Downstream Process Development Research Articles

Unveiling the pyrolysis mechanisms of cellulose: Experimental and theoretical studies

Biomass pyrolysis is a renewable technology for the production of bio-oil and chemicals. The present study aims to develop a fundamental and plausible model for cellulose pyrolysis. The product formation characteristics of cellulose pyrolysis were investigated via Py-GC/MS and TGA-FTIR spectroscopy. The density functional theory method was employed to calculate the energy barriers of different pathways, and the formation and interaction mechanisms of the three most abundant components, levoglucosan, hydroxyacetaldehyde, and pyruvaldehyde, were compared. Levoglucosan is most likely formed via a concerted mechanism; hydroxyacetaldehyde can be formed from C1-C2 and C5-C6 via a ring-opening reaction and dehydration, while pyruvaldehyde is produced via ring opening, followed by dehydration and retro-aldol reactions. Related to the variations in products, the formation of hydroxyacetaldehyde and pyruvaldehyde was suggested to compete with that of levoglucosan. Based on the experimental and theoretical results, a plausible reaction scheme was proposed. By accounting for these main reactions, approximately 80% (in relative area) of the products of cellulose pyrolysis were included. This investigation is crucial to downstream process development for biomass and provides guidance for product control.

Investigation of fouling mechanisms of virus filters during the filtration of protein solutions using a high throughput filtration screening device.

The downstream process development of novel antibodies (Abs) is often challenged by virus filter fouling making a better understanding of the underlying mechanisms highly desirable. The present study combines the protein characterization of different feedstreams with their virus filtration performance using a novel high throughput filtration screening system. Filtration experiments with Ab concentrations of up to 20 g/L using either low interacting or hydrophobically interacting pre-filters indicate the existence of two different fouling mechanisms, an irreversible and a reversible one. At the molecular level, size exclusion chromatography revealed that the presence of large amount of high molecular weight species-considered as irreversible aggregates-correlates with irreversible fouling that caused reduced Ab throughput. Results using dynamic light scattering show that a concentration dependent increase of the mean hydrodynamic diameter to the range of dimers (17 nm at 20 g/L) together with a negative DLS interaction parameter kD (-18 mL/g) correlate with the propensity to form reversible aggregates and to cause reversible fouling, probably by a decelerated Ab transport velocity within the virus filter. The two fouling mechanisms are further supported by buffer flush experiments. Finally, concepts for reversible and irreversible fouling mechanisms are discussed together with strategies for respective fouling mitigation. © 2019 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2776, 2019.

Packing quality, protein binding capacity and separation efficiency of pre-packed columns ranging from 1 mL laboratory to 57 L industrial scale

Pre-packed chromatography columns are routinely used in downstream process development and scale-down studies. In recent years they have also been widely adopted for large scale, cGMP manufacturing of biopharmaceuticals. Despite columns being qualified at their point of manufacture before release for sale, the suitability of pre-packed chromatography columns for protein separations at different scales has not yet been demonstrated. In this study, we demonstrated that the performance results obtained with small scale columns (0.5 cm diameter × 5 cm length, 1 mL column volume) are scalable to production sized columns (60 cm diameter × 20 cm length, 57 L column volume). The columns were characterized with acetone and blue dextran pulses to determine the packing density and packed bed consistency. Chromatography performance was evaluated with breakthrough curves including capacity measurements and with separation of a ternary protein mixture (lysozyme, cytochrome C and RNase A) with a step gradient. The equilibrium binding capacity and dynamic binding capacity were equivalent for all columns. The step gradient separation of the ternary protein mixture displayed similar peak profiles when normalized in respect to column volume and the eluted protein pools had the same purities for all scales. Scalable performance of pre-packed columns is demonstrated but as with conventionally packed columns the influence of extra column volume and system configurations, especially buffer mixing, must be taken into account when comparing separations at different scales.

Quality Control and Downstream Processing of Therapeutic Enzymes.

Therapeutic enzymes are a commercially minor but clinically important area of biopharmaceuticals. An array of therapeutic enzymes has been developed for a variety of human diseases, including leukaemia and enzyme-deficiency diseases such as Gaucher's disease. Production and testing of therapeutic enzymes is strictly governed by regulatory bodies in each country around the world, and batch-to-batch consistency is crucially important. Manufacture of a batch starts with the fermentation or cell culture stage. After expression of the therapeutic enzyme in a cell culture bioreactor, robust and reproducible protein purification, or downstream processing (DSP) of the target product, is critical to ensuring safe delivery of these medicines. Modern processing technology, including the use of disposable processing equipment, has greatly improved the DSP development pathway in terms of robustness and speed to clinic. Once purified, the drug substance undergoes rigorous quality control (QC) testing according to current regulatory guidance, to enable release to the clinic and patient. QC testing is conducted to ensure the safety, purity, identity, potency and strength of the medicinal product, requiring multiple analytical methods that are rigorously validated and monitored for robust performance. Several case studies, including L-asparaginase and asfotase alfa, are discussed to illustrate the methods described herein.

A Direct Approach for Process Development Using Single Column Experiments Results in Predictable Streamlined Multi-Column Chromatography Bioprocesses.

An emphasis on continuous monoclonal antibody (mAb) bioprocessing in the pharmaceutical industry necessitates effective approaches for downstream process development (PD). With a PD strategy, the process parameters are optimized to directly develop streamlined three-step continuous chromatography processes. A design of experiment (DoE) approach with single column (batch mode) is used to simulate a multi-column (continuous mode) purification method and characterize each chromatography step: Protein A capture, anion exchange, and mixed mode cation exchange. A novel and targeted approach to quickly characterize a DoE design space was employed and empirical modeling was used to define the capacity for multi-column chromatography to accurately transfer the batch process to continuous mode of purification. This PD approach effectively mimics the continuous mode of operation and provides the flexibility to develop multiple continuous processes with target mAb recovery and purity. By implementing this PD strategy and the process parameters defined in batch mode, two robust and predictable continuous bioprocesses were developed within 7 weeks of investigation, which resulted in a total product yield of recovery at or above 74%, host cell protein (HCP) content below 5 ppm, and aggregate content below 1%.

Progression of continuous downstream processing of monoclonal antibodies: Current trends and challenges.

Rapid advances in intensifying upstream processes for biologics production have left downstream processing as a bottleneck in the manufacturing scheme. Biomanufacturers are pursuing continuous downstream process development to increase efficiency and flexibility, reduce footprint and cost of goods, and improve product consistency and quality. Even after successful laboratory trials, the implementation of a continuous process at manufacturing scale is not easy to achieve. This paper reviews specific challenges in converting each downstream unit operation to a continuous mode. Key elements of developing practical strategies for overcoming these challenges are detailed. These include equipment valve complexity, favorable column aspect ratio, protein-A resin selection, quantitative assessment of chromatogram peak size and shape, holistic process characterization approach, and a customized process economic evaluation. Overall, this study provides a comprehensive review of current trends and the path forward for implementing continuous downstream processing at the manufacturing scale.

Glycine-betaine ionic liquid analogues as novel phase-forming components of aqueous biphasic systems.

Given the biotechnology advances observed in recent years in terms of upstream, the development of effective downstream processes becomes mandatory to decrease the associated costs of biotechnological-based products. Although a large interest has been devoted to ionic-liquid-based aqueous biphasic systems (IL-based ABS) as tailored separation platforms, imidazolium-based ILs have been the preferred choice as phase-forming agents. To overcome some toxicity and biodegradability issues associated to imidazolium-based ILs, novel ABS composed of ILs analogues of glycine-betaine (AGB-ILs) are here proposed and investigated. Five AGB-ILs were synthesized, characterized in terms of ecotoxicity, and applied toward the development of novel ABS formed with Na2 SO4 . Three commercial ILs were also investigated for comparison purposes. The respective ABS ternary phase diagrams, as well as the tie-lines and tie-line lengths, were determined at 25°C. Finally, their performance as extraction strategies was evaluated with five amino acids (L-tryptophan, L-phenylalanine, D-phenylalanine, L-tyrosine and L-3,4-dihydroxyphenylalanine/L-dopa). In all studied systems amino acids preferentially migrate to the IL-rich phase, and with AGB-ILs, the amino acid extraction efficiencies to the IL-rich phase range between 65% and 100%, obtained in a single-step. Furthermore, the studied AGB-ILs display a higher ability to form ABS and to extract amino acids than ABS composed of more traditional and commercial ILs. In summary, novel ABS composed of AGB-ILs can be formed and used as separation routes of value-added compounds of biotechnological interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1205-1212, 2018.

Impact Factors in the Process Development for Therapeutic Antibodies

The research and development of therapeutic antibodies in the world is rapidly rising in recent years. It is a huge challenge to obtain a robust process and achieve the consistency of consistent product quality, because most of the antibody drugs are produced from mammalian cells of which the production processes are long and complex. To develop a robust and high yield production process for a biotherapeutic, one needs to have a general consideration of entire R&D project working flow. First of all, it is necessary to start with a good drug candidate, which not only meets the requirement for both the effectiveness and drug safety, but also suits for manufacture. Then, the expression system and host cell line used for the product expression need to be determined based on availability and regulatory requirements. Cell line development needs to combine with early upstream and downstream process developments as well as product quality assessment. Once a suitable stable cell line is selected, the Master Cell Bank (MCB) and the Working Cell Bank (WCB) are established according to the regulatory requirements, and tested for growth, production stability and cellular safety. Finally, process optimization and scale-up production are carried out to obtain a consistent production. Upstream process development is mainly to optimize a variety of process parameters, including physical parameters, chemical parameters, metabolite levels, and feed strategy and foam control; downstream process development focuses on product quality, recovery rate, and cost efficiency. For scale-up production, enterprises need to make decisions whether to use traditional stainless steel system or the single-use disposable system based on demands on product market potential and available technical supports. In this review, combined with the development of new technologies in recent years and the authors’ personal working experience, the factors that need to be considered in process development are discussed.

Recovery of intracellular ectoine from Halomonas salina cells with poly(propylene) glycol/salt aqueous biphasic system

Abstract Ectoine is a compatible cell protectant sourced from halophilic and halotolerant microorganisms. Various biotechnological applications of ectoine have urged the development of cost-effective downstream processing of ectoine. Aqueous biphasic system (ABS) composed of poly(propylene) glycol (PPG) 425 and salt was adopted for the recovery of intracellular ectoine from Halomonas salina DSM5928T fermentation. Phosphate, citrate and sulfate salts were evaluated for their effects on the recovery of intracellular ectoine using the PPG/salt ABS. In this study, ectoine exhibited a salt-rich bottom phase preference in the PPG/salt ABS because of the hydrophilic surface properties of ectoine. The effects of concentrations of PPG 425 and salts, crude load, pH and concentrations of neutral salts (NaCl and KCl) on the recovery of ectoine were investigated. Optimum recovery of intracellular ectoine was obtained with ABS (pH 5.5) composed of 30% (w/w) PPG 425 and 20% (w/w) sulfate; 15% (w/w) crude load; and 1.5% (w/w) of KCl. High yield of 94.7% of intracellular ectoine was successfully recovered from H. salina cells with an enrichment factor (EF) of 1.7 and a purity of 87.03%.

Universal label-free in-process quantification of influenza virus-like particles.

Virus-like particles (VLPs) are becoming established as vaccines, in particular for influenza pandemics, increasing the interest in the development of VLPs manufacturing bioprocess. However, for complex VLPs, the analytical tools used for quantification are not yet able to keep up with the bioprocess progress. Currently, quantification for Influenza relies on traditional methods: hemagglutination assay or Single Radial Immunodiffusion. These analytical technologies are time-consuming, cumbersome, and not supportive of efficient downstream process development and monitoring. Hereby we report a label-free tool that uses Biolayer interferometry (BLI) technology applied on an Octet platform to quantify Influenza VLPs at all stages of bioprocess. Human (α2,6-linked sialic acid) and avian (α2,3-linked sialic acid) biotinylated receptors associated with streptavidin biosensors were used, to quantify hemagglutinin content in several mono- and multivalent Influenza VLPs. The applied method was able to quantify hemagglutinin from crude samples up to final bioprocessing VLP product. BLI technology confirmed its value as a high throughput analytical tool with high sensitivity and improved detection limits compared to traditional methods. This simple and fast method allowed for real-time results, which are crucial for in-line monitoring of downstream processing, improving process development, control and optimization.

Evolución del modelo de zonas francas permanentes en el mundo: Con un énfasis en el caso Latinoamericano

Las zonas francas han sido un importante polo dinamizador del desarrollo económico mundial contemporáneo. El artículo presenta las diferentes tipologías de las zonas, y describe su evolución en el mundo, su impacto económico y social y los factores distintivos de su desarrollo en la región Latinoamérica. Los resultados muestran que la región tiene una de las más altas dinámica de desarrollo de las ZFP tanto en número como en empleo, no obstante, el impacto es bajo en el desarrollo de valor tanto en el aspecto humano como en la innovación. Es necesario para potenciar el modelo de ZFP que los gobiernos incentiven el desarrollo de procesos productivos ascendentes y de procesos de soporte asociados, tanto en el aumento de valor y mejora de la calidad de vida del capital humano, como en los mecanismos legales en pro del comercio internacional para mantener la dinámica de desarrollo.

Peptidliganden zur Aufreinigung kamelider Schwere-Ketten-Antikörper

Highly specific peptides that bind a given target protein are useful ligands for the affinity purification of such proteins from complex biological matrices. The capability to provide this kind of peptidic ligands for the affinity purification of virtually any protein can simplify and enhance the downstream process development. The general applicability, scalability, and comparatively low costs of peptidic ligands are key features for their application in downstream purification processes.

Construct design, production, and characterization of Plasmodium falciparum 48/45 R0.6C subunit protein produced in Lactococcus lactis as candidate vaccine

BackgroundThe sexual stages of Plasmodium falciparum are responsible for the spread of the parasite in malaria endemic areas. The cysteine-rich Pfs48/45 protein, exposed on the surface of sexual stages, is one of the most advanced antigens for inclusion into a vaccine that will block transmission. However, clinical Pfs48/45 sub-unit vaccine development has been hampered by the inability to produce high yields of recombinant protein as the native structure is required for the induction of functional transmission-blocking (TB) antibodies. We have investigated a downstream purification process of a sub-unit (R0.6C) fragment representing the C-terminal 6-Cys domain of Pfs48/45 (6C) genetically fused to the R0 region (R0) of asexual stage Glutamate Rich Protein expressed in Lactococcus lactis.ResultsA series of R0.6C fusion proteins containing features, which aim to increase expression levels or to facilitate protein purification, were evaluated at small scale. None of these modifications affected the overall yield of recombinant protein. Consequently, R0.6C with a C-terminal his tag was used for upstream and downstream process development. A simple work-flow was developed consisting of batch fermentation followed by two purification steps. As such, the recombinant protein was purified to homogeneity. The composition of the final product was verified by HPLC, mass spectrometry, SDS-PAGE and Western blotting with conformation dependent antibodies against Pfs48/45. The recombinant protein induced high levels of functional TB antibodies in rats.ConclusionsThe established production and purification process of the R0.6C fusion protein provide a strong basis for further clinical development of this candidate transmission blocking malaria vaccine.

Optimization of biopharmaceutical downstream processes supported by mechanistic models and artificial neural networks.

Downstream process development is a major area of importance within the field of bioengineering. During the design of such a downstream process, important decisions have to be made regarding the type of unit operations as well as their sequence and their operating conditions. Current computational approaches addressing these issues either show a high level of simplification or struggle with computational speed. Therefore, this article presents a new approach that combines detailed mechanistic models and speed-enhancing artificial neural networks. This approach was able to simultaneously optimize a process with three different chromatographic columns toward yield with a minimum purity of 99.9%. The addition of artificial neural networks greatly accelerated this optimization. Due to high computational speed, the approach is easily extendable to include more unit operations. Therefore, it can be of great help in the acceleration of downstream process development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:696-707, 2017.

High-throughput downstream process development for cell-based products using aqueous two-phase systems (ATPS) - A case study.

The availability of preparative-scale downstream processing strategies for cell-based products presents a critical juncture between fundamental research and clinical development. Aqueous two-phase systems (ATPS) present a gentle, scalable, label-free, and cost-effective method for cell purification, and are thus a promising tool for downstream processing of cell-based therapeutics. Here, the application of a previously developed robotic screening platform that enables high-throughput cell partitioning analysis in ATPS is reported. In the present case study a purification strategy for two model cell lines based on high-throughput screening (HTS)-data and countercurrent distribution (CCD)-modeling, and validated the CCD-model experimentally is designed. The obtained data are shown an excellent congruence between CCD-model and experimental data, indicating that CCD-models in combination with HTS-data are a powerful tool in downstream process development. Finally, the authors are shown that while cell cycle phase significantly influences cell partitioning, cell type specific differences in surface properties are the main driving force in charge-dependent separation of HL-60 and L929 cells. In order to design a highly robust purification process it is, however, advisable to maintain constant growth conditions.

Integrated process design for biocatalytic synthesis by a Leloir Glycosyltransferase: UDP‐glucose production with sucrose synthase

Nucleotide sugar-dependent ("Leloir") glycosyltransferases (GTs), represent a new paradigm for the application of biocatalytic glycosylations to the production of fine chemicals. However, it remains to be shown that GT processes meet the high efficiency targets of industrial biotransformations. We demonstrate in this study of uridine-5'-diphosphate glucose (UDP-glc) production by sucrose synthase (from Acidithiobacillus caldus) that a holistic process design, involving coordinated development of biocatalyst production, biotransformation, and downstream processing (DSP) was vital for target achievement at ∼100 g scale synthesis. Constitutive expression in Escherichia coli shifted the recombinant protein production mainly to the stationary phase and enhanced the specific enzyme activity to a level (∼480 U/gcell dry weight ) suitable for whole-cell biotransformation. The UDP-glc production had excellent performance metrics of ∼100 gproduct /L, 86% yield (based on UDP), and a total turnover number of 103 gUDP-glc /gcell dry weight at a space-time yield of 10 g/L/h. Using efficient chromatography-free DSP, the UDP-glc was isolated in a single batch with ≥90% purity and in 73% isolated yield. Overall, the process would allow production of ∼0.7 kg of isolated product/L E. coli bioreactor culture, thus demonstrating how integrated process design promotes the practical use of a GT conversion. Biotechnol. Bioeng. 2017;114: 924-928. © 2016 Wiley Periodicals, Inc.

Development of downstream processing to minimize beta‐glucan impurities in GMP‐manufactured therapeutic antibodies

The presence of impurities or contaminants in biological products such as monoclonal antibodies (mAb) could affect efficacy or cause adverse reactions in patients. ICH guidelines (Q6A and Q6B) are in place to regulate the level of impurities within clinical drug products. An impurity less often reported and, therefore, lacking regulatory guideline is beta-glucan. Beta-glucans are polysaccharides of d-glucose monomers linked by (1-3) beta-glycosidic bonds, and are produced by prokaryotic and eukaryotic organisms, including plants. They may enter manufacturing processes via raw materials such as cellulose-based membrane filters or sucrose. Here we report the detection of beta-glucan contamination of a monoclonal IgE antibody (MOv18), manufactured in our facility for a first-in-human, first-in-class clinical trial in patients with cancer. Since beta-glucans have potential immunostimulatory properties and can cause symptomatic infusion reactions, it was of paramount importance to identify the source of beta-glucans in our product and to reduce the levels to clinically insignificant concentrations. We identified beta-glucans in sucrose within the formulation buffer and within the housing storage buffer of the virus removal filter. We also detected low level beta-glucan contamination in two of four commercially available antibodies used in oncology. Both formulation buffers contained sucrose. We managed to reduce levels of beta-glucan in our product 10-fold, by screening all sucrose raw material, filtering the sucrose by Posidyne® membrane filtration, and by incorporating extra wash steps when preparing the virus removal filter. The beta-glucan levels now lie within a range that is unlikely to cause clinically significant immunological effects. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1494-1502, 2016.

Energy Efficiency Analysis towards Early Decision‐Making in Downstream Process Development

Chemie Ingenieur TechnikVolume 88, Issue 9 p. 1223-1223 Poster Energy Efficiency Analysis towards Early Decision-Making in Downstream Process Development M. Ostermann, Corresponding Author M. Ostermann marie-christine.ostermann@bci.tu-dortmund.de TU Dortmund, Fakultät Bio- und Chemieingenieurwesen, Lehrstuhl für Anlagen- und Prozesstechnik, Emil-Figge-Straße 70, 44227 Dortmund, GermanyTU Dortmund, Fakultät Bio- und Chemieingenieurwesen, Lehrstuhl für Anlagen- und Prozesstechnik, Emil-Figge-Straße 70, 44227 Dortmund, GermanySearch for more papers by this authorP. Totchum-Simo, P. Totchum-Simo TU Dortmund, Fakultät Bio- und Chemieingenieurwesen, Lehrstuhl für Anlagen- und Prozesstechnik, Emil-Figge-Straße 70, 44227 Dortmund, GermanySearch for more papers by this authorProf. Dr. G. Schembecker, Prof. Dr. G. Schembecker TU Dortmund, Fakultät Bio- und Chemieingenieurwesen, Lehrstuhl für Anlagen- und Prozesstechnik, Emil-Figge-Straße 70, 44227 Dortmund, GermanySearch for more papers by this authorDr. K. Wohlgemuth, Dr. K. Wohlgemuth TU Dortmund, Fakultät Bio- und Chemieingenieurwesen, Lehrstuhl für Anlagen- und Prozesstechnik, Emil-Figge-Straße 70, 44227 Dortmund, GermanySearch for more papers by this authorDr. C. Bramsiepe, Dr. C. Bramsiepe TU Dortmund, Fakultät Bio- und Chemieingenieurwesen, Lehrstuhl für Anlagen- und Prozesstechnik, Emil-Figge-Straße 70, 44227 Dortmund, GermanySearch for more papers by this author M. Ostermann, Corresponding Author M. Ostermann marie-christine.ostermann@bci.tu-dortmund.de TU Dortmund, Fakultät Bio- und Chemieingenieurwesen, Lehrstuhl für Anlagen- und Prozesstechnik, Emil-Figge-Straße 70, 44227 Dortmund, GermanyTU Dortmund, Fakultät Bio- und Chemieingenieurwesen, Lehrstuhl für Anlagen- und Prozesstechnik, Emil-Figge-Straße 70, 44227 Dortmund, GermanySearch for more papers by this authorP. Totchum-Simo, P. Totchum-Simo TU Dortmund, Fakultät Bio- und Chemieingenieurwesen, Lehrstuhl für Anlagen- und Prozesstechnik, Emil-Figge-Straße 70, 44227 Dortmund, GermanySearch for more papers by this authorProf. Dr. G. Schembecker, Prof. Dr. G. Schembecker TU Dortmund, Fakultät Bio- und Chemieingenieurwesen, Lehrstuhl für Anlagen- und Prozesstechnik, Emil-Figge-Straße 70, 44227 Dortmund, GermanySearch for more papers by this authorDr. K. Wohlgemuth, Dr. K. Wohlgemuth TU Dortmund, Fakultät Bio- und Chemieingenieurwesen, Lehrstuhl für Anlagen- und Prozesstechnik, Emil-Figge-Straße 70, 44227 Dortmund, GermanySearch for more papers by this authorDr. C. Bramsiepe, Dr. C. Bramsiepe TU Dortmund, Fakultät Bio- und Chemieingenieurwesen, Lehrstuhl für Anlagen- und Prozesstechnik, Emil-Figge-Straße 70, 44227 Dortmund, GermanySearch for more papers by this author First published: 29 August 2016 https://doi.org/10.1002/cite.201650450AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume88, Issue9Special Issue: ProcessNet-Jahrestagung und 32. DECHEMA-Jahrestagung der Biotechnologen 2016September, 2016Pages 1223-1223 RelatedInformation

Towards bacterial lipopeptide products for specific applications — a review of appropriate downstream processing schemes

Abstract Lipopeptides are versatile molecules that are viable and potential replacements for synthetic surfactants in agricultural, pharmaceutical, food and cosmetic industries. While process optimization and intensification approaches have significantly improved lipopeptide production in terms of yield and productivity, downstream processing options to produce tailor-made lipopeptide products of different degrees of purity for specific applications have received considerably less attention. The use of conventional downstream methods such as solvent extraction, membrane filtration, adsorption and size exclusion has satisfactorily addressed the demand of lipopeptide mixtures to some extent, but the lack of well-established downstream techniques for these molecules still withholds their complete commercial realization. Moreover, fractionation of lipopeptide mixtures into families or individual isoforms is undeveloped, significantly limiting the use of lipopeptides for high end applications. This review highlights the recent developments in downstream processing of lipopeptides and discusses their pertinence on a case-to case basis in obtaining lipopeptides of appropriate purity for distinct and diverse applications.

High-throughput downstream process development for cell-based products using aqueous two-phase systems

As the clinical development of cell-based therapeutics has evolved immensely within the past years, downstream processing strategies become more relevant than ever. Aqueous two-phase systems (ATPS) enable the label-free, scalable, and cost-effective separation of cells, making them a promising tool for downstream processing of cell-based therapeutics. Here, we report the development of an automated robotic screening that enables high-throughput cell partitioning analysis in ATPS. We demonstrate that this setup enables fast and systematic investigation of factors influencing cell partitioning. Moreover, we examined and optimized separation conditions for the differentiable promyelocytic cell line HL-60 and used a counter-current distribution-model to investigate optimal separation conditions for a multi-stage purification process. Finally, we show that the separation of CD11b-positive and CD11b-negative HL-60 cells is possible after partial DMSO-mediated differentiation towards the granulocytic lineage. The modeling data indicate that complete peak separation is possible with 30 transfers, and >93% of CD11b-positive HL-60 cells can be recovered with >99% purity. The here described screening platform facilitates faster, cheaper, and more directed downstream process development for cell-based therapeutics and presents a powerful tool for translational research.