Analytical and preparative chiral supercritical fluid chromatography resolutions using crown ether-derived column.

In this study, crown ether-derived column Crownpak® CR-I (+) was evaluated under SFC conditions using 12 primary amines, and the chromatographic results were compared against eight immobilized polysaccharide-based columns. Crownpak® CR-I (+) achieved a significantly higher success rate. It was found that the addition of 5% water to the modifier dramatically improved the peak shape for chiral separation of primary amines on Crownpak® CR-I (+). The first reported preparative SFC separations on Crownpak® CR-I (+) are shown, offering a new approach for the preparative resolution of primary amines. The case studies demonstrate that Crownpak® CR-I (+) is a very useful column in the chiral separation of challenging compounds that contain a primary amine group in the pharmaceutical industry.

Creation of a versatile automated two-step purification system with increased throughput capacity for preclinical mAb material generation.

The ever-increasing speed of biotherapeutic drug discovery has driven the development of automated and high throughput purification capabilities. Typically, purification systems require complex flow paths or third-party components that are not found on a standard fast protein liquid chromatography instrument (FPLC) (e.g., Cytiva's ÄKTA) to enable higher throughput. In early mAb discovery there is often a trade-off between throughput and scale where a high-throughput process requires miniaturized workflows necessitating a sacrifice in the amount of material generated. At the interface of discovery and development, flexible automated systems are required that can perform purifications in a high-throughput manner, while also generating sufficient quantities of preclinical material for biophysical, developability, and preclinical animal studies. In this study we highlight the engineering efforts to generate a highly versatile purification system capable of balancing the purification requirements between throughput, chromatographic versatility, and overall product yields. We incorporated a 150 mL Superloop into an ÄKTA FPLC system to expand our existing purification capabilities. This allowed us to perform a range of automated two-step tandem purifications including primary affinity captures (protein A (ProA)/immobilized metal affinity chromatography (IMAC)/antibody fragment (Fab)) followed by secondary polishing with either size exclusion (SEC) or cation exchange (CEX) chromatography. We also integrated a 96 deep-well plate fraction collector into the ÄKTA FPLC system with purified protein fractions being analyzed by a plate based high performance liquid chromatography instrument (HPLC). This streamlined automated purification workflow allowed us to process up to 14 samples within 24 h, enabling purification of ∼1100 proteins, monoclonal antibodies (mAbs), and mAb related protein scaffolds during a 12-month period. We purified a broad range of cell culture supernatant volumes, between 0.1 and 2 L, with final purification yields up to 2 g. The implementation of this new automated, streamlined protein purification process greatly expanded our sample throughput and purification versatility while also enabling the accelerated production of greater quantities of biotherapeutic candidates for preclinical in vivo animal studies and developability assessment.

Advances and applications of chiral resolution in pharmaceutical field.

The attention to chiral drugs has been raised to an unprecedented level as drug discovery and development strategies grow rapidly. However, separation of enantiomers is still a huge task, which leads to an increasing significance to equip a wider range of expertise in chiral separation science to meet the current and future challenges. In the last few decades, remarkable progress of chiral resolution has been achieved. This review summarizes and classifies chiral resolution methods in analytical scale and preparative scale systematically and comprehensively, including crystallization-based method, inclusion complexation, chromatographic separation, capillary electrophoresis, kinetic resolution, liquid-liquid extraction, membrane-based separation, and especially one bold new progress based on chiral-induced spin selectivity theory. The advances and recent applications will be presented in detail, in which the contents may bring more thinking to wide-ranging readers in various professional fields, from analytical chemistry, pharmaceutical chemistry, natural medicinal chemistry, to manufacturing of drug production.

Chiral Separations in Preparative Scale: A Medicinal Chemistry Point of View.

Enantiomeric separation is a key step in the development of a new chiral drug. Preparative liquid chromatography (LC) continues to be the technique of choice either during the drug discovery process, to achieve a few milligrams, or to a scale-up during the clinical trial, needing kilograms of material. However, in the last few years, instrumental and technical developments allowed an exponential increase of preparative enantioseparation using other techniques. Besides LC, supercritical fluid chromatography (SFC) and counter-current chromatography (CCC) have aroused interest for preparative chiral separation. This overview will highlight the importance to scale-up chiral separations in Medicinal Chemistry, especially in the early stages of the pipeline of drugs discovery and development. Few examples within different methodologies will be selected, emphasizing the trends in chiral preparative separation. The advantages and drawbacks will be critically discussed.

Screening and isolation of cyclooxygenase-2 inhibitors from the stem bark of Phellodendron amurense Ruprecht by ultrafiltration with liquid chromatography and tandem mass spectrometry, and complex chromatography.

Nonsteroidal anti-inflammatory drugs appear to reduce the risk of developing cancer. One mechanism through which nonsteroidal anti-inflammatory drugs act to prevent carcinogenesis is inhibition of the activity of the enzyme cyclooxygenase-2. The cyclooxygenase-2 inhibitors are widely used to reduce the risk of developing cancer. Natural products are considered to be a promising source of several novel cyclooxygenase-2 inhibitors. Ultrafiltration with liquid chromatography and mass spectrometry is an efficient method that can be applied to rapidly screen and identify the ligands from the barks of Phellodendron amurense Ruprecht. A continuous online method comprised of pressurized liquid extraction, countercurrent chromatography, and semi-preparative liquid chromatography was developed for the efficient scaled-up production of eight compounds with high purities. The bioactivities of the separated compounds were assessed by an in vitro enzyme inhibition assay. The use of bioactivity screening method combined with preparation method of bioactive compounds and an in vitro enzyme inhibition assay facilitated the efficient screening and isolation of the cyclooxygenase-2 inhibitors from complex samples. This could be used as an efficient method for the large-scale production of functional ingredients.

Enantioseparation of 5-chloro-2-{2-[3,4-dihydroisoquinoline-2(1H)-yl]ethyl}-2-methyl-2,3-dihydro-1H-inden-1-one (SYA 40247), a high-affinity 5-HT7 receptor ligand, by HPLC-PDA using amylose tris-(3, 5- dimethylphenylcarbamate) as a chiral stationary phase.

In previous structure-activity relationship studies to identify new and selective 5-HT7 receptor (5-HT7 R) ligands, we identified the chiral compound, 5-chloro-2-{2-[3,4-dihydroisoquinoline-2(1H)-yl]ethyl}-2-methyl-2,3-dihydro-1H-inden-1-one (SYA 40247), with high-affinity binding to the 5-HT7 R. Thus, it was of interest to separate the enantiomers in order to evaluate their affinity at the 5-HT7 R. To achieve this separation, a normal-phase analytical method using HPLC-PDA and a 4.6 × 250 mm Chiralpak AD-H column was developed. Optimized isocratic conditions of 1.00 mL/min 95:5:0.1 v/v/v hexane-ethanol-diethylamine and a 254 nm analysis wavelength yielded a 6.07 min baseline separation. The method was scaled up to a 10 × 250 mm Chiralpak AD-H column, allowing 3 mg of racemate to be separated with a single injection, and 6 mg for an overlapping double injection in the same run. The separated enantiomers were reinjected into the analytical HPLC system, peak identities confirmed by retention time and PDA UV spectra, and the enantiomeric purities determined to be 100% for peak 1 and 100% for peak 2. A Jasco P-1020 polarimeter was used to determine the specific rotation [α] of the enantiomers of peaks 1 and 2, which were -86.2 and +93.3 (deg mL)/(g dm) respectively. No racemization was observed, and the enantiomeric purity remained at 100% for each peak.

Preparative scale extraction of mangiferin and lupeol from mango (Mangifera indica L.) leaves and bark by different extraction methods.

High biological value compounds are very important in the food and pharmaceutical sectors. The leading research interests are seeking efficient methods for extracting these substances. The objective of this study was to evaluate different extraction methods to obtain mangiferin and lupeol at preparative scale from leaves and bark of mango tree varieties Ataulfo and Autochthonous from Nayarit, Mexico. Four extraction techniques were evaluated such as maceration, Soxhlet, sonication (UAE) and microwave (MAE). Sonication gave the highest concentration of mangiferin and lupeol, demonstrating that extraction assisted by ultrasound could be an effective alternative to conventional extraction techniques because it is a low cost, simple and reliable process. Finally, mangiferin and lupeol were obtained at preparative scale with a higher concentration of bioactive compounds, 1.45 g 100 g-1 y 0.92 mg 100 g-1 sample on (d.b.), respectively. The barks from Ataulfo and Autochthonous mango trees turned out to be favourable sources for obtaining mangiferin and lupeol.

Preparative separation of enantiomers based on functional nucleic acids modified gold nanoparticles.

The preparative-scale separation of chiral compounds is vitally important for the pharmaceutical industry and related fields. Herein we report a simple approach for rapid preparative separation of enantiomers using functional nucleic acids modified gold nanoparticles (AuNPs). The separation of DL-tryptophan (DL-Trp) is demonstrated as an example to show the feasibility of the approach. AuNPs modified with enantioselective aptamers were added into a racemic mixture of DL -Trp. The aptamer-specific enantiomer (L-Trp) binds to the AuNPs surface through aptamer-L-Trp interaction. The separation of DL-Trp is then simply accomplished by centrifugation: the precipitate containing L-Trp bounded AuNPs is separated from the solution, while the D-Trp remains in the supernatant. The precipitate is then redispersed in water. The aptamer is denatured under 95 °C and a second centrifugation is then performed, resulting in the separation of AuNPs and L-Trp. The supernatant is finally collected to obtain pure L-Trp in water. The results show that the racemic mixture of DL-Trp is completely separated into D-Trp and L-Trp, respectively, after 5 rounds of repeated addition of fresh aptamer-modified AuNPs to the DL-Trp mixture solution. Additionally, the aptamer-modified AuNPs can be repeatedly used for at least eight times without significant loss of its binding ability because the aptamer can be easily denatured and renatured in relatively mild conditions. The proposed approach could be scaled up and extended to the separation of other enantiomers by the adoption of other enantioselective aptamers.

Preparative Production of Functionalized (N- and O-Heterocyclic) Polycyclic Aromatic Hydrocarbons by Human Cytochrome P450 3A4 in a Bioreactor.

Polycyclic aromatic hydrocarbons (PAHs) and their N- and O-containing derivatives (N-/O-PAHs) are environmental pollutants and synthetically attractive building blocks in pharmaceuticals. Functionalization of PAHs can be achieved via C-H activation by cytochrome P450 enzymes (e.g., P450 CYP3A4) in an environmentally friendly manner. Despite its broad substrate scope, the contribution of CYP3A4 to metabolize common PAHs in humans was found to be small. We recently showcased the potential of CYP3A4 in whole-cell biocatalysis with recombinant yeast Komagataella phaffii (Pichia pastoris) catalysts for the preparative-scale synthesis of naturally occurring metabolites in humans. In this study, we aimed at exploring the substrate scope of CYP3A4 towards (N-/O)-PAHs and conducted a bioconversion experiment at 10 L scale to validate the synthetic potential of CYP3A4 for the preparative-scale production of functionalized PAH metabolites. Hydroxylated products were purified and characterized using HPLC and NMR analysis. In total, 237 mg of fluorenol and 48 mg of fluorenone were produced from 498 mg of fluorene, with peak productivities of 27.7 µmol/L/h for fluorenol and 5.9 µmol/L/h for fluorenone; the latter confirmed that CYP3A4 is an excellent whole-cell biocatalyst for producing authentic human metabolites.

Preparative scale mass spectrometry: A brief history of the calutron.

Calutrons were developed in the laboratory of E. O. Lawrence at the University of California at Berkeley. They were a modification of the cyclotrons he had invented and used in his Nobel Prize-winning investigations of the atomic nucleus. At the time their construction was undertaken, calutrons represented the only certain means of preparing enriched uranium isotopes for the construction of a fission bomb. The effort was successful enough that every atom of the 42 kg of 235 U used in the first uranium bomb had passed through at least one stage of calutron separation. At peak production, the first stage separators, α tanks, yielded an aggregate 258-g/day 235 U enriched to about 10 at. % from its natural abundance level of 0.72 at. %. The second stage separators, ß tanks, used the 10 at. % material as feedstock and produced a total 204-g/day 235 U enriched to at least 80 at. %. The latter, weapons grade, material was used in fission bombs. Under typical operating conditions, each α tank operated at a uranium beam intensity at the collectors of approximately 20 mA and each ß tank at a beam intensity of approximately 215 mA at the collectors. Bulk separation of isotopes for bomb production ceased in 1945. Since that time calutrons have been used to separate stable isotopes, but on a more limited scale than wartime weapons production. Stable isotope separations since 1960 have taken place using one modified ß tank.

Combined Use of Deep Eutectic Solvents, Macroporous Resins, and Preparative Liquid Chromatography for the Isolation and Purification of Flavonoids and 20-Hydroxyecdysone from Chenopodium quinoa Willd.

: Deep eutectic solvents (DESs) were used in combination with macroporous resins to isolate and purify flavonoids and 20-hydroxyecdysone from Chenopodium quinoa Willd by preparative high-performance liquid chromatography (HPLC). The extraction performances of six DESs and the adsorption/desorption performances of five resins (AB-8, D101, HPD 400, HPD 600, and NKA-9) were investigated using the total flavonoid and 20-hydroxyecdysone extraction yields as the evaluation criteria, and the best-performing DES (choline chloride/urea, DES-6) and macroporous resin (D101) were further employed for phytochemical extraction and DES removal, respectively. The purified extract was subjected to preparative HPLC, and the five collected fractions were purified in a successive round of preparative HPLC to isolate three flavonoids and 20-hydroxyecdysone, which were identified by spectroscopic techniques. The use of a DES in this study significantly facilitated the preparative-scale isolation and purification of polar phytochemicals from complex plant systems.

Whole-cell biocatalysis platform for gram-scale oxidative dearomatization of phenols.

Technologies enabling new enzyme discovery and efficient protein engineering have spurred intense interest in the development of biocatalytic reactions. In recent years, whole-cell biocatalysis has received attention as a simple, efficient, and scalable biocatalytic reaction platform. Inspired by these developments, we have established a whole-cell protocol for oxidative dearomatization of phenols using the flavin-dependent monooxygenase, TropB. This approach provides a scalable biocatalytic platform for accessing gram-scale quantities of chiral synthetic building blocks.

Keeping pace with the increasing demand for high quality drug candidates in pharmaceutical research: Development of a new two-step preparative tandem high performance chromatographic system for the purification of antibodies.

In the area of biological drug development, high throughput (HT) technologies are key to identifying the most promising therapeutic candidate in a time-efficient and market-competitive manner. While efficient cloning and expression methods exist, HT downstream processing mainly relies on liquid handling workstations applying miniaturized chromatography columns or resin-based 96-well plates to shorten process development time. However, there is still a lack of generic, preparative chromatographic methods devoid of aggregates and endotoxins with sufficient throughput. The only truly generic antibody purification strategy including an efficient dimer removal consists of Protein A capture followed by size exclusion chromatography (SEC) as a polishing step. Other polishing methods, including IEX, HIC, and CHT, require an antibody-specific fine tuning. However, standard preparative SEC setups tend to be rather time-consuming, and so limit throughput. In this work, we devised a unique chromatography setup enabling an unattended two-step purification of IgGs on the milligram scale directly from 35 mL clarified cell supernatants, processing up to 48 samples in 44.0 h. By introducing a silica-based SEC column, preparative SEC could be accelerated. By further developing an HT two-step preparative Protein A/alternating column regeneration SEC system using Agilent 1260 Infinity LC components, mAbs can be purified generically by two chromatographic steps in 55 min. In this way, by using a 2-position/10-port valve and two quaternary pumps, two SEC columns can be run in parallel, excluding the cleaning and equilibrating phase from the actual cycle time. By further applying a third pump, the Protein A step can be run independently, resulting in a time-optimized process nesting. By introducing a CETAC ASX-520 autosampler, 48 samples can be run automatically without any user intervention over two working days. The developed system is highly reproducible for all tested human IgG1 antibodies, easily generating milligram scale material sufficient for full characterization of the antibodies and for their use in in vitro and in vivo activity assessments.

Hybrid optimization of preparative chromatography for a ternary monoclonal antibody mixture.

In the purification of monoclonal antibodies, ion-exchange chromatography is typically used among the polishing steps to reduce the amount of product-related impurities such as aggregates and fragments, whilst simultaneously reducing HCP, residual Protein A and potential toxins and viruses. When the product-related impurities are difficult to separate from the products, the optimization of these chromatographic steps can be complex and laborious. In this paper, we optimize the polishing chromatography of a monoclonal antibody from a challenging ternary feed mixture by introducing a hybrid approach of the simplex method and a form of local optimization. To maximize the productivity of this preparative bind-and-elute cation-exchange chromatography, wide ranges of the three critical operational parameters-column loading, the initial salt concentration, and gradient slope-had to be considered. The hybrid optimization approach is shown to be extremely effective in dealing with this complex separation that was subject to multiple constraints based on yield, purity, and product breakthrough. Furthermore, it enabled the generation of a large knowledge space that was subsequently used to study the sensitivity of the objective function. Increased design space understanding was gained through the application of Monte Carlo simulations. Hence, this work proposes a powerful hybrid optimization method, applied to an industrially relevant process development challenge. The properties of this approach and the results and insights gained, make it perfectly suited for the rapid development of biotechnological unit operations during early-stage bioprocess development.

Preparative Recovery of Carotenoids from Microalgal Biomass.

Carotenoids are widespread substances with important physiological roles, and some of them, such as lutein, astaxanthin, or vaucherioxanthin, are high-value products that can be used as high-quality food color and antioxidants, and some have an alleged role in the prevention of disorders such as AMD. Carotenoid extracts are currently obtained from plant sources, but microalgae have been demonstrated to be a competitive source likely to become an alternative. The extraction of carotenoids from microalgae possesses specific problems that arise from the different structure and composition of the source biomass. Here is presented a method for the recovery of carotenoid extracts from microalgal biomass in the kilogram scale.

Analytical and Preparative Scale Separation of Enantiomers of Chiral Drugs by Chromatography and Related Methods.

While the amino acids, enzymes and hormones are chiral, chirality plays significant role in the life of plants, animals, as well as the human being. Chirality of molecules is important in various industries, such as pharmaceutical, agricultural, food, electronics, etc. Chiral drugs may have different bioavailability, distribution, biotransformation and excretion, as well as quantitatively and/or qualitatively different pharmacological or toxic properties. Enantiomerically pure chiral drugs have been increasingly developed for the pharmaceutical market due to their superiority from the viewpoints of potency and safety. This is supported by the development of new methods for enantioselective production of the chiral compounds, as well as by the capability of the enantioselective analytical methods to allow a detection and quantification of minor enantiomeric impurity in the presence of another enantiomer in a large excess. The aim of the present review is to provide a short summary of the basic principles of chiral separations on an analytical and preparative scale. In addition, some selected applications for analytical techniques, such as gas chromatography, supercritical fluid chromatography, high performance liquid chromatography, capillary electrophoresis and capillary electrochromatography for the separation of enantiomers of chiral pharmaceuticals published in the last two years are also discussed.

Lipase-catalysed esters synthesis of cafestol and kahweol.

Cafestol and kahweol (C&K), two coffee diterpene alcohols with structural similarity which exhibit anticarcinogenic effects, were isolated from green coffee Arabica beans, followed by their lipase-catalysed esterification and purification by preparative high-performance liquid chromatography (HPLC). The isolation and enzymatic synthesis parameters of C&K esters were studied, with the latter optimised by a Central Composite Design; both procedures were monitored by gas chromatography. Scale up and improved isolation conditions resulted in 1.29 g of C&K, with 98% purity from 300 g of green Arabica beans. The highest C&K ester yields were observed using an alcohol:fatty acid molar ratio of 1:5, 73.3 mg mL-1 of CAL-B enzyme, 70 °C and 240 rpm for 3 days in toluene, leading to 85-88% conversion among a variety of tested C&K esters, including n-C14:0-C20:0, C18:1, C18:2 and C18:3.

Laboratory-Scale Hydroxylation of Steroids by P450BM3 Variants.

This chapter describes the asymmetric hydroxylation of steroids on laboratory preparative scale, using engineered variants of P450BM3 (CYP102A1) as enzyme catalyst. The following protocol covers the creation of an Escherichia coli BL21-Gold (DE3) expression strain, including necessary control experiments like plasmid preparation, test expression, and creation of storage cultures, to verify successful experimental access to recombinant expressed P450BM3 variants. The recombinant expressed P450BM3 variants are obtained as cleared cell lysate and used in a biotransformation setup to hydroxylate 2.8 mg and up to 15 mg testosterone in the presented protocol. Since P450BM3 depends on NADPH as an electron source for the reaction, a glucose and glucose dehydrogenate based recycling system is added to the reaction. The protocol further includes liquid-liquid extraction of hydroxytestosterone and directs the experimenter to compound purification via column chromatography.

Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro oxindole alkaloids from Uncaria macrophylla.

Increasing challenge arising from configurational interconversion in aqueous solvent renders it rather difficult to isolate high-purity tautomeric reference standards and thus largely hinders the holistic quality control of traditional Chinese medicine (TCM). Spiro oxindole alkaloids (SOAs), as the markers for the medicinal Uncaria herbs, can easily isomerize in polar or aqueous solvent via a retro-Mannich reaction. In the present study, supercritical fluid chromatography (SFC) is utilized to separate and isolate two pairs of 7-epimeric SOAs, including rhynchophylline (R) and isorhynchophylline (IR), corynoxine (C) and corynoxine B (CB), from Uncaria macrophylla. Initially, the solvent that can stabilize SOA epimers was systematically screened, and acetonitrile was used to dissolve and as the modifier in SFC. Then, key parameters of ultra-high performance SFC (ultra-performance convergence chromatography, UPC2), comprising stationary phase, additive in modifier, column temperature, ABPR pressure, and flow rate, were optimized in sequence. Two isocratic UPC2 methods were developed on the achiral Torus 1-AA and Torus Diol columns, suitable for UV and MS detection, respectively. MCI gel column chromatography fractionated the U. macrophylla extract into two mixtures (R/IR and C/CB). Preparative SFC, using a Viridis Prep Silica 2-EP OBD column and acetonitrile-0.2% diethylamine in CO2 as the mobile phase, was finally employed for compound purification. As a result, the purity of four SOA compounds was all higher than 95%. Different from reversed-phase HPLC, SFC, by use of water-free mobile phase (inert CO2 and aprotic modifier), provides a solution to rapid analysis and isolation of tautomeric reference standards for quality control of TCM.

Preparative supercritical fluid chromatography: A powerful tool for chiral separations.

In 2012, the 4 biggest pharmaceutical blockbusters were pure enantiomers and separating racemic mixtures is now frequently a key step in the development of a new drug. For a long time, preparative liquid chromatography was the technique of choice for the separation of chiral compounds either during the drug discovery process to get up to a hundred grams of a pure enantiomer or during the clinical trial phases needing kilograms of material. However the advent of supercritical Fluid Chromatography (SFC) in the 1990s has changed things. Indeed, the use of carbon dioxide as the mobile phase in SFC offers many advantages including high flow rate, short equilibration time as well as low solvent consumption. Despite some initial teething troubles, SFC is becoming the primary method for preparative chiral chromatography. This article will cover recent developments in preparative SFC for the separation of enantiomers, reviewing several aspects such as instrumentation, chiral stationary phases, mobile phases or purely preparative considerations including overloading, productivity or large scale chromatography.