Biologics Formulation Factors Affecting Metal Leachables from Stainless Steel

An area of increasing concern and scientific scrutiny is the potential contamination of drug products by leachables entering the product during manufacturing and storage. These contaminants may either have a direct safety impact on the patients or act indirectly through the alteration of the physicochemical properties of the product. In the case of biotherapeutics, trace amounts of metal contaminants can arise from various sources, but mainly from contact with stainless steel (ss). The effect of the various factors, buffer species, solution fill volume per unit contact surface area, metal chelators, and pH, on metal leachables from contact with ss over time were investigated individually. Three major metal leachables, iron, chromium, and nickel, were monitored by inductively coupled plasma–mass spectrometry because they are the major components of 316L ss. Iron was primarily used to evaluate the effect of each factor since it is the most abundant. It was observed that each studied factor exhibited its own effect on metal leachables from contact with ss. The effect of buffer species and pH exhibited temperature dependence over the studied temperature range. The metal leachables decreased with the increased fill volume (mL) per unit contact ss surface area (cm²) but a plateau was achieved at approximately 3 mL/cm². Metal chelators produced the strongest effect in facilitating metal leaching. In order to minimize the metal leachables and optimize biological product stability, each formulation factor must be evaluated for its impact, to balance its risk and benefit in achieving the target drug product shelf life.     

Biosorption of heavy metals by Bacillus thuringiensis strain OSM29 originating from industrial effluent contaminated north Indian soil

The study was navigated to examine the metal biosorbing ability of bacterial strain OSM29 recovered from rhizosphere of cauliflower grown in soil irrigated consistently with industrial effluents. The metal tolerant bacterial strain OSM29 was identified as Bacillus thuringiensis following 16S rRNA gene sequence analysis. In the presence of the varying concentrations (25–150 mgl⁻¹) of heavy metals, such as cadmium, chromium, copper, lead and nickel, the B. thuringiensis strain OSM29 showed an obvious metal removing potential. The effect of certain physico-chemical factors such as pH, initial metal concentration, and contact time on biosorption was also assessed. The optimum pH for nickel and chromium removal was 7, while for cadmium, copper and lead, it was 6. The optimal contact time was 30 min. for each metal at 32 ± 2 °C by strain OSM29. The biosorption capacity of the strain OSM29 for the metallic ions was highest for Ni (94%) which was followed by Cu (91.8%), while the lowest sorption by bacterial biomass was recorded for Cd (87%) at 25 mgl⁻¹ initial metal ion concentration. The regression coefficients obtained for heavy metals from the Freundlich and Langmuir models were significant. The surface chemical functional groups of B. thuringiensis biomass identified by Fourier transform infrared (FTIR) were amino, carboxyl, hydroxyl, and carbonyl groups, which may be involved in the biosorption of heavy metals. The biosorption ability of B. thuringiensis OSM29 varied with metals and was pH and metal concentration dependent. The biosorption of each metal was fairly rapid which could be an advantage for large scale treatment of contaminated sites.     

EDTA chelation effects on urinary losses of cadmium, calcium, chromium, cobalt, copper, lead, magnesium, and zinc

The efficacy of a chelating agent in binding a given metal in a biological system depends on the binding constants of the chelator for the particular metals in the system, the concentration of the metals, and the presence and concentrations of other ligands competing for the metals in question. In this study, we make a comparison of the in vitro binding constants for the chelator, ethylenediaminetetraacetic acid, with the quantitative urinary excretion of the metals measured before and after EDTA infusion in 16 patients. There were significant increases in lead, zinc, cadmium, and calcium, and these increases roughly corresponded to the expected relative increases predicted by the EDTA-metal-binding constants as measured in vitro. There were no significant increases in urinary cobalt, chromium, or copper as a result of EDTA infusion. The actual increase in cobalt could be entirely attributed to the cobalt content of the cyanocobalamin that was added to the infusion. Although copper did increase in the post-EDTA specimens, the increase was not statistically significant. In the case of magnesium, there was a net retention of approximately 85% following chelation. These data demonstrate that EDTA chelation therapy results in significantly increased urinary losses of lead, zinc, cadmium, and calcium following EDTA chelation therapy. There were no significant changes in cobalt, chromium, or copper and a retention of magnesium. These effects are likely to have significant effects on nutrient concentrations and interactions and partially explain the clinical improvements seen in patients undergoing EDTA chelation therapy.     

Heavy metal biosorption by biomass of Ochrobactrum anthropi producing exopolysaccharide in activated sludge

The removal of chromium, cadmium and copper, toxic metals of high environmental priority due to their toxicity, from dilute aqueous solutions has been studied in the present work, applying a dead exopolysaccharide producing bacterium, Ochrobactrum anthropi, isolated from activated sludge. Particularly, the effect of pH, metal concentration and the effects of contact time were considered. Optimum adsorption pH values of chromium(VI), cadmium(II) and copper(II) were 2.0, 8.0 and 3.0 respectively. Experimental results also showed the influence of initial metal concentration on the metal uptake for dried biomass. Both the Freundlich and Langmuir adsorption models were suitable for describing the short-term biosorption of chromium(VI), cadmium(II) and copper(II) by O. anthropi.     

The impacts of heavy metals on oxidative stress and growth of spring barley

Oxidative stress is accepted to play a significant role in stress symptoms, caused by different stressors in a variety of organisms. In this study seedlings of spring barley (Hordeum vulgare L.) were exposed to a wide range of copper, zinc, chromium, nickel, lead and cadmium concentrations in order to determine the relationships between heavy metals-induced oxidative stress and plant growth inhibition. All investigated heavy metals induced an essential increase in lipid peroxidation and a reduction of dry biomass along with an increase in metal concentration in the nutrient solution. A very close and statistically significant exponential relationship between lipid peroxidation and growth inhibition was detected in this study. According to the results of analysis of variance (ANOVA), the intensity of nonspecific oxidative stress is identified as the main factor of barley growth inhibition, explaining 75% of total variance. Almost 10% of growth inhibition is attributed to the specific impact of heavy metals. The most pronounced increase of malondialdehyde content and growth inhibition was observed in Cu and Cd treatments, whereas the lowest changes in observed indicators were detected after exposure to Zn and Pb.     

The use of flocculating brewer's yeast for Cr(III) and Pb(II) removal from residual wastewaters

The use of inexpensive biosorbents to sequester heavy metals from aqueous solutions, is one of the most promising technologies being developed to remove these toxic contaminants from wastewaters. Considering this challenge, the viability of Cr(III) and Pb(II) removal from aqueous solutions using a flocculating brewer's yeast residual biomass from a Portuguese brewing industry was studied. The influence of physicochemical factors such as medium pH, biomass concentration and the presence of a co-ion was characterised. Metal uptake kinetics and equilibrium were also analysed, considering different incubation temperatures. For both metals, uptake increased with medium pH, being maximal at 5.0. Optimal biomass concentration for the biosorption process was determined to be 4.5?g dry weight/l. In chromium and lead mixture solutions, competition for yeast binding sites was observed between the two metals, this competition being pH dependent. Yeast biomass showed higher selectivity and uptake capacity to lead. Chromium uptake kinetic was characterised as having a rapid initial step, followed by a slower one. Langmuir model describes well chromium uptake equilibrium. Lead uptake kinetics suggested the presence of mechanisms other than biosorption, possibly including its precipitation.     

A Risk-Based Approach to Management of Leachables Utilizing Statistical Analysis of Extractables

To incorporate quality by design concepts into the management of leachables, an emphasis is often put on understanding the extractable profile for the materials of construction for manufacturing disposables, container-closure, or delivery systems. Component manufacturing processes may also impact the extractable profile. An approach was developed to (1) identify critical components that may be sources of leachables, (2) enable an understanding of manufacturing process factors that affect extractable profiles, (3) determine if quantitative models can be developed that predict the effect of those key factors, and (4) evaluate the practical impact of the key factors on the product. A risk evaluation for an inhalation product identified injection molding as a key process. Designed experiments were performed to evaluate the impact of molding process parameters on the extractable profile from an ABS inhaler component. Statistical analysis of the resulting GC chromatographic profiles identified processing factors that were correlated with peak levels in the extractable profiles. The combination of statistically significant molding process parameters was different for different types of extractable compounds. ANOVA models were used to obtain optimal process settings and predict extractable levels for a selected number of compounds. The proposed paradigm may be applied to evaluate the impact of material composition and processing parameters on extractable profiles and utilized to manage product leachables early in the development process and throughout the product lifecycle.KEY WORDS: design of experiments, extractables, injection molding, leachables, process parameters, quality by design     

Transition metal abnormalities in progressive dementias

Abnormal distributions of transition metals inside the brain are potential diagnostic markers for several central nervous system diseases, including Alzheimer’s disease (AD), Parkinson’s disease, dementia with Lewy bodies (DLB), bipolar disorders and depression. To further explore this possibility, the total concentrations of iron, zinc, copper, manganese, aluminum, chromium and cadmium were measured in post-mortem hippocampus and amygdala tissues taken from AD, DLB and Control patients. A statistically significant near fifty percent reduction in the total copper levels of AD patients was observed in both the hippocampus and amygdala. The statistical power of the hippocampus and amygdala copper analysis was found to be 86 and 74% respectively. No statistically significant deviations in the total metal concentrations were found for zinc, manganese, chromium or aluminum. Iron was found to be increased by 38% in AD amygdala tissues, but was unchanged in AD hippocampus tissues. Accounting for differences in tissue water content, as a function of both tissue type and disease state, revealed more consistencies with previous literature. To aid in the design of future experiments, the effect sizes for all tissue types and metals studied are also presented.     

Effects of copper and cadmium on heavy metal polluted waterbody restoration by duckweed (Lemna minor).

Aquatic plants have been identified as a potentially useful group for accumulating and bioconcentrating heavy metals. In the study, we investigated changes in the contents of soluble protein and photosynthetic pigments as well as the activity of antioxidant enzymes caused by copper sulfate and cadmium dichloride, respectively in duckweed (Lemna minor) during concentration-dependent exposure (0.05-20 mg l(-1)) to metal salt. The results demonstrated that exposure to high concentration heavy metals (Cu>10 mg l(-1), Cd>0.5 mg l(-1)) could result the disintegration of antioxidant system in duckweed. Also, the significant decrease of contents of soluble protein and photosynthetic pigments was observed to high-level metal stress. Additionally, cadmium was found to be more toxic than copper on plants. The outcome of this study corroborate that Lemna minor is a suitable candidate for the phytoremediation of low-level copper and cadmium contaminated waterbody.     

Effects of Chromium Ore Tailings on Growth and Physiological Activities of Mesua ferrea L.

A greenhouse pot experiment was conducted to evaluate the feasibility of using a native ornamental plant, Mesua ferrea L. as phytostabilizer for chromium ore tailings (COT) and to assess the metal accumulation capacity. Different ratios of soil and COT were taken in pots and sowed with seeds of M. ferrea. Plants were harvested at various intervals and separated into roots and shoots for analysis of metal concentrations and physiological characteristics of the plants. The study revealed that the plant has great tolerance and stronger ability to accumulate Cr. The results suggested an increase in growth, chlorophyll content, antioxidant activities, as well as metal accumulation capacity of M. ferrea with increasing proportion of COT in the soil. This indicates the plant's efficiency to overcome any stress generated due to excess of chromium as well as other heavy metals. The order of accumulation of heavy metals was observed to be Fe>Cr>Ni>Cd>Co. The accumulation of Cr was higher in root compared to that in shoot. M. ferrea has found to be potential as a native species candidate for phytostabilization of chromium mine tailings.     

RNA polymerase II from wheat germ contains tightly bound zinc

Atomic absorption studies indicate that the DNA-dependent RNA polymerase II from wheat germ contains about 7 tightly bound zinc atoms per enzyme molecule. This value has been repeatedly obtained with a number of enzyme preparations subjected to varying conditions of purification and dialysis. However, prolonged dialysis of the enzyme with the metal chelator $\text{o}$-phenanthroline results in the loss of enzyme activity and extraction of the bound zinc. Other metals including copper, cobalt, manganese, magnesium, chromium, nickel and iron were not present in significant amounts.     

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin.

The interaction of hydroxypyridinones with human serum transferrin and ovotransferrin has been studied by analyzing the distribution of iron between the chelator and the proteins as a function of both ligand concentration and transferrin saturation. The kinetics of iron removal by 3-hydroxypyridin-4-ones from both transferrins is slow; in ovotransferrin it appears to be monophasic, in contrast to that observed for serum transferrin. After 24 hours incubation at a 40:1 chelator:protein molar ratio, the percentage of iron removed from Fe(III)-ovotransferrin is 50%-60%, and is somewhat higher in the case of serum transferrin, in line with the respective affinity constants for the metal. The 3-hydroxypyridin-2-ones and the 3-hydroxypyran-4-ones, both of which have lower affinities for Fe(III), remove smaller proportions of the metal. The percentage of desaturation obtained with bidentate and hexadentate pyridinones appears to be similar for both transferrin classes at chelator:protein molar ratios from 40:1. The degree of transferrin saturation influences the extent of chelator mediated iron mobilization in the case of serum transferrin, but not of ovotransferrin. 59Fe competition studies demonstrate that bidentate pyridin-4-ones are capable of donating iron to serum apotransferrin; the relative concentrations of ligand and protein influence the distribution of iron because their effective binding constants (at pH 7.4) for Fe(III) are similar.     

In situ Carica papaya stem matrix and Fusarium oxysporum (NCBT-156) mediated bioremediation of chromium

Removal of heavy metal chromium was carried out using the fungus Fusarium oxysporum NCBT-156 strain isolated from soil of leather tanning effluent in in situ condition using potassium dichromate solution with 10 per cent Czapek-dox liquid medium. Biosorbent matrix was developed using Carica papaya plant dry stem to colonize the fungal strain to facilitate bioabsorption process. Bioabsorption of chromium was by metabolically mediated intracellular accumulation process. Maximum efficiency of chromium removal by biosorption upto 90 per cent was achieved at the end of 5th day of incubation (120 h of contact time) for 100 and 200 ppm concentration, upto 80 per cent for 300 and 400 ppm, and upto 65 per cent for 500 ppm to 1000 ppm concentrations with pH ranging from 5.8, 5.6, 5.5, 5.4 and 5.2, respectively for 100, 200, 300, 400, 500-1000 ppm concentration. SDS-PAGE protein profile showed significant difference in 34 kDa protein band after chromium absorption by the fungus. FTIR spectroscopic analysis revealed that the main functional groups involved in the uptake of chromium by F. oxysporium strain were carbonyl, carboxyl, amino and hydroxyl groups.     

Single and Combined Effects of Exposure Concentration and Duration on Biological Responses of Ceratophyllum demersum L. Exposed to Cr Species

This study aimed to demonstrate the ways in which two chromium species, Cr (III) and Cr (VI), can affect various physiological and biochemical parameters in the plant Ceratophyllum demersum L., and to evaluate the single and combined impact of exposure concentration and duration. C. demersum was exposed to Cr (III) and Cr (VI) at a variety of concentrations (1, 2, 5, and 10 mM) and for differing durations (1, 2, 4, and 7 days), after which Cr accumulation, relative growth rate (RGR), malondialdehyde (MDA) content, electrical conductivity (EC), photosynthetic pigmentation, proline content and antioxidant enzyme activities were examined. The single and combined effects of exposure duration and Cr concentration on each parameter were determined using a two-way analysis of variance. For both the Cr (III) and Cr (VI) applications, it was observed that concentration had a significant effect on all parameters assessed. However, duration had no statistically significant effect on proline content in the Cr (III) application, or on MDA and protein content in the Cr (VI) application. It was determined that concentration exerted greater effects than duration for both Cr species studied. In addition, the results indicated that duration and concentration had a synergistic effect on variations of RGR, EC, protein content, and antioxidant enzyme activities in both the Cr (III) and Cr (VI) applications. These results may be useful when planning further phytoremediation and plant biotechnology studies.     

A study of the kinetics of iron and copper binding to hen ovotransferrin.

The kinetics of iron and copper binding to hen's-egg apo-ovotransferrin were studied by using citrate chelates of these metals at pH9.3 in borate buffer in the presence of bicarbonate. The kinetics of the absorbance change associated with the formation of the final product show a fast process, which is pseudo-first-order, where the reagents are in excess with respect to the protein, and the citrate concentration is higher than 25mM. At lower citrate concentration, the progress curves are clearly biphasic. There is marked dependence of the rate of the reaction on bicarbonate concentration, which may be interpreted as a displacement reaction of the ligand-metal-protein ternary complex. The kinetics have been interpreted in the framework of a reaction scheme which involves bimolecular reaction of a metal chelate to the protein and subsequent colour development by displacement of the chelator by bicarbonate. The pH-dependence of this reaction supports the belief that tyrosine residues are involved in the process of iron-binding. The overall similarity of kinetics for iron and copper binding, notwithstanding their different co-ordination preferences, suggests that the process of metal-binding or chromophore development for the two metal complexes must be similar.     

Interspecific differences in metal bioaccumulation and plant-water concentration ratios in five aquatic bryophytes

We studied the capacity of five species of aquatic bryophyte to accumulate metals, and the relationship between plant metal content and water composition, on the basis of 170 samples taken from 32 rivers in Galicia (NW Spain). In all cases, only the final two centimetres of the apex were analysed. Scapania undulata was the species with the highest accumulatory capacity, and Fissidens polyphyllus was that with the lowest. Fontinalis antipyretica, Rhynchostegium riparioides and Brachythecium rivulare displayed intermediate capacities for metal accumulation, but showed a broader range of variation in body concentration in comparison with similar contamination levels. This resolution capacity, together with a greater resistance to pollution and, in the study region, a wider distribution and higher abundance, suggests that the latter two species are the most useful for bioindication studies. Bioaccumulation factors were high for all metals studied, tending to increase with increasing body concentration but decreasing with increasing water concentration. The relationship between metal in plant and filtrable metal in water was low, but statistically significant for all the metals studied except Co in F. antipyretica and Cd, Pb and Co in S. undulata, F. polyphllyllus and B. rivulare. The influence of physical and chemical variables of the water on bioaccumulation was evaluated using step-wise multiple correlation analysis. Bioaccumulation is largely governed by physical and chemical factors, by the concentration of metal in the water and by the bioaccumulation factor of the bryophyte species. Sulphate concentration, pH and to a lesser extent nitrite, ammonia and FRP (filtrable reactive phosphate) appear to be the most important physical and chemical variables governing metal bioavailability.     

Role of Iron-Oxidizing Microorganism in Bioleaching of Heavy Metals From Sewage Sludge Contaminated with Textile Dyes

This article reports the role of indigenous iron-oxidizing microorganisms in bioleaching of heavy metals from anaerobically digested sewage sludge in presence of toxic dyes namely, methylene blue (MB), Remazol black B (RBB) and mixture of both the dyes (DM). To achieve this goal, different concentrations of dyes (0, 500, 10,000, 15,000 and 25,000 mg/L) were added to the sewage sludge (initial pH ～ 7) and subsequently, the solubilization of heavy metals (Cu, Ni, Zn and Cr) was measured at time intervals of 48 h for 16 days. The results showed that an increase in dye concentration adversely affected the population of indigenous iron-oxidizing microorganisms, thereby decreased the bioleaching of metals. The metal solubilization from sludge is more adversely affected in presence of RBB and DM than MB. In majority of cases, the maximum metal solubilization was recorded at dye concentration of 5,000 mg/L. Two factor analyses (ANOVA) suggests the impact of both factors: dye concentration and dye type on metal leaching rate in sludge system.     

Effects of work schedule and period of exposure on changes in urinary chromium and nickel excretion among rotating shift workers in a stainless-steel plant

ABSTRACT

We investigated the association between the period of exposure and changes in urinary excretion of chromium and nickel among rotating shift workers in a stainless-steel plant. The study participants were composed of two groups: the workers who were occupationally exposed to metals (“exposed group”) and those who were not occupationally exposed to metals (“unexposed group”). The exposed and unexposed groups consisted of 56 and 40 male rotating shift workers, respectively. Urine samples were collected immediately before and immediately after the day shift, evening shift, and night shift. Urinary chromium and nickel were measured using inductively coupled plasma mass spectrometry. To correct for variations in urine dilution, urinary metal concentrations were expressed as a ratio to urinary creatinine concentration. In the exposed group, post-shift urinary excretion of chromium was significantly higher than pre-shift excretion. However, although urinary chromium excretion clearly increased after the day and night shift [63% (p < .0001) and 87% (p < .0001), respectively], urinary chromium excretion after the evening shift was only slightly higher than that measured before the evening shift (8%, p = .028). Similar patterns were found for urinary nickel excretion (p = .0001, 0.20, and 0.18 for the day, evening, and night shifts, respectively). Non-uniform urinary excretion of metals between the day shift, evening shift, and night shift were observed in the exposed group; specifically, urinary metal excretion increased only slightly during the evening shift. In the unexposed group, no significant increase or decrease was found in median urinary chromium or nickel excretion (p= .63–0.87). Work shift-specific permissible exposure level would be necessary.     

Changes of chromium behavior in soil during phenanthrene removal by <Emphasis Type="Italic">Penicillium frequentans</Emphasis>

Soil contamination due to polycyclic aromatic hydrocarbons is often associated with the presence of high levels of potentially toxic metals. Bioremediation is an important option for the clean up of this type of contamination. Changes of chromium fluxes and concentrations during the phenanthrene removal by Penicillium frequentans in soil were investigated. During the bioremediation process, changes in chromium behavior were monitored by Diffusive Gradients in Thin-films (DGT) and by filtration in both sterilized and non-sterilized soils. DGT provided absolute data on fluxes from the solid phase and relative trends of concentrations of the most labile metal species. Filtration provided data on the concentrations of Cr in the solution phase. Together the data provided information about the physical and chemical metal behavior. Results showed that the highest phenanthrene removal was observed in non-sterilized soil (which included the autochthonous microorganisms and P. frequentans inoculum), with a phenanthrene removal of 73 ± 3.2%. However, in all cases microbial activity increased chromium fluxes and chromium soil solution concentration. The bioremediation of soil by P. frequentans increased the lability and mobility of chromium in soil, with potential consequences for plant uptake and for increased movement of metals into the human food chain. Published online December 2004     

The Effects of Antihypertensive Drugs on Chromium Status, Glucose Metabolism, and Antioxidant and Inflammatory Indices in Spontaneously Hypertensive Rats

The long-term use of hypotensive drugs may cause side effects, including impaired glucose metabolism and mineral status. This study tested the hypothesis that some hypotensive drugs can affect tissular chromium levels and indices of glucose metabolic and antioxidant potential in rats. The experiment was performed on 40 male spontaneously hypertensive rats (SHRs), which were assigned to five groups: control (C), with perindopril (PR), with metoprolol (MT), with indapamide (ID), and with amlodipine (AM). All rats were provided ad libitum standard diet (with or without drugs) and distilled water for 45 days. Glucose and insulin levels, along with total antioxidant status (TAS) and concentrations of TNF-alpha and C-reactive protein, were assayed in serum. Chromium concentrations in the liver and kidney were determined using the flame atomic absorption spectrometry method. Detailed statistical analysis was performed using Statistica for Windows 10.0 (StatSoft, Poland). One-way analysis of variance (ANOVA), followed by a post hoc Tukey test, was used to compare the data between groups. Treatment with indapamide and amlodipine resulted in significantly higher chromium concentrations in the liver and kidney (AM) of the rats, compared with the control group. A markedly higher concentration of glucose was found in the ID group. Treatment with amlodipine significantly increased TAS levels in serum and decreased TNF-alpha concentration in serum of the rats. A significant positive correlation between chromium concentration in tissues and serum TAS level was observed, as was a significant negative correlation between chromium concentration in the kidneys, and TNF-alpha and glucose levels in serum. In conclusion, the administration of amlodipine may lead to an increase in chromium accumulation in the internal organs, which is associated with increased antioxidant status and suppression of the inflammatory response of cells in SHRs.