Container Closure Integrity of a Glass Prefillable Syringe in Deep Frozen Storage Conditions.

Development of novel pharmaceutical drug modalities has created a need for frozen storage and transportation. Accurate and easy assessment of container closure integrity (CCI) in frozen conditions remains a challenge. Thus, container closure systems (CCS) suitable for low temperatures have been primarily restricted to vials despite the growing popularity of prefillable syringes (PFS) for parenteral administration. A new dye ingress test method, suitable for testing at low temperatures, was developed and applied to PFS across a range of deep-frozen temperatures. The method is versatile and can easily be extended to other common CCS formats over a wide range of temperatures including storage on dry ice (-80 °C). This new method was paired with an orthogonal technique, laser-based CO2 headspace gas analysis, to evaluate the CCI of a glass PFS at temperatures from -50 °C to -80 °C. Both test methods showed comparable results and consistent CCI failure below a temperature of -70 °C. The primary mode of failure was the plunger-to-barrel interface, likely attributable to dimensional changes and loss of elasticity. This study demonstrates the temperature dependent CCI behavior of glass PFS and underscores the importance of thorough characterization of package integrity for deep frozen drug products.

Bio-transfer and bioaccumulation patterns of heavy metals in mine site-inhabiting ants and grasshoppers, across mine site restoration chronosequence.

Soil is known to serve as a significant sink for heavy metals in coal mine sites, thus also influence the plant and other organisms of that area. Hence, the presence of heavy metals in coal mine soil is of concern to land managers. Insects occupy different trophic positions in the food chains, thus many insect species accumulate large amounts of heavy metals in their bodies and this is a matter of concern. In the present study, we investigated biotransfer and bioaccumulation of heavy metals from soil, grass species Cynodon dactylon, Vetiveria zizanioides, grasshopper species Gastrimargus africanus, Choroedocus robustus, ant species Cataglyphis longipedem and Camponotus compressus in six different ages (2, 4, 6, 8, 10 and 12 year old) of coal mine sites. Our study revealed that at some extent the heavy metal content and BAF patterns of heavy metals along different pathways (from soil to grass, soil to grasshoppers and from grass to grasshoppers) were not consistent and did not reflect the soil pollution status for all the metals, related to the mine spoil dump age. However, in contrast, ants successfully reflected a consistent pattern in the bioaccumulation of heavy metals via soil, thereby indicating the pollution status of the soil along with the restoration age of mine spoil dumps. Our study showed that ant species can successfully forecast the presence of heavy metals of coal mine spoils along with their restoration.

Specific Targeting and Labeling of Colonic Polyps in CPC-APC Mice with Mucin 5AC Fluorescent Antibodies: A Model for Detection of Early Colon Cancer.

Poor visualization of polyps can limit colorectal cancer screening. Fluorescent antibodies to mucin5AC (MUC5AC), a glycoprotein upregulated in adenomas and colorectal cancer, could improve screening colonoscopy polyp detection rate. Adenomatous polyposis coli flox mice with a Cdx2-Cre transgene (CPC-APC) develop colonic polyps that contain both dysplastic and malignant tissue. Mice received MUC5AC-IR800 or IRdye800 as a control IV and were sacrificed after 48 h for near-infrared imaging of their colons. A polyp-to-background ratio (PBR) was calculated for each polyp by dividing the mean fluorescence intensity of the polyp by the mean fluorescence intensity of the background tissue. The mean 25 µg PBR was 1.70 (±0.56); the mean 50 µg PBR was 2.64 (±0.97); the mean 100 µg PBR was 3.32 (±1.33); and the mean 150 µg PBR was 3.38 (±0.87). The mean PBR of the dye-only control was 2.22 (±1.02), significantly less than the 150 µg arm (p-value 0.008). The present study demonstrates the ability of fluorescent anti-MUC5AC antibodies to specifically target and label colonic polyps containing high-grade dysplasia and intramucosal adenocarcinoma in CPC-APC mice. This technology can potentially improve the detection rate and decrease the miss rate of advanced colonic neoplasia and early cancer at colonoscopy.

Regulation of Glucose Insulinotropic Peptide and Intestinal Glucose Transporters in the Diet-Induced Obese Mouse.

Our recent studies have shown that glucose-dependent insulinotropic polypeptide (GIP), but not glucagon-like peptide 1 (GLP-1), augments Na-glucose transporter 1- (SGLT1-) mediated glucose absorption in mouse jejunum. Na-dependent glucose absorption sharply rose and peaked in 3 months of high-fat (i.e., obese) compared to normal (i.e., normal weight) diet fed animals. Previous studies have shown that GIP-augmented SGLT1 and PEPT1 (peptide transporter 1) are regulated by protein kinase A (PKA) signaling in mouse jejunum. Additional studies have indicated that cAMP and PI3 kinase signaling augment PEPT1 through EPAC and AKT activation pathways, respectively, through increased apical PEPT1 trafficking in intestinal epithelial cells. However, little is known about how the signaling glucose transport paradigm is altered over a long period. Early on, increased glucose absorption occurs through SGLT1, but as the obesity and diabetes progress, there is a dramatic shift towards a Na-independent mechanism. Surprisingly, at the peak of glucose absorption during the fifth month of the progression of obesity, the SGLT1 activity was severely depressed, while a Na-independent glucose absorptive process begins to appear. Since glucose transporter 2 (GLUT2) is expressed on the apical membrane of the small intestine in obese patients and animal models of obesity, it was hypothesized to be the new more efficient route. Western blot analyses and biotinylation of the apical membrane revealed that the GIP expression increases in the obese animals and its trafficking to the apical membrane increases with the GIP treatment.

Artificial Intelligence-Based Assessment of Colorectal Polyp Histology by Elastic-Scattering Spectroscopy.

BACKGROUND: Colonoscopic screening and surveillance for colorectal cancer could be made safer and more efficient if endoscopists could predict histology without the need to biopsy and perform histopathology on every polyp. Elastic-scattering spectroscopy (ESS), using fiberoptic probes integrated into standard biopsy tools, can assess, both in vivo and in real time, the scattering and absorption properties of tissue related to its underlying pathology. AIMS: The objective of this study was to evaluate prospectively the potential of ESS to predict polyp pathology accurately. METHODS: We obtained ESS measurements from patients undergoing screening/surveillance colonoscopy using an ESS fiberoptic probe integrated into biopsy forceps. The integrated forceps were used for tissue acquisition, following current standards of care, and optical measurement. All measurements were correlated to the index pathology. A machine learning model was then applied to measurements from 367 polyps in 169 patients to prospectively evaluate its predictive performance. RESULTS: The model achieved sensitivity of 0.92, specificity of 0.87, negative predictive value (NPV) of 0.87, and high-confidence rate (HCR) of 0.84 for distinguishing 220 neoplastic polyps from 147 non-neoplastic polyps of all sizes. Among 138 neoplastic and 131 non-neoplastic polyps ≤ 5 mm, the model achieved sensitivity of 0.91, specificity of 0.88, NPV of 0.89, and HCR of 0.83. CONCLUSIONS: Results show that ESS is a viable endoscopic platform for real-time polyp histology, particularly for polyps ≤ 5 mm. ESS is a simple, low-cost, clinically friendly, optical biopsy modality that, when interfaced with minimally obtrusive endoscopic tools, offers an attractive platform for in situ polyp assessment.

Stress-dependent Flexibility of a Full-length Human Monoclonal Antibody: Insights from Molecular Dynamics to Support Biopharmaceutical Development.

After several decades of advancements in drug discovery, product development of biopharmaceuticals remains a time- and resource-consuming endeavor. One of the main reasons is associated to the lack of fundamental understanding of conformational dynamics of such biologic entities, and how they respond to various stresses encountered during manufacturing, storage, and shipping. In this work, we have studied the conformational dynamics of human IgG1κ b12 monoclonal antibody (mAb) using molecular dynamics simulations. The hundreds of nanoseconds long trajectories reveal that b12 mAb is highly flexible. Its variable domains show greater conformational fluctuations than the constant domains. Additionally, it collapses towards a more globular shape in response to thermal stress, leading to decrease in the total solvent exposed surface area and radius of gyration. This behavior is more pronounced for the deglycosylated b12 mAb, and it appears to correlate with increase in inter-domain contacts between specific regions of the antibody. Conformational fluctuations also cause transient formation and disruption of hydrophobic and charged patches on the antibody surface, which is particularly important for the prediction of CMC properties during development phases of antibody-based biotherapeutics. The insights gained through these simulations may help the development of biologic drugs.

Real-time artificial intelligence-based histologic classification of colorectal polyps with augmented visualization.

BACKGROUND AND AIMS: Artificial intelligence (AI)-based computer-aided diagnostic (CADx) algorithms are a promising approach for real-time histology (RTH) of colonic polyps. Our aim is to present a novel in situ CADx approach that seeks to increase transparency and interpretability of results by generating an intuitive augmented visualization of the model's predicted histology over the polyp surface. METHODS: We developed a deep learning model using semantic segmentation to delineate polyp boundaries and a deep learning model to classify subregions within the segmented polyp. These subregions were classified independently and were subsequently aggregated to generate a histology map of the polyp's surface. We used 740 high-magnification narrow-band images from 607 polyps in 286 patients and over 65,000 subregions to train and validate the model. RESULTS: The model achieved a sensitivity of .96, specificity of .84, negative predictive value (NPV) of .91, and high-confidence rate (HCR) of .88, distinguishing 171 neoplastic polyps from 83 non-neoplastic polyps of all sizes. Among 93 neoplastic and 75 non-neoplastic polyps ≤5 mm, the model achieved a sensitivity of .95, specificity of .84, NPV of .91, and HCR of .86. CONCLUSIONS: The CADx model is capable of accurately distinguishing neoplastic from non-neoplastic polyps and provides a histology map of the spatial distribution of localized histologic predictions along the delineated polyp surface. This capability may improve interpretability and transparency of AI-based RTH and offer intuitive, accurate, and user-friendly guidance in real time for the clinical management and documentation of optical histology results.

Advanced endoscopic imaging for detecting and guiding therapy of early neoplasias of the esophagus.

Esophageal cancers, largely adenocarcinoma in Western countries and squamous cell cancer in Asia, present a significant burden of disease and remain one of the most lethal of cancers. Key to improving survival is the development and adoption of new imaging modalities to identify early neoplastic lesions, which may be small, multifocal, subsurface, and difficult to detect by standard endoscopy. Such advanced imaging is particularly relevant with the emergence of ablative techniques that often require multiple endoscopic sessions and may be complicated by bleeding, pain, strictures, and recurrences. Assessing the specific location, depth of involvement, and features correlated with neoplastic progression or incomplete treatment may optimize treatments. While not comprehensive of all endoscopic imaging modalities, we review here some of the recent advances in endoscopic luminal imaging, particularly with surface contrast enhancement using virtual chromoendoscopy, highly magnified subsurface imaging with confocal endomicroscopy, optical coherence tomography, elastic scattering spectroscopy, angle-resolved low-coherence interferometry, and light scattering spectroscopy. While there is no single ideal imaging modality, various multimodal instruments are also being investigated. The future of combining computer-aided assessments, molecular markers, and improved imaging technologies to help localize and ablate early neoplastic lesions shed hope for improved disease outcome.

Freezing of Biologicals Revisited: Scale, Stability, Excipients, and Degradation Stresses.

Although many biotech products are successfully stored in the frozen state, there are cases of degradation of biologicals during freeze storage. These examples are discussed in the Perspective to emphasize the fact that stability of frozen biologicals should not be taken for granted. Frozen-state degradation (predominantly, aggregation) has been linked to crystallization of a cryoprotector in many cases. Other factors, for example, protein unfolding (either due to cold denaturation or interaction of protein molecules with ice crystals), could also contribute to the instability. As a hypothesis, additional freezing-related destabilization pathways are introduced in the paper, that is, air bubbles formed on the ice crystallization front, and local pressure and mechanical stresses due to volume expansion during water-to-ice transformation. Furthermore, stability of frozen biologicals can depend on the sample size, via its impact on the freezing kinetics (i.e., cooling rates and freezing time) and cryoconcentration effects, as well as on the mechanical stresses associated with freezing. We conclude that, although fundamentals of freezing processes are fairly well described in the current literature, there are important gaps to be addressed in both scientific foundations of the freezing-related manufacturing processes and implementation of the available knowledge in practice.

Computer-assisted assessment of colonic polyp histopathology using probe-based confocal laser endomicroscopy.

INTRODUCTION: Probe-based confocal laser endomicroscopy (pCLE) is a promising modality for classifying polyp histology in vivo, but decision making in real-time is hampered by high-magnification targeting and by the learning curve for image interpretation. The aim of this study is to test the feasibility of a system combining the use of a low-magnification, wider field-of-view pCLE probe and a computer-assisted diagnosis (CAD) algorithm that automatically classifies colonic polyps. METHODS: This feasibility study utilized images of polyps from 26 patients who underwent colonoscopy with pCLE. The pCLE images were reviewed offline by two expert and five junior endoscopists blinded to index histopathology. A subset of images was used to train classification software based on the consensus of two GI histopathologists. Images were processed to extract image features as inputs to a linear support vector machine classifier. We compared the CAD algorithm's prediction accuracy against the classification accuracy of the endoscopists. RESULTS: We utilized 96 neoplastic and 93 non-neoplastic confocal images from 27 neoplastic and 20 non-neoplastic polyps. The CAD algorithm had sensitivity of 95%, specificity of 94%, and accuracy of 94%. The expert endoscopists had sensitivities of 98% and 95%, specificities of 98% and 96%, and accuracies of 98% and 96%, while the junior endoscopists had, on average, a sensitivity of 60%, specificity of 85%, and accuracy of 73%. CONCLUSION: The CAD algorithm showed comparable performance to offline review by expert endoscopists and improved performance when compared to junior endoscopists and may be useful for assisting clinical decision making in real time.

Comparing Physical Container Closure Integrity Test Methods and Artificial Leak Methodologies.

The sterility of drug products intended for parenteral administration is a critical quality attribute (CQA) because it serves to ensure patient safety and is thus a key requirement by health authorities. While sterility testing is a probabilistic test, the assurance of sterility is a holistic concept including adequate design of manufacturing facilities, process performance, and product design. Container closure integrity testing (CCIT) is necessary to confirm the integrity of a container closure system (CCS), until the end of a product's shelf life. The new and revised United States Pharmacopeia (USP) General Chapter <1207> is a comprehensive guidance on CCI. Nevertheless, practical considerations including the choice of CCIT methods, the acceptance criteria, or the positive control samples (artificial leaks) must be addressed by the pharmaceutical manufacturer.This study is the first to provide a systematic comparison of four commonly used physical CCIT (pCCIT) methods [Helium (He) leak, vacuum decay, laser-based headspace analysis (HSA), and dye ingress] and four commonly used modes of creating artificial leaks (laser-drilled micro holes, copper wire introduced leaks, and two types of capillary leaks).The results from these experiments provide comprehensive data to allow a direct comparison of the capabilities of the individual methods. The results confirmed that the He leak detection method, which is considered the "gold-standard" for pCCIT regarding method sensitivity, indeed demonstrates the highest detection sensitivity (lowest detection limit). In comparison to the dye ingress method, HSA and vacuum decay also demonstrated better detection sensitivity in our study.Capillary leaks with orifice diameter (capillary leak with flow according to an ideal orifice) and micro holes yielded similar leak rates, whereas capillaries with nominal diameters yielded significantly lower leak rates. In conclusion, method sensitivity cannot be compared by means of a leak diameter, but requires the consideration of multiple impacting factors (e.g., path length, uniformity).LAY ABSTRACT: Sterility of drug products intended for parenteral administration is a critical quality attribute to ensure patient's safety and is thus a key requirement by health authorities. The absence of microbial contamination must be demonstrated by container closure integrity (CCI) of the container closure system (CCS). Currently, the revised United States Pharmacopeia (USP) General Chapter <1207> provides the most extensive guidance on how CCI should be assessed. Nevertheless, practical considerations on the choice of an appropriate CCIT method, artificial leaks or the choice of an acceptance criteria are lacking and must be addressed by the pharmaceutical manufacturer.This study provides a systematic comparison of four commonly used physical CCIT (pCCIT) methods [Helium (He) leak, vacuum decay, laser-based headspace analysis (HSA) and dye ingress] and four commonly used modes of creating artificial leaks (laser-drilled micro holes, copper wire introduced leaks, and two types of capillary leaks).

In Silico Prediction of Diffusion Interaction Parameter (kD), a Key Indicator of Antibody Solution Behaviors.

PURPOSE: To develop resource-sparing in silico approaches that aim to reduce experimental effort and material required by developability assessments (DA) of monoclonal antibody (mAb) drug candidates. METHODS: A battery of standardized biophysical experiments was performed on high concentration formulations of 16 drug product development stage mAbs using a platform buffer. Full-length molecular models of these mAbs were also generated via molecular modeling. These models were used to computationally estimate molecular descriptors of these 16 mAbs. Pairwise and multi-parameter correlations among experimentally measured biophysical attributes and calculated molecular descriptors were obtained via statistical analyses. RESULTS: Diffusion interaction parameter (kD) showed statistically significant pairwise correlations (p-values <0.005) with thermal stability, viscosity, isoelectric point, and apparent solubility of the antibodies in our dataset. kD also showed statistically significant pairwise correlations (p-values <0.005) with several computationally calculated molecular descriptors (pI, net charge, charge on the Fv region, and zeta potential.) These pairwise correlations were further refined by multivariate analyses. These analyses yielded several useful equations for prediction of kD from antibody sequences, structural models, and experimentally measured biophysical attributes. CONCLUSIONS: Diffusion interaction parameter (kD) was found to be a key biophysical property for the mAbs in our dataset. It connects conformational heterogeneity of an antibody with its colloidal and rheological behaviors. The equations derived in this work shall enable rapid, resource-sparing, and cost-effective DAs of biologic drug candidates.

Are Injection Site Reactions in Monoclonal Antibody Therapies Caused by Polysorbate Excipient Degradants?

Injection site reactions (ISRs) and other adverse side effects are commonly observed during therapy with biologics. These hypersensitivity-related side effects can vary from simple rash to life-threatening anaphylactic reaction and may be linked to the immunogenicity of the drug including formation of antidrug antibodies. Reactions can also occur as a consequence of excipients in the product. We report the case of a patient who developed erythematous ISRs to both commercial PCSK9i formulations and had to go off therapy even though efficacy was not impacted. Skin testing showed that the patient was reacting to the polysorbates. Polysorbates are added to stabilize the biotherapeutic. Polysorbates can also activate complement and lead to a range of acute hypersensitivity and systemic immunostimulation reactions. Oxidative degradation products can function as haptens by reacting with proteins at the injection site. Reactive degradation products may even form adducts with the biologic itself, creating a potential neoantigen. Further research is needed to understand the fundamental causes of ISRs. It is critical that only the highest quality raw material is used, and proper storage conditions are employed to minimize degradation of polysorbates in the product. Although complete elimination of ISRs is unlikely, all efforts must be made to minimize them.

Considerations for the Use of Polysorbates in Biopharmaceuticals.

PURPOSE: Polysorbates are commonly added to protein formulations and serve an important function as stabilizers. This paper reviews recent literature detailing some of the issues seen with the use of polysorbate 80 and polysorbate 20 in protein formulations. Based on this knowledge, a development strategy is proposed that leads to a control strategy for polysorbates in protein formulations. METHODS: A consortium of Biopharmaceutical scientists working in the area of protein formulations, shared experiences with polysorbates as stabilizers in their formulations. RESULTS: Based on the authors experiences and recent published literature, a recommendation is put forth for a development strategy which will lead into the appropriate control strategy for these excipients. CONCLUSIONS: An appropriate control strategy may comprise one or more elements of raw material, in-process and manufacturing controls. Additionally, understanding the role, if any, polysorbates play during stability will require knowledge of the criticality of the excipient, based upon its impact on CQAs due to variations in concentration and degradation level.

Container Closure Integrity Testing of Prefilled Syringes.

Prefilled syringes (PFSs) are increasingly preferred over vials as container closure systems (CCSs) for injectable drug products when facilitated or self-administration is required. However, PFSs are more complex compared to CCSs consisting of vial, rubber stopper, and crimp cap. Container closure integrity (CCI) assurance and verification has been a specific challenge for PFSs as they feature several sealing areas. A comprehensive understanding of the CCS is necessary for an appropriate CCI assessment as well as for packaging development and qualification. A comprehensive CCI assessment of 6 different PFSs from 3 different manufacturers (including 1 polymeric PFS) was conducted using helium leak testing. PFS components were manipulated to systematically assess the contribution of the different sealing areas to CCI, namely rigid needle shield (RNS)/needle, RNS/tip cone, and the individual ribs of a syringe plunger. The polymeric PFS required an equilibrium measurement for accurate container closure integrity testing. The different sealing areas and a single plunger rib were shown to provide adequate CCI. Acceptable tip cap movement until the point of CCI failure was estimated. The assessment of acceptable tip cap movement demonstrated the importance of considering the RNS/tip cone seal design to ensure CCI of the PFS upon post assembly possesses and shipment.

Probing the Conformation of an IgG1 Monoclonal Antibody in Lyophilized Solids Using Solid-State Hydrogen-Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS).

Therapeutic proteins are often formulated as lyophilized products to improve their stability and prolong shelf life. The stability of proteins in the solid-state has been correlated with preservation of native higher order structure and/or molecular mobility in the solid matrix, with varying success. In the studies reported here, we used solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS) to study the conformation of an IgG1 monoclonal antibody (mAb) in lyophilized solids and related the extent of ssHDX to aggregation during storage in the solid phase. The results demonstrate that the extent of ssHDX correlated better with aggregation rate during storage than did solid-state Fourier-transform infrared (ssFTIR) spectroscopic measurements. Interestingly, adding histidine to sucrose at different formulation pH conditions decreased aggregation of the mAb, an effect that did not correlate with structural or conformational changes as measured by ssFTIR or ssHDX-MS. Moreover, peptide-level ssHDX-MS analysis in four selected formulations demonstrated global changes across the structure of the mAb when lyophilized with sucrose, trehalose, or mannitol, whereas site-specific changes were observed when lyophilized with histidine as the sole excipient.

Effects of Drying Process on an IgG1 Monoclonal Antibody Using Solid-State Hydrogen Deuterium Exchange with Mass Spectrometric Analysis (ssHDX-MS).

PURPOSE: Lyophilization and spray drying are widely used to manufacture solid forms of therapeutic proteins. Lyophilization is used to stabilize proteins vulnerable to degradation in solution, whereas spray drying is mainly used to prepare inhalation powders or as an alternative to freezing for storing bulk drug substance. Both processes impose stresses that may adversely affect protein structure, stability and bioactivity. Here, we compared lyophilization with and without controlled ice nucleation, and spray drying for their effects on the solid-state conformation and matrix interactions of a model IgG1 monoclonal antibody (mAb). METHODS: Solid-state conformation and matrix interactions of the mAb were probed using solid-state hydrogen-deuterium exchange with mass spectrometric analysis (ssHDX-MS), and solid-state Fourier transform infrared (ssFTIR) and solid-state fluorescence spectroscopies. RESULTS: mAb conformation and/or matrix interactions were most perturbed in mannitol-containing samples and the distribution of states was more heterogeneous in sucrose and trehalose samples that were spray dried. CONCLUSIONS: The findings demonstrate the sensitivity of ssHDX-MS to changes weakly indicated by spectroscopic methods, and support the broader use of ssHDX-MS to probe formulation and process effects on proteins in solid samples.

Biopharmaceutical Informatics: supporting biologic drug development via molecular modelling and informatics.

OBJECTIVES: The purpose of this article is to introduce an emerging field called 'Biopharmaceutical Informatics'. It describes how tools from Information technology and Molecular Biophysics can be adapted, developed and gainfully employed in discovery and development of biologic drugs. KEY FINDINGS: The findings described here are based on literature surveys and the authors' collective experiences in the field of biologic drug product development. A strategic framework to forecast early the hurdles faced during drug product development is weaved together and elucidated using chemical degradation as an example. Efficiency of translating biologic drug discoveries into drug products can be significantly improved by combining learnings from experimental biophysical and analytical data on the drug candidates with molecular properties computed from their sequences and structures via molecular modeling and simulations. SUMMARY: Biopharmaceutical Informatics seeks to promote applications of computational tools towards discovery and development of biologic drugs. When fully implemented, industry-wide, it will enable rapid materials-free developability assessments of biologic drug candidates at early stages as well as streamline drug product development activities such as commercial scale production, purification, formulation, analytical characterization, safety and in vivo performance.

Heavy metal accumulation and ecosystem engineering by two common mine site-nesting ant species: implications for pollution-level assessment and bioremediation of coal mine soil.

The present study focuses on the abundance, heavy metal content, and the impact of ecosystem engineering activities of two coal mine site-inhabiting ant species, Cataglyphis longipedem and Camponotus compressus. The abundance of Ct. longipedem increased while that of C. compressus decreased, with increasing soil pollution. Correspondence analysis reveals a close association between soil heavy metal concentrations and Ct. longipedem abundance, but this association is lacking in the case of C. compressus. Cataglyphis ants which occupy stress-characterized niches appear to be pre-adapted to tolerate heavy metal pollution. Higher concentrations of Zn and Mn in Ct. longipedem may contribute to the strengthening of the cuticular structures, necessary for nest excavation in the hard, arid soil and for single load carrying. C. compressus ants appear to be pollution sensitive. Their higher Fe content may be related to metal uptake via plant-derived liquids and species-specific regulatory mechanisms. The metal pollution index and biota-to-soil accumulation factors, calculated by using the ant body metal content of the two species, indicate an overall decrease of soil heavy metal concentrations with increase of the site age, which reflects the degree of pollution related to the mine site age. The concentrations of total and available heavy metals (Fe, Zn, Mn, Pb, and Cu) were significantly lower in the ant nest debris soil as compared to the reference soil. The results of the present study highlight the role of ants as bioindicators and in bioremediation of contaminated soil.