Multistage continuous countercurrent diafiltration for formulation of monoclonal antibodies

There is growing interest in the development of fully integrated and continuous biomanufacturing processes for the production of monoclonal antibody products. A recent study has demonstrated the feasibility of using a two-stage countercurrent diafiltration (DF) process for continuous product formulation, but this system did not provide sufficient levels of buffer exchange for most applications. The objective of this study was to design and test a three-stage countercurrent DF system that could achieve at least 99.9% buffer exchange over 24 hr of continuous operation. Experimental data were obtained using concentrated solutions of human immunoglobulin G as a model protein, with the extent of vitamin B₁₂ removal used to track the extent of DF. Pall Cadence™ inline concentrators with Delta 30 kD regenerated cellulose membranes were used in the three stages to achieve high conversion in a single pass. The three-stage system showed stable operation with >99.9% vitamin B₁₂ removal and a minimal increase in pressure over the full 24 hr. Modules were effectively cleaned using sodium hydroxide, with nearly complete recovery of water permeability. A simple economic analysis was presented that accounts for the trade-offs between quantity of buffer used and membrane costs for this type of countercurrent staged DF process. The results provide important insights to the design and operation of a continuous process for antibody formulation.     

Countercurrent staged diafiltration for formulation of high value proteins

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.     

Enzymatic synthesis of glucuronides using lipophilic hollow fiber membranes

A lipophilic hollow fiber membrane preparation was used for the enzymatic glucuronidation of lipophilic aromatic compounds. A crude solubilized microsomal enzyme preparation was circulated on the external side of the lipophilic membrane while the phenol containing buffer solution was circulated through the internal side of the hollow fiber membrane. Phenols, which accumulate in and penetrate the lipophilic membrane, were converted by UDP-glucuronyltransferase to the corresponding glucuronides. During this process the lipophilic compounds are converted to hydrophilic substances, which are not able to rediffuse through the lipophilic membrane into the donor side of the hollow fiber module. The produced glucuronide is separated by means of a coupled dialysis with a module of hydrophilic surface (cellulose acetate), while the enzyme protein is retained.On the stripping side of the dialysing module the glucuronide can be separated by solid phase extraction (Lichroprep RP-18) while a continuous substitution of cofactor into this compartment is possible. UDPGA follows its own concentration gradient and migrates into the enzymatic mixture, where it is utilized. This new technique using hollow fiber modules offers completely new possibilities for long-term high-capacity, highly specific glucuronidation of phenolic compounds. Fields of application are not only the economical production of special glucuronides, but also the specific elimination of phenols from waste fluids or from serum and blood of patients.For the production of glucuronides by this technique the use of highly purified enzymes is not essential. Cheap crude enzyme preparations are quite adequate for an optimal reaction. Using a crude enzyme preparation with a specific batch activity of 13 nMol/min per mg of protein, the activity in the reactor system was observed to be 4.6 nMol/min of 2-naphtol glucuronide formed per mg of protein. This corresponds to 3.6 nMol/h of product formed per mg of protein per cm² of hollow fiber surface.Using a membrane surface of 0.5 m² the production of 18 mMol of glucuronide per h and mg protein can be achieved.     

Urocanic acid production using whole cells immobilized in a hollow fiber reactor

The feasibility of using hollow fiber membrane dialyzers (C-DAK) for immobilization of microbial whole cells was investigated. The cells are located on the shell side of the dialyzer, while substrates and products are free to diffuse across the hollow fiber membranes. The biochemical reaction studied was the conversion of L -histidine to urocanic acid and catalyzed by L -histidine ammonia-lyase. C-DAK dialyzers containing a heat-treated suspension of Pseudomonas fluorescens ATCC 11299b (with L -histidine ammonia–lyase activity) were incorporated into constant volume recycle reactor systems for continuous product formation. A simple model successfully correlated the data and predicted performance. It was found that the reaction was not likely to be diffusion limited, and such a cell immobilization scheme is convenient and workable for continuous production of biochemicals.     

Development of a generic transient transfection process at 100 L scale

We have developed a generic transient transfection process at 100 L scale, using HEK293-EBNA cells and PEI as the transfection reagent for the production of recombinant IgG. The process, including large-scale plasmid preparation, expression at bioreactor scale, capture, purification and, if necessary, endotoxin removal allows reproducible production of more than 0.5 g IgG for in vitro and in vivo studies. We compared the performance of two HEK cell lines, investigated the effect of conditioned medium, optimized the DNA:PEI ratio and implemented a feed strategy to prolong the culture time to increase product yield. The transient transfection protocol developed enables a closed process from seeding culture to protein capture. The challenge of performing a medium exchange before transfection at large scale is solved by applying a continuous centrifugation step between the seeding bioreactor and the production bioreactor. After 7–8 days the harvest and capture is performed in a one-step operation using a Streamline expanded bed chromatography system. Following a polishing step the purified antibody is transferred to the final formulation buffer. The method has shown to be reproducible at 10, 50, and 100 L scale expressing between 5 and 8 mg L⁻¹ IgG.     

Sludge exchange process on two serial CSTRs anaerobic digestions: process failure and recovery

The sludge exchange process using two anaerobic digesters (CSTRs) in series was investigated under the mesophilic condition (36-38 °C). At first, the digesting sludge of the CSTRs in series with different TVFA/alkalinity ratios was tested in the laboratory by mixing the digesting sludge of two CSTRs from 6.5% to 50% based on volume. The sludge exchange test was then performed using the same CSTRs under batch and continuous processes. The change in the TVFA/alkalinity ratio was found to be linear with the digesting sludge exchange volume. The CSTR of TVFA/alkalinity ratio 1.970 recovered completely failed within 11 days for the batch process and the CSTR of TVFA/alkalinity ratio 1.514 within 3 weeks for the continuous feeding process at a sludge exchange volume of 13%. The reactor operation was stable when the TVFA/alkalinity ratio was less than 1.0 and when the TVFA concentration was lower than 10,000 mg L⁻¹.     

Strategies for the depyrogenation of contaminated immunoglobulin G solutions by histidine-immobilized hollow fiber membrane

The depyrogenation of different IgG solutions using the histidine-linked hollow fiber membrane developed in our laboratory is presented here. Three strategies for endotoxin (ET) removal were investigated according to the immobilized histidine's ability to bind different immunoglobulins: (1) ET removal from 1 mg/ml non histidine-binding mouse monoclonal IgG₁ (MabCD4) solution was achieved in the presence of acetate buffer (pH 5.0) without any protein loss. (2) For contaminated human IgG, combined adsorption of ET and IgG in the presence of MOPS or Tris buffer was tested, followed by differential elution using increasing salt concentrations. This attempt was not successful since ET were quantitatively found in the IgG elution fraction. (3) Alternatively, it was proposed to adsorb selectively ET in the presence of acetate buffer (pH 5.0) under non binding conditions for human IgG. Human IgG could then be purified if necessary with the same membrane in the presence of MOPS buffer (pH 6.5). With a 1 m² histidine-PEVA module under these operating conditions, it is estimated that the depyrogenation of 3 1 of 1 mg/ml IgG (human or murine) solution containing 80 EU/ml of ET should be possible.     

A continuous single organic phase process for the lipase catalyzed synthesis of peroxy acids increases productivity

The design of an optimal process is particularly crucial when the reactants deactivate the biocatalyst. The reaction cascades of the chemo‐enzymatic epoxidation where the intermediate peroxy acid is produced by an enzyme are still limited by enzyme inhibition and deactivation by hydrogen peroxide. To avoid additional effects caused by interfaces (aq/org) and to reduce the process limiting deactivation by the substrate hydrogen peroxide, a single‐phase concept was applied in a fed‐batch and a continuous process (stirred tank), without the commonly applied addition of a carrier solvent. The synthesis of peroxyoctanoic acid catalyzed by Candida antarctica lipase B was chosen as the model reaction. Here, the feasibility of this biocatalytic reaction in a single‐phase system was shown for the first time. The work shows the economic superiority of the continuous process compared to the fed‐batch process. Employing the fed‐batch process reaction rates up to 36 mmol h^?1 per gram_cat, and a maximum yield of 96 % was achieved, but activity dropped quickly. In contrast, continuous operation can maintain long‐term enzyme activity. For the first time, the continuous enzymatic reaction could be performed for 55 h without any loss of activity and with a space‐time yield of 154 mmol L^?1 h^?1, which is three times higher than in the fed‐batch process. The higher catalytic productivity compared to the fed‐batch process (34 vs. 18 g_Prod g^?1_cat) shows the increased enzyme stability in the continuous process.     

Life cycle assessment of vitamin D3 synthesis: from batch to photo-high p,T

Purpose

Novel process windows allow the development of faster, flexible, and greener processes. Therefore, novel process windows were applied to develop a greener process for the synthesis of vitamin D₃. In this study the environmental impacts of several batch pathways to obtain vitamin D₃ are benchmarked against the continuous microflow process, where novel process windows such as high temperature and pressure were applied. To evaluate the environmental impact of these processes, life cycle assessments were conducted.

Methods

A new process concept was developed to optimize and simplify the synthesis of crystalline vitamin D₃. This process was conducted in microflow by combining UV photoirradiation and high-p,T (photo-high-p,T) processing. Microreactors allow a high photon flux and enable the harsh conditions, respectively. The process was coupled with an integrated continuous crystallization, and its feasibility has been proven and reported before. The potential environmental impacts were assessed from a cradle-to-gate perspective. Both processes, continuous and batch, were modeled in Aspen Plus using foreground data from the experimental continuous setup, and background data from different patents. The assessment was performed in the software Umberto NXL LCA using the ReCiPe Midpoint 2008 method.

Results and discussion

The continuous process has a significantly lower environmental impact than the batch processes. This lower impact is largely due to the fact that fewer amounts of material, particularly solvents, are used. Moreover, the continuous process is faster and has fewer steps, i.e., process-simplified. Among the industrial processes, the synthesis conducted in isopropanol has the lowest environmental impact, although, even in this case, the impact is between 20 and 30 times higher—depending on the conditions—compared with the continuous process. When the batch process is conducted in benzene, the worst environmental impact is obtained. Finally, recycle of the solvent for the best batch case was assessed. This improved the batch process to make it comparable with the continuous process.

Conclusions

The continuous production of vitamin D₃ leads to an interesting alternative to the industrial process. Continuous manufacturing of vitamin D₃ is faster, requires fewer steps, and uses less solvents compared with the industrial synthesis. However, although the environmental impact of this continuous process is already lower than that of the batch processes, the continuous process can still benefit from further optimization, particularly the introduction of a recycle loops for the solvents methyl tert-butyl ether and acetonitrile.

     

Countercurrent tangential chromatography for large-scale protein purification

Recent advances in cell culture technology have created significant pressure on the downstream purification process, leading to a "downstream bottleneck" in the production of recombinant therapeutic proteins for the treatment of cancer, genetic disorders, and cardiovascular disease. Countercurrent tangential chromatography overcomes many of the limitations of conventional column chromatography by having the resin (in the form of a slurry) flow through a series of static mixers and hollow fiber membrane modules. The buffers used in the binding, washing, and elution steps flow countercurrent to the resin, enabling high-resolution separations while reducing the amount of buffer needed for protein purification. The results obtained in this study provide the first experimental demonstration of the feasibility of using countercurrent tangential chromatography for the separation of a model protein mixture containing bovine serum albumin and myoglobin using a commercially available anion exchange resin. Batch uptake/desorption experiments were used in combination with critical flux data for the hollow fiber filters to design the countercurrent tangential chromatography system. A two-stage batch separation yielded the purified target protein at >99% purity with 94% recovery. The results clearly demonstrate the potential of using countercurrent tangential chromatography for the large-scale purification of therapeutic proteins.     

High-performance countercurrent membrane purification for host cell protein removal from monoclonal antibody products

The transition to continuous biomanufacturing has led to renewed interest in alternative approaches for downstream processing of monoclonal antibody (mAb) products. In this study, we examined the potential of using high-performance countercurrent membrane purification (HPCMP) for the removal of host cell proteins (HCPs) derived from Chinese Hamster Ovary cells in the purification of a mAb. Initial studies used several model proteins to identify appropriate operating conditions for the hollow fiber membrane modules. HPCMP was then used for mAb purification, with mAb yield >95% and more than 100-fold reduction in HCP. Stable operation was maintained for 48 h for feeds that were first prefiltered through the 3M^TM Harvest RC chromatographic clarifier to remove DNA and other foulants. In addition, the Process Mass Intensity for HPCMP can be much less than that for alternative HCP separation processes. These results highlight the potential of using HPCMP as part of a fully continuous mAb production process.     

Behavior of human immunoglobulin G adsorption onto immobilized Cu(II) affinity hollow‐fiber membranes

Iminodiacetic acid (IDA) and tris(2‐aminoethyl)amine (TREN) chelating ligands were immobilized on poly(ethylene vinyl alcohol) (PEVA) hollow‐fiber membranes after activation with epichlorohydrin or butanediol diglycidyl ether (bisoxirane). The affinity membranes complexed with Cu(II) were evaluated for adsorption of human immunoglobulin G (IgG). The effects of matrix activation and buffer system on adsorption of IgG were studied. Isotherms of batch IgG adsorption onto finely cut membranes showed that neither of the chelates, IDA‐Cu(II) or TREN‐Cu(II), had a Langmuirean behavior with negative cooperativity for IgG binding. A comparison of equilibrium and dynamic maximum capacities showed that the dynamic capacity for a mini‐cartridge in a cross‐flow filtration mode (52.5 and 298.4 mg g^?1 dry weight for PEVA‐TREN‐Cu(II) and PEVA‐IDA‐Cu(II), respectively) was somewhat higher than the equilibrium capacity (9.2 and 73.3 mg g^?1 dry weight for PEVA‐TREN‐Cu(II) and PEVA‐IDA‐Cu(II), respectively). When mini‐cartridges were used, the dynamic adsorption capacity of IDA‐Cu(II) was the same for both mini‐cartridge and agarose gel. Copyright © 2013 John Wiley & Sons, Ltd.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Monoclonal antibody production in hollow fiber bioreactors using serum-free medium

Murine hybridoma cells that produce monoclonal antibody directed against human fibronectin have been cultured in VITAFIBER II and VITAFIBER V hollow fiber bioreactors using defined, serum-free WRC 935 medium. During a two-week growth period, following inoculation of the bioreactors, the cells proliferated to an extent where the bioreactor was filled with cultured cells. Using a 5 sq. ft. VITAFIBER V bioreactor, over 15 grams of antibody were produced during the 40 days of the experiment. This antibody was greater than 95% IgG. During the production period, this packed mass of cells produced 579 +/- 15 mg IgG per day. Because the medium is formulated for air equilibration and high cell densities, WRC 935 medium is especially useful for production of gram quantities of monoclonal antibodies using continuous feed hollow fiber bioreactor cell culture systems.     

Continuous Hydrolysis of Olive Oil by Lipase in Microporous Hydrophobic Hollow Fiber Bioreactor

Olive oil was hydrolyzed continuously at 40ºC by Candida cylindracea lipase in a small hollow fiber bioreactor (total area of hollow fibers was about 0.11 m²) in which the hollow fibers were made of microporous polypropylene. The lipase could be adsorbed easily onto oil-impregnated hollow fibers from its aqueous solution. The continuous feedings of olive oil inside the hollow fibers and of the buffer solution containing 18% glycerol as a stabilizer outside the hollow fibers were started after the enzyme-glycerol solution was removed from the bioreactor and the buffer-glycerol solution was added. An unvaried half life of 14 days of the adsorbed enzyme was observed when increasing amounts of the enzyme (1.0~5.0mg/ml) were put in, but its half-life was lowered to 6 days when the amount of the added enzyme was less (0.05mg/ml). Free enzymes in the enzyme solutions with and without 18% glycerol retained their initial activities equally for at least 3 months at temperatures below 4ºC. This suggests the feasibility of reuse of the enzyme-glycerol solution that was used in the preceding adsorption procedure after the solution was stored and supplemented with fresh enzyme. It was demonstrated by three successive cleanings and continuous hydrolyses that the used hollow fibers were regenerable. The productivity number was 0.81 mg-(unit)^-1 -h^-1, which was 26 times as great as that of the gel-entrapped lipase.     

Use of hollow fiber systems for rapid and direct scale up of antibody production from hybridoma cell lines cultured in CL-1000 flasks using BD Cell MAb medium

The combination of BD Cell MAb medium with the CL-1000 flask is increasingly being used to generate a few hundred milligram of antibody for early stage research projects. Cells are inoculated at 2 million per ml, and the antibody is harvested after 15 days or when the antibody concentration reaches above 10 mg ml⁻¹, whichever comes first. Currently, there is no means to scale up beyond this production level using this technology. In this study, we evaluated hollow fiber technology as the scale up alternative. The hollow fiber system was run in batch mode to mimic the method used for the CL-1000 with BD MAb medium. The FL-NS murine hybridoma cell line was simultaneously inoculated at 2 million cells per ml in a CL-1000 and the Maximizer hollow fiber bioreactor system, a 21-fold theoretical scale up over the CL-1000. The Maximizer produced 23-fold more antibody, very close to the expected theoretical amount. However, production was complete after 9 days in the Maximizer, while the CL-1000 required the full 15 days for production. In summary, these results demonstrate successful scale up of antibody production from the CL-1000 to a hollow fiber system. This revised version was published online in July 2006 with corrections to the Cover Date.     

High density culture of hybridoma cells in a dual hollow fiber bioreactor

Summary Hybridoma cells were cultured for two months in the dual hollow fiber bioreactor (DHFBR) which had been successfully used for high cell density cultures of various microbial cells. In batch suspension culture the concentration of monoclonal antibody (Mab) against human Chorionic Gonadotropin (hCG) and the cell density of Alps 25-3 hybridoma cells were obtained in 30 μg/mL and 2.35×10⁶ cells/mL, respectively. The continuous culture with DHFBR produced Mab of 100–130 μg/mL for 30 days and the estimated cell density in the extracapillary space of DHFBR was 1.87×10⁸ cells/mL based on the antibody production rate. The productivity of Mab was 205 mg/day per litre of the total reactor volume while that of the batch suspension culture was only 10 mg/L day.     

Performance of electron acceptors in catholyte of a two-chambered microbial fuel cell using anion exchange membrane

The performance of the cathodic electron acceptors (CEA) used in the two-chambered microbial fuel cell (MFC) was in the following order: potassium permanganate (1.11 V; 116.2 mW/m²) > potassium persulfate (1.10 V; 101.7 mW/m²) > potassium dichromate, K₂Cr₂O₇ (0.76 V; 45.9 mW/m²) > potassium ferricyanide (0.78 V; 40.6 mW/m²). Different operational parameters were considered to find out the performance of the MFC like initial pH in aqueous solutions, concentrations of the electron acceptors, phosphate buffer and aeration. Potassium persulfate was found to be more suitable out of the four electron acceptors which had a higher open circuit potential (OCP) but sustained the voltage for a much longer period than permanganate. Chemical oxygen demand (COD) reduction of 59% was achieved using 10 mM persulfate in a batch process. RALEX™ AEM-PES, an anion exchange membrane (AEM), performed better in terms of power density and OCP in comparison to Nafion®117 Cation Exchange Membrane (CEM).     

Ethanol production in a hollow fiber bioreactor using Saccharomyces cerevisiae

Summary A new approach for continuous production of ethanol was developed using a Hollow fiber fermentor (HFF). Saccharomyces cerevisiae cells were packed into the shell-side of a hollow fiber module. Using 100 g/l glucose in the feed gave an optimum ethanol productivity, based on total HFF volume, of 40 g ethanol/l/h at a dilution rate of 3.0 h^-1. Under these conditions, glucose utilization was 30%. However, at 85% glucose utilization the productivity was 10 g ethanol/l/h. This compares to batch fermentor productivity of 2.1 g ethanol/l/h at 100% glucose utilization.     

Optimization of growth and production of toxins by three dinoflagellates in photobioreactor cultures

A bioreactor system for biotoxin production was appraised against traditional methods of growing dinoflagellate cultures. In an optimised bioreactor culture (5.4?L) operated in batch mode, growth of Karenia selliformis was more efficient than in 15-L bulk carboy culture in terms of growth rate (μ?=?0.07?day^?1 versus 0.05?day^?1) and growth maximum (G _max, 169.10⁶ versus 41.10⁶ cells L^?1). Maximal gymnodimine concentration (1200?μg L^?1) in bioreactor culture was 8-fold higher than in bulk carboy culture, and the yield per cell (pg cell^?1) was 2-fold higher. Similarly the bioreactor batch culture of Alexandrium ostenfeldii performed more efficiently than carboy cultures in terms of growth rate (1.6-fold higher), growth maximum (15-fold higher) and desmethyl C spirolide (SPX-desMe-C) yield (5-fold higher [μg L^?1], though the yield [pg cell^?1basis] was lower). When bioreactor cultures of K. selliformis were operated in continuous mode, the yield of gymnodimine was substantially higher than a carboy or the bioreactor run in batch mode to growth max (793?μg day^?1 over 58?days in continuous culture was achieved versus an average of 60?μg day^?1 [carboy over 40?days] or 249?μg day^?1 [batch mode] over 26?days). Likewise in continuous bioreactor cultures of A. ostenfeldii run over 25?days, the yield of SPX-desMe-C (29?μg day^?1) was substantially higher than in same cultures run in batch mode or carboys (10.2 day^?1 and 7.7?μg day^?1 respectively). Similarly 5.4?L bioreactor batch cultures of K. brevisulcata reached 3.8-fold higher cell densities than carboy cultures, and when operated in continuous mode, the brevisulcatic acids were more efficiently produced than in batch culture (12?μg day^?1 versus 7?μg day^?1). When the bioreactor system was upscaled to 52?L, the maximum cell densities and toxin yields of K. brevisulcata cultures were somewhat less than those achieved in the smaller reactor, which was attributed to reduced light penetration.