Exploiting Principal Component Analysis (PCA) to reveal temperature, buffer and metal ions' role in neuromelanin (NM) synthesis by dopamine (DA) oxidative polymerization.

Neuromelanin (NM) plays a well-established role in neurological disorders pathogenesis; the mechanism of action is still discussed and the investigations in this field are limited by NM's complex and heterogeneous composition, insolubility, and low availability from human brains. An alternative can be offered by synthetic NM obtained from dopamine (DA) oxidative polymerization; however, a deep knowledge of the influence of both physicochemical parameters (T, pH, ionic strength) and other compounds in the reaction media (buffer, metal ions, other catecholamines) on DA oxidation process and, consequently, on synthetic NM features is mandatory to develop reliable NM preparation methodologies. To partially fulfill this aim, the present work focuses on defining the role of temperature, buffer and metal ions on both DA oxidation rate and DA oligomer size. DA oxidation in the specific conditions is monitored by UV-Vis spectroscopy and Principal Component Analysis (PCA) is run either on the raw spectra to model the background absorption increase, related to small DA oligomers formation, or on their first derivative to rationalize DA consumption. After having studied three case studies, 3-Way PCA is applied to directly evaluate the effect of temperature and buffer type on DA oxidation in the presence of different metal ions. Despite the proof-of-concept nature of the work and the number of compounds still to be included in the investigation, the preliminary results and the possibility to further expand the chemometric approach represent an interesting contribution to the field of in vitro simulation of NM synthesis.

Association between sleep pattern, salivary cariogenic bacteria and fungi populations, pH and buffering capacity in children: A comparative study.

BACKGROUND: Sleep quality has a significant impact on a child's health and is linked to oral and systemic diseases. It affects the circadian rhythm, which plays a crucial role in regulating the balance of the endocrine and hormonal systems. Current research has focused on exploring its role in the development of caries, which is influenced by inherent oral factors such as the composition of the oral microbiome and pH levels. OBJECTIVES: This study aimed to investigate the relationship between bacterial population, pH, and buffering properties of saliva and sleep patterns in 8- to 12-year-old children. MATERIAL AND METHODS: This cross-sectional study was conducted on 85 elementary school children aged 8-12 years. After obtaining written consent, non-stimulating saliva samples were collected using the spitting method. The participants' sleep pattern information was obtained with the use of the Persian version of the Children's Sleep Habits Questionnaire (CSHQ). Based on the results of the CSHQ, the participants were divided into 2 groups: those with appropriate sleep patterns; and those with inappropriate sleep patterns. The study compared the bacterial population of Streptococcus mutans, Lactobacillus spp. and Candida albicans, as well as the buffering capacity and pH of the saliva between the 2 groups. The statistical analysis employed the χ2 test, the independent samples t-test and Spearman's correlation. RESULTS: The group with inappropriate sleep patterns had significantly lower pH and buffering capacity (p < 0.001) and significantly higher colony counts of Lactobacillus and S. mutans (p < 0.001 and p = 0.012, respectively). There was no association between C. albicans and sleep patterns (p = 0.121). CONCLUSIONS: Inappropriate sleep patterns increase the population of caries-causing bacteria and reduce salivary pH and buffering capacity. This can be a significant factor in the development of dental caries in children aged 8-12 years.

Rapid Enrichment of a Native Multipass Transmembrane Protein via Cell Membrane Electrophoresis through Buffer pH and Ionic Strength Adjustment.

Supported membrane electrophoresis is a promising technique for collecting membrane proteins in native bilayer environments. However, the slow mobility of typical transmembrane proteins has impeded the technique's advancement. Here, we successfully applied cell membrane electrophoresis to rapidly enrich a 12-transmembrane helix protein, glucose transporter 1 with antibodies (GLUT1 complex), by tuning the buffer pH and ionic strength. The identified conditions allowed the separation of the GLUT1 complex and a lipid probe, Fast-DiO, within a native-like environment in a few minutes. A force model was developed to account for distinct electric and drag forces acting on the transmembrane and aqueous-exposed portion of a transmembrane protein as well as the electroosmotic force. This model not only elucidates the impact of size and charge properties of transmembrane proteins but also highlights the influence of pH and ionic strength on the driving forces and, consequently, electrophoretic mobility. Model predictions align well with experimentally measured electrophoretic mobilities of the GLUT1 complex and Fast-DiO at various pH and ionic strengths as well as with several lipid probes, lipid-anchored proteins, and reconstituted membrane proteins from previous studies. Force analyses revealed the substantial membrane drag of the GLUT1 complex, significantly slowing down electrophoretic mobility. Besides, the counterbalance of similar magnitudes of electroosmotic and electric forces results in a small net driving force and, consequently, reduced mobility under typical neutral pH conditions. Our results further highlight how the size and charge properties of transmembrane proteins influence the suitable range of operating conditions for effective movement, providing potential applications for concentrating and isolating membrane proteins within this platform.

Unraveling the Microscopic Mechanism of Molecular Ion Interaction with Monoclonal Antibodies: Impact on Protein Aggregation.

Understanding and predicting protein aggregation represents one of the major challenges in accelerating the pharmaceutical development of protein therapeutics. In addition to maintaining the solution pH, buffers influence both monoclonal antibody (mAb) aggregation in solution and the aggregation mechanisms since the latter depend on the protein charge. Molecular-level insight is necessary to understand the relationship between the buffer-mAb interaction and mAb aggregation. Here, we use all-atom molecular dynamics simulations to investigate the interaction of phosphate (Phos) and citrate (Cit) buffer ions with the Fab and Fc domains of mAb COE3. We demonstrate that Phos and Cit ions feature binding mechanisms, with the protein that are very different from those reported previously for histidine (His). These differences are reflected in distinctive ion-protein binding modes and adsorption/desorption kinetics of the buffer molecules from the mAb surface and result in dissimilar effects of these buffer species on mAb aggregation. While His shows significant affinity toward hydrophobic amino acids on the protein surface, Phos and Cit ions preferentially bind to charged amino acids. We also show that Phos and Cit anions provide bridging contacts between basic amino acids in neighboring proteins. The implications of such contacts and their connection to mAb aggregation in therapeutic formulations are discussed.

Fluorinated Ethylamines as Electrospray-Compatible Neutral pH Buffers for Native Mass Spectrometry.

Native electrospray ionization mass spectrometry (ESI-MS) has emerged as a potent tool for examining the native-like structures of macromolecular complexes. Despite its utility, the predominant "buffer" used, ammonium acetate (AmAc) with pKa values of 4.75 for acetic acid and 9.25 for ammonium, provides very little buffering capacity within the physiological pH range of 7.0-7.4. ESI-induced redox reactions alter the pH of the liquid within the ESI capillary. This can result in protein unfolding or weakening of pH-sensitive interactions. Consequently, the discovery of volatile, ESI-compatible buffers, capable of effectively maintaining pH within a physiological range, is of high importance. Here, we demonstrate that 2,2-difluoroethylamine (DFEA) and 2,2,2-trifluoroethylamine (TFEA) offer buffering capacity at physiological pH where AmAc falls short, with pKa values of 7.2 and 5.5 for the conjugate acids of DFEA and TFEA, respectively. Native ESI-MS experiments on model proteins cytochrome c and myoglobin electrosprayed with DFEA and TFEA demonstrated the preservation of noncovalent protein-ligand complexes in the gas phase. Protein stability assays and collision-induced unfolding experiments further showed that neither DFEA nor TFEA destabilized model proteins in solution or in the gas phase. Finally, we demonstrate that multisubunit protein complexes such as alcohol dehydrogenase and concanavalin A can be studied in the presence of DFEA or TFEA using native ESI-MS. Our findings establish DFEA and TFEA as new ESI-compatible neutral pH buffers that promise to bolster the use of native ESI-MS for the analysis of macromolecular complexes, particularly those sensitive to pH fluctuations.

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.

A Novel Approach to Monitor the Concentration of Phosphate Buffers in the Range of 1 M to 0.1 M Using a Silicon-Based Impedance Sensor.

We present a novel and easy approach using a silicon-based impedance chip to determine the concentration of the given aqueous buffer solution. An accurate determination of the post-dilution concentration of the buffers is necessary for ensuring optimal buffer capacity, pH stability, and to assess solution reproducibility. In this study, we focused on phosphate buffer as the test liquid to achieve precise post-dilution concentration determinations. The impedance chip consisting of a top gold ring electrode, where a test volume of 20 µL to 30 µL of phosphate buffer was introduced for impedance measurements within the frequency range of 40 Hz to 1 MHz. For impedance investigation, we used phosphate buffers with three different pH values, and the impedance was measured after diluting the phosphate buffers to a concentration of 1.00 M, 0.75 M, 0.50 M, 0.25 M, 0.10 M, 0.05 M, and 0.01 M. In order to analyze the distinctive changes in the measured impedance, an equivalent circuit was proposed and modeled. From the impedance modeling, we report that the circuit parameter RAu/Si showed exponential dependence on the concentration of phosphate buffer and no dependence on the pH values of the phosphate buffer and on the added volume inside the ring electrode. The proposed silicon-based impedance chip is quick and uses reduced liquid volume for post-dilution concentration measurements of buffers and has perspective applications in the pharmaceutical and biological domains for regulating, monitoring, and quality control of the buffers.

Strategies to Stabilize Dalbavancin in Aqueous Solutions; Section-1: the Effects of Metal Ions and Buffers.

PURPOSE: The effect of monovalent (Na+ and K+) and divalent (Ca2+, Mg2+, and Zn2+) metal ions combined with citrate or acetate buffers (pH 4.5) on the stability of dalbavancin in aqueous solutions was investigated. METHOD: RP-HPLC and HP-SEC were used to evaluate the stability of aqueous solutions of dalbavancin in different combinations of buffers and metal ions after four weeks of storage at 5°C and 55°C. A long-term study of formulations with divalent metal ions was conducted over six months at 5°C., 25°C and 40°C using RP-HPLC. RESULTS: All formulations in citrate buffered solutions precipitated. Dalbavancin solutions in 10 mM acetate buffer at 55°C were more stable in 10 mM CaCl2, 5 mM ZnCl2 and 10 mM MgCl2 than those containing 2 mM NaCl or 5 mM KCl, although the MgCl2 formulations precipitated slightly. No significant effect was observed for any of the divalent metal ions at 40°C for six months. CONCLUSION: Dalbavancin's stability in solution was improved by a combination of acetate and divalent metal ions at 55°C for four weeks. No effect was observed with acetate or metal ions alone, and no effect was observed after six months at 40°C suggesting that acetate and divalent metal ions together interact with dalbavancin via a thermally activated step to inhibit hydrolysis of the drug.

Phosphate buffer interferes dissolution of prazosin hydrochloride in compendial dissolution testing.

The purpose of this study was to elucidate the lack of supersaturation behavior in the dissolution profile of prazosin hydrochloride (PRZ-HCl) in the compendial dissolution test. The equilibrium solubility was measured by a shake-flask method. Dissolution tests were performed by a compendial paddle method with a phosphate buffer solution (pH 6.8, 50 mM phosphate). The solid form of the residual particles was identified by Raman spectroscopy. In the pH range below 6.5, the equilibrium solubility in phosphate buffer was lower than that in the unbuffered solutions (pH adjusted by HCl and NaOH). Raman spectra showed that the residual solid was a phosphate salt of PRZ. In the pH range above 6.5, the pH-solubility profiles in the phosphate buffer solutions and the unbuffered solutions were the same. The residual solid was a PRZ freebase (PRZ-FB). In the dissolution test, PRZ-HCl particles first changed to a phosphate salt within 5 min, then gradually changed to PRZ-FB after several hours. Since the intestinal fluid is buffered by the bicarbonate system in vivo, the dissolution behavior in vivo may not be properly evaluated using a phosphate buffer solution. For drugs with a low phosphate solubility product, it is necessary to consider this aspect.

Investigating freezing-induced acidity changes in citrate buffers.

Citrate buffers are commonly utilized in the field of biomolecule stabilization. We investigate their applicability in the frozen state within a range of initial pHs (2.5 to 8.0) and concentrations (0.02 to 0.60 M). Citrate buffer solutions subjected to various cooling and heating temperatures are examined in terms of the freezing-induced acidity changes, revealing that citrate buffers acidify upon cooling. The acidity is assessed with sulfonephthalein molecular probes frozen in the samples. Optical cryomicroscopy combined with differential scanning calorimetry was employed to investigate the causes of the observed acidity changes. The buffers partly crystallize and partly vitrify in the ice matrix; these processes influence the resulting pH and allow designing the optimal storage temperatures in the frozen state. The freezing-induced acidification apparently depends on the buffer concentration; at each pH, we suggest pertinent concentration, at which freezing causes minimal acidification.

Investigation of excipients impact on polysorbate 80 degradation in biopharmaceutical formulation buffers.

A study on the polysorbate 80 stability in various formulation buffers commonly used in biopharmaceuticals was performed, to investigate the excipients influence on polysorbate 80 degradation. Polysorbate 80 is a common excipient in biopharmaceutical products. However, its degradation will potentially impact the drug product quality, and may trigger protein aggregation and particles formation. Due to the heterogeneity of the polysorbates and the mutual effects with other formulation compositions, the study of polysorbate degradation is challenging. Herein, a real-time stability study was designed and performed. The polysorbate 80 degradation trend was monitored by fluorescence micelle-based assay (FMA), reversed-phase-ultra-performance liquid chromatography-evaporative light scattering detector (RP-UPLC-ELSD) assay, and LC-MS assay. These assays provide orthogonal results to reveal both the micelle-forming capability and the compositional changes of polysorbate 80 in different buffer systems. The degradation occurred after a period of storage under 25 °C in different trend, which indicates the excipients could impact the degradation kinetics. Upon comparison, the degradation is prone to happen in histidine buffer than in acetate, phosphate or citrate buffers. LC-MS confirms oxidation as an independent degradation pathway with detection of the oxidative aldehyde. Thus, it is necessary to pay more attention to the excipients selection and their potential impact on polysorbate 80 stability to achieve longer shelf life for the biopharmaceuticals. Besides, the protective roles of several additives were figured out, which could be applied as potential industrial solutions to the polysorbate 80 degradation issues.

Physiology of intracellular calcium buffering.

Calcium signaling underlies much of physiology. Almost all the Ca2+ in the cytoplasm is bound to buffers, with typically only ∼1% being freely ionized at resting levels in most cells. Physiological Ca2+ buffers include small molecules and proteins, and experimentally Ca2+ indicators will also buffer calcium. The chemistry of interactions between Ca2+ and buffers determines the extent and speed of Ca2+ binding. The physiological effects of Ca2+ buffers are determined by the kinetics with which they bind Ca2+ and their mobility within the cell. The degree of buffering depends on factors such as the affinity for Ca2+, the Ca2+ concentration, and whether Ca2+ ions bind cooperatively. Buffering affects both the amplitude and time course of cytoplasmic Ca2+ signals as well as changes of Ca2+ concentration in organelles. It can also facilitate Ca2+ diffusion inside the cell. Ca2+ buffering affects synaptic transmission, muscle contraction, Ca2+ transport across epithelia, and the killing of bacteria. Saturation of buffers leads to synaptic facilitation and tetanic contraction in skeletal muscle and may play a role in inotropy in the heart. This review focuses on the link between buffer chemistry and function and how Ca2+ buffering affects normal physiology and the consequences of changes in disease. As well as summarizing what is known, we point out the many areas where further work is required.

A step closer to continuous buffer preparation from solids: Predicting powder compaction and how to prevent it.

The preparation of buffer solutions used in the biopharmaceutical industry is typically performed manually by the addition of one or multiple buffering reagents to water. Recently, the adaptation of powder feeders for continuous solid feeding was demonstrated for continuous buffer preparation. However, the intrinsic characteristics of powders can change the stability of the process, due to the hygroscopic nature of some substances and humidity-induced caking and compaction behavior, but there is no simple and easy methodology available for predicting this behavior for buffer species. To predict which buffering reagents are suitable without special precautions and investigate their behavior, force displacement measurements were conducted with a customized rheometer over 18 h. While most of the eight investigated buffering reagents indicated uniform compaction, especially sodium acetate and dipotassium hydrogen phosphate (K2HPO4) showed a significant increase in yield stress after 2 h. Experiments conducted with a 3D printed miniaturized screw conveyor confirmed the increased yield stress measurements by visible compaction and failure of the feeding. By taking additional precautions and adjusting the design of the hopper, we demonstrated a highly linear profile of all buffering reagents over a duration of 12 and 24 h. We showed that force displacement measurements accurately predict the behavior of buffer components in continuous feeding devices for continuous buffer preparation and are a valuable tool to identify buffer components that need special precautions. Stable, precise feeding of all tested buffer components was demonstrated, highlighting the importance of identifying buffers that need a specialized setup with a rapid methodology.

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.

Screening of Buffers and Additives for Protein Stabilization by Thermal Shift Assay: A Practical Approach.

Thermal shift assay (TSA), also commonly designed by differential scanning fluorimetry (DSF) or ThermoFluor, is a technique relatively easy to implement and perform, useful in a myriad of applications. In addition to versatility, it is also rather inexpensive, making it suitable for high-throughput approaches. TSA uses a fluorescent dye to monitor the thermal denaturation of the protein under study and determine its melting temperature (Tm). One of its main applications is to identify the best buffers and additives that enhance protein stability.Understanding the TSA operating mode and the main methodological steps is a central key to designing effective experiments and retrieving meaningful conclusions. This chapter intends to present a straightforward TSA protocol, with different troubleshooting tips, to screen effective protein stabilizers such as buffers and additives, as well as data treatment and analysis. TSA results provide conditions in which the protein of interest is stable and therefore suitable to carry out further biophysical and structural characterization.

An automated buffer management system for small-scale continuous downstream bioprocessing.

Buffer management for biopharmaceutical purification processes include buffer preparation, storage of buffers and restocking the buffers when needed. This is usually performed manually by the operators for small scale operations. However, buffer management can become a bottleneck when running integrated continuous purification processes for prolonged times, even at small scale. To address this issue, a buffer management system for the application in continuous lab-scale bioprocessing is presented in this paper. For this purpose, an ÄKTA™ explorer chromatography system was reconfigured to perform the buffer formulation. The system formulated all buffers from stock solutions and water according to pre-specified recipes. A digital twin of the physical system was introduced in the research software Orbit, written in python. Orbit was also used for full automation and control of the buffer system, which could run independently without operator input and handle buffer management for one or several connected buffer-consuming purification systems. The developed buffer management system performed automatic monitoring of buffer volumes, buffer order handling as well as buffer preparation and delivery. To demonstrate the capability of the developed system, it was integrated with a continuous downstream process and supplied all 9 required buffers to the process equipment during a 10-day operation. The buffer management system processed 55 orders and delivered 38 L of buffers, corresponding to 20% of its capacity. The pH and conductivity profiles observed during the purification steps were consistent across the cycles. The deviation in conductivity and pH from the measured average value was within ±0.89% in conductivity and ±0.045 in pH, well within the typical specification for buffer release, indicating that the prepared buffers had the correct composition. The operation of the developed buffer management system was robust and fully automated, and provides one solution to the buffer management bottleneck on lab scale for integrated continuous downstream bioprocessing.

A Systematic Degradation Kinetics Study of Dalbavancin Hydrochloride Injection Solutions.

The degradation kinetics of the glycopeptide antibiotic dalbavancin in solution are systematically evaluated over the pH range 1-12 at 70°C. The decomposition rate of dalbavancin was measured as a function of pH, buffer composition, temperature, ionic strength, and drug concentration. A pH-rate profile was constructed using pseudo first-order kinetics at 70°C after correcting for buffer effects; the observed pH-rate profile could be fitted with standard pseudo first order rate laws. The degradation reactions of dalbavancin were found to be strongly dependent on pH and were catalyzed by protons or hydroxyl groups at extreme pH values. Dalbavancin shows maximum stability in the pH region 4-5. Based on the Arrhenius equation, dalbavancin solution at pH 4.5 is predicted to have a maximum stability of thirteen years under refrigerated conditions, eight months at room temperature and one month at 40°C. Mannosyl Aglycone (MAG), the major thermal and acid degradation product, and DB-R6, an additional acid degradation product, were formed in dalbavancin solutions at 70°C due to hydrolytic cleavage at the anomeric carbons of the sugars. Through deamination and hydrolytic cleavage of dalbavancin, a small amount of DB-Iso-DP2 (RRT-1.22) degradation product was also formed under thermal stress at 70°C. A greater amount of the base degradation product DB-R2 forms under basic conditions at 70°C due to epimerization of the alpha carbon of phenylglycine residue 3.