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Hollow fiber‐based membrane filtration has emerged as the dominant technology for cell retention in perfusion processes yet significant challenges in alleviating filter fouling remain unsolved. In this work, the benefits of co‐current filtrate flow applied to a tangential flow filtration (TFF) module to reduce or even completely remove Starling recirculation caused by the axial pressure drop within the module was studied by pressure characterization experiments and perfusion cell culture runs. Additionally, a novel concept to achieve alternating Starling flow within unidirectional TFF was investigated. Pressure profiles demonstrated that precise flow control can be achieved with both lab‐scale and manufacturing‐scale filters. TFF systems with co‐current flow showed up to 40% higher product sieving compared to standard TFF. The decoupling of transmembrane pressure from crossflow velocity and filter characteristics in co‐current TFF alleviates common challenges for hollow fiber‐based systems such as limited crossflow rates and relatively short filter module lengths, both of which are currently used to avoid extensive pressure drop along the filtration module. Therefore, co‐current filtrate flow in unidirectional TFF systems represents an interesting and scalable alternative to standard TFF or alternating TFF operation with additional possibilities to control Starling recirculation flow. [ABSTRACT FROM AUTHOR]

There is significant interest in identifying the preferred binding domains of biological products to various chromatographic materials. In this work, we develop a biophysical technique that uses diethyl pyrocarbonate (DEPC) based covalent labeling in concert with enzymatic digestion and mass spectrometry to identify the binding patches for proteins bound to commercially available multimodal (MM) cation exchange chromatography resins. The technique compares the changes in covalent labeling of the protein in solution and in the bound state and uses the differences in this labeling to identify residues that are sterically shielded upon resin binding and, therefore, potentially involved in the resin binding process. Importantly, this approach enables the labeling of many amino acids and can be carried out over a pH range of 5.5–7.5, thus enabling the protein surface mapping at conditions of interest in MM cation exchange systems. The protocol is first developed using the model protein ubiquitin and the results indicate that lysine residues located on the front face of the protein show dramatic changes in DEPC labeling while residues present on other regions have minimal or no reductions. This indicates that the front face of ubiquitin is likely involved in resin binding. In addition, surface property maps indicate that the hypothesized front face binding region consists of overlapping positively charged and hydrophobic patches. The technique is then employed with an IgG1 FC and the results indicate that residues on the CH2–CH3 interface and the hinge are significantly sterically shielded upon binding to the resin. Further, these regions are again associated with significant overlap of positively charged and hydrophobic patches. On the other hand, while, residues on the CH2 and the front face of the IgG1 FC also exhibited some changes in DEPC labeling upon binding, these regions have less distinct charged and hydrophobic patches. Importantly, the hypothesized binding patches identified for both ubiquitin and FC using this approach are shown to be consistent with previously reported NMR studies. In contrast to NMR, this new approach enables the identification of preferred binding regions without the need for isotopically labeled proteins or chemical shift assignments. The technique developed in this work sets the stage for the evaluation of the binding domains of a wide range of biological products to chromatographic surfaces, with important implications for designing biomolecules with improved biomanufacturability properties. [ABSTRACT FROM AUTHOR]

In an effort to increase selectivity among proteins with crossflow ultrafiltration, we offer and demonstrate a comprehensive approach to fractionate proteins of similar molecular weight and relatively close p I values. This multidimensional approach involves optimizing membrane charge type and density together with operating conditions such as precise control of pH, ionic strength, and transmembrane pressure for reduced membrane fouling. Each filtration experiment was performed in cross-flow configuration for ∼20 min, allowing fast screening for optimal separation as determined by maximum selectivity, Ψ, and purity, P. Using our comprehensive approach for fractionating mixtures RNase A-lysozyme and BSA-hemoglobin, we obtained values of Ψ = 9.1, P = 95.7%, and Ψ = 6.5, P = 62.1%, respectively. Biotechnol. Bioeng. 2013; 110: 1704-1713. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Bioprocess intensification can be achieved through high cell density perfusion cell culture with continuous protein capture integration. Protein passage and cell retention are commonly accomplished using tangential flow filtration systems consisting of microporous membranes. Significant challenges, including low efficiency and decaying product sieving over time, are commonly observed in these cell retention devices. Here, we demonstrate that a macroporous membrane overcomes the product sieving challenges when comparing to several other membrane chemistries and pore sizes within the microporous range. This way, variable chromatography column loading is avoided. The macroporous membrane yielded a 13,000 L/m2 volumetric throughput. The membrane's cut‐off size results in an increased permeate turbidity due to particles passage, such as cell debris, through pores ranging from 1 to 4 µm. In addition, successful chromatography column plugging mitigation was achieved by employing depth filtration before the chromatographic step. Depth filtration volumetric throughputs were between 600 and 1,000 L/m2. Combing a macroporous cell retention device with a depth filter not only provided an alternative to address the challenge of undesired long protein residence times in the bioreactor due to product sieving decay, but also exhibited a throughput increase, making the integration of multicolumn capture chromatography with a perfusion cell culture a more robust process. [ABSTRACT FROM AUTHOR]

A combined pore blockage and cake filtration model was applied to the virus filtration of an Fc‐fusion protein using the three commercially available filters, F‐1, F‐2, and F‐3 in a range of buffer conditions including sodium‐phosphate and tris‐acetate buffers with and without 200 mM NaCl at pH 7.5. The fouling behaviors of the three filters for the feed solutions spiked with minute virus of mice were described well by this combined model for all the solution conditions. This suggests that fouling of the virus filters is dominated by the pore blockage mechanism during the initial stage of the filtration and transformed to the cake filtration mechanism during the later stage of the filtration. Both flux and transmembrane resistance can be described well by this model. The pore blockage rate and the rate of increase of protein layer resistance over blocked pores are found to be affected by membrane properties as well as the solution conditions resulting from the modulation of interactions between virus, protein, and membrane by the solution conditions. [ABSTRACT FROM AUTHOR]

We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N‐terminal Lys‐Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B‐block threonine, which is now seen to play a causative role not only in ground‐state destabilization of the scissile N‐terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N‐terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing. We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N‐terminal Lys–Cys splice junction. We identified conditions to stablize the thiazoline ring and provide the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. [ABSTRACT FROM AUTHOR]

Alternating tangential flow (ATF) filtration has been successfully adopted as a low shear cell separation device in many perfusion‐based processes. The reverse flow per cycle is used to minimize fouling compared with tangential flow filtration. Currently, modeling of the ATF system is based on empirically derived formulas, leading to oversimplification of model parameters. In this study, an experimentally validated porous computational fluid dynamic (CFD) model was used to predict localized fluid behavior and pressure profiles in the ATF membrane for both water and supernatant solutions. The results provided numerical evidence of Starling flow phenomena that has been theorized but not previously proven for the current operating parameters. Additionally, feed cross flow velocity was shown to significantly impact the localized flux distribution; higher feed cross flow rates lead to an increased localized permeate flux as well as irreversible and reversible fouling resistance. Further, the small average permeate flux values of 2 L·m−2·h−1 traditionally used in perfusion bioreactor membranes lead to approximately 50% of the membrane length utilized for permeate flow during each pressure and exhaust phase, leading to a full membrane utilization during one ATF cycle. Our preliminary CFD results demonstrate that local flux and resistance distribution further elucidate the dynamics of ATF membrane fouling in a perfusion‐based system. Alternating tangential flow filtration has been successfully adopted as a low shear cell separation device in many perfusion‐based processes. [ABSTRACT FROM AUTHOR]

The use of cell-free systems to produce recombinant proteins has grown rapidly over the past decade. In particular, cell-free protein synthesis (CFPS) systems based on mammalian cells provide alternative methods for the production of many proteins, including those that contain disulfide bonds, glycosylation, and complex structures such as monoclonal antibodies. In the present study, we show robust production of turbo green fluorescent protein (tGFP) and streptokinase in a cell-free system using instrumented mini-bioreactors for highly reproducible protein production. We achieved recombinant protein production (∼600 μg/ml of tGFP and 500 μg/ml streptokinase) in 2.5 hr of expression time, comparable to previously reported yields for cell-free protein expression. Also, we demonstrate the use of two different affinity tags for product capture and compare those to a tag-free self-cleaving intein capture technology. The intein purification method provided a product recovery of 86%, compared with 52% for conventionally tagged proteins, while resulting in a 30% increase in total units of activity of purified recombinant streptokinase compared with conventionally tagged proteins. These promising beneficial features combined with the intein technology makes feasible the development of dose-level production of therapeutic proteins at the point-of-care. [ABSTRACT FROM AUTHOR]

ABSTRACT Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue toward energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (−24 to +16 total charges) and total charge density (0.28-0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases. Biotechnol. Bioeng. 2017;114: 740-750. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT There is considerable interest in developing microscale (i.e., high-throughput) methods that enable multiple filtration experiments to be run in parallel with smaller sample amounts and thus reduce the overall required time and associated cost to run the filtration tests. Previous studies to date have focused on simply evaluating the filtration capacity, not the separation performance. In this work, the stirred-well filtration (SWF) method was used in combination with design-of-experiment (DOE) methods to optimize the separation performance for three binary mixtures of bio-molecules: protein-protein, protein-polysaccharide, and protein-DNA. Using the parallel based format of the SWF method, eight constant-flux ultrafiltration experiments were conducted at once to study the effects of stirring conditions, permeate flux, and/or solution conditions (pH, ionic strength). Four separate filtration tests were conducted for each combination of process variables; in total, over 100 separate tests were conducted. The sieving coefficient and selectivity results are presented to match the DOE design format and enable a greater understanding of the effects of the different process variables that were studied. The method described herein can be used to rapidly determine the optimal combination of process factors that give the best separation performance for a range of membrane-based separations applications and thus obviate the need to run a large number of traditional lab-scale tests. Biotechnol. Bioeng. 2016;113: 2131-2139. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT During fermentation processes, in situ product recovery (ISPR) using submerged membranes allows a continuous operation mode with effective product removal. Continuous recovery reduces product inhibition and organisms in the reactor are not exposed to changing reaction conditions. For an effective in situ product removal, submerged membrane systems should have a sufficient large membrane area and an anti-fouling concept integrated in a compact device for the limited space in a lab-scale bioreactor. We present a new membrane stirrer with integrated filtration membranes on the impeller blades as well as an integrated gassing concept in an all-in-one device. The stirrer is fabricated by rapid prototyping and is equipped with a commercial micromesh membrane. Filtration performance is tested using a yeast cell suspension with different stirring speeds and aeration fluxes. We reduce membrane fouling by backflushing through the membrane with the product stream. Biotechnol. Bioeng. 2015;112: 331-338. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT In this work, a proof of concept elastin-like polypeptide-Z domain fusion (ELP-Z) based affinity precipitation process is developed for monoclonal antibody (mAb) purification from industrial harvest feeds. Greater than 99% mAb recoveries are obtained during the initial binding step of the process for both pure mAb and the mAb harvest feeds. Great than 90% overall mAb yields are also obtained for the subsequent elution step of the process with no measurable mAb aggregation. The process is shown to result in more than 2 logs of host cell protein (HCP) and more than 4 logs of DNA clearance from the harvest feed. While the overall mAb yield and HCP clearance for the affinity precipitation process was comparable to Protein A chromatography the DNA clearance was clearly superior. Performance is maintained for mAb final elution concentrations up to 20 g/L, demonstrating the ability of the process to both concentrate and purify the mAb. Effective ELP-Z regeneration is also demonstrated using 0.1 M NaOH with no adverse effect on subsequent capture efficiency. Finally, the reusability of the ELP-Z construct and robustness of the process is demonstrated for up to three purification-regeneration cycles with minimal product and impurity carryover and high yields and purity. This work demonstrates that the ELP-Z based precipitation approach can be successfully employed as an affinity capture step for industrial mAbs. Biotechnol. Bioeng. 2014;111: 1595-1603. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT We present a feasibility study for an antibody capturing process from clarified cell culture fluid using semi-selective protein precipitation with salt-tolerant copolymers. Protein precipitation is mediated by hydrophobic and electrostatic interactions with the copolymer that can be customized for the respective target. Precipitation yield with different copolymers at ionic strength of 2-22.5 mS cm−1 and pH 5.0-pH 5.7 was evaluated using pure monoclonal antibody solutions. Optimized parameters were used to elucidate yield and purity of various antibodies precipitated at physiological conditions from cell culture fluid of CHO, NS0, and SP2/0 cell culture fluid. Precipitated protein was easily redissolved in small volume, enabling concentrating monoclonal antibodies (mAb) more than 40-fold and up to 100-fold, while residual polymer was removed to >98% using cationic polymer attached to silica flakes. mAb recovery of >90% and host cell protein clearance of >80% were achieved, not requiring any pre-dilution of cell culture fluid. Precipitation showed no impact on mAb binding affinity when compared to non-precipitated mAb. The obtained yield and purity were lower compared to a protein A based purification and loss of mAb was factor 1.5-3.0 higher. Yet, for high titer mAb purification processes being implemented in the future, precipitation is an attractive option due to its ease of scalability and cost-effectiveness. Biotechnol. Bioeng. 2013;110: 2915-2927. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT This study provides a detailed investigation into the performance of a stimuli responsive ELP-Z based process for monoclonal antibody (mAb) affinity precipitation. A multidimensional high-throughput screening (HTS) protocol was developed and employed to investigate the effects of a variety of operating conditions on mAb yield and aggregation during the process. Precipitation efficiency of ELP-Z in the absence of mAb was first determined as a function of temperature and sodium sulfate concentration and conditions producing high yields were identified. HTS was then employed to determine appropriate conditions for the initial capture and co-precipitation of mAbs at high yields using ELP-Z. mAb elution from ELP-Z was then examined using HTS and the mAb yields and aggregate content of the overall process were determined. It was observed that mAb aggregation was sensitive primarily to the elution conditions and that this behavior was antibody specific and a strong function of operating temperature and elution pH. Importantly, for both mAbs examined in this study, the results indicated that room temperature operation and appropriate elution pH could be readily employed to produce both high mAb yields and low aggregate content using this approach. This study demonstrates the ability of ELP-Z based affinity precipitation for mAb purification and shows that HTS can be successfully employed to rapidly develop a robust and high yield process. Biotechnol. Bioeng. 2013;110: 2664-2676. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Rapid and efficient tag removal remains a significant problem in recombinant protein purification. Using an engineered DnaE intein from Nostoc punctiforme, we developed a split intein mediated ultra-rapid purification (SIRP) method for the purification of tagless recombinant protein from E. coli lysate in less than 1 h. This system exhibits extraordinarily rapid thio-induced C-terminal cleavage with about 50% completion within 30 s at both 22°C and 6°C. This is the fastest C-terminal cleavage activity reported to date for inteins. Although the reaction kinetics slow down after the first minute, >90% cleavage completion is achieved within 30 min at 22°C, or within 3 h at 6°C. The ultra-rapid cleavage kinetics are made possible by the positioning of the purification tag at the split junction to the C-terminus of the intein N-fragment, thus avoiding potential steric hindrance of the critical interaction between the N- and C-extein. Target proteins are cleaved to >72% completion after 1 h of intein reaction regardless of the identity of the N-terminal amino acid except in the cases of threonine (50% cleavage) and proline. The C-terminal cleavage reaction can be effectively inhibited by divalent Zn2+ under non-reducing conditions. Importantly, the association of the intein N- and C-fragments is reversible, enabling the column-bound intein N-fragment bait protein to be regenerated for multiple usages and further reducing the cost of protein purification. SIRP technology should provide a useful tool for the purification of tagless proteins and peptides. Biotechnol. Bioeng. 2013; 110:2471-2481. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The unregulated activity of inteins during expression and consequent side reactions during work-up limits their widespread use in biotechnology and chemical biology. Therefore, we exploited a mechanism-based approach to regulate intein autocatalysis for biotechnological application. The system, inspired by our previous structural studies, is based on reversible trapping of the intein's catalytic cysteine residue through a disulfide bond. Using standard mutagenesis, the disulfide trap can be implemented to impart redox control over different inteins and for a variety of applications both in vitro and in Escherichia coli. Thereby, we first enhanced the output for bioconjugation in intein-mediated protein ligation, also referred to as expressed protein ligation, where precursor recovery and product yield were augmented fourfold to sixfold. Second, in bioseparation experiments, the redox trap boosted precursor recovery and product yield twofold. Finally, the disulfide-trap intein technology stimulated development of a novel bacterial redox sensor. This sensor reliably identified hyperoxic E. coli harboring mutations that disrupt the reductive pathways for thioredoxin and glutathione, against a background of wild-type cells. Biotechnol. Bioeng. 2013; 110: 1565-1573. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

While protein purification has long been dominated by standard chromatography, the relatively high cost and complex scale-up have promoted the development of alternative non-chromatographic separation methods. Here we developed a new non-chromatographic affinity method for the purification of proteins expressed in Escherichia coli. The approach is to genetically fuse the target proteins with an affinity tag. Direct purification and recovery can be achieved using a thermo-responsive elastin-like protein (ELP) scaffold containing the capturing domain. Naturally occurring cohesin-dockerin pairs, which are high-affinity protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the model. By exploiting the highly specific interaction between the dockerin and cohesin domain from Clostridium thermocellum and the reversible aggregation property of ELP, highly purified and active dockerin-tagged proteins, such as the endoglucanase CelA, chloramphenicol acetyl transferase (CAT), and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single thermal precipitation step with yields achieving over 90%. Incorporation of a self-cleaving intein domain enabled rapid removal of the affinity tag from the target proteins, which was subsequently removed by another cycle of thermal precipitation. This method offers great flexibility as a wide range of affinity tags and ligands can be used. Biotechnol. Bioeng. 2012; 109: 2829-2835. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The characterization of host cell protein (HCP) content during the production of therapeutic recombinant proteins is an important aspect in the drug development process. Despite this, key components of the HCP profile and how this changes with processing has not been fully investigated. Here we have investigated the supernatant HCP profile at different times throughout culture of a null and model GS-CHO monoclonal antibody producing mammalian cell line grown in fed-batch mode. Using 2D-PAGE and LC-MS/MS we identify a number of intracellular proteins (e.g., protein disulfide isomerise; elongation factor 2; calreticulin) that show a significant change in abundance relative to the general increase in HCP concentration observed with progression of culture. Those HCPs that showed a significant change in abundance across the culture above the general increase were dependent on the cell line examined. Further, our data suggests that the majority of HCPs in the supernatant of the cell lines investigated here arise through lysis or breakage of cells, associated with loss in viability, and are not present due to the secretion of protein material from within the cell. SELDI-TOF and principal components analysis were also investigated to enable rapid monitoring of changes in the HCP profile. SELDI-TOF analysis showed the same trends in the HCP profile as observed by 2D-PAGE analysis and highlighted biomarkers that could be used for process monitoring. These data further our understanding of the relationship between the HCP profile and cell viability and may ultimately enable a more directed development of purification strategies and the development of cell lines based upon their HCP profile. Biotechnol. Bioeng. 2012; 109:971-982. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian cell line that maintains stability of production. Problems with process yield, lack of effective use of costly resources, and a possible delay in obtaining regulatory approval of the product may ensue otherwise. Therefore the stability of expression in a number of Chinese hamster ovary (CHO) derived production cell lines that were isolated using the glutamine synthetase (GS) selection system was investigated by defining a culture as unstable if the titer (which is a measure of productivity) of a cell line expressing an antibody or antibody-fusion protein declined by 20-30% or more as it underwent 55 population doublings. Using this criterion, a significant proportion of the GS-selected CHO production cell lines were observed to be unstable. Reduced antibody titers correlated with the gradual appearance of a secondary, less productive population of cells as detected with flow cytometric analysis of intracellular antibody content. Where tested, it was observed that the secondary population arose spontaneously from the parental population following multiple passages, which suggested inherent clonal instability. Moreover, the frequency of unstable clones decreased significantly if the host cell line from which the candidate production cell lines were derived was apoptotic-resistant. This data suggested that unstable cell lines were more prone to apoptosis, which was confirmed by the fact that unstable cell lines had higher levels of Annexin V and caspase 3 activities. This knowledge has been used to develop screening protocols that identify unstable CHO production cell lines at an early stage of the cell line development process, potentially reducing the cost of biotherapeutic development. Biotechnol. Bioeng. 2012; 109:1016-1030. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The presence of even minute amounts of protein aggregates in solution can significantly alter the kinetics of amyloid formation. Removal of such pre-existing aggregates is critical for reproducible analysis of amyloid formation. Here we examine the effects of membrane filtration on insulin fibrillization. We find that filtration of insulin with large pore membranes (≥100 nm) generally slows fibril formation relative to unfiltered solutions by removing pre-aggregated protein. Unexpectedly, filtration with small pore membranes (<100 nm) showed no beneficial effect and, in some cases, accelerated insulin fibril formation. This effect may be due to fragmentation of pre-existing aggregates during filtration through small pore membranes, which can increase the number of amyloid-forming precursors. These findings reveal the complexity of removing protein aggregates via filtration and suggest optimal filtration protocols for conducting fibril formation analysis of insulin and similar amyloidogenic proteins. Biotechnol. Bioeng. 2012; 109:840-845. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The Zonal Rate Model (ZRM) has previously been shown to accurately account for contributions to elution band broadening, including external flow nonidealities and radial concentration gradients, in ion-exchange membrane (IEXM) chromatography systems operated under nonbinding conditions. Here, we extend the ZRM to analyze and model the behavior of retained proteins by introducing terms for intra-column mass transfer resistances and intrinsic binding kinetics. Breakthrough curve (BTC) data from a scaled-down anion-exchange membrane chromatography module using ovalbumin as a model protein were collected at flow rates ranging from 1.5 to 20 mL min−1. Through its careful accounting of transport nonidealities within and external to the membrane stack, the ZRM is shown to provide a useful framework for characterizing putative protein binding mechanisms and models, for predicting BTCs and complex elution behavior, including the common observation that the dynamic binding capacity can increase with linear velocity in IEXM systems, and for simulating and scaling separations using IEXM chromatography. Global fitting of model parameters is used to evaluate the performance of the Langmuir, bi-Langmuir, steric mass action (SMA), and spreading-type protein binding models in either correlating or fundamentally describing BTC data. When combined with the ZRM, the bi-Langmuir, and SMA models match the chromatography data, but require physically unrealistic regressed model parameters to do so. In contrast, for this system a spreading-type model is shown to accurately predict column performance while also providing a realistic fundamental explanation for observed trends, including an observed increase in dynamic binding capacity with flow rate. Biotechnol. Bioeng. 2012; 109:615-629. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

This contribution describes the preparation of strong anion-exchange membranes with higher protein binding capacities than the best commercial resins. Quaternary amine (Q-type) anion-exchange membranes were prepared by grafting polyelectrolyte nanolayers from the surfaces of macroporous membrane supports. A focus of this study was to better understand the role of polymer nanolayer architecture on protein binding. Membranes were prepared with different polymer chain graft densities using a newly developed surface-initiated polymerization protocol designed to provide uniform and variable chain spacing. Bovine serum albumin and immunoglobulin G were used to measure binding capacities of proteins with different size. Dynamic binding capacities of IgG were measured to evaluate the impact of polymer chain density on the accessibility of large size protein to binding sites within the polyelectrolyte nanolayer under flow conditions. The dynamic binding capacity of IgG increased nearly linearly with increasing polymer chain density, which suggests that the spacing between polymer chains is sufficient for IgG to access binding sites all along the grafted polymer chains. Furthermore, the high dynamic binding capacity of IgG (>130 mg/mL) was independent of linear flow velocity, which suggests that the mass transfer of IgG molecules to the binding sites occurs primarily via convection. Overall, this research provides clear evidence that the dynamic binding capacities of large biologics can be higher for well-designed macroporous membrane adsorbers than commercial membrane or resin ion-exchange products. Specifically, using controlled polymerization leads to anion-exchange membrane adsorbers with high binding capacities that are independent of flow rate, enabling high throughput. Results of this work should help to accelerate the broader implementation of membrane adsorbers in bioprocess purification steps. Biotechnol. Bioeng. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

This study reveals that residence time distribution (RTD) analysis with pH monitoring after acid bolus injection can be used to globally study the flow dynamics of a perfusion bioreactor, while fluorescence microscopy and magnetic resonance imaging (MRI) were used to locally investigate mass transport within a hydrogel scaffold seeded or not with cells. The bioreactor used in this study is a close-loop tubular reactor. A dispersion model in one dimension has been used to describe the non-ideal behavior of the reactor. From open-loop experiments (single-cycle analysis), the presence of stagnant zones and back mixing were observed. The impact of the flow rate, the compliance chamber volume and mixing were investigated. Intermediate flows (30, 45, 60, and 90 mL min−1) had no effect over RTD function expressed in reduced time ( θ). Lower flow rates (5 and 15 mL min−1) were associated to smaller extent of dispersion. The compliance chamber volume greatly affected the dynamics of the RTD function, while the effects of mixing and flow were small to non-significant. An empirical equation has been proposed to localize minima of the RTD function and to predict Per. Finally, cells seeded in a gelatin gel at a density of 800,000 cells mL−1 had no effect over the permeability and the apparent diffusion coefficient, as revealed by fluorescent microscopy and MRI experiments. Biotechnol. Bioeng. 2011;108: 2488-2498. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Under conditions relevant to the manufacturing of insulin (e.g., pH 3, room temperature), biosynthetic human insulin (BHI), and Lispro insulin (Lispro) require a nucleation step to initiate aggregation. However, upon seeding with preformed aggregates, both insulins rapidly aggregate into nonnative fibrils. Far ultraviolet circular dichroism (far-UV CD) and second derivative Fourier transform infrared (2D-FTIR) spectroscopic analyses show that the fibrillation process involves a change in protein secondary structure from α-helical in native insulin to predominantly β-sheet in the nonnative fibrils. After seeding, Lispro aggregates faster than BHI, likely because of a reduced propensity to reversibly self-associate. Composition gradient multi-angle light scattering (CG-MALS) analyses show that Lispro is more monomeric than BHI, whereas their conformational stabilities measured by denaturant-induced unfolding are statistically indistinguishable. For both BHI and Lispro, as the protein concentration increases, the apparent first-order rate constant for soluble protein loss decreases. To explain these phenomena, we propose an aggregation model that assumes fibril growth through monomer addition with competitive inhibition by insulin dimers. Biotechnol. Bioeng. 2011;108: 2359-2370. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The article analyzes the adsorption reversibility and viscoelastic properties of ribonuclease A adsorbed to hydrophobic self-assembled monolayers using a quartz microbalance with dissipative monitoring (QCM-D). Changes in frequency and dissipation were linked with increased viscosity during protein introduction. The difference in viscosity between protein-free and concentrated protein solutions using rheological measurements was also explored. Increased bulk protein solution concentrations showed increase in adsorption reversibility.