High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures.

Rhizosecretion of recombinant pharmaceuticals from in vitro hydroponic transgenic plant cultures is a simple, low cost, reproducible and controllable production method. Here, we demonstrate the application and adaptation of this manufacturing platform to a human antivitronectin IgG1 monoclonal antibody (mAb) called M12. The rationale for specific growth medium additives was established by phenotypic analysis of root structure and by LC-ESI-MS/MS profiling of the total protein content profile of the hydroponic medium. Through a combination of optimization approaches, mAb yields in hydroponic medium reached 46 µg/mL in 1 week, the highest figure reported for a recombinant mAb in a plant secretion-based system to date. The rhizosecretome was determined to contain 104 proteins, with the mAb heavy and light chains the most abundant. This enabled evaluation of a simple, scalable extraction and purification protocol and demonstration that only minimal processing was necessary prior to protein A affinity chromatography. MALDI-TOF MS revealed that purified mAb contained predominantly complex-type plant N-glycans, in three major glycoforms. The binding of M12 purified from hydroponic medium to vitronectin was comparable to its Chinese hamster ovary (CHO)-derived counterpart. This study demonstrates that in vitro hydroponic cultivation coupled with recombinant protein rhizosecretion can be a practical, low-cost production platform for monoclonal antibodies.

A comprehensive classification of solvent systems used for natural product purifications in countercurrent and centrifugal partition chromatography.

Using both library paper copies and modern electronic copies, every known, published, English-language journal paper that employs either countercurrent or centrifugal partition chromatography solvent systems for natural product purifications has been studied and the solvent systems classified in a comprehensive database. Papers were studied from the earliest found examples containing natural product separations in 1984 until the end of 2014. In total, 2594 solvent systems have been classified, of which 272 are gradient systems. To observe any trends or patterns in the data, the natural product solutes were divided into 21 classes and the solvent systems into 7 different types. The complete database, sorted according to natural product class, is available for download to assist separation scientists in future liquid-liquid chromatography purifications.

Counter-current chromatography for the separation of terpenoids: a comprehensive review with respect to the solvent systems employed.

Natural products extracts are commonly highly complex mixtures of active compounds and consequently their purification becomes a particularly challenging task. The development of a purification protocol to extract a single active component from the many hundreds that are often present in the mixture is something that can take months or even years to achieve, thus it is important for the natural product chemist to have, at their disposal, a broad range of diverse purification techniques. Counter-current chromatography (CCC) is one such separation technique utilising two immiscible phases, one as the stationary phase (retained in a spinning coil by centrifugal forces) and the second as the mobile phase. The method benefits from a number of advantages when compared with the more traditional liquid-solid separation methods, such as no irreversible adsorption, total recovery of the injected sample, minimal tailing of peaks, low risk of sample denaturation, the ability to accept particulates, and a low solvent consumption. The selection of an appropriate two-phase solvent system is critical to the running of CCC since this is both the mobile and the stationary phase of the system. However, this is also by far the most time consuming aspect of the technique and the one that most inhibits its general take-up. In recent years, numerous natural product purifications have been published using CCC from almost every country across the globe. Many of these papers are devoted to terpenoids-one of the most diverse groups. Naturally occurring terpenoids provide opportunities to discover new drugs but many of them are available at very low levels in nature and a huge number of them still remain unexplored. The collective knowledge on performing successful CCC separations of terpenoids has been gathered and reviewed by the authors, in order to create a comprehensive document that will be of great assistance in performing future purifications.

Two-step separation of nostotrebin 6 from cultivated soil cyanobacterium (Nostoc sp.) by high performance countercurrent chromatography.

High performance countercurrent chromatography (HPCCC) was successfully applied for the separation of nostotrebin 6 from cultivated soil cyanobacteria in a two-step operation. A two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (4:5:4:5, v/v/v/v) was employed for the HPCCC separation. In the first-step operation, its neutral upper phase was used as stationary phase and its basic lower phase (1% NH3 in lower phase) was employed as mobile phase at a flow rate of 1 mL/min. In the second operation step, its neutral upper phase was used as stationary phase, whereas both its neutral lower phase and basic lower phase were employed as mobile phase with a linear gradient elution at a flow rate of 0.8 mL/min. The revolution speed and temperature of the separation column were 1,000 rpm and 30 °C, respectively. Using HPCCC followed by clean-up on Sephadex LH-20 gel, 4 mg of nostotrebin 6 with a purity of 99% as determined by HPLC/DAD-ESI-HRMS was obtained from 100 mg of crude extract. The chemical identity of the isolated compound was confirmed by comparing its spectroscopic data (UV, ESI-HRMS, ESI-HRMS2) with those of an authentic standard and data available in the literature.

Changes in the mobile phase composition on a stepwise counter-current chromatography elution for the isolation of flavonoids from Siparuna glycycarpa.

This paper describes the isolation of flavonoids and other aromatic compounds from an ethyl acetate extract of leaves of Siparuna glycycarpa using stepwise elution counter-current chromatography (CCC). The elution profile yielded the following compounds: diglycosylated flavonoids, quercetin 3-O-rutinoside and quercetin 7-O-rutinoside, followed by monoglycosylated flavonoids, kaempferol-3-O-ß-glucopyranoside, kaempferol-3-O-ß-rhamnopiranoside, kaempferol-3-O-ß-6''(p-coumaroyl) glucopyranoside, and quercetin-3-O-ß-glucopyranoside, and then free phenolics, protocatechuic acid, and 2',6'-dihydroxy-4, 4'-dimethoxydihydrochalcone, which shows that this type of elution covers a broader range of polarity than the traditional isocratic mode. This makes it more suitable to perform separations of mixtures containing large differences in hydrophobicity. A GC analysis of a blank CCC run was performed to determine if changes in the mobile phase composition affect the chromatographic process. Results showed a gradual variation of the composition of the mobile phase emerging after the step gradient, favoring the selectivity of the solvent system.

Isolation of the minor and rare constituents from fruits of Peucedanum alsaticum L. using high-performance counter-current chromatography.

A high-performance counter-current chromatography (HPCCC) method was applied for the first time for the preparative separation and purification of three rare compounds which occur as minor constituents in the fruits of Peucedanum alsaticum L.: 5-substituted coumarin notoptol and two dihydropyranochromones: divaricatol and ledebouriellol. A scale-up process from analytical to preparative in a very short time was developed. In order to purify a range of rare and minor compounds with different polarity two separate experiments were performed, one in reverse phase, the other in normal phase, using the same crude extract. A two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (1:1:1:1) was developed. The components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 0.7 mg of notoptol, 1.46 mg of ledebouriellol at purity of 99.5%, and 10 mg of mixtures of divaricatol, alsaticol and alsaticocoumarin, where divaricatol present 22% by peak area. These amounts were obtained from 1 g of the crude extract in a single run. This is the first time when minor notoptol, ledebouriellol, and divaricatol were isolated in a single run using HPCCC method and first time when these were identified in plant from Peucedanum genus.

Improved hydrophilic interaction chromatography method for the identification and quantification of glucosinolates.

An improved hydrophilic interaction liquid chromatography (HILIC) method has been developed to separate members of a closely related family of chemoprotective phytochemicals called glucosinolates. This method exploits the emergence of a second generation of HILIC chemistry, using a silica-based permanently zwitterionic stationary phase. These columns are more robust, durable, and glucosinolates separations are more reproducible than with the original polyhydroxyethyl aspartamide columns. Furthermore, the HILIC system that we report herein permits much greater alteration of the mobile phase composition for customized separation of glucosinolates from plant extracts, across a wide spectrum of polarity.

Counter-current chromatography separation scaled up from an analytical column to a production column.

An analytical separation was performed on an analytical J-type counter-current chromatography (CCC) instrument using a 5.4 ml column, with a 1 ml/min mobile phase flow rate. This separation had a resolution of 0.69 and was achieved in 10 min. The same separation was performed using two 2300 ml columns connected in series at a flow rate of 850 ml/min using a production scale J-type centrifuge. This production scale separation was also obtained in 10 min with a resolution of 0.71. This represents an 850 times increase in productivity. This paper presents these separations and the underlying scale up theory.

Rapid purification and scale-up of honokiol and magnolol using high-capacity high-speed counter-current chromatography.

In this paper, a rapid separation approach has been developed using high-capacity high-speed counter-current chromatography (high-capacity HSCCC) to isolate and purify honokiol and magnolol, which are the main bioactive constituents from Houpu. The optimization of the solvent selection process, sample loading volume and flow rate is systematically studied using analytical high-capacity HSCCC. The optimized parameters obtained rapidly at analytical scale were used for a 1000 x scale-up preparative run using pilot scale high-capacity HSCCC in a MAXI-DE centrifuge. A crude sample of 43 g was successfully separated and the fractions were analysed by high-performance liquid chromatography (HPLC). This large scale preparative single step run yielded 16.9 and 19.4 g of honokiol and magnolol with purities of 98.6 and 99.9%, in only 20 min. This is the first time that high-performance counter-current chromatography has been used to purify multiple gram grade bioactive compounds in less than 1h and at such high concentrations of final products (10.8 g/l for magnolol and 7.0 g/l for honokiol).

Sample injection strategy to increase throughput in counter-current chromatography: Case study of Honokiol purification.

Counter-current chromatography (CCC) has been widely used as a preparative separation method to purify natural products from plant extracts and fermentation broths. Traditionally, throughput optimization in CCC has focused on sample concentration and sample volume. In this paper sample injection was considered as consisting of three variables: injection flow rate, post-injection flow rate and sample solvent. The effects of these parameters were studied using a honokiol purification from a Magnolia officinalis bark extract as a case study aiming to achieve the highest throughput/yield ratio for greater than 99% purity of this potential anti-cancer drug obtained for submission to the Chinese FDA. An injection method was established that increased the throughput of honokiol by 46.5% (from 3.05g/h to 4.47g/h), and decreased the solvent consumption of mobile phase and stationary phase per gram of honokiol by 40.0% (from 0.68L/g to 0.41L/g) and 48.4% (from 0.40L/g to 0.21L/g) respectively. These results show the importance of understanding the whole injection process when optimizing a given CCC separation.

Schinus terebinthifolius countercurrent chromatography (Part II): Intra-apparatus scale-up and inter-apparatus method transfer.

Countercurrent chromatography (CCC) is being widely used across the world for purification of various materials, especially in natural product research. The predictability of CCC scale-up has been successfully demonstrated using specially designed instruments of the same manufacturer. The reality is that the most of CCC users do not have access to such instruments and do not have enough experience to transfer methods from one CCC column to another. This unique study of three international teams is based on innovative approach to simplify the scale-up between different CCC machines using fractionation of Schinus terebinthifolius berries dichloromethane extract as a case study. The optimized separation methodology, recently developed by the authors (Part I), was repeatedly performed on CCC columns of different design available at most research laboratories across the world. Hexane - ethyl acetate - methanol - water (6:1:6:1, v/v/v/v) was used as solvent system with masticadienonic and 3ß-masticadienolic acids as target compounds to monitor stationary phase retention and calculate peak resolution. It has been demonstrated that volumetric, linear and length scale-up transfer factors based on column characteristics can be directly applied to different i.d., volume and length columns independently on instrument make in an intra-apparatus scale-up and inter-apparatus method transfer.

Biomechanical parameters in lower limbs during natural walking and Nordic walking at different speeds.

PURPOSE: Nordic Walking (NW) is a sport that has a number of benefits as a rehabilitation method. It is performed with specially designed poles and has been often recommended as a physical activity that helps reduce the load to limbs. However, some studies have suggested that these findings might be erroneous. STUDY AIM: The aim of this paper was to compare the kinematic, kinetic and dynamic parameters of lower limbs between Natural Walking (W) and Nordic Walking (NW) at both low and high walking speeds. METHODS: The study used a registration system, BTS Smart software and Kistler platform. Eleven subjects walked along a 15-metre path at low (below 2 m⋅s-1) and high (over 2 m⋅s-1) walking speeds. The Davis model was employed for calculations of kinematic, kinetic and dynamic parameters of lower limbs. RESULTS: With constant speed, the support given by Nordic Walking poles does not make the stroke longer and there is no change in pelvic rotation either. The only change observed was much bigger pelvic anteversion in the sagittal plane during fast NW. There were no changes in forces, power and muscle torques in lower limbs. CONCLUSIONS: The study found no differences in kinematic, kinetic and dynamic parameters between Natural Walking (W) and Nordic Walking (NW). Higher speeds generate greater ground reaction forces and muscle torques in lower limbs. Gait parameters depend on walking speed rather than on walking style.

Using quantitative structure activity relationship models to predict an appropriate solvent system from a common solvent system family for countercurrent chromatography separation.

Countercurrent chromatography (CCC) is a form of liquid-liquid chromatography. It works by running one immiscible solvent (mobile phase) over another solvent (stationary phase) being held in a CCC column using centrifugal force. The concentration of compound in each phase is characterised by the partition coefficient (Kd), which is the concentration in the stationary phase divided by the concentration in the mobile phase. When Kd is between approximately 0.2 and 2, it is most likely that optimal separation will be achieved. Having the Kd in this range allows the compound enough time in the column to be separated without resulting in a broad peak and long run time. In this paper we report the development of quantitative structure activity relationship (QSAR) models to predict logKd. The QSAR models use only the molecule's 2D structure to predict the molecular property logKd.

New solvent systems for gradient counter-current chromatography in separation of betanin and its derivatives from processed Beta vulgaris L. juice.

Betalains, natural plant pigments, are beneficial compounds due to their antioxidant and possible chemoprotective properties. A mixture of betalains: betanin/isobetanin, decarboxybetanins and neobetanin from processed red beet roots (Beta vulgaris L.) juice was separated in food-grade, gradient solvent systems using high-performance counter-current chromatography (HPCCC). The decarboxylated and dehydrogenated betanins were obtained by thermal degradation of betanin/isobetanin from processed B. vulgaris L. juice under mild conditions. Two solvent systems (differing in their composition by phosphoric acid and ethanol volume gradient) consisting of BuOH-EtOH-NaClsolution-H2O-H3PO4 (v/v/v/v/v, 1300:200-1000:1300:700:2.5-10) in the 'tail-to-head' mode were run. The flow rate of the mobile phase (organic phase) was 1.0 or 2.0 ml/min and the column rotation speed was 1,600 rpm (20°C). The retention of the solvent system stationary phase (aqueous phase) was ca. 80%. The system with the acid and ethanol volume gradient consisting of BuOH-EtOH-NaClsolution-H2O-H3PO4 (v/v/v/v/v, 1300:200-240:1300:700:2.5-4.5) pumped at 2.0 ml/min was the most effective for a separation of betanin/isobetanin, 17-decarboxy-betanin/-isobetanin, 2-decarboxy-betanin/-isobetanin, 2,17-bidecarboxy-betanin/-isobetanin pairs as well as neobetanin. The pigments were detected by LC-DAD and LC-MS. The results are crucial in the application of completely food-grade solvent systems in separation of food-grade compounds as well, and the systems can possibly be extended to other ionizable and polar compounds with potential health benefits. In particular, the method is applicable for the isolation and purification of betalains present in such rich sources as B. vulgaris L. roots as well as cacti fruits and Amaranthaceae flowering plants due to modification possibilities of the solvent systems polarity.

Schinus terebinthifolius scale-up countercurrent chromatography (Part I): High performance countercurrent chromatography fractionation of triterpene acids with off-line detection using atmospheric pressure chemical ionization mass spectrometry.

'Countercurrent chromatography' (CCC) is an ideal technique for the recovery, purification and isolation of bioactive natural products, due to the liquid nature of the stationary phase, process predictability and the possibility of scale-up from analytical to preparative scale. In this work, a method developed for the fractionation of Schinus terebinthifolius Raddi berries dichloromethane extract was thoroughly optimized to achieve maximal throughput with minimal solvent and time consumption per gram of processed crude extract, using analytical, semi-preparative and preparative 'high performance countercurrent chromatography' (HPCCC) instruments. The method using the biphasic solvent system composed of n-heptane-ethyl acetate-methanol-water (6:1:6:1, v/v/v/v) was volumetrically scaled up to increase sample throughput up to 120 times, while maintaining separation efficiency and time. As a fast and specific detection alternative, the fractions collected from the CCC-separations were injected to an 'atmospheric pressure chemical ionization mass-spectrometer' (APCI-MS/MS) and reconstituted molecular weight MS-chromatograms of the APCI-ionizable compounds from S. terebinthifolius were obtained. This procedure led to the direct isolation of tirucallane type triterpenes such as masticadienonic and 3ß-masticadienolic acids. Also oleanonic and moronic acids have been identified for the first time in the species. In summary, this approach can be used for other CCC scale-up processes, enabling MS-target-guided isolation procedures.

The influence of a bicycle commuter's appearance on drivers' overtaking proximities: an on-road test of bicyclist stereotypes, high-visibility clothing and safety aids in the United Kingdom.

This study looked at whether drivers overtaking a bicyclist changed the proximities of their passes in response to the level of experience and skill signalled by the bicyclist's appearance. Seven outfits were tested, ranging from a stereotypical sport rider's outfit, portraying high experience and skill, to a vest with 'novice cyclist' printed on the back, portraying low experience. A high-visibility bicycling jacket was also used, as were two commercially available safety vests, one featuring a prominent mention of the word 'police' and a warning that the rider was video-recording their journey, and one modelled after a police officer's jacket but with a letter changed so it read 'POLITE'. An ultrasonic distance sensor recorded the space left by vehicles passing the bicyclist on a regular commuting route. 5690 data points fulfilled the criteria for the study and were included in the analyses. The only outfit associated with a significant change in mean passing proximities was the police/video-recording jacket. Contrary to predictions, drivers treated the sports outfit and the 'novice cyclist' outfit equivalently, suggesting they do not adjust overtaking proximity as a function of a rider's perceived experience. Notably, whilst some outfits seemed to discourage motorists from passing within 1m of the rider, approximately 1-2% of overtakes came within 50 cm no matter what outfit was worn. This suggests there is little riders can do, by altering their appearance, to prevent the very closest overtakes; it is suggested that infrastructural, educational or legal measures are more promising for preventing drivers from passing extremely close to bicyclists.

Phase system selection with fractional factorial design for purification of recombinant cyanovirin-N from a hydroponic culture medium using centrifugal partition chromatography.

Centrifugal partition chromatography (CPC) with an aqueous two-phase system (ATPS) was used to purify recombinant cyanovirin-N (CV-N) from other proteins which were co-secreted into a hydroponic plant medium in a rhizosecretion process. To achieve satisfactory protein concentration, the purification was preceded by ultrafiltration performed on a 5 kDa filter. ATPS, because of their gentle nature, were selected as the phase system for CPC. A systematic phase system selection was applied. This involved studying the effect of seven parameters of ATPS: polymer type, salt type, the polymer and salt concentration, the polymer molecular weight, pH, and presence of two additional salts; NaCl and NaClO4, which all together gave 320 combinations. design of experiment (DoE) software allowed the reduction of this number to 46. Having tested partitioning of cyanovirin-N and impurities in 46 ATPS, the three best potential phase systems generated by the programme were then tested on the CPC. Out of these three, 13/13% PEG4000 sodium phosphate, pH 3.0, proved to be most effective phase system in the purification of cyanovirin-N, judged by ELISA and SDS-PAGE analysis, as it eliminated most of the impurities from the final cyanovirin-N preparation.

Versatile solvent systems for the separation of betalains from processed Beta vulgaris L. juice using counter-current chromatography.

Two mixtures of decarboxylated and dehydrogenated betacyanins from processed red beet roots (Beta vulgaris L.) juice were fractionated by high performance counter-current chromatography (HPCCC) producing a range of isolated components. Mixture 1 contained mainly betacyanins, 14,15-dehydro-betanin (neobetanin) and their decarboxylated derivatives while mixture 2 consisted of decarboxy- and dehydro-betacyanins. The products of mixture 1 arose during thermal degradation of betanin/isobetanin in mild conditions while the dehydro-betacyanins of mixture 2 appeared after longer heating of the juice from B. vulgaris L. Two solvent systems were found to be effective for the HPCCC. A highly polar, high salt concentration system of 1-PrOH-ACN-(NH4)2SO4 (satd. soln)-water (v/v/v/v, 1:0.5:1.2:1) (tail-to-head mode) enabled the purification of 2-decarboxy-betanin/-isobetanin, 2,17-bidecarboxy-betanin/-isobetanin and neobetanin (all from mixture 1) plus 17-decarboxy-neobetanin, 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, 2-decarboxy-neobetanin and 2,15,17-tridecarboxy-neobetanin (from mixture 2). The other solvent system included heptafluorobutyric acid (HFBA) as ion-pair reagent and consisted of tert-butyl methyl ether (TBME)-1-BuOH-ACN-water (acidified with 0.7% HFBA) (2:2:1:5, v/v/v/v) (head-to-tail mode). This system enabled the HPCCC purification of 2,17-bidecarboxy-betanin/-isobetanin and neobetanin (from mixture 1) plus 2,15,17-tridecarboxy-2,3-dehydro-neobetanin, 2,17-bidecarboxy-2,3-dehydro-neobetanin and 2,15,17-tridecarboxy-neobetanin (mixture 2). The results of this research are crucial in finding effective isolation methods of betacyanins and their derivatives which are meaningful compounds due their colorant properties and potential health benefits regarding antioxidant and cancer prevention. The pigments were detected by LC-DAD and LC-MS/MS techniques.

Scalable technology for the extraction of pharmaceutics: outcomes from a 3 year collaborative industry/academia research programme.

This paper reports on some of the key outcomes of a 3 year £1.5m Technology Strategy Board (TSB) funded research programme to develop a small footprint, versatile, counter-current chromatography purification technology and methodology which can be operated at a range of scales in both batch and continuous modes and that can be inserted into existing process plant and systems. Our consortium, integrates technology providers (Dynamic Extractions) and the scientific development team (Brunel) with end user needs (GSK & Pfizer), addressing major production challenges aimed at providing flexible, low capital platform technology driving substantial cost efficiency in both drug development and drug manufacturing processes. The aims of the Technology Strategy Board's high value manufacturing programme are described and how the academic/industry community were challenged to instigate step changes in the manufacturing of high value pharmaceuticals. This paper focusses on one of the themes of the TSB research programme, "Generate a Comprehensive Applications Portfolio". It outlines 15 applications from this portfolio that can be published in the public domain and gives four detailed case studies illustrating the range of application of the technology on the separation of (1) isomers, (2) polar compounds, (3) crude mixtures and (4) on the removal of impurities. Two of these case studies that were scaled up demonstrate between 10 and 20% lower solvent usage and were projected to have significant cost savings compared to conventional solid phase silica gel chromatography at procss scale demonstrating that the latest high performance countercurrent chromatography technology is a competitive platform technolgy for the pharmaceutical industry.

Bioactivity-guided fractionation of the volatile oil of Angelica sinensis radix designed to preserve the synergistic effects of the mixture followed by identification of the active principles.

In natural product research, it is a common experience that fractionation of biologically-active crude extracts can lead to the loss of their original activity. This is attributed to synergistic effects, where two or more components are required to be present together for full activity of the sample. Our previous study showed that a volatile oil of Angelica sinensis radix (VOAS) inhibited endothelial cell proliferation in culture. Here we have used a bioactivity-guided fractionation method to preserve any synergistic effects of VOAS combining countercurrent chromatography (CCC), the MTS cell viability assay and gas chromatography (GC). Using a two-phase CCC solvent system (heptane-ethyl acetate-methanol-water at a volume ratio of 27:23:27:23%), forty-five fractions were isolated, nine of which exhibited anti-endothelial properties. GC analysis showed two bioactive alkylphthalides, Z-ligustilide and n-butylidenephthalide (BP) were the major compounds detected in the bioactive fractions, and were absent in non-bioactive fractions. Our results indicate that Z-ligustilide and BP are the main constituents responsible for the anti-endothelial properties of VOAS. This rapid and reliable approach in preserving sample activity while isolating and identifying its active compounds suggests that this protocol can be a powerful tool for drug discovery from natural products.