, we found that dissimilarity of bacterial communities associated with two plant species and the percentage of specific OTUs in quartz sand were significantly higher than those in soil. Moreover, several biomarkers enriched by plants in quartz sand turned to be general taxa in soil, which indicated that SDC attenuated the regulation of bacterial community by PDC. Taken together, these results suggest that SDC interacted with PDC and the root-associated microbial community, thus acted as soil buffering component of biological process contributing to soil resilience. The importance of PDC in structuring rhizosphere bacterial community needs to be reconsidered in the context of wider contribution of other C pool, such as SDC.

In-situ gradient formation by direct solid addition of buffer components

Buffer preparation and storage requires a significant facility footprint in large scale bioprocessing and together with the costs of supply chain management can have a substantial economic impact. In-line buffer mixing in chromatography is commonly performed by blending different buffer solutions using at least two pumps and a static or dynamic mixer. We developed a device for an in-line gradient delivery of buffering agents directly from solids to be applied for chromatographic separation processes. A solid feeding device with a screw conveyor and a hold tank for the solids was designed and a miniaturized system was 3D printed. The coefficient of variation for the precision of the solid feeding of 5 different buffering agents was below 5% even for very small solid flow rates necessary for lab-scale chromatography. Stability was demonstrated by a constant linear solid feed at a very low dosing rate of 0.05 g.min−1 over 24 hours. We demonstrated the suitability for chromatography by directly connecting the system to a standard chromatography workstation for protein chromatography. The solids were fed into a miniaturized continuously stirred tank reactor connected to an ÄKTA purification system. The performance of the in-line gradient delivery of buffering agents directly from solids was compared to conventional in-line buffer mixing. We were able to achieve highly linear gradients for elution using only one pump of a chromatographic system, generating the gradient by the direct addition of solids avoiding the necessity of additional pumps and hold tanks. By direct conditioning of buffers and the addition of solids a simple, just in time, at site preparation of buffers was possible. The design of the feeding unit for solid addition for buffer preparation is easily scalable and adaptable to work with or as a replacement for already existing in-line dilution or conditioning units.

Optimization of a molecular diagnostic strategy to verify SARS-CoV-2 infections by RT-qPCR

Abstract Objectives Fast and precise detection of SARS-CoV-2 RNA in infected patients is essential for treatment decisions. Methods A diagnostic strategy by analyzing nasopharyngeal swabs to detect SARS-CoV-2 RNA in individuals was established. The negative impacts of the individual buffer components on RT-qPCR analysis was reviewed and overcome by RNA purification. To investigate the functionality of the improved protocol we compared the novel diagnostic strategy to a Bead-based RNA extraction method using previously positive tested samples. Results A method to extract purify RNA molecules from SARS-CoV-2 was established. We examined the significance of nucleic acid purification and the need for an RNase inhibitor. Evaluation of 3,664 samples from March 23rd until May 18th in 2020 showed the incidence of COVID-19 infections in Thuringia, Germany. Additionally, benefits and limits of three RT-qPCR kits were assessed. Conclusions Our study suggests that virus capsid lysis combined with nucleic acid purification enables a viable alternative for the molecular diagnostics of SARS-CoV-2 infections. Due to the current delivery delays from different companies, this method offers the possibility to continue diagnosis and to handle the large number of samples.

Binary (Split) Light‐up Aptameric Sensors

AbstractThis Minireview discusses the design and applications of binary (also known as split) light‐up aptameric sensors (BLAS). BLAS consist of two RNA or DNA strands and a fluorogenic organic dye added as a buffer component. When associated, the two strands form a dye‐binding site, followed by an increase in fluorescence of the aptamer‐bound dye. The design is cost‐efficient because it uses short oligonucleotides and does not require conjugation of organic dyes with nucleic acids. In some applications, BLAS design is preferable over monolithic sensors because of simpler assay optimization and improved selectivity. RNA‐based BLAS can be expressed in cells and used for the intracellular monitoring of biological molecules. BLAS have been used as reporters of nucleic acid association events in RNA nanotechnology and nucleic‐acid‐based molecular computation. Other applications of BLAS include the detection of nucleic acids, proteins, and cancer cells, and potentially they can be tailored to report a broad range of biological analytes.