Impact of micro and macroporous TFF membranes on product sieving and chromatography loading for perfusion cell culture

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.     

Understanding and modeling alternating tangential flow filtration for perfusion cell culture

Alternating tangential flow (ATF) filtration has been used with success in the Biopharmaceutical industry as a lower shear technology for cell retention with perfusion cultures. The ATF system is different than tangential flow filtration; however, in that reverse flow is used once per cycle as a means to minimize fouling. Few studies have been reported in the literature that evaluates ATF and how key system variables affect the rate at which ATF filters foul. In this study, an experimental setup was devised that allowed for determination of the time it took for fouling to occur for given mammalian (PER.C6) cell culture cell densities and viabilities as permeate flow rate and antifoam concentration was varied. The experimental results indicate, in accordance with D'Arcy's law, that the average resistance to permeate flow (across a cycle of operation) increases as biological material deposits on the membrane. Scanning electron microscope images of the post‐run filtration surface indicated that both cells and antifoam micelles deposit on the membrane. A unique mathematical model, based on the assumption that fouling was due to pore blockage from the cells and micelles in combination, was devised that allowed for estimation of sticking factors for the cells and the micelles on the membrane. This model was then used to accurately predict the increase in transmembane pressure during constant flux operation for an ATF cartridge used for perfusion cell culture. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1291–1300, 2014     

Modulation of transmembrane pressure in manufacturing scale tangential flow filtration N-1 perfusion seed culture

Mammalian cells were grown to high density in a 3,000 L culture using perfusion with hollow fibers operated in a tangential flow filtration mode. The high-density culture was used to inoculate the production stage of a biomanufacturing process. At constant permeate flux operation, increased transmembrane pressures (TMPs) were observed on the final day of the manufacturing batches. Small scale studies suggested that the filters were not irreversibly fouled, but rather exposed to membrane concentration polarization that could be relieved by tangential sweeping of the hollow fibers. Studies were undertaken to analyze parameters that influence the hydrodynamic profile within hollow fibers; including filter area, cell density, recirculation flow rate, and permeate flow rate. Results indicated that permeate flow rate had the greatest influence on modulating TMP. Further evaluation showed a significant decrease in TMP when permeate flow was reduced, and this occurred without any negative effect on cell growth or viability. Hence, a 30% reduction of permeate flow rate was implemented at manufacturing scale. A stable operation was achieved as TMP was successfully reduced by 75% while preserving all critical factors for performance in the perfusion bioreactor.     

Evaluation of membranes for use in on-line cell separation during mammalian cell perfusion processes

In this study two microporous hollow fibre membranes were evaluated for their use as cell retention device in continuous perfusion systems. A chemically modified permanent hydrophillic PTFE membrane and a hydrophilized PP membrane were tested. To investigate the filtration characteristics under process conditions each membrane was tested during a long term perfusion cultivation of a hybridoma cell line. In both cultivations the conditions influencing membrane filtration (e.g. transmembrane flux) were kept constant. Filtration behaviour was investigated by monitoring transmembrane pressure and protein permeability. Transmembrane pressure was measured on-line with an autoclavable piezo-resistive pressure sensor. Protein permeability was determined by quantitative evaluation of unreduced, Coomassie stained SDS-PAGE. The membrane fouling process influences the filtration characteristics of both membranes in a different way. After fermentation the PP membrane was blocked by a thick gel layer located in the big outer pores of the asymmetric membrane structure. The hydraulic resistance was higher but the protein permeability was slightly better than of the PTFE membrane. For this reason the PP membrane should be preferred. On the other hand, transmembrane pressure decreases slower when the PTFE membrane is used, which favours this membrane for long term cultivations, especially when low molecular weight proteins (<30 KD) are produced.Abbreviations PP Polypropylene - PTFE Polytetrafluoroethylene     

Long-term perfusion culture of hybridoma: a "grow or die" cell cycle system

In order to elucidate the hybridoma life cycle and the limiting factors in perfusion systems, we performed cultures in a stirred tank bioreactor, coupled to an external tangential flow filtration unit. Cell density and antibody production in perfusion were consistent with previous studies. The average life span of the cells (2.1-2.2 days), antibody, productivity per cell produced (30-38 mg/10(9) cells) and cell size diameter evolution appeared similar to values observed in batch cultures. These observations highly suggest a similar "grow or die" life cycle. Cell and antibody production, strictly related to the medium perfusion rate, seem to be under the control of the nutrient availability. A hypothesis to explain such a life cycle of hybridoma cells in perfusion systems and a model for viable and dead cell density is proposed.     

Investigating the combination of single-pass tangential flow filtration and anion exchange chromatography for intensified mAb polishing

There is growing interest within the biopharmaceutical industry to improve manufacturing efficiency through process intensification, with the goal of generating more product in less time with smaller equipment. In monoclonal antibody (mAb) purification, a unit operation that can benefit from intensification is anion exchange (AEX) polishing chromatography. Single-pass tangential flow filtration (SPTFF) technology offers an opportunity for process intensification by reducing intermediate pool volumes and increasing product concentration without recirculation. This study evaluated the performance of an AEX resin, both in terms of host cell protein (HCP) purification and viral clearance, following concentration of a mAb feed using SPTFF. Results show that preconcentration of AEX feed material improved isotherm conditions for HCP binding, resulting in a fourfold increase in resin mAb loading at the target HCP clearance level. Excellent clearance of minute virus of mouse and xenotropic murine virus was maintained at this higher load level. The increased mAb loading enabled by SPTFF preconcentration effectively reduced AEX column volume and buffer requirements, shrinking the overall size of the polishing step. In addition, the suitability of SPTFF for extended processing time operation was demonstrated, indicating that this approach can be implemented for continuous biomanufacturing. The combination of SPTFF concentration and AEX chromatography for an intensified mAb polishing step which improves both manufacturing flexibility and process productivity is supported.     

The effects of alternating tangential flow (ATF) residence time,hydrodynamic stress,and filtration flux on high-density perfusion cell culture

In this study, we investigated the effects of alternating tangential flow (ATF) cell separation on high-density perfusion cultures. We have developed methods to estimate theoretical residence times of cells in the ATF system and discovered that long residence times (above 75 s) correlate with decreased growth, metabolism, and productivity. We have calculated energy dissipation rates in the ATF transfer line and filter and empirically studied the impacts of increased exchange rates on cell culture, determining that increased hydrodynamic stress can lead to decreased cell size, lactate production, and specific productivity. Finally, we have conducted experiments to understand the relationship between filtration fluxes and ATF membrane fouling, finding that at fluxes above 60 L·m^–2·day ^–1, protein sieving coefficients see significant rates of decrease (greater than 1% per day). While most of these studies have been conducted with one cell line at one target viable cell density (40 million cells/ml), the general, directional knowledge arising from this study should be applicable to other conditions and programs, ultimately leading to more robust and well-designed perfusion processes.     

Recent advances in bioprocessing application of membrane chromatography

Compared to traditional chromatography using resins in packed-bed columns, membrane chromatography is a relatively new and immature bioseparation technology based on the integration of membrane filtration and liquid chromatography into a single-stage operation. Over the past decades, advances in membrane chemistry have yielded novel membrane devices with high binding capacities and improved mass transfer properties, significantly increasing the bioprocessing efficiency for purification of biomolecules. Due to the disposable nature, low buffer consumption, and reduced equipment costs, membrane chromatography can significantly reduce downstream bioprocessing costs. In this review, we discuss technological merits and disadvantages associated with membrane chromatography as well as recent bioseparation applications with a particular attention on purification of large biomolecules.     

Development of a novel and efficient cell culture flocculation process using a stimulus responsive polymer to streamline antibody purification processes

Recent advances in mammalian cell culture processes have significantly increased product titers, but have also resulted in substantial increases in cell density and cellular debris as well as process and product related impurities. As such, with improvements in titer, corresponding improvements in downstream processing are essential. In this study we have developed an alternative antibody harvest process that incorporates flocculation using a novel stimulus responsive polymer, benzylated poly(allylamine), followed by depth filtration. As tested on multiple antibodies, this process demonstrates high process yield, improved clearance of cells and cell debris, and efficient reduction of aggregates, host cell proteins (HCP) and DNA. A wide operating window was established for this novel flocculation process through design of experiments condition screening and optimization. Residual levels of impurities in the Protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. In addition, efficient clearance of residual polymer was demonstrated using a fluorescence tagged polymer in the presence of a stimulus reagent. The mechanism of HCP and aggregates removal during flocculation was also explored. This novel and efficient process can be easily integrated into current mAb purification platforms, and may overcome downstream processing challenges. Biotechnol. Bioeng. 2013;110: 2928–2937. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Flow cytometry and two-dimensional electrophoresis (2-DE) for system evaluation of long term continuous perfused animal cell cultures in macroporous beads

Immobilization of r-CHO cells at high density using macroporous polyethylene carriers in a modular fluidized bed reactor is demonstrated. Specific growth rates of the cells are measured by incorporation of BrdU. At a cell density of about 10⁸ cells/ml a stable growth rate of 0.004 h⁻¹ was established. Total release of proteins into the culture supernatant during protein-free perfusion was analyzed by 2-DE in various phases of the long-term culture showing very similar patterns indicating a constant pattern of gene expression.