How are we handling protein drugs in hospitals? A human factors and systems engineering approach to compare two hospitals and suggest a best practice.

Biopharmaceuticals are complex biological molecules that require careful storage and handling to ensure medication integrity. In this study, a work system analysis of real-world protein drug (PD) handling was performed with the following goals: identify main barriers and facilitators for successful adherence to accepted recommendations in PD handling, analyse differences in two organizations, and define a Best Current Practice in the real-life handling of PDs based on the results of the work system analysis. Observational study was held in two university hospitals in Spain and Sweden. Based on the Systems Engineering Initiative for Patient Safety (SEIPS) model, the tools chosen were: the PETT scan, in order to indicate the presence of barriers or facilitators for the PETT components (People, Environment, Tools, Tasks); the Tasks and tools matrices to construct a checklist to record direct observations during the real-life handling of biopharmaceuticals, and the Journey map to depict the work process. Observations were performed between March and November 2022. Each episode of direct observation included a single protein drug in some point of the supply chain and considered all the elements in the work system. Based on the results of the work system analysis and the literature review, the authors propose a list of items which could be assumed as Best Current Practice for PDs handling in hospitals. There were a total of 34 observations involving 19 PDs. Regarding People involved in the work process, there was a diversity of professionals with different previous training and knowledge, leading to an information gap. With respect to Environment, some structural and organizational differences between hospitals lead to risks related to the time exposure of PDs to room temperature and mechanical stress. Some differences also existed in the Tools and Tasks involved in the process, being especially relevant to the lack of compatibility information of PDs with new technologies, such as pneumatic tube system, robotic reconstitution, or closed-system transfer devices. Finally, 15 suggestions for best current practice are proposed. Main barriers found for compliance with accepted recommendations were related to the information gap detected in professionals involved in the handling of protein drugs, unmonitored temperature, and the lack of compatibility information of protein drugs with some new technologies. By applying a Human Factors and Systems Engineering Approach, the comparison of two European hospitals has led to a suggested list of Best Current Practices in the handling of protein drugs in a hospital.

Examination of the Protein Drug Supply Chain in a Swedish University Hospital: Focus on Handling Risks and Mitigation Measures.

Protein drugs, such as monoclonal antibodies, have proved successful in treating cancer and immune system diseases. The structural complexity of these molecules requires careful handling to ensure integrity and stability of the drug. In this study, a failure mode and effects analysis was performed based on a Gemba Walk method in a Swedish University Hospital. The Gemba Walk is focused on pharmacists observing the actual supply process steps from distributor, pharmacy cleanroom to patient administration. Relevant protein drugs are chosen based on sales statistics within the hospital and the corresponding wards were observed. Further is the Double Diamond design method used to identify major risks and deliver mitigation strategies. The study identified potential stress factors such as temperature, shock by impact, shaking, vibration and light exposure. There were also risks associated with porters' and healthcare professionals' lack of awareness and access to information. These risk factors may cause loss of efficacy and quality of the protein drug, potentially leading to patient safety concerns. In this study, a simulation is also performed to list measures that theoretically should be in place to ensure the quality of the protein drug, for example validated and protocol-based compounding in cleanroom, training and validated transports.

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.

Characterization of binding between model protein GA-Z and human serum albumin using asymmetrical flow field-flow fractionation and small angle X-ray scattering.

Protein-based drugs often require targeted drug delivery for optimal therapy. A successful strategy to increase the circulation time of the protein in the blood is to link the therapeutic protein with an albumin-binding domain. In this work, we characterized such a protein-based drug, GA-Z. Using asymmetrical flow field-flow fractionation coupled with multi-angle light scattering (AF4-MALS) we investigated the GA-Z monomer-dimer equilibrium as well as the molar binding ratio of GA-Z to HSA. Using small angle X-ray scattering, we studied the structure of GA-Z as well as the complex between GA-Z and HSA. The results show that GA-Z is predominantly dimeric in solution at pH 7 and that it binds to monomeric as well as dimeric HSA. Furthermore, GA-Z binds to HSA both as a monomer and a dimer, and thus, it can be expected to stay bound also upon dilution following injection in the blood stream. The results from SAXS and binding studies indicate that the GA-Z dimer is formed between two target domains (Z-domains). The results also indicate that the binding of GA-Z to HSA does not affect the ratio between HSA dimers and monomers, and that no higher order oligomers of the complex are seen other than those containing dimers of GA-Z and dimers of HSA.

Formation and Out-of-Equilibrium, High/Low State Switching of a Nitroaldol Dynamer in Neutral Aqueous Media.

The nitroaldol reaction is demonstrated as an efficient dynamic covalent reaction in phosphate buffers at neutral pH. Rapid equilibration was recorded with pyridine-based aldehydes, and dynamic oligomerization could be achieved, leading to nitroaldol dynamers of up to 17 repeating units. The dynamers were applied in a coherent stimuli-responsive molecular system in which larger dynamers transiently existed out-of-equilibrium in a neutral aqueous system rich in formaldehyde, controlled by nitromethane.

Adsorption of lysozyme, beta-casein and their layer-by-layer formation on hydrophilic surfaces: Effect of ionic strength.

The adsorbed amount and layer structure of lysozyme, beta-casein and mixed layers of the two proteins were studied on hydrophilic silica and quartz surfaces using the following techniques: ellipsometry, quartz crystal microbalance with dissipation monitoring (QCM-D) and total internal reflection fluorescence (TIRF). Particular emphasis was put on the effect of solution ionic strength on the layer formation. Both lysozyme and beta-casein showed a higher affinity for the silica surface when adsorbed from a solution of low ionic strength even though beta-casein and silica are negatively charged at the pH used. No beta-casein remained adsorbed after rinsing with a 150mM buffer solution. The adsorbed amount of lysozyme on silica exceeded a monolayer coverage irrespective of the solution conditions and displayed a rigid structure. beta-Casein forms more than a single layer on pre-adsorbed lysozyme; an inner flat layer and an outer layer with an extended structure, which largely desorbs on rinsing. The build-up through sequential adsorption of lysozyme and beta-casein is favoured at intermediate and high ionic strength. The total adsorbed amount increased slightly with each deposition cycle and the mixed lysozyme/beta-casein layers contain higher amounts of protein compared to those of pure lysozyme or beta-casein. Sequential adsorption gives rise to a proteinaceous layer consisting of both lysozyme and beta-casein. The protein layers are probably highly interpenetrated with no clear separation between them.

Studies on the exchange of early pellicle proteins by mucin and whole saliva.

Adsorption of small pellicle proteins statherin or proline-rich protein 1 (PRP1), respectively, and subsequent adsorption of human whole saliva (HWS) or salivary mucin MUC5B, respectively, was studied using ellipsometry and total internal reflectance fluorescence. Differences in elution (using sodium dodecyl sulphate (SDS) solutions) between mixed and single protein films were also investigated. On both hydrophilic and hydrophobized surfaces HWS and MUC5B were found to adsorb to pre-adsorbed layers of statherin and PRP1, respectively. Statherin adsorption on both substrate types showed no or minor exchange by HWS or MUC5B and no change in SDS elution between mixed and single protein films. Small amounts of PRP1 were exchanged by HWS on both surface types and the SDS elutable fractions were similar or larger for mixed films compared to single protein films. PRP1 and MUC5B in sequence showed minor exchange of PRP1 on hydrophilic surfaces, while no exchange could be established on hydrophobized substrates. SDS elutable fractions decreased for PRP1 and MUC5B mixed films compared to single protein films. In conclusion, minor amounts of statherin and PRP1 are exchanged during the time course of the experiments, which indicates that these proteins may to a large extent remain incorporated in the pellicle.

Adsorption and activity of Thermomyces lanuginosus lipase on hydrophobic and hydrophilic surfaces measured with dual polarization interferometry (DPI) and confocal microscopy.

The adsorption and activity of Thermomyces lanuginosus lipase (TLL) was measured with dual polarization interferometry (DPI) and confocal microscopy at a hydrophilic and hydrophobic surface. In the adsorption isotherms, it was evident that TLL both had higher affinity for the hydrophobic surface and adsorbed to a higher adsorbed amount (1.90 mg/m(2)) compared to the hydrophilic surface (1.40-1.50mg/m(2)). The thickness of the adsorbed layer was constant (approximately 3.5 nm) on both surfaces at an adsorbed amount >1.0mg/m(2), but decreased on the hydrophilic surface at lower surface coverage, which might be explained by partially unfolding of the TLL structure. However, a linear dependence of the refractive index of the adsorbed layer on adsorbed amount of TLL on C18 surfaces indicated that the structure of TLL was similar at low and high surface coverage. The activity of adsorbed TLL was measured towards carboxyfluorescein diacetate (CFDA) in solution, which upon lipase activity formed a fluorescent product. The surface fluorescence intensity increase was measured in a confocal microscope as a function of time after lipase adsorption. It was evident that TLL was more active on the hydrophilic surface, which suggested that a larger fraction of adsorbed TLL molecules were oriented with the active site facing the solution compared to the hydrophobic surface. Moreover, most of the activity remained when the TLL surface coverage decreased. Earlier reports on TLL surface mobility on the same surfaces have found that the lateral diffusion was highest on hydrophilic surfaces and at low surface coverage of TLL. Hence, a high lateral mobility might lead to a longer exposure time of the active site towards solution, thereby increasing the activity against a water-soluble substrate.

Protein-surfactant interactions at hydrophobic interfaces studied with total internal reflection fluorescence correlation spectroscopy (TIR-FCS).

The aim of this work was to study the dynamics of proteins near solid surfaces in the presence or absence of competing surfactants by means of total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Two different proteins were studied, bovine serum albumin (BSA) and Thermomyces lanuginosus lipase (TLL). A nonionic/anionic (C12E6/LAS) surfactant composition was used to mimic a detergent formulation and the surfaces used were C18 terminated glass. It was found that with increasing surfactant concentrations the term in the autocorrelation function (ACF) representing surface binding decreased. This suggested that the proteins were competed off the hydrophobic surface by the surfactant. When fitting the measured ACF to a model for surface kinetics, it was seen that with raised C12E6/LAS concentration, the surface interaction rate increased for both proteins. Under these experimental conditions this meant that the time the protein was bound to the surface decreased. At 10 microM C12E6/LAS the surface interaction was not visible for BSA, whereas it was still distinguishable in the ACF for TLL. This indicated that TLL had a higher affinity than BSA for the C18 surface. The study showed that TIR-FCS provides a useful tool to quantify the surfactant effect on proteins adsorption.

Tracking single lipase molecules on a trimyristin substrate surface using quantum dots.

The mobility of single lipase molecules has been analyzed using single molecule tracking on a trimyristin substrate surface. This was achieved by conjugating lipases to quantum dots and imaging on spin-coated trimyristin surfaces by means of confocal laser scanning microscopy. Image series of single lipase molecules were collected, and the diffusion coefficient was quantified by analyzing the mean square displacement of the calculated trajectories. During no-flow conditions, the lipase diffusion coefficient was (8.0+/-5.0)x10(-10) cm2/s. The trajectories had a "bead on a string" appearance, with the lipase molecule restricted in certain regions of the surface and then migrating to another region where the restricted diffusion continued. This gave rise to clusters in the trajectories. When a flow was applied to the system, the total distance and average step length between the clusters increased, but the restricted diffusion in the cluster regions was unaffected. This can be explained by the lipase operating in two different modes on the surface. In the cluster regions, the lipase is likely oriented with the active site toward the surface and hydrolyzes the substrate. Between these regions, a diffusion process is proposed where the lipase is in contact with the surface but affected by the external flow.

Salivary mucin MUC5B could be an important component of in vitro pellicles of human saliva: an in situ ellipsometry and atomic force microscopy study.

This paper describes a combined investigation of the salivary and MUC5B films structure and topography in conditions similar to those found in the oral cavity in terms of ionic strength, pH, and protein concentration. AFM and ellipsometry were successfully used to give a detailed picture of the film structure and topography both on hydrophilic and on hydrophobic substrata. Regardless of the substrata, the salivary film can be described as having a two sublayer structure in which an inner dense layer is decorated by large aggregates. However, the shape and height of these larger aggregates largely depend on the type of substrata used. Additionally, we show that the adsorption of MUC5B is controlled by the type of substrata and the MUC5B film topography is similar to that of the larger aggregates present in the salivary films, especially on hydrophobic substrates. Therefore, we conclude that MUC5B is a major component in the salivary film when formed on hydrophobic substrates. Furthermore, we studied how resistant the salivary and MUC5B films are against elutability by buffer rinsing and addition of SDS solution. We conclude that the adsorbed proteins contain fractions with varying binding strengths to the two types of surfaces. Specifically, we have shown that the large MUC5B biomacromolecules on the hydrophobic substrates are especially resistant to both elution with buffer solution and SDS. Therefore, these large mucins can be responsible for the increased resistance of HWS films on hydrophobic substrates and can protect the intraoral surfaces against surface-active components present in oral health care products.

A method for microbial cell surface fingerprinting based on surface plasmon resonance.

A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli mutants have been used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cell mutants and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the mutants were observed. At the same time, the physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and compared to the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA).

Mobility of Thermomyces lanuginosus lipase on a trimyristin substrate surface.

We have studied the mobility of active and inactive Thermomyces lanuginosus lipase (TLL) on a spin-coated trimyristin substrate surface using fluorescence recovery after photobleaching (FRAP) in a confocal microscopy setup. By photobleaching a circular spot of fluorescently labeled TLL adsorbed on a smooth trimyristin surface, both the diffusion coefficient D and the mobile fraction f could be quantified. FRAP was performed on surfaces with different surface density of lipase and as a function of time after adsorption. The data showed that the mobility of TLL was significantly higher on the trimyristin substrate surfaces compared to our previous studies on hydrophobic model surfaces. For both lipase variants, the diffusion decreased to similar rates at high relative surface density of lipase, suggesting that crowding effects are dominant with higher adsorbed amount of lipase. However, the diffusion coefficient at extrapolated infinite surface dilution, D0, was higher for the active TLL compared to the inactive (D0 = 17.9 x 10(-11) cm2/s vs D0 = 4.1 x 10(-11) cm2/s, data for the first time interval after adsorption). Moreover, the diffusion decreased with time after adsorption, most evident for the active TLL. We explain the results by product inhibition, i.e., that the accumulation of negatively charged fatty acid products decreased the diffusion rate of active lipases with time. This was supported by sequential adsorption experiments, where the adsorbed amount under flow conditions was studied as a function of time after adsorption. A second injection of lipase led to a significantly lower increase in adsorbed amount when the trimyristin surface was pretreated with active TLL compared to pretreatment of inactive TLL.

A comparison between dual polarization interferometry (DPI) and surface plasmon resonance (SPR) for protein adsorption studies.

This work was performed with the aim of comparing protein adsorption results obtained from the recently developed dual polarization interferometry (DPI) with the well-established surface plasmon resonance (SPR) technique. Both techniques use an evanescent field as the sensing element but completely different methods to calculate the adsorbed mass. As a test system we used adsorption of the lipase from Thermomyces lanuginosus (TLL) on C18 surfaces. The adsorbed amount calculated with both techniques is in good agreement, with both adsorption isotherms saturating at 1.30-1.35 mg/m(2) at TLL concentrations of 1000 nM and above. Therefore, this supports the use of both SPR and DPI as tools for studying protein adsorption, which is very important when comparing adsorption data obtained from the use different techniques. Due to the spot sensing in SPR, this technique is recommended for initial kinetic studies, whereas DPI is more accurate when the refractive index and thickness of the adsorbed layer is of more interest.

Immobilization of enamel matrix derivate protein onto polypeptide multilayers. Comparative in situ measurements using ellipsometry, quartz crystal microbalance with dissipation, and dual-polarization interferometry.

The buildup of biodegradable poly(L-glutamic acid) (PGA) and poly(L-lysine) (PLL) multilayers on silica and titanium surfaces and the immobilization of enamel matrix derivate (EMD) protein was followed by utilizing in situ ellipsometry, quartz crystal microbalance with dissipation, and dual-polarization interferometry (DPI). The use of the relatively new DPI technique validated earlier published ellipsometry measurements of the PLL-PGA polypeptide films. The hydrophobic aggregating EMD protein was successfully immobilized both on top of and within the multilayer structures at pH 5.0. DPI measurements further indicated that the immobilization of EMD is influenced by the flow pattern during adsorption. The formed polypeptide-EMD multilayer films are of interest since it is known that EMD is able to trigger cell response and induce biomineralization. The multilayer films thus have potential to be useful as bioactive and biodegradable coatings for future dental implants.

Adsorption of chlorhexidine and black tea onto in vitro salivary pellicles, as studied by ellipsometry.

The adsorption from 0.2% (w/w) chlorhexidine and black tea solutions onto an in vitro pellicle from whole unstimulated saliva on hydroxyapatite discs was studied by ellipsometry. It was found that chlorhexidine adsorbed to the pellicle and caused a modification of the pellicle properties, leading to a subsequent increase in adsorption of salivary and black tea components. There was a distinct order-of-addition effect, whereby chlorhexidine followed by black tea gave an overall greater adsorption of components compared with black tea followed by chlorhexidine. This increase in adsorption resulted in a concomitant increase in color or stain, as measured by a reflectance chromameter. The increase in adsorbed amounts and stain was modified, in part, by the adsorption of salivary fractions between the chlorhexidine and black tea treatments. In all cases, the chlorhexidine and black tea-modified pellicles were not readily removed by either phosphate or sodium dodecyl sulfate rinses. Thus, following exposure to chlorhexidine, the accelerated adsorption of salivary and black tea components can ultimately lead to increased staining of the pellicle.

Adsorption and mobility of a lipase at a hydrophobic surface in the presence of surfactants.

With the aim of being able to manipulate the processes involved in interfacial catalysis, we have studied the effects of a mixture of nonionic/anionic surfactants, C12E6/LAS (1:2 mol %), on the adsorption and surface mobility of a lipase obtained from Thermomyces lanuginosus (TLL). Surface plasmon resonance (SPR) and ellipsometry were used to analyze the competitive adsorption process between surfactants and TLL onto hydrophobic model surfaces intended to mimic an oily substrate for the lipase. We obtained the surface diffusion coefficient of a fluorescently labeled TLL variant on silica silanized with octadecyltrichlorosilane (OTS) by fluorescence recovery after photobleaching (FRAP) on a confocal laser scanning microscope. By means of ellipsometry we calibrated the fluorescence intensity with the surface density of the lipase. The TLL diffusion was measured at different surface densities of the enzyme and at two time intervals after coadsorption with different concentrations of C12E6/LAS. The surfactant concentrations were chosen to represent concentrations below the critical micelle concentration (CMC), in the CMC region, and above the CMC. The apparent TLL surface diffusion was extrapolated to infinite surface dilution, D0. We found that the presence of surfactants strongly modulated the surface mobility of TLL: with D(0) = 0.8 x 10(-11) cm2/s without surfactants and D0 = 13.1 x 10(-11) cm2/s with surfactants above the CMC. The increase in lipase mobility on passing the CMC was also accompanied by a 2-fold increase in the mobile fraction of TLL. SPR analysis revealed that surface bound TLL was displaced by C12E6/LAS in a concentration-dependent manner, suggesting that the observed increase in surface mobility imparts bulk-mediated diffusion and so-called rebinding of TLL to the surface. Our combined results on lipase/surfactant competitive adsorption and lipase surface mobility show how surfactants may play an important role in regulating interfacial catalysis from physiological digestion to technical applications such as detergency.

Self-assembly/aggregation behavior and adsorption of enamel matrix derivate protein to silica surfaces.

Adsorption of the amelogein protein mixture enamel matrix derivate (EMD) to silica surfaces has been studied by in situ ellipsometry and quartz crystal microbalance with dissipation (QCM-D). The protein was found to adsorb as nanospheres in mono- or multilayers, depending on the concentration of "free" nanospheres available in solution. The concentration of free nanospheres is determined by the competitive processes of adsorption and rapid aggregation into microscopic particles, measured by dynamic light scattering (DLS). Multilayers could also be formed by sequential injections of fresh EMD solution. At higher temperature, an up to 6 times thicker gel-like film was formed on the substrate surface, and decreasing the pH lead to disruption of the multilayer/aggregate formation and a decreased amount adsorbed.

Interfacial adsorption of insulin conformational changes and reversibility of adsorption.

The adsorption of human insulin to Teflon particles was studied with respect to conformational changes and the reversibility of adsorption was examined by total internal reflection fluorescence (TIRF). Adsorption isotherms for the adsorption of human insulin indicated high affinity adsorption, even at electrostatic repulsive conditions. The plateau value for adsorption was in accordance with a protein layer consisting primarily of insulin monomers. Conformational changes of the insulin upon adsorption, was investigated by circular dicroism (CD) and fluorescence spectroscopy. The results suggested unfolding of adsorbed insulin, as observed by a decrease in alpha-helix and increase in random coil conformation. The changes in protein structure was not only related to the adsorbed species being monomeric, since CD and fluorescence results were different for adsorbed insulin compared to a monomeric analog of human insulin. Furthermore, the thermal stability in the adsorbed state was changed compared to insulin in solution. On the basis of the TIRF studies with FITC-labelled insulin it was not possible to firmly conclude whether exchange between human insulin in the adsorbed state and in solution takes place, due to the limited time range investigated. However, the desorption mechanism appeared to be different with unlabelled insulin in the bulk solution compared to phosphate buffer.

Lipase surface diffusion studied by fluorescence recovery after photobleaching.

We have analyzed surface diffusion properties of a variant of Thermomyces lanuginosa lipase (TLL) on hydrophilic silica and silica methylated with dichlorodimethylsilane (DDS) or octadecyltrichlorosilane (OTS). For this study a novel method for analysis of diffusion on solid surfaces was developed. The method is based on fluorescence recovery after photobleaching using confocal microscopy. When a rectangular area of the sample was photobleached, fluorescence recovery could be analyzed as one-dimensional diffusion, resulting in simplified mathematical expressions for fitting the data. The method was initially tested by measuring bovine serum albumin diffusion on glass, which led to a diffusion coefficient in good correspondence to earlier reports. For the analysis of TLL diffusion, ellipsometry data of TLL adsorption were used to calibrate fluorescence intensity to surface density of lipase, enabling measurements of the diffusion coefficient at different surface densities. The average diffusion coefficient was calculated in two time intervals after adsorption. Mobile fraction and diffusion coefficient were lowest on the OTS surface, when extrapolated to infinite surface dilution. Moreover, the diffusion rate decreased with time on the hydrophobic surfaces. Our observations can be explained by the surface dependence on the distribution of orientations and conformations of adsorbed TLL, where the transition from the closed to the catalytically active open and more hydrophobic structure is important.