Bacterial extracellular vesicles (BEVs) have extraordinary biotechnological potential, but traditional purification methods lack desirable scalability and commonly co-isolate protein impurities, limiting clinical translation. Anion exchange chromatography (AEC) separates molecules based on differences in net charge and is widely used for industrial biomanufacturing of protein therapeutics. Recently, AEC has recently been applied for purification of EVs from both mammalian and bacterial sources. Since most bacteria produce BEVs with a negative surface membrane change, AEC can potentially be widely used for BEV purification. Here, we describe a method utilizing high-performance AEC (HPAEC) in tandem with size-based tangential flow filtration for improved BEV purification. We have previously found this method can reduce co-isolated protein impurities and potentiate anti-inflammatory bioactivity of probiotic BEVs. Thus, this method holds promise as a scalable alternative for improved BEV purification.

Extracellular vesicles (EVs) are relatively recently discovered biological nanoparticles that mediate intercellular communication. The development of new methods for the isolation and characterization of EVs is crucial to support further studies on these small and structurally heterogenous vesicles. New scalable production methods are also needed to meet the needs of future therapeutic applications. A reliable inline detection method for the EV manufacturing process is needed to ensure reproducibility and to identify any possible variations in real time. Here, we demonstrate the use of an inline Raman detector in conjunction with anion exchange chromatography for the isolation of EVs from human platelets. Anion-exchange chromatography can be easily coupled with multiple inline detectors and provides an alternative to size-based methods for separating EVs from similar-sized impurities, such as lipoprotein particles. Raman spectroscopy enabled us to identify functional groups in EV samples and trace EVs and impurities in different stages of the process. Our results show a notable separation of impurities from the EVs during anion-exchange chromatography and demonstrate the power of inline Raman spectroscopy. Compared to conventional EV analysis methods, the inline Raman approach does not require hands-on work and can provide detailed, real-time information about the sample and the purification process.

The recent FDA approval of several adeno-associated virus (AAV)-based gene therapies is driving demand for AAV production. One of the biggest AAV manufacturing challenges is removing \"empty\" capsids, which do not contain the gene of interest. Anion exchange chromatography has emerged as the leading solution for scalable full capsid enrichment. Here we develop a process for the baseline separation of empty and full AAV capsids using anion exchange membrane chromatography. This process development approach utilized AAV serotypes 8 and 9 and traverses initial screening of separation conditions up to manufacturing-scale processes. Process development of a two-step elution was performed via response surface DoE, exploring conductivity and the length of the first elution step. The results from response surfaces were used to construct statistical models of the process operating space. These models provide optimal conditions for recovery and purity, both of which can exceed 70 %. Model predictions were then validated at small scale prior to scale-up. We present the results from our scale-up purification and show that purity and yield are consistent with the results obtained from the response surface model.

Tobacco flavor, an important tobacco additive, is an essential raw material in cigarette production that can effectively improve the quality of tobacco products, add aroma and taste, and increase the suction flavor. The quality consistency of tobacco flavors affects the quality stability of branded cigarettes. Therefore, the quality control of tobacco flavors is a major concern for cigarette and flavor manufacturers. Physical and chemical indices, odor similarity, and sensory efficacy are employed to evaluate the quality of tobacco flavors, and the analysis of chemical components in tobacco flavors is usually conducted using gas chromatography (GC) and high performance liquid chromatography (HPLC). However, because the composition of tobacco flavors is complex, their quality cannot be fully reflected using a single component or combination of components. Therefore, establishing an objective analytical method for the quality control of tobacco flavors is of extreme importance. Chromatographic fingerprint analysis is routinely used for the discriminative analysis of tobacco flavors. Chromatographic fingerprints refer to the general characteristics of the concentration profiles of different chemical compounds. In the daily procurement process, fingerprints established by GC and HPLC are effective for the evaluation and identification of tobacco flavors. However, given continuous improvements in aroma-imitation technology, some flavors with high similarity cannot be directly distinguished using existing methods. In this study, a method for the determination of organic acids and inorganic anions in tobacco flavors based on ion chromatography (IC) was developed to ensure the quality consistency of tobacco flavors. A 1.0 g sample of tobacco flavors and 10 mL of deionized water were mixed and vibrated for 30 min. The aqueous sample solution was passed through a 0.45 μm membrane filter and RP pretreatment column in succession to eliminate interferences and then subjected to IC. Standard solutions containing nine organic acids and seven inorganic anions were used to identify the anions in the tobacco flavors, and satisfactory reproducibility was obtained. The relative standard deviations (RSDs) for retention times and peak areas were <0.71% and <6.02%, respectively. The chromatographic fingerprints of four types of tobacco flavors (samples A-D) from five different batches were obtained. Nine tobacco flavor samples from different manufacturers (samples AY1-AY3, BY1-BY2, CY1-CY2, DY1-DY2) were also analyzed to obtain their chromatographic fingerprints. Hierarchical cluster and similarity analyses were used to evaluate the quality of tobacco flavors from different manufacturers. Hierarchical clustering refers to the process of subdividing a group of samples into clusters that exhibit a high degree of intracluster similarity and intercluster dissimilarity. The dendrograms obtained using SPSS 12.0 indicated good quality consistency among the samples in different batches. Samples AY3, BY2, CY2, and DY1 clustered with the batches of standard tobacco flavors. Therefore, hierarchical cluster analysis can effectively distinguish the quality of products from different manufacturers. The Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (version 2.0) was used to evaluate the similarity between the standard tobacco flavors and products from different manufacturers. Among the samples analyzed, samples AY3, BY2, CY2, and DY1 showed the highest similarity values (>97.7%), which was consistent with the results of the hierarchical cluster analysis. This finding indicates that IC combined with chromatographic fingerprint analysis could accurately determine the quality of tobacco flavors. GC combined with ultrasonic-assisted liquid-liquid extraction was also used to analyze the tobacco flavors and verify the accuracy of the proposed method. Compared with GC coupled with ultrasonic-assisted liquid-liquid extraction, IC demonstrated more significant quality differences among certain tobacco flavors.

Hydrogen sulfide (H2S) is a pervasive gaseous pollutant that emits the characteristic odor of rotten gas, even at low concentrations. It is generated during various industrial processes, including petroleum and natural gas refining, mining operations, wastewater treatment activities, and refuse disposal practices. According to statistics from the World Health Organization (WHO), over 70 occupations are exposed to H2S, rendering it a key monitoring factor in occupational disease detection. Although H2S has legitimate uses in the chemical, medical, and other fields, prolonged exposure to this gas can cause severe damage to the respiratory and central nervous systems, as well as other organs in the human body. Moreover, the substantial release of H2S into the environment can lead to significant pollution. This noxious substance has the potential to impair soil, water, and air quality, while disrupting the equilibrium of the surrounding ecosystems. Therefore, sulfide has become one of the most commonly measured substances for environmental monitoring worldwide. Achieving the stable enrichment and accurate detection of low-level H2S is of great significance. Common methods for detecting this gas include spectrophotometry, chemical analysis, gas chromatography, rapid field detection, and ion chromatography. Although these methods provide relatively reliable results, they suffer from limitations such as high detection cost, low recovery, lack of environmental friendliness, and imprecise quantification of low-concentration H2S. Furthermore, the sampling processes involved in these methods are complex and require specialized equipment and electrical devices. Additionally, approximately 20% of the sulfides in a sample are lost after 2 h in a conventional alkaline sodium hydroxide solution, causing difficulties in preservation and detection. In this study, an accurate, efficient, and cost-saving method based on ion chromatography-pulse amperometry was developed for H2S determination. A conventional IonPac AS7 (250 mm×4 mm) anion-exchange column was employed, and a new eluent based on sodium hydroxide and sodium oxalate was used to replace the original sodium hydroxide-sodium acetate eluent. The main factors influencing the separation and detection performance of the proposed method, including the pulse amperage detection potential parameters and integration time, as well as the type and content of additives in the stabilizing solution, were optimized. The results showed that the proposed method had a good linear relationship between 10 and 3000 μg/L, with correlation coefficients (r2) of up to 0.999. The limits of detection (S/N=3) and quantification (S/N=10) were 1.53 and 5.10 μg/L, respectively. The relative standard deviations (RSDs) of the peak area and retention time of sulfides were less than 0.2% (n=6). The new method exhibited excellent stability, with up to 90% reduction in reagent costs. Compared with conventional ion chromatography-pulse amperometry, this method is more suitable for detecting low concentrations of sulfides in actual samples. Sulfides in a 250 mmol/L sodium hydroxide-0.8% (mass fraction) ethylenediaminetetraacetic acid disodium salt solution were effectively maintained for over 10 h. The new stabilizer significantly improved the reliability of both large-scale and long-term detection. The recovery of the proposed method was investigated by combining the system with a badge-type passive sampler. This sampling method requires no power devices; it is inexpensive, simple to operate, and can realize long-term sampling without the need for skilled personnel. Moreover, it can overcome the influence of short-term changes in pollutant concentration. The sampling results have high reference value for large-scale intervention-less pollutant monitoring in ultraclean rooms, museum counters, and other places. The results demonstrated that the recovery of the proposed method was greater than 95% for the blank sample and 80% for the sample plus standard solution. Finally, the newly established method was applied to determine H2S levels in air samples collected via passive sampling at school garbage stations. The measured results did not exceed the national limit.

Ion chromatography and related techniques have been the most popular separation methods used in the determination of organic and inorganic anions and cations, predominantly in water and wastewater samples. Making progress in their development and introducing new stationary phases, methods of detection and preparation of samples for analyses have given rise to the broadening of their analytical range. Nowadays, they are also used for substances that are not ionic by nature but can convert to such forms under certain conditions. These encompass, among others, carbohydrates, whose role and significance in humans\' lives and environment is invaluable. Their presence in the air is mostly due to the industrial burning of biomass for energy production purposes. In addition, the content of sugars in plants, fruits and vegetables, constituting the base of human diets, affects our health condition. Given that, there is not only a need for their determination by means of routine methods but also for searching for novel analytical solutions. Based on literature data from the past decade, this paper presents the possibilities and examples of applications regarding ion chromatography and related techniques for the determination of carbohydrates in environmental samples, biomass and plants constituting food or raw materials for food production. Attention has been paid to the virtues and limitations of the discussed separation methods in this respect. Moreover, perspectives on their development have been defined.

This paper discusses the development of an analytical method by an alternative separation approach, sequential elution liquid chromatography (SE-LC), to separate permanently charged ions (anions), weak acids, and neutral compounds using anion exchange and reversed-phase columns in tandem. SE-LC separates classes of compounds by group by employing two or more elution modes. Advantages to using SE-LC over conventional HPLC are a greater peak capacity and a reduced separation disorder. Importantly, the same HPLC as used for a conventional HPLC separation may be used to afford a successful SE-LC separation. Mobile phase selection and gradient optimization are integral for a successful SE-LC class separation of permanent anions, weak acids, and neutral compounds and will be discussed in detail in this paper. The most successful (best resolution and repeatability) SE-LC separation was achieved by applying isocratic elution at low pH to elute the weak acids, followed by an acetonitrile gradient to elute the neutral compounds, and last a sodium methanesulfonate gradient to elute the anionic compounds using a superficially porous C18 column coupled with a strong anion exchange (SAX) column. Repeatability (RSD) in the retention times and peak areas of the analytes was less than 0.25 % and 1.5 %, respectively.

Plasmid DNA (pDNA) is an essential tool in genetic engineering that has gained prevalence in cell and gene therapies. Plasmids exist as supercoiled (SC), open circular (OC), and linear forms. Plasmid multimerization can also occur during the manufacturing process. Even though the SC forms are thought to provide optimal knock-in (KI) efficiency, there is no strong consensus on the effect of the topological forms and multimers on the functional activity. In addition, the results obtained for conventional pDNAs (>5 kbp) do not necessarily translate to smaller pDNAs (∼3 kbp). In this study, a workflow was developed for the analytical and functional characterization of pDNA topological forms and multimers. An anion exchange chromatography (AEC) method was first developed to quantify the topological forms and multimers. Four AEC columns were initially compared, one of which was found to provide superior chromatographic performance. The effect of mobile phase pH, various salts, column temperature, and acetonitrile content on the separation performance was systematically studied. The method performance, including precision and accuracy, was evaluated. The final AEC method was compared to capillary gel electrophoresis (CGE) by analyzing several pDNA sequences and lots. A forced degradation study revealed unexpectedly high degradation of the SC forms. Finally, the KI efficiency was compared for the SC and OC forms, and the multimers.

Real-time state estimation in chromatography is a useful tool to improve monitoring of biopharmaceutical downstream processes, combining mechanistic model predictions with real-time data acquisition to obtain an estimation that surpasses that of either approach individually. One common technique for real-time state estimation is Kalman filtering. However, non-linear adsorption isotherms pose a significant challenge to Kalman filters, which are dependent on fast algorithm execution to function. In this work, we apply Kalman filtering of non-constant elution conditions using a non-linear adsorption isotherm using a novel approach where dual Kalman filters are used to estimate the states of the adsorption modifier, salt, and the components to be separated. We performed offline tuning of the Kalman filters on real chromatogram data from a linear gradient, ion-exchange separation of two proteins. The tuning was then validated by running the Kalman filters in parallel with a chromatographic separation in real time. The resulting, tuned, dual Kalman filters improved the L2 norm by 53 % over the open-loop model prediction, when compared to the true elution profiles. The Kalman filters were also applicable in real-time with a signal sampling frequency of 5 s, enabling accurate and robust estimation and paving the way for future applications beyond monitoring, such as real-time optimal pooling control.

As an advanced analytical technology, Ion Chromatography (IC) has been widely used in various fields. At present, it is faced with the challenges of sample complexity and instrument precision. It is necessary to select appropriate pretreatment methods to achieve sample preparation and protect the instruments. Therefore, this paper reviews several commonly used sample pretreatment technologies in IC, focusing on sample digestion and purification techniques. Additionally, we introduce some advanced IC technologies and automatic sample processing devices. We provide a comprehensive summary of the basic principles, primary applications and the advantages and disadvantages of each method. Pretreatment methods should be carefully selected and optimized on the specific characteristics of the sample and the ions to be measured, in order to achieve better analysis results.