Techno-economic analysis of a transient plant-based platform for monoclonal antibody production

Plant-based biomanufacturing of therapeutic proteins is a relatively new platform with a small number of commercial-scale facilities, but offers advantages of linear scalability, reduced upstream complexity, reduced time to market, and potentially lower capital and operating costs. In this study we present a detailed process simulation model for a large-scale new “greenfield” biomanufacturing facility that uses transient agroinfiltration of Nicotiana benthamiana plants grown hydroponically indoors under light-emitting diode lighting for the production of a monoclonal antibody. The model was used to evaluate the total capital investment, annual operating cost, and cost of goods sold as a function of mAb expression level in the plant (g mAb/kg fresh weight of the plant) and production capacity (kg mAb/year). For the Base Case design scenario (300 kg mAb/year, 1 g mAb/kg fresh weight, and 65% recovery in downstream processing), the model predicts a total capital investment of $122 million dollars and cost of goods sold of $121/g including depreciation. Compared with traditional biomanufacturing platforms that use mammalian cells grown in bioreactors, the model predicts significant reductions in capital investment and >50% reduction in cost of goods compared with published values at similar production scales. The simulation model can be modified or adapted by others to assess the profitability of alternative designs, implement different process assumptions, and help guide process development and optimization.     

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.     

The future of biomanufacturing in Singapore

Singapore's vision is to become a global biomedical sciences (BMS) hub and to develop the BMS industry as the fourth manufacturing pillar of Singapore's economy. The biomanufacturing industry has been a significant growth area for Singapore's BMS industry, with four major biologics investments totaling close to US$1 billion being committed in less than 2 years and the setting up of local biomanufacturing companies. In parallel, resources in biomedical research and bioprocess manpower capabilities are being built up for this knowledge-intensive industry. Located at the Biopolis, the epicenter of biomedical R&D, the Bioprocessing Technology Institute (BTI) has been developing bioprocess expertise and programs to support the manpower needs of the growing biomanufacturing sector. With a strong commitment to provide the biomanufacturing industry with research and manpower support, the future looks promising and Singapore is well positioned to become a leading biomanufacturing hub in Asia Pacific.     

Impacts of intentional mycoplasma contamination on CHO cell bioreactor cultures

Mycoplasma contamination events in biomanufacturing facilities can result in loss of production and costly cleanups. Mycoplasma may survive in mammalian cell cultures with only subtle changes to the culture and may penetrate the 0.2 µm filters often used in the primary clarification of harvested cell culture fluid. Culture cell-based and indicator cell-based assays that are used to detect mycoplasma are highly sensitive but can take up to 28 days to complete and cannot be used for real-time decision making during the biomanufacturing process. To support real-time measurements of mycoplasma contamination, there is a push to explore nucleic acid testing. However, cell-based methods measure growth or colony forming units and nucleic acid testing measures genome copy number; this has led to ambiguity regarding how to compare the sensitivity of the methods. In addition, the high risk of conducting experiments wherein one deliberately spikes mycoplasma into bioreactors has dissuaded commercial groups from performing studies to explore the multiple variables associated with the upstream effects of a mycoplasma contamination in a manufacturing setting. Here we studied the ability of Mycoplasma arginini to persist in a single-use, perfusion rocking bioreactor system containing a Chinese hamster ovary (CHO) DG44 cell line expressing a model monoclonal immunoglobulin G1 (IgG1) antibody. We examined M. arginini growth and detection by culture methods, as well as the effects of M. arginini on mammalian cell health, metabolism, and productivity. We compared process parameters and controls normally measured in bioreactors including dissolved oxygen, gas mix, and base addition to maintain pH, to examine parameter changes as potential indicators of contamination. Our work showed that M. arginini affects CHO cell growth profile, viability, nutrient consumption, oxygen use, and waste production at varying timepoints after M. arginini introduction to the culture. Importantly, how the M. arginini contamination impacts the CHO cells is influenced by the concentration of CHO cells and rate of perfusion at the time of M. arginini spike. Careful evaluation of dissolved oxygen, pH control parameters, ammonia, and arginine over time may be used to indicate mycoplasma contamination in CHO cell cultures in a bioreactor before a read-out from a traditional method.     

Pharmaceutical-grade albumin: impaired drug-binding capacity <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Albumin is the most abundant protein in blood plasma, and due to its ligand binding properties, serves as a circulating depot for endogenous and exogenous (e.g. drugs) compounds. Hence, the unbound drug is the pharmacologically active drug. Commercial human albumin preparations are frequently used during surgery and in critically ill patients. Recent studies have indicated that the use of pharmaceutical-grade albumin is controversial in critically ill patients. In this in vitro study we investigated the drug binding properties of pharmaceutical-grade albumins (Baxter/Immuno, Octapharma, and Pharmacia & Upjohn), native human serum, and commercially available human serum albumin from Sigma Chemical Company.     

Processing of plasmid DNA with ColE1-like replication origin

With the increasing utilization of plasmid DNA as a biopharmaceutical drug, there is a rapidly growing need for high quality plasmid DNA for drug applications. Although there are several different kinds of replication origins, ColE1-like replication origin is the most extensively used origin in biotechnology. This review addresses problems in upstream and downstream processing of plasmid DNA with ColE1-like origin as drug applications. In upstream processing of plasmid DNA, regulation of replication of ColE1-like origin was discussed. In downstream processing of plasmid DNA, we analyzed simple, robust, and scalable methods, which can be used in the efficient production of pharmaceutical-grade plasmid DNA.     

Colchicine and the mitotic spindle of the aquatic phycomycete Allomyces

Summary Exposure of germlings of Allomyces neo-moniliformis to colchicine for 0 to 5 min after zoospore encystment was found to block 30% of germlings derived from flagellated zoospores and 55% of germlings derived from deflagellated zoospores in C-metaphase configurations at the first mitotic division. The zoospore lacks a pool of colchicine binding protein, and protein synthesis is absent during the time when colchicine first becomes effective in inducing C-metaphase. From these observations it is concluded that the microtubule subunit protein of the spindle apparatus of the first mitotic division to a large extent is derived from the depolymerization of the cytoplasmic microtubules of the zoospore. GTP, Mg²⁺, and ATP were observed to be antagonistic to the action of colchicine in vivo. It is suggested that these compounds may compete with colchicine for binding to the subunit protein in vivo. Germlings derived from flagellated zoospores are appreciably less subject to the action of colchicine in the presence of the antagonistic compounds than are germlings derived from deflagellated zoospores. This differential sensitivity to colchicine is interpreted as reflecting a difference in the quantity of microtubule subunit protein present at the time of exposure to colchicine.     

High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles, have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation coisolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). Escherichia coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.     

Studies on a Morphologically Distinct Colchicine-Resistant Variant of Entamoeba sp.*

Colchicine has a temperature-dependent cytotoxic effect on Entamoeba sp. (Laredo isolate) that is most apparent when the drug is applied during the initiation of cultures at a concentration of 7.5 mM or higher. Continued transfer of cultures in medium containing progressively increasing concentrations of colchicine has resulted in a variant that grows prolifically in the presence of colchicine (7.5 mM) with a generation time comparable to that of the parent stock. Comparison of a number of parameters of the 2 variants revealed that colchicine resistance was accompanied by a change in cell shape, a reduced membrane permeability, which could partially be overcome by the addition of dimethyl sulfoxide (DMSO), and a reduced tolerance to osmotic stress. However, the parent strain and resistant variant were equally susceptible to cycloheximide and puromycin suggesting that the acquired colchicine resistance may not be explained on the basis of an entirely unspecific generalized reduced ability for drug uptake. Colchicine resistance and altered structure were found to be stable over a long period of time. The possible interdependence of these 2 parameters and their relation to cell motility in Entamoeba sp. are discussed.     

The effect of colchicine on pyrin and pyrin interacting proteins

MEFV which encodes pyrin, cause familial Mediterranean fever (FMF), the most common auto‐inflammatory disease. Pyrin is believed to be a regulator of inflammation, though the nature of this regulatory activity remains to be identified. Prophylactic treatment with colchicine, a microtubule toxin, has had a remarkable effect on disease progression and outcome. It has been thought that, inhibition of microtubule polymerization is the main mechanism of action of colchicine. But, the exact cellular mechanism explaining the efficacy of colchicine in suppressing FMF attacks is still unclear. Given the ability of colchicine treatment to be considered as a differential diagnosis criteria of FMF, we hypothesized that colchicine may have a specific effect on pyrin and pyrin interacting proteins. This study showed that colchicine prevents reticulated fibrils formed by PSTPIP1 filaments and reduces ASC speck rates in transfected cells. We further noted that, colchicine down‐regulates MEFV expression in THP‐1 cells. We also observed that colchicine causes re‐organization of actin cytoskeleton in THP‐1 cells. Pyrin is an actin‐binding protein that specifically localizes with polymerizing actin filaments. Thus, MEFV expression might be affected by re‐organization of actin cytoskeleton. The data presented here reveal an important connection between colchicine and pyrin which might explain the remarkable efficacy of colchicine in preventing FMF attacks. J. Cell. Biochem. 113: 3536–3546, 2012. © 2012 Wiley Periodicals, Inc.     

Colchicine-induced microspore embryogenesis in coffee

A protocol for the induction of androgenesis and plant regeneration from C. arabica cv. Caturra isolated microspores in vitro using colchicine pretreatment has been developed. Microspores were mechanically isolated and then carefully purified. Before colchicine pretreatment, microspores were cultured in a semi-solid medium for further develop and regeneration. Different times of colchicine exposure as well as different concentrations were tested. The best androgenic response was found when microspores were precultured in 100 mg l^–1 colchicine for 48 h. The microspore developmental stages responsive to colchicine were late-uninucleated and early binucleated pollen. Flow cytometry and morphological analyses revealed that 95% of regenerated plants were dihaploids (2n=2x=22). However, some doubled dihaploid plants (2n=4x=44) were also obtained, suggesting that not only androgenic induction but also chromosome duplication could be expected as result of colchicine exposure of coffee microspores. This report represents a new approach in the coffee pollen culture, as well as a major step forward to the utilization of haploid technology in coffee breeding.     

Enhanced expression of a biosimilar monoclonal antibody with a novel NS0 platform

The precise product quality and lower cost of goods demands of the growing biosimilars industry are driving biomanufacturing innovation. Biosimilar cell lines that produce complex glycoproteins such as monoclonal antibodies must be both highly productive and express a product with critical quality attributes closely matching those of the innovator reference. In this work, a biomanufacturing platform is described that harnesses the commercially‐established NS0 host cell in new ways to create stable, highly productive cell lines with characteristics meeting the current demands. A cholesterol metabolic selection marker and implementation strategy that can be generically applied are shown to yield high expressing cell lines as well as eliminate the need for cholesterol addition, which has been a significant barrier in both stainless steel reactors as well as in single‐use plastic systems. Additionally, for the first time, a multiplex selection strategy was implemented that served to increase NS0 cell line specific productivity >10‐fold and volumetric yields >6‐fold. The best overall performing cell line had a Qp of 28.5 picograms per cell per day and was rapidly adapted to a lean production medium. Yields in l ‐glutamine fed‐batch shaker cultures exceeded 500 mg/L. An initial screening of four feeding strategies resulted in a final 13‐day yield of over 1.4 g/L in small shaker culture. Overall, this work shows both the strategy to develop biosimilar cell lines and the commercial potential of a novel expression system highly suited for the manufacture of biosimilars of reference biologics currently produced in murine cells. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:455–462, 2018     

Cucurbitacine als stathmokinetische Wirkstoffe

Summary A synergism of colchicine and different cucurbitacins has been shown, to be a cause in the formation of ball-metaphases in Bryonia species. The bitter principles cucurbitacin L and I, dihydrocucurbitacin B, and tetrahydrocucurbitacin I, which were examined more closely with respect to their karyological effects, were shown to give rise to C-metaphases in Hordeum and Vicia at concentrations of about 0.016 per cent. On the one hand the number of mitotic stages is reduced, and on the other hand there is an increase of metaphase stages. Higher concentrations lead to pycnotic degenerations of nuclei and are toxic. When colchicine is applied together with cucurbitacins, ball-metaphases result. This could be demonstrated in plants lacking colchicine and cucurbitacins (Hordeum) when both substances were applied at the same time. Ball-metaphases also appear in plants containing cucurbitacins (Bryonia alba, B. dioica, Citrullus colocythis, Iberis amara) when they are treated with colchicine.     

四乙酰基植物鞘氨醇生物合成的研究进展

四乙酰基植物鞘氨醇(tetraacetyl phytosphingosine, TAPS)是一种性能卓越的天然护肤品原料,经去乙酰化后生成的植物鞘氨醇可作为前体合成保湿护肤品神经酰胺,因此广泛应用于护肤化妆品行业。非常规酵母威克汉姆西弗酵母(Wickerhamomyces ciferrii)是已知的唯一可天然分泌四乙酰基植物鞘氨醇的微生物,目前已成为四乙酰基植物鞘氨醇工业生产的宿主。本文介绍了四乙酰基植物鞘氨醇的发现、功能及其生物合成途径,综述了近年来利用单倍体筛选、诱变育种和代谢工程改造威克汉姆西弗酵母高产四乙酰基植物鞘氨醇的研究进展,并展望了实现四乙酰基植物鞘氨醇工业生产的未来发展方向。     

Effetto Della Colchicina Su Enzimi Solfidrilici

Abstract

The effect of colchicine on sulfhydryl enzymes. — The effect of colchicine has been tested on mithocondrial ATPase and rat-liver LDH. The colchicine inhibits ATPase for 40% and LDH for 100%, therefore its action is aspecific.     

Heterogeneity in Cell Behaviour in Primordia ofVicia faba

The response of cells of small primordia ofVicia faba to³H-TdR and colchicine is discussed. The delayed uptake of³H-TdR shown by cells of small primordia appears to be a property of only 50% of the cells; the remaining never become capable of incorporating³H-TdR. Prom the labeled cells and polyploid cells induced by colchicine the shortest cycle time in small primordia is estimated to be 12 hours. Within a period equal to 1 mitotic cycle, 31–35% of all mitoses are tetraploid, following treatment with colchicine. The remainder are diploid and diploid mitoses were seen for up to 30 hours. These observations are indicative of a heterogeneity for mitotic cycle time in populations consisting of up to 1,500 cells. The percentage labeled mitosis curve of diploid cells was changed in primordia treated with colchicine; higher peaks were found. These results show that even small populations of cells, at the beginning of a morphogenetic system, are very heterogeneous for key cell properties. This research has been supported by the U.S.A.B.C. [AT (11-1) 1625-21].     

合成生物制造2022

本文对2022年《生物工程学报》发表的与合成生物制造相关的综述和研究论文进行了评述,重点讨论了DNA测序、DNA合成、DNA编辑、基因表达调控和数学细胞模型等底层技术,酶的设计、改造和应用技术,化学品生物催化、氨基酸及其衍生物、有机酸、天然化合物、抗生素与活性肽、功能多糖、功能蛋白质等重要产品的生物制造技术,一碳化合物和生物质原料利用技术以及合成微生物组技术,以帮助读者从一个侧面了解合成生物制造相关技术和产业的发展情况。     

Bioseparation in antibody manufacturing: the good, the bad and the ugly

Improvements in upstream production have boosted productivity in the biomanufacturing industry, but this is leading to bottlenecks in downstream processing as current technology platforms reach their limits of throughput and scalability. Although chromatography remains an indispensible component of downstream processing due to its simplicity and high resolving power (The Good), there is virtually no economy of scale effect so more product translates almost linearly into greater production costs. Bind-and-elute processes (such as the initial capture step in antibody manufacturing) are volume-driven and therefore have knock-on effects that impact on the entire production facility since the space required for preparation, storage, and cleaning steps has to be similarly adapted (The Bad). During long-term operations with multiple cycles, thorough cleaning is necessary to prevent progressive fouling and microbial contamination (The Ugly). Innovative solutions are required, which may include revisiting simpler and less expensive separation technologies, the use of disposable modules, and the integration of improved processes that are scalable to cope with increased demands. Among the alternatives that have been put forward, membrane adsorbers are beginning to make a real impact on the industry, particularly for flow-through applications such as polishing and viral clearance.     

Sustaining an efficient and effective CHO cell line development platform by incorporation of 24‐deep well plate screening and multivariate analysis

Efficient and effective cell line screening is paramount toward a successful biomanufacturing program. Here we describe the implementation of 24‐deep well plate (24‐DWP) screening of CHO lines as part of the cell line development platform at AbbVie. Incorporation of this approach accelerated the identification of the best candidate lines for process development. In an effort to quantify and predict process performance comparability, we compared cell culture performance in and in shake flasks, for a panel of Chinese Hamster Ovary cell lines expressing a monoclonal antibody. The results in 24‐DWP screening showed reduced growth profiles, but comparable viability profiles. Slow growers in 24‐DWP achieved the highest productivity improvement upon scaling‐up to shake flasks. Product quality of the protein purified from shake flasks and 24‐DWP were also compared. The 24‐DWP culture conditions were found to influence the levels of acidic species, reduce the G0 N‐glycan species, and increase the high‐mannose N‐glycan species. Nevertheless, the identification of undesirable profiles is executed consistently with the scaled‐up culture. We further employed multivariate data analysis to capture differences depending on the two scales and we could demonstrate that cell line profiles were adequately clustered, regardless of the vessel used for the development. In conclusion, the 24‐DWP platform was reasonably predictive of the parameters crucial for upstream process development activities, and has been adapted as part of the AbbVie cell line development platform. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:175–186, 2018