Hemoglobin‐based oxygen carrier and convection enhanced oxygen transport in a hollow fiber bioreactor

A mathematical model was developed to study O₂ transport in a convection enhanced hepatic hollow fiber (HF) bioreactor, with hemoglobin‐based O₂ carriers (HBOCs) present in the flowing cell culture media stream of the HF lumen. In this study, four HBOCs were evaluated: PEG‐conjugated human hemoglobin (MP4), human hemoglobin (hHb), bovine hemoglobin (BvHb) and polymerized bovine hemoglobin (PolyBvHb). In addition, two types of convective flow in the HF extra capillary space (ECS) were considered in this study. Starling flow naturally occurs when both of the ECS ports are closed. If one of the ECS ports is open, forced convective flow through the ECS will occur due to the imposed pressure difference between the lumen and ECS. This type of flow is referred to as cross‐flow in this work, since some of the fluid entering the HF lumen will pass across the HF membrane and exit via the open ECS port. In this work, we can predict the dissolved O₂ concentration profile as well as the O₂ transport flux in an individual HF of the bioreactor by solving the coupled momentum and mass transport equations. Our results show that supplementation of the cell culture media with HBOCs can dramatically enhance O₂ transport to the ECS (containing hepatocytes) and lead to the formation of an in vivo‐like O₂ spectrum for the optimal culture of hepatocytes. However, both Starling flow and cross‐flow have a very limited effect on O₂ transport in the ECS. Taken together, this work represents a novel predictive tool that can be used to design or analyze HF bioreactors that expose cultured cells to defined overall concentrations and gradients of O₂. Biotechnol. Bioeng. 2009;102: 1603–1612. © 2008 Wiley Periodicals, Inc.     

Perfluorocarbon facilitated O2 transport in a hepatic hollow fiber bioreactor

A mathematical model describing O₂ transport in a hepatic hollow fiber (HF) bioreactor supplemented with perfluorocarbons (PFCs) in the circulating cell culture media was developed to explore the potential of PFCs in properly oxygenating a bioartificial liver assist device (BLAD). The 2‐dimensional model is based on the geometry of a commercial HF bioreactor operated under steady‐state conditions. The O₂ transport model considers fluid motion of a homogeneous mixture of cell culture media and PFCs, and mass transport of dissolved O₂ in a single HF. Each HF consists of three distinct regions: (1) the lumen (conducts the homogeneous mixture of cell culture media and PFCs), (2) the membrane (physically separates the lumen from the extracapillary space (ECS), and (3) the ECS (hepatic cells reside in this compartment). In a single HF, dissolved O₂ is predominantly transported in the lumen via convection in the axial direction and via diffusion in the radial direction through the membrane and ECS. The resulting transport equations are solved using the finite element method. The calculated O₂ transfer flux showed that supplementation of the cell culture media with PFCs can significantly enhance O₂ transport to the ECS of the HF when compared with a control with no PFC supplementation. Moreover, the O₂ distribution and subsequent analysis of ECS zonation demonstrate that limited in vivo‐like O₂ gradients can be recapitulated with proper selection of the operational settings of the HF bioreactor. Taken together, this model can also be used to optimize the operating conditions for future BLAD development that aim to fully recapitulate the liver's varied functions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Simulation of oxygen carrier mediated oxygen transport to C3A hepatoma cells housed within a hollow fiber bioreactor

A priori knowledge of the dissolved oxygen (O2) concentration profile within a hepatic hollow fiber (HF) bioreactor is important in developing an effective bioartificial liver assist device (BLAD). O2 provision is limiting within HF bioreactors and we hypothesize that supplementing a hepatic HF bioreactor's circulating media with bovine red blood cells (bRBCs), which function as an O2 carrier, will improve oxygenation. The dissolved O2 concentration profile within a single HF (lumen, membrane, and representative extra capillary space (ECS)) was modeled with the finite element method, and compared to experimentally measured data obtained on an actual HF bioreactor with the same dimensions housing C3A hepatoma cells. Our results (experimental and modeling) indicate bRBC supplementation of the circulating media leads to an increase in O2 consumed by C3A cells. Under certain experimental conditions (pO2,IN) = 95 mmHg, Q = 8.30 mL/min), the addition of bRBCs at 5% of the average in vivo human red blood cell concentration (% hRBC) results in approximately 50% increase in the O2 consumption rate (OCR). By simply adjusting the operating conditions (pO2,IN) = 25 mmHg, Q = 1.77 mL/min) and increasing bRBC concentration to 25% hRBC the OCR increase is approximately 10-fold. However, the improved O2 concentration profile experienced by the C3A cells could not duplicate the full range of in vivo O2 tensions (25-70 mmHg) typically experienced within the liver sinusoid with this particular HF bioreactor. Nonetheless, we demonstrate that the O2 transport model accurately predicts O2 consumption within a HF bioreactor, thus setting up the modeling framework for improving the design of future hepatic HF bioreactors.     

Next-generation polymerized human hemoglobins in hepatic bioreactor simulations

Hepatic hollow fiber (HF) bioreactors can be used to provide temporary support to patients experiencing liver failure. Before being connected to the patient's circulation, cells in the bioreactor must be exposed to a range of physiological O₂ concentrations as observed in the liver sinusoid to ensure proper performance. This zonation in cellular oxygenation promotes differences in hepatocyte phenotype and may better approximate the performance of a real liver within the bioreactor. Polymerized human hemoglobin (PolyhHb) locked in the tense quaternary state (T-state) has the potential to both supply and regulate O₂ transport to cultured hepatocytes in the bioreactor due to its low O₂ affinity. In this study, T-state PolyhHb production and purification processes were optimized to minimize the concentration of low-molecular-weight PolyhHb species in solution. Deconvolution of size-exclusion chromatography spectra was performed to calculate the distribution of polymeric Hb species in the final product. Fluid flow and mass transport within a single fiber of a hepatic HF bioreactor was computationally modeled with finite element methods to simulate the effects of employing T-state PolyhHb to facilitate O₂ transport in a hepatic bioreactor system. Optimal bioreactor performance was defined as having a combined hypoxic and hyperoxic volume fraction in the extracapillary space of less than 0.05 where multiple zones were observed. The Damköhler number and Sherwood number had strong inverse relationships at each cell density and fiber thickness combination. These results suggest that targeting a specific Damköhler number may be beneficial for optimal hepatic HF bioreactor operation.     

Erythrocytic ATP release in the presence of modified cell-free hemoglobin

The red blood cell (RBC) has been proposed as an O₂ sensor through a direct link between the desaturation of intracellular hemoglobin (Hb) and ATP release, leading to vasodilation. We hypothesized that the addition of cell-free Hb to the extracellular space provides a supplementary O₂ source that reduces RBC desaturation and, consequently, ATP release. In this study, the saturation of RBC suspensions was lowered by additions of deoxygenated hemoglobin-based oxygen carrier (HBOC) and then assayed for extracellular ATP. When an acellular human Hb intramolecularly cross-linked between α subunits (ααHb, p50 = 33 mmHg) was added to the red cell suspension, ATP production was significantly less than that in the presence of a lower p50 HBOC (Hb cross-linked between β subunits, ββHb, p50 = 8 mmHg). These results provide a potential mechanism for the O₂ affinity of HBOCs to interfere with a vasodilatory signal.     

Effect of the interfacial surfactant layer on oxygen transfer through the oil/water phase boundary in perfluorocarbon emulsions

The effect of interfacial surfactant molecules on oxygen transfer through oil/water phase boundary has been studied in FlurO(2) (TM) emulsions, i.e., perfluorocarbon (PFC) emulsions developed as oxygen carriers in cell culture. Measurements of oxygen permeability were made with a polarographic oxygen electrode in pure PFCs and in emulsions with various PFC volume fractions. Comparison of the experimental results with the theoretically derived values of relative oxygen permeability clearly indicates that the mass transfer resistance caused by the interfacial surfactant layer in PFC emulsions is insignificant. Therefore, oxygen dissolved in the enclosed PFC phase is readily available to cells growing in the aqueous media and FlurO(2) emulsions with very fine emulsion particles (< 0.2 mum) can be used to effectively enhance gas/liquid interfacial oxygen transfer in bioreactors. The inadequacy in describing mass transfer in heterogeneous systems, such as the PFC emulsions, by conventional concentration-based oxygen diffusion coefficients has also been discussed.     

Use of perfluorocarbons to enhance the performance of perfused three‐dimensional hepatic cultures

Bioartificial liver devices (BALs) are extracorporeal systems designed to temporarily bridge patients until a suitable donated liver is available for transplantation and also have value for pharmaceutical testing applications. Yet critical issues exist that limit the functional performance of their current designs. One of these concerns scale up issues connected to oxygen (O₂) delivery to the cells housed within their three‐dimensional (3D) configurations, and its consequences to device performance. As primary blood substitute candidates with extraordinarily high O₂ capacity, perfluorocarbons (PFCs) offer hope as one strategy for addressing the O₂ delivery issue encountered when scaling up the tissue space of current BAL designs. This study utilizes a PFC‐based second‐generation O₂ carrier OXYCYTE®, as an additive to regular nutrient medium, for augmenting O₂ delivery in a customized 3D tissue assembly system. The results demonstrate that the addition of PFCs significantly increases the O₂ capacity of regular medium and that net cytochrome P450 activity levels are considerably increased under flow in PFC‐treated systems, as compared to controls. This work thus clarifies the benefits of using PFCs to enhance the functional performance of 3D liver systems. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:718–726, 2013     

Characterization of oxygen transfer in a membrane-aerated hollow-fibre bioreactor using modified microcoaxial needle electrodes

In this work the volumetric O₂ transfer coefficient (OTC) through a membrane of a miniaturized hollow-fibre bioreactor was measured by the use of modified O₂ microaxial needle electrodes. Before measurment, available electrodes were modified by inserting and gluering them in thin galss capillaries to avoid damage. No differences in the behaviour of the electrodes occurred in comparison to the non-modified electrodes. These modified electrodes allowed O₂ partial pressure measurement in the 0.8-mm-high extracapillary space (ECS) of the bioreactor with high sensitivity and reliability. O₂ measurements were carried out the two ports of the ECS at different insertion depths. The results of the measurements showed a homogenous O₂ supply during variation of the radial co-coordinate of the electrode. In addition to these results, an increase in the local supply in the direction of medium flow was observed . The calculated mean OTC (47–63 h^–1) gave extremely improved O₂ transfer due to membrane aeration compared to conventional hallow-fibre systems and other bioreactors used in animal-cell culture technology. The improved OTC and the small ECS volume (4.3 ml) makes this culture system suitable for the cultivation of primary cells with tissue-like densities.     

Targeted oxygen delivery within hepatic hollow fiber bioreactors via supplementation of hemoglobin-based oxygen carriers

Hepatic hollow fiber bioreactors are considered a promising class of bioartificial liver assist device (BLAD). Unfortunately, limited oxygen (O(2)) transport to hepatocytes within this device hinders further development. Hepatocytes in vivo (in the liver sinusoid) experience a wide range of oxygen tensions (pO(2) = 25-70 mmHg), which is important for development of proper differentiated function (zonation). Previously, we observed that bovine red blood cell (bRBC) supplementation of the circulating media stream enhanced oxygenation of cultured C3A hepatoma cells compared to a culture with no O(2) carrier (Gordon, J.; Palmer, A. F. Artif. Cells, BloodSubstitutes, Biotechnol. 2006, 33 (3), 297-306). Despite this success, the cells were not exposed to the desired in vivo O(2) spectrum (Sullivan, J.; Gordon, J.; Palmer, A. Biotechnol. Bioeng. 2006, 93 (2) 306-317). We hypothesize that altering the kinetics of O(2) binding/release to/from hemoglobin-based O(2) carriers (HBOCs) could potentially target O(2) delivery to cell cultures. High P(50) (low O(2) affinity) HBOCs preferentially targeted O(2) delivery at high inlet pO(2) values. Conversely, low P(50) (high O(2) affinity) HBOCs targeted O(2) delivery at low inlet pO(2) values. Additionally, inlet pO(2), flow rate, and HBOC concentration were varied to find optimal bioreactor operating conditions. Our results demonstrate that HBOCs can enhance O(2) delivery to cultured hepatocytes, while exposing them to in vivo-like O(2) tensions, which is critical to create a fully functional BLAD.     

Theoretical analysis of effects of blood substitute affinity and cooperativity on organ oxygen transport.

Hemoglobin-based O(2) carriers (HBOCs), which are developed as an alternative to blood transfusion, provide O(2) delivery. At present, there is no model to predict the O(2) transport for a red blood cell-HBOC mixture on a whole organ basis. On the basis of the first principles of mass balance, a model of O(2) transport for an organ was derived to calculate venous Po(2) (Pv(O(2))) for a given inlet arterial Po(2) (Pa(O(2))), blood flow, and oxygen consumption. The model was validated by using several in vivo animal studies on HBOC administration for a wide range of HBOC oxygen-binding parameters and predicted Pv(O(2)) for various Pa(O(2)) in the same species. The model was also used to predict the effect of HBOC affinity and cooperativity on Pv(O(2)) for humans. The results indicate that Pv(O(2)) can be increased at a constant blood flow-to-oxygen consumption ratio by reducing the affinity of HBOC for normoxia and mild hypoxia; however, a high-affinity HBOC would be more efficient in maintaining higher Pv(O(2)) for severe hypoxia (Pa(O(2)) < 40 Torr).     

Hemoglobin-based O2 carrier O2 affinity and capillary inlet pO2 are important factors that influence O2 transport in a capillary

Hemopure (Biopure; Cambridge, MA) and PolyHeme (Northfield Laboratories; Evanston, IL) are two acellular hemoglobin-based O2 carriers (HBOCs) currently in phase III clinical trials for use as red blood cell substitutes. The most common adverse side effect that these HBOCs exhibit is increased vasoconstriction. Autoregulatory theory has been presented as a possible explanation for this physiological effect, where it is hypothesized that low-affinity HBOCs over-deliver O2 to tissues surrounding arterioles, thereby eliciting vasoconstriction. In this paper, we wanted to investigate HBOC oxygenation of tissue surrounding a capillary, which is the smallest element of the circulatory system. An a priori model has been developed in which the performance of mixtures of acellular HBOCs (synthesized by our group and others) and human red blood cells (hRBCs) has been simulated using a Krogh tissue cylinder model (KTCM) comprising a capillary surrounded by a capillary membrane and skeletal muscle tissue in cylindrical coordinates with specified tissue O2 consumption rates and Michaelis-Menten kinetics. In this study, the total hemoglobin (hRBCs and HBOCs) concentration was kept constant. The HBOCs studied possessed O2 affinities that were higher and lower compared to hRBCs (P50's spanned 5-55 mmHg), and the equilibrium binding/release of oxygen to/from the HBOCs was modeled using the Adair equation. At normoxic inlet pO2's, there was no correlation between O2 flux out of the capillary and the O2 affinity of the HBOC. However, a correlation was found between the average pO2 tension in the capillary and the O2 affinity of the HBOC. Additionally, we studied the change in the O2 equilibrium curve of HBOCs with different O2 affinities over a wide range of inlet pO2's and found that changing the inlet pO2 greatly affected which HBOC, having a unique O2 affinity, best delivered O2 to the surrounding tissue. The analysis of oxygen transport presented could lead to a better prediction of which acellular HBOC is best suited for a specific transfusion application that many times depends on the capillary inlet pO2 tension.     

Tangential flow filtration facilitated fractionation and PEGylation of low and high-molecular weight polymerized hemoglobins and their biophysical properties

Various types of hemoglobin (Hb)-based oxygen carriers (HBOCs) have been developed as red blood cell substitutes for treating blood loss when blood is not available. Among those HBOCs, glutaraldehyde polymerized Hbs have attracted significant attention due to their facile synthetic route, and ability to expand the blood volume and deliver oxygen. Hemopure®, Oxyglobin®, and PolyHeme® are the most well-known commercially developed glutaraldehyde polymerized Hbs. Unfortunately, only Oxyglobin® was approved by the FDA for veterinary use in the United States, while Hemopure® and PolyHeme® failed phase III clinical trials due to their ability to extravasate from the blood volume into the tissue space which facilitated nitric oxide scavenging and tissue deposition of iron, which elicited vasoconstriction, hypertension and oxidative tissue injury. Fortunately, conjugation of poly (ethylene glycol) (PEG) on the surface of Hb is capable of reducing the vasoactivity of Hb by creating a hydration layer surrounding the Hb molecule, which increases its hydrodynamic diameter and reduces tissue extravasation. Several commercial PEGylated Hbs (MP4®, Sanguinate®, Euro-PEG-Hb) have been developed for clinical use with a longer circulatory half-life and improved safety compared to Hb. However, all of these commercial products exhibited relatively high oxygen affinity compared to Hb, which limited their clinical use. To dually address the limitations of prior generations of polymerized and PEGylated Hbs, this current study describes the PEGylation of polymerized bovine Hb (PEG-PolybHb) in both the tense (T) and relaxed (R) quaternary state via thiol-maleimide chemistry to produce an HBOC with low or high oxygen affinity. The biophysical properties of PEG-PolybHb were measured and compared with those of commercial polymerized and PEGylated HBOCs. T-state PEG-PolybHb possessed higher hydrodynamic volume and P₅₀ than previous generations of commercial PEGylated Hbs. Both T- and R-state PEG-PolybHb exhibited significantly lower haptoglobin binding rates than the precursor PolybHb, indicating potentially reduced clearance by CD163 + monocytes and macrophages. Thus, T-state PEG-PolybHb is expected to function as a promising HBOC due to its low oxygen affinity and enhanced stealth properties afforded by the PEG hydration shell.     

Development of a scale-up strategy for Chinese hamster ovary cell culture processes using the kLa ratio as a direct indicator of gas stripping conditions

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO₂) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO₂ was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the k_La values of O₂ (k_La[O₂]) and CO₂ [k_La(CO₂)] in the SS bioreactors as a scale-up factor for dCO₂ reduction. The k_La ratios [k_La(CO₂)/k_La(O₂)] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the k_La ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO₂ trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This k_La ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The k_La ratios for the SUBs were evaluated and conditions that provided k_La ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the k_La ratio is a powerful scale-up factor useful to control dCO₂ during fed-batch cultures.     

Modified CelliGen-packed bed bioreactors for hybridoma cell cultures

This study describes two packed bed bioreactor configurations which were used to culture a mouse-mouse hybridoma cell line (ATCC HB-57) which produces an IgG₁ monoclonal antibody. The first configuration consists of a packed column which is continuously perfused by recirculating oxygenated media through the column. In the second configuration, the packed bed is contained within a stationary basket which is suspended in the vessel of a CelliGen^™ bioreactor. In this configuration, recirculation of the oxygenated media is provided by the CelliGen Cell Lift impeller. Both configurations are packed with disk carriers made from a non-woven polyester fabric. During the steady-state phase of continuous operation, a cell density of 10⁸ cells per cm³ of bed volume was obtained in both bioreactor configurations. The high levels of productivity (0.5 gram MAb per 1 of packed bed per day) obtained in these systems demonstrates that the culture conditions achieved in these packed bed bioreactors are excellent for the continuous propagation of hybridomas using media which contains low levels (1 %) of serum as well as serum-free media. These packed bed bioreactors allow good control of pH, dissolved oxygen and temperature. The media flows evenly over the cells and produces very low shear forces. These systems are easy to set up and operate for prolonged periods of time. The potential for scale-up using Fibra-cel carriers is enhanced due to the low pressure drop and low mass transfer resistance, which creates high void fraction approaching 90% in the packed bed.     

A symbiotic gas exchange between bioreactors enhances microalgal biomass and lipid productivities: taking advantage of complementary nutritional modes

This paper describes the association of two bioreactors: one photoautotrophic and the other heterotrophic, connected by the gas phase and allowing an exchange of O₂ and CO₂ gases between them, benefiting from a symbiotic effect. The association of two bioreactors was proposed with the aim of improving the microalgae oil productivity for biodiesel production. The outlet gas flow from the autotrophic (O₂ enriched) bioreactor was used as the inlet gas flow for the heterotrophic bioreactor. In parallel, the outlet gas flow from another heterotrophic (CO₂ enriched) bioreactor was used as the inlet gas flow for the autotrophic bioreactor. Aside from using the air supplied from the auto- and hetero-trophic bioreactors as controls, one mixotrophic bioreactor was also studied and used as a model, for its claimed advantage of CO₂ and organic carbon being simultaneously assimilated. The microalga Chlorella protothecoides was chosen as a model due to its ability to grow under different nutritional modes (auto, hetero, and mixotrophic), and its ability to attain a high biomass productivity and lipid content, suitable for biodiesel production. The comparison between heterotrophic, autotrophic, and mixotrophic Chlorella protothecoides growth for lipid production revealed that heterotrophic growth achieved the highest biomass productivity and lipid content (>22%), and furthermore showed that these lipids had the most suitable fatty acid profile in order to produce high quality biodiesel. Both associations showed a higher biomass productivity (10–20%), when comparing the two separately operated bioreactors (controls) which occurred on the fourth day. A more remarkable result would have been seen if in actuality the two bioreactors had been inter-connected in a closed loop. The biomass productivity gain would have been 30% and the lipid productivity gain would have been 100%, as seen by comparing the productivities of the symbiotic assemblage with the sum of the two bioreactors operating separately (controls). These results show an advantage of the symbiotic bioreactors association towards a cost-effective microalgal biodiesel production.     

Numerical simulation of oxygen delivery to muscle tissue in the presence of hemoglobin-based oxygen carriers

This work represents a culmination of research on oxygen transport to muscle tissue, which takes into account oxygen transport due to convection, diffusion, and the kinetics of simultaneous reactions between oxygen and hemoglobin and myoglobin. The effect of adding hemoglobin-based oxygen carriers (HBOCs) to the plasma layer of blood in a single capillary surrounded by muscle tissue based on the geometry of the Krogh tissue cylinder is examined for a range of HBOC oxygen affinity, HBOC concentration, capillary inlet oxygen tension (pO(2)), and hematocrit. The full capillary length of the hamster retractor muscle was modeled under resting (V(max) = 1.57 x 10(-4) mLO(2) mL(-1) s(-1), cell velocity (v(c)) = 0.015 cm/s) and working (V(max) = 1.57 x 10(-3) mLO(2) mL(-1) s(-1), v(c) = 0.075 cm/s) conditions. Two spacings between the red blood cell (RBC) and the capillary wall were examined, corresponding to a capillary with and without an endothelial surface layer. Simulations led to the following conclusions, which lend physiological insight into oxygen transport to muscle tissue in the presence of HBOCs: (1) The reaction kinetics between oxygen and myoglobin in the tissue region, oxygen and HBOCs in the plasma, and oxygen and RBCs in the capillary lumen should not be neglected. (2) Simulation results yielded new insight into possible mechanisms of oxygen transport in the presence of HBOCs. (3) HBOCs may act as a source or sink for oxygen in the capillary and may compete with RBCs for oxygen. (4) HBOCs return oxygen delivery to muscle tissue to normal for varying degrees of hypoxia (inlet capillary pO(2) < 30 mmHg) and anemia (hematocrit < 46%) for the hamster model.     

A strategy to determine operating parameters in tissue engineering hollow fiber bioreactors

The development of tissue engineering hollow fiber bioreactors (HFB) requires the optimal design of the geometry and operation parameters of the system. This article provides a strategy for specifying operating conditions for the system based on mathematical models of oxygen delivery to the cell population. Analytical and numerical solutions of these models are developed based on Michaelis–Menten kinetics. Depending on the minimum oxygen concentration required to culture a functional cell population, together with the oxygen uptake kinetics, the strategy dictates the model needed to describe mass transport so that the operating conditions can be defined. If c_min ≫ K_m we capture oxygen uptake using zero‐order kinetics and proceed analytically. This enables operating equations to be developed that allow the user to choose the medium flow rate, lumen length, and ECS depth to provide a prescribed value of c_min. When , we use numerical techniques to solve full Michaelis–Menten kinetics and present operating data for the bioreactor. The strategy presented utilizes both analytical and numerical approaches and can be applied to any cell type with known oxygen transport properties and uptake kinetics. Biotechnol. Bioeng. 2011; 108:1450–1461. © 2011 Wiley Periodicals, Inc.     

A practical approach in bioreactor scale‐up and process transfer using a combination of constant P/V and vvm as the criterion

Bioreactor scale‐up is a critical step in the production of therapeutic proteins such as monoclonal antibodies (MAbs). With the scale‐up criterion such as similar power input per volume or O₂ volumetric mass transfer coefficient ( ), adequate oxygen supply and cell growth can be largely achieved. However, CO₂ stripping in the growth phase is often inadequate. This could cascade down to increased base addition and osmolality, as well as residual lactate increase and compromised production and product quality. Here we describe a practical approach in bioreactor scale‐up and process transfer, where bioreactor information may be limited. We evaluated the sparger and (CO₂ volumetric mass transfer coefficient) from a range of bioreactor scales (3–2,000 L) with different spargers. Results demonstrated that for oxygen is not an issue when scaling from small‐scale to large‐scale bioreactors at the same gas flow rate per reactor volume (vvm). Results also showed that sparging CO₂ stripping, , is dominated by the gas throughput. As a result, a combination of a minimum constant vvm air or N₂ flow with a similar specific power was used as the general scale‐up criterion. An equation was developed to determine the minimum vvm required for removing CO₂ produced from cell respiration. We demonstrated the effectiveness of using such scale‐up criterion with five MAb projects exhibiting different cell growth and metabolic characteristics, scaled from 3 to 2,000 L bioreactors across four sites. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1146–1159, 2017     

Polynitroxyl hemoglobin: a pharmacokinetic study of covalently bound nitroxides to hemoglobin platforms

Adding antioxidant activities to hemoglobin-based oxygen carriers (HBOCs) represents a means of reducing cell-free hemoglobin-mediated oxidative cascades. We have covalently bound nitroxides, a class of antioxidant enzyme mimetics, to HBOCs. The objectives of this study were (1) to evaluate the pharmacokinetic (PK) effects of administering nitroxide covalently bound to HBOCs compared to those of free nitroxide coadministered with HBOCs and (2) to elucidate the effects of differing molecular weight HBOCs on the PK of bound nitroxide in a conscious guinea pig model of 25% blood exchange transfusion. Two HBOC platforms were used, intramolecular cross-linked hemoglobin (XLHb) and dextran polymerized/conjugated XLHb (PolyHb). Polynitroxylation was achieved by reacting 4-(2-bromoacetamido)-2,2,6,6,-tetramethylpiperidine-1-oxyl with XLHb or PolyHb to form polynitroxylated XLHb and polynitroxylated PolyHb, respectively, whereas a physical mixture of XLHb or PolyHb with 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl was prepared to reflect a molar equivalence to HBOC-bound nitroxide. Plasma concentrations of two redox states, nitroxide and hydroxylamine, were determined by electron paramagnetic resonance spectroscopy. Results are presented to illustrate the influence of covalent labeling and HBOC molecular weight on nitroxide PK. The therapeutic potential of polynitroxylation of HBOCs as it relates to observations from the current and previously reported studies is discussed.     

Comparison of growth properties of carrot hairy root in various bioreactors

Summary Growth properties of carrot hairy root cells in various bioreactors were investigated. A turbine-blade reactor and an immobilized rotating drum reactor were found to be advantageous for the hairy root culture because of a high oxygen transfer coefficient (k in L a). After 30 days of culture, 10 g/l of dry hairy root cells were obtained in both bioreactors and maximum growth rates (V _m ) were found to be 0.63 and 0.61 g/l per day for the turbine-blade reactor and immobilized rotating drum reactor, respectively. Specific growth rates () at various cultivation times were observed to be linearly proportional to X/k _l a for both bioreactor configurations where X is the cell concentration. The estimated specific oxygen uptake rate of 0.34 mmol O₂/g dry cells per hour compares fairly well with an experimental value of 0.3.