Low-cost and open-source strategies for chemical separations.

In recent years, a trend toward utilizing open access resources for laboratory research has begun. Open-source design strategies for scientific hardware rely upon the use of widely available parts, especially those that can be directly printed using additive manufacturing techniques and electronic components that can be connected to low-cost microcontrollers. Open-source software eliminates the need for expensive commercial licenses and provides the opportunity to design programs for specific needs. In this review, the impact of the "open-source movement" within the field of chemical separations is described, primarily through a comprehensive look at research in this area over the past five years. Topics that are covered include general laboratory equipment, sample preparation techniques, separations-based analysis, detection strategies, electronic system control, and software for data processing. Remaining hurdles and possible opportunities for further adoption of open-source approaches in the context of these separations-related topics are also discussed.

A low-cost, open-source digital stripchart recorder for chromatographic detectors using a Raspberry Pi.

One of the most critical aspects of chromatographic analysis is effective data acquisition and processing. Typical approaches include software platforms designed for specific instruments or commercial data acquisition hardware boards, both of which require expensive licenses to use and operate. To increase the access and affordability of chromatographic data acquisition, especially for systems in which software control has become obsolete or must be written in-house, an open-source digital stripchart recorder has been developed. This system is built upon a Raspberry Pi single-board computer and a plug-in printed circuit board with the necessary integrated circuits for data acquisition. Using an open-source software called Processing, a complete user interface to control the system was developed that enables the acquisition, filtering, and processing of chromatographic data. The system performs comparably to more expensive platforms, with calculated values for peak area, retention time, and plate count all within 3% of the values calculated by a widely used commercial chromatography data software package.

Integrated continuous biomanufacturing platform with ATF perfusion and one column chromatography operation for optimum resin utilization and productivity.

A new integrated continuous biomanufacturing platform for continuous production of antibodies at fixed cell volumes and cell concentrations for extended periods with immediate capture is presented. Upstream antibody production has reached technological maturity, however, the bottleneck for continuous biomanufacturing remains the efficient and cost-effective capture of therapeutic antibodies in an initial chromatography step. In this study, the first successful attempt at using one-column continuous chromatography (OCC) for the continuous capture of therapeutic antibodies produced through alternating tangential flow perfusion is presented. By performing upstream media optimizations, the upstream perfusion rate was reduced to one vessel volume per day (vv/d), increasing antibody titer and reducing the volume of perfusate. In addition, process improvements were performed to increase productivity by 80% over previously reported values. In addition, a real-time method for evaluating column performance to make column switching decisions was developed. This improved productivity coupled with the use of a single-column improved process monitoring and control in OCC compared to multi-column systems. This approach is the first report on using a single column for the implementation of an integrated continuous biomanufacturing platform and offers a cost-effective and flexible platform process for the manufacture of therapeutic proteins.

Integrated continuous bioprocessing: Economic, operational, and environmental feasibility for clinical and commercial antibody manufacture.

This paper presents a systems approach to evaluating the potential of integrated continuous bioprocessing for monoclonal antibody (mAb) manufacture across a product's lifecycle from preclinical to commercial manufacture. The economic, operational, and environmental feasibility of alternative continuous manufacturing strategies were evaluated holistically using a prototype UCL decisional tool that integrated process economics, discrete-event simulation, environmental impact analysis, operational risk analysis, and multiattribute decision-making. The case study focused on comparing whole bioprocesses that used either batch, continuous or a hybrid combination of batch and continuous technologies for cell culture, capture chromatography, and polishing chromatography steps. The cost of goods per gram (COG/g), E-factor, and operational risk scores of each strategy were established across a matrix of scenarios with differing combinations of clinical development phase and company portfolio size. The tool outputs predict that the optimal strategy for early phase production and small/medium-sized companies is the integrated continuous strategy (alternating tangential flow filtration (ATF) perfusion, continuous capture, continuous polishing). However, the top ranking strategy changes for commercial production and companies with large portfolios to the hybrid strategy with fed-batch culture, continuous capture and batch polishing from a COG/g perspective. The multiattribute decision-making analysis highlighted that if the operational feasibility was considered more important than the economic benefits, the hybrid strategy would be preferred for all company scales. Further considerations outside the scope of this work include the process development costs required to adopt continuous processing. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:854-866, 2017.

Linear Quantitative Profiling Method Fast Monitors Alkaloids of Sophora Flavescens That Was Verified by Tri-Marker Analyses.

The present study demonstrated the use of the Linear Quantitative Profiling Method (LQPM) to evaluate the quality of Alkaloids of Sophora flavescens (ASF) based on chromatographic fingerprints in an accurate, economical and fast way. Both linear qualitative and quantitative similarities were calculated in order to monitor the consistency of the samples. The results indicate that the linear qualitative similarity (LQLS) is not sufficiently discriminating due to the predominant presence of three alkaloid compounds (matrine, sophoridine and oxymatrine) in the test samples; however, the linear quantitative similarity (LQTS) was shown to be able to obviously identify the samples based on the difference in the quantitative content of all the chemical components. In addition, the fingerprint analysis was also supported by the quantitative analysis of three marker compounds. The LQTS was found to be highly correlated to the contents of the marker compounds, indicating that quantitative analysis of the marker compounds may be substituted with the LQPM based on the chromatographic fingerprints for the purpose of quantifying all chemicals of a complex sample system. Furthermore, once reference fingerprint (RFP) developed from a standard preparation in an immediate detection way and the composition similarities calculated out, LQPM could employ the classical mathematical model to effectively quantify the multiple components of ASF samples without any chemical standard.

Evaluation of center-cut separations applying simulated moving bed chromatography with 8 zones.

Different multi-column options to perform continuous chromatographic separations of ternary mixtures have been proposed in order to overcome limitations of batch chromatography. One attractive option is given by simulated moving bed chromatography (SMB) with 8 zones, a process that offers uninterrupted production, and, potentially, improved economy. As in other established ternary separation processes, the separation sequence is crucial for the performance of the process. This problem is addressed here by computing and comparing optimal performances of the two possibilities assuming linear adsorption isotherms. The conclusions are presented in a decision tree which can be used to guide the selection of system configuration and operation.

Selecting the column configuration with lowest media replacement cost for small adsorption systems.

A framework was developed for preliminary evaluation of the relative media replacement costs of three alternative column configurations used for adsorption systems with two vessels, such as those serving small systems. The media replacement cost is the cost of fresh media and the replacement service cost (including transportation, labor, and other non-material costs). Cost normalization methods were developed in part based on the data from US EPA Arsenic Treatment Technology Demonstration Program. Adsorption equilibrium and kinetics were modeled using the PSDM model and breakthrough curves were normalized using the target effluent to influent concentration ratio (C/Co) and the mass transfer zone fraction (%MTZBT). Two factors were found to be important for the relative replacement cost of each configuration - the frequency which at least one column needed replacement of media, and the cycle replacement cost (CRCost) which is a combination of the fresh media cost and the replacement service cost. The lead-lag configuration has the lowest annual replacement cost at low target C/Co, high %MTZBT, and high CRCost ratios. The parallel configuration performs better at high target C/Co, high %MTZBT, and high CRCost ratios. Although the single configuration (two columns operated in tandem and replaced simultaneously) has higher media consumption compared to lead-lag and parallel, it can result in the lowest replacement cost at short %MTZBT and very low CRCost ratios due to savings in the replacement service cost.

Economic analysis of uricase production under uncertainty: Contrast of chromatographic purification and aqueous two-phase extraction (with and without PEG recycle).

Uricase is the enzyme responsible for the breakdown of uric acid, the key molecule leading to gout in humans, into allantoin, but it is absent in humans. It has been produced as a PEGylated pharmaceutical where the purification is performed through three sequential chromatographic columns. More recently an aqueous two-phase system (ATPS) was reported that could recover Uricase with high yield and purity. Although the use of ATPS can decrease cost and time, it also generates a large amount of waste. The ability, therefore, to recycle key components of ATPS is of interest. Economic modelling is a powerful tool that allows the bioprocess engineer to compare possible outcomes and find areas where further research or optimization might be required without recourse to extensive experiments and time. This research provides an economic analysis using the commercial software BioSolve of the strategies for Uricase production: chromatographic and ATPS, and includes a third bioprocess that uses material recycling. The key parameters that affect the process the most were located via a sensitivity analysis and evaluated with a Monte Carlo analysis. Results show that ATPS is far less expensive than chromatography, but that there is an area where the cost of production of both bioprocesses overlap. Furthermore, recycling does not impact the cost of production. This study serves to provide a framework for the economic analysis of Uricase production using alternative techniques.

Alternative chromatographic system for the quality control of lipophilic technetium-99m radiopharmaceuticals such as [(99m)Tc(MIBI)6].

Knowledge of the radiochemical purity of radiopharmaceuticals is mandatory and can be evaluated by several methods and techniques. Planar chromatography is the technique normally employed in nuclear medicine since it is simple, rapid and usually of low cost. There is no standard system for the chromatographic technique, but price, separation efficiency and short time for execution must be considered. We have studied an alternative system using common chromatographic stationary phase and alcohol or alcohol:chloroform mixtures as the mobile phase, using the lipophilic radiopharmaceutical [(99m)Tc(MIBI)6]⁺ as a model. Whatman 1 modified phase paper and absolute ethanol, Whatman 1 paper and methanol:chloroform (25:75), Whatman 3MM paper and ethanol:chloroform (25:75), and the more expensive ITLC-SG and 1-propanol:chloroform (10:90) were suitable systems for the direct determination of radiochemical purity of [(99m)Tc(MIBI)6]⁺ since impurities such as (99m)Tc-reduced-hydrolyzed (RH), (99m)TcO(4)(-) and [(99m)Tc(cysteine)2]⁻ complex were completely separated from the radiopharmaceutical, which moved toward the front of chromatographic systems while impurities were retained at the origin. The time required for analysis was 4 to 15 min, which is appropriate for nuclear medicine routines.

Alternative chromatographic system for the quality control of lipophilic technetium-99m radiopharmaceuticals such as [99mTc(MIBI)6]+

Knowledge of the radiochemical purity of radiopharmaceuticals is mandatory and can be evaluated by several methods and techniques. Planar chromatography is the technique normally employed in nuclear medicine since it is simple, rapid and usually of low cost. There is no standard system for the chromatographic technique, but price, separation efficiency and short time for execution must be considered. We have studied an alternative system using common chromatographic stationary phase and alcohol or alcohol:chloroform mixtures as the mobile phase, using the lipophilic radiopharmaceutical [99mTc(MIBI)6]+ as a model. Whatman 1 modified phase paper and absolute ethanol, Whatman 1 paper and methanol:chloroform (25:75), Whatman 3MM paper and ethanol:chloroform (25:75), and the more expensive ITLC-SG and 1-propanol:chloroform (10:90) were suitable systems for the direct determination of radiochemical purity of [99mTc(MIBI)6]+ since impurities such as99mTc-reduced-hydrolyzed (RH),99mTcO4- and [99mTc(cysteine)2]-complex were completely separated from the radiopharmaceutical, which moved toward the front of chromatographic systems while impurities were retained at the origin. The time required for analysis was 4 to 15 min, which is appropriate for nuclear medicine routines.

Parabens determination in cosmetic and personal care products exploiting a multi-syringe chromatographic (MSC) system and chemiluminescent detection.

Parabens are widely used in dairy products, such as in cosmetics and personal care products. Thus, in this work a multi-syringe chromatographic (MSC) system is proposed for the first time for the determination of four parabens: methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP) in cosmetics and personal care products, as a simpler, practical, and low cost alternative to HPLC methods. Separation was achieved using a 5mm-long precolumn of reversed phase C18 and multi-isocratic separation, i.e. using two consecutive mobile phases, 12:88 acetonitrile:water and 28:72 acetonitrile:water. The use of a multi-syringe buret allowed the easy implementation of chemiluminescent (CL) detection after separation. The chemiluminescent detection is based on the reduction of Ce(IV) by p-hydroxybenzoic acid, product of the acid hydrolysis of parabens, to excite rhodamine 6G (Rho 6G) and measure the resulting light emission. Multivariate designs combined with the concepts of multiple response treatments and desirability functions have been employed to simultaneously optimize and evaluate the responses. The optimized method has proved to be sensitive and precise, obtaining limits of detection between 20 and 40 µg L(-1) and RSD <4.9% in all cases. The method was satisfactorily applied to cosmetics and personal care products, obtaining no significant differences at a confidence level of 95% comparing with the HPLC reference method.

Recent advances in sample preparation methods for analysis of endocrine disruptors from various matrices.

Due to the high toxicity of endocrine disruptors (EDs), studies are being undertaken to design effective techniques for separation and detection of EDs in various matrices. Recently, research activities in this area have shown that a diverse range of chromatographic techniques are available for the quantification and analysis of EDs. Therefore, on the basis of significant, recent original publications, we aimed at providing an overview of different separation and detection methods for the determination of trace-level concentrations of selected EDs. The biological effects of EDs and current pretreatment techniques applied to EDs are also discussed. Various types of chromatographic techniques are presented for quantification, highlighting time- and cost-effective techniques that separate and quantify trace levels of multiple EDs from various environmental matrices. Reports related to methods for the quantification of EDs from various matrices primarily published since 2008 have been cited.

Simultaneously sizing and quantitating zeptomole-level DNA at high throughput in free solution.

Determining the sizes and measuring the quantities of DNA molecules are fundamental tasks in molecular biology. DNA sizes are usually evaluated by gel electrophoresis, but this method cannot simultaneously size and quantitate a DNA at low zeptomole (zmol) levels of concentration. We have recently developed a new technique, called bare-narrow-capillary/hydrodynamic-chromatography or BaNC-HDC, for resolving DNA based on their sizes without using any sieving matrices. In this report, we utilize BaNC-HDC for measuring the sizes and quantities of DNA fragments at zmol to several-molecule levels of concentration. DNA ranging from a few base pairs to dozens of kilo base pairs are accurately sized and quantitated at a throughput of 15 samples per hour, and each sample contains dozens of DNA strands of different lengths. BaNC-HDC can be a cost-effective means and an excellent tool for high-throughput DNA sizing and quantitation at extremely low quantity level.

Economics of recombinant antibody production processes at various scales: Industry-standard compared to continuous precipitation.

Standard industry processes for recombinant antibody production employ protein A affinity chromatography in combination with other chromatography steps and ultra-/diafiltration. This study compares a generic antibody production process with a recently developed purification process based on a series of selective precipitation steps. The new process makes two of the usual three chromatographic steps obsolete and can be performed in a continuous fashion. Cost of Goods (CoGs) analyses were done for: (i) a generic chromatography-based antibody standard purification; (ii) the continuous precipitation-based purification process coupled to a continuous perfusion production system; and (iii) a hybrid process, coupling the continuous purification process to an upstream batch process. The results of this economic analysis show that the precipitation-based process offers cost reductions at all stages of the life cycle of a therapeutic antibody, (i.e. clinical phase I, II and III, as well as full commercial production). The savings in clinical phase production are largely attributed to the fact that expensive chromatographic resins are omitted. These economic analyses will help to determine the strategies that are best suited for small-scale production in parallel fashion, which is of importance for antibody production in non-privileged countries and for personalized medicine.

Continuous antibody purification using precipitation: an important step forward.

Recent advances in cell culture have reduced upstream costs, and made downstream processing the more expensive component of biologics manufacturing. Within downstream processing, the chromatographic steps have usually been the most expensive, on the one hand because chromatographic steps are traditionally run in batch mode and on the other because resins are typically more expensive per unit mass of product than other bioproduct-contact materials [1, 2]. There have been many attempts to consider alternatives to chromatography [3, 4], but the comparatively high resolution of chromatography, combined with the bioprocess industry's wealth of experience in using the various chromatographic modes, have made it hard to replace in a manufacturing setting. In this issue of Biotechnology Journal Hammerschmid et al. [5] make several original contributions to this discussion.

Preparative chromatography and natural products discovery.

Methodology is reviewed which significantly enhances the economics and efficiency of larger scale preparative normal phase chromatography. Maintenance of hydration of the silica media and regeneration and re-equilibration of the column after each separation is demonstrated to allow repeated use of the column without loss of performance or requirement of repacking.

Development of chromatofocusing techniques employing mixed-mode column packings for protein separations.

Recent studies reported in the literature using mixed-mode chromatography (MMC) column packings have shown that multiple modes of interactions between the column packing and proteins can be usefully exploited to yield excellent resolution as well as salt-tolerant adsorption of the target protein. In this study, a mixed-mode separation method using commercially available column packings was explored which combines the techniques of hydrophobic-interaction chromatography and chromatofocusing. Two different column packings, one based on mercapto-ethyl-pyridine (MEP) and the other based on hexylamine (HEA) were investigated with regard to their ability to separate proteins when using internally generated, retained pH gradients. The effects of added salt and urea on the behavior of the retained pH gradient and the protein separation achieved when using MMC column packings for chromatofocusing were also investigated. Numerical simulations using methods developed in previous work were shown to agree with experimental results when using reasonable physical parameters. These numerical simulations were also shown to be a useful qualitative method to select the compositions of the starting and elution buffers in order to achieve desired shapes for the pH and ionic strength gradients. The use of the method to fractionate blood serum was explored as a prototype example application.

Designing cost-effective biopharmaceutical facilities using mixed-integer optimization.

Chromatography operations are identified as critical steps in a monoclonal antibody (mAb) purification process and can represent a significant proportion of the purification material costs. This becomes even more critical with increasing product titers that result in higher mass loads onto chromatography columns, potentially causing capacity bottlenecks. In this work, a mixed-integer nonlinear programming (MINLP) model was created and applied to an industrially relevant case study to optimize the design of a facility by determining the most cost-effective chromatography equipment sizing strategies for the production of mAbs. Furthermore, the model was extended to evaluate the ability of a fixed facility to cope with higher product titers up to 15 g/L. Examination of the characteristics of the optimal chromatography sizing strategies across different titer values enabled the identification of the maximum titer that the facility could handle using a sequence of single column chromatography steps as well as multi-column steps. The critical titer levels for different ratios of upstream to dowstream trains where multiple parallel columns per step resulted in the removal of facility bottlenecks were identified. Different facility configurations in terms of number of upstream trains were considered and the trade-off between their cost and ability to handle higher titers was analyzed. The case study insights demonstrate that the proposed modeling approach, combining MINLP models with visualization tools, is a valuable decision-support tool for the design of cost-effective facility configurations and to aid facility fit decisions. 2013.