Tangential flow filtration of hemoglobin

Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS‐PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P₅₀) and cooperativity coefficient (n) were regressed from the measured oxygen‐RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC‐MS was used to measure the molecular weight of the alpha (α) and beta (β) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the α and β globin chains agreed well with the calculated theoretical mass of the α‐ and β‐ globin chains. Taken together, our results demonstrate that HPLC‐grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb‐based oxygen carriers. © 2008 American Institute of Chemical Engineers, 2009     

Functional comparison of hemoglobin purified by different methods and their biophysical implications

Hemoglobin (Hb) that is purified from red blood cells (RBCs) is commonly subjected to harsh processing conditions, such as high temperatures and extensive column separation, which may damage the Hb by altering the heme prosthetic group and/or the Hb protein structure. In this study, bovine and human Hb purified by tangential flow filtration (TFF) was compared to commercial preparations of human Hb (Hemosol, Inc., Toronto, Canada) and bovine Hb (Biopure, Inc., Cambridge, MA). Purified Hbs were characterized by measuring their overall purity (SDS–PAGE, SEC, and ESI‐MS), susceptibility to oxidation (k_ox), responses to physiological conditions (pH, [Cl^?], [IHP], and T), and ligand binding kinetics (O₂, NO, and CO). All Hbs evaluated possessed comparable biophysical properties, however, a small amount of catalase was detected in the TFF‐purified Hbs that reduced the rate of autoxidation. Mass changes observed by mass spectrometry suggest that structural alterations may be introduced into Hbs that are purified by extensive chromatographic separations. These results demonstrate that TFF is a suitable process for the purification of Hb from RBCs with a quality equivalent to that of commercial Hb preparations that employ more extensive purification strategies. This work also shows that TFF can yield highly pure Hb which can be used for Hb‐based O₂ carrier synthesis. Biotechnol. Bioeng. 2010; 106: 76–85. © 2010 Wiley Periodicals, Inc.     

A PEGylated bovine hemoglobin as a potent hemoglobin‐based oxygen carrier

Hemoglobin (Hb)‐based oxygen carriers (HBOCs) have been used as blood substitutes in surgery medicine and oxygen therapeutics for ischemic stroke. As a potent HBOC, the PEGylated Hb has received much attention for its oxygen delivery and plasma expanding ability. Two PEGylated Hbs, Euro‐Hb, and MP4 have been developed for clinical trials, using human adult hemoglobin (HbA) as the original substrate. However, HbA was obtained from outdated human blood and its quantity available from this source may not be sufficient for mass production of PEGylated HbA. In contrast, bovine Hb (bHb) has no quantity constraints for its ample resource. Thus, bHb is of potential to function as an alternative substrate to obtain a PEGylated bHb (bHb‐PEG). bHb‐PEG was prepared under the same reaction condition as HbA‐PEG, using maleimide chemistry. The structural, functional, solution and physiological properties of bHb‐PEG were determined and compared with those of HbA‐PEG. bHb‐PEG showed higher hydrodynamic volume, colloidal osmotic pressure, viscosity and P₅₀ than HbA‐PEG. The high P₅₀ of bHb can partially compensate the PEGylation‐induced perturbation in the R to T state transition of HbA. bHb‐PEG was non‐vasoactive and could efficiently recover the mean arterial pressure of mice suffering from hemorrhagic shock. Thus, bHb‐PEG is expected to function as a potent HBOC for its high oxygen delivery and strong plasma expanding ability. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:252–260, 2017     

Comprehensive characterization of tense and relaxed quaternary state glutaraldehyde polymerized bovine hemoglobin as a function of cross-link density

Previously, our lab developed high molecular weight (MW) tense (T) quaternary state glutaraldehyde polymerized bovine hemoglobins (PolybHbs) that exhibited reduced vasoactivity in several small animal models. In this study, we prepared PolybHb in the T and relaxed (R) quaternary state with ultrahigh MW (>500 kDa) with varying cross-link densities, and investigated the effect of MW on key biophysical properties (i.e., O₂ affinity, cooperativity (Hill) coefficient, hydrodynamic diameter, polydispersity, polymer composition, viscosity, gaseous ligand-binding kinetics, auto-oxidation, and haptoglobin [Hp]-binding kinetics). To further optimize current PolybHb synthesis and purification protocols, we performed a comprehensive meta-data analysis to evaluate correlations between procedural parameters (i.e., cross-linker:bovine hemoglobin (bHb) molar ratio, gas-liquid exchange time, temperature during sodium dithionite addition, and number of diafiltration cycles) and the biophysical properties of both T- and R-state PolybHbs. Our results showed that, the duration of the fast-step auto-oxidation phase of R-state PolybHb increased with decreasing glutaraldehyde:bHb molar ratio. Additionally, T-state PolybHbs exhibited significantly higher bimolecular rate constants for binding to Hp and unimolecular O₂ offloading rate constants compared to R-state PolybHbs. The methemoglobin (metHb) level in the final product was insensitive to the molar ratio of glutaraldehyde to bHb for all PolybHbs. During tangential flow filtration processing of the final product, 14 diafiltration cycles was found to yield the lowest metHb level.     

Theoretical analysis of excipient concentrations during the final ultrafiltration/diafiltration step of therapeutic antibody

Diafiltration of a protein solution into a new buffer is a common final step in biopharmaceutical manufacturing. However, the excipient concentrations in the retentate are not always equal to their corresponding concentrations in the new buffer (diafiltration buffer). This phenomenon was observed repeatedly during diafiltration of different therapeutic monoclonal antibodies in which the concentrations of histidine and either sorbitol or sucrose (depending on which was chosen for the diafiltration buffer) in the retentate were lower than in the diafiltration buffer. Experimental studies and theoretical analyses of the ultrafiltration/diafiltration (UF/DF) step were carried out to determine the primary causes of the phenomenon and to develop a mathematical model capable of predicting retentate excipient concentrations. The analyses showed that retentate histidine concentration was low primarily because of repulsive charge interactions between positively‐charged histidine molecules and positively‐charged protein molecules, and that volume exclusion effects were secondary for like‐charged molecules. The positively‐charged protein molecules generate an electrical potential that cause an uneven distribution of charged histidine molecules. This interaction was used to construct a mathematical model based on the Poisson‐Boltzmann equation. The model successfully predicted the final histidine concentration in the diafiltered product (retentate) from the UF/DF development and production runs, with good agreement across a wide range of protein and histidine concentrations for four therapeutic monoclonal antibodies. The concentrations of uncharged excipients (sorbitol or sucrose) were also successfully predicted using previously established models, with volume exclusion identified as the primary cause of differences in uncharged excipient concentrations in the retentate and diafiltration buffer. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Synthesis, biophysical properties, and oxygenation potential of variable molecular weight glutaraldehyde-polymerized bovine hemoglobins with low and high oxygen affinity

In a recent study, ultrahigh molecular weight (Mw ) glutaraldehyde-polymerized bovine hemoglobins (PolybHbs) were synthesized with low O2 affinity and exhibited no vasoactivity and a slight degree of hypertension in a 10% top-load model.(1) In this work, we systematically investigated the effect of varying the glutaraldehyde to hemoglobin (G:Hb) molar ratio on the biophysical properties of PolybHb polymerized in either the low or high O2 affinity state. Our results showed that the Mw of the resulting PolybHbs increased with increasing G:Hb molar ratio. For low O2 affinity PolybHbs, increasing the G:Hb molar ratio reduced the O2 affinity and CO association rate constants in comparison to bovine hemoglobin (bHb). In contrast for high O2 affinity PolybHbs, increasing the G:Hb molar ratio led to increased O2 affinity and significantly increased the CO association rate constants compared to unmodified bHb and low O2 affinity PolybHbs. The methemoglobin level and NO dioxygenation rate constants were insensitive to the G:Hb molar ratio. However, all PolybHbs displayed higher viscosities compared to unmodified bHb and whole blood, which also increased with increasing G:Hb molar ratio. In contrast, the colloid osmotic pressure of PolybHbs decreased with increasing G:Hb molar ratio. To preliminarily evaluate the ability of low and high O2 affinity PolybHbs to potentially oxygenate tissues in vivo, an O2 transport model was used to simulate O2 transport in a hepatic hollow fiber (HF) bioreactor. It was observed that low O2 affinity PolybHbs oxygenated the bioreactor better than high O2 affinity PolybHbs. This result points to the suitability of low O2 affinity PolybHbs for use in tissue engineering and transfusion medicine. Taken together, our results show the quantitative effect of varying the oxygen saturation of bHb and G:Hb molar ratio on the biophysical properties of PolybHbs and their ability to oxygenate a hepatic HF bioreactor. We suggest that the information gained from this study can be used to guide the design of the next generation of hemoglobin-based oxygen carriers (HBOCs) for use in tissue engineering and transfusion medicine applications.     

A nonchromatographic process for purification of secretory immunoglobulins from caprine whey

Secretory immunoglobulins are an important antibody class being primarily responsible for immunoprotection of mucosal surfaces. A simple, non‐chromatographic purification process for secretory immunoglobulins from caprine whey was developed. In the first process step whey was concentrated 30–40‐fold on a 500 kDa membrane, thereby increasing the purity from 3% to 15%. The second step consisted of a fractionated PEG precipitation, in which high molecular weight impurities were removed first and in the second stage the secretory immunoglobulins were precipitated, leaving a majority of the low molecular weight proteins in solution. The re‐dissolved secretory immunoglobulin fraction had a purity of 43% which could then be increased to 72% by diafiltration at a volume exchange factor of 10. Further increase of purity was only possible at the expense of very high buffer consumption. If diafiltration was performed directly after ultrafiltration, followed by precipitation, the yield was higher but purity was only 54%. Overall, filtration performance was characterized by high concentration polarization, therefore process conditions were set to low trans‐membrane pressure and moderate protein concentration. As such purity and to a lesser extent throughput were the major objectives rather than yield, since whey, as a by‐product of the dairy industry, is a cheap raw material of almost unlimited supply. Ultra‐/diafiltration performance was described well by correlations using dimensionless numbers. Compared with a theoretical model (Graetz/Leveque solution) the flux was slightly overestimated. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:642–653, 2017     

Purification of bovine hemoglobin via fast performance liquid chromatography

Bovine hemoglobin (bHb) was purified from bovine red blood cells (bRBCs) via anion exchange chromatography preceded by dialysis. This is a fast and effective way to obtain bHb from bRBCs using Q Sepharose XL, a strong anion exchange resin. This resin had double the binding capacity for bHb compared to three other anion exchange resins that were studied in this work. Methemoglobin levels remained below 2% with bHb concentrations between 0.7 and 1.7 mM. The high purity of bHb was confirmed via SDS-PAGE and size exclusion chromatography (SEC).     

Structural,functional and physiochemical properties of dextran-bovine hemoglobin conjugate as a hemoglobin-based oxygen carrier

As a hemoglobin (Hb)-based oxygen carrier (HBOC), Hb suffers from the disadvantages of short half-life, renal toxicity and vasoactivity. Because dextran is a macromolecule that can be easily derivatized with various chemical moieties, conjugation of Hb with dextran can effectively increase the size of Hb and overcome the disadvantages of Hb. Thus, a dextran-bovine Hb (bHb) conjugate (dex-bHb) was prepared by conjugation of bHb with periodate-oxidized dextran here. As an important functional amino acid residue of bHb, Cys-93(β) was reversibly protected by 4,4′-dithiodipyridine to avoid reaction with periodate-oxidized dextran. Dex-bHb showed significantly higher hydrodynamic volume and higher viscosity than bHb. Conjugation with dextran stabilized the R state of bHb and slightly altered the heme environment of bHb. Conjugation with dextran decreased the P₅₀ of bHb, lowered the sensitivity to the allosteric effectors and slightly decreased the autoxidation rate of bHb. Thus, dex-bHb was expected to act as a potent HBOC with low oxidative toxicity.     

Small molecule clearance in ultrafiltration/diafiltration in relation to protein interactions: Study of citrate binding to a Fab

Ultrafiltration/diafiltration (UFDF) is commonly utilized in the purification of recombinant proteins to concentrate and buffer exchange the product. It is often the final step in the purification process, placing the protein in its final formulation and clearing small molecules introduced in upstream purification steps. This article presents a case study of reduced small molecule clearance in ultrafiltration/diafiltration of an antigen‐binding fragment of a monoclonal antibody. Citrate, a commonly utilized small molecule in downstream processes, is shown to have reduced clearance due to specific interactions with the protein product. The study presents process solutions and utilizes a simple model to characterize clearance of small molecules which exhibit interactions with product protein. Biotechnol. Bioeng. 2009;102: 1718–1722. © 2008 Wiley Periodicals, Inc.     

Very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE bioreactor™—part II: Applications for antibody production and cryopreservation

A high cell density perfusion process of monoclonal antibody (MAb) producing Chinese hamster ovary (CHO) cells was developed in disposable WAVE Bioreactor? using external hollow fiber (HF) filter as cell separation device. Tangential flow filtration (TFF) and alternating tangential flow (ATF) systems were compared and process applications of high cell density perfusion were studied here: MAb production and cryopreservation. Operations by perfusion using microfiltration (MF) or ultrafiltration (UF) with ATF or TFF and by fed‐batch were compared. Cell densities higher than 10⁸ cells/mL were obtained using UF TFF or UF ATF. The cells produced comparable amounts of MAb in perfusion by ATF or TFF, MF or UF. MAbs were partially retained by the MF using ATF or TFF but more severely using TFF. Consequently, MAbs were lost when cell broth was discarded from the bioreactor in the daily bleeds. The MAb cell‐specific productivity was comparable at cell densities up to 1.3 × 10⁸ cells/mL in perfusion and was comparable or lower in fed‐batch. After 12 days, six times more MAbs were harvested using perfusion by ATF or TFF with MF or UF, compared to fed‐batch and 28× more in a 1‐month perfusion at 10⁸ cells/mL density. Pumping at a recirculation rate up to 2.75 L/min did not damage the cells with the present TFF settings with HF short circuited. Cell cryopreservation at 0.5 × 10⁸ and 10⁸ cells/mL was performed using cells from a perfusion run at 10⁸ cells/mL density. Cell resuscitation was very successful, showing that this system was a reliable process for cell bank manufacturing. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:768–777, 2013     

High O2 affinity hemoglobin-based oxygen carriers synthesized via polymerization of hemoglobin with ring-opened 2-chloroethyl-beta-D-fructopyranoside and 1-o-octyl-beta-D-glucopyranoside

Second generation hemoglobin-based O(2) carriers (HBOCs) are being developed with high O(2) affinity (low P(50)) in order to suppress vasoconstriction elicited by over-oxygenating tissues, a problem associated with low O(2) affinity first generation HBOCs. Our group has previously investigated the polymerization of hemoglobin (Hb) with dialdehydes as a strategy for engineering high O(2) affinity HBOCs. In this study, two novel reactive dialdehydes were synthesized by ring-opening 2-chloroethyl-beta-D-fructopyranoside (2-CEFP) and 1-o-octyl-beta-D-glucopyranoside (1-OGP) at the 1,2-diol position, respectively, to yield novel Hb polymerizing reagents. High-affinity polymerized HBOCs were synthesized by reacting R-state bovine hemoglobin (bHb) with ring-opened 2-CEFP and 1-OGP at cross-linker to bHb molar ratios ranging from 10:1 to 30:1. The resulting polymerized bovine HBOCs (bHBOCs) displayed P(50)s ranging from 7 to 18 mmHg, cooperativities ranging from 0.8 to 1.4, and methemoglobin (metHb) levels ranging from 3% to 10%. The cross-linking reaction also stabilized the third stepwise Adair coefficient for bHbs reacted with ring-opened 1-OGP at cross-linker to bHb molar ratios of 20:1 and 30:1 and for bHbs reacted with ring-opened 2-CEFP at molar ratios of 30:1. Additionally, the number-averaged molecular weight, M(n), of each polymerized bHBOC was larger compared to bHb. Molecular weight distributions leaning towards larger molecular weight bHBOCs were obtained by increasing the cross-linker to bHb molar ratio. Taken together, the results of this study have identified novel Hb polymerization reagents that are easy to synthesize, and that are capable of yielding bHBOCs with higher O(2) affinities and weight-averaged molecular weights compared to bHb.     

Recombinant murine growth hormone from E. coli inclusion bodies: Expression,high‐pressure solubilization and refolding,and characterization of activity and structure

We expressed recombinant murine growth hormone (rmGH) in E. coli as a cost‐effective way to produce large quantities (gram scale) of the protein for use in murine studies of immunogenicity to therapeutic proteins. High hydrostatic pressure was used to achieve high solubility and high refolding yields of rmGH protein produced in E. coli inclusion bodies. A two‐step column purification protocol was used to produce 99% pure monomeric rmGH. Secondary and tertiary structures of purified rmGH were investigated using circular dichroism and 2D‐UV spectroscopy. The purified rmGH produced was found to be biologically active in hypophysectomized rats. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The quaternary structure of tetrameric hemoglobin regulates the oxygen affinity of polymerized hemoglobin

This study focuses on the effect of the initial quaternary structure of bovine hemoglobin (Hb) on the physical properties of glutaraldehyde polymerized Hb (PolyHb) solutions. Tense (T) state PolyHb was synthesized by maintaining the pO₂ of Hb before and after polymerization at 0 mm Hg. In contrast, relaxed (R) state PolyHb was generated by maintaining the pO₂ of Hb before and after polymerization to >749 mm Hg. PolyHb solutions were characterized by measuring the pO₂, methemoglobin (metHb) level, molecular weight distribution, O₂ affinity and cooperativity coefficient. The metHb level of all PolyHb solutions was low (<2%). Analysis of the molecular weight distribution of PolyHb solutions indicates that in general, the molecular weight of PolyHb solutions increased with increasing cross‐link density. T‐state PolyHb solutions exhibited lower O₂ affinity compared to unmodified Hb, whereas R‐state PolyHb solutions exhibited higher O₂ affinity compared to unmodified Hb. In addition, the polymerization reaction resulted in a significant decrease in cooperativity that was more pronounced at higher cross‐link densities. All of these results were explained in terms of the quaternary structure of Hb. Taken together, our results yield more insight into the importance Hb's quaternary structure plays in defining the physical properties of glutaraldehyde PolyHb solutions. This information will be useful in designing optimized glutaraldehyde PolyHb oxygen carriers for various applications in transfusion medicine. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009     

Purification of human antibodies from transgenic corn using aqueous two‐phase systems

A recombinant human antibody expressed in corn was purified using aqueous two‐phase extraction. The antibody was an immunoglobulin G fully unglycosylated. Using systems of different compositions and/or pHs in each of one or two partitioning stages followed by one more stage in which the antibody was precipitated at the liquid/liquid interface facilitated the removal of different impurities in each stage. The best system yields a product 72% pure (22‐fold purification) with a yield of 49%. The optimum extraction was done in two partitioning stages followed by an interfacial precipitation stage using poly(ethylene)glycol/potassium phosphate systems. NaCl was added to the first stage to eliminate large molecular weight impurities. The pH in the first stage was kept at 6 but a pH of 8 was used in the second stage and in the precipitation stage. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Investigation of proteins and peptides from yeastolate and subsequent impurity testing of drug product

Hydrolysates play an important role in modern biological production. These mixtures are mostly undefined and contain a mixture of proteins, peptides, and amino acids along with other non–amino acid‐based components. Recently, there has been an interest in defining and sequencing proteins and peptides in these hydrolysates to subsequently develop an assay to ensure removal during product purification. This work investigates an ultrafiltrate of yeastolate to determine whether any protein is present. Size exclusion chromatography indicated a possible high molecular weight component (>10 kDa). This suspected high molecular weight fraction was collected and investigated. It was determined that this fraction consists of nucleic acids; and no protein was detected using sensitive modern techniques including HPLC, mass spectrometry, and SDS‐PAGE. Next, five unique, yeast‐specific peptides were identified, sequenced, and confirmed. Finally, an impurity assay for any residual yeast specific peptides was developed and the analytical metrics were determined including accuracy, precision, linearity, range, and limits of detection and quantitation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Amino-acid sequence of human and bovine brain myelin proteolipid protein (lipophilin) is completely conserved

Proteolipid protein (PLP) was isolated from white matter of human brain by chloroform/methanol extraction and further purified by chromatography. Performic acid oxidation yielded a product homogeneous in NaDodSO4-polyacrylamide electrophoresis with a molecular mass of 30 kDa. The carboxymethylated PLP was chemically cleaved with cyanogen bromide into four fragments: CNBr I 22-24 kDa, CNBr II 5 kDa, CNBr III 1.4 kDa and CNBr IV 0.7 kDa. HBr/dimethylsulfoxide cleavage at tryptophan residues released four fragments: Trp I 14-16 kDa, Trp II 2.0 kDa, Trp III 5 kDa and Trp IV 7 kDa. Hydrophilic fragments were enriched in 50% formic acid (CNBr II, III, IV and Trp II and III), whereas hydrophobic peptides precipitated from this solvent were CNBr I, Trp I and IV. The fragments were separated by gel filtration with 90% formic acid as solvent and finally purified by gel permeation HPLC (Si 60 and Si 100) for automated liquid and solid-phase Edman degradation. Large fragments were further cleaved with different proteinases (trypsin, V8-proteinase, endoproteinase Lys-C and thermolysin). We used an improved strategy in the sequencing of the human proteolipid protein compared with our approach to the structural elucidation of bovine brain PLP. The amino-acid sequence of human PLP contains 276 residues, the same as found in bovine proteolipid protein. The two sequences proved to be identical. The possible importance of the conservative structure of this integral membrane protein is discussed.     

An alternate diafiltration strategy to mitigate protein precipitation for low solubility proteins

Application of the minimum diafiltration (DF) time solution for a monoclonal antibody resulted in a 20‐h process time rather than the expected 12 h. Further investigation indicated high turbidity associated with a product solubility issue that caused a flux decline. As a result, the gel flux model and the associated minimum DF time were not predictive. Multiwell plate solubility screening confirmed that the protein passed through a region of low solubility during the ultrafiltration step. Multiple approaches to address this issue were considered and a new strategy involving variable volume diafiltration (VVDF) was developed. Process modeling and simulation were used to predict performance and to select a value of the DF ratio control parameter (buffer flow/permeate flow = 0.65). Feasibility testing at the bench and pilot scales confirmed that the new strategy reduced solubility issues, fit within existing manufacturing tank volume and system area constraints, matched model predictions, and did not present significant implementation issues. Recommendations are made regarding the general value of this strategy, when it should be used, and how to implement it. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:646–655, 2014     

Large scale demonstration of a process analytical technology application in bioprocessing: Use of on‐line high performance liquid chromatography for making real time pooling decisions for process chromatography

Process Analytical Technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community because of the potential for continuous real time quality assurance resulting in improved operational control and compliance. In previous publications, we have demonstrated feasibility of applications involving use of high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) for real‐time pooling of process chromatography column. In this article we follow a similar approach to perform lab studies and create a model for a chromatography step of a different modality (hydrophobic interaction chromatography). It is seen that the predictions of the model compare well to actual experimental data, demonstrating the usefulness of the approach across the different modes of chromatography. Also, use of online HPLC when the step is scaled up to pilot scale (a 2294 fold scale‐up from a 3.4 mL column in the lab to a 7.8 L column in the pilot plant) and eventually to manufacturing scale (a 45930 fold scale‐up from a 3.4 mL column in the lab to a 158 L column in the manufacturing plant) is examined. Overall, the results confirm that for the application under consideration, online‐HPLC offers a feasible approach for analysis that can facilitate real‐time decisions for column pooling based on product quality attributes. The observations demonstrate that the proposed analytical scheme allows us to meet two of the key goals that have been outlined for PAT, i.e., “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions”. The application presented here can be extended to other modes of process chromatography and/or HPLC analysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

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.