Oligonucleotide separation techniques for purification and analysis: What can we learn for today's tasks?

Nucleic acids are the blueprint of life. They are not only the construction plan of the single cell or higher associations of them, but also necessary for function, communication and regulation. Due to the pandemic, the attention shifted in particular to their therapeutic potential as a vaccine. As pharmaceutical oligonucleotides are unique in terms of their stability and application, special delivery systems were also considered. Oligonucleotide production systems can vary and depend on the feasibility, availability, price and intended application. To achieve good purity, reliable results and match the strict specifications in the pharmaceutical industry, the separation of oligonucleotides is always essential. Besides the separation required for production, additional and specifically different separation techniques are needed for analysis to determine if the product complies with the designated specifications. After a short introduction to ribonucleic acids (RNAs), messenger RNA vaccines, and their production and delivery systems, an overview regarding separation techniques will be provided. This not only emphasises electrophoretic separations but also includes spin columns, extractions, precipitations, magnetic nanoparticles and several chromatographic separation principles, such as ion exchange chromatography, ion-pair reversed-phase, size exclusion and affinity.     

Rapid purification and size separation of gold nanoparticles via diafiltration

Purification and size-based separation of nanoparticles remain significant challenges in the preparation of well-defined materials for fundamental studies and applications. Diafiltration shows considerable potential for the efficient and convenient purification and size separation of water-soluble nanoparticles, allowing for the removal of small-molecule impurities and for the isolation of small nanoparticles from larger nanostructures in a single process. Herein, we report studies aimed at assessing the suitability of diafiltration for (i) the purification of water-soluble thiol-stabilized 3-nm gold nanoparticles, (ii) the separation of a bimodal distribution of nanoparticles into the corresponding fractions, and (iii) the separation of a polydisperse sample into fractions of differing mean core diameter. NMR, thermogravimetric analysis (TGA), and X-ray photoelectron spectroscopy (XPS) measurements demonstrate that diafiltration produces nanoparticles with a much higher degree of purity than is possible by dialysis or a combination of solvent washes, chromatography, and ultracentrifugation. UV-visible spectroscopic and transmission electron microscopic (TEM) analyses show that diafiltration offers the ability to separate nanoparticles of disparate core size. These results demonstrate the applicability of diafiltration for the rapid and green preparation of high-purity gold nanoparticle samples and the size separation of heterogeneous nanoparticle samples. They also suggest the development of novel diafiltration membranes specifically suited to high-resolution nanoparticle size separation.     

Determination of lipid content and stability in lipid nanoparticles using ultra high-performance liquid chromatography in combination with a Corona Charged Aerosol Detector

For many years, lipid nanoparticles (LNPs) have been used as delivery vehicles for various payloads (especially various oligonucleotides and mRNA), finding numerous applications in drug and vaccine development. LNP stability and bilayer fluidity are determined by the identities and the amounts of the various lipids employed in the formulation and LNP efficacy is determined in large part by the lipid composition which usually contains a cationic lipid, a PEG-lipid conjugate, cholesterol, and a zwitterionic helper phospholipid. Analytical methods developed for LNP characterization must be able to determine not only the identity and content of each individual lipid component (i.e., the parent lipids), but also the associated impurities and degradants. In this work, we describe an efficient and sensitive reversed-phase chromatographic method with charged aerosol detection (CAD) suitable for this purpose. Sample preparation diluent and mobile phase pH conditions are critical and have been optimized for the lipids of interest. This method was validated for its linearity, accuracy, precision, and specificity for lipid analysis to support process and formulation development for new drugs and vaccines.     

纳米粒子毛细管电泳/微流控芯片新技术及其在手性分离中的应用

纳米粒子因其具有较大的比表面积和良好的生物相容性等特点,已广泛应用于分离科学领域。纳米粒子毛细管电泳/微流控芯片技术是纳米材料技术与毛细管电泳/微流控芯片技术相结合的产物。纳米粒子可以被吸附或键合到毛细管壁作为固定相与被分析物发生相互作用;也可以作为假固定相参与样品在柱内的分配和保留,从而提高柱效,改善分离。手性是自然界的本质属性之一,开发新的快速、高效、灵敏的手性分离分析方法对于对映体的立体选择性合成、药理研究、手性纯度检测和环境检测都具有重要的意义。本文主要综述了近些年来几种不同类型纳米粒子(聚合物纳米粒子、磁性纳米粒子、金纳米粒子、碳纳米管和其他类型纳米粒子)用于毛细管电泳/微流控芯片进行手性分离的现状,并对该领域今后的发展进行了展望。     

Current status of multiple antigen-presenting peptide vaccine systems: Application of organic and inorganic nanoparticles

Many studies are currently investigating the development of safe and effective vaccines to prevent various infectious diseases. Multiple antigen-presenting peptide vaccine systems have been developed to avoid the adverse effects associated with conventional vaccines (i.e., live-attenuated, killed or inactivated pathogens), carrier proteins and cytotoxic adjuvants. Recently, two main approaches have been used to develop multiple antigen-presenting peptide vaccine systems: (1) the addition of functional components, e.g., T-cell epitopes, cell-penetrating peptides, and lipophilic moieties; and (2) synthetic approaches using size-defined nanomaterials, e.g., self-assembling peptides, non-peptidic dendrimers, and gold nanoparticles, as antigen-displaying platforms. This review summarizes the recent experimental studies directed to the development of multiple antigen-presenting peptide vaccine systems.     

Alum colloid encapsulated inside β-glucan particles enhance humoral and CTL immune responses of MUC1 vaccine

We have developed a MUC1 antigen-based antitumor vaccine loaded on alum colloid encapsulated insideβ-glucan particles(GP-Al). The constructed vaccine induced strong MUC1 antigen specific Ig G antibody titers and enhanced CD8~+ T cells cytotoxic effect to kill tumor cells. These results indicated that GP-Al can be served as an efficient delivery system and adjuvant for the development of cancer vaccines especially small molecule antigens based cancer vaccines.     

An innovative method for quality control of conjugated Haemophilus influenzae vaccines: A short review of two-dimensional nanoparticle electrophoresis

This article provides an overview of a 2D agarose electrophoretic procedure for the characterization of semi-synthetic Haemophilus influenzae type b meningitis vaccines that were prepared for the immunization of small children. The analysis of such vaccines has been particularly challenging because the vaccine particles (i) are highly negatively charged, (ii) are as large as or even larger than intact viruses, and (iii) have a continuous (polydisperse) size distribution because of randomizing steps in the vaccine production (sonification and crosslinking). As a result of these characteristics, 1D electrophoresis of the vaccines produced smears without discernable peaks, but with a second dimension of separation a characteristic vaccine fingerprint was obtained. Whereas O’Farrell gels can accomplish a 2D separation according to size and charge for samples with protein-sized particles, nondenaturing 2D agarose electrophoresis achieves a similar result for much larger virus-sized particles. The separation principle, however, is different. Even though the 2D electrophoretic method was developed from 1983 to 1995, it remains a promising tool for vaccine quality control and for predicting vaccine effectiveness. Modern technology makes the analysis significantly more practical and affordable than it was more than 10 years ago, and the method is applicable to a variety of conjugated vaccines and complex mixtures of virus-sized particles.     

Integrity profiling of high throughput screening hits using LC-MS and related techniques

Integrity profiling of HTS hits is valuable for verification of the hit identity and purity. This provides early discovery researchers with more confident SAR theories. Methodology for integrity profiling of HTS hits must be high throughput, consume little material, and selectively provide structure-based data. Analytical techniques that can be utilized for integrity profiling methods are reviewed for their appropriateness in sample preparation, component separation, detection, purity quantitation, identity confirmation, and follow-up.     

Liquid chromatographic determination of total and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid in lathyrus sativus seeds using both refractive index and bioelectrochemical detection

The development of a stability-indicating assay for ranitidine hydrochloride using a mobile phase added ion-interaction reagent was achieved. The assay easily separated all known and unknown impurities/degradants. This assay may be used for the determination of purity, identity and strength for the active ingredient and finished dosage forms. Placebo samples were analyzed for all of the dosage forms and did not interfere with the separation. The system was found to be linear over a range of 0.056 to 44.4 microg/g, with a limit of detection of 0.028 microg/g and a limit of quantitation of 0.056 microg/g. The system precision was determined to be 0.7%. The development of the stability-indicating assay and the effect that each chromatographic variable had on the separation will be discussed.     

Polysaccharide conjugate vaccine:A kind of vaccine with great development potential

Diseases caused by microbial bacteria such as Haemophilus influenzae type b(Hib),Streptococcus pneumoniae and Neisseria meningitidis are still very serious disease,which has brought a lot of burden to many countries.Development of vaccine has brought hope for the prevention of such diseases.Polysaccharide conjugate vaccines have been shown to have very good effects in preventing such diseases.The polysaccharide co njugate vaccine adds the positive characteristics of protein antigens to the polysaccharide antigen,thereby improving the immunogenicity of the polysaccharide antigen,solving the problem that the polysaccharide vaccine cannot be effectively applicated in toddler or children,which greatly promoting the development of this vaccine.This review introduces the progress of polysaccharide conjugate vaccines.We introduce the typical polysaccharide conjugate vaccines currently on the market firstly,and then elucidate the protein carriers,the coupling chemistry methods and quality control that required in the preparation of polysaccharide conjugate vaccines.We can see that polysaccharide conjugate vaccine is a kind of vaccine with great development potential,which can be a sharp edge for us to prevent diseases.     

Development of an imaged capillary isoelectric focusing method for characterizing the surface charge of mRNA lipid nanoparticle vaccines

Lipid nanoparticles (LNPs) have been employed for drug delivery in small molecules, siRNA, mRNA, and pDNA for both therapeutics and vaccines. Characterization of LNPs is challenging because they are heterogeneous mixtures of large complex particles. Many tools for particle size characterization, such as dynamic and static light scattering, have been applied as well as morphology analysis using electron microscopy. CE has been applied for the characterization of many different large particles such as liposomes, polymer, and viruses. However, there have been limited efforts to characterize the surface charge of LNPs and CIEF has not been explored for this type of particle. Typically, LNPs for delivery of oligonucleotides contain at least four different lipids, with at least one being an ionizable cationic lipid. Here, we describe the development of an imaged capillary isoelectric focusing method used to measure the surface charge (i.e., pI) of an LNP‐based mRNA vaccine. This method is capable of distinguishing the pI of LNPs manufactured with one or more different ionizable lipids for the purpose of confirming LNP identity in a manufacturing setting. Additionally, the method is quantitative and stability‐indicating making it suitable for both process and formulation development.     

Analytical methods for separating and isolating magnetic nanoparticles

Despite the large body of literature describing the synthesis of magnetic nanoparticles, few analytical tools are commonly used for their purification and analysis. Due to their unique physical and chemical properties, magnetic nanoparticles are appealing candidates for biomedical applications and analytical separations. Yet in the absence of methods for assessing and assuring their purity, the ultimate use of magnetic particles and heterostructures is likely to be limited. In this review, we summarize the separation techniques that have been initially used for this purpose. For magnetic nanoparticles, it is the use of an applied magnetic flux or field gradient that enables separations. Flow based techniques are combined with applied magnetic fields to give methods such as magnetic field flow fractionation and high gradient magnetic separation. Additional techniques have been explored for manipulating particles in microfluidic channels and in mesoporous membranes. Further development of these and new analytical tools for separation and analysis of colloidal particles is critically important to enable the practical use of these, particularly for medicinal purposes.     

Injectable Biodegradable Chitosan‐Alginate 3D Porous Gel Scaffold for mRNA Vaccine Delivery

mRNA vaccines have proven to be more stable, effective, and specific than protein/peptide‐based vaccines in stimulating both humoral and cellular immune response. However, mRNA's fast degradation rate and low‐transfection efficiency in vivo impede its potential in vaccination. Recent research in gene delivery has focused on nonviral vaccine carriers and either implantable or injectable delivery systems to improve transgene expression in vivo. Here, an injectable chitosan‐alginate gel scaffold for the local delivery of mRNA vaccines is reported. Gel scaffold biodegradation rates and biocompatibility are quantified. Scaffold‐mediated mRNA in vivo transgene expression as well as ovalbumin antigen specific cellular and humoral immune responses are evaluated in vivo. Luciferase reporter protein expression resulting from mRNA lipoplex‐loaded gel scaffolds is five times higher than systemic injection. Compared to systemic injections of naked mRNA or mRNA:lipoplexes, elevated levels of T cell proliferation and IFN‐γ secretion are seen with in vivo scaffold‐mediated mRNA lipoplex delivery. Furthermore, a humoral response (ovalbumin antigen specific IgG levels) is observed as early as week 1 for scaffold‐mediated mRNA lipoplex delivery, while protein‐based immunization did not elicit IgG production until 2 weeks post‐injection. Results suggest that injectable scaffold mRNA vaccine delivery maybe a viable alternative to traditional nucleic acid immunization methods.     

Electrophoretic methods for separation of nanoparticles

This article reviews progress in the application of electrophoretic techniques for the separation of nanoparticles. Numerous types of nanoparticles have recently been synthesised and integrated into different products and procedures. Consequently, analytical methods for the efficient characterisation of nanoparticles are now required. Several studies have revealed that gel electrophoresis can readily be used for separating nanoparticles according to their size or shape. However, many other studies focused on separation of nanoparticles by CE. In some cases nanoparticles could be separated by CZE, simply using pure buffer as the BGE. In other studies, buffer additives (most often SDS) were used, enabling fast separations of metallic nanoparticles by size. Other CE methods also allowed for separation of nanoparticle conjugates with biomolecules. Dielectrophoresis is yet another electrophoretic technique useful in separation and characterisation of nanoparticles; particularly nanotubes. Detection methods often used after electrophoretic separation include UV/Vis absorption and fluorescence spectroscopy. Examples of recent and relevant older reports are presented here. The authors conclude that electrophoretic methods for nanoanalysis can provide inexpensive and efficient tools for quality assurance and safety control; and as a consequence, they can augment transfer of nanotechnologies from research to industry.     

超顺纳米磁颗粒在生化分离分析中的研究进展

超顺纳米磁颗粒由于具有超顺磁性、单分散性好、磁饱和强度大、分离速度快等优点,不仅可以作为磁免疫亲和载体,而且可以作为磁信号传感元件和信号的放大系统,因此在生化分析领域中得到了广泛的应用。该文介绍了超顺纳米磁颗粒在分离纯化、生物传感器、免疫学检测、分子生物学等生化分析领域方面的最新研究进展,并对超顺纳米磁颗粒在生化分析领域中的应用潜力进行了展望。     

DNA vaccines for cervical cancer: from bench to bedside

More than 99% of cervical cancers have been associated with human papillomaviruses (HPVs), particularly HPV type 16. The clear association between HPV infection and cervical cancer indicates that HPV serves as an ideal target for development of preventive and therapeutic vaccines. Although the recently licensed preventive HPV vaccine, Gardasil, has been shown to be safe and capable of generating significant protection against specific HPV types, it does not have therapeutic effect against established HPV infections and HPV-associated lesions. Two HPV oncogenic proteins, E6 and E7, are consistently co-expressed in HPV-expressing cervical cancers and are important in the induction and maintenance of cellular transformation. Therefore, immunotherapy targeting E6 and/or E7 proteins may provide an opportunity to prevent and treat HPV-associated cervical malignancies. It has been established that T cell-mediated immunity is one of the most crucial components to defend against HPV infections and HPV-associated lesions. Therefore, effective therapeutic HPV vaccines should generate strong E6/E7-specific T cell-mediated immune responses. DNA vaccines have emerged as an attractive approach for antigen-specific T cell-mediated immunotherapy to combat cancers. Intradermal administration of DNA vaccines via a gene gun represents an efficient way to deliver DNA vaccines into professional antigen-presenting cells in vivo. Professional antigen-presenting cells, such as dendritic cells, are the most effective cells for priming antigen-specific T cells. Using the gene gun delivery system, we tested several DNA vaccines that employ intracellular targeting strategies for enhancing MHC class I and class II presentation of encoded model antigen HPV-16 E7. Furthermore, we have developed a strategy to prolong the life of DCs to enhance DNA vaccine potency. More recently, we have developed a strategy to generate antigen-specific CD4(+) T cell immune responses to further enhance DNA vaccine potency. The impressive pre- clinical data generated from our studies have led to several HPV DNA vaccine clinical trials.     

Gas separation membrane cascades I. One-compressor cascades with minimal exergy losses due to mixing

In this first part of this two part paper, attention is paid to the one-compressor cascades with minimal exergy loss due to mixing of streams with different compositions. The desire to avoid mixing losses leads to the development of several new binary gas separation process schemes with no exergy losses due to mixing of streams with different compositions. These schemes are simulated and compared with the “series type two unit separation cell” for higher purity/recovery separations, and with other conventional schemes for enrichment or rejection of the more permeable component. These comparisons show that schemes with no mixing losses are much more efficient than conventional schemes in most cases, although they are only marginally more efficient for low pressure side feed schemes with high permeate purity requirements.     

Sixteen capillary electrophoresis applications for viral vaccine analysis

A broad range of CE applications from our organization is reviewed to give a flavor of the use of CE within the field of vaccine analyses. Applicability of CE for viral vaccine characterization, and release and stability testing of seasonal influenza virosomal vaccines, universal subunit influenza vaccines, Sabin inactivated polio vaccines (sIPV), and adenovirus vector vaccines were demonstrated. Diverse CZE, CE-SDS, CGE, and cIEF methods were developed, validated, and applied for virus, protein, posttranslational modifications, DNA, and excipient concentration determinations, as well as for the integrity and composition verifications, and identity testing (e.g., CZE for intact virus particles, CE-SDS application for hemagglutinin quantification and influenza strain identification, chloride or bromide determination in process samples). Results were supported by other methods such as RP-HPLC, dynamic light scattering (DLS), and zeta potential measurements. Overall, 16 CE methods are presented that were developed and applied, comprising six adenovirus methods, five viral protein methods, and methods for antibodies determination of glycans, host cell-DNA, excipient chloride, and process impurity bromide. These methods were applied to support in-process control, release, stability, process- and product characterization and development, and critical reagent testing. Thirteen methods were validated. Intact virus particles were analyzed at concentrations as low as 0.8 pmol/L. Overall, CE took viral vaccine testing beyond what was previously possible, improved process and product understanding, and, in total, safety, efficacy, and quality.     

Immunological Evaluation of Co-Assembling a Lipidated Peptide Antigen and Lipophilic Adjuvants: Self-Adjuvanting Anti-Breast-Cancer Vaccine Candidates

Co-assembling vaccines composed of a lipidated HER2-derived antigenic CH401 peptide and either a lipophilic adjuvant, Pam₃CSK₄, α-GalCer, or lipid A 506, were evaluated as breast cancer vaccine candidates. This vaccine design was aimed to inherit both antigen multivalency and antigen-specific immunostimulation properties, observed in reported self-adjuvanting vaccine candidates, by using self-assembly and adjuvant-conjugated antigens. Under vaccination concentrations, respective lipophilic adjuvants underwent co-assembly with lipidated CH401, which boosted the anti-CH401 IgG and IgM production. In particular, α-GalCer was responsible for the most significant immune activation. Therefore, the newly developed vaccine design enabled the optimization of adjuvants against the antigenic CH401 peptide in a simple preparatory manner. Overall, the co-assembling vaccine design opens the door for efficient and practical self-adjuvanting vaccine development.     

Efficient separation of high‐purity compounds from Oxytropis falcata using two‐dimensional preparative chromatography

The separation of high‐purity compounds from traditional Tibetan medicines plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of traditional Tibetan medicines. In this work, an offline two‐dimensional reversed‐phase preparative method was successfully developed for the separation of high‐purity compounds from Oxytropis falcata . Based on the analysis results, an ODS C18 prep column was used for first‐dimensional preparation, and 14.8 g of the crude sample was separated into five fractions with a recovery of 74.6%. Then, an XAqua C18 prep column was used to isolate high‐purity compounds in the second‐dimensional preparation because its separation selectivity is different with the ODS C18 stationary phase. As a result, eight compounds in the crude sample were isolated in more than 98% purity. This is the first report of trans‐cinnamic acid ( 1 ) and trifolirhizin ( 2 ) from Oxytropis falcata . This method has the potential to be an efficient separation method of high‐purity compounds from Oxytropis falcata and it shows great promise for the separation of high‐purity compounds from complex samples.