Common Buffers Research Articles

In Vitro Characterization and Rescue of VX Metabolism in Human Liver Microsomes.

Venomous agent X (VX) is an organophosphate acetylcholinesterase (AChE) inhibitor, and although it is one of the most toxic AChE inhibitors known, the extent of metabolism in humans is not currently well understood. The known metabolism in humans is limited to the metabolite identification from a single victim of the Osaka poisoning in 1994, which allowed for the identification of several metabolic products. VX has been reported to be metabolized in vitro by paraoxonase-1 and phosphotriesterase, although their binding constants are many orders of magnitude above the LD50, suggesting limited physiologic relevance. Using incubation with human liver microsomes (HLMs), we have now characterized the metabolism of VX and the formation of multiple metabolites as well as identified a Food and Drug Administration-approved drug [ethylenediaminetetraacetic acid (EDTA)] that enhances the metabolic rate. HLM incubation alone shows a pronounced increase in the metabolism of VX compared with buffer, suggesting that cytochrome P450-mediated metabolism of VX is occurring. We identified a biphasic decay with two distinct rates of metabolism. The enhancement of VX metabolism in multiple buffers was assessed to attempt to mitigate the effect of hydrolysis rates. The formation of VX metabolites was shown to be shifted with HLMs, suggesting a pathway enhancement over simple hydrolysis. Additionally, our investigation of hydrolysis rates in various common buffers used in biologic assays discovered dramatic differences in VX stability. The new human in vitro VX metabolic data reported points to a potential in vivo treatment strategy (EDTA) for rescue in individuals that are poisoned though enhancement of metabolism alongside existing treatments. SIGNIFICANCE STATEMENT: Venomous agent X (VX) is a potent acetylcholinesterase inhibitor and chemical weapon. To date, we do not possess a clear understanding of its metabolism in humans that would assist us in treating those exposed to it. This study now describes the human liver microsomal metabolism of VX and identifies ethylenediaminetetraacetic acid, which appears to enhance the rate of metabolism. This may provide a potential treatment option for human VX poisoning.

Predicting Colloidal Stability of High-Concentration Monoclonal Antibody Formulations in Common Pharmaceutical Buffers Using Improved Polyethylene Glycol Induced Protein Precipitation Assay.

Colloidal stability is an important consideration when developing high concentration mAb formulations. PEG-induced protein precipitation is a commonly used assay to assess the colloidal stability of protein solutions. However, the practical usefulness and the current theoretical model for this assay have yet to be verified over a large formulation space across multiple mAbs and mAb-based modalities. In the present study, we used PEG-induced protein precipitation assays to evaluate colloidal stability of 3 mAbs in 24 common formulation buffers at 20 and 5 °C. These prediction assays were conducted at low protein concentration (1 mg/mL). We also directly characterized high concentration (100 mg/mL) formulations for cold-induced phase separation, turbidity, and concentratibility by ultrafiltration. This systematic study allowed analysis of the correlation between the results of low concentration assays and the high concentration attributes. The key findings of this study include the following: (1) verification of the usefulness of three different parameters (Cmid, μB, and Tcloud) from PEG-induced protein precipitation assays for ranking colloidal stability of high concentration mAb formulations; (2) a new method to implement PEG-induced protein precipitation assay suitable for high throughput screening with low sample consumption; (3) improvement in the theoretical model for calculating robust thermodynamic parameters of colloidal stability (μB and εB) that are independent of specific experimental settings; (4) systematic evaluation of the effects of pH and buffer salts on colloidal stability of mAbs in common formulation buffers. These findings provide improved theoretical and practical tools for assessing the colloidal stability of mAbs and mAb-based modalities during formulation development.

Serial and multi-level proteome analysis for microscale protein samples

Post-translational modifications (PTMs), such as phosphorylation and ubiquitination, play key roles in signal transduction and protein homeostasis. The crosstalk of PTMs greatly expands the components of proteome and protein functions. Multi-level proteome analysis, which involves proteome investigations of total lysate and PTMs in this context, provides a comprehensive approach to explore the PTM crosstalk of a biological system under diverse disturbances. However, multi-level proteome practice remains technically challenging. Here we intended to build a strategy for multi-level proteome analysis, in which we focus on the serial profiling the total proteome, ubiquitinome and phosphoproteome from the microscale of starting material. We started by evaluating five common lysis buffers and found that the sodium deoxycholate buffer provided the best overall performance. We then developed an approach for serial enrichment and profiling of the multi-level proteome. To expand the depth of identification, we customized the variable windows to perform data-independent acquisition (DIA) sequencing for each proteome. In total, we identified 6465 proteins, ∼20,000 GlyGly sites (class 1), and ∼ 19,000 phosphosites (class 1) sequentially using 1 mg of HeLa digest by three DIA measurements. We applied this strategy to analyze MG132-treated HeLa cells and observed the crosstalk between ubiquitination and phosphorylation. Our method can be referenced for other multi-level proteome studies with microscale samples. SignificanceLysis buffer containing sodium deoxycholate provided the best overall performance in multi-level proteome analysis. One step of ubiquitination enrichment before phosphorylation enrichment does not reduce the reproducibility of phosphoproteome. Customized isolation windows were established for DIA analysis on each level of proteome. Combined the serial enrichment approach and the customized single-shot DIA method enabled the multi-level proteome of microscale protein samples.

Mannich Base PIP-199 Is a Chemically Unstable Pan-Assay Interference Compound.

Mannich base PIP-199 is the only reported small-molecule inhibitor of the Fanconi anemia complementation group M-RecQ-mediated genome instability protein (FANCM-RMI), a protein-protein interaction that governs genome instability in the genetic disorders Fanconi anemia and Bloom's syndrome. PIP-199 and analogues with the same indole-derived Mannich base scaffold have been used as tool compounds in diverse biological studies. We report the first published synthesis of PIP-199 and its analogues, demonstrating that PIP-199 immediately decomposes in common aqueous buffers and some organic solvents. Neither PIP-199 nor its more hydrolytically stable analogues show any observable activity in binding and competitive biophysical assays for FANCM-RMI. We conclude that PIP-199 is not an effective tool compound for biological studies and that apparent cellular activity likely arises from the nonspecific toxicity of breakdown products. More generally, apparent inhibitors that share this Mannich scaffold potentially represent a new family of pan-assay interference compounds (PAINS) that should be thoroughly assessed for aqueous stability prior to use in biological studies.

Virus Inactivation by Formaldehyde and Common Lysis Buffers.

Numerous mammalian viruses are routinely analyzed in clinical diagnostic laboratories around the globe or serve as indispensable model systems in viral research. Potentially infectious viral entities are handled as blood, biopsies, or cell and tissue culture samples. Countless protocols describe methods for virus fixation and inactivation, yet for many, a formal proof of safety and completeness of inactivation remains to be shown. While modern nucleic acid extraction methods work quite effectively, data are largely lacking on possible residual viral infectivity, e.g., when assessed after extended culture times, which maximizes the sensitivity for low levels of residual infectiousness. Therefore, we examined the potency and completeness of inactivation procedures on virus-containing specimens when applying commonly used fixatives like formaldehyde or nucleic acid extraction/lysis buffers. Typical representatives of different virus classes, including RNA and DNA viruses, enveloped and non-enveloped, such as adenovirus, enterovirus, lentivirus, and coronavirus, were used, and the reduction in the in vitro infectiousness was assessed for standard protocols. Overall, a 30-minute incubation with formaldehyde at room temperature effectively inactivated all tested enveloped and non-enveloped viruses. Full inactivation of HIV-1 and ECHO-11 was also achieved with all buffers in the test, whereas for SARS-CoV-2 and AdV-5, only five of the seven lysis buffers were fully effective under the tested conditions.

Buffer Solutions of known Ionic Strength

pH buffer solutions are those in which minimal pH variations occur when moderate amounts of strong acids or bases are added or diluted. The most common buffers are those used in the intermediate pH zone and are made up of an acid-base conjugate pair (HA/A-), with Ca and Cb as analytical concentrations of acid and base respectively. The buffer capacity of a solution is the measure of its effectiveness in preserving the pH value when adding an acid or a base. Three new programs working under the Windows 10 environment have been developed. The first one, the BUFFER program, allows to prepare buffers of known ionic strength without the need of adding an inert electrolyte, calculating the pH and buffering capacity. On the other hand, the BRÖMSTED method allows calculating the pH of conjugated acid-base systems applying the Newton-Raphson method. In this work two more programs are described, one applying the Brömsted method to monoprotic acids and another new one to diprotic acids.

Interactions of Common Biological Buffers with Iron Oxide Nanoparticles.

Iron oxide nanoparticles (IONPs) have been studied extensively for biomedical applications, which require that they be aqueous-stable at physiological pH. The structures of some of these buffers, however, may also allow for binding to surface iron, thus potentially exchanging with functionally relevant ligands, and altering the desired properties of the nanoparticles. We report here on the interactions of five common biologically relevant buffers (MES, MOPS, phosphate, HEPES, and Tris) with iron oxide nanoparticles through spectroscopic studies. The IONPs in this study are capped with 3,4-dihydroxybenzoic acid (3,4-DHBA) to serve as models for IONP functionalized with catechol ligands. Unlike previous studies, which relied exclusively on dynamic light scattering (DLS) and ζ-potential measurements to characterize buffer interactions with IONPs, we use Fourier transform infrared (FTIR) and ultraviolet-visible (UV-visible) spectroscopic techniques to characterize the IONP surface to demonstrate binding of buffers and etching of the IONP surface. Our findings establish that phosphate and Tris bind to the IONP surface, even in the presence of strongly bound catechol ligands. We further observe significant etching of IONPs in Tris buffer, with the release of surface Fe into solution. Minor etching is noted in HEPES, and to a lesser degree, in MOPS, while no etching is observed in MES. Our findings suggest that, while morpholino buffers, such as MES and MOPS, may be more appropriate for use with IONPs, proper buffer selection should always be considered on a case-by-case basis.

Rapid, Label-Free Screening of Diverse Biotransformations by Flow-Injection Mass Spectrometry.

Mass spectrometry-based high-throughput screening methods combine the advantages of photometric or fluorometric assays and analytical chromatography, as they are reasonably fast (throughput ≥1 sample/min) and broadly applicable, with no need for labelled substrates or products. However, the established MS-based screening approaches require specialised and expensive hardware, which limits their broad use throughout the research community. We show that a more common instrumental platform, a single-quadrupole HPLC-MS, can be used to rapidly analyse diverse biotransformations by flow-injection mass spectrometry (FIA-MS), that is, by automated infusion of samples to the ESI-MS detector without prior chromatographic separation. Common organic buffers can be employed as internal standard for quantification, and the method provides readily validated activity and selectivity information with an analytical run time of one minute per sample. We report four application examples that cover a broad range of analyte structures and concentrations (0.1-50 mM before dilution) and diverse biocatalyst preparations (crude cell lysates and whole microbial cells). Our results establish FIA-MS as a versatile and reliable alternative to more traditional methods for screening enzymatic reactions.

Nanopore Liberates G-Quadruplexes from Biochemical Buffers for Accurate Mass Spectrometric Examination.

Achieving accurate measurements of G-quadruplexes (G4s), especially the characterization of their complicated non-covalent interactions with various components (such as metal ions and ligands) under physiological conditions, is of fundamental significance in unveiling their biological roles and developing antitumor drugs. By employing a nanopore ion emitter (∼30 nm), we demonstrated for the first time that G4 ions, which are free of non-specific adduction and meanwhile maintaining their pre-existing specific bindings with metal ions or ligands, can be directly liberated from common biochemical buffers (consisting of concentrated non-volatile salts of >150 mM) for mass spectrometric examination. Notably, the intermediate complexes of G4s with mixed di-cation coordination formed during the Na+/K+ exchange were successfully observed by mass spectrometry, whose structures were also revealed by the reconstructed circular dichroism spectra. We believe the nanopore-based ion emitters have built a solid bridge between native G4s in aqueous buffers and their accurate stoichiometries obtained by mass spectrometric examination.

New insight into the importance of formulation variables on parenteral growth hormone preparations: potential effect on the injection-site pain

Reducing injection-site pain (ISP) in patients with chronic conditions such as growth hormone deficiency is a valuable strategy to improve patient compliance and therapeutic efficiency. Thus understanding different aspects of pain induction following subcutaneous injection of biotherapeutics and identifying the responsible factors are vital. Here we have discussed the effects of formulation’s viscosity, concentration, osmolality, buffering agents, pH, and temperature as well as injection volume, dosing frequency, and different excipients on ISP following subcutaneous injection of commercially available recombinant human growth hormone products. Our literature review found limited available data on the effects of different components of parenteral rhGH products on ISP. This may be due to high cost associated with conducting various clinical trials to assess each excipient in the formulation or to determine the complex interactions of different components and its impact on ISP. Recently, conducting molecular dynamics simulation studies before formulation design has been recommended as an alternative and less-expensive approach. On the other hand, the observed inconsistencies in the available data is mainly due to different pain measurement approaches used in each study. Moreover, it is difficult to translate data obtained from animal studies to human subjects. Despite all these limitations, our investigation showed that components of parenteral rhGH products can significantly contribute to ISP. We suggest further investigation is required for development of long acting, buffer-free, preservative-free formulations. Besides, various excipients are currently being investigated for reducing ISP which can be used as alternatives for common buffers, surfactants or preservatives in designing future rhGH formulations.

Leveraging Biological Buffers for Efficient Messenger RNA Delivery via Lipid Nanoparticles.

Lipid nanoparticles containing messenger RNA (mRNA-LNPs) have launched to the forefront of nonviral delivery systems with their realized potential during the COVID-19 pandemic. Here, we investigate the impact of commonly used biological buffers on the performance and durability of mRNA-LNPs. We tested the compatibility of three common buffers─HEPES, Tris, and phosphate-buffered saline─with a DLin-MC3-DMA mRNA-LNP formulation before and after a single controlled freeze-thaw cycle. We hypothesized that buffer composition would affect lipid-aqueous phase separation. Indeed, the buffers imposed structural changes in LNP morphology as indicated by electron microscopy, differential scanning calorimetry, and membrane fluidity assays. We employed in vitro and in vivo models to measure mRNA transfection and found that Tris or HEPES-buffered LNPs yielded better cryoprotection and transfection efficiency compared to PBS. Understanding the effects of various buffers on LNP morphology and efficacy provides valuable insights into maintaining the stability of LNPs after long-term storage.

When the times get tough the toughs get funny: Means by which humor buffers against death anxiety emerged during COVID-19 outbreak.

According to Terror Management Theory (TMT), there are three common buffers that minimize the anxiety of mortality salience: affirmation of a) one’s cultural worldview, b) the self and one’s personal values, and c) one’s significance in the context of close personal relationships. The current study aimed at examining the contents of memes, which were distributed on social media during the COVID-19 outbreak, to explore the means by which humor buffers against death anxiety. A deductive and inductive thematic analysis captured three means by which humor buffers against death anxiety, a) humor as a means for connecting to cultural worldviews; b) humor as a means for inclusion in group; c) humor as a means to gain a sense of control. These findings are discussed through the theoretical lens of TMT.

Bioorthogonal click and release: A general, rapid, chemically revertible bioconjugation strategy employing enamine N-oxides

A chemically revertible bioconjugation strategy featuring a new bioorthogonal dissociative reaction employing enamine N-oxides is described. The reaction is rapid, complete, directional, traceless, and displays a broad substrate scope. Reaction rates for cleavage of fluorophores from proteins are on the order of 82 M-1s-1, and the reaction is relatively insensitive to common aqueous buffers and pHs between 4 and 10. Diboron reagents with bidentate and tridentate ligands also effectively reduce the enamine N-oxide to induce dissociation and compound release. This reaction can be paired with the corresponding bioorthogonal hydroamination reaction to afford an integrated system of bioorthogonal click and release via an enamine N-oxide linchpin with a minimal footprint. The tandem associative and dissociative reactions are useful for the transient attachment of proteins and small molecules with access to a discrete, isolable intermediate. We demonstrate the effectiveness of this revertible transformation on cells using chemically cleavable antibody-drug conjugates.

StarBright Dyes:-Build bigger better panels with superior dyes excitable by the violet and ultraviolet lasers.

Abstract StarBright UltraViolet and StarBright Violet Dyes are the brightest ultraviolet and violet laser excitable fluorescent dyes specifically developed for flow cytometry. Their narrow excitation and emission mean that despite the increased brightness there is no loss of resolution due to excessive spillover and spreading. StarBright Dyes can be pre-mixed, fixed and have no requirement for a special buffer when multiplexing, providing researchers with the flexibility to build bigger, better panels. Here we demonstrate this flexibility by comparing freshly made and premixed antibody cocktails from Bio-Rad Antibodies to immunophenotype human peripheral blood. Cells were stained using common staining buffers with acquisition on the ZE5 Cell Analyzer to show how bigger multiplex panels can be built with ease.

Displacement Statistics of Unhindered Single Molecules Show no Enhanced Diffusion in Enzymatic Reactions.

Recent studies have sparked debate over whether catalytic reactions enhance the diffusion coefficients D of enzymes. Through high statistics of the transient (600 μs) displacements of unhindered single molecules freely diffusing in common buffers, we here quantify D for four enzymes under catalytic turnovers. We thus formulate how ∼ ±1% precisions may be achieved for D, and show no changes in diffusivity for catalase, urease, aldolase, and alkaline phosphatase under the application of wide concentration ranges of substrates. Our single-molecule approach thus overcomes potential limitations and artifacts underscored by recent studies to show no enhanced diffusion in enzymatic reactions.

Improved yield, stability, and cleavage reaction of a novel tobacco etch virus protease mutant.

The protease catalytic subunit of the nuclear inclusion protein A from tobacco etch virus (TEVp) is widely used to remove tags and fusion proteins from recombinant proteins. Some intrinsic drawbacks to its recombinant production have been studied for many years, such as low solubility, auto-proteolysis, and instability. Some point mutations have been incorporated in the amino acid protease sequence to improve its production. Here, a comprehensive review of each mutation reported so far has been made to incorporate them into a mutant called TEVp7M with a total of seven changes. This mutant with a His7tag at N-terminus was produced with remarkable purification yields (55mg/L of culture) from the soluble fraction in a single step affinity purification. The stability of His7-TEVp7M was analyzed and compared with the single mutant TEVp S219V, making evident that His7-TEVp7M shows very constant thermal stability against pH variation, whereas TEVp S219V is highly sensitive to this change. The cleavage reaction was optimized by determining the amount of protease that could cleave a 100-fold excess substrate in the shortest possible time at 30°C. Under these conditions, His7-TEVp7M was able to cleave His-tag in the buffers commonly used for affinity purification. Finally, a structural analysis of the mutations showed that four of them increased the polarity of the residues involved and, consequently, showed increased solubility of TEVp and fewer hydrophobic regions exposed to the solvent. Taken together, the seven changes studied in this work improved stability, solubility, and activity of TEVp producing enough protease to digest large amounts of tags or fusion proteins. KEY POINTS: • Production of excellent yields of a TEVp (TEVp7M) by incorporation of seven changes. • His-tag removal in an excess substrate in the common buffers used for purification. • Incorporated mutations improve polarity, stability, and activity of TEVp7M.

"Out of the Depths Have I Called Thee": Jewish Israeli Undertakers' Management of Death Anxiety.

According to Terror Management Theory, there are three common buffers that minimize the anxiety of mortality salience: affirmation of one's cultural worldview, the self and one's personal values, and one's significance in the context of close personal relationships. The current study aimed to explore the manner by which Jewish Israeli undertakers manage their constant exposure to death and buffer against death anxiety. A deductive and inductive thematic analysis captured a dialectical movement between, and within, two conflicting worldviews participants were engaged in, in their attempt to manage the mortality salience effect they experience and buffer against death anxiety.

Toward Mechanistic Design of Surrogate Buffers for Dissolution Testing of pH-Dependent Drug Delivery Systems.

The in vivo dissolution of enteric-coated (EC) products is often overestimated by compendial in vitro dissolution experiments. It is of great interest to mimic the in vivo conditions as closely as possible in vitro in order to predict the in vivo behavior of EC dosage forms. The reason behind this is the overly high buffering capacity of the common compendial buffers compared to the intestinal bicarbonate buffer. However, a bicarbonate-based buffer is technically difficult to handle due to the need for continuous sparging of the media with CO2 to maintain the desired buffer pH. Therefore, bicarbonate buffers are not commonly used in routine practice and a non-volatile alternative is of interest. A mathematical mass transport modelling approach was previously found to enable accurate calculation of surrogate buffer molarities for small molecule compounds; however, the additional complexity of polymeric materials makes this difficult to achieve for an enteric coat. In this work, an approach was developed allowing relatively rapid screening of potential surrogate buffers for enteric coating. It was found that the effective buffering pKa of bicarbonate at the surface of a dissolving enteric polymer tended to be around 5.5, becoming higher when the dissolving enteric polymer formed a gel of greater firmness/viscosity and vice versa. Using succinate (pKa 5.2 under physiological ionic strength) and/or citrate (pKa 5.7 under physiological ionic strength) at conjugate base molarities corresponding to bicarbonate molarities in the intestinal segments of interest as an initial “guess” can minimize the number of experimental iterations necessary to design an appropriate surrogate.

Neuronal Tau Protein Film Stability on Gold Surface: Electrochemical Impedance Spectroscopy of Partial and Complete Surfaces

Tau is a neuronal protein responsible for microtubule formation [1]. It is also the pathological protein active in the Alzheimer’s Disease (AD) pathway. When Tau begins to aggregate as a result of misfolding, the neuronal cells die due to the lack of microtubule formation and can take on prion-like properties [2]. Therefore, detection of aggregated Tau can serve as a powerful tool in the early diagnosis of AD and dementia related diseases. As has been previously demonstrated, Tau 441 can be detected with an electrochemical immunoassay based on the electrochemical impedance spectroscopy (EIS) by using gold electrode [3]. The EIS was also used to monitor interactions between Tau protein and Heparin [4]. Due to the sensitive nature of protein molecules, buffers of physiological pH need to be employed to maintain native protein structure. However, not all buffer solutions are appropriately suited for surface studies. Herein, we describe use of EIS to monitor and detect surface stability of Tau protein films on gold electrode in two common buffers: MES buffer and Phosphate Buffer (PB). Additionally, the stability of partial and complete surfaces was also monitored. The partial surface was completed by backfilling with a solution of bovine serum albumin (BSA) and the complete surface was backfilled using 1-butylamine and hexanethiol. After modifying the surface of the electrode with activated lipoic acid and Tau, a series of further incubation steps were performed to test surface stability under various conditions. The EIS was measured as a function of Tau or Heparin concentrations, and incubation time and temperature. The control experiments included, Tau-free surface, Heparin-free solution, and buffer solutions in order to minimize non-specific interactions. The stability of the surfaces was measured as a signal change in EIS after incubations and it was discovered that certain buffered solutions and partial surfaces induced large changes in tau protein films on surfaces.

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.