Development and validation of a quantitative wipe sampling method to determine platinum contamination from antineoplastic drugs on surfaces in workplaces at Swedish hospitals.

INTRODUCTION: Antineoplastic drugs (ADs) are frequently used pharmaceuticals in the healthcare, and healthcare workers can be occupationally exposed to ADs. Monitoring of surface contamination is a common way to assess occupational exposure to ADs. The objective was to develop and validate a sensitive and quantitative monitoring method to determine surface contaminations of Pt as a marker for Pt-containing ADs. The surface contaminations of Pt-containing ADs were monitored at four Swedish hospital workplaces. METHODS: An analytical method was developed based on inductively coupled plasma mass spectrometry. The wipe sampling procedure was validated regarding different surface materials. The stability of collected wipe samples was investigated. Workplace surfaces were monitored by wipe sampling to determine contaminations of Pt-containing ADs. RESULTS: A wipe sampling and analytical method with a limit of detection of 0.1 pg Pt/cm2 was developed. Pt was detected in 67% of the wipe samples collected from four workplaces, and the concentrations ranged from <0.10 to 21100 pg/cm2. In 4% of samples, the detected surface contaminations of Pt in three hospital wards were above proposed hygienic guidance value (HGV) of Pt. In the hospital pharmacy, 9% of the detected surface contaminations of Pt were above lowest proposed HGV. CONCLUSIONS: A user-friendly, specific, and sensitive method for determination of surface contaminations of Pt from ADs in work environments was developed and validated. A large variation of contaminations was observed between detected surface contaminations of Pt in samples collected in wards, and it likely reflects differences in amounts handled and work practices between the wards.

Investigating thermally induced aggregation of Somatropin- new insights using orthogonal techniques.

Three orthogonal techniques were used to provide new insights into thermally induced aggregation of the therapeutic protein Somatropin at pH 5.8 and 7.0. The techniques were Dynamic Light Scattering (DLS), Asymmetric Flow-Field Flow-Fractionation (AF4), and the TEM-based analysis system MiniTEM™. In addition, Differential Scanning Calorimetry (DSC) was used to study the thermal unfolding and stability. DSC and DLS were used to explain the initial aggregation process and aggregation rate at the two pH values. The results suggest that less electrostatic stabilization seems to be the main reason for the faster initial aggregation at pH 5.8, i.e., closer to the isoelectric point of Somatropin. AF4 and MiniTEM were used to investigate the aggregation pathway further. Combining the results allowed us to demonstrate Somatropin's thermal aggregation pathway at pH 7.0. The growth of the aggregates appears to follow two steps. Smaller elongated aggregates are formed in the first step, possibly initiated by partly unfolded species. In the second step, occurring during longer heating, the smaller aggregates assemble into larger aggregates with more complex structures.

Investigation of native and aggregated therapeutic proteins in human plasma with asymmetrical flow field-flow fractionation and mass spectrometry.

Physiochemical degradation of therapeutic proteins in vivo during plasma circulation after administration can have a detrimental effect on their efficacy and safety profile. During drug product development, in vivo animal studies are necessary to explore in vivo protein behaviour. However, these studies are very demanding and expensive, and the industry is working to decrease the number of in vivo studies. Consequently, there is considerable interest in the development of methods to pre-screen the behaviour of therapeutic proteins in vivo using in vitro analysis. In this work, asymmetrical flow field-flow fractionation (AF4) and liquid chromatography-mass spectrometry (LC-MS) were combined to develop a novel analytical methodology for predicting the behaviour of therapeutic proteins in vivo. The method was tested with two proteins, a monoclonal antibody and a serum albumin binding affibody. After incubation of the proteins in plasma, the method was successfully used to investigate and quantify serum albumin binding, analyse changes in monoclonal antibody size, and identify and quantify monoclonal antibody aggregates.

Revisiting the dynamics of proteins during milk powder hydration using asymmetric flow field-flow fractionation (AF4).

The dynamics of ß-casein and casein micelles in the reconstitution of skim milk were revisited in this study. ß-casein migrates into casein micelles upon an increase in temperatures due to an increase in the hydrophobic effect and lower calcium-phosphate cluster solubility. This process can be reversed upon cooling. These phenomena are well known in fresh milk and are not yet clearly established for reconstituted milk powder. As milk powder is commonly used as a functional ingredient in food products, it is of interest to investigate the migration of casein micelle ß-casein to and from the serum phase in reconstituted milk. This study aimed to use asymmetrical flow field flow fractionation (AF4) in combination with various detectors to revisit the dynamics of ß-casein when reconstituting skim milk at different temperatures. Fluorescence-labelled ß-casein was added to fresh and reconstituted skim milk and rapid transport of ß-casein into the outer shell of the casein micelles could be observed already after 5 â€‹min of reconstitution at 50 â€‹°C. This process stabilized after approximately 5 â€‹h, which indicates that an equilibrium of ß-casein between the serum and the micellar phase was reached. Similar results were found for fresh milk. The apparent density of the casein micelles in the skim milk samples was also found to increase during reconstitution at 50 â€‹°C. During cold reconstitution of milk powders, the migration of ß-casein to the serum was not observed. The results suggest that ß-casein was already present in the serum phase upon reconstitution at 6 â€‹°C. When a sample was reconstituted for 180 â€‹min at 50 â€‹°C, the migration of ß-casein back into the serum was observed upon cooling the same sample to 6 â€‹°C. The size of casein micelles in reconstituted milk at 6 â€‹°C was larger compared to reconstitution at 50 â€‹°C. With AF4 and the multi-detector approach, the change in concentration and size of casein micelles can be readily investigated and the migration of ß-casein can be tracked simultaneously. Therefore, the method is a valuable tool for studies of the properties and changes in various milk samples.

Investigating the effect of powder manufacturing and reconstitution on casein micelles using asymmetric flow field-flow fractionation (AF4) and transmission electron microscopy.

Milk powders are commonly used for a variety of food products in which among others the milk proteins add to the properties of the products. Processing of milk can, depending on the processing parameters, change the size and structure of the proteins. These changes can be difficult to measure due to the polydispersity of milk components, which makes it a challenge to obtain direct information about the individual proteins. In this paper, the results from an investigation of casein micelle size,size distribution, and structure in reconstituted skim milk and the comparison with raw and pasteurized skim milk are reported. The investigation used asymmetrical flow field-flow fractionation (AF4) in combination with online UV, multi-angle light scattering (MALS), and refractive index (RI) detection and the results were confirmed by transmission electron microscopy (TEM). The results show that there is a difference in casein micelle size distribution between the differently processed milk samples. The casein micelles of the reconstituted milk were found to have a z-average radius of gyration of 72 nm and the casein micelles in the raw and pasteurized skim milk were 58 and 62 nm respectively. The AF4 and TEM data suggest that the cause of the larger casein micelle size is a layer of aggregated whey proteins associated with the casein micelles surface. Moreover, the TEM investigation showed that a larger proportion of the casein micelles are aggregated in reconstituted milk compared to raw and fresh skim milk. Investigation of the effect of reconstitution time shows that the amount of aggregated casein micelles decreases during the first 20 min of reconstitution. The results show that the AF4-method can provide detailed insights into the reconstitution process and properties of different milk samples. Hence, it can be used as a reference or validation for more indirect methods to track the reconstitution of milk powders.

Asymmetric flow field-flow fractionation coupled to surface plasmon resonance detection for analysis of therapeutic proteins in blood serum.

Coupling of surface plasmon resonance (SPR) detection to asymmetric flow field-flow fractionation (AF4) offers the possibility to study active fractions of bio-separations on real samples, such as serum and saliva, including the assessment of activity of possibly aggregated species. The coupling of SPR with AF4 requires the possibility to select fractions from a fractogram and redirect them to the SPR. The combination of SPR with chromatography-like methods also requires a mechanism for regeneration of the receptor immobilised onto the SPR sensor surface. In recent work, the combination of size exclusion chromatography (SEC) with SPR was pioneered as a successful methodology for identification, characterisation and quantification of active biocomponents in biological samples. In this study, the approach using AF4 is evaluated for the antibody trastuzumab in buffer and serum. The particular object of this study was to test the feasibility of using AF4 in combination with SPR to detect and quantify proteins and aggregates in complex samples such as blood serum. Also, in the investigation, three different immobilisation methods for the receptor HER-2 were compared, which involved (1) direct binding via EDC/NHS, the standard approach; (2) immobilisation via NTA-Ni-Histag complexation; and (3) biotin/avidin-linked chemistry using a regenerable form of avidin. The highest specific activity was obtained for the biotin-avidin method, while the lowest specific activity was observed for the NTA-Ni-Histag linkage. The data show that AF4 can separate trastuzumab monomers and aggregates in blood serum and that SPR has the ability to selectively monitor the elution. This is an encouraging result for automated analysis of complex biological samples using AF4-SPR.

Proteins and antibodies in serum, plasma, and whole blood-size characterization using asymmetrical flow field-flow fractionation (AF4).

The analysis of aggregates of therapeutic proteins is crucial in order to ensure efficacy and patient safety. Typically, the analysis is performed in the finished formulation to ensure that aggregates are not present. An important question is, however, what happens to therapeutic proteins, with regard to oligomerization and aggregation, after they have been administrated (i.e., in the blood). In this paper, the separation of whole blood, plasma, and serum is shown using asymmetric flow field-flow fractionation (AF4) with a minimum of sample pre-treatment. Furthermore, the analysis and size characterization of a fluorescent antibody in blood plasma using AF4 are demonstrated. The results show the suitability and strength of AF4 for blood analysis and open new important routes for the analysis and characterization of therapeutic proteins in the blood.

Size separations of starch of different botanical origin studied by asymmetrical-flow field-flow fractionation and multiangle light scattering.

Asymmetrical-flow field-flow fractionation combined with multiangle light scattering and refractive index detection has been revealed to be a powerful tool for starch characterization. It is based on size separation according to the hydrodynamic diameter of the starch components. Starch from a wide range of different botanical sources were studied, including normal starch and high-amylose and high-amylopectin starch. The starch was dissolved by heat treatment at elevated pressure in a laboratory autoclave. This gave clear solutions with no granular residues. Amylose retrogradation was prevented by using freshly dissolved samples. Programmed cross flow starting at 1.0 mL min(-1) and decreasing exponentially with a half-life of 4 min was utilised. The starches showed two size populations representing mainly amylose and mainly amylopectin with an overlapping region where amylose and amylopectin were possibly co-eluted. Most of the first population had molar masses below 10(6) g mol(-1), and most of the second size population had molar masses above 10(7) g mol(-1). Large differences were found in the relative amounts of the two populations, the molar mass, and hydrodynamic diameters, depending on the plant source and its varieties.

Asymmetrical flow field-flow fractionation coupled with multi-angle light scattering and refractive index detections for characterization of ultra-high molar mass poly(acrylamide) flocculants.

The molar mass distributions of ultra-high molar mass polyacrylamide-based flocculants were measured using asymmetrical flow field-flow fractionation (AFFFF) coupled with multi-angle light scattering and refractive index detectors. The mass load onto the separation channel was found to be critical in obtaining a good size separation. The detailed investigation with ultra-high molar mass polyacrylamides found that the injected amount should be

Competitive adsorption of a polydisperse polymer during emulsification: experiments and modeling.

In this paper we study the selective adsorption of a high molar mass polymer, OSA-starch, at the cyclohexane/water interface during emulsification. This was made possible through the use of AsFlFFF-MALS-RI which enables us to characterize the size and molar mass of polydisperse ultrahigh molar mass polymers. The results show that the high molar mass components in the molar mass distribution of the polymer were selectively adsorbed. The selective adsorption is most likely due to convective mass transport in the turbulent flow fields, during formation of the emulsion, which favors transport to the interface of the high molar mass polymers in the sample. The rapid adsorption that occurs during emulsification is likely to give rise to nonequilibrium effects and jamming at the interface. Furthermore, we describe the adsorption process and illustrate its selective nature through a turbulent flow collision model.

Programmed cross flow asymmetrical flow field-flow fractionation for the size separation of pullulans and hydroxypropyl cellulose.

Different functions for the programming of the cross flow in asymmetrical flow field-flow fractionation were studied with the aim to find the flow conditions most suitable for the molar mass distribution analysis of high molecular weight polysaccharides. A mixture of four differently sized pullulans covering the molar mass range 5.8 x 10(3)-1.6 x 10(6) g mol(-1) were used as a model sample. Two types of programs were studied, linear and exponential decays, both with and without initial periods of a constant cross flow. For comparison, nonprogrammed runs, i.e. using constant cross flow, were studied. It was found that exponentially decaying cross flow gave the most uniform molar mass selectivity across the fractogram. The programmed cross flow was applied to the molar mass distribution analysis of a technical quality of hydroxypropyl cellulose.

Mechanical degradation and changes in conformation of hydrophobically modified starch.

In this paper, we study the mechanical degradation and changes in conformation of a branched ultrahigh molar mass biomacromolecule, hydrophobically modified starch, as caused by high-pressure homogenization. The characterization was performed with asymmetrical flow field-flow fractionation (AsFlFFF) with multiangle light scattering (MALS) and refractive index detection. The starch which had been chemically modified with octenyl succinate anhydride (OSA) proved to be very large and polydisperse. Upon high-pressure homogenization, the molar mass and rms radius (r(rms)) decreased, and the extent of these changes was related to the turbulent flow conditions during homogenization. The treatment also induced an increase and scaling with size in the apparent density of the macromolecules. To further study the changes in conformation, it was necessary to calculate the hydrodynamic radii (r(h)). This can be determined numerically from the elution times in the analysis and the flow conditions in the AsFlFFF channel. The results showed that the treatment can cause a dramatic decrease in the quotient between r(rms) and r(h), suggesting major conformational changes. These results together could be interpreted as degradation and "crumpling" of the macromolecule, which would give a decrease in r(rms) and an increase in apparent density, together with a "fraying" of more outer parts of the macromolecule, which could give rise to the increase in r(h).

Size characterization of green fluorescent protein inclusion bodies in E. coli using asymmetrical flow field-flow fractionation-multi-angle light scattering.

The goal of this study was to investigate the applicability of asymmetrical flow field-flow fractionation-multi-angle light scattering (AsFlFFF-MALS) for size analysis of green fluorescent protein inclusion bodies (GFPIBs). The size distributions of GFPIBs prepared by various culture conditions were determined. For GFPIBs prepared at 37 degrees C the peak maximum hydrodynamic diameter (d(H)) first increased and then decreased with the increase of the induction times in the presence of 0.1 and 2 mM isopropyl-beta-D-thiogalactoside (IPTG). For GFPIBs prepared at 30 degrees C the peak maximum d(H) was constant at about 700 nm irrespectively of the induction times and IPTG concentrations.