Truly continuous low pH viral inactivation for biopharmaceutical process integration

Continuous virus inactivation (VI) has received little attention in the efforts to realize fully continuous biomanufacturing in the future. Implementation of continuous VI must assure a specific minimum incubation time, typically 60 min. To guarantee the minimum incubation time, we implemented a packed bed continuous viral inactivation reactor (CVIR) with narrow residence time distribution (RTD) for low pH incubation. We show that the RTD does not broaden significantly over a wide range of linear flow velocities—which highlights the flexibility and robustness of the design. Prolonged exposure to acidic pH has no impact on bed stability, assuring constant RTD throughout long term operation. The suitability of the packed bed CVIR for low pH inactivation is shown with two industry-standard model viruses, that is xenotropic murine leukemia virus and pseudorabies virus. Controls at neutral pH showed no system-induced VI. At low pH, significant VI is observed, even after only 15 min. Based on the low pH inactivation kinetics, the continuous process is equivalent to traditional batch operation. This study establishes a concept for continuous low pH inactivation and, together with previous reports, highlights the versatility of the packed bed reactor for continuous VI, regardless of the inactivation method.     

Continuous Solvent/Detergent Virus Inactivation Using a Packed‐Bed Reactor

Continuous virus inactivation (VI) remains one of the missing pieces while the biopharma industry moves toward continuous manufacturing. The challenges of adapting VI to the continuous operation are two‐fold: 1) achieving fluid homogeneity and 2) a narrow residence time distribution (RTD) for fluid incubation. To address these challenges, a dynamic active in‐line mixer and a packed‐bed continuous virus inactivation reactor (CVIR) are implemented, which act as a narrow RTD incubation chamber. The developed concept is applied using solvent/detergent (S/D) treatment for inactivation of two commonly used model viruses. The in‐line mixer is characterized and enables mixing of the viscous S/D chemicals to ±1.0% of the target concentration in a small dead volume. The reactor's RTD is characterized and additional control experiments confirm that the VI is due to the S/D action and not induced by system components. The CVIR setup achieves steady state rapidly before two reactor volumes and the logarithmic reduction values of the continuous inactivation process are identical to those obtained by the traditional batch operation. The packed‐bed reactor for continuous VI unites fully continuous processing with very low‐pressure drop and scalability.     

Removal of aqueous phenol using immobilized enzymes in a bench scale and pilot scale three-phase fluidized bed reactor

The main objective of this work was to investigate the removal of aqueous phenol using immobilized enzymes in both bench scale and pilot scale three-phase fluidized bed reactors. The enzyme used in this application was a fungal tyrosinase [E.C. 1.14.18.1] immobilized in a system of chitosan and alginate. The immobilization matrix consisted of a chitosan matrix cross-linked with glutaraldehyde with an aliginate-filled pore space. This support matrix showed superior mechanical properties along with retaining the unique adsorptive characteristics of the chitosan. Adsorption of the o-quinone product by the chitosan reduced tyrosinase inactivation that is normally observed for this enzyme under these conditions. This approach allowed reuse of the enzyme in repeated batch applications. For the bench scale reactor (1.2-l capacity) more than 92% of the phenol could be removed from the feed water using an immobilized enzyme volume of 18.5% and a residence time of the liquid phase of 150 min. Removal rates decreased with subsequent batch runs. For the pilot scale fluidized bed (60 l), 60% phenol removal was observed with an immobilized enzyme volume of 5% and a residence time of the liquid phase of 7 h. Removal decreased to 45% with a repeat batch run with the same immobilized enzyme.     

Leveraging flow mechanics to determine critical process and scaling parameters in a continuous viral inactivation reactor

A continuous viral inactivation (CVI) chamber has been designed to operate with acceptable residence time distribution (RTD) characteristics. However, altering the CVI's geometry and operation to accommodate the scale was not obvious. In this work, we elucidate the influence of Dean vortices and leverage the transition into the weak turbulent regime to establish relationships between input variables and process outputs. This study was targeted to understand and quantify the impact of viscosity, Dean number, internal diameter, and path length on the RTD. When the Dean number exceeds 70, radial mixing generated by the Dean vortices began to consistently alter the axial dispersive effects experienced by the pulse injection. Increasing to a Dean number of >100, the axial dispersive effects were dominated by the Dean vortices which allowed the calculation of the minimum and maximum residence time to be generated. This work provides a method to calculate operational solutions for a tubular incubation reactor in terms of path length, internal diameter, flow rate, and target minimum and maximum residence time specifications that assures both viral residence times while also establishing criteria to maximize product quality during continuous operation.     

Mixing characteristics and liquid circulation in a new multi-environment bioreactor

The theoretical and experimental aspects of the hydrodynamics and mixing in a new multi-environment bioreactor that uses the air-lift design were investigated. This study focused on the mixing characteristics, residence time distribution, liquid circulation between three zones of aerobic, microaerophilic and anoxic, and liquid displacement in the bioreactor at influent flow rates of 720–1,450 L/day and air flow rates of 15–45 L/min. The theoretical analysis of liquid displacement led to the estimation of the specific rate of liquid discharge from the bioreactor at any given influent flow rate, and the number of liquid circulations between various bioreactor zones before the discharge of a given quantity of wastewater. The ratio of mean residence time to the overall hydraulic retention time (t _m/HRT) decreased with the increase of air flow rate at any given influent flow rate, and approached unity at higher air flow rates. Mixing was characterized in terms of the axial dispersion coefficient and Bodenstein number, demonstrating a linear relationship with the superficial gas velocity. A correlation was developed between the Bodenstein number and the Froude number. The study of liquid circulation between the zones showed that less than 1.5 % of the circulating liquid escapes circulation at each cycle and flows towards the outer clarifier, while the percentage of escaped liquid decreases with increasing air flow rate at a given influent flow rate. The specific rate of liquid discharge from the bioreactor increased from 0.19 to 0.69 h^?1 with the increase of air and influent flow rates from 15 to 45 L/min and 500 to 1,450 L/day, respectively. Under the examined operating conditions, mixed liquor circulates between 364 and 1,698 times between the aerobic, microaerophilic and anoxic zones before 99 % of its original volume is replaced by the influent wastewater.     

Residence time distributions of gas flowing through rotating drum bioreactors

Residence time distribution studies of gas through a rotating drum bioreactor for solid-state fermentation were performed using carbon monoxide as a tracer gas. The exit concentration as a function of time differed considerably from profiles expected for plug flow, plug flow with axial dispersion, and continuous stirred tank reactor (CSTR) models. The data were then fitted by least-squares analysis to mathematical models describing a central plug flow region surrounded by either one dead region (a three-parameter model) or two dead regions (a five-parameter model). Model parameters were the dispersion coefficient in the central plug flow region, the volumes of the dead regions, and the exchange rates between the different regions. The superficial velocity of the gas through the reactor has a large effect on parameter values. Increased superficial velocity tends to decrease dead region volumes, interregion transfer rates, and axial dispersion. The significant deviation from CSTR, plug flow, and plug flow with axial dispersion of the residence time distribution of gas within small-scale reactors can lead to underestimation of the calculation of mass and heat transfer coefficients and hence has implications for reactor design and scale-up.     

Design,Construction, and Starting-up of an Anaerobic Reactor for the Stabilisation,Handling, and Disposal of Excess Biological Sludge Generated in a Wastewater Treatment Plant

Design, construction, and starting-up of an upflow anaerobic sludge blanket reactor was carried out. This system was proposed for excess sludge stabilisation, particularly that generated at an activated sludge wastewater treatment facility installed in a sugarcane mill. The upflow anaerobic sludge blanket (UASB) reactor built, had a working volume of 22.3 m³and a hydraulic residence time of 22 days. Methane production was at a maximum of 79% volume with an average of 60% for this treatment. For starting up the anaerobic reactor, a suitable inoculum from a neighboring plant was used. As the waste characteristics in both plants were different, an acclimation procedure was followed to achieve granulation. Control and stability of anaerobic reactions were monitored with alkalinity data, using the so-called ‘alfa alkalinity’ to try to keep its value at around 0.4. Once pseudosteady-state conditions were reached (chemical oxygen demand reduction and methane-rich biogas production within ±10 percent), the organic load was steadily increased up to feeding 100% excess sludge. The UASB reactor used to stabilise the excess biomass generated a sludge with a much lower volume than that originally fed. Its design ensured adequate hydraulic flow and biogas production with a high methane content. The bacteria were attached constituting spheres and very minor maintenance operations were required.     

Kinetics of Encapsulated Yeast Alcohol Dehydrogenase Dispersed in an Organic Solvent

Microcapsules dispersed in organic solvents provide a suitable environment for conducting enzyme reactions involving cofactors and hydrophobic substrates. Encapsulated YADH is active and stable in cyclohexane provided the pH is adjusted appropriately. Mass transfer does not influence batch reaction rates. Conversion in a fluidized-bed reactor containing encapsulated YADH/NAD⁺ and employing cyclohexane as the continuous phase depends strongly on residence time and inlet cinnamyl alcohol concentration. However, interpretation of these results is complicated by enzyme inactivation by the product, cinnamaldehyde, and interference from residual encapsulating agents.     

Optimization and modeling of lactose hydrolysis in a packed bed system using immobilized β-galactosidase from Aspergillus oryzae

This work studied the hydrolysis of lactose using β-galactosidase from Aspergillus oryzae immobilized with a combination of adsorption and glutaraldehyde cross-linking onto the ion exchange resin Duolite A568 as a carrier. A central composite design (CCD) was used to study the effects of lactose concentration and feed flow rate on the average hydrolysis reaction rate and lactose conversion in a fixed bed reactor operating continuously with an upflow at a temperature of 35 ± 1 °C. The optimal conditions for the average hydrolysis reaction rate and the lactose conversion included a lactose concentration of 50 g/L and a feed flow rate of 6 mL/min. The average reaction rate and conversion reached 2074 U and 65%, respectively. The immobilized enzyme activity was maintained during the 30 days of operation in a fixed bed reactor with a 0.3 mL/min feed flow rate of a 50 g/L lactose solution at room temperature. Feed flows ranging from 0.6 to 12 mL/min were used to determine the distribution of residence times and the kinetics of the fixed bed reactor. A non-ideal flow pattern with the formation of a bypass flow in the fixed bed reactor was identified. The conditions used for the kinetics study included a lactose solution concentration of 50 g/L at pH 4.5 and a temperature of 35 ± 1 °C. Kinetic models using a PFR and axial dispersion methods were used to describe the lactose hydrolysis in the fixed bed reactor, thus accounting for the competitive inhibition by galactose. To increase the lactose conversion, experiments were performed for two fixed bed reactors in series, operating in continuous duty with upflow, with the optimal conditions determined using the CCD for a fixed bed reactor. The total conversion for the two reactors in series was 82%.     

FLAVOR MODIFICATION OF MILK CHOCOLATE BY CONCHING IN A TWIN-SCREW, CO-ROTATING, CONTINUOUS MIXER

Short-time continuous conching of chocolate offers significant economic advantages over traditional long-time batch methods. High-shear continous mixers can affect conching in a matter of minutes as compared to hours for traditional systems. Milk chocolate conched in two twin-screw, co-rotating, continous mixers operating in series was compared by a variety of sensory methods to chocolate conched by a batch method. A significant difference (P < 0.05) in flavor was found between chocolates conched at 60C by the continuous and batch methods, yet there was no preference for either chocolate. Chocolate conched by the batch method (23 h at 60C) had stronger caramel flavor (P < .10) than chocolate conched continuously, but there were no significant differences (P < .10) in sweet, chocolate or milk flavor. In the continuous system, caramel flavor generally increased with conching temperature and residence time; although, at the highest temperature (95C) and the longest residence time (7.5 min) caramel flavor decreased. Increasing the temperature of continuous conching from 70 to 90C produced chocolate significantly (P < .10) more like chocolate conched in a batch system for 21.5 h at 60C.     

Kinetics of Encapsulated Yeast Alcohol Dehydrogenase Dispersed in an Organic Solvent

Microcapsules dispersed in organic solvents provide a suitable environment for conducting enzyme reactions involving cofactors and hydrophobic substrates. Encapsulated YADH is active and stable in cyclohexane provided the pH is adjusted appropriately. Mass transfer does not influence batch reaction rates. Conversion in a fluidized-bed reactor containing encapsulated YADH/NAD⁺ and employing cyclohexane as the continuous phase depends strongly on residence time and inlet cinnamyl alcohol concentration. However, interpretation of these results is complicated by enzyme inactivation by the product, cinnamaldehyde, and interference from residual encapsulating agents.     

Effect of mixing on the washout and steady-state performance of continuous cultures

The effects of mixing on the critical mean holding time for washout and the steady state performance of growth processes in continuous flow reactors are investigated. Macromixing, micromixing, and cell recycle arc considered. The tanks-in-series model composed of N completely mixed flow reactors, the dispersion model, the plug flow model, and a combined model composed of a plug flow reactor and a continuous stirred tank flow reactor connected in series arc used to represent the macro-mixing or residence time distribution. The extreme cases of micromixing, namely, complete segregation and maximum mixedness, as well as intermediate states of micromixing are investigated to determine their effects on washout and the occurence of multiple steady states. A technique for predicting the maximum mixedness washout condition from a knowledge of the residence time distribution is presented and used to determine the washout condition for the dispersion model under maximum mixedness conditions.     

An immobilized cell reactor with simultaneous product separation. II. Experimental reactor performance

The simultaneous separation of volatile fermentation products from product-inhibited fermentations can greatly increase the productivity of a bioreactor by reducing the product concentration in the bioreactor, as well as concentrating the product in an output stream free of cells, substrate, or other feed impurities. The Immobilized Cell Reactor-Separator (ICRS) consists of two column reactors: a cocurrent gas-liquid "enricher" followed by a countercurrent "stripper" The columns are four-phase tubular reactors consisting of (1) an inert gas phase, (2) the liquid fermentation broth, (3) the solid column internal packing, and (4) the immobilized biological catalyst or cells. The application of the ICRS to the ethanol-from-whey-lactose fermentation system has been investigated. Operation in the liquid continuous or bubble flow regime allows a high liquid holdup in the reactor and consequent long and controllable liquid residence time but results in a high gas phase pressure drop over the length of the reactor and low gas flow rates. Operation in the gas continuous regime gives high gas flow rates and low pressure drop but also results in short liquid residence time and incomplete column wetting at low liquid loading rates using conventional gas-liquid column packings. Using cells absorbed to conventional ceramic column packing (0.25-in. Intalox saddles), it was found that a good reaction could be obtained in the liquid continuous mode, but little separation, while in the gas continuous mode there was little reaction but good separation. Using cells sorbed to an absorbant matrix allowed operation in the gas continuous regime with a liquid holdup of up to 30% of the total reactor volume. Good reaction rates and product separation were obtained using this matrix. High reaction rates were obtained due to high density cell loading in the reactor. A dry cell density of up to 92 g/L reactor was obtained in the enricher. The enricher ethanol productivity ranged from 50 to 160 g/L h while the stripper productivity varied from 0 to 32 g/L h at different feed rates and concentrations. A separation efficiency of as high as 98% was obtained from the system.     

Optimization and mathematical modeling of the transtubular bioreactor for the production of monoclonal antibodies from a hybridoma cell line

This report describes the use of a transtubular bioreactor to study the relative effects of diffusion versus perfusion of medium on antibody production by a hybridoma cell line. The study was performed with a high-density cell culture maintained in a serum-free, low-protein medium for 77 days. It was determined that the reactor possessed a macro-mixing pattern residence time distribution similar to a continuous stirred tank reactor (CSTR). However, due to the arrangement of the medium lines in the reactor, the flow patterns for nutrient distribution consist of largely independent medium path lengths ranging from short to long. When operated with cyclic, reversing, transtubular medium flow, some regions of the reactor (with short residence times) are more accessible to medium than others (with long residence times). From this standpoint, the reactor can be divided into three regions: a captive volume, which consists of medium primarily delivered via diffusion; a lapped volume, which provides nutrients through unilateral convection; and a swept volume, which operates through bilateral convection. The relative sizes of these three volumes were modified experimentally by changing the period over which the direction of medium flow was reversed from 15 min (larger captive volume) to 9 h (larger swept volume). The results suggest that antibody concentration increases as the size of the diffusion-limited (captive) volume is increased to a maximum at around 30 min with a sharp decrease thereafter. As reflected by changes in measured consumption of glucose and production of lactate, no significant difference in cellular metabolism occurred as the reactor was moved between these different states. These results indicate that the mode of operation of the transtubular bioreactor may influence antibody productivity under serum-free, low-protein conditions with minimal effects on cellular metabolism.     

Hydrolysis of liquefied corn starch in a membrane reactor

The objective of this study was to develop a continuous hydrolysis process for the enzymatic saccharification of liquefied corn starch using a membrane reactor. A residence time distribution study confirmed that the membrane reactor could be modeled as a simple continuous stirred tank reactor (CSTR). Kinetic studies indicated that the continuous reactor operated in the first-order region with respect to substrate concentration at substrate concentrations greater than 200 g/L. At a residence time of 1 h and an enzyme concentration of 1 g/L, the maximum reaction velocity (V(m)) was 3.86 g glucose/L min and the apparent Michaelis constant (K(m) (')) was 562 g/L. The K(m) (') value for the continuous reactor was 2-7 times greater than that obtained in a batch reactor.Kinetic data were fit to a model based on the Michaelis-Menten rate expression and the design equation for a CSTR. Application of the model at low reactor space times was successful. At space times of 6 min or less, the model predicted the reactor's performance reasonably well. Additional work on the detection and quantitation of reversion products formed by glucoamylase is required. Isolation, detection, and quantitation of reversion products by HPLC was difficult. Detailed analysis on the formation of these reversion products could lead to better reactor designs in the future.     

Characterization of a pilot plant airlift tower loop bioreactor: II. Evaluation of global mixing properties of the gas phase during yeast cultivation

Saccharomyces cerevisiae was cultivated in a 4-m(3) pilot plant airlift tower loop reactor with a draft tube in batch and continuous operations and for comparison in a laboratory airlift tower loop reactor of 0.08 m(3) volume. The reactors were characterized during and after the cultivation by measuring the distributions of the residence times of the gas phase with pseudostochastic tracer signals and mass spectrometer and by evaluating the mixing in the liquid phase with a pulse-shaped volatile tracer signal and mass spectrometer as a detector. The mean residence times and the intensities of the axial mixing in the riser and downcomer, the circulation times of the gas phase, and the fraction of the recirculated gas phase were evaluated and compared.     

Virus study for continuous low pH viral inactivation inside a coiled flow inverter

Continuous processing for the production of monoclonal antibodies (mAb) gains more and more importance. Several solutions exist for all the necessary production steps, leading to the possibility to build fully continuous processes. Low pH viral inactivation is a part of the standard platform process for mAb production. Consequently, Klutz et al. introduced the coiled flow inverter (CFI) as a tool for continuous low pH viral inactivation. Besides theoretical calculations of viral reduction, no viral clearance study has been presented so far. In addition, the validation of continuous viral clearance is often neglected in the already existing studies for continuous processing. This study shows in detail the development and execution of a virus study for continuous low pH viral inactivation inside a CFI. The concept presented is also valid for adaptation to other continuous viral clearance steps. The development of this concept includes the technical rationale for an experimental setup, a valid spiking procedure, and finally a sampling method. The experimental results shown represent a viral study using xenotropic murine leukemia virus as a model virus. Two different protein A (ProtA) chromatography setups with varying pH levels were tested. In addition, one of these setups was tested against a batch experiment utilizing the same process material. The results show that sufficient low pH viral inactivation (decadic logarithm reduction value >4) was achieved in all experiments. Complete viral inactivation took place within the first 14.5 min for both continuous studies and the batch study, hence showing similar results. This study therefore represents a successful virus study concept and experiment for a continuous viral inactivation step. Moreover, it was shown that the transfer from batch results to the continuous process is possible. This is accomplished by the narrow residence time distribution of the CFI, showing how close the setup approaches the ideal plug flow and with that batch operation.     

Performance test of a 6-stage continuous reactor for palm methyl ester production

Effects of residence time (3-12 min), stirrer speed (0-800 rpm), and NaOH concentration (0.25-1.0 wt% of oil) on the production performance of the designed 6-stage continuous reactor (2.272 l) for transesterification of palm oil were investigated at molar ratio of methanol to oil of 6:1 and temperature of 60 degrees C. Higher stirrer speed increased the reaction rate up to an appropriate speed but excessive stirrer speed decreased the reaction rate. Inappropriate stirrer speed runs dramatically decreased the production capacity of the reactor. Higher NaOH concentration significantly increased reaction rate and production capacity of the reactor. The reactor had a residence time distribution equivalent to 5.98 ideal CSTRs in series and a production performance equivalent to a plug flow reactor. At NaOH of 1.0 wt% of oil, the reactor could produce saleable biodiesel within residence time of 6 min in which a production capacity was 17.3 l/h and a power consumption of stirrer was 0.6 kW/m(3).     

Investigation on macrokinetics of heterogeneous process of monosaccharide isomerization using non-growing cells of a glucoisomerase producer <Emphasis Type="Italic">Arthrobacter nicotianae</Emphasis> immobilized inside SiO<Subscript>2</Subscript>-xerogel

Macrokinetic peculiarites of heterogeneous process of monosaccharide (glucose/fructose) isomerization using biocatalysts prepared by incorporation of non-growing cells of a glucose isomerase-producing strain Arthobacter nicotianae inside SiO₂-xerogel have been investigated. It was shown that the process proceeds in kinetic regime without diffusion limitation and biocatalyst activities at 60 and 75°C were 19 and 32 U/g, respectively. Time of equilibrium in the reaction of monosaccharide isomerization was a function of starting (“triggering”) glucose isomerase activity in a unit of reaction volume. When the activity exceeds 10 U/ml, equilibrium equimolar mixture of glucose and fructose was produced within a few hours. It was established that a continuous process carried out in a plug-flow packed-bed reactor is more efficient than a batch process accompanied with recycling, first of all, to significant improvement of operation stability of the designed biocatalysts. Under model conditions of industrial heterogeneous process of producing glucose-fructose syrups, the half-life time of inactivation of the biocatalysts was more than 500 h at (65 ± 5)°C.     

Enhancing operational stability and exhibition of enzyme activity by removing water in the immobilized lipase‐catalyzed production of erythorbyl laurate

Erythorbyl laurate was continuously synthesized by esterification in a packed‐bed enzyme reactor with immobilized lipase from Candida antarctica. Response surface methodology based on a five‐level three‐factor central composite design was adopted to optimize conditions for the enzymatic esterification. The reaction variables, such as reaction temperature (10–70°C), substrate molar ratio ([lauric acid]/[erythorbic acid], 5–15), and residence time (8–40 min) were evaluated and their optimum conditions were found to be 56.2°C, 14.3, and 24.2 min, respectively. Under the optimum conditions, the molar conversion yield was 83.4%, which was not significantly different (P < 0.05) from the value predicted (84.4%). Especially, continuous water removal by adsorption on an ion‐exchange resin in a packed‐bed enzyme reactor improved operational stability, resulting in prolongation of half‐life (2.02 times longer compared to the control without water‐removal system). Furthermore, in the case of batch‐type reactor, it exhibited significant increase in initial velocity of molar conversion from 1.58% to 2.04%/min. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:882–889, 2013