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N‐glycans present in milk glycoproteins (e.g. lactoferrin and immunoglobulin G) can provide infants with nutritional and protective effects similar to breast milk. In this study, we enriched N‐glycoproteins from WPC 70 using ultrafiltration and anion exchange chromatography on DEAE Sepharose Fast Flow column, with N‐glycan content and N‐glycoprotein purity as evaluation indicators. The results showed that changing feed flow rate and molecular weight cut‐off was more effective to improve ultrafiltration separation than changing feed concentration. When using a 100 kDa ultrafiltration membrane with a feed flow rate of 10 rpm and a feed concentration of 10 mg/mL, N‐glycan content could be increased by 200%. However, anion exchange chromatography was more efficient for purification, increasing N‐glycan content by 700%. This study provided a reliable method for N‐glycan enrichment and a scientific basis for further research and potential application of N‐glycans in infant formula and whey products. [ABSTRACT FROM AUTHOR]

While bioactivity and a favorable safety profile for biotherapeutics is of utmost importance, manufacturability is also worth of consideration to ease the manufacturing process. Manufacturability in the scientific literature is mostly related to stability of formulated drug substances, with limited focus on downstream process‐related manufacturability, that is, how easily can a protein be purified. Process‐related impurities or biological impurities like viruses and host cell proteins (HCP) are present in the harvest which have mostly acid isoelectric points and need to be removed to ensure patient safety. Therefore, during molecule design, the surface charge of the target molecule should preferably differ sufficiently from the surface charge of the impurities to enable an efficient purification strategy. In this feasibility study, we evaluated the possibility of improving manufacturability by adapting the surface charge of the target protein. We generated several variants of a GLP1‐receptor‐agonist‐Fc‐domain‐FGF21‐fusion protein and demonstrated proof of concept exemplarily for an anion exchange chromatography step which then can be operated at high pH values with maximal product recovery allowing removal of HCP and viruses. Altering the surface charge distribution of biotherapeutic proteins can thus be useful allowing for an efficient manufacturing process for removing HCP and viruses, thereby reducing manufacturing costs. [ABSTRACT FROM AUTHOR]

The 3,3′‐linked cyclic dinucleotides (CDNs) of adenosine monophosphate (AMP) and guanosine monophosphate (GMP), cyclic di‐AMP (c‐di‐AMP), cyclic di‐GMP (c‐di‐GMP), and c‐GMP–AMP (cGAMP) are second messenger molecules in bacteria that regulate processes, such as biofilm formation, motility, virulence, stress response, and cell wall homeostasis. To analyze the profiles of the three CDNs together with their breakdown and precursor molecules, 5′‐phosphoadenylyl‐(3′ → 5′)adenine (pApA), 5′‐guanylyl‐(3′ → 5′)guanine (pGpG), 5′‐AMP, 3′‐ and 5′‐GMP, adenosine triphosphate (ATP), and GTP, we established an LC–MS/MS‐based approach for semi‐quantification and profiling. Weak anion exchange solid‐phase extraction was employed to improve selectivity and instrumental signal/noise of CDNs as well as pApA and pGpG. CDNs were analyzed using reverse‐phase UHPLC–MS/MS, whereas all other nucleotides were analyzed using hydrophilic interaction chromatography (HILIC)–MS/MS. The instrument limit of quantification ranged from 0.72 (c‐di‐AMP) to 60 nM (ATP and GTP). We applied this method to the analysis of the nine nucleotides in eight bacterial strains and found that the profiles varied widely in terms of both absolute and relative concentrations. Thus, CDN concentrations were generally <1 pmol/mg biomass, and the hydrolysis products, pApA and pGpG, were detected at lower pmol/mg concentrations. The presented method is a relatively simple and straightforward approach to profiling nucleotides with the rationale of comparing their relative levels between populations of bacterial strains. [ABSTRACT FROM AUTHOR]

The application of biotechnology through the utilization of enzymes is considered an easy and inexpensive method of producing valuable products from waste resources. The present study describes the production of a keratinolytic enzyme from Pseudomonas aeruginosa S-04, which showed efficiency for feather biodegradation. The production of extracellular keratinase was improved 1.3-fold by optimizing various parameters through the one factor at a time (OFAT) approach. The keratinase was purified to homogeneity by ammonium sulfate precipitation and DEAE-cellulose anion exchange chromatography. The purified keratinase (35.5 kDa) displayed optimal activity at 60 °C and pH 9.5 and was stable between pH 7 and 9.5 and temperatures ranging from 4 to 40 °C for 2 h. The catalytic activity of keratinase was enhanced in the presence of Fe3+ and Mn2+ ions, Triton X-100, Tween 20, DMSO, isopropyl alcohol and ethanol, but reduced activity was recorded in the presence of methanol and acetone. Furthermore, the enzyme activity was deactivated by EDTA, suggesting that this keratinase belongs to a metalloprotease family. The Km and Vmax of the purified keratinase were 7.62 mg/ml and 200 U/mg protein, respectively. The partially purified keratinase displayed remarkable feather degradation capabilities (93% after 24 h), and the nutritional properties of the resulting feather hydrolysate make it a promising candidate for use as a poultry feed ingredient. Thus, thermostable alkaline keratinase from P. aeruginosa S-04 is a promising candidate that converts low-cost chicken feather waste into a value-added product, indicating a novel approach to feather waste treatment and utilization. [ABSTRACT FROM AUTHOR]

ABSTRACT While bioactivity and a favorable safety profile for biotherapeutics is of utmost importance, manufacturability is also worth of consideration to ease the manufacturing process. Manufacturability in the scientific literature is mostly related to stability of formulated drug substances, with limited focus on downstream process‐related manufacturability, that is, how easily can a protein be purified. Process‐related impurities or biological impurities like viruses and host cell proteins (HCP) are present in the harvest which have mostly acid isoelectric points and need to be removed to ensure patient safety. Therefore, during molecule design, the surface charge of the target molecule should preferably differ sufficiently from the surface charge of the impurities to enable an efficient purification strategy. In this feasibility study, we evaluated the possibility of improving manufacturability by adapting the surface charge of the target protein. We generated several variants of a GLP1‐receptor‐agonist‐Fc‐domain‐FGF21‐fusion protein and demonstrated proof of concept exemplarily for an anion exchange chromatography step which then can be operated at high pH values with maximal product recovery allowing removal of HCP and viruses. Altering the surface charge distribution of biotherapeutic proteins can thus be useful allowing for an efficient manufacturing process for removing HCP and viruses, thereby reducing manufacturing costs.

Carboxylic acids (CAs) are key players in human and animal metabolism. As they are hardly retained under reversed-phase liquid chromatography (RP-LC) conditions in their native form, derivatization is an option to make them accessible to RP-LC and simultaneously increase their response for mass spectrometric detection. In this work, two RP-LC tandem mass spectrometry-based methods using aniline or 3-nitrophenylhydrazine (3-NPH) as derivatization agents were compared with respect to several factors including completeness of derivatization, apparent recoveries (RAs) in both cow feces and ruminal fluid, and concentrations obtained in feces and ruminal fluid of cows. Anion exchange chromatography coupled to high-resolution mass spectrometry (AIC-HR-MS) served as reference method. Derivatization efficiencies were close to 100% for 3-NPH derivatization but variable (20–100%) and different in solvent solutions and matrix extracts for aniline derivatization. Likewise, average RAs of 13C-labeled short-chain fatty acids as internal standards were around 100% for 3-NPH derivatization but only 45% for aniline derivatization. Quantification of CAs in feces and ruminal fluid of cows initially fed a forage-only diet and then transitioned to a 65% high-grain diet which yielded similar concentrations for 3-NPH derivatization and AIC-HR-MS, but concentrations determined by aniline derivatization were on average five times lower. For these reasons, derivatization with aniline is not recommended for the quantitative analysis of CAs in animal samples. [ABSTRACT FROM AUTHOR]

A highly efficient separation method for trace plutonium from high uranium plutonium ratio samples was established by combining two Reillex anion exchange chromatographic columns. The effects of resin type, wash condition, valence state adjustment on plutonium separation were investigated, and the conditions of separation process were determined. When the mass ratio of uranium to plutonium in the feed liquid is 106, after two-stage Reillex column separation, the average yield of plutonium is 99.35% and the decontamination factor of uranium is greater than 107. Using this process, high purity trace 239Pu is separated from irradiated uranium target, and the atomic ratio of (238U+238Pu)/239Pu is (3.60±1.15)×10-5(n=6).

Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. The present work studied the effect of genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension-adapted HEK293 cells via triple transfection using AAV plasmids containing the same GFP transgene with DNA stuffers for variable-size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography, and the results suggest that the triple transfection condition significantly affects the AEX retention time and resolution between the full and empty capsid peaks. Charge detection-mass spectrometry was performed on all AEX full-capsid peak samples showing a wide distribution of empty, partial, full length, and copackaged DNA in the capsids. The AEX-purified samples were then analyzed by differential scanning fluorimetry, and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content.

Increasing plasmid demand for both production of viral and gene therapies as well as nucleic acid based vaccines has highlighted bottlenecks in production. One bottleneck is traditional bead‐based chromatography as a capture step. To meet the needs of fast‐growing markets, new production solutions are needed. These solutions must enable efficient capture of a diverse range of plasmid types and excellent clearance of bacterial host impurities, such as endotoxin. Enhanced endotoxin clearance during chromatographic purification has previously been demonstrated with detergents such as Triton™ X‐100. However, degradation products of Triton™ X‐100 are known to have a negative environmental impact, and more sustainable, environmentally benign alternatives have been identified. This work establishes an efficient, intensified plasmid capture using convective anion exchange (AEX) chromatography. The feasibility of the intensified capture approach was assessed with different membrane and a monolith AEX supports. Various detergents from different physico‐chemical classes were evaluated with different AEX technologies. Purification efficiency evaluated endotoxin and host cell protein (HCP) clearance, plasmid yield, potential interference of the detergents with analytical in‐process control assays, and overall process compatibility. This comprehensive screening approach provides valuable insights to intensified plasmid production. [ABSTRACT FROM AUTHOR]

Adeno‐associated virus‐based gene therapies have demonstrated substantial therapeutic benefit for the treatment of genetic disorders. In manufacturing processes, viral capsids are produced with and without the encapsidated gene of interest. Capsids devoid of the gene of interest, or "empty" capsids, represent a product‐related impurity. As a result, a robust and scalable method to enrich full capsids is crucial to provide patients with as much potentially active product as possible. Anion exchange chromatography has emerged as a highly utilized method for full capsid enrichment across many serotypes due to its ease of use, robustness, and scalability. However, achieving sufficient resolution between the full and empty capsids is not trivial. In this work, anion exchange chromatography was used to achieve empty and full capsid resolution for adeno‐associated virus serotype 5. A salt gradient screen of multiple salts with varied valency and Hofmeister series properties was performed to determine optimal peak resolution and aggregate reduction. Dual salt effects were evaluated on the same product and process attributes to identify any synergies with the use of mixed ion gradients. The modified process provided as high as ≥75% AAV5 full capsids (≥3‐fold enrichment based on the percent full in the feed stream) with near baseline separation of empty capsids and achieved an overall vector genome step yield of >65%. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated viral vector (rAAV) mediated gene therapy is gaining traction in treating genetic disorders. Current rAAV production systems yield a mixture of capsids largely devoid of the transgene (empty capsid) compared with the desired therapeutic product (full capsid). Anion exchange chromatography (AEX) is an attractive method for separating empty and full AAV capsids because of its scalability. Resin types and buffer composition are key considerations for AEX and must support capsid stability to be suitable for downstream processing. We examined the impact of binding durations (0–8 h) using various binding ionic strengths (15–75 mM), pH (7.5–9.0), resin chemistry (POROS XQ, POROS HQ, POROS I, and BIA QA monolith), and proprietary Q resins with different ligand densities for effects on capsid stability. Empty capsids were altered upon extended binding, leading to retention time shifts and loss of resolution between empty and full capsids. Viral capsid protein analysis reveals that full capsids have more viral capsid protein 3 (VP3) proteins than empty capsids. Analytical hydrophilic liquid chromatography showed that empty capsid retention time shift is accompanied by changes to the empty capsid’s native VP3 protein. Among the potential stabilizing additives considered, magnesium chloride was the most effective at reducing negative impacts caused by extended binding.

BACKGROUND:Activated almonds are raw almonds that have been soaked in water for 12-24 h at room temperature, sometimes followed by a 24 h drying period at low temperature (50 ± 5 °C). This treatment is thought to enhance the nutrient bioavailability of almonds by degrading nutrient inhibitors, such as phytic acid or d-myo-inositol hexaphosphate (InsP6 ), through the release of phytase or passive diffusion of InsP6 into the soaking water. Over a wide pH range, InsP6 is a negatively charged compound that limits the absorption of essential nutrients by forming insoluble complexes with minerals such as iron and zinc. It is hypothesized that hydrating the seed during soaking triggers InsP6 degradation into lower myo-inositol phosphates with less binding capacity. RESULTS:Anion-exchange chromatography coupled with tandem mass spectrometry was used to quantify myo-inositol mono-, di-, tris-, tetra-, penta-, and hexaphosphates (InsP1-6 ) in raw pasteurized activated almonds. At least 24 h of soaking at ambient temperature was required to reduce InsP6 content from 14.71 to 14.01 µmol g-1 . CONCLUSIONS:The reduction in InsP6 is statistically significant (P

Newcastle disease virus (NDV), belonging to the species avian orthoavulavirus 1, genus Orthoavulavirus, and family Paramyxoviridae, is responsible for Newcastle disease in poultry and other avian species. It has shown significant potential as an oncolytic virus and as a vector for vaccine delivery. NDV from infected biological serum is usually isolated or purified using density gradient ultracentrifugation. However, it has many disadvantages, including the fact that it is time consuming and can process only a limited quantity of sample at one time. In our study, native agarose gel electrophoresis and dynamic light scattering (DLS) analysis showed that NDV carried a net negative surface charge. Thus, we purified the virus using a HiTrap Q Sepharose Fast Flow anion exchange column with salt elution. Hemagglutination assay and plaque assay showed that the procedure yielded high‐purity NDV particles with a recovery of more than 80%, and the process was fast and simple. The purity of the virus was confirmed using sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blot analysis. The hydrodynamic volume and 'dry state' diameter of the purified NDV were analyzed using dynamic light scattering and transmission electron microscopy and were to be in the range of 200–300 nm. The viruses did not exhibit any deviation from their known physical properties. The genome of the virus was also detected by amplifying a 423‐bp region using reverse transcription‐polymerase chain reaction. Our study confirmed that NDV could be effectively purified using an anion exchange column. In addition, the procedure could be easily upscaled or downscaled based on the experimental requirements. [ABSTRACT FROM AUTHOR]

Abstract Background Since many research works have been carried out to identify the pharmacological properties of conotoxins from many Conus sp. The pharmacological properties of higher molecular weight toxin from the venom of Conus sp. have been poorly elucidated. Therefore, the present investigation was made to isolate the higher molecular weight toxin from the venom of Conus virgo and to test its antitumor effect on rat glioma cell line. Results The partially purified fraction 1 showed better activity (IC50 50 μg/ml) than fraction 2 (IC50 150 µg/ml) after 24 h incubation. The decreased elevation of creatine kinase was documented on fraction 1treated C6 cells. Vero cells showed minimal cell damage at 150 μg/ml 24 h as well. The nuclear morphology and nuclear fragmentation showed the presence of nuclear damage in C6 cells. Western blot analysis revealing the upregulation of the p53-mediated intrinsic apoptotic pathway was documented. Conclusions The present investigation emphasizes that the partially purified fraction 1 has triggered p53 mediated apoptosis by synergism of two higher molecular weight toxins in Rat glioma cell.

Adeno-associated virus (AAV)-based gene therapy is experiencing a rapid growth in the field of medicine and holds great promise in combating a wide range of human diseases. For successful development of AAV-based products, comprehensive thermal stability studies are often required to establish storage conditions and shelf life. However, as a relatively new modality, limited studies have been reported to elucidate the chemical degradation pathways of AAV products under thermal stress conditions. In this study, we first presented an intriguing difference in charge profile shift between thermally stressed AAV8 and AAV1 capsids when analyzed by anion exchange chromatography. Subsequently, a novel and robust peptide mapping protocol was developed and applied to elucidate the underlying chemical degradation pathways of thermally stressed AAV8 and AAV1. Compared to the conventional therapeutic proteins, the unique structure of AAV capsids also led to some key differences in how modifications at specific sites may impact the overall charge properties. Finally, despite the high sequency identity, the analysis revealed that the opposite charge profile shifts between thermally stressed AAV8 and AAV1 could be mainly attributed to a single modification unique to each serotype. • Opposite charge profile shifts observed for thermally stressed AAV8 and AAV1. • An optimized one-pot tryptic digestion protocol developed for AAV peptide mapping. • The location of modifications largely impacts the AAV retention on AEX. • A unique PTM specific to each AAV serotype is mainly responsible for the observed charge profile shift. [ABSTRACT FROM AUTHOR]

• Purification of an oligonucleotide through Integrated Batch process. • Performance comparison between the Integrated Batch process and Single Column. • Comparison between performance obtained with AEX-RP and RP-AEX sequences. Therapeutic oligonucleotides represent a recent breakthrough in the pharmaceutical industry due to their ability to regulate gene expression with great specificity. This aspect allows treatment of a wide range of diseases. However, since oligonucleotides are used for therapeutic purposes, the Active Pharmaceutical Ingredient (API) must fulfill strict purity levels which require intensive purification steps. For oligonucleotides, and biomolecules in general, preparative liquid chromatography is the technique of choice to perform large scale purifications, typically in batch mode, i.e. using a single column. Specifically, since ONs are mainly large, hydrophilic and charged molecules, Anion Exchange chromatography (AEX) and Ion Pair Reversed Phase chromatography (IP-RP) are the preferred chromatographic modes for their downstream processing. Nevertheless, these approaches suffer from a purity-yield trade-off, and for this reason, more than one purification step is usually required. The two chromatographic modes can therefore be used consequently to remove different groups of impurities, thanks to their orthogonality. In this work, a multidimensional and orthogonal approach on a (semi)preparative scale, namely "Integrated Batch process", was applied for the purification of a single-stranded DNA oligonucleotide. This process combines two chromatographic steps without any hold step, operator intervention or sampling of the first step. The performance parameters of the Integrated Batch were compared to those obtained in the single batch runs under different experimental conditions (chromatographic mode, eluent systems), showing the potential of this integrated approach. This proof-of-concept study illustrates how this technique can considerably reduce overall production time and how it allows to increase the robustness and reproducibility of the method, since the process is highly automated. [Display omitted] [ABSTRACT FROM AUTHOR]

The burgeoning requirement for purified biomacromolecules in biopharmaceutical industry has amplified the exigency for advanced chromatographic separation techniques. Herein, macroporous cellulose microspheres (CCMs) with micron-sized pores are produced by a facile regulation via carbon nanotubes (CNTs). In this strategy, the incorporation of CNTs breaks the homogeneous regeneration of the cellulose, thus providing anisotropic phase force to produce macropores. The CCMs have manifested a faster mass transfer rate and more available adsorption sites owing to well-defined macropores (2.69 ± 0.57 μm) and high specific surface area (147.47 m2 g−1). Further, CCMs are functionalized by quaternary ammonium salts (GTAc-CCMs) and utilized as anion adsorbents to adsorb pancreatic kininogenase (PK). The prepared GTAc-CCMs show rapid adsorption kinetics for PK at pH 6.0, reaching 90 % equilibrium within 60 min. Also, GTAc-CCMs for PK exhibit high adsorptive capacity (632.50 mg g−1), excellent recyclability (> 80 % removal amount after 10 cycles) and selectivity especially at pH 6.0. Notably, the GTAc-CCMs have been successfully applied in a fixed-bed chromatography process, indicating their potential as an effective chromatographic medium for rapid separation of biomacromolecules. [Display omitted] [ABSTRACT FROM AUTHOR]

• Mechanistic model enables accurate simulation of virus removal. • Flow rate dependent LRVs of mock virus particles are shown. • Verification of simulation at different NaCl concentrations and residence times. • Simulation of combinatorial effects of NaCl concentration and residence times. Biopharmaceutical products are often produced in Chinese hamster ovary (CHO) cell cultures that are vulnerable to virus infections. Therefore, it is a regulatory requirement that downstream purification steps for biopharmaceuticals can remove viruses from feedstocks. Anion exchange chromatography (AEX) is one of the downstream unit operations that is most frequently used for this purpose and claimed for its capability to remove viruses. However, the impact of various process parameters on virus removal by AEX is still not fully understood. Mechanistic modeling could be a promising way to approach this gap, as these models require comparatively few experiments for calibration. This makes them a valuable tool to improve understanding of viral clearance, especially since virus spiking studies are costly and time consuming. In this study, we present how the virus clearance of a MVM mock virus particle by Q Sepharose FF resin can be described by mechanistic modeling. A lumped kinetic model was combined with a steric mass action model and calibrated at micro scale using three linear gradient experiments and an incremental step elution gradient. The model was subsequently verified for its capability to predict the effect of different sodium chloride concentrations, as well as residence times, on virus clearance and was in good agreement with the LRVs of the verification runs. Overall, models like this could enhance the mechanistic understanding of viral clearance mechanisms and thereby contribute to the development of more efficient and safer biopharmaceutical downstream processes. [ABSTRACT FROM AUTHOR]

In the past few years, the production of shrimp shell waste from the seafood processing industries has confronted a significant surge. Furthermore, insignificant dumping of waste has dangerous effects on both nature and human well-being. This marine waste contains a huge quantity of chitin which has several applications in different fields. The chitinase enzyme can achieve degradation of chitin, and the chitin itself can be used as the substrate as well for production of chitinase. In the current study, the chitinase enzyme was produced by Thermomyces lanuginosus. The extracellular chitinase was purified from crude extract using ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography and Sephadex G-100 gel filtration chromatography. The stability and activity of chitinase with different pH, temperature, different times for a reaction, in the presence of different metal ions, and different concentration of enzyme and substrate were analyzed. The chitinase activity was found to be highest at pH 6.5, 50 °C, and 60 min after the reaction began. and the chitinase showed the highest activity and stability in the presence of β-mercaptoethanol (ME). The SDS-PAGE of denatured purified chitinase showed a protein band of 18 kDa. The characterization study concludes that Cu2+, Hg2+, and EDTA have an inhibitory effect on chitinase activity, whereas β-ME acts as an activator for chitinase activity. The utilization of chitin to produce chitinase and the degradation of chitin using that chitinase enzyme would be an opportunity for bioremediation of shrimp shell waste. [ABSTRACT FROM AUTHOR]

Hemophilia A is treated with human plasma coagulation factor VIII (FVIII) replacement therapy and Hemophilia B with coagulation factor IX, which is purified from prothrombin complex concentrate (PCC). In this paper we evaluated the separation of FVIII and PCC by directly loading raw thawed plasma to an anion exchange resin (AEX). Under this relatively high ionic strength, most of the plasma proteins such as albumin, immunoglobulins and others were not adsorbed. Five resins commonly used in protein purification (plasma fractionation) were tested. With all resins, PCC was eluted by pseudoaffinity in a calcium gradient step. Afterwards, FVIII could be recovered with a good yield and high purification factor in the salt gradient step with 400–500 mM NaCl. Using ANX Sepharose FF and Q Sepharose FF, the CaCl2 elution step was introduced after the intermediate wash with 200 mM NaCl, whereas using DEAE Sepharose FF, Fractogel EMD TMAE and Fractogel EMD DEAD, PCC eluted after the wash of the unbound proteins. Our results indicate that three important fractions: (1) albumin, immunoglobulin etc.; (2) PCC; and (3) FVIII can be separated in one chromatographic AEX column and the delicate and troublesome cryoprecipitation can be eliminated, making the purification of blood products faster and cheaper. [ABSTRACT FROM AUTHOR]

This study aimed to propose a methodology for developing a mechanistic model for viral clearance of the minute virus of mice (MVM) on flow-through anion exchange (AEX) chromatography. Protein surface analysis was applied to investigate the possibility of molecular interaction between the recombinant biotherapeutic and MVM. The protein product-free Tris buffers were spiked with MVM, and the MVM elution profile from AEX chromatography was quantitatively analyzed using quantitative polymerase chain reaction (qPCR) for pooled fractions. GoSilico™ Chromatography Modeling Software was applied to develop the mechanistic models for MVM species. For evaluating the visual fit of the developed model, the R 2 of intact MVM virions and uncoated capsids between the simulated and measured amount in each fraction are 0.880 and 0.948, respectively. Response surface plots of logarithmic reduction values (LRV) against pH and conductivity in loaded sample were generated to show the range for suitable loaded sample conditions for achieving a good LRV. To evaluate the applicability of the developed MVM elution model to a recombinant biotherapeutic, two demonstrations of AEX chromatography purification were performed with a loaded sample of a model monoclonal antibody. The peaks of the MVM species in the elution step of both runs were accurately simulated by the developed model. In addition, to assess the possibility of molecular interaction between the virus and the target protein significantly affecting the MVM elution behavior, the antibody's surface was evaluated in terms of hydrophobicity/hydrophilicity using surface analysis., (© 2024 American Institute of Chemical Engineers.)

Adeno-associated viruses (AAV) are widely used viral vectors for in vivo gene therapy. The purification of AAV, particularly the separation of genome-containing from empty AAV capsids, is usually time-consuming and requires expensive equipment. In this study, we present a novel laboratory scale anion exchange flow-through polishing method designed to separate full and empty AAV. Once the appropriate conditions are defined, this method eliminates the need for a chromatography system. Determination of optimal polishing conditions using a chromatography system revealed that the divalent salt MgCl 2 resulted in better separation of full and empty AAV than the monovalent salt NaCl. The efficacy of the method was demonstrated for three distinct AAV serotypes (AAV8, AAV5, and AAV2) on two different stationary phases: a membrane adsorber and a monolith, resulting in a 4- to 7.5-fold enrichment of full AAV particles. Moreover, the method was shown to preserve the AAV capsids' functional potency and structural integrity. Following the successful establishment of the flow-through polishing approach, it was adapted to a manual syringe-based system. Manual flow-through polishing using the monolith or membrane adsorber achieved 3.6- and 5.4-fold enrichment of full AAV, respectively. This study demonstrates the feasibility of separating full and empty AAV without complex linear or step gradient elution and the necessity of specialized equipment. Flow-through polishing provides a rapid and easy-to-perform platform for polishing multiple vector preparations, addressing a critical aspect in the research and development of novel gene therapies., (© 2024 The Author(s). Biotechnology Journal published by Wiley‐VCH GmbH.)

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In the present study, the shade dried powdered leaves of Rhinacanthus nasutus (L.) Kurz., was subjected to maceration with ethyl acetate, concentrated and evaporated to dryness. The ethyl acetate extract was subjected to anion exchange column chromatography and the isolated rhinacanthin enriched extract was analyzed by thin layer chromatography and fourier-transform infrared studies. In the present study, the in vitro cytotoxic activity of the rhinacanthin rich extract of Rhinacanthus nasutus (L) Kurz., over SH-SY5Y human neuroblastoma cell line was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide assay and compared with the standard drug doxorubicin. The preliminary phytochemical screening revealed the presence of alkaloids, quinones, glycosides, carbohydrates, amino acids and tannins. The thin layer chromatography profiling showed the presence of secondary metabolites in various mobile phase systems. Fourier-Transform Infrared analysis of rhinacanthin enriched extract confirmed the presence of functional groups including 1, 4-quinone carbonyl component. The results of in vitro cell line studies revealed a dose dependent cytotoxic effect on human neuroblastoma cell lines with an IC50 value of 88.9 µg/ml. Thus the results validate the potential of rhinacanthin enriched extract against neuroblastoma cancer and based on this initial screening, further studies at elucidation of its molecular mechanism as cancer therapeutics can be undertaken. [ABSTRACT FROM AUTHOR]

Polyamidamine (PAMAM) dendrimer-functionalized hydrothermal nanosized carbonaceous spheres (HNCSs) were prepared and utilized as latexes for agglomerated anion exchange chromatography (AEC) stationary phase. The high-concentration and scalable production of monodisperse HNCSs (73–98 nm) was accomplished via the polyquaternium-7-assisted hydrothermal carbonization of fructose. The novel PAMAM-based quaternizations of HNCSs were designed by the amidation with PAMAM and epoxy-amine addition reaction with glycidol in aqueous solution. The mild functionalization condition leads to well-kept morphology of HNCSs, which forms one even latex layer on the sulfonated surface of polystyrene-divinylbenzene microbeads for the construction of AEC packing. Under isocratic elution, seven common inorganic anions and five organic acids were baseline separated in 9 min on prepared packing with efficiencies of 54,000–79,800 plates m−1 and asymmetry factor (As) of 1.02–1.12. The obtained separation efficiency, peak symmetry, and analysis time were superior to reported or typical commercial counterparts. The quick separation of polarizable anions in 7 min and carbohydrates in 5 min could also be carried out with symmetrical peaks (As: 1.00–1.18) and high efficiencies (49,700–62,100 N/m). Favorable stability and reproducibility were proved by continuous flushing and injection. The constructed packings were further applied to the determination of thiosulfate and sulfate in water reducer, galacturonic acid in Angelica polysaccharide hydrolysate, and fluoride samples in 4 min. [ABSTRACT FROM AUTHOR]

Background: Since many research works have been carried out to identify the pharmacological properties of conotoxins from many Conus sp. The pharmacological properties of higher molecular weight toxin from the venom of Conus sp. have been poorly elucidated. Therefore, the present investigation was made to isolate the higher molecular weight toxin from the venom of Conus virgo and to test its antitumor effect on rat glioma cell line. Results: The partially purified fraction 1 showed better activity (IC50 50 μg/ml) than fraction 2 (IC50 150 µg/ml) after 24 h incubation. The decreased elevation of creatine kinase was documented on fraction 1treated C6 cells. Vero cells showed minimal cell damage at 150 μg/ml 24 h as well. The nuclear morphology and nuclear fragmentation showed the presence of nuclear damage in C6 cells. Western blot analysis revealing the upregulation of the p53-mediated intrinsic apoptotic pathway was documented. Conclusions: The present investigation emphasizes that the partially purified fraction 1 has triggered p53 mediated apoptosis by synergism of two higher molecular weight toxins in Rat glioma cell. [ABSTRACT FROM AUTHOR]

Abstract Background Although our knowledge about diatom photosynthesis has made huge progress over the last years, many aspects about their photosynthetic apparatus are still enigmatic. According to published data, the spatial organization as well as the biochemical composition of diatom thylakoid membranes is significantly different from that of higher plants. Results In this study the pigment protein complexes of the diatom Thalassiosira pseudonana were isolated by anion exchange chromatography. A step gradient was used for the elution process, yielding five well-separated pigment protein fractions which were characterized in detail. The isolation of photosystem (PS) core complex fractions, which contained fucoxanthin chlorophyll proteins (FCPs), enabled the differentiation between different FCP complexes: FCP complexes which were more closely associated with the PSI and PSII core complexes and FCP complexes which built-up the peripheral antenna. Analysis by mass spectrometry showed that the FCP complexes associated with the PSI and PSII core complexes contained various Lhcf proteins, including Lhcf1, Lhcf2, Lhcf4, Lhcf5, Lhcf6, Lhcf8 and Lhcf9 proteins, while the peripheral FCP complexes were exclusively composed of Lhcf8 and Lhcf9. Lhcr proteins, namely Lhcr1, Lhcr3 and Lhcr14, were identified in fractions containing subunits of the PSI core complex. Lhcx1, Lhcx2 and Lhcx5 proteins co-eluted with PSII protein subunits. The first fraction contained an additional Lhcx protein, Lhcx6_1, and was furthermore characterized by high concentrations of photoprotective xanthophyll cycle pigments. Conclusion The results of the present study corroborate existing data, like the observation of a PSI-specific antenna complex in diatoms composed of Lhcr proteins. They complement other data, like e.g. on the protein composition of the 21 kDa FCP band or the Lhcf composition of FCPa and FCPb complexes. They also provide interesting new information, like the presence of the enzyme diadinoxanthin de-epoxidase in the Lhcx-containing PSII fraction, which might be relevant for the process of non-photochemical quenching. Finally, the high negative charge of the main FCP fraction may play a role in the organization and structure of the native diatom thylakoid membrane. Thus, the results present an important contribution to our understanding of the complex nature of the diatom antenna system.

Alpha-1-acid glycoprotein (AGP) is a heterogeneous glycoprotein fulfilling key roles in many biological processes, including transport of drugs and hormones and modulation of inflammatory and immune responses. The glycoform profile of AGP is known to change depending on (patho)physiological states such as inflammatory diseases or pregnancy. Besides complexity originating from five N-glycosylation sites, the heterogeneity of the AGP further expands to genetic variants. To allow in-depth characterization of this intriguing protein, we developed a method using anion exchange chromatography (AEX) coupled to mass spectrometry (MS) revealing the presence of over 400 proteoforms differing in their glycosylation or genetic variants. More precisely, we could determine that AGP mainly consists of highly sialylated higher antennary structures with on average 16 sialic acids and 0 or 1 fucose per protein. Interestingly, a slightly higher level of fucosylation was observed for AGP1 variants compared to that of AGP2. Proteoform assignment was supported by integrating data from complementary MS-based approaches, including AEX-MS of an exoglycosidase-treated sample and glycopeptide analysis after tryptic digestion. The developed analytical method was applied to characterize AGP from plasma of women during and after pregnancy, revealing differences in glycosylation profiles, specifically in the number of antennae, HexHexNAc units, and sialic acids.

Fresh meat quality is adversely affected by protein oxidation. However, a fundamental understanding of the diverse factors that influence protein oxidation in postmortem muscle remains elusive. Peroxiredoxin-2 (Prdx2), an antioxidant protein, is more abundant in tough meat based on instrumental tenderness; however, the role of Prdx2 in postmortem skeletal muscle is unknown. Therefore, the objective was to develop a method to purify Prdx2 from the diaphragm, psoas major, and longissimus lumborum. Proteins soluble at low ionic strength were extracted, dialyzed, clarified, and loaded onto a Q-Sepharose anion exchange column equilibrated with TEM (pH 7.4). In all preparations, Prdx2 eluted between about 75 and 115 mM NaCl. Immunoreactive fractions were dialyzed against TEM (pH 8.0), clarified, and loaded onto a DEAE-650S anion exchange column. In all preparations, Prdx2 eluted between approximately 55 and 75 mM NaCl. Immunoreactive fractions were concentrated and loaded onto a Superose-12 size exclusion column. Prdx2 was detected between 14 and 16 mL, and these fractions were concentrated and reduced with 0.5% 2-mercaptoethanol. A final pass over the Superose-12 column was conducted, and Prdx2 was detected in 2 peaks from 11–12 mL and 15–16 mL. Fractions 15–16 were pooled and retained for further experiments. The elution profile of Prdx2 in all 3 muscles was similar. The iden- tification of the primary protein was confirmed with liquid chromatography with tandem mass spectrometry. The purity of Prdx2 off the final Superose-12 column was approximately 33%, 52%, and 47% pure in the diaphragm, psoas major, and longissimus lumborum, respectively. This is the first report of a method to partially purify Prdx2 from skeletal muscle.

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Hemophilia A is treated with human plasma coagulation factor VIII (FVIII) replacement therapy and Hemophilia B with coagulation factor IX, which is purified from prothrombin complex concentrate (PCC). In this paper we evaluated the separation of FVIII and PCC by directly loading raw thawed plasma to an anion exchange resin (AEX). Under this relatively high ionic strength, most of the plasma proteins such as albumin, immunoglobulins and others were not adsorbed. Five resins commonly used in protein purification (plasma fractionation) were tested. With all resins, PCC was eluted by pseudoaffinity in a calcium gradient step. Afterwards, FVIII could be recovered with a good yield and high purification factor in the salt gradient step with 400–500 mM NaCl. Using ANX Sepharose FF and Q Sepharose FF, the CaCl2 elution step was introduced after the intermediate wash with 200 mM NaCl, whereas using DEAE Sepharose FF, Fractogel EMD TMAE and Fractogel EMD DEAD, PCC eluted after the wash of the unbound proteins. Our results indicate that three important fractions: (1) albumin, immunoglobulin etc.; (2) PCC; and (3) FVIII can be separated in one chromatographic AEX column and the delicate and troublesome cryoprecipitation can be eliminated, making the purification of blood products faster and cheaper.

Adzuki bean is high in 5′-phosphodiesterase (5′-PDE), an enzyme that can form flavor enhancing 5′-nucleotides. In this work, 5′-PDE from adzuki bean is purified. Acetone precipitation (1 crude extract:1.25 cold acetone) is used as the first purification step as it is commonly used in enzyme purification to reduce unwanted proteins, before advancing into an application of the Fast Protein Liquid Chromatography system (FPLC). Three major techniques of purification were carried out in sequence as follows: desalting (using a Hi Prep 26/10 desalting column), anion exchange chromatography (using a Hi-Trap DEAE Fast Flow column), and gel filtration chromatography (using a Superose 12 10/300 GL column). The purification protocol adopted yielded 13.37% 5′-PDE with a purification fold of 6.7 and specific activity of 35.2 µmol p-nitrophenol/min/mg protein. The molecular weight of adzuki bean 5′-PDE was estimated using gel filtration chromatography by comparing the fraction that contained the highest 5′-PDE activity with the molecular weights of standard proteins (1.35–669 kDa). Native PAGE was conducted to determine the homogeneity of proteins in the gel filtration fraction showed one band. SDS-PAGE analysis indicated that the enzyme was composed of two identical polypeptide chains (subunits), each with a molecular weight of 62 kDa. Therefore, the estimated molecular weight of the native protein using SDS-PAGE was 124 kDa. [ABSTRACT FROM AUTHOR]

Ion exchange chromatography is a powerful and ubiquitous unit operation in the purification of therapeutic proteins. However, the performance of an ion‐exchange process depends on a complex interrelationship between several parameters, such as protein properties, mobile phase conditions, and chromatographic resin characteristics. Consequently, batch variations of ion exchange resins play a significant role in the robustness of these downstream processing steps. Ligand density is known to be one of the main lot‐to‐lot variations, affecting protein adsorption and separation performance. The use of a model‐based approach can be an effective tool for comprehending the impact of parameter variations (e.g., ligand density) and their influence on the process. The objective of this work was to apply mechanistic modeling to gain a deeper understanding of the influence of ligand density variations in anion exchange chromatography. To achieve this, 13 prototype resins having the same support as the strong anion exchange resin Fractogel® EMD TMAE (M), but differing in ligand density, were analyzed. Linear salt gradient elution experiments were performed to observe the elution behavior of a monoclonal antibody and bovine serum albumin. A proposed isotherm model for ion exchange chromatography, describing the dependence of ligand density variations on protein retention, was successfully applied. [ABSTRACT FROM AUTHOR]

Assessment of critical quality attributes of the biopharmaceutical erythropoietin (EPO) prior to product release requires the use of several analytical methods. We developed an MS-compatible anion exchange (AEX) method for monitoring multiple quality attributes of EPO biopharmaceuticals. AEX was performed using a stationary phase with quaternary ammonium functional groups and a pH gradient for elution. Baseline separation of charge variants and high-quality MS data were achieved using 30 mM ammonium formate pH 5.5 and 30 mM formic acid pH 2.5 as mobile phases. In a single experiment, assessment of critical quality attributes, such as charge heterogeneity, sialic acid content and number of N -acetyllactosamine units, was possible while providing additional information on other modifications such as O -acetylation and deamidation. In addition, good repeatability and robustness for the relative areas of the individual glycoforms and average number of Neu5Ac per EPO molecule were observed. The results were comparable to common pharmacopeia and standard methods with the advantage of requiring fewer analytical methods and less sample treatment saving time and costs. Image 1 • AEX-MS permits monitoring multiple quality attributes of EPO pharmaceuticals. • AEX-MS charge variant distribution is equivalent to the pharmacopeia method. • Additional information on O -acetylation and deamidation is obtained. • Applicability is demonstrated for EPO molecules with altered glycosylation. • Fewer methods and sample treatment are needed reducing time and cost. [ABSTRACT FROM AUTHOR]

This work describes a streamlined approach to the recovery and purification of 234U from aged 238Pu. Key modifications to conventional techniques are described that result in an efficient uranium and plutonium separation process that successfully produced tens of grams of ultrahigh purity 234U at Oak Ridge National Laboratory. A new processing scheme was developed using an anion exchange column run in a cycling mode with HNO3 media as the primary uranium purification process. This was followed with a uranyl peroxide precipitation step to purify and solidify the uranium before conversion to U3O8. Both the traditional uranyl peroxide precipitation method and a new cascade uranyl peroxide precipitation process were used for final purification of the 234U. [ABSTRACT FROM AUTHOR]

Many manufacturers of biopharmaceuticals are moving from batch to continuous processing. While this approach offers advantages over batch processing, demonstration of viral clearance for continuous processes is challenging. Fluctuating output from a continuous process chromatography column results in a nonhomogeneous load for the subsequent column and must be considered when designing viral clearance studies. One approach to clearance studies is to downscale the connected unit operations and introduce virus by in‐line spiking. This is challenging to be implemented at the contract research organization performing the clearance study given the complexity of systems and level of expertise required. Alternately, each unit operation could be evaluated in traditional batch mode but the spiking and loading conditions be modified to mimic the variance introduced by the transition between two connected columns. Using a standard chromatography system, we evaluated a flow‐through anion exchange chromatography step in a monoclonal antibody (mAb) manufacturing process using five different methods to introduce the virus to the column. Our data show that whether the virus or the mAbs were introduced in concentrated peaks, or as a homogeneous batch, the clearance of mouse minute virus was similar. This study introduces an alternative way to evaluate viral clearance in a continuous process and demonstrates the robustness of anion exchange chromatography unit operating in continuous processing. [ABSTRACT FROM AUTHOR]

The baculovirus expression vector system is a very powerful tool to produce virus‐like particles and gene‐therapy vectors, but the removal of coexpressed baculovirus has been a major barrier for wider industrial use. We used chimeric human immunodeficiency virus‐1 (HIV‐1) gag influenza‐hemagglutin virus‐like particles produced in Tnms42 insect cells using the baculovirus insect cell expression vector system as model virus‐like particles. A fast and simple purification method for these virus‐like particles with direct capture and purification within one chromatography step was developed. The insect cell culture supernatant was treated with endonuclease and filtered, before it was directly loaded onto a polymer‐grafted anion exchanger and eluted by a linear salt gradient. A 4.3 log clearance of baculovirus from virus‐like particles was achieved. The absence of the baculovirus capsid protein (vp39) in the product fraction was additionally shown by high performance liquid chromatography‐mass spectrometry. When considering a vaccination dose of 109 particles, 4200 doses can be purified per L pretreated supernatant, meeting the requirements for vaccines with <10 ng double‐stranded DNA per dose and 3.4 µg protein per dose in a single step. The process is simple with a very low number of handling steps and has the characteristics to become a platform for purification of these types of virus‐like particles. [ABSTRACT FROM AUTHOR]

The characterisation of relative copper isotope amount ratios (δ65Cu) helps constrain a variety of geochemical processes occurring in the geosphere, biosphere and hydrosphere. The accurate and precise determination of δ65Cu in matrix reference materials is crucial in the effort to validate measurement methods. With the goal of expanding the number and variety of available geological and biological materials, we have characterised the δ65Cu values of ten reference materials by MC‐ICP‐MS using C‐SSBIN model for mass bias correction. SGR‐1b (Green River shale), DOLT‐5 (dogfish liver), DORM‐4 (fish protein), TORT‐3 (lobster hepatopancreas), MESS‐4 (marine sediment) and PACS‐3 (marine sediment) have for the first time been characterised for δ65Cu. Additionally, four reference materials (with published δ65Cu values) have been characterised: BHVO‐1 (Hawaiian basalt), BIR‐1 (Icelandic basalt), W‐2a (diabase) and Seronorm™ Trace Elements Serum L‐1 (human serum). The reference materials measured in this study possess complex and varied matrices with copper mass fractions ranging from 1.2 µg g−1 to 497 µg g−1 and δ65Cu values ranging from −0.20‰ to 0.52‰ with a mean expanded uncertainty of ± 0.07‰ (U, k = 2), covering much of the natural copper isotope variability observed in the environment. [ABSTRACT FROM AUTHOR]

Computational methods increasingly provide access for Quantitative-Structure-Activity Relationship (QSAR) models to molecular descriptors that have minimal identifiable or experimentally accessible chemical/physical properties. However, molecular information inherent in chromatographic data suggests chromatography as a convenient experimental platform for legitimizing newly proposed molecular descriptors. In this study, liquid chromatographic retention data collected on a trimethylammonium (TAM) anion exchange stationary phase was used to evaluate two new molecular descriptors, P− and P+ developed to account for contributions from ion-π interactions. In the Linear Solvation Energy Relationship (LSER) model, the P− and P+ parameter replaced E. Incorporation of the new parameters was most impactful on S (sconv= −0.173 ± 0.070 vs. smod -0.115 ± 0.081). The remaining coefficients (v, b, c) were within experimental error of their respective coefficients in the conventional model. Nonparametric statistical analysis revealed comparable goodness of fits for the conventional and modified models. Finally, comparison of the contribution of individual interaction modes for substituted benzenes (log kbenzene-X) relative to benzene (log kbenzene) conducted with the new model was consistent with chemical intuition for the model-attributed ion − π and other individual interaction modes. Thus, adoption of P descriptors into the LSER model provides a convenient means for exploring models for weak ion − π interactions. [ABSTRACT FROM AUTHOR]

Bovine κ-casein glycomacropeptide (GMP) found in cheese whey is a sialylated phosphorylated peptide which is thought to be a potential ingredient for functional food as well as dietetic food. This study was undertaken to determine whether high purity GMP can be isolated from soluble whey fraction (SWF) using column chromatography on food grade anion exchange resin and chitin as an adsorbent. Samples of commercially available anion exchange resin (resin A, resin B and resin C) and those of chitin (chitin A, chitin B and chitin C) were examined in this experiment. The GMP fraction obtained from each column was analyzed for amino acid composition which reflects the purity of the peptide. Major findings for commercial anion exchange resin were that: (1) the proportion of GMP monitored as sialic acid in total recovered sialic acid was similar among the three samples of resin accounting for average 78% of recovered sialic acid; (2) the GMP fraction from resin A or resin B contained undetectable level of contaminating amino acids including phenylalanine, histidine, arginine and tyrosine; (3) the GMP fraction from resin C contained small amounts (<1 mol%) of contaminating amino acids, arginine, phenylalanine and tyrosine; and (4) the GMP binding capacity expressed as mg/100 mg dry weight of resin was more than 2.5 times higher in resin C (average 22.9) than in resin A or resin B with no difference between resin A and resin B averaging 8.7. Results obtained for chitin A, chitin B and chitin C were in general similar to those found with resin A and resin B. Since chitin has a remarkable GMP binding capacity averaging 8.6 mg/100 mg dry weight of chitin, it may be a useful adsorbent for whey fractionation. Further research is needed to develop an efficient inexpensive method to purify GMP. [ABSTRACT FROM AUTHOR]

A heteroleptic Am3+ coordination complex of oxalate and diglycolate was isolated and characterized by single crystal X-ray diffraction and single crystal UV–vis-NIR spectroscopy. Long, B. N.; Pace, K. A.; Adelman, S. L.; Anderson, N. H.; Arko, B. T.; Bowes, E. G.; Kalin, T. A.; Kozimor, S. A.; Mocko, V.; Piedmonte, I. D. [Display omitted] Improving f -element separations is important for actinide(III) (An3+) and lanthanide(III) (Ln3+) based technologies. Unfortunately, An3+ and Ln3+ ions are difficult to separate from one another because they have similar chemical characteristics. One successful separation method utilizes anion exchange chromatography. This approach exploits differences in An3+ and Ln3+ Lewis acidities and their varying abilities to attract anionic complexing agents, like oxalates (C 2 O 4 2–) and diglycolates (ODA2–). The resulting negatively charged complexes are then separated using an anion exchange resin. To better understand how this anion exchange separation works, we reacted Am3+ (aq) (aq designates Am3+ dissolved in water) with the anion exchange complexing agents (H 2 C 2 O 4 and H 2 ODA). The resulting Am(ODA)(C 2 O 4)(H 2 O) 3 product was characterized using single crystal X-ray diffraction and UV-Vis-NIR spectroscopy. The Am(ODA)(C 2 O 4)(H 2 O) 3 structure was similar to that established previously for Ln3+ analogues, namely Ln(ODA)(C 2 O 4)(H 2 O) x. These compounds were all isomorphous, had bridging C 2 O 4 2– and ODA2– ligands, and crystallized as 2-dimensional extended solids. In addition, the Am3+–O bond distances could be predicted based on relative differences in Am3+ and Ln3+ 9-coordinate metal ionic radii. Overall, isolation of Am(ODA)(C 2 O 4)(H 2 O) 3 showcased similarities in complexation and crystallization chemistry for Am3+ and Ln3+. [ABSTRACT FROM AUTHOR]

Development of clinically desirable adeno-associated virus (AAV) vectors with optimal genome design requires rapid and accurate analytical methods to assess AAV quality. Anion-exchange (AEX) chromatography provides a powerful analytical method for full/empty AAV capsid ratio determination. However, the current AEX methodology for separation of empty and full AAV capsids largely relies on the use of the highly toxic tetramethylammonium chloride (TMAC). Here, we describe a novel analytical AEX method for separation of empty and full AAV capsids that uses only non-toxic, choline-type compounds that contain structural similarity to the quaternary ammonium ligand present on the surface of AEX resin. Choline-Cl gradient, combined with sensitive fluorescence detection, allowed a safe and effective separation of empty and full AAV capsids with reproducible empty/full ratio determination. The choline-based assay was suitable for commonly used serotypes, AAV2, AAV5, AAV6, and AAV8. The limit of detection was ∼3.9 × 108 virus particles in the assay. A gradient-hold step-gradient elution with choline-Cl resulted in enhanced baseline separation of empty and full AAV8 capsids. In summary, the use of choline-Cl in the AEX assay is recommended for empty/full capsid ratio determination and other applications in AAV production, and it eliminates the necessity of using toxic TMAC. [Display omitted] • Novel analytical anion-exchange method for adeno-associated viral vectors. • Choline-type compounds for separation of empty and full viral capsids. • Applicable to a variety of clinically important AAV serotypes. • Non-toxic and environmentally friendly alternative method for AAVs. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. The present work studied the effect of genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension-adapted HEK293 cells via triple transfection using AAV plasmids containing the same GFP transgene with DNA stuffers for variable-size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography, and the results suggest that the triple transfection condition significantly affects the AEX retention time and resolution between the full and empty capsid peaks. Charge detection-mass spectrometry was performed on all AEX full-capsid peak samples showing a wide distribution of empty, partial, full length, and copackaged DNA in the capsids. The AEX-purified samples were then analyzed by differential scanning fluorimetry, and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content., Competing Interests: N.I. is a full-time employee at Sarepta Therapeutics, a company with interest in developing AAV technology, and B.E.D. is a full time employee at Megadalton Solutions, a company with interest in developing CD-MS technology. C.W.L. declares no competing interests., (© 2024 The Authors.)