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.     

应用TFF微孔过滤澄清白喉、破伤风毒素培养液及超滤浓缩的试验

用ＴＦＦ微孔过滤系统对白喉、破伤风毒素培养液分别进行了两次澄清过滤试验,澄清过滤后的毒素上清液立即用ＴＦＦ超滤系统浓缩至原培养液体积的１／１０－１／２０。白喉毒素总回收率分别为８６％、７６．６％,破伤风毒素总回收率为９４．１％、９２％。澄清过滤中白喉毒素平均Ｆ１ｕｘ分别为２３．４、１４．３Ｌ／ｍ２／ｈ,破伤风毒素平均Ｆｌｕｘ为２４及２２．９Ｌ／ｍ２／ｈ。结果表明破伤风两次试验有很好的一致性,白喉毒素回收率差别不甚明显,而两次试验Ｆｌｕｘ相差较大,ＴＦＦ微孔过滤系统用于澄清白喉培养液,滤膜使用后的清洗程序仍需改良,以提高膜滤过功能的恢复。     

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     

Impact of clarification strategy on chromatographic separations: Pre-processing of cell homogenates

This research focused on how the extent and type of primary solid-liquid separation can affect the performance of guard filtration and chromatography, in this instance hydrophobic interaction chromatography. The system used in the study was yeast (Saccharomyces cerevisiae) with the target molecule being an intracellular protein; alcohol dehydrogenase (ADH). As expected, loading more poorly clarified suspensions (both centrates and primary filtrates) required proportionally larger guard filtration areas. In addition for feed suspensions prepared by centrifugation, increased clarification led to greater column capacity. However, where filtration was used to achieve similar clarification considerably lower column capacity was achieved. These results were attributed to centrifugation leading to the aggregation of lipids and their subsequent removal in this form before application to the column. Clarification by filtration leaves such lipids in their original "soluble" state and hence they are not removed. The importance of the need to examine such interactive effects in bioprocess studies is discussed. This observation was confirmed with further analytical work into the nature of the aggregated material formed in the supernatant under centrifugation conditions. This material was only soluble in an organic solvent, and identified as phophatidylcholine and ergosterol as among the components removed by centrifugation and guard filtration as opposed to filtration and guard filtration.     

Wide-surface pore microfiltration membrane drastically improves sieving decay in TFF-based perfusion cell culture and streamline chromatography integration for continuous bioprocessing

Although several compelling benefits for bioprocess intensification have been reported, the need for a streamlined integration of perfusion cultures with capture chromatography still remains unmet. Here, a robust solution is established by conducting tangential flow filtration-based perfusion with a wide-surface pore microfiltration membrane. The resulting integrated continuous bioprocess demonstrated negligible retention of antibody, DNA, and host cell proteins in the bioreactor with average sieving coefficients of 98 ± 1%, 124 ± 28%, and 109 ± 27%, respectively. Further discussion regarding the potential membrane fouling mechanisms is also provided by comparing two membranes with different surface pore structures and the same hollow fiber length, total membrane area, and chemistry. A cake-growth profile is reported for the narrower surface pore, 0.65-µm nominal retention perfusion membrane with final antibody sieving coefficients ≤70%. Whereas the sieving coefficient remained ≥85% during 40 culture days for the wide-surface pore, 0.2-µm nominal retention rating membrane. The wide-surface pore structure, confirmed by scanning electron microscopy imaging, minimizes the formation of biomass deposits on the membrane surface and drastically improves product sieving. This study not only offers a robust alternative for integrated continuous bioprocess by eliminating additional filtration steps while overcoming sieving decay, but also provides insight into membranes' fouling mechanism.     

Development of isoporous microslit silicon nitride membranes for sterile filtration applications

The widely used 0.2/0.22 µm polymer sterile filters were developed for small molecule and protein sterile filtration but are not well-suited for the production of large nonprotein biological therapeutics, resulting in significant yield loss and production cost increases. Here, we report on the development of membranes with isoporous sub-0.2 μm rectangular prism pores using silicon micromachining to produce microslit silicon nitride (MSN) membranes. The very high porosity (~33%) and ultrathin (200 nm) nature of the 0.2 µm MSN membranes results in a dramatically different structure than the traditional 0.2/0.22 µm polymer sterile filter, which yielded comparable performance properties (including gas and hydraulic permeance, maximum differential pressure tolerance, nanoparticle sieving/fouling behavior). The results from bacteria retention tests, conducted according to the guidance of regulatory agencies, demonstrated that the 0.2 µm MSN membranes can be effectively used as sterile filters. It is anticipated that the results and technologies presented in this study will find future utility in the production of non-protein biological therapeutics and in other biological and biomedical applications.     

Enhancing Protein A performance in mAb processing: A method to reduce and rapidly evaluate host cell DNA levels during primary clarification

The use and impact of 3M™ Emphaze™ AEX Hybrid Purifier, a single-use, fully synthetic chromatographic product, was explored to reduce host cell DNA (HC-DNA) concentration during the primary clarification of a monoclonal antibody (mAb). An approximately 5-log reduction in HC-DNA was achieved at an Emphaze AEX Hybrid Purifier throughput of 200 L/m². The appreciable reduction in HC-DNA achieved during primary clarification enhanced Protein A chromatography performance, resulting in a sharper and narrower elution profile. In addition, a 24× improvement in host cell protein (HCP) removal and fewer impurities nonspecifically bound to the Protein A column were observed compared to those resulting from the use of depth filtration for clarification. The use of a rapid, qualitative acidification assay to facilitate HC-DNA monitoring was also investigated. This assay involves the acidification-induced precipitation of HC-DNA, enabling the easy and rapid detection of DNA breakthrough across purification media such as Emphaze AEX Hybrid Purifier by means of turbidimetric and particle size measurements.     

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.     

Single-stage chromatographic clarification of Chinese Hamster Ovary cell harvest reduces cost of protein production

A single-stage clarification was developed using a single-use chromatographic clarification device (CCD) to recover a recombinant protein from Chinese Hamster Ovary (CHO) harvest cell culture fluid (HCCF). Clarification of a CHO HCCF is a complex and costly process, involving multiple stages of centrifugation and/or depth filtration to remove cells and debris and to reduce process-related impurities such as host cell protein (HCP), nucleic acids, and lipids. When using depth filtration, the filter train consists of multiple filters of varying ratios, layers, pore sizes, and adsorptive properties. The depth filters, in combination with a 0.2-micron membrane filter, clarify the HCCF based on size-exclusion, adsorptive, and charge-based mechanisms, and provide robust bioburden control. Each stage of the clarification process requires time, labor, and utilities, with product loss at each step. Here, use of the 3M™ Harvest RC Chromatographic Clarifier, a single-stage CCD, is identified as an alternative strategy to a three-stage filtration train. The CCD results in less overall filter area, less volume for flushing, and higher yield. Using bioprocess cost modeling, the single-stage clarification process was compared to a three-stage filtration process. By compressing the CHO HCCF clarification to a single chromatographic stage, the overall cost of the clarification process was reduced by 17%–30%, depending on bioreactor scale. The main drivers for the cost reduction were reduced total filtration area, labor, time, and utilities. The benefits of the single-stage harvest process extended throughout the downstream process, resulting in a 25% relative increase in cumulative yield with comparable impurity clearance.     

Using the F/R-ratio for an evaluation of the ability of the demosponge Halichondria panicea to nourish solely on phytoplankton versus free-living bacteria in the sea

The F/R-ratio (litres of water filtered per ml of oxygen respired) was determined for the filter-feeding demosponge Halichondria panicea to be 15.5?l?H₂O?(ml O₂)^?1 which was used to evaluate the potential of the sponge to nourish solely on nano- (2–20?µm) and micro- (20–200?µm) phytoplankton cells in the sea. It was estimated that in order to balance the maintenance requirement of H. panicea the necessary content of suspended particulate organic carbon must be at least 0.03?mg?C?l^?1, which may be compared with actually reported values of 0.04 to 0.2?mg?C?l^?1 thus implying that H. panicea may be able to nourish on a sole diet of phytoplankton in nature. However, the amount of carbon represented by free-living heterotrophic bacteria, cyanobacteria and other small (0.2–2?µm) picoplankton which are also accessible to the sponge lies in the range of 0.05–0.10?mg?C?l^?1, and therefore bacteria seem to be an important, although in many cases apparently a somewhat insufficient food source relative to phytoplankton. Video-microscope observations of the osculum cross-sectional area (OSA) and simultaneous measurement of the filtration rate of H. panicea showed that the filtration rate varied considerably over time concurrently with often pronounced variations in the OSA caused by disturbance when the aquarium through-flow was stopped during filtration rate measurements in the laboratory. It is concluded that the optimal and undisturbed filtration rate may be considerably higher than measured here, i.e. 6.1?ml water (ml sponge)^?1 min^?1, thus increasing the F/R-ratio to > 15.5?l?H₂O (ml O₂)^?1, which is comparable to values for more advanced eumetazoan filter-feeding marine invertebrates grazing on phytoplankton.     

Extracellular biosynthesis,characterization, optimization of silver nanoparticles (AgNPs) using Bacillus mojavensis BTCB15 and its antimicrobial activity against multidrug resistant pathogens

Abstract

Biosynthesis of metal nanoparticles is an area of interest among researchers because of its eco-friendly approach. Current study focuses at biosynthesis of silver nanoparticles (AgNPs) and optimization of physico-chemical conditions to obtain mono-dispersed and stable AgNPs having antimicrobial activity. Initially Bacillus mojavensis BTCB15 produced silver nanoparticles (AgNPs) of 105?nm. Silver nanoparticles (AgNPs) were characterized by particle size analyzer, UV-Vis Spectroscopy, Fourier transforms infrared spectroscopy (FTIR), Atomic force microscopy (AFM), and X-ray diffraction (XRD). Whereas, under optimal conditions of temperature 55?°C, pH 8, addition of surfactant Tween 20, and metal ion K₂SO₄, about 104% size reduction was achieved with average size of 2.3nm. Molecular characterization revealed 98% sequence homology with Bacillus mojavensis. AgNPs exhibited antibacterial activity at concentrations ranging from 0.5 to 2.5?µg/µl against Escherichia coli BTCB03, Klebsiella pneumonia BTCB04, Acinetobacter sp. BTCB05, and Pseudomonas aeruginosa BTCB01 but none against Staphylococcus aureus BTCB02. Highest antibacterial activity was observed at 0.27?µg/µl and lowest at 0.05?µg/µl of AgNPs indicated by zone of inhibition. Conclusively, under optimum conditions, Bacillus mojavensis BTCB15 was able to produce AgNPs of 2.3?nm size and had antibacterial activity against multi drug resistant pathogens.     

Picoplankton size distributions in marine and fresh waters: problems with filter fractionation studies

Abstract The retention of algal picoplankton by Nuclepore polycarbonate filters of 0.2, 1.0, 2.0 and 3.0 μm pore size was tested in 2 marine and 3 freshwater sites. When 1 μm Nuclepore filters were used, the percentage of the total cyanobacterial cells passing the filter varied between sites and with increasing depth within sites. As much as 99% of the Synechococcus -like cells was retained by a 1 μm filter. This could lead to an underestimation of the picoplanktonic contribution or, more seriously, an apparent distribution pattern that is an artifact of the choice of filter pore size. Filter retention was also dependent on vaccum pressure during filtration. This study emphasizes the need for direct observation of picoplankton numbers in filter fractionation studies.     

Virus harvesting in perfusion culture: Choosing the right type of hollow fiber membrane

The use of bioreactors coupled to membrane-based perfusion systems enables very high cell and product concentrations in vaccine and viral vector manufacturing. Many virus particles, however, are not stable and either lose their infectivity or physically degrade resulting in significant product losses if not harvested continuously. Even hollow fiber membranes with a nominal pore size of 0.2 µm can retain much smaller virions within a bioreactor. Here, we report on a systematic study to characterize structural and physicochemical membrane properties with respect to filter fouling and harvesting of yellow fever virus (YFV; ~50 nm). In tangential flow filtration perfusion experiments, we observed that YFV retention was only marginally determined by nominal but by effective pore sizes depending on filter fouling. Evaluation of scanning electron microscope images indicated that filter fouling can be reduced significantly by choosing membranes with (i) a flat inner surface (low boundary layer thickness), (ii) a smooth material structure (reduced deposition), (iii) a high porosity (high transmembrane flux), (iv) a distinct pore size distribution (well-defined pore selectivity), and (v) an increased fiber wall thickness (larger effective surface area). Lowest filter fouling was observed with polysulfone (PS) membranes. While the use of a small-pore PS membrane (0.08 µm) allowed to fully retain YFV within the bioreactor, continuous product harvesting was achieved with the large-pore PS membrane (0.34 µm). Due to the low protein rejection of the latter, this membrane type could also be of interest for other applications, that is, recombinant protein production in perfusion cultures.     

The effects of membrane filters used in biopharmaceutical processes on the concentration and composition of polysorbate 20

Polysorbate 20 (PS‐20) is often included in the formulation for therapeutic proteins to reduce protein aggregation and surface adsorption. During the production process of therapeutic proteins, various membrane filters are used to filter product pools containing PS‐20. The purpose of this study is to quantify the effects of these membrane filtration processes on the concentration and composition of PS‐20. A quantitative understanding of this process provides the knowledge base for better controlling the consistency of formulation excipients in drug products. PS‐20 solutions (without protein) were filtered through either 0.2 µm sterilizing filters or membrane filters with 30 kDa MWCO. The concentration of PS‐20 was measured by a mixed‐mode chromatography method and a nuclear magnetic resonance spectroscopy (NMR) assay. The composition of PS‐20 was characterized by ¹H‐NMR and a reverse‐phase chromatography method. Non‐specific adsorption of PS‐20 on both the sterilizing filter and 30 kDa MWCO membrane filter was quantified. Composition of PS‐20 was altered after 30 kDa MWCO membrane filtration, possibly because the different interactions between heterogeneous PS‐20 components and the 30 kDa MWCO membrane were not uniform. As a result, the retentate after the 30 kDa MWCO membrane filtration step contains no POE sorbitan and increased amount of POE sorbitan di‐esters and tri‐esters. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1503–1511, 2013     

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     

The use of chitosan as a flocculant in mammalian cell culture dramatically improves clarification throughput without adversely impacting monoclonal antibody recovery

Flocculants have been employed for many years as aides in the clarification of wastewater, chemicals and food. Flocculants aggregate and agglutinate fine particles resulting in their settling from the liquid phase and a reduction in solution turbidity. These materials have not been widely used in the clarification of mammalian cell culture harvest. In this paper we examined chitosan as a flocculent of cells and cell particulates in NS0 culture harvest and the subsequent further clarification of this material by continuous flow centrifugation followed by depth and absolute filtration. Chitosan is an ideal flocculant for biotechnology applications as it is produced from non-mammalian sources (typically arthropod shells) and is also available in a highly purified form that is low in heavy metals, volatile organics and microbial materials. Chitosan is a polymer of deacetylated chitin. The deacetylation imparts limited solubility on insoluble chitin and the amino groups on the polymer result in a polycationic material at acidic and neutral pH that can interact with polyanions, such as DNA and cell culture debris (typically negatively charged). Likely the interaction of chitosan with cell culture particulate forms a germinal center for further interaction and agglomeration of particulates thereby reducing the solubility of these materials resulting in their settling out into the solid phase. Chitosan improved the clarification throughput six to seven folds without a deleterious effect on monoclonal antibody recovery or purity. The procedure for utilizing chitosan is facile, easily implemented, and highly effective in improving material clarity and increasing material throughput.     

Scaled‐Up Inertial Microfluidics: Retention System for Microcarrier‐Based Suspension Cultures

Recently, particle concentration and filtration using inertial microfluidics have drawn attention as an alternative to membrane and centrifugal technologies for industrial applications, where the target particle size varies between 1 µm and 500 µm. Inevitably, the bigger particle size (>50 µm) mandates scaling up the channel cross‐section or hydraulic diameter (D_H > 0.5 mm). The Dean‐coupled inertial focusing dynamics in spiral microchannels is studied broadly; however, the impacts of secondary flow on particle migration in a scaled‐up spiral channel is not fully elucidated. The mechanism of particle focusing inside scaled‐up rectangular and trapezoidal spiral channels (i.e., 5–10× bigger than conventional microchannels) with an aim to develop a continuous and clog‐free microfiltration system for bioprocessing is studied in detail. Herein, a unique focusing based on inflection point without the aid of sheath flow is reported. This new focusing mechanism, observed in the scaled‐up channels, out‐performs the conventional focusing scenarios in the previously reported trapezoidal and rectangular channels. Finally, as a proof‐of‐concept, the utility of this device is showcased for the first time as a retention system for a cell–microcarrier (MC) suspension culture.     

Modeling flux in tangential flow filtration using a reverse asymmetric membrane for Chinese hamster ovary cell clarification

Tangential flow filtration is advantageous for bioreactor clarification as the permeate stream could be introduced directly to the subsequent product capture step. However, membrane fouling coupled with high product rejection has limited its use. Here, the performance of a reverse asymmetric hollow fiber membrane where the more open pore structure faces the feed stream and the barrier layer faces the permeate stream has been investigated. The open surface contains pores up to 40 μm in diameter while the tighter barrier layer has an average pore size of 0.4 μm. Filtration of Chinese hamster ovary cell feed streams has been investigated under conditions that could be expected in fed batch operations. The performance of the reverse asymmetric membrane is compared to that of symmetric hollow fiber membranes with nominal pore sizes of 0.2 and 0.65 μm. Laser scanning confocal microscopy was used to observe the locations of particle entrapment. The throughput of the reverse asymmetric membrane is significantly greater than the symmetric membranes. The membrane stabilizes an internal high permeability cake that acts like a depth filter. This stabilized cake can remove particulate matter that would foul the barrier layer if it faced the feed stream. An empirical model has been developed to describe the variation of flux and transmembrane pressure drop during filtration using reverse asymmetric membranes. Our results suggest that using a reverse asymmetric membrane could avoid severe flux decline associated with fouling of the barrier layer during bioreactor clarification.     

New cyclopentaquinoline hybrids with multifunctional capacities for the treatment of Alzheimer’s disease

Alzheimer’s disease (AD) is the most common progressive form of brain neurodegeneration and the most prevailing cause of dementia. Unfortunately, the aetiology of AD is not completely studied but different factors are associated with the development of AD such as among others low level of acetylcholine, aggregation of β-amyloid (Aβ), hyperphosphorylated tau protein, oxidative stress, and inflammation. The study encompass organic syntheses of 2,3-dihydro-1H-cyclopenta[b]quinoline with 5,6-dichloronicotinic acid and suitable linkers derivatives as multifunctional agents for AD treatment. Afterwards self-induced amyloid beta aggregation, inhibition studies of acetylcholinesterase and butyrylcholinesterase and molecular docking studies were performed. The results showed that 3b compound exhibited the best acetylcholinesterase inhibitory activity, with IC₅₀ value of 0.052?µM which is lower compared to references. Besides, all synthesised compounds showed good butyrylcholinesterase inhibitory activity with IC₅₀ values from 0.071 to 0.797?µM. Compound 3b exhibited strong Aβ_1–42 aggregation inhibitory effect with 25.7% at 5?µM to 92.8% at 100?µM as well as good anti-inflammatory effect. Thus, new compounds could create new perspectives for further development as a multi-target-directed agent for AD treatment.     

Soil Ingestion Estimates for Children in Anaconda Using Trace Element Concentrations in Different Particle Size Fractions

This investigation assessed the effect of soil particle size on soil ingestion estimates of children residing at a superfund site. Earlier research indicated that wide intertracer variability in soil ingestion estimates are based on soil concentrations with a soil particle size of 0 to 2?µm was markedly reduced when the estimates were based on soil tracer concentrations for a soil particle size of 0-250?µm. The reduced intertracer variation was principally attributed to changes in soil concentrations of only three of the soil tracers (i.e., Ce, La, Nd) which became concentrated in the finer particle size by approximately 2.5 to 4.0-fold. It was hypothesized that the intertracer agreement in soil ingestion estimates may continue to improve if the estimates are based on concentrations of tracers at finer particle sizes assuming that children ingest finer particles and that the above three tracers would continue to be further concentrated in the finer sized soil particles. The principal findings indicate: 1. The soil concentrations of Al, Si, and Ti do not increase at the two finer particle size ranges measured. 2. The soil concentrations of Ce, La, and Nd increased by a factor 2.5 to 4.0 in the 100 to 250?µm particle size range when compared with the 0 to 2?µm particle size range. No further substantial increase in concentration was observed in the 53 to 100 |jm particle size range. 3. The soil ingestion estimates are consistently and markedly changed only between the estimates based in 0 to 2?µm and 100 to 250?µm for Ce, La, and Nd. These changes reduced the intertracer variability in estimating soil ingestion, suggesting that the children eat finer soil particle sizes. 4. Because the particle sizes for all tracers (except Zr) were only modestly affected at the 53 to 100?µm range, it was not possible to confidently resolve the particle size of soil ingested by the children. 5. Residual intertracer variability in soil ingestion estimates based on Ce, La, Nd are likely to be significantly affected by non-food, non-soil sources of these tracers (i.e., source error). 6. Soil ingestion estimates of this study will be more reliable when derived from the finer-sized particles.