Abstract
A continuous downstream process of monoclonal antibody was developed based on the process characterization. Periodic‐counter current chromatography (PCCC) with two protein A columns was used ...for the capture step. For low pH virus inactivation (VI), a batch reactor was employed, which can work as a surge (buffer) tank. Flow‐through chromatography (FTC) with two connected columns of different separation modes (anion‐mixed mode and cation exchange) was designed as a polish step. After 24 h PCCC run, the collected pool was processed for VI. After adjusting pH and electric conductivity, the solution was fed to the two connected FTC columns for 24 h. Virus filter was also connected to the exit of the connected‐column. PCCC and FTC were run in parallel. Six runs of different feed rates (0.5–10 L/day) and feed concentrations (1–3.2 g/L) were performed with protein A columns of 1–5 mL and FTC columns of 3–10 mL. The largest run (feed rate 10 L/day, feed concentration 2 g/L) was carried out at a GMP facility with 15 mL protein A columns and 100 mL FTC columns. Good recovery and purity values were obtained for all runs. The process was found to be flexible and stable for feed fluctuations. Only three surge or pool tanks were needed in addition to the final product pool tank.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
•First report of a scalable complete purification process for ORF virus.•Overall virus yield: 64 % (>70 % clarification and >90 % 2-step chromatography)•100 % protein removal and 96–98 % DNA ...depletion (1 ngDNA per 1E + 06 infective virus).•Platform process - robustness shown for two different ORF virus genotypes.•Confirmed virus integrity and infectivity after the downstream process.
The large demand for safe and efficient viral vector-based vaccines and gene therapies against both inherited and acquired diseases accelerates the development of viral vectors. One outstanding example, the Orf virus, has a wide range of applications, a superior efficacy and an excellent safety profile combined with a reduced pathogenicity compared to other viral vectors. However, besides these favorable attributes, an efficient and scalable downstream process still needs to be developed. Recently, we screened potential chromatographic stationary phases for Orf virus purification. Based on these previous accomplishments, we developed a complete downstream process for the cell culture-derived Orf virus. The described process comprises a membrane-based clarification step, a nuclease treatment, steric exclusion chromatography, and a secondary chromatographic purification step using Capto® Core 700 resin. The applicability of this process to a variety of diverse Orf virus vectors was shown, testing two different genotypes. These studies render the possibility to apply the developed downstream scheme for both genotypes, and lead to overall virus yields of about 64 %, with step recoveries of >70 % for the clarification, and >90 % for the chromatography train. Protein concentrations of the final product are below the detection limits, and the final DNA concentration of about 1 ng per 1E + 06 infective virus units resembles a total DNA depletion of 96–98 %.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Host cell proteins (HCPs) are process-related impurities in a therapeutic protein expressed using cell culture technology. This review presents biopharmaceutical industry trends in terms of both HCPs ...in the bioprocessing of monoclonal antibodies (mAbs) and the capabilities for HCP clearance by downstream unit operations. A comprehensive assessment of currently implemented and emerging technologies in the manufacturing processes with extensive references was performed. Meta-analyses of published downstream data were conducted to identify trends. Improved analytical methods and understanding of "high-risk" HCPs lead to more robust manufacturing processes and higher-quality therapeutics. The trend of higher cell density cultures leads to both higher mAb expression and higher HCP levels. However, HCP levels can be significantly reduced with improvements in operations, resulting in similar concentrations of approx. 10 ppm HCPs. There are no differences in the performance of HCP clearance between recent enhanced downstream operations and traditional batch processing. This review includes best practices for developing improved processes.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, PNG, SBCE, SBMB, UL, UM, UPUK
The alternative fuel butanol can be produced via acetone–butanol–ethanol (ABE) fermentation from biomass. The high costs for the separation of ABE from the dilute fermentation broth have so far ...prohibited the industrial-scale production of bio-butanol. In order to facilitate an effective and energy-efficient product removal, we suggest a hybrid extraction–distillation downstream process with ABE extraction in an external column. By means of computer-aided molecular design (CAMD), mesitylene is identified as novel solvent with excellent properties for ABE extraction from the fermentation broth. An optimal flowsheet is developed by systematic process synthesis which combines shortcut and rigorous models with rigorous numerical optimization. Optimization of the flowsheet structure and the operating point, consideration of heat integration, and the evaluation of the minimum energy demands are covered. It is shown that the total annualized costs of the novel process are considerably lower compared to the costs of alternative hybrid or pure distillation processes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
An optimal purification process for biopharmaceutical products is important to meet strict safety regulations, and for economic benefits. To find the global optimum, it is desirable to screen the ...overall design space. Advanced model-based approaches enable to screen a broad range of the design-space, in contrast to traditional statistical or heuristic-based approaches. Though, chromatographic mechanistic modeling (MM), one of the advanced model-based approaches, can be speed-limiting for flowsheet optimization, which evaluates every purification possibility (e.g., type and order of purification techniques, and their operating conditions). Therefore, we propose to use artificial neural networks (ANNs) during global optimization to select the most optimal flowsheets. So, the number of flowsheets for final local optimization is reduced and consequently the overall optimization time. Employing ANNs during global optimization proved to reduce the number of flowsheets from 15 to only 3. From these three, one flowsheet was optimized locally and similar final results were found when using the global outcome of either the ANN or MM as starting condition. Moreover, the overall flowsheet optimization time was reduced by 50% when using ANNs during global optimization. This approach accelerates the early purification process design; moreover, it is generic, flexible, and regardless of sample material's type.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Virus filtration in biopharmaceutical downstream process ensures viral safety with removal efficiency greater than 99.99%. Despite its robust performance, it has been reported that viruses can escape ...through the membrane under certain conditions. This study aimed to investigate virus breakthrough using Viresolve® Pro membrane with PP7 bacteriophage under high bacteriophage titer, high protein concentration, flow interruption, and low-flux operation. The results show high virus removal performance (LRV >6.8) up to 109 PFU/mL for 200 L/m2, with the first phage breakthrough observation over 1012 PFU/m2 of phage challenge in the membrane. As the protein concentration increased in the coexistence condition, a greater number of phages passed through the membrane. During the post-buffer flush and the operation at low filtrate flux, diffusion plays an important role in the escape of phages, which were detected as high as 1012 PFU/m2, but as low as 109 PFU/m2. These results suggest that the virus-retentive membrane may become susceptible to undesired virus breakthrough when exposed to phage challenges of > 1012 PFU/mL, providing important key factors to ensure viral safety during the downstream process of biopharmaceutical production.
•Virus breakthrough during filtration was investigated under harsh conditions.•Viresolve® Pro membrane showed robust virus removal performance as LRV > 6.8.•The first phage breakthrough occurred at ∼1012 PFU/m2 of phage challenge.•As the protein concentration increased, more phages escaped.•Diffusion plays an important role in virus breakthrough.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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•Recent advances in carboxylic acids recovery methods were systematically reviewed.•Integrated process could overcome drawbacks of sole separation method.•Application via biological ...processes is more competitive than chemical process.•Novel desorption methods need to be developed to reduce agent consumption.•Future work should focus on carbon footprint calculation and life cycle assessment.
Recovering carboxylic acids derived from organic wastes from fermentation broth is challenging. To provide a reference for future study and industrial application, this review summarized recent advances in recovery technologies of carboxylic acids including precipitation, extraction, adsorption, membrane-based processes, etc. Meanwhile, applications of recovered carboxylic acids are summarized as well to help choose suitable downstream processes according to purity requirement. Integrated processes are required to remove the impurities from the complicated fermentation broth, at the cost of loss and expense. Compared with chemical processes, biological synthesis is better options due to low requirements for the substrates. Generally, the use of toxic agents, consumption of acid/alkaline and membrane fouling hamper the sustainability and scale-up of the downstream processes. Future research on novel solvents and materials will facilitate the sustainable recovery and reduce the cost of the downstream processes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The use of biopharmaceuticals dates from the 19th century and within 5–10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry ...experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN α, β, and γ) and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Virus filtration process is used to ensure viral safety in the biopharmaceutical downstream processes with high virus removal capacity (i.e., >4 log10). However, it is still constrained by protein ...fouling, which results in reduced filtration capacity and possible virus breakthrough. This study investigated the effects of protein fouling on filtrate flux and virus breakthrough using commercial membranes that had different symmetricity, nominal pore size, and pore size gradients. Flux decay tendency due to protein fouling was influenced by hydrodynamic drag force and protein concentration. As the results of prediction with the classical fouling model, standard blocking was suitable for most virus filters. Undesired virus breakthrough was observed in the membranes having relatively a large pore diameter of the retentive region. The study found that elevated levels of protein solution reduced virus removal performance. However, the impact of prefouled membranes was minimal. These findings shed light on the factors that influence protein fouling during the virus filtration process of biopharmaceutical production.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
•Only small subset (10%) of mAbs exhibited significant HCP copurification issue.•High-HCP mAbs share many common HCPs and similar copurification mechanism.•HCP copurification correlates with mAb's ...self-association propensity.•Mab-clustering strengthens interactions between mAb with HCPs.•Breaking mAb-clusters effectively prevents HCP copurification.
Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs show significant HCP copurification. In this study, we carried out systematic studies using 4 different mAbs to explore the HCP copurification mechanism. HCP identification results revealed that the 3 high-HCP mAbs had many common HCPs which do not copurify with the low-HCP mAb, suggesting a similar mechanism is at play. Through wash evaluation, surface patch analysis, chain-swapping, domain evaluation, and structure-guided mutations, several charged residues in each mAb were found which correlated with HCP copurification. Surprisingly, these residues are also critical for self-association propensity. We observed an inverse correlation between diffusion interaction parameter and HCP copurification. Each of the high-HCP mAbs could form dynamic clusters consisting of 3∼6 mAb molecules. Therefore, a mAb cluster can exhibit higher net positive charges on the order of 3 to 6, compared with the individual mAb. In Protein A chromatography, high-HCP mAbs had elution tailing which contained high level of HCPs. Addition of Arginine-HCl or point mutations preventing cluster formation effectively reduced HCP copurification and elution tailing. Based on these results, we propose a novel HCP-copurification mechanism that formation of mAb clusters strengthens charge-charge interactions with HCPs and thus compromises HCP removal by Protein A chromatography. Besides arginine, histidine under acidic pH conditions prevented cluster formulation and resulted in effective HCP removal. Finally, structure-guided protein engineering and solution screening by using cluster size as indicator are useful tools for managing mAbs with high-HCP issues.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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