Robust depth filter sizing for centrate clarification

Cellulosic depth filters embedded with diatomaceous earth are widely used to remove colloidal cell debris from centrate as a secondary clarification step during the harvest of mammalian cell culture fluid. The high cost associated with process failure in a GMP (Good Manufacturing Practice) environment highlights the need for a robust process scale depth filter sizing that allows for (1) stochastic batch‐to‐batch variations from filter media, bioreactor feed and operation, and (2) systematic scaling differences in average performance between filter sizes and formats. Matched‐lot depth filter media tested at the same conditions with consecutive batches of the same molecule were used to assess the sources and magnitudes of process variability. Depth filter sizing safety factors of 1.2–1.6 allow a filtration process to compensate for random batch‐to‐batch process variations. Matched‐lot depth filter media in four different devices tested simultaneously at the same conditions was used with a common feed to assess scaling effects. All filter devices showed <11% capacity difference and the Pod format devices showed no statistically different capacity differences. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1542–1550, 2015     

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/m² equivalent gravity settling area per hour followed by a resultant required depth filter area of 0.07 m²/L supernatant). A range of USD predictions was verified at the pilot scale for centrifugation followed by depth filtration. © 2016 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 32:382–392, 2016     

Depth filter material process interaction in the harvest of mammalian cells

Upstream advances have led to increased mAb titers above 5 g/L in 14-day fed-batch cultures. This is accompanied by higher cell densities and process-related impurities such as DNA and Host Cell Protein (HCP), which have caused challenges for downstream operations. Depth filtration remains a popular choice for harvesting CHO cell culture, and there is interest in utilizing these to remove process-related impurities at the harvest stage. Operation of the harvest stage has also been shown to affect the performance of the Protein A chromatography step. In addition, manufacturers are looking to move away from natural materials such as cellulose and Diatomaceous Earth (DE) for better filter consistency and security of supply. Therefore, there is an increased need for further understanding and knowledge of depth filtration. This study investigates the effect of depth filter material and loading on the Protein A resin lifetime with an industrially relevant high cell density feed material (40 million cells/ml). It focuses on the retention of process-related impurities such as DNA and HCP through breakthrough studies and a novel confocal microscopy method for imaging foulant in-situ. An increase in loading of the primary-synthetic filter by a third, led to earlier DNA breakthrough in the secondary filter, with DNA concentration at a throughput of 50 L/m² being more than double. Confocal imaging of the depth filters showed that the foulant was pushed forward into the filter structure with higher loading. The additional two layers in the primary-synthetic filter led to better pressure profiles in both primary and secondary filters but did not help to retain HCP or DNA. Reduced filtrate clarity, as measured by OD600, was 1.6 fold lower in the final filtrate where a synthetic filter train was used. This was also associated with precipitation in the Protein A column feed. Confocal imaging of resin after 100 cycles showed that DNA build-up around the outside of the bead was associated with synthetic filter trains, leading to potential mass transfer problems.     

Process cost and facility considerations in the selection of primary cell culture clarification technology

The bioreactor volume delineating the selection of primary clarification technology is not always easily defined. Development of a commercial scale process for the manufacture of therapeutic proteins requires scale‐up from a few liters to thousands of liters. While the separation techniques used for protein purification are largely conserved across scales, the separation techniques for primary cell culture clarification vary with scale. Process models were developed to compare monoclonal antibody production costs using two cell culture clarification technologies. One process model was created for cell culture clarification by disc stack centrifugation with depth filtration. A second process model was created for clarification by multi‐stage depth filtration. Analyses were performed to examine the influence of bioreactor volume, product titer, depth filter capacity, and facility utilization on overall operating costs. At bioreactor volumes <1,000 L, clarification using multi‐stage depth filtration offers cost savings compared to clarification using centrifugation. For bioreactor volumes >5,000 L, clarification using centrifugation followed by depth filtration offers significant cost savings. For bioreactor volumes of ～2,000 L, clarification costs are similar between depth filtration and centrifugation. At this scale, factors including facility utilization, available capital, ease of process development, implementation timelines, and process performance characterization play an important role in clarification technology selection. In the case study presented, a multi‐product facility selected multi‐stage depth filtration for cell culture clarification at the 500 and 2,000 L scales of operation. Facility implementation timelines, process development activities, equipment commissioning and validation, scale‐up effects, and process robustness are examined. © 2013 The Authors. American Institute of Chemical Engineers Biotechnol. Prog., 29:1239–1245, 2013     

An effective,simple and low-cost pretreatment for culture clarification in tetanus toxoid production

Abstract

Chemically inactivated tetanus toxin (tetanus toxoid, TT), purified from cultures of a virulent Clostridium tetani strain, is the active pharmaceutical ingredient of anti-tetanus vaccines. Culture clarification for TT production and is usually performed by filtration-based techniques. Final clarification of the culture supernatant is achieved by passage through 0.2?µm pore size filtering membranes. Large particles removal (primary clarification) before final filtration (secondary clarification) reduces costs of the overall clarification process. With this aim, chitosan-induced particle aggregation was assessed as an alternative for primary clarification. Three chitosan variants were tested with similar results. Optimal clarification of culture supernatant was achieved by the addition of 8?mg chitosan per l of culture. Extrapolation analysis of filter sizing results indicate that 100?l of chitosan-treated supernatant can be finally filtered with a 0.6 m2 normal filtration cartridge of 0.45?+?0.2?µm pore size. The clarified material is compatible with current standard downstream processing techniques for TT purification. Thus, chitosan-induced particle aggregation is a suitable operation for primary clarification.     

Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth

The increased cell density and product titer in biomanufacturing have led to greater use of depth filtration as part of the initial clarification of cell culture fluid, either as a stand-alone unit operation or after centrifugation. Several recent studies have shown that depth filters can also reduce the concentration of smaller impurities like host cell proteins (HCP) and DNA, decreasing the burden on subsequent chromatographic operations. The objective of this study was to evaluate the HCP removal properties of the Pall PDH4 depth filter media, a model depth filter containing diatomaceous earth, cellulose fibers, and a binder. Experiments were performed with both cell culture fluid (CCF) and a series of model proteins with defined pI, molecular weight, and hydrophobicity chosen to match the range of typical HCP. The location of adsorbed (fluorescently labeled) proteins within the depth filters was determined using confocal scanning laser microscopy. Protein binding was greater for proteins that were positively charged and more hydrophobic, consistent with adsorption to the negatively charged diatomaceous earth. The lowest degree of binding was seen with proteins near their pI, which were poorly removed by this filter. These results provide new mechanistic insights into the factors governing the filter capacity and performance characteristics of depth filters containing diatomaceous earth that are widely used in the clarification of CCF.     

Upflow biological filtration with floating filter media

An aerobic biological filter with floating filter media was tested using domestic wastewater to determine the optimum operating and backwashing parameters in this study. This system was designed for a small-scale joint treatment plant and 4 mm diameter polystyrene foam pellets were used as the filter media. As the backwash is the most important point for using a floating biofilter, “air shot” system, a turbulent-flow backwashing utilising the air reverse syphon and the buoyancy of the filter media, was developed for this purpose. Through the test, the optimum position of the diffuser tube and the structure of the backwashing device were determined. It was confirmed that the system could achieve effluent biological oxygen demand (BOD) of under 10 mg/l, and a nitrification rate of over 86% at a BOD loading of 0.7 kg/m³ per day and “total-nitrogen (T-N) loading” of 0.16 kg/m³ per day. The backwashing frequency should be as many times as possible per cycle to remove the sloughed sludge thoroughly in order to maintain good effluent quality after backwashing.     

Pretreatments for enhancing clarification efficiency of depth filtration during production of monoclonal antibody therapeutics

High cell density, high product titer mammalian cell culture is the new paradigm for production of recombinant proteins. While the typical motivation is to get a high product titer, additional undesirable outcomes often include an increase in percentage solids in the cell culture fluid (cellular debris and sub-micron colloids), thereby offering new challenges to downstream processing. This article focuses on scouting and comparison of different approaches used for clarification of cell culture fluid. The approaches include centrifugation followed by depth filtration, direct depth filtration without centrifugation and feed pretreatment with use of specially designed density gradient filtration to improve efficiency of clarification and removal of process contaminants from feed stream. The work also evaluates impact of three different pretreatment approaches, namely pH adjustment to acidic condition, metal cation (calcium phosphate) flocculation, and polycationic polymer flocculation (using polymer-I and polymer-II). The results obtained indicate that the use of pretreatment significantly improves the clarification efficiency of depth filtration. Pretreatment options like polycationic polymer-I based flocculation resulted in a >5 fold reduction in filter area requirement as well as >6 fold reduction in HCDNA while retaining acceptable recovery of the IgG (>98%). Thus, pretreatment offers a significant reduction in the depth filtration footprint (~5–6 fold decrease in filter area requirement). However, one must take into consideration the process development time required, capital cost, consumable cost, cost of the pretreatment chemical, cost of testing to demonstrate clearance of treatment agent, ease of scale-ability, and process robustness when finalizing the optimal clarification approach.     

Clarification of recombinant proteins from high cell density mammalian cell culture systems using new improved depth filters

Increasingly high cell density, high product titer cell cultures containing mammalian cells are being used for the production of recombinant proteins. These high productivity cultures are placing a larger burden on traditional downstream clarification and purification operations due to higher product and impurity levels. Controlled flocculation and precipitation of mammalian cell culture suspensions by acidification or using polymeric flocculants have been employed to enhance clarification throughput and downstream filtration operations. While flocculation is quite effective in agglomerating cell debris and process related impurities such as (host cell) proteins and DNA, the resulting suspension is generally not easily separable solely using conventional depth filtration techniques. As a result, centrifugation is often used for clarification of cells and cell debris before filtration, which can limit process configurations and flexibility due to the investment and fixed nature of a centrifuge. To address this challenge, novel depth filter designs were designed which results in improved primary and secondary direct depth filtration of flocculated high cell density mammalian cell cultures systems feeds, thereby providing single‐use clarification solution. A framework is presented here for optimizing the particle size distribution of the mammalian cell culture systems with the pore size distribution of the gradient depth filter using various pre‐treatment conditions resulting in increased depth filter media utilization and improved clarification capacity. Feed conditions were optimized either by acidification or by polymer flocculation which resulted in the increased average feed particle‐size and improvements in throughput with improved depth filters for several mammalian systems. Biotechnol. Bioeng. 2013; 110: 1964–1972. © 2013 Wiley Periodicals, Inc.     

A membrane filter direct count technique for enumeratingBdellovibrio

A membrane filter direct count method was devised for enumeratingBdellovibrio cells in “clean” suspensions. The procedure involves filtering a specified volume of a diluted, Trisbuffered suspension ofBdellovibrio cells through a known area of a 100-nm-pore-size Millipore brand membrane filter. A clarification solvent was used to render the filter transparent, so that the bdeloyvibrios on the filter could be photomicrographed and counted either visually or by means of a Quantimet 720 Image Analyzing Computer. The number ofBdellovibrio cells per milliliter in the undituted suspension could be calculated from the mean number of cells per unit area of the filter, the dilution factor, and the volume of diluted suspension filtered. TheBdellovibrio cells were distributed on the filters in a Poisson manner when there were not more than about 3.5 cells per 100 μm² of filter surface. The membrane filter direct counts correlated well with direct counts obtained by the Petroff-Hausser method. The correlation of direct counts with plaque (“viable”) counts showed that 80 to 95% of the direct-countedBdellovibrio cells in the clean suspensions were capable of forming plaques on lawns of suitable substrate bacteria. *** DIRECT SUPPORT *** A01R4002 00007     

Cyanobacteria passage through drinking water filters during perturbation episodes as a function of cell morphology, coagulant and initial filter loading rate

Eight pilot-scale in-line filtration trials were performed to evaluate the passage of cyanobacterial cells through drinking water filters after sudden increases in hydraulic loading rates. Trials were performed at 30 °C using two coagulant combinations (aluminum sulfate and cationic polymer or ferric chloride and cationic polymer), two initial filter loading rates (7 or 10 m/h) and two species of morphologically different cyanobacteria (Microcystis aeruginosa or Anabaena flos aquae). The filter was perturbed by instantaneously increasing the hydraulic loading rate by 50%. Filter influent and effluent water qualities were characterized by measuring turbidity, particles and chlorophyll a. The observed post-perturbation filter effluent chlorophyll a peaks were 1.6–48 times greater than the pre-perturbation averages. Chlorophyll a peaks were larger for M. aeruginosa than for A. flos aquae. Chlorophyll a peaks were also larger for the higher (10 m/h) than for the lower (7 m/h) initial filter loading rate. The post-perturbation effluent turbidity peaks were 1.4–7.2 times greater than the pre-perturbation averages. The post-perturbation effluent particle peaks were 6.5–25 times greater than the pre-perturbation averages. These results indicate that particles were a more sensitive indicator of cyanobacterial passage than turbidity.     

The use of artificial neural networks to reduce data collection demands in determining spine loading: a laboratory based analysis

The extensive data requirements of three-dimensional inverse dynamics and joint modelling to estimate spinal loading prevent the implementation of these models in industry and may hinder development of advanced injury prevention standards. This work examines the potential of feed forward artificial neural networks (ANNs) as a data reduction approach and compared predictions to rigid link and EMG-assisted models. Ten males and ten females performed dynamic lifts, all approaches were applied and comparisons of predicted joint moments and joint forces were evaluated. While the ANN under- predicted peak extension moments (p = 0.0261) and joint compression (p < 0.0001), predictions of cumulative extension moments (p = 0.8293) and cumulative joint compression (p = 0.9557) were not different. Therefore, the ANNs proposed may be used to obtain estimates of cumulative exposure variables with reduced input demands; however they should not be applied to determine peak demands of a worker's exposure.     

Prediction of viral filtration performance of monoclonal antibodies based on biophysical properties of feed

Controlling viral contamination is an important issue in the process development of monoclonal antibodies (MAbs) produced from mammalian cell lines. Virus filtration (VF) has been demonstrated to be a robust and effective clearance step which can provide ≥4 logs of reduction via size exclusion. The minimization of VF area by increasing flux and filter loading is critical to achieving cost targets as VFs are single use and often represent up to 10% of total purification costs. The research presented in this publication describes a development strategy focused on biophysical attributes of product streams that are directly applicable to VF process performance. This article summarizes a case study where biophysical tools (high‐pressure size exclusion chromatography, dynamic light scattering, and absolute size exclusion chromatography) were applied to a specific MAb program to illustrate how changes in feed composition (pH, sodium chloride concentration, and buffer salt type) can change biophysical properties which correlate with VF performance. The approach was subsequently refined and expanded over the course of development of three MAbs where performance metrics (i.e., loading and flux) were evaluated for two specific virus filters (Viresolve Pro and Planova 20N) during both unspiked control runs and virus clearance experiments. The analyses of feed attributes can be applied to a decision tree to guide the recommendation of a VF filter and operating conditions for use in future MAb program development. The understanding of the biophysical properties of the feed can be correlated to virus filter performance to significantly reduce the mass of product, time, and costs associated with virus filter step development. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:765–774, 2015     

Flocculation increases the efficacy of depth filtration during the downstream processing of recombinant pharmaceutical proteins produced in tobacco

Flocculation is a cost‐effective method that is used to improve the efficiency of clarification by causing dispersed particles to clump together, allowing their removal by sedimentation, centrifugation or filtration. The efficacy of flocculation for any given process depends on the nature and concentration of the particulates in the feed stream, the concentration, charge density and length of the flocculant polymer, the shear rate, the properties of the feed stream (e.g. pH and ionic strength) and the properties of the target products. We tested a range of flocculants and process conditions using a design of experiments approach to identify the most suitable polymers for the clarification step during the production of a HIV‐neutralizing monoclonal antibody (2G12) and a fluorescent marker protein (DsRed) expressed in transgenic tobacco leaves. Among the 23 different flocculants we tested, the greatest reduction in turbidity was achieved with Polymin P, a branched, cationic polyethylenimine with a charge density of 13.0 meq/g. This flocculant reduced turbidity by more than 90% under a wide range of process conditions. We developed a model that predicted its performance under different process conditions, and this enabled us to increase the depth filter capacity three–sevenfold depending on the process scale, depth filter type and plant species. The costs of filter consumables were reduced by more than 50% compared with a process without flocculant, and there was no loss of recovery for either 2G12 or DsRed.     

Differentiation of probiotic and environmental Saccharomyces cerevisiae strains in animal feed

Aims: To establish an identification system for probiotic Saccharomyces cerevisiae strains based on artificial neural network (ANN)–assisted Fourier‐transform infrared (FTIR) spectroscopy to improve quality control of animal feed. Methods and Results: The ANN‐based system for differentiating environmental from probiotic S. cerevisiae strains comprises five authorized feed additive strains plus environmental strains isolated from different habitats. A total of 108 isolates were used as reference strains to create the ANN. DHPLC analysis and δ‐PCR were used as reference methods to type probiotic yeast isolates. The performance of the FTIR‐ANN was tested in an internal validation using unknown spectra of each reference strain. This validation step yielded a classification rate of 99·1 %. For an external validation, a test data set comprising 965 spectra of 63 probiotic and environmental S. cerevisiae isolates unknown to the ANN was used, resulting in a classification rate of 98·2 %. Conclusions: Our results demonstrate that probiotic S. cerevisiae strains in feed can be differentiated successfully from environmental isolates using both genotypic approaches and ANN‐based FTIR spectroscopy. Significance and Impact of the Study: FTIR‐based artificial neural network analysis provides a rapid and inexpensive technique for yeast identification both at the species and at the strain level in routine diagnostic laboratories, using a single sample preparation.     

Predictive modelling of Fe(III) precipitation in iron removal process for bioleaching circuits

In this study, the applicability of three modelling approaches was determined in an effort to describe complex relationships between process parameters and to predict the performance of an integrated process, which consisted of a fluidized bed bioreactor for Fe³⁺ regeneration and a gravity settler for precipitative iron removal. Self-organizing maps were used to visually evaluate the associations between variables prior to the comparison of two different modelling methods, the multiple regression modelling and artificial neural network (ANN) modelling, for predicting Fe(III) precipitation. With the ANN model, an excellent match between the predicted and measured data was obtained (R ² = 0.97). The best-fitting regression model also gave a good fit (R ² = 0.87). This study demonstrates that ANNs and regression models are robust tools for predicting iron precipitation in the integrated process and can thus be used in the management of such systems.     

Nitrification of synthetic wastewater in a cross flow medium trickling filter

The nitrification performance of a synthetic wastewater was investigated in a cross flow medium trickling filter. The attachment of biomass on packing medium was studied. The reactor was operated continuously for three different hydraulic loading rates 5, 9 and 13?m³/m²?·?d under various influent ammonium concentrations. The maximum specific surface nitrification rate was about 1.21?g/m²?·?d. There was a good agreement between actual and predicted alkalinity consumption. The ammonium conversion along the filter depth was also investigated. The external surface of the nitrifying biofilm was captured by scanning electron microscope. The results are found to be satisfactory.     

Phytoextraction potential of water fern (Azolla pinnata) in the removal of a hazardous dye,methyl violet 2B: Artificial neural network modelling

This study investigated the potential of Azolla pinnata (AP) in the removal of toxic methyl violet 2B (MV) dye wastewater using the phytoextraction approach with the inclusion of an Artificial Neural Network (ANN) modelling. Parameters examined included the effects of dye concentration, pH and plant dosage. The highest removal efficiency was 93% which was achieved at a plant dosage of 0.8 g (dye volume = 200 mL, initial pH = 6.0, initial dye concentration = 10 mg L^?1). A significant decrease in relative frond number (RFN), a growth rate estimator, observed at a dye concentration of 20 mg L^?1 MV indicated some toxicity, which coincided with the plant pigments studies where the chlorophyll a content was lower than the control. There were little differences in the plant pigment contents between the control and those in the presence of dye (5 to 15 mg L^?1) indicating the tolerance of AP to MV at lower concentrations. A three-layer ANN model was optimized (6 neurons in the hidden layer) and successfully predicted the phytoextraction of MV (R = 0.9989, RMSE = 0.0098). In conclusion, AP proved to be a suitable plant that could be used for the phytoextraction of MV while the ANN modelling has shown to be a reliable method for the modelling of phytoextraction of MV using AP.     

Leveraging mathematical models for optimizing filter utility at manufacturing scale

In the production of biopharmaceuticals depth filters followed by sterile filters are often employed to remove residual cell debris present in the feed stream. In the back drop of a global pandemic, supply chains associated with the production of biopharmaceuticals have been constrained. These constraints have limited the available amount of depth filters for the manufacture of biologics. This has placed manufacturing facilities in a difficult position having to choose between running processes with reduced number of depth filters and risking a failed batch or the prospect of plants going into temporary shutdown until the depth filter resources are replenished. This communication describes a modeling based method that leverages manufacturing scale filtration data to predict the depth filter performance with a reduced number of filters and an increased operational flux. This method can be used to quantify the acceptable level of area reduction before which the filtration process performance is affected. This enables facilities to manage their filter inventory avoiding potential plant shutdowns and reduces the risks of negative depth filter performance.