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.

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.

A fast and inexpensive procedure for the isolation of synthetic cannabinoids from 'Spice' products using a flash chromatography system.

In the age of the Internet, the variety of drugs offered online is constantly increasing, and new drugs emerge every month. One group of drugs showing such an enormous increase is that of synthetic cannabinoids. Since their first identification in 'herbal mixtures', new structural modifications continue to appear on the market. In order to keep up with this process, toxicological screening methods need to be up to date. This can become extremely difficult if no reference material is available. In this article, a fast and effective way to extract and purify synthetic cannabinoids from 'herbal mixtures' is presented. This method opens a new opportunity for a timely reaction by obtaining reference material straight out of the 'herbal mixtures' ordered via the Internet. Isolation was carried out on a flash chromatography system with gradient elution on a C18 column using methanol and 0.55 % formic acid as mobile phases. The obtained purity of all compounds exceeded 99 %. In addition to the isolation of single compounds, the method proved to be suitable for the separation of various synthetic cannabinoids in one mixture, including the diastereomers cis- and trans-CP-47,497-C8. This approach for obtaining pure standards of new drugs proved to be effective, inexpensive and much quicker than waiting for the substances to be commercially available as reference material.

Using a single hydrophobic-interaction chromatography to purify pharmaceutical-grade supercoiled plasmid DNA from other isoforms.

CONTEXT: The recent developments in non-viral gene therapy and DNA vaccine have fostered the development of efficient plasmid DNA (pDNA) purification processes. OBJECTIVES: This work aimed to establish a cost-effective purification process for the large-scale production of plasmid DNA for gene therapy and DNA vaccine. MATERIALS AND METHODS: E. coli DH5α harboring pCDNA3.1-GFP (7200 base pairs) was used as a model plasmid. Hydrophobic-interaction chromatography (HIC) was employed to purify supercoiled plasmid DNA (sc pDNA). RESULTS: With this method, not only host contaminants, but also open circular plasmid DNA (oc pDNA) could be removed from sc pDNA. Anion-exchange HPLC analysis proved that the recovery of HIC could reach 75%. The plasmid DNA exhibited high purity with supercoiled percentage of 98 ± 1.2% and undetectable residual endotoxins, genomic DNA, RNA and protein. The purity of pDNA had nothing to do with the flow rate in the range at least up to 400 cm/h. Liposomes transfection experiment prove that the purified pDNA in this article had higher transfection efficiency than the control pDNA. DISCUSSION AND CONCLUSION: In the present work, we confirmed the possibility of separation of sc pDNA from oc pDNA and other host contaminants using a single HIC chromatography.

Microfluidic platforms for single-cell analysis.

Microfluidics, the study and control of the fluidic behavior in microstructures, has emerged as an important enabling tool for single-cell chemical analysis. The complex procedures for chemical cytometry experiments can be integrated into a single microfabricated device. The capability of handling a volume of liquid as small as picoliters can be utilized to manipulate cells, perform controlled cell lysis and chemical reactions, and efficiently minimize sample dilution after lysis. The separation modalities such as chromatography and electrophoresis within microchannels are incorporated to analyze various types of intracellular components quantitatively. The microfluidic approach offers a rapid, accurate, and cost-effective tool for single-cell biology. We present an overview of the recent developments in microfluidic technology for chemical-content analysis of individual cells.

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.

Simulated moving bed chromatography for the separation of enantiomers.

Simulated moving bed (SMB) chromatography, a continuous multi-column chromatographic process, has become one of the preferred techniques for the separation of the enantiomers of a chiral compound. Several active pharmaceutical ingredients, including blockbuster drugs, are manufactured using the SMB technology. Compared to single column preparative chromatography, SMB separations achieve higher productivity and purity, while reducing the solvent consumption. The SMB technology has found applications both at small and large scales. Design methods have been developed for robust operation and scale-up, using data obtained from analytical experiments. In the last few years, rapid developments have been made in the areas of design, improved process schemes, optimization and robust control. This review addresses these developments, as well as both the fundamentals of the SMB science and technology and some practical issues concerning the operation of SMB units. Particular emphasis is placed on the consolidation of the "triangle theory", a design tool that is used both in the academia and industry for the design of SMB processes.

General methods for flash chromatography using disposable columns.

As technology has evolved available guidelines for normal-phase flash chromatography have become less relevant. Years of experience performing chromatography with disposable columns have been condensed into simple guidelines useful for translating TLC results into either isocratic- or gradient-flash chromatography. The described studies should provide researchers with a means of selecting adequate columns and guidelines to reduce the waste of solvents, silica, time, and money.

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.

Working without a blindfold: the critical role of diagnostics in malaria control.

Diagnostic testing for malaria has for many years been eschewed, lest it be an obstacle to the delivery of rapid, life-saving treatment. The approach of treating malaria without confirmatory testing has been reinforced by the availability of inexpensive treatment with few side effects, by the great difficulty of establishing quality-assured microscopy in rural and resource-poor settings, and by the preeminence of malaria as a cause of important fever in endemic regions. Within the last decade, all three of these factors have changed. More expensive artemisinin combination therapy (ACT) has been widely introduced, simple immunochromatographic tests for malaria have been developed that can be used as an alternative to microscopy by village health workers, and recognition of the health cost of mismanaging non-malarial fever is growing. In most of the world a small fraction of fever is due to malaria, and reflex treatment with ACT does not make medical or economic sense. Global malaria control efforts have been energized by the availability of new sources of funding, and by the rapid reduction in malaria prevalence in a number of settings where bed nets, indoor residual spraying with insecticides, and ACT have been systematically deployed. This momentum has been captured by a new call for malaria elimination. Without wide implementation of accurate and discriminating diagnostic testing, and reporting of results, most fever will be inappropriately managed, millions of doses of ACT will be wasted, and malaria control programmes will be blindfolded to the impact of their efforts.

Optimal economic design and operation of single- and multi-column chromatographic processes.

Single-column chromatography is widely used in the biopharmaceutical industries, although multi-column alternatives in the form of simulated moving bed (SMB) processes are now emerging. It may be difficult, however, to determine which column alternative will be best suited for a given application, and this work sets out to address this issue. A systematic approach is presented that is based on a full economic appraisal of each process alternative based on an optimization of the net annual profit. Single-column processes with and without recycling are considered, as are both the SMB and the Varicol process. The cyclic steady state for the SMB and Varicol processes is determined directly by complete discretization. The approach is applied to a case study based on a linear isotherm where it is found that for this particular system, a recycling policy is not necessary for the single column. When comparing the single-column process with the multi-column alternatives, the single column is the most economical provided the life time of the project is short; however, the economic benefits of the more capital-intensive multi-column processes are greater if the life time of the project is over 5 years. The SMB process is found to perform marginally better than the Varicol process over 15 years; however, this may be because not all extra degrees of freedom for the Varicol process were considered.

[Concordance between thick blood smear, immunochromatography and polymerase chain reaction for malaria diagnosis]. / Concordancia entre gota gruesa, inmunocromatografía y reacción en cadena de la polimerasa para el diagnóstico de malaria.

INTRODUCTION: The rapid and effective diagnosis of malaria is the determining condition for an appropriate treatment and control of the disease. OBJECTIVE: The sensitivity, specificity and the positive and negative predictive values were evaluated in cases of suspected malaria in Colombia in a comparison of a rapid diagnostic test. the PCR test and the thick blood smear-the traditional gold standard. MATERIALS AND METHODS: A group of 100 patients with symptoms compatible with malaria, were included in the study. They were selected from the following Colombian regions: Urabá, Córdoba, lower Cauca, and relatively fewer from other malaria endemic areas of Colombia including the provinces of Valle, Chocó in the central west of Colombia and Vichada to the east. To each patient the following three tests were performed: the rapid OptiMAL test, the PCR identification and the thick blood smear. The PCR amplified specific DNA sequences with primers designed to identify the genus Plasmodium, and the two species present in Colombia, P. falciparum and P. vivax. RESULTS: The sensitivity of the rapid test versus the thick smear, for the diagnosis of both species of Plasmodium was 93.9% (95% CI: 87-100%) and the specificity was 94.3% (95% CI:.253 85-100%). The PCR compared with the thick smear showed a sensitivity of 100% (95% CI: 99-100%) and a specificity of 97.1% (95% CI: 90-100%). CONCLUSIONS: The sensitivity and specificity of the three tests did not present statistically significant differences. However, the thick blood smear was recommended as the standard test, mainly due to its low cost.

Very large scale monoclonal antibody purification: the case for conventional unit operations.

Technology development initiatives targeted for monoclonal antibody purification may be motivated by manufacturing limitations and are often aimed at solving current and future process bottlenecks. A subject under debate in many biotechnology companies is whether conventional unit operations such as chromatography will eventually become limiting for the production of recombinant protein therapeutics. An evaluation of the potential limitations of process chromatography and filtration using today's commercially available resins and membranes was conducted for a conceptual process scaled to produce 10 tons of monoclonal antibody per year from a single manufacturing plant, a scale representing one of the world's largest single-plant capacities for cGMP protein production. The process employs a simple, efficient purification train using only two chromatographic and two ultrafiltration steps, modeled after a platform antibody purification train that has generated 10 kg batches in clinical production. Based on analyses of cost of goods and the production capacity of this very large scale purification process, it is unlikely that non-conventional downstream unit operations would be needed to replace conventional chromatographic and filtration separation steps, at least for recombinant antibodies.

Development of immunochromatography strip-test using nanocolloidal gold-antibody probe for the rapid detection of aflatoxin B1 in grain and feed samples.

An immunochromatography (ICG) strip test using a nanocolloidal gold-antibody probe was developed and optimized for the rapid detection of aflatoxin B1 (AFB1). A monoclonal antibody specific to AFB1 was produced from the cloned hybridoma cell (AF78), coupled with nanocolloidal gold, and distributed on the conjugate pad of the ICG strip test. The visual detection limit of the ICG strip test was 0.5 ng/ml, and this method showed a cross-reaction to aflatoxin B2, G1, and G2. In total, 172 grain and feed samples were collected and analyzed by both the ICG strip test and HPLC. The results of the ICG strip test showed a good agreement with those obtained by HPLC. These results indicated that the ICG strip test has a potential use as a rapid and cost-effective screening tool for the determination of AFB1 in real samples and could be applied to the preliminary screening of mycotoxin in food and agricultural products, generating results within 15 min without complicated steps.

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.

Basic concepts in Q membrane chromatography for large-scale antibody production.

The large-scale production of recombinant human monoclonal antibodies demands economical purification processes with high throughputs. In this article we briefly describe a common antibody process and evaluate the Q membrane adsorber for process-scale antibody production as an alternative to a Q-packed-bed column in a flow-through mode. The scientific concepts underlining Q membrane technology and its application are reviewed. The disadvantages and advantages of using Q membrane chromatography as a purification unit in large-scale production are discussed, including problems initially seen with the Q membrane scale-down model but solved with the invention of a new scale-down model. The new Q-membrane unit operation has a process capacity greater than 3,000 g/m(2) or 10.7 kg/L with a LRV over 5 for four model viruses. In this Review, a cost analysis illustrates that Q membrane chromatography is a viable alternative to Q column chromatography as a polishing step in a flow-through mode for process-scale antibody production.

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.

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.

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.

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.