Pharmacokinetic study of minipooled solvent/detergent-filtered cryoprecipitate factor VIII.

Eighteen cryoprecipitate minipools, each made of 30 units of low volume, concentrated cryoprecipitate, have been treated by solvent-detergent and filtration (S/D-F) in a single-use CE-marked bag system. The S/D-F cryoprecipitate contained a mean of 10.5 IU mL⁻¹ factor VIII (FVIII), 17 mg mL⁻¹ clottable fibrinogen, and >10 IU mL⁻¹ von Willebrand factor ristocetin co-factor, and anti-A and anti-B isoagglutinins were undetectable. The products have been infused in 11 severe (FVIII <1%) haemophilia A patients (mean age: 17.4 years; mean weight: 57.6 kg) at a dose close to 40 IU kg⁻¹. Patients were hospitalized for at least 36 h to determine FVIII recovery, half-life and clearance. They were also closely monitored for possible adverse events. None of the infused patients demonstrated reactions or adverse events even though they did not receive anti-allergic drugs or corticosteroids prior to infusion. The mean recovery of FVIII 10 min postinfusion was 69.7%. Mean FVIII half-life was 14.2 h and clearance was 2.6 mL h⁻¹ kg⁻¹. All patients had a bleeding-free interval of 8-10 days postS/D-F cryoprecipitate infusion. The data show that S/D-F cryoprecipitate FVIII presents a normal pharmacokinetics profile, and support that it could be safely used for the control of acute and chronic bleeding episodes in haemophilia A patients.

Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance.

Intravenous immunoglobulin G (IVIG) is now the leading product obtained by fractionation of human plasma. It is the standard replacement therapy in primary and acquired humoral deficiency, and is also used for immunomodulatory therapy in various autoimmune disorders and transplantation. Over the last 30 years, the production processes of IVIG have evolved dramatically, gradually resulting in the development of intact IgG preparations safe to administer intravenously, with normal half-life and effector functions, prepared at increased yield, and exhibiting higher pathogen safety. This article reviews the developments that have led to modern IVIG preparations, the current methods used for plasma collection and fractionation, the safety measures implemented to minimize the risks of pathogen transmission and the major quality control tests that are available for product development and as part of mandatory batch release procedures.

Solvent-detergent filtered (S/D-F) fresh frozen plasma and cryoprecipitate minipools prepared in a newly designed integral disposable processing bag system.

Solvent-detergent (S/D) viral inactivation was recently adapted to the treatment of single plasma donations and cryoprecipitate minipools. We present here a new process and a new bag system where the S/D reagents are removed by filtration and the final products subjected to bacterial (0.2 microm) filtration. Recovered and apheresis plasma for transfusion (FFP) and cryoprecipitate minipools (400 +/- 20 mL) were subjected to double-stage S/D viral inactivation, followed by one oil extraction and a filtration on a S/D and phthalate [di(2-ethylhexyl) phthalate (DEHP)] adsorption device and a 0.2 microm filter. The initial and the final products were compared for visual appearance, blood cell count and cell markers, proteins functional activity, von Willebrand factor (VWF) multimers and protein profile by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Tri (n-butyl) phosphate (TnBP) was quantified by gas chromatography and Triton X-45 and DEHP by high-performance-liquid chromatography (HPLC). General safety tests were by 6.5 mL/kg intravenous injection in rats. The treated plasmas and cryoprecipitates were very clear and the protein content and functionality, VWF multimers and SDS-PAGE profiles were well preserved. TnBP and Triton X-45 were < 1 and <25 ppm, respectively, and DEHP (about 5 ppm) was less than it was in the starting materials. Blood cell counts and CD45, CD61 and glycophorin A markers were negative. There was no enhanced toxicity in rats. Thus, plasma and cryoprecipitate can be S/D-treated in this new CE-marked disposable integral processing system under conditions preserving protein function and integrity, removing blood cells, S/D agents and DEHP, and ensuring bacterial sterility. This process may offer one additional option to blood establishments for the production of virally inactivated plasma components.

Properties of a concentrated minipool solvent-detergent treated cryoprecipitate processed in single-use bag systems.

Cryoprecipitate is still used to treat factor VIII (FVIII), von Willebrand factor (VWF) and/or fibrinogen deficiency. Recently a solvent-detergent (S/D) process of minipools of cryoprecipitate performed in a closed bag system has been designed to improve its viral safety. Still, cryoprecipitate has other drawbacks, including low concentration in active proteins, and presence of haemolytic isoagglutinins. We report here the biochemical evaluation of S/D-treated minipools of cryoprecipitates depleted of cryo-poor plasma. Cryoprecipitates were solubilized by 8 mL of a sterile glucose/saline solution, pooled in batches of 40 donations and subjected to S/D treatment in a plastic bag system using either 2% TnBP or 1% TnBP-1%Triton X-45, followed by oil extractions (n = 10). Mean (+/-SD) FVIII and fibrinogen content was 8.86 (+/-1.29) IU mL(-1) and 16.02 (+/-1.98) mg mL(-1), and 8.92 (+/-1.05) IU mL(-1) in cryoprecipitate minipools treated with 2% TnBP, and 17.26 (+/-1.71) mg mL(-1), in those treated by TnBP-Triton X-45, respectively. The WWF antigen, ristocetin cofactor and collagen binding activities were close to 10, 7 and 8 IU mL(-1), respectively, and were not affected by either SD treatment. VWF multimeric pattern of SD-treated cryoprecipitates were similar to that of normal plasma, and the >15 mers and >10 mers content was identical to that of the starting cryoprecipitates. The anti-A and anti-B titre was 0-1 and 0-1/8, respectively. Therefore, it is possible to prepare virally inactivated cryoprecipitate minipools depleted of isoagglutinins and enriched in functional FVIII, VWF and clottable fibrinogen.

[Solvent-detergent viral inactivation of minipools of plasma for transfusion, cryoprecipitate and cryo-poor plasma in single-use bag systems]. / Inactivation virale par procédé solvant-détergent de minipools de plasma, de cryoprécipité et de surnageant de cryoprécipité dans des dispositifs à usage unique.

Non-virally inactivated plasma, cryoprecipitate and cryoprecipitate-poor plasma, prepared by blood establishments, are still used in many countries in the world, in both the developing world and industrialized countries, for the treatment of various hematological disorders. In the absence of viral inactivation treatment, these fractions may be involved, in spite of increasingly sensitive viral detection methods, into the transmission of plasma-borne viruses, most critically HIV and Hepatitis B (HBV) or C (HCV). We have adapted the well-established industrial solvent-detergent (SD) viral inactivation treatment to allow its application in a small scale using a single-use plastic bag system. The procedure can be used by blood establishments, without the need to build an industrial-scale manufacturing facility. Results show a good recovery of the functional activity of plasma proteins, including coagulation factors (such as factor VIII and coagulable fibrinogen) and/or protease inhibitors (such as alpha 2-antiplasmin). Viral validation studies revealed reduction factors greater than 4.17, greater than 4.73 and greater than 4.72 for HIV, BVDV and PRV, respectively, within a few minutes of treatment. A single-use SD treatment and SD-elimination system is currently under development to allow standardized use of the procedure by blood establishments or national or regional service centers.

Reducing the risk of infection from plasma products: specific preventative strategies.

Collection and testing procedures of blood and plasma that are designed to exclude donations contaminated by viruses provide a solid foundation for the safety of all blood products. Plasma units may be collected from a selected donor population, contributing to the exclusion of individuals at risk of carrying infectious agents. Each blood/plasma unit is individually screened to exclude donations positive for a direct (e.g., viral antigen) or an indirect (e.g. anti-viral antibodies) viral marker. As infectious donations, if collected from donors in the testing window period, can still be introduced into manufacturing plasma pools, the production of pooled plasma products requires a specific approach that integrates additional viral reduction procedures. Prior to the large-pool processing, samples of each donation for fractionation are pooled ('mini-pool') and subjected to a nucleic acid amplification test (NAT) by, for example, the polymerase chain reaction (PCR) to detect viral genomes (in Europe: HCV RNA plasma pool testing is now mandatory). Any individual donation found PCR positive is discarded before the industrial pooling. The pool of eligible plasma donations (which may be 2000 litres or more) may be subjected to additional viral screening tests, and then undergoes a series of processing and purification steps that, for each product, comprise one or several reduction treatments to exclude HIV, HBV HCV and other viruses. Viral inactivation treatments most commonly used are solvent-detergent incubation and heat treatment in liquid phase (pasteurization). Nanofiltration (viral elimination by filtration), as well as specific forms of dry-heat treatments, have gained interest as additional viral reduction steps coupled with established methods. Viral reduction steps have specific advantages and limits that should be carefully balanced with the risks of loss of protein activity and enhancement of epitope immunogenicity. Due to the combination of these overlapping strategies, viral transmission events of HIV, HBV, and HCV by plasma products have become very rare. Nevertheless, the vulnerability of the plasma supply to new infectious agents requires continuous vigilance so that rational and appropriate scientific countermeasures against emerging infectious risks can be implemented promptly.

Splenic abscess: another look at an old disease.

OBJECTIVE: To study the changes in the incidence, causes, bacteriologic profile, and management of a splenic abscess. DESIGN: Retrospective case study. SETTING: Tertiary, university referral center. PATIENTS: Thirty-nine patients with a splenic abscess. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Demographics, signs and symptoms, causes, risk factors, diagnostic methods, bacteriologic profile, treatment, and outcome. RESULTS: Patients presented at a mean age of 43 years (range, 2-83 years), after a mean symptomatic period of 16 days, with fever (69%), abdominal pain (56%), nausea and vomiting (38%), and splenomegaly (31%). The majority of abscesses represented metastatic infection (n=19), and 11 were secondary to immunosuppression. Twelve patients had human immunodeficiency virus disease and 9 used intravenous drugs. In patients who underwent computed tomography, all had abnormal scans (n=33), with a well-defined abscess(es) in 28. Nine abscesses were polymicrobial; monomicrobial isolates included gram-positive organisms (23%), gram-negative organisms (31%), fungi (23%), and mycobacteria (23%). Patients presenting before 1989 (1981-1988) (n=15) and those presenting after 1989 (1989-1996) (n=24) differed in risk factors (intravenous drug abuse, 0% vs 47% [P=.02]; hematologic malignancy, 43% vs 9% [P=.04]) and gram-positive isolates (18% vs 64%; P=.06). Patients underwent splenectomy (n=18), open drainage (n=4), medical therapy (n=10), or percutaneous drainage (n=5) with respective survival rates of 94%, 50%, 70%, and 100%. CONCLUSIONS: In 1996, splenic abscesses are increasingly common. Intravenous drug abuse and human immunodeficiency virus disease are significant risk factors, and the diagnosis should be considered in a patient with fever and abdominal pain who uses intravenous drugs. Antimicrobial agents should be broad since 36% of abscesses were polymicrobial, and should include coverage of gram-positive organisms.

Safety of recombinant and plasma-derived medicinals for the treatment of coagulopathies.

The introduction of advanced technologies (PCR testing, chromatography, and specific viral inactivation and removal techniques) has led to remarkable improvements in the quality and efficacy of biopharmaceutical products. The current safety strategies for both recombinant protein and PDP products depend on the extensive screening of the source material for infectious agents and the use of mild purification methods, specific mild viral-reduction techniques, GMP, QC, and QA. An appropriate system of pharmacologic vigilance is also an integral element for assuring product quality and safety in the marketplace. Such precautions make available high quality therapeutic recombinant proteins and PDP products. The risks in the clinical setting and the cost/benefit ratio must be considered in choosing a product for therapeutic use. The choice should be based on the analysis of data available for a specific product, because some variations in quality and safety can be observed in different brands. Overall, a much finer control of infectious risks has been achieved, and improvement will continue. With the new products, thrombotic episodes have become rare. Reducing immunogenic potential and improving yield to increase product supply could be the next challenges for producers of biopharmaceuticals.

Chromatographic purification and properties of a therapeutic human protein C concentrate.

Protein C deficiency (inherited and acquired) has a relatively high incidence rate in the general population worldwide. For many years, protein C deficient patients have been treated with fresh frozen plasma, prothrombin complex concentrates, heparin or oral anticoagulants, which all have clinical drawbacks. We report the production process of a highly purified human protein C concentrate from 1500 l of cryo-poor plasma by a four-step chromatographic procedure. After DEAE-Sephadex adsorption, protein C was separated from clotting factors II, VII and IX by DEAE-Sepharose FF and further purified, using a new strategy, by an on-line chromatographic system combining DMAE-Fractogel and heparin-Sepharose CL-6B. In addition, the product was treated against viral risks by solvent-detergent and nanofiltration on 15-nm membranes. The protein C concentrate was essentially free of other vitamin K-dependent proteins. Proteolytic activity was undetectable. Neither activated protein C, prekallikrein activator, nor activated vitamin K-dependent clotting factors were found resulting in good stability of the protein C activity. In vitro and in vivo animal tests did not reveal any sign of potential thrombogenicity. The final freeze-dried product had a mean protein C concentration of 58 IU/ml and a mean specific activity of 215 IU/mg protein, corresponding to over 12000-fold purification from plasma. Therefore, this concentrate appears to be of potential benefit for the treatment of protein C deficiency.

Large-scale preparation of highly purified human C1-inhibitor for therapeutic use.

A two-step chromatographic procedure has been developed to purify human C1-inhibitor from cryoprecipitate-poor plasma after removal of vitamin K-dependent proteins and antithrombin III. The procedure, which is fully compatible with modern plasma fractionation schemes, includes anion-exchange chromatography on DMAE-Fractogel EMD, viral inactivation by solvent-detergent treatment, adsorption on SO3-Fractogel EMD and viral removal by nanofiltration on 35- and 15-nm pore size membranes. Overall yields were about 45% and 58% for antigen and activity, respectively, providing 60-70 mg of highly purified inhibitor per litre of plasma. The purified inhibitor had a specific activity of 6.5 +/- 0.5 units/mg protein, representing a more than 400-fold increase in purity compared with plasma. C1-inhibitor purity with respect to total protein was greater than 80%. The main contaminant was complement component C3 which accounted for 4-10% of the total protein. Minor contaminants included low amounts of IgM, IgG, IgA, fibrinogen and albumin. Complement component C4 was undetectable. The purified inhibitor was stable throughout the purification process and for more than 24 h at room temperature after reconstitution of the freeze-dried material. Animal tests in rats and mice demonstrated that the C1-inhibitor concentrate was well tolerated at relatively high doses.

Affinity chromatography in the industrial purification of plasma proteins for therapeutic use.

Affinity chromatography is a powerful technique for the purification of many proteins in human plasma. Applications cover the isolation of proteins for research purposes but also, to a large extent, for the production of therapeutic products. In industrial plasma fractionation, affinity chromatography has been found to be particularly advantageous for fine and rapid capture of plasma proteins from industrial plasma fractions pre-purified by ethanol fractionation or by ion-exchange chromatography. To date, affinity chromatography is being used in the production of various licensed therapeutic plasma products, such as the concentrates of Factor VIII, Factor IX, von Willebrand Factor, Protein C, Antithrombin III, and Factor XI. Most commonly used ligands are heparin, gelatin, murine antibodies, and, to a lesser extent, Cu(2+). Possible development of the use of affinity chromatography in industrial plasma fractionation should be associated to the current development of phage display and combinatorial chemistry. Both approaches may lead to the development of tailor-made synthetic ligands that would allow implementation of protein capture technology, providing improved productivity and yield for plasma products.

Phenanthrene degradation in soils co-inoculated with phenanthrene-degrading and biosurfactant-producing bacteria.

Contaminant sorption within the soil matrix frequently limits biodegradation. However, contaminant bioavailability can be species-specific. This study investigated bioavailability of phenanthrene (PHE) to two PHE-degrading bacteria (Pseudomonas strain R and isolate P5-2) in the presence of rhamnolipid biosurfactant and/or a biosurfactant-producing bacterium, Pseudomonas aeruginosa ATCC 9027. Pseudomonas strain R mineralized more soil-sorbed PHE than strain P5-2, but in aqueous cultures the rate and extent of PHE mineralization by P5-2 exceeded that by P. strain R. In Fallsington sandy loam (fine-loamy, mixed, active, mesic Typic Endoaquult) (high PHE-sorption capacity) the addition of rhamnolipid increased PHE mineralization by P. strain R. Phenanthrene mineralization in soils inoculated with P5-2 was minimal and no enhancement in PHE degradation was observed when biosurfactant was added. Co-inoculation of Fallsington sandy loam with the biosurfactant producer did not affect PHE mineralization by isolate P5-2, but significantly enhanced PHE mineralization by P. strain R. The enhancement of PHE mineralization could not be explained by P. aeruginosa-mediated PHE degradation. The addition of rhamnolipid at concentrations above the critical micelle concentration (CMC) resulted in enhanced PHE release from test soils. These results suggest that the PHE-degrading strains were able to access different pools of PHE and that the biosurfactant-enhanced release of PHE from soils did not result in enhanced biodegradation. The results also demonstrated that bacteria with the catabolic potential to degrade sorbed hydrophobic contaminants could interact commensally with surfactant-producing strains by an unknown mechanism to hasten the biodegradation of aromatic hydrocarbons. Thus, understanding interactions among microbes may provide opportunities to further enhance biodegradation of soil-bound organic contaminants.

[Viral safety of intravenous immunoglobulins G for therapeutic use]. / Sécurité virale des préparations d'immunoglobulines G intraveineuses à usage thérapeutique.

Even though IV IgG concentrates are considered to be among the safest products derived from human plasma, some preparations have been associated with the sporadic transmission of NANB hepatitis and, specifically of hepatitis C. The risk of transmission may have decreased markedly for several IgG preparations since the availability of an immunological test to detect the antibodies against HCV in the starting plasma, but it has not been fully eradicated. Thus, in addition to established viral inactivation treatments, such as acid pH incubation, new methods have been (or are being) implemented to further reduce the risk of HCV infection through IV IgG concentrates. Among these methods are the solvent-detergent treatment already shown to be highly effective for the inactivation of HCV and other enveloped viruses in clotting factor concentrates, and nanofiltration for the specific removal of viruses on the basis of their size. Also, chromatographic methods have helped to improve the overall safety of the product not only by removing viruses but also by improving purity and thus favoring a better in vivo tolerance. This paper reviews the reported cases of HCV transmission and the viral validation data for various IV IgG processing steps and current specific viral inactivation methods. An overview of the present safety status of IV IgG concentrates is presented as well as the recent introduction of new promising techniques for the overall improvement of the safety of this plasma derivative.

A minipool process for solvent-detergent treatment of cryoprecipitate at blood centres using a disposable bag system.

BACKGROUND AND OBJECTIVES: Single-donor or small-pool cryoprecipitates are produced by blood establishments, mostly in developing countries, for substitute therapy in haemophilia A, von Willebrand disease and fibrinogen deficiency, as well as for the manufacture of fibrin sealant. As cryoprecipitate may be contaminated with pathogenic plasma-borne viruses, there is an urgent need to develop a simple method for the viral inactivation of cryoprecipitate. MATERIALS AND METHODS: Cryoprecipitate was obtained according to standard procedures. Ten minipools of five or six donations of cryoprecipitate were prepared and subjected, in sterile closed bags, to a viral inactivation treatment using either 2% tri(n-)butyl phosphate (TnBP) for 4 h at 37 degrees C or the combination of 1% TnBP and 1% Triton X-45 for 4 h at 31 degrees C. The cryoprecipitates were subsequently extracted three times in their processing bags by mixing and decantation using 7.5% sterile ricinus oil. The TnBP-treated cryoprecipitates were further subjected to a clarifying centrifugation step at 3800 g for 30 min. The final products were dispensed into individual bags and frozen at -30 degrees C or lower. RESULTS: The cryoprecipitates treated with either 2% TnBP or 1% TnBP + 1% Triton X-45 showed excellent (> 93%) mean recovery of coagulant factor VIII (FVIII), ristocetin cofactor Von Willebrand factor (VWF:RCo), and clottable fibrinogen activity. Prothrombin time, international normalized ratio and activated partial thromboplastin time increased during solvent-detergent treatment but returned to initial values after oil extractions. The final content of TnBP and Triton X-45 was < 10 and 50 ppm, indicating excellent removal by the oil-extraction procedure. CONCLUSIONS: Viral inactivation treatment by TnBP, with or without Triton X-45, can be applied to minipools of cryoprecipitate, with good recovery of FVIII, VWF and fibrinogen. The viral inactivation and solvent-detergent removal process can be performed in a closed bag system and using simple blood establishment techniques and equipment. This technology could be considered for the improved viral safety of cryoprecipitate which is used to treat haemophilia A, von Willebrand disease or fibrinogen deficiency, or to prepare fibrin sealant.

New methodology for viability testing in environmental samples.

Environmental samples can be complex and are comprised of microorganisms and a matrix of decaying organic matter as well as an inorganic phase such as sand or precipitated material (waste water, sludge, soils, etc.). Nucleic acid dyes have recently been developed to address the growing need for environmental analyses (cell staining, counting, viability testing and specific organism identification). However, certain dyes may not be ideally suited for testing of environmental samples, because they readily adhere to the substrate material as well as their target molecule, resulting in increased non-specific binding and background fluorescence. The aim of this study was to address the limitations of the widely used and commercially available Live/Dead BacLight Bacterial Viability kit (Molecular Probes, Eugene, OR). A new combination of nucleic acid dyes, i.e. SYTO13 and SYTOX Orange (Molecular Probes, Eugene, OR), was proposed as an alternative. The dyes were carefully chosen for their spectral separation and increase of fluorescence quantum yield. A protocol for this combination was first designed and optimized and the two staining assays were compared against suspensions of live and dead E. coli, mixed in different proportions and it was shown that both protocols performed equally on pure cultures. However, when testing activated sludge samples, the commercial kit showed greater background fluorescence and non-specific binding than the alternate combination. Therefore, the proposed dye combination and its corresponding protocol are deemed more suitable for use on complex environmental samples than the Live/Dead BacLight Bacterial Viability kit.

Research and development commitments in an integrated plasma collection and plasma fractionation environment.

Plasma fractionation has emerged as one of the most scientifically demanding fields in the biopharmaceutical area. Producing safe plasma derivatives implies the development, use, and proper understanding of sensitive testing technologies to detect infection markers in starting plasma. It also requires the implementation of carefully selected, nondenaturing, efficient plasma protein purification and viral reduction technologies that do not alter the physiological functions and clinical potential of plasma proteins. Success in this field can be achieved only by a strong commitment to sustain constant research and development of projects targeting the production of safer and innovative plasma products.

Nanofiltration of plasma-derived biopharmaceutical products.

This review presents the current status on the use and benefits of viral removal filtration systems--known as nanofiltration--in the manufacture of plasma-derived coagulation factor concentrates and other biopharmaceutical products from human blood origin. Nanofiltration of plasma products has been implemented at a production scale in the early 1990s to improve margin of viral safety, as a complement to the viral reduction treatments, such as solvent-detergent and heat treatments, already applied for the inactivation of human immunodeficiency virus, hepatitis B and hepatitis C virus. The main reason for the introduction of nanofiltration was the need to improve product safety against non-enveloped viruses and to provide a possible safeguard against new infectious agents potentially entering the human plasma pool. Nanofiltration has gained quick acceptance as it is a relatively simple manufacturing step that consists in filtering protein solution through membranes of a very small pore size (typically 15-40 nm) under conditions that retain viruses by a mechanism largely based on size exclusion. Recent large-scale experience throughout the world has now established that nanofiltration is a robust and reliable viral reduction technique that can be applied to essentially all plasma products. Many of the licensed plasma products are currently nanofiltered. The technology has major advantages as it is flexible and it may combine efficient and largely predictable removal of more than 4 to 6 logs of a wide range of viruses, with an absence of denaturing effect on plasma proteins. Compared with other viral reduction means, nanofiltration may be the only method to date permitting efficient removal of enveloped and non-enveloped viruses under conditions where 90-95% of protein activity is recovered. New data indicate that nanofiltration may also remove prions, opening new perspectives in the development and interest of this technique. Nanofiltration is increasingly becoming a routine step in the manufacture of biopharmaceutical products.

Inhibition of atrazine degradation by cyanazine and exogenous nitrogen in bacterial isolate M91-3.

A variety of s-triazine herbicides and nitrogen fertilizers frequently occur as co-contaminants at pesticide manufacturing and distribution facilities. The degradation of atrazine and cyanazine by the bacterial isolate M91-3 was investigated in washed-cell suspensions and crude cellular extracts. Cyanazine competitively inhibited atrazine degradation. The maximum atrazine degradation rate (Vmax) was 41 times higher and the half-saturation constant for the inhibitor (Ki) was 1.3 times higher in the crude cellular extract than in the washed-cell suspension, suggesting that cellular uptake influenced degradation of the s-triazines. Cultures that had received prior exposure to atrazine and simazine exhibited comparable atrazine degradation rates, while cells exposed to cyanazine, propazine, ametryne, cyanuric acid, 2-hydroxyatrazine, biuret, and urea exhibited a lack of atrazine-degradative activity. Growth in the presence of exogenous inorganic nitrogen inhibited subsequent atrazine-degradative activity in washed-cell suspensions, suggesting that regulation of s-triazine and nitrogen metabolism are linked in this bacterial isolate. These findings have significant implications for the environmental fate of s-triazines in agricultural settings since these herbicides are frequently applied to soils receiving N fertilizers. Furthermore, these results suggest that bioremediation of s-triazine-contaminated sites (common at pesticide distribution facilities in the cornbelt) may be inhibited by the presence of N fertilizers that occur as co-contaminants.