Significance: Bioactive proteins represent the most important component class in biopharmaceutical products for therapeutic applications. Their production is most often biotechnologically realized by genetically engineered microorganisms. For the quality assurance of insulins as representatives of life-saving pharmaceuticals, analytical methods are required that allow more than total protein quantification in vials or batches. Chemical and physical factors such as unstable temperatures or shear rate exposure under storage can lead to misfolding, nucleation, and subsequent fibril forming of the insulins. The assumption is valid that these processes go parallel with a decrease in bioactivity.
Aim: Infrared (IR) spectroscopy has been successfully utilized for secondary structure analysis in cases of protein misfolding and fibril formation.
Approach: A reliable method for the quantification of the secondary structure changes has been developed using insulin dry-film Fourier-transform IR spectroscopy in combination with the attenuated total reflection (ATR) technique and subsequent data analyses such as band-shift determination, spectral band deconvolution, and principal component analysis.
Results: A systematic study of insulin spectra was carried out on model insulin specimens, available either as original formulations or as hormones purified by ultrafiltration. Insulin specimens were stored at different temperatures, i.e., 0°C, 20°C, and 37°C, respectively, for up to three months. Weekly ATR-measurements allowed the monitoring of hormone secondary structure changes, which are supposed to be negatively correlated with insulin bioactivity.
Conclusions: It could be shown that IR-ATR spectroscopy offers a fast and reliable analytical method for the determination of secondary structural changes within insulin molecules, as available in pharmaceutical insulin formulations and therefore challenges internationally established measurement techniques for quality control regarding time, costs, and effort of analysis.
In biopharmaceutical products for therapeutic usage, proteins represent the most important substance class. For the quality control of insulins as representatives of life saving pharmaceuticals, analytical methods are needed allowing more than a total protein quantification in vials. Chemical and physical influences such as unstable temperatures or shear rate exposure under storage lead to misfolding, nucleation and subsequent fibril forming of the insulins. The hypothesis is that these processes go parallel with a decrease in bioactivity. Infrared spectroscopy has been successfully utilized for secondary structure analysis in cases of protein folding and fibril formation. A reliable method for the quantification of the secondary structure changes has been developed by using insulin dry-film Fourier-Transform infrared spectroscopy in combination with the attenuated total reflection (ATR) technique and subsequent data analyses such as band-shift determination, spectral band deconvolution and principal component analysis. A systematic study of insulin spectra was carried out with model insulin specimens, available either as original formulations or as hormones purified by ultrafiltration, stored at 0°C, 20°C and 37 °C, respectively, for up to three months. Weekly ATR-measurements allowed the monitoring of the hormone secondary structure changes, which are supposedly negatively correlated with the insulin bioactivity.
Dependent on various factors such as pH, temperature and shear forces, therapeutic insulins undergo a continuous process of chemical degradation during manufacturing and storage until administered by patients. Consequently, changes in secondary up to quaternary structures of the protein appear, with the consequence of a decrease in biological activity due to partial misfolding of the monomers and finally their aggregation to fibrils. Infrared spectrometry has been applied for quantifying chemical degradation processes of therapeutic insulins, based on changes in secondary structure. For the determination of insulin potency, the glucose metabolism rate of cells from the human monocytic cell line MONOMAC-6 has been monitored under standardized conditions, providing a measure of biological insulin activity. For cell culture monitoring with a focus on substrate and metabolite concentrations, microdialysis has been used in combination with infrared spectrometry of the continuously sampled dialysates with duration up to 48 h. The dialysate spectra were analyzed by a classical least-squares (CLS) method with appropriate reference spectra, including the determination of microdialysis recovery rates as obtained from perfusate losses of mannitol, which had been used as internal standard. By analysing the time dependent glucose utilization, the potency of tested insulins can be assessed without patient clamp experiments or animal testing.
Spectroscopic analysis of different body fluids has been realized by using tapered flat silver halide fiber elements as infrared biosensors. Here, a specially functionalized sensor is presented, which had been prepared by an Nhydroxysuccinimide (NHS) ester derivative containing a reactive thiol group. NHS esters are often used as coupling agents to covalently bind amine-containing biomolecules (e.g., enzymes, antibodies or peptides) for the preparation of bioanalytical sensors of high selectivity. Recently, an immuno-infrared-sensor for Alzheimer disease (AD) screening has been presented based on infrared ATR-measurements with antibody-immobilized Ge-element surfaces for the extraction and analysis of Amyloid-beta (Aβ)-monomers, oligomers, and fibrils from blood plasma and cerebrospinal fluid (CSF). Thereby the biomarker amide I maximum frequency was used for AD classification. Here, for functionalizing the silver halide surfaces different procedures have been investigated, which consider the exchange reaction of the halogen atoms by the thiol-group as one option. Other preparation methods use the chemical reduction of silver ions, either from aqueous salt solutions or of the fiber material itself. A further method uses a first printing of silver nanoparticles on top of the flattened fiber sections. The combination of specific protein immobilization via functionalized silver halide fibers with recently introduced quantum cascade laser spectrometers is very promising for device miniaturization suited for implementation into hospital laboratories or general practitioners’ offices.
Mid-infrared spectroscopy hyphenated with micro-dialysis is an excellent method for monitoring metabolic blood parameters as it enables the concurrent, reagent-free and precise measurement of multiple clinically relevant substances such as glucose, lactate and urea in micro-dialysates of blood or interstitial fluid. For a marketable implementation, quantum cascade lasers (QCL) seem to represent a favourable technology due to their high degree of miniaturization and potentially low production costs. In this work, an external cavity (EC) - QCL-based spectrometer and two Fourier-transform infrared (FTIR) spectrometers were benchmarked with regard to the precision, accuracy and long-term stability needed for the monitoring of critically ill patients. For the tests, ternary aqueous solutions of glucose, lactate and mannitol (the latter for dialysis recovery determination) were measured in custom-made flow-through transmission cells of different pathlengths and analyzed by Partial Least Squares calibration models. It was revealed, that the wavenumber tuning speed of the QCL had a severe impact on the EC-mirror trajectory due to matching the digital-analog-converter step frequency with the mechanical resonance frequency of the mirror actuation. By selecting an appropriate tuning speed, the mirror oscillations acted as a hardware smoothing filter for the significant intensity variations caused by mode hopping. Besides the tuning speed, the effects of averaging over multiple spectra and software smoothing parameters (Savitzky-Golay-filters and FT-smoothing) were investigated. The final settings led to a performance of the QCL-system, which was comparable with a research FTIR-spectrometer and even surpassed the performance of a small FTIR-mini-spectrometer.
Spectroscopic analysis of different biofluids and bodyfluid-like media has been realized by using tapered flat silver halide fiber elements as infrared biosensors. Optical stability and biocompatibility testing of the sensor elements have been performed with in-vitro samples under representative physiological conditions. After improving the reproducibility of manufacturing the sensor elements, the incoupling of radiation and the general handling including their chemical composition characterization, the fiber sensors were further optimized for the experiments. Stability tests in physiological solutions as well as porcine blood have shown that best results for biospectroscopic applications are available for the mid-IR fingerprint region, with the most stable behaviour as analyzed by the single-beam spectra. Despite several contrary reports, the silver halide material tested is toxic to cell lines chosen from the DIN standard specification for biocompatibility testing. Spectral changes as well as the results based on the DIN standard showed that pretreatment of the fibers is unavoidable to prevent direct contact of cells or human tissue and the silver halide material. Further applications of tapered flat silver halide fibers for the quantification of analytes in bodyfluids have also been tested by ensheathing the fiber-optic sensor element with a dialysis membrane. With the successfully produced prototype, results of diffusion rates and performance of a membrane-ensheathed fiber probe have been obtained. An invitro monitoring fiber sensor was developed aiming at the implantation of a microdialysis system for the analytical quantification of biomolecules such as glucose, lactate and others.
Blood glucose monitoring has been realised by biosensors in combination with micro-dialysis, using either subcutaneously or intravascularly implanted catheters. Another alternative is ex-vivo micro-dialysis of continuously sampled heparinized whole blood available from the patient even under critical care conditions. However, most devices suffer from inaccuracies due to variable recovery rates. Infrared spectrometry has been suggested for analyte quantification, since besides glucose other clinically relevant analytes can be simultaneously determined that are, e.g., important for intensive care patients. Perfusates with acetate and mannitol have been investigated as recovery markers (internal standards). In contrast to the previously used acetate, an almost linear dependency between mannitol loss and glucose recovery was observed for micro-dialysis of glucose spiked aqueous albumin solutions or porcine heparinized whole blood when testing flat membranes within a custom-made micro-dialysator. By this, a straightforward compensation of any dialysis recovery rate variation during patient monitoring is possible. The combination of microdialysis with infrared spectrometry provides a calibration-free assay for accurate continuous glucose monitoring, as reference spectra of dialysate components can be a-priori allocated.
Micro-dialysis can be used for continuously harvesting body fluids, while a multi-component analysis of the dialysates
by infrared spectrometry offers splendid opportunities for monitoring substrates and metabolites such as glucose, lactate
and others small enough to penetrate the semi-permeable dialysis membranes. However, a drawback of this process are
variable recovery rates, which can be observed especially for subcutaneously implanted catheters in human subjects.
Isotonic perfusates were investigated with acetate and mannitol as recovery markers for the dialysis of human serum at
37°C to mimic in vivo patient monitoring. The latter non-ionic substance has been suggested for application when other
ionic substances such as bicarbonate or pH are also to be determined. Simultaneously for acetate and mannitol, the
depletion of the marker substances from the perfusates using different micro-dialysis devices was investigated under
various flow-rates. Relationships between relative dialysate marker concentrations and glucose recovery rates were
determined based on multivariate calibrations. For quantification, classical least squares with reference spectra for
modelling the serum dialysates was used, rendering a basis for reliable blood glucose and lactate measurements.
Micro-dialysis has been established in the clinical environment for continuously harvesting body fluids, but a drawback of this process are variable recovery rates, which can be observed especially for subcutaneously implanted catheters. Perfusates with either acetate or mannitol have been investigated as recovery markers. The latter substance is suggested for application with external cavity tuneable quantum cascade lasers, rendering a limited wavenumber interval in contrast to FTIR-spectrometers. Despite the overlap of mannitol and glucose spectra, their simultaneous quantification was successful. By investigating the depletion of the marker substances from the perfusates using different micro-dialysis devices, the theoretical nonlinear relationship between the relative dialysate marker concentration and glucose recovery rate was confirmed for the marker substance-analyte pair of acetate and glucose, rendering a basis for reliable blood glucose measurements. For the pair of mannitol and glucose an almost linear dependency was expected for the microdialysate catheters and experimentally verified, which provides a straightforward correction of any dialysis recovery rate variation during patient monitoring.
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