Application of monoliths for plasmid DNA purification development and transfer to production

The demand of high-purity plasmid DNA (pDNA) for gene-therapy and genetic vaccination is still increasing. For the large scale production of pharmaceutical grade plasmids generic and economic purification processes are needed. Most of the current processes for pDNA production use at least one chromatography step, which always constitutes as the key-step in the purification sequence. Monolithic chromatographic supports are an alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. Anion-exchange chromatography is the most popular chromatography method for plasmid separation, since polynucleotides are negatively charged independent of the buffer conditions. For the implementation of a monolith-based anion exchange step into a pDNA purification process detailed screening experiments were performed. These studies included supports, ligand-types and ligand-densities and optimization of resolution and productivity. For this purpose model plasmids with a size of 4.3 and 6.9 kilo base pairs (kbp) were used. It could be shown, that up-scaling to the production scale using 800 ml CIM Convective Interaction Media radial flow monoliths is possible under low pressure conditions. CIM DEAE was successfully implemented as intermediate step of the cGMP pDNA manufacturing process. Starting from 2001 fermentation aliquots pilot scale purification runs were performed in order to prove scale-up and to predict further up-scaling to 8 1 tube monolithic columns. The analytical results obtained from these runs confirmed suitability for pharmaceutical applications.     

Library selection approaches to engineering enhanced retroviral and lentiviral vectors

Retroviral and lentiviral based gene delivery vectors have been used in numerous pre-clinical studies and clinical trials due to their advantages, including stable and prolonged expression of therapeutic transgenes and minimal immune responses against the vector. Despite such advantages, however, retroviral vectors also have several limitations for gene therapy applications. For example, they can suffer from a lack of efficient or targeted gene delivery to key cell types. In addition, retroviral vector stability can be compromised by their envelope proteins. This review briefly describes how such limitations have been overcome by recently developed library selection approaches that borrow a lesson from nature: the ability of evolution to generate biomolecules with novel function. These library selection approaches are based on the construction of retroviral libraries where the sequences encoding natural viral components are partially randomized using a variety of methods in order to generate diverse libraries that can be selected to create improved or novel functions. These high throughput, library-based approaches provide a strong complement to rational engineering of viral components for the rapid development of efficient and safe retroviral and lentiviral vector systems for gene therapy.     

Reactive separation processes for the production of PEGylated proteins

Protein PEGylation plays an important role in the whole panel of reactions used for the modification and improvement of therapeutic proteins. The classic production process consists of a batch reactor followed by a purification unit. In this review, we show that other processes smartly combining the separation and the reaction unit operations within a single process can bring a competitive advantage in terms of yield, conversion and productivity. This represents an alternative approach to the development of new ad-hoc specific chemistries. In the following, in addition to provide an overview of processes described in the literature and discuss advantages and disadvantages of each, we propose innovative solutions which include continuous manufacturing technologies.     

Electrodialysis with ultrafiltration membranes for peptide separation

A number of bioactive peptides find their potential applications in food or pharmaceutical industry; however, there arise some limitations of their large-scale production to satisfy market demands. Although pressure-driven membrane processes are able of continuous production and separation of peptides, these technologies often demonstrate insufficient selectivity. Electrophoresis is a well-known purification process characterized by high resolution of separated species but it is limited by relatively low production capacity. On the other hand, electromembrane processes offer high production capacity but their limitation is the size of separated molecules. Electrodialysis with inserted ultrafiltration membranes is an alternative method of peptide separation into fractions, their concentration and possibly demineralization at the same time to achieve large production quantities. It is a hybrid process combining conventional electrodialysis and electrophoresis principles using ultrafiltration membranes. These membranes serve as a molecular barrier separating two types of solution while the driving force remains electric potential difference. This article offers state-of-the-art summary in the field of bioactive peptide separation and fractionation by electrodialysis with ultrafiltration membranes.     

Artificial evolution with adeno-associated viral libraries

After attracting the attention of the scientific community due to a number of favourable characteristics that make it an attractive vector for human gene therapy [1,2], AAV has been thoroughly investigated in the past two decades. Standard technologies for the manipulation of the viral genome and for efficient packaging and purification protocols have paved the road for trial and error manipulation by educated guesses to study viral infectious biology by reverse genetics and to generate improved vectors for human gene transfer. However, despite remarkable progress, our limited knowledge of molecular mechanisms implicated in virus-cell interactions has been a limiting factor. Combinatorial engineering and high-throughput selection techniques hold the potential to boost technological improvement by offering the possibility to screen large numbers of randomly generated clones by appropriate selection protocols. These approaches not only require lesser knowledge of viral biology, but can also be employed as valuable tools to investigate molecular mechanisms that drive the infection process. In this review we recapitulate the rationale for employment of combinatorial methods in AAV vector development and the accomplishments achieved so far, discussing current limitations and interesting developments that are in sight.     

Separation of influenza virus‐like particles from baculovirus by polymer‐grafted anion exchanger

The baculovirus expression vector system is a very powerful tool to produce virus‐like particles and gene‐therapy vectors, but the removal of coexpressed baculovirus has been a major barrier for wider industrial use. We used chimeric human immunodeficiency virus‐1 (HIV‐1) gag influenza‐hemagglutin virus‐like particles produced in Tnms42 insect cells using the baculovirus insect cell expression vector system as model virus‐like particles. A fast and simple purification method for these virus‐like particles with direct capture and purification within one chromatography step was developed. The insect cell culture supernatant was treated with endonuclease and filtered, before it was directly loaded onto a polymer‐grafted anion exchanger and eluted by a linear salt gradient. A 4.3 log clearance of baculovirus from virus‐like particles was achieved. The absence of the baculovirus capsid protein (vp39) in the product fraction was additionally shown by high performance liquid chromatography‐mass spectrometry. When considering a vaccination dose of 10⁹ particles, 4200 doses can be purified per L pretreated supernatant, meeting the requirements for vaccines with <10 ng double‐stranded DNA per dose and 3.4 µg protein per dose in a single step. The process is simple with a very low number of handling steps and has the characteristics to become a platform for purification of these types of virus‐like particles.     

Application of a novel affinity adsorbent for the capture and purification of recombinant Factor VIII compounds

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13 kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.     

A survey on cobalt metallurgical processes and its application

The Cobalt (Co) transition element in the recent years have been a new research area with increased trends in inorganic chemistry. The Co is used in several areas for example ceramics, technologies, additive manufacturing, rechargeable batteries in manufacturing firms. The magnets, and alloys, digital processing, bio-essentiality, Healthcare computing, sustainable development, storage of renewable energy, biogas amongst others are entailed. In this study, the Co Metallurgical processes are presented and how they are used. This is because of its low resistance in smaller geometries and capacity to work through a thinner barrier. This paper avails the purification process of Co from its ores. Presented a diagrammatic illustration of Co within an adverse aspect. This in return avails all fundamental research content that can facilitate the researchers in opposing models and frame works that are state-of-art in elements purification processes.     

Vector and Cell Line Engineering Technologies Toward Recombinant Protein Expression in Mammalian Cell Lines

The rapid growth of global biopharmaceutical market in the recent years has been a good indication of its significance in biotechnology industry. During a long period of time in recombinant protein production from 1980s, optimizations in both upstream and downstream processes were launched. In this regard, one of the most promising strategies is expression vector engineering technology based on incorporation of DNA opening elements found in the chromatin border regions of vectors as well as targeting gene integration. Along with these approaches, cell line engineering has revealed convenient outcomes in isolating high-producing clones. According to the fact that more than 50% of the approved therapeutic proteins is being manufactured in mammalian cell lines, in this review, we focus on several approaches and developments in vector and cell line engineering technologies in mammalian cell culture.     

The distinctive separation attributes of mixed-mode resins and their application in monoclonal antibody downstream purification process

Increased upstream productivity and the continuous pressure to deliver high quality drug product have resulted in the development of new separation technologies and platform strategies for downstream purification processes of monoclonal antibodies (mAb). In this study, the separation attributes of three mixed-mode resins, Mercapto-Ethyl-Pyridine (MEP) hydrophobic charge induction resin, Capto adhere multi-modal anion exchange resin, and ceramic hydroxyapatite/fluoroapatite (CHT/CFT) resins, were investigated to define their roles in monoclonal antibody purification processes. We demonstrated that the multi-modal nature of ligands on mixed-mode resins allows the separation resolution to be honed, either through a single dominant mechanism or through mix-modal balanced purification strategies. In addition, the three mixed-mode resins present different purification powers for different types of impurities. We also demonstrated that besides enhancing chromatography separation and improve product quality, especially for high molecular weight (HMW) aggregate reduction, mixed-mode resins can also help to improve process efficiency in industrial-scale mAb drug manufacturing. Our results underscore the importance of selecting appropriate chromatography resins during DSP design to obtain the best overall process outcome.     

Polyethylenimine-Based Transfection Method as a Simple and Effective Way to Produce Recombinant Lentiviral Vectors

HIV-1-derived lentiviral vectors (LvV) are within the most attractive gene delivery vehicles in the context of both dividing and quiescent cells. LvV is currently produced by the conventional calcium phosphate precipitation method. Nevertheless, this procedure is highly susceptible to variations in pH and impurities, which lead to inconsistencies in LvV production. Here, we present a simple and robust procedure for LvV production using branched 25 kDa polyethylenimine, with a transfection efficiency of over 90% and viral titer yields of about 1 × 10⁷ infective lentiviral particles per milliliter. The procedure outlined is simple, consistent, and as inexpensive as the CaPO₄-based method.     

Synthetic, self-assembly ABCD nanoparticles; a structural paradigm for viable synthetic non-viral vectors

Gene therapy research is still in trouble owing to a paucity of acceptable vector systems to deliver nucleic acids to patients for therapy. Viral vectors are efficient but may be too dangerous. Synthetic non-viral vectors are inherently safer but are currently not efficient enough to be clinically viable. The solution for gene therapy lies with improved synthetic non-viral vectors systems. This review is focused on synthetic cationic liposome/micelle-based non-viral vector systems and is a critical review written to illustrate the increasing importance of chemistry in gene therapy research. This review should be of primary interest to synthetic chemists and biomedical researchers keen to appreciate emerging technologies, but also to biological scientists who remain to be convinced about the relevance of chemistry to biology.     

Immobilized metal affinity chromatography of histidine-tagged lentiviral vectors using monolithic adsorbents

Histidine-tagged lentiviral vectors were separated from crude cell culture supernatant using labscale monolithic adsorbents by immobilized metal affinity chromatography. The capture capacity, concentration factor, purification factor, and elution efficiency of a supermacroporous cryogel monolith were evaluated against the BIA Separations convective interaction media (CIM) disc, which is a commercial macroporous monolith. The morphology of the polymeric cryogel material was characterised by scanning electron microscopy. Iminodiacetic acid was used as the metal chelating ligand in both monoliths and the chelating capacity for metal ions was found to be comparable. The CIM-IDA-Ni(2+) adsorbent had the greatest capture capacity (6.7 x 10(8) IU/ml of adsorbent), concentration factor (1.3-fold), and elution efficiency (69%). Advantages of the cryogel monoliths included rapid, low pressure processing as well low levels of protein and DNA in the final purified vector preparations.     

Adenoviral vectors

Gene therapy is a promising tool for treatment of the human diseases that cannot be cured by rational therapies, and its primary success depends on suitable vectors to deliver therapeutic genes. Adenoviruses (Ads) are among the most commonly used vectors for gene therapy, second only to retroviruses. During the last decade, remarkable progress has been made in the development of Ad vectors and in the understanding of the toxicity related to the Ad vector system. Ad vector has certain advantages such as high transduction efficiency for different quiescent and dividing cell types and high levels of short-term expression to provide therapeutic benefits. However, researchers are facing the challenges associated with tissue-specific targeting of vectors and the vector-mediated immunogenicity. This review mainly focuses on the studies that have employed methods to improve Ad vectors and reduce viral toxicity for different applications. These methods include minimization or elimination of viral genes, retargeting of vector to the tissue of interest, and generation of immunocompromised recombinant vectors that lead to safer use of Ad vector systems that improve persistence of transgene expression. Moreover, the therapeutic applications of Ad vectors for liver-targeted gene therapy, suicide gene therapy, delivery of small interfering RNA, and production of recombinant vaccine under regulated conditions used in clinical trials are discussed.     

Molecular Mechanisms behind Acetylene Adsorption and Selectivity in Functional Porous Materials

Since its first industrial production in 1890s, acetylene has played a vital role in manufacturing a wide spectrum of materials. Although current methods and infrastructures for various segments of acetylene industries are well-established, with emerging functional porous materials that enabled desired selectivity toward target molecules, it is of timely interest to develop new efficient technologies to promote safer acetylene processes with a higher energy efficiency and lower carbon footprint. In this Minireview, we, from the perspective of materials chemistry, review state-of-the-art examples of advanced porous materials, namely metal–organic frameworks and decorated zeolites, that have been applied to the purification and storage of acetylene. We also discuss the challenges on the roadmap of translational research in the development of new solid sorbent-based separation technologies and highlight areas which require future research efforts.     

Tangential flow filtration for virus purification

Purification of virus particles and viral vectors for viral vaccines and gene therapy applications is a major large-scale separations challenge. Purification of parvovirus particles such as adeno-associated virus, the leading candidate for gene therapy applications, is particularly challenging given their small size, typically 18–26 nm. We have investigated the use of ultrafiltration for purification of Aedes aegypti densonucleosisvirus, a mosquito parvovirus.     

Polyethylene glycol

The increasing demand of synthetic oligonucleotides for therapeutic and diagnostic purposes can be hardly satisfied by simply upscaling the commercial synthesizers. The introduction of a liquid-phase method that utilizes a polymeric support soluble into the reaction media can overcome the shortcomings related to the heterogeneity of the solid phase and allow a convenient large-scale process. Recently we have proposed a new synthetic approach for the oligonucleotide production that utilizes polyethylene glycol or PEG as soluble supporting polymer. We call this method High-Efficiency Liquid Phase or HELP. This approach preserves the advantages of a homogeneous synthesis in solution and adds an easy purification step of all the intermediates, mimicking the solid-phase procedure. In fact, reagent excess and byproducts can be eliminated by a simple precipitation-and-filtration step at the end of each synthetic cycle. Since all the reactions take place in solution, the scale-up of the process is easily predictable. Various synthetic protocols have been tested and optimized for the oligonucleotide production, up to the antisense-size level. After the phosphotriester and the phosphoramidite chemistry, the H-phosphonate approach is now under development. The possibility of an efficient automation of the whole process is also investigated.     

Green metrics for biologics

Biologics are the fastest growing segment of the pharmaceutical market, therefore, the environmental impact of manufacturing these drugs needs to be fully characterized. For monoclonal antibodies, in particular, several metrics have been identified as the manufacturing process is quite standardized for batch processes. This paper will provide an overview of carbon footprint analysis, process mass intensity (PMI), water related impact of energy (WARIEN) and life cycle assessment (LCA) as they have been applied to monoclonal antibody production. Further development and standardization of these tools will allow the industry to identify and implement new technologies that significantly lower the environmental impact of not only monoclonal antibody production but other modalities as well.     

亲和色谱纯化蛋白质新进展

通过对３５篇文献的综述,介绍了亲和色谱技术的新进展     

Purification of monoclonal antibody from tobacco extract using membrane-based bioseparation techniques

Transgenic plants offer a promising system for large-scale production of therapeutic proteins such as monoclonal antibodies (mAbs). This paper describes a membrane-based process suitable for purification of a humanized mAb expressed in tobacco. Most monoclonal antibody purification schemes rely on the use of Protein A as the affinity ligand for antibody capture. The main objective of our work was to develop non-Protein A-based purification methods to avoid some of the problems and limitations associated with this ligand, e.g. cost, immunotoxicity, and antibody aggregation during elution. Ion exchange membrane chromatography (IEMC) was used for primary capture and preliminary purification of the mAb from tobacco juice. Hydrophobic interaction membrane chromatography (HIMC) was then used for high-resolution purification, followed by ultrafiltration for polishing, desalting and buffer exchange. Using this scheme, both high mAb purity (single peak in size exclusion chromatogram, i.e., ca. 100% purity) and high recovery (77% of mAb spiked into the tobacco extract) were achieved. Membrane chromatography is generally considered unsuitable for resolving bound proteins by gradient elution and is therefore commonly used in the bind and elute mode with a single-step change of mobile phase. We show that the gradient elution process in the HIMC step can be optimized to increase the resolution and thereby obtain product of high purity.