RESUMO
Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2-3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts.
Assuntos
Polipeptídeos Semelhantes à Elastina , Seda , Seda/genética , Proteínas de Artrópodes , Elastina/genética , Elastina/química , Elastina/metabolismo , Nicotiana/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
This research utilized a customized laboratory setup to compare the filtration performance and fouling buildup during microfiltration with polymeric membranes of skim milk using 2 diafiltration media: ultrafiltration permeate and ultrapure water. Two filtration modes were evaluated: in stage 1, the diafiltration media was added in a 1:1 ratio, with the collection of permeate continuing until the initial protein concentration was restored. In stage 2, retentates and permeates were recycled to simulate fouling accumulation in a steady-state without altering the retentate composition. Utilizing water as the diafiltration medium resulted in higher flux and lower resistance values compared with using ultrafiltration permeate, irrespective of the filtration mode. The concentration had a significant impact on membrane resistance, with no noticeable time-dependent effect on fouling layer development after 60 min of filtration when the retentate composition remained constant. The protein composition of the permeate and extracted foulants were comparable between the 2 media, with caseins predominating in the fouling layer.
RESUMO
Processing temperature has a significant influence on the composition and functionality of the resulting streams following microfiltration (MF) of skim milk. In this study, MF and diafiltration (DF) were performed at 4 or 50°C to produce ß-casein (ß-CN)-depleted and nondepleted (i.e., native casein profile) micellar casein isolate retentates, respectively. Microfiltration combined with extensive DF resulted in a 40% depletion of ß-CN at 4°C, whereas no ß-CN depletion occurred at 50°C. Microfiltration at 4°C led to higher transmission of calcium into permeates, with retentate generated at 4°C containing less total calcium compared with retentate generated at 50°C, based on the volume of retentate remaining. Higher heat stability at 120°C was measured for retentates generated at 4°C compared with those at 50°C, across all pH values measured. Retentates generated at 4°C also had significantly lower ionic calcium values at each pH compared with those generated at 50°C. Higher apparent viscosities at 4°C were measured for retentates generated at 4°C compared with retentates generated at 50°C, likely due to increased voluminosity of ß-CN-depleted casein micelles. The results of this study provide new information on how changing the composition of MF retentate, by appropriate control of processing temperature and DF, can alter physicochemical properties of casein micelles, with potential implications for ingredient functionality.
Assuntos
Caseínas , Micelas , Animais , Caseínas/química , Temperatura , Cálcio/análise , Manipulação de Alimentos/métodos , Filtração/métodos , Filtração/veterinária , Leite/química , Proteínas do Leite/análiseRESUMO
Diafiltration (DF) is a separation method used to separate and concentrate macromolecules, such as polysaccharides and proteins. To obtain high-purity target molecules by DF, appropriate conditions should be used. In this study, a mathematical model was developed to suggest appropriate ultra-filtration (UF) membrane modules for the separation of phycocyanin (PC) by multistep DF. PC is a protein produced by microalgae. The contribution of each UF membrane module to PC productivity and purity at each stage of the multistep DF process was quantified by the proposed model. The parameters required as model inputs (k, Fα1, and Fα2) were experimentally determined by permeating PC-containing solution through UF membrane modules (150, 30, and 10 kDa cutoffs). The resulting analytical solutions and those predicted by the model were in close agreement. The PC purity increased from 0.20 to 0.30 when a 10 kDa UF membrane module was used in two-step DF. An orthogonal table was used to determine the combination of UF membrane modules needed to achieve higher purity of PC. The model predicted that the 30 kDa UF membrane module would have the highest contribution to PC productivity and purity at any position in a three-step DF. The developed model can help identify appropriate conditions for separating macromolecules by DF.
Assuntos
Nostoc commune , Ultrafiltração , Ultrafiltração/métodos , Ficocianina , Proteínas , PolissacarídeosRESUMO
BACKGROUND AND OBJECTIVES: Red blood cell (RBC) units in hypothermic storage degrade over time, commonly known as the RBC storage lesion. These older RBC units can cause adverse clinical effects when transfused, as older RBCs in the unit lyse and release cell-free haemoglobin (Hb), a potent vasodilator that can elicit vasoconstriction, systemic hypertension and oxidative tissue injury after transfusion. In this study, we examined a novel method of washing ex vivo stored single RBC units to remove accumulated cellular waste, specifically cell-free Hb, using tangential flow filtration (TFF) driven by a centrifugal pump. MATERIALS AND METHODS: The TFF RBC washing system was run under hypothermic conditions at 4°C, at a constant system volume with 0.9 wt% saline as the wash solution. The RBC washing process was conducted on 10 separate RBC units. For this proof-of-concept study, RBC units were expired at the time of washing (60-70 days old). Cell-free Hb was quantified by UV-visible absorbance spectroscopy and analysed via the Winterbourn equations. Pre- and post-wash RBC samples were analysed by Hemox Analyser, Coulter counter and Brookfield rheometer. The RBC volume fraction in solution was measured throughout the wash process by standard haematocrit (HCT) analysis. RESULTS: No substantial decrease in the HCT was observed during the TFF RBC washing process. However, there was a significant decrease in RBC concentration in the first half of the TFF RBC wash process, with no significant change in RBC concentration during the second half of the TFF cell wash process with an 87% overall cell recovery compared with the total number of cells before initiation of cell washing. Utilization of the extinction coefficients and characteristic peaks of each Hb species potentially present in solution was quantified by Winterbourn analysis on retentate and permeate samples for each diacycle to quantify Hb concentration during the washing process. Significant cell-free Hb reduction was observed within the first four diacycles with a starting cell-free Hb concentration in the RBC unit of 0.105 mM, which plateaus to a constant Hb concentration of 0.01 mM or a total extracellular Hb mass of 0.2 g in the resultant washed unit. The oxygen equilibrium curve showed a significant decrease in P50 between the initial and final RBC sample cell wash with an initial P50 of 15.6 ± 1.8 mm Hg and a final P50 of 14 ± 1.62 mm Hg. Cooperativity increased after washing from an initial Hill coefficient of 2.37 ± 0.19 compared with a final value of 2.52 ± 0.12. CONCLUSION: Overall, this study investigated the proof-of-concept use of TFF for washing single RBC units with an emphasis on the removal of cell-free Hb from the unit. Compared with traditional cell washing procedures, the designed system was able to more efficiently remove extracellular Hb but resulted in longer wash times. For a more complete investigation of the TFF RBC washing process, further work should be done to investigate the effects of RBC unit storage after washing. The designed system is lightweight and transportable with the ability to maintain sterility between uses, providing a potential option for bedside ex vivo transfusion in clinical applications.
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Preservação de Sangue , Eritrócitos , Preservação de Sangue/métodos , Filtração , Hematócrito , Hemoglobinas/análise , Humanos , Solução SalinaRESUMO
Since it is known that hyaluronic acid contributes to soft tissue growth, elasticity, and scar reduction, different strategies of producing HA have been explored in order to satisfy the current demand of HA in pharmaceutical products and formulations. The current interest deals with production via bacterial and yeast fermentation and extraction from animal sources; however, the main challenge is the right extraction technique and strategy since the original sources (e.g., fermentation broth) represent a complex system containing a number of components and solutes, which complicates the achievement of high extraction rates and purity. This review sheds light on the main pathways for the production of HA, advantages, and disadvantages, along with the current efforts in extracting and purifying this high-added-value molecule from different sources. Particular emphasis has been placed on specific case studies attempting production and successful recovery. For such works, full details are given together with their relevant outcomes.
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Ácido Hialurônico , Animais , Fermentação , Ácido Hialurônico/metabolismoRESUMO
This study aimed to investigate the production of acid-coagulated fresh cheese by using slightly acid diafiltered (DF) microfiltered (MF) casein concentrates (8% protein). Three different acidifying agents were tested during DF: carbon dioxide, lactic acid, and citric acid. Fresh cheese was manufactured using acid-DF casein concentrates, or casein concentrates DF with just water, and compared with cheese manufactured using MF casein concentrates without DF. The fresh cheeses were characterized for composition, rheological, and sensorial properties. Acid-DF casein concentrates improved acidification kinetics during cheesemaking and reduced casein leakage to cheese whey, compared with cheese from regular MF casein concentrate. Among the rheological properties investigated in this study, the storage modulus of the fresh cheese was higher when DF of the casein concentrate was performed with nonacidified DF water or when DF water was acidified with citric acid. However, fresh cheese made from casein concentrate diafiltered with DF water acidified by citric acid was most liked in a sensory ranking test.
Assuntos
Queijo , Animais , Caseínas , Queijo/análise , Manipulação de Alimentos , Leite/química , Soro do Leite/química , Proteínas do Soro do Leite/análiseRESUMO
BACKGROUND/PURPOSE: The pharmacokinetics of vancomycin in patients who undergo sustained low efficiency daily diafiltration (SLEDD-f) is not clear. This study aimed to determine the appropriate vancomycin dosage regimen for patients receiving SLEDD-f. METHODS: This prospectively observational study enrolled critically ill patients older than 18 years old that used SLEDD-f as renal replacement therapy and received vancomycin treatment. An 8-h SLEDD-f was performed with FX-60 (high-flux helixone membrane, 1.4 m2). Serial blood samples were collected before, during, and after SLEDD-f to analyse vancomycin serum concentrations. Effluent fluid samples (a mixture of dialysate and ultrafiltrate) were also collected to determine the amount of vancomycin removal. RESULTS: Seventeen patients were enrolled, and 10 completed the study. The amount of vancomycin removal was 447.4 ± 88.8 mg (about 78.4 ± 18.4% of the dose administered before SLEDD-f). The vancomycin concentration was reduced by 57.5 ± 14.9% during SLEDD-f, and this reduction was followed by a rebound with duration of one to three hours. The elimination half-life of vancomycin decreased from 64.1 ± 35.7 h before SLEDD-f to 7.0 ± 3.0 h during SLEDD-f. CONCLUSION: Significant amount of vancomycin removed during SLEDD-f. Despite the existence of post-dialysis rebound, a sufficient supplemental dose is necessary to maintain therapeutic range.
Assuntos
Terapia de Substituição Renal Híbrida , Injúria Renal Aguda , Adolescente , Antibacterianos , Estado Terminal , Humanos , Estudos Prospectivos , VancomicinaRESUMO
Several models have been developed to describe the shifts in pH and excipient concentrations seen during diafiltration of monoclonal antibody (mAb) products accounting for both Donnan equilibrium and electroneutrality constraints. However, these models have assumed that the mAb charge is either constant or only a function of pH, assumptions that will not be valid when formulating highly concentrated mAbs using bufferless or low-buffered media due to the change in local H+ concentration at the protein surface. The objective of this study was to incorporate the effects of both pH and ionic strength on the mAb charge, through the use of a charge regulation model based on the amino acid sequence of the mAb, into an appropriate mass balance model to describe the pH and excipient profiles during diafiltration. The model involves no adjustable parameters, with the protein charge evaluated directly from the protonation/deprotonation of the ionizable amino acids accounting for the electrostatic interactions between the charged mAb and the H+ ions. Model predictions are in excellent agreement with experimental data for the pH and ion concentrations during diafiltration of a mAb and fusion protein with different isoelectric points and different formulation conditions. Model simulations are then used to obtain fundamental insights into the factors controlling the diafiltration behavior as well as guidelines for development of diafiltration processes to achieve target bufferless formulation conditions.
Assuntos
Anticorpos Monoclonais/metabolismo , Técnicas de Cultura de Células/métodos , Meios de Cultura/metabolismo , Ultrafiltração/métodos , Anticorpos Monoclonais/análise , Meios de Cultura/química , Excipientes , Concentração de Íons de Hidrogênio , Concentração Osmolar , Eletricidade EstáticaRESUMO
Cheese made from microfiltration (MF) retentate may suffer from textural defects due to a high Ca concentration. The reduction of colloidal minerals by the acidification of milk before MF at pH below 6.0 has been well documented in the literature. This process, however, creates less valuable side streams to the MF process and induces changes in the casein micelles that negatively affect their coagulation properties. The objective of this study was to determine whether a minor reduction in pH by using different acidifiers in the diafiltration (DF) water could induce changes in composition and renneting properties of the MF retentate. A 2-stage filtration process was used, with the first designed to increase the casein concentration to 8% and the second to slightly reduce the casein concentrate by 0.1 pH unit by DF, without influencing the total protein concentration. Four acidifying agents were tested during DF: lactic acid, hydrochloric acid, citric acid, and carbon dioxide. Diafiltration with water was used as a reference. At the start of DF, the retentates of acid DF had a slightly reduced pH, with an average of 0.09, whereas the pH of the reference retentate increased by an average of 0.07 unit. The reference retentate regained its starting pH by the end of DF. The carbonated retentate gradually increased in pH during processing, whereas the pH of the lactic, hydrochloric, and citric acid retentates remained constant. The permeate from the lactic acid and carbonated treatments had a reduced whey protein content compared with the reference. The total P and inorganic phosphate were lowered in the retentate by using carbonation. The total amount of Mg and Na were lowered in the retentate by using citric acid. The ionic Ca content in the retentate increased with use of lactic or hydrochloric acid. The type of acidifier used reduced the rennet clotting time. Combined acidified diafiltration with a slight reduction affects the permeate composition and improves the retentate clotting time despite the minimal mineral modification.
Assuntos
Caseínas/química , Queijo/análise , Quimosina/química , Manipulação de Alimentos , Animais , Filtração/métodos , Manipulação de Alimentos/métodos , Concentração de Íons de Hidrogênio , Micelas , Leite/química , Água , Proteínas do Soro do Leite/análiseRESUMO
Using membrane filtration it is possible to selectively concentrate proteins and, in the case of microfiltration, concentrate casein micelles. During filtration, water is often added and this practice, called diafiltration, causes further release of permeable components and maintains filtration efficiency. Filtration causes changes in composition of the protein as well as the soluble phase, including soluble calcium, which is a critical factor controlling the gelation properties of the casein micelles in milk. It was hypothesized that concentrates obtained using membrane filtration with or without diafiltration would have different gelation behavior. To test this hypothesis, two concentrates of similar casein micelle volume fraction were prepared, using spiral wound polymeric microfiltration membranes with a 800 kDa molecular weight cutoff, with or without diafiltration. The concentrates showed a gelation behavior comparable to that of skim milk, with a similar gelation time and with a higher firmness, due to the higher number of protein linkages in the network. In contrast, the hydrolysis of κ-casein by chymosin and casein aggregation were inhibited in diafiltered casein micelle suspensions. When the concentrates were recombined with the original skim milk to a final concentration of 5% protein, which re-established a similar soluble phase composition, differences in gelation behavior were no longer observed: both treatments showed similar gelation time and gel firmness. These results confirmed that membrane filtration can result in concentrates with different functionality, and that ionic environmental conditions are critical to the aggregation behavior of casein micelles. This is of particular significance in industrial settings where these fractions are used as a way to standardize proteins in cheese making.
Assuntos
Caseínas/química , Caseínas/isolamento & purificação , Filtração/métodos , Géis/química , Micelas , Água/química , Animais , Cálcio/análise , Cálcio/química , Caseínas/metabolismo , Quimosina/metabolismo , Leite/química , Proteínas do Leite/análise , Proteínas do Leite/química , Proteínas do Leite/metabolismo , Fosfatos/análise , Fosfatos/química , Reologia , SolubilidadeRESUMO
Continuous precipitation is a new unit operation for the continuous capture of antibodies. The capture step is based on continuous precipitation with PEG6000 and Zn++ in a tubular reactor integrated with a two-stage continuous tangential flow filtration unit. The precipitate cannot be separated with centrifugation, because a highly compressed sediment results in poor resolubilization. We developed a new two-stage tangential flow microfiltration method, where part of the concentrated retentate of the first stage was directly fed to the second stage, together with the wash buffer. Thus, the precipitate was concentrated and washed in a continuous process. We obtained 97% antibody purity, a 95% process yield during continuous operation, and a fivefold reduction in pre-existing high-molecular-weight impurities. For other unit operations, surge tanks are often required, due to interruptions in the product mass flow out of the unit operation (e.g., the bind/elute mode in periodic counter-current chromatography). Our setup required no surge tanks; thus, it provided a truly continuous antibody capture operation with uninterrupted product mass flow. Continuous virus inactivation and other flow-through unit operations can be readily integrated downstream of the capture step to create truly continuous, integrated, downstream antibody processing without the need for hold tanks.
Assuntos
Anticorpos Monoclonais , Precipitação Fracionada , Imunoglobulina G , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Células CHO , Cricetulus , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Polietilenoglicóis/química , UltrafiltraçãoRESUMO
Conjugated vaccines prepared from the capsular polysaccharide of Streptococcus pneumoniae can provide immunization against invasive pneumococcal disease, meningitis, and otitis media. One of the critical steps in the production of these vaccines is the removal of free (unreacted) polysaccharides from the protein-polysaccharide conjugate. Experimental studies were performed to evaluate the effects of membrane pore size, filtrate flux, and solution conditions on the transmission of both the conjugate and free polysaccharide through different ultrafiltration membranes. Conjugate purification was done using diafiltration performed in a linearly-scalable tangential flow filtration cassette. More than 98% of the free polysaccharide was removed within a 5-diavolume diafiltration process, which is a significant improvement over previously reported results for purification of similar conjugated vaccines. These results clearly demonstrate the opportunities for using ultrafiltration/diafiltration for the final purification of conjugated vaccine products.
Assuntos
Cápsulas Bacterianas/química , Vacinas Bacterianas/isolamento & purificação , Polissacarídeos Bacterianos/isolamento & purificação , Ultrafiltração/métodos , Vacinas Conjugadas/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Vacinas Bacterianas/química , Porosidade , Streptococcus pneumoniae/química , Vacinas Conjugadas/químicaRESUMO
The availability of material for experimental studies is a key constraint in the development of full-scale bioprocesses. This is especially true for the later stages in a bioprocess sequence such as purification and formulation, where the product is at a relatively high concentration and traditional scale-down models can require significant volumes. Using a combination of critical flow regime analysis, bioprocess modelling, and experimentation, ultra scale-down (USD) methods can yield bioprocess information using only millilitre quantities before embarking on highly demanding full-scale studies. In this study the performance of a pilot-scale tangential flow filtration (TFF) system based on a membrane flat-sheet cassette using pumped flow was predicted by devising an USD device comprising a stirred cell using a rotating disc. The USD device operates with just 2.1 cm2 of membrane area and, for example, just 1.7 mL of feed for diafiltration studies. The novel features of the design involve optimisation of the disc location and the membrane configuration to yield an approximately uniform shear rate. This is characterised using computational fluid dynamics for a defined layer above the membrane surface. A pilot-scale TFF device operating at ~500-fold larger feed volume and membrane area was characterised in terms of the shear rate derived from flow rate-pressure drop relationships for the cassette. Good agreement was achieved between the USD and TFF devices for the flux and resistance values at equivalent average shear rates for a monoclonal antibody diafiltration stage.
Assuntos
Anticorpos Monoclonais , Ultrafiltração/instrumentação , Ultrafiltração/métodos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Simulação por Computador , Desenho de Equipamento , Membranas ArtificiaisRESUMO
Objective: To study the efficacy and safety of sustained low-efficiency diafiltration (SLEDf) versus hemodialysis (HD) for patients with wasp stings who developed stage III acute kidney injury (AKI). Methods: We retrospectively analyzed the clinical data of consecutive patients who developed AKI following wasp stings. All eligible patients received renal replacement therapy in combination with hemoperfusion. Thereafter, blood purification therapy and HD were performed with a volumetrically controlled machine and 1.7 m2 surface, Fresenius Polysulfone HD filter and SLEDf was undertaken with a volumetrically controlled machine and 1.3 m2 surface, Fresenius Polysulfone HD filter. Results: Forty patients developed stage III AKI following wasp stings, including 14 patients that received SLEDf and 26 patients underwent HD. Thirteen patients were aged less than 60 years and underwent HD (group I), 27 patients were aged at least 60 years, including 13 patients undergoing HD (group II) and 14 patients receiving SLEDf (group III). Groups I and II completed 150 and 162 sessions of HD, respectively, and group III completed 156 sessions of sustained low-efficiency blood purification therapy, including 50 sessions of SLEDf. The time to return to normal serum creatinine levels was 38.8 ± 2.7 days for group I, 47.2 ± 5.3 days for group II, and 39.2 ± 3.3 days for group III. A statistically significant difference was observed in time to normal serum creatinine levels among the three groups. Conclusion: Elderly wasp victims have more severe illness than younger wasp victims and SLEDf is safe and superior to HD in recovery of renal function of elderly wasp victims.
Assuntos
Injúria Renal Aguda/terapia , Mordeduras e Picadas de Insetos/complicações , Rim/fisiopatologia , Diálise Renal/métodos , Vespas , APACHE , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/fisiopatologia , Idoso , Animais , Creatinina/sangue , Feminino , Humanos , Mordeduras e Picadas de Insetos/diagnóstico , Mordeduras e Picadas de Insetos/imunologia , Rim/imunologia , Masculino , Pessoa de Meia-Idade , Recuperação de Função Fisiológica , Estudos Retrospectivos , Resultado do Tratamento , Venenos de Vespas/imunologiaRESUMO
Concentration of milk proteins by ultrafiltration (UF) and diafiltration (DF) processes during manufacturing of milk protein concentrate (MPC) powders alter their natural milk protein stabilization system. Increasing calcium and protein contents often leads to poor functional properties in MPC powders. The pH adjustment using disodium phosphate (DSP, Na2HPO4) and DF with 150 mM NaCl solution of UF retentate were hypothesized to produce desirable changes in various properties of resulted MPC powders. Addition of Na2HPO4 followed by homogenization; DF of 5 × UF retentate with 150 mM NaCl solution resulted in significant improvement in the dispersibility, wettability, flowability, solubility, heat stability, buffer index, emulsification and foaming and water and oil binding capacities of the MPC powders. The solubility of developed MPC powders was significantly higher than MPC-C powder in fresh as well as even after 90 days of storage at 25 ± 1 °C. Rheological behaviour of reconstituted MPC was best explained by Herschel Bulkley model. Scanning electron microscopy micrograph indicated that MPC powders were having smooth surfaced, intact and separate smaller particles compared to rough, larger, infused aggregates with dents in MPC-C. Technological interventions applied are easier to adopt, cost-effective and efficient in producing excellent quality MPC powders that may find applications in wide range of novel food formulations.
RESUMO
A number of groups have studied the application of continuous bioreactors and continuous chromatographic systems as part of efforts to develop an integrated continuous biomanufacturing process. The objective of this study was to examine the feasibility of using a countercurrent staged diafiltration process for continuous protein formulation with reduced buffer requirements. Experiments were performed using a polyclonal immunoglobulin (IgG) with Cadence™ Inline Concentrators. Model equations were developed for the product yield, impurity removal, and buffer requirements as a function of the number of stages and the stage conversion (ratio of permeate to feed flow rate). Data from a countercurrent two-stage system were in excellent agreement with model calculations, demonstrating the potential of using countercurrent staged diafiltration for protein formulation. Model simulations demonstrated the importance of the countercurrent staging on both the extent of buffer exchange and the amount of buffer required per kg of formulated product. The staged diafiltration process not only provides for continuous buffer exchange, it could also provide significant reductions in the number of pump passes while providing opportunities for reduced buffer requirements.
Assuntos
Biotecnologia/métodos , Filtração/métodos , Imunoglobulina G/isolamento & purificação , Soro/químicaRESUMO
The concept of continuous manufacturing has gained significant interest from the biopharmaceutical industry over the past several years. Benefits include increased manufacturing productivity, improved quality control, reduction in plant footprint, and more flexible management of facility capacity. There are several technologies currently available that enable continuous processing for chromatography and ultrafiltration. However, a single pass diafiltration design that meets the required small molecule clearance and has been integrated into a fully continuous monoclonal antibody purification process has not been previously published. Here, the theory and design of a 3-stage single pass diafiltration step is presented. Buffer exchange greater than 99.75% was experimentally demonstrated. Several critical design aspects were incorporated to minimize system complexity and reduce the buffer volume requirements. Lastly, single pass diafiltration was demonstrated in a pilot scale continuous process with uninterrupted flow from the bioreactor through the formulation step. This work illustrates the feasibility of incorporating a single pass diafiltration step into an end-to-end continuous protein purification process. This article is protected by copyright. All rights reserved.
RESUMO
The concept of continuous manufacturing has gained significant interest from the biopharmaceutical industry over the past several years. Benefits include increased manufacturing productivity, improved quality control, reduction in plant footprint, and more flexible management of facility capacity. There are several technologies currently available that enable continuous processing for chromatography and ultrafiltration. However, a single pass diafiltration design that meets the required small molecule clearance and has been integrated into a fully continuous monoclonal antibody purification process has not been previously published. Here, the theory and design of a 3-stage single pass diafiltration step is presented. Buffer exchange greater than 99.75% was experimentally demonstrated. Several critical design aspects were incorporated to minimize system complexity and reduce the buffer volume requirements. Lastly, single pass diafiltration was demonstrated in a pilot scale continuous process with uninterrupted flow from the bioreactor through the formulation step. This work illustrates the feasibility of incorporating a single pass diafiltration step into an end-to-end continuous protein purification process.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Biotecnologia/métodos , Filtração/métodos , Fatores Imunológicos/isolamento & purificação , Tecnologia Farmacêutica/métodos , Antígenos de HelmintosRESUMO
Macrophage activation syndrome (MAS) is a life-threating complication of systemic juvenile idiopathic arthritis (s-JIA). Steroid and cyclosporine (CsA) are effective for MAS, but, treatment for steroid- and CsA-resistant patients is still challenging. We report the case of steroid and CsA resistant s-JIA associated MAS misdiagnosed as Kawasaki disease (KD), who was successfully treated with the combination of plasma exchange (PE) and leukocytapheresis (LCAP) followed by plasma diafiltration (PDF). PE + LCAP effectively removed proinflammatory cytokines and reduced the number of peripheral white blood cells. Furthermore, PDF also removed proinflammatory cytokines as effectively as PE + LCAP. Early diagnosis of s-JIA is necessary to avoid developing MAS. The measurement of serum ferritin and IL-18 levels are useful for differentiating s-JIA from KD. Apheresis therapies are an alternative option to induce remission for severe patients with steroid- or CsA-resistant MAS.