Liposome Sterile Filtration Characterization via X-ray Computed Tomography and Confocal Microscopy.

Two high resolution, 3D imaging techniques were applied to visualize and characterize sterilizing grade dual-layer filtration of liposomes, enabling membrane structure to be related with function and performance. Two polyethersulfone membranes with nominal retention ratings of 650 nm and 200 nm were used to filter liposomes of an average diameter of 143 nm and a polydispersity index of 0.1. Operating conditions including differential pressure were evaluated. X-ray computed tomography at a pixel size of 63 nm was capable of resolving the internal geometry of each membrane. The respective asymmetry and symmetry of the upstream and downstream membranes could be measured, with pore network modeling used to identify pore sizes as a function of distance through the imaged volume. Reconstructed 3D digital datasets were the basis of tortuous flow simulation through each porous structure. Confocal microscopy visualized liposome retention within each membrane using fluorescent dyes, with bacterial challenges also performed. It was found that increasing pressure drop from 0.07 MPa to 0.21 MPa resulted in differing fluorescent retention profiles in the upstream membrane. These results highlighted the capability for complementary imaging approaches to deepen understanding of liposome sterilizing grade filtration.

The use of a surface active agent in the protection of a fusion protein during bioprocessing.

The bioprocessing of a fusion protein is characterised by low yields and at a series of recovery and purification stages that leads to an overall 90% loss. Much of this apparent loss is due to the denaturation of a protein, missing a vital affinity ligand. However, there is evidence of the protection of degradation products which occurs in the presence of shear plus air/liquid interfaces. This study seeks out to characterise the loss and use ultra-scale-down studies to predict its occurrence and hence shows these may be diminished by the use of protective reagents such as Pluronic F68.

Evaluation of options for harvest of a recombinant E. Coli fermentation producing a domain antibody using ultra scale-down techniques and pilot-scale verification.

Ultra scale-down (USD) methods operating at the millilitre scale were used to characterise full-scale processing of E. coli fermentation broths autolysed to different extents for release of a domain antibody. The focus was on the primary clarification stages involving continuous centrifugation followed by depth filtration. The performance of this sequence was predicted by USD studies to decrease significantly with increased extents of cell lysis. The use of polyethyleneimine reagent was studied to treat the lysed cell broth by precipitation of soluble contaminants such as DNA and flocculation of cell debris material. The USD studies were used to predict the impact of this treatment on the performance and here it was found that the fermentation could be run to maximum productivity using an acceptable clarification process (e.g., a centrifugation stage operating at 0.11 L/m(2) equivalent gravity settling area per hour followed by a resultant required depth filter area of 0.07 m(2) /L supernatant). A range of USD predictions was verified at the pilot scale for centrifugation followed by depth filtration. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:382-392, 2016.

Enhancing the selective extracellular location of a recombinant E. coli domain antibody by management of fermentation conditions.

The preparation of a recombinant protein using Escherichia coli often involves a challenging primary recovery sequence. This is due to the inability to secrete the protein to the extracellular space without a significant degree of cell lysis. This results in the release of nucleic acids, leading to a high viscosity, difficulty to clarify, broth and also to contamination with cell materials such as lipopolysaccharides and host cell proteins. In this paper, we present different fermentation strategies to facilitate the recovery of a V H domain antibody (13.1 kDa) by directing it selectively to the extracellular space and changing the balance between domain antibody to nucleic acid release. The manipulation of the cell growth rate in order to increase the outer cell membrane permeability gave a small ~1.5-fold improvement in released domain antibody to nucleic acid ratio without overall loss of yield. The introduction during fermentation of release agents such as EDTA gave no improvement in the ratio of released domain antibody to nucleic acid and a loss of overall productivity. The use of polyethyleneimine (PEI) during fermentation was with the aim to (a) permeabilise the outer bacterial membrane to release selectively domain antibody and (b) remove selectively by precipitation nucleic acids released during cell lysis. This strategy resulted in up to ~4-fold increase in the ratio of domain antibody to soluble nucleic acid with no reduction in domain antibody overall titre. In addition, a reduction in host cell protein contamination was achieved and there was no increase in endotoxin levels. Similar results were demonstrated with a range of other antibody products prepared in E. coli.

Ultra scale-down characterization of the impact of conditioning methods for harvested cell broths on clarification by continuous centrifugation-Recovery of domain antibodies from rec E. coli.

The processing of harvested E. coli cell broths is examined where the expressed protein product has been released into the extracellular space. Pre-treatment methods such as freeze-thaw, flocculation, and homogenization are studied. The resultant suspensions are characterized in terms of the particle size distribution, sensitivity to shear stress, rheology and solids volume fraction, and, using ultra scale-down methods, the predicted ability to clarify the material using industrial scale continuous flow centrifugation. A key finding was the potential of flocculation methods both to aid the recovery of the particles and to cause the selective precipitation of soluble contaminants. While the flocculated material is severely affected by process shear stress, the impact on the very fine end of the size distribution is relatively minor and hence the predicted performance was only diminished to a small extent, for example, from 99.9% to 99.7% clarification compared with 95% for autolysate and 65% for homogenate at equivalent centrifugation conditions. The lumped properties as represented by ultra scale-down centrifugation results were correlated with the basic properties affecting sedimentation including particle size distribution, suspension viscosity, and solids volume fraction. Grade efficiency relationships were used to allow for the particle and flow dynamics affecting capture in the centrifuge. The size distribution below a critical diameter dependent on the broth pre-treatment type was shown to be the main determining factor affecting the clarification achieved.

Bioengineering the liver: scale-up and cool chain delivery of the liver cell biomass for clinical targeting in a bioartificial liver support system.

Acute liver failure has a high mortality unless patients receive a liver transplant; however, there are insufficient donor organs to meet the clinical need. The liver may rapidly recover from acute injury by hepatic cell regeneration given time. A bioartificial liver machine can provide temporary liver support to enable such regeneration to occur. We developed a bioartificial liver machine using human-derived liver cells encapsulated in alginate, cultured in a fluidized bed bioreactor to a level of function suitable for clinical use (performance competence). HepG2 cells were encapsulated in alginate using a JetCutter to produce ∼500 µm spherical beads containing cells at ∼1.75 million cells/mL beads. Within the beads, encapsulated cells proliferated to form compact cell spheroids (AELS) with good cell-to-cell contact and cell function, that were analyzed functionally and by gene expression at mRNA and protein levels. We established a methodology to enable a ∼34-fold increase in cell density within the AELS over 11-13 days, maintaining cell viability. Optimized nutrient and oxygen provision were numerically modeled and tested experimentally, achieving a cell density at harvest of >45 million cells/mL beads; >5×10(10) cells were produced in 1100 mL of beads. This process is scalable to human size ([0.7-1]×10(11)). A short-term storage protocol at ambient temperature was established, enabling transport from laboratory to bedside over 48 h, appropriate for clinical translation of a manufactured bioartificial liver machine.

An ultra scale-down approach to study the interaction of fermentation, homogenization, and centrifugation for antibody fragment recovery from rec E. coli.

Escherichia coli is frequently used as a microbial host to express recombinant proteins but it lacks the ability to secrete proteins into medium. One option for protein release is to use high-pressure homogenization followed by a centrifugation step to remove cell debris. While this does not give selective release of proteins in the periplasmic space, it does provide a robust process. An ultra scale-down (USD) approach based on focused acoustics is described to study rec E. coli cell disruption by high-pressure homogenization for recovery of an antibody fragment (Fab') and the impact of fermentation harvest time. This approach is followed by microwell-based USD centrifugation to study the removal of the resultant cell debris. Successful verification of this USD approach is achieved using pilot scale high-pressure homogenization and pilot scale, continuous flow, disc stack centrifugation comparing performance parameters such as the fraction of Fab' release, cell debris size distribution and the carryover of cell debris fine particles in the supernatant. The integration of fermentation and primary recovery stages is examined using USD monitoring of different phases of cell growth. Increasing susceptibility of the cells to disruption is observed with time following induction. For a given recovery process this results in a higher fraction of product release and a greater proportion of fine cell debris particles that are difficult to remove by centrifugation. Such observations are confirmed at pilot scale.

An ultra scale-down characterization of low shear stress primary recovery stages to enhance selectivity of fusion protein recovery from its molecular variants.

Fusion proteins offer the prospect of new therapeutic products with multiple functions. The primary recovery is investigated of a fusion protein consisting of modified E2 protein from hepatitis C virus fused to human IgG1 Fc and expressed in a Chinese hamster ovary (CHO) cell line. Fusion protein products inevitably pose increased challenge in preparation and purification. Of particular concerns are: (i) the impact of shear stress on product integrity and (ii) the presence of product-related contaminants which could prove challenging to remove during the high resolution purification steps. This paper addresses the use of microwell-based ultra scale-down (USD) methods to develop a bioprocess strategy focused on the integration of cell culture and cell removal operations and where the focus is on the use of operations which impart low shear stress levels even when applied at eventual manufacturing scale. An USD shear device was used to demonstrate that cells exposed to high process stresses such as those that occur in the feed zone of a continuous non-hermetic centrifuge resulted in the reduction of the fusion protein and also the release of glycosylated intracellular variants. In addition, extended cell culture resulted in release of such variants. USD mimics of low shear stress, hydrohermetic feed zone centrifugation and of depth filtration were used to demonstrate little to no release during recovery of these variants with both results verified at pilot scale. Furthermore, the USD studies were used to predict removal of contaminants such as lipids, nucleic acids, and cell debris with, for example, depth filtration delivering greater removal than for centrifugation but a small (~10%) decrease in yield of the fusion protein. These USD observations of product recovery and carryover of contaminants were also confirmed at pilot scale as was also the capacity or throughput achievable for continuous centrifugation or for depth filtration. The advantages are discussed of operating a lower yield cell culture and a low shear stress recovery process in return for a considerably less challenging purification demand.

Use of focused acoustics for cell disruption to provide ultra scale-down insights of microbial homogenization and its bioprocess impact--recovery of antibody fragments from rec E. coli.

An ultra scale-down (USD) device that provides insight of how industrial homogenization impacts bioprocess performance is desirable in the biopharmaceutical industry, especially at the early stage of process development where only a small quantity of material is available. In this work, we assess the effectiveness of focused acoustics as the basis of an USD cell disruption method to mimic and study high-pressure, step-wise homogenization of rec Escherichia coli cells for the recovery of an intracellular protein, antibody fragment (Fab'). The release of both Fab' and of overall protein follows first-order reaction kinetics with respect to time of exposure to focused acoustics. The rate constant is directly proportional to applied electrical power input per unit volume. For nearly total protein or Fab' release (>99%), the key physical properties of the disruptate produced by focused acoustics, such as cell debris particle size distribution and apparent viscosity show good agreement with those for homogenates produced by high-pressure homogenization operated to give the same fractional release. The only key difference is observed for partial disruption of cells where focused acoustics yields a disruptate of lower viscosity than homogenization, evidently due to a greater extent of polynucleic acids degradation. Verification of this USD approach to cell disruption by high-pressure homogenization is achieved using USD centrifugation to demonstrate the same sedimentation characteristics of disruptates prepared using both the scaled-down focused acoustic and the pilot-scale homogenization methods for the same fraction of protein release.

Sub-population analysis of human cancer vaccine cells--ultra scale-down characterization of response to shear.

The allogeneic whole cell therapy field presents a novel challenge for cell line selection. Large numbers of candidates will he screened for desirable traits including cell growth rates and maintenance of critical product attributes. Screening cell lines for susceptibility to large scale processing conditions has become of increasing importance; innovators do not wish to face the quandary of clinically efficacious cell lines that show poor manufacturability. We have previously described an ultra scale-down method for assessing the impact of the hydrodynamic environment on human cells (McCoy et al., 2009). This body of work describes the validation of this experimental approach using a second, clinically tested, human prostate cancer cell line and describes an additional level of sub-population characterisation. The small-scale conditions set were similar to those expected in downstream process, formulation and vial filling operations with maximum shear rates ranging from 30 x 10(3) to 90 x 10(3) s(-1)(equivalent maximum power dissipation rates of 1.5 x 10(3) to 14 x 10(3) W kg(-1)). Changes to critical cell quality attributes such as membrane integrity retention, cell surface marker staining levels (CD9, CD59, CD147) and cell surface marker density is described. Evaluation of two sub-populations which are formed in response to shear showed distinct staining profiles. Identification of these phenotypically distinct populations, derived in response to the same hydrodynamic environment, poses an interesting bio-processing challenge with regards to both maintenance of product quality, but also highlights the potential benefit of process manipulation through shear-rate intervention with regards to product efficacy.

Mimic of a large-scale diafiltration process by using ultra scale-down rotating disc filter.

Ultra scale-down (USD) approach is a powerful tool to predict large-scale process performance by using very small amounts of material. In this article, we present a method to mimic flux and transmission performance in a labscale crossflow operation by an USD rotating disc filter (RDF). The Pellicon 2 labscale system used for evaluation of the mimic can readily be related to small pilot and industrial scale. Adopted from the pulsed sample injection technique by Ghosh and Cui (J Membr Sci. 2000;175:5-84), the RDF has been modified by building in inserts to allow the flexibility of the chamber volume, so that only 1.5 mL of processing material is required for each diafiltration experiment. The reported method enjoys the simplicity of dead-end mode operation with accurate control of operation conditions that can mimic well the crossflow operation in large scale. Wall shear rate correlations have been established for both the labscale cassette and the USD device, and a mimic has been developed by operating both scales under conditions with equivalent averaged shear rates. The studies using E. coli lysate show that the flux vs. transmembrane pressure profile follows a first-order model, and the transmission of antibody fragment (Fab') is independent of transmembrane pressure. Predicted flux and transmission data agreed well with the experimental results of a labscale diafiltration where the cassette resistance was considered.

Ultra scale-down studies of the effect of shear on cell quality; Processing of a human cell line for cancer vaccine therapy.

Whole cell therapy is showing potential in the clinic for the treatment of many chronic diseases. The translation of laboratory-scale methods for cell harvesting and formulation to commercial-scale manufacturing offers major bioprocessing challenges. This is especially the case when the cell properties determine the final product effectiveness. This study is focused on developing an ultra scale-down method for assessing the impact of the hydrodynamic environment on human cells that constitute the therapeutic product. Small volumes of a prostate cancer cell line, currently being developed in late phase II clinical trials as an allogeneic whole cell vaccine therapy for prostate cancer, were exposed to hydrodynamic shear rates similar to those present in downstream process, formulation and vial filling operations. A small scale rotating disc shear device (20 mL) was used over a range of disc speeds to expose cells to maximum shear rates ranging from 90 x 10(3) to 175 x 10(3) s(-1) (equivalent maximum power dissipation rates of 14 x 10(3) to 52 x 10(3) W kg(-1)). These cells were subsequently analyzed for critical cell quality attributes such as the retention of membrane integrity and cell surface marker profile and density. Three cell surface markers (CD9, CD147, and HLAA-C) were studied. The cell markers exhibited different levels of susceptibility to hydrodynamic shear but in all cases this was less than or equal to the loss of membrane integrity. It is evident that the marker, or combination or markers, which might provide the required immunogenic response, will be affected by hydrodynamic shear environment during bioprocessing, if the engineering environment is not controlled to within the limits tolerated by the cell components.

Large-scale plasmid DNA processing: evidence that cell harvesting and storage methods affect yield of supercoiled plasmid DNA.

The effect of bacterial-cell centrifugation and handling on the initial stages of plasmid processing was investigated. Escherichia coli cells containing either a 6 or 20 kb plasmid were grown in 75- and 450-litre bioreactors, and the process yield of the early recovery stages was characterized in terms of SC pDNA (supercoiled plasmid DNA) recovered. In all cases, the cells were totally recovered using either a continuous-feed, intermittent-solids-discharge, disc-stack centrifuge or a continuous-feed, batch-discharge, solid-bowl centrifuge. The cells were then either processed immediately or stored frozen. The centrifugation method considerably affected the yield of SC pDNA, and there was evidence that the intermittent discharge of cells from a centrifuge operating at high speed led to a sediment containing lysed cells and degraded pDNA. This led to estimated plasmid yield losses of up to 40% as compared with cells recovered from laboratory or solid-bowl centrifuges, where there is evidently no cell stress on discharge. By inference, the cell stress on feed to either of the continuous centrifuges studied was not implicated in product loss. Freezing of the recovered cells gives a convenient hold stage prior to further processing. In all cases, this extra freeze-thaw stage led to loss of SC pDNA, and this was in addition to the loss attributed to cell lysis during centrifugation discharge. Only average yields can be gained from pilot plant-scale studies; separate laboratory-based experiments indicated that this loss of SC pDNA is determined by the time and temperature for which the resuspended cells are held.

Regenerative medicine bioprocessing: building a conceptual framework based on early studies.

This paper reviews early studies of regenerative medicine using human cells and engineered tissues progressing from a laboratory-centered manual procedure toward automated manufacture. It then examines the distinctive bioprocesses by which autologous human material must be produced, the degree of simplification allowed by use of allogeneic cell lines and engineered tissue derived from them, and issues that affect both cell types. The paper concludes by drawing upon this discussion to suggest some factors that will determine how regenerative medicine bioprocessing can progress to provide many units of material economically.

Studies on aerosol delivery of plasmid DNA using a mesh nebulizer.

Aerosol delivery of plasmid DNA therapeutic solutions is promising for the treatment of respiratory diseases. However, it poses challenges, most significantly the need to protect the delicate supercoiled (sc) structure of plasmid during aerosolization. Nebulizers for liquid aerosolization using meshes appear a better method for delivery than conventional jet and ultrasonic nebulizers. This paper explores their application to the delivery of plasmid DNA. A computational fluid dynamics model of the dynamics of fluid flow through the nozzle of the MicroAIR mesh nebulizer indicated high strain rates (>10(5) s(-1)) near the nozzle exit capable of causing damage to the shear-sensitive plasmid DNA. Knowledge of the strain rates predicted using CFD and molecule size determined using atomic force microscopy (AFM) enabled estimation of the hydrodynamic force and whether damage of shear-sensitive therapeutics was likely. Plasmids of size 5.7 and 20 kb were aerosolized in the mesh nebulizer. The sc structure of the 5.7-kb plasmid was successfully delivered without damage, while aerosolization of the 20-kb plasmid led to disintegration of the pDNA sc structure as observed in AFM. Subsequent formulation of the sc 20-kb plasmid with PEI resulted in successful aerosol delivery. The maximum hydrodynamic forces computed for the aerosolization of structures of the size of 5.7-kb and PEI formulated 20-kb plasmids were less than the forces reported to damage the structure of double-stranded DNA. A combination of CFD analysis and structure analysis may be used to predict successful aerosol delivery in such a mesh nebulizer.

A methodology for centrifuge selection for the separation of high solids density cell broths by visualisation of performance using windows of operation.

Expression systems capable of growing to high cell densities are now readily available and are popular due to the benefits of increased product concentration. However, such high solids density cultures pose a major challenge for bioprocess engineers as choosing the right separation equipment and operating it at optimal conditions is crucial for efficient recovery. This study proposes a methodology for the rapid determination of suitable operating conditions for the centrifugal recovery of high cell density fermentation broths. An ultra scale-down (USD) approach for the prediction of clarification and dewatering levels achieved in a range of typical high-speed centrifuges is presented. Together with a visualisation tool, a Window of Operation, this provides for the rapid analysis of separation performance and evaluation of the available operating conditions, as an aid in the selection of the centrifuge equipment most appropriate for a given process duty. A case study examining centrifuge selection for the processing of a high cell density Pichia pastoris culture demonstrates the method. The study examines semi-continuous disc-stack centrifuges and batch-operated machines such as multi-chamber bowls and Carr Powerfuges. Performance is assessed based on the variables of clarification, dewatering and product yield. Inclusion of limits imposed by the centrifuge type and design, and operation itself, serve to constrain the process and to define the Windows of Operation. The insight gained from the case study provides a useful indication of the utility of the methodology presented and illustrates the challenges of centrifuge selection for the demanding case of high solids concentration feed streams.

A capillary cytometer method to quantitate viable virus particles based on early detection of viral antigens and cellular events within single cells.

The traditional plaque forming and TCID(50) methods to determine replication competent virus titres rely on several cycles of replication and infection to generate a plaque with an incubation period of 24-72 h post-infection typically required. We developed a method to quantify infective viral particles based on early detection of cellular events by capillary cytometry. The method uses a capillary cytometer as a precise cell counter that can discriminate infected from non-infected cells. The general protocol was developed using a Guava PCA, genetically modified HSV-1 virus and polyclonal antibodies against antigens expressed on the cell membrane. Infection was detected after 1 h incubation and a plateau in the number of infected cells was observed between 7 and 9 h. A good correlation between titres obtained by the plaque forming method and the proposed method was observed for a ratio of infected to total cells between 0.5 and 0.05. The rapid and automated analysis (10 s/1000 events acquired per sample) makes the method particularly useful for high-throughput applications. The proposed method can be extended easily to determine the titre of other viruses providing a powerful tool for virology and antiviral screening.

Effect of fouling on the capacity and breakthrough characteristics of a packed bed ion exchange chromatography column.

This study examined the impact of fouling with yeast homogenate on capacity and breakthrough performance of an ion exchange packed bed column. Column performance was assessed by analysis of breakthrough curves obtained with BSA as a test protein. The overall impact of fouling on breakthrough performance depended heavily on the level of clarification of the feed stream. Challenging the column with particulate-free homogenate caused no change in column performance. Loading successive small volumes of poorly clarified homogenate, interspersed with frequent column salt washes, did not alter significantly the column capacity. By contrast, when the column was challenged with an equivalent cumulative volume of poorly clarified homogenate, dynamic binding capacity decreased significantly and changes in breakthrough curves suggested increased intraparticle and external mass transfer limitations. These changes were ascribed to deposition of solid particulates in void spaces in the bed and colloidal contaminants in the bead pores.