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The use of antidepressants (ADs) has increased significantly as a result of COVID-19 and its consequences. However, there are some notable differences in the relative levels of use between geographical areas and population groups. The aim of this work is to assess the impact of COVID-19 on the consumption of ADs in the Canary Islands, focusing on the islands of Gran Canaria, Fuerteventura and Lanzarote, by analyzing the trends in prescriptions of ADs during the pandemic period (2020) compared to the pre-pandemic period (2016–2020). Data were extracted from the community pharmacy wholesaler at a population level. Consumption patterns are expressed as the number of defined daily doses per 1000 inhabitant/day. The overall consumption of DIDs was higher in Gran Canaria, mainly in urban areas and the capital. It was similar in both Lanzarote and Fuerteventura, but particularly localized in the capital, which are considered semi-urban areas. Lanzarote and Fuerteventura present the same pattern of prescription ADs use, whereas Gran Canaria is notably different. This finding was also observed in the more consumed active pharmaceutical ingredients, although small inter-island variations in the ranking and percentages were observed. Sertraline and escitalopram are two of the most prescribed N06AB ADs, whereas the most recent N06AX ADs such as venlafaxine, mirtazapine and desvenlafaxine are more commonly prescribed. These differences in prescription ADs can be explained by demographical characteristics, population size, the fact of living in an urban area and general medical practice. In this context, the COVID-19 pandemic did not have an impact on the overall trend of the use of ADs between 2016 and 2020 in the islands under study.

This study is an evaluation of prescription opioid use on the island of La Gomera, a mainly rural area, during the period 2016–2019 at various levels. Data were extracted from the wholesalers who supply the community pharmacies at the population level. Prescription opioid use was measured as defined daily doses per 1,000 inhabitants/day (DID) and by the number of units sold per 1,000 inhabitants and year (units sold). This provided an island total of La Gomera’s overall prescription of opioids and its rate of change, as well as differences in prescribing at the municipal and health area level. Tramadol with acetaminophen and tramadol in monotherapy were the most consumed by “units sold” parameter, which accounted for 69.48% and 18.59% of the total. The situation was similar for DID, although with lower percentages, but a significant increase was observed in the use of fentanyl and buprenorphine, around 15% in each case. The balance between the uses of weak or strong opioids was different in La Gomera compared to that of Spain as a whole. In Spain, almost 70% of the prescriptions were for weak opioids compared to 58.67% in La Gomera. Fentanyl was the most used strong opioid (16.10%) followed by tapentadol and buprenorphine, around 5% each, whereas in La Gomera, buprenorphine was the most consumed (15.75%) followed by fentanyl (14.87%) and tapentadol (5.82%). These differences in prescription opioid use are most likely explained by prescriber characteristics, whereas the population age, socioeconomic status, or living in rural/urban area are not decisive determinants.

Statins are effective sterol lowering agents with high amoebicidal activity. Nevertheless, due to their poor aqueous solubility, they remain underused especially in eye drop formulation. The aim of the present study is to develop Pitavastatin loaded nanoparticles suitable for ophthalmic administration and designed for the management of Acanthamoeba Keratitis. These nanocarriers are aimed to solve both the ophthalmic route-associated problems and the limited aqueous drug solubility issues of Pitavastatin. Nanoparticles were obtained by a nanoprecipitation-solvent displacement method and their amoebicidal activity was evaluated against four strains of Acanthamoeba: A. castellanii Neff, A. polyphaga, A. griffini and A. quina. In Acanthamoeba polyphaga, the effect of the present nanoparticles was investigated with respect to the microtubule distribution and several programmed cell death features. Nanoparticles were able to eliminate all the tested strains and Acanthamoeba polyphaga was determined to be the most resistance strain. Nanoparticles induced chromatin condensation, autophagic vacuoles and mitochondria dysfunction.

Geodiversity is recognized as one of the most important drivers of ecosystem characteristics and biodiversity globally. However, in the northern Neotropics, the contribution of highly diverse landscapes, environmental conditions, and geological history in structuring large-scale patterns of aquatic environments and aquatic species associations remains poorly understood. We evaluated the relationships among geodiversity, limnological conditions, and freshwater ostracodes from southern Mexico to Nicaragua. A cluster analysis (CA), based on geological, geochemical, mineralogical, and water-column physical and chemical characteristics of 76 aquatic ecosystems (karst, volcanic, tectonic) revealed two main limnological regions: (1) karst plateaus of the Yucatán Peninsula and northern Guatemala, and (2) volcanic terrains of the Guatemalan highlands, mid-elevation sites in El Salvador and Honduras, and the Nicaraguan lowlands. In addition, seven subregions were recognized, demonstrating a high heterogeneity of aquatic environments. Principal component analysis (PCA) identified water chemistry (ionic composition) and mineralogy as most influential for aquatic ecosystem classification. Multi-parametric analyses, based on biological data, revealed that ostracode species associations represent disjunct faunas. Five species associations, distributed according to limnological regions, were recognized. Structural equation modeling (SEM) revealed that geodiversity explains limnological patterns of the study area. Limnology further explained species composition, but not species richness. The influence of conductivity and elevation were individually evaluated in SEM and were statistically significant for ostracode species composition, though not for species richness. We conclude that geodiversity has a central influence on the limnological conditions of aquatic systems, which in turn influence ostracode species composition in lakes of the northern Neotropical region.

Geodiversity is recognized as one of the most important drivers of ecosystems characteristics and biodiversity globally. However, in the northern Neotropics, the contribution of highly diverse landscapes and geological history in structuring large-scale patterns of limnology and aquatic species associations remain poorly understood. We evaluate the interaction between geodiversity, limnology and freshwater ostracode assemblages to explore drivers of aquatic ecosystem structure from southern Mexico to Nicaragua. Cluster analysis based on geological, limnological, sedimentological and mineralogical characteristics of 76 aquatic ecosystems (karst, volcanic, tectonic) reveal two main limnological regions: (1) Karst terraces from the Yucatán Peninsula and northern Guatemala, and (2) volcanic terrains of the Guatemalan highlands, El Salvador and Honduras mid-elevations, and Nicaraguan lowlands. In addition, seven limnological subregions were recognized, attesting high limnological heterogeneity. Principal Component Analysis identifies water ionic and sediment composition as most influential for aquatic ecosystem classification and given their source of formation, we recognized geology as the fundamental determinant for large-scale limnological patterns. Multiparametric analyses based on biological data revealed that species association represent disjunct faunas. For instance, five species associations are closely related to limnological regions. Structural equation modelling reveals a strong influence of limnology and elevation on the biological composition, but geodiversity resulted in the most important driver. The confinement of species associations is attributed to regional lake geochemistry. We deduce a linear, continuous and primary influence of geodiversity for limnological structure, geochemical properties and aquatic biological composition in Lakes of the northern Neotropical region.

Analytical biosimilarity assessment relies on two implicit conditions. First, the analytical method must meet a set of requirements known as fit for intended use related to trueness and precision. Second, the manufacture of the reference drug product must be under statistical quality control, i.e., the between-batch variability is not larger than the expected within-batch variability. In addition, the quality range (QR) method is based on one sample per batch to avoid biased standard deviations in unbalanced studies. This, together with the small number of reference drug product batches, leads to highly variable QR bounds. In this paper, we propose to set the QR bounds from variance components estimated using a two-level nested linear model, accounting for between- and within-batch variances of the reference drug product. In this way, the standard deviation used to set QR is equal to the square root of the sum of between-batch variance plus the within-batch variance estimated by the maximum likelihood method. The process of this method, which we call QRML, is as follows. First, the condition of statistical quality control of the manufacture process is tested. Second, confidence intervals for QR bounds lead to an analysis of the reliability of the biosimilarity assessment. Third, after analyzing the molecular weight and dimer content of seven batches of a commercial bevacizumab drug product, we concluded that the QRML method was more reliable than QR.

This study aimed to describe the consumption of opioid drugs in the island of La Gomera over a four-year period (2016–2019) at various levels (island, township and health areas). Data were extracted from the wholesaler in community pharmacies at a population level. Consumption patterns were expressed as number of Defined Daily Doses per 1000 inhabitant /day (DID) and by the number of sold units per 1000 inhabitants and year. A common classification system (ATC) and the two units of measurement proposed enabled comparisons at various levels. Major differences between island, township, and health areas are presented. Little is known about the reasons for these differences. The opioids belonging to the N02AJ groups and N02AX were the most consumed according to the “sold units” parameter, which accounted for 69.48% and 18.59%, respectively. The situation was similar in terms of DID, although with lower percentages, but a significative increase was observed in the use of fentanyl and buprenorphine, around 15% each. The balance between the uses of weak or strong opioids showed differences in La Gomera compared to those nationwide. In mainland Spain, almost 70% of the consumption came from weak opioids versus that of 58.67% in La Gomera. Fentanyl was the most frequently used strong opioid (16.10%) followed by tapentadol and buprenorphine, around 5% each, whereas in La Gomera buprenorphine was the most consumed (15.75%) followed by fentanyl (14.87%) and tapentadol (5.82%). These differences in opioids consumption are most likely explained by different changes on the prescribing habits and sociodemographic characteristics.

Different control charts in combination with the process capability indices, Cp, Cpm and Cpk, as part of the control strategy, were evaluated, since both are key elements in determining whether the method or process is reliable for its purpose. All these aspects were analyzed using real data from unitary processes and analytical methods. The traditional x-chart and moving range chart confirmed both analytical method and process are in control and stable and therefore, the process capability indices can be computed. We applied different criteria to establish the specification limits (i.e., analyst/customer requirements) for fixed method or process performance (i.e., process or method requirements). The unitary process does not satisfy the minimum capability requirements for Cp and Cpk indices when the specification limit and control limits are equal in breath. Therefore, the process needs to be revised; especially, a greater control in the process variation is necessary. For the analytical method, the Cpm and Cpk indices were computed. The obtained results were similar in both cases. For example, if the specification limits are set at ±3% of the target value, the method is considered “satisfactory” (1.22

Recently in 2019, the United States Food and Drug Administration (FDA) circulated a new draft guidance for comparative analytical assessment. They suggest the use of quality range (QR) methods. In this article, selection of the k value, and the effect of mean shifts and relative variability are evaluated. These are expressed as a ratio between the two standard deviations of the tested product and the reference product, σT/σR. In a second step, the two modified versions of the QR method proposed by Son et al. (2020) are also analysed under several scenarios, through simulation studies using real data from a biotechnology company and our own data for bevacizumab. Results indicate that k has a great impact on the probability of passing similarity tests. Pass rates higher than 90 % can be achieved for small relative variabilities (σT/σR ≤ 0.6) and large mean shifts (≈4%) by using k = 3. The situation is totally different for k = 2: the pass rate is higher than 90 % for scenarios with small (

In addition to the analytical challenges related to the size and complexity of biopharmaceutical drugs, the inherent variability that arises due to their manufacturing process requires monitoring throughout the production process to ensure the safety and efficacy of the finished product. In this step, validation data should demonstrate that the process is controlled and reproducible, whereas the manufacturing process must ensure the quality and consistency of the product. For this, the manufacturer sets specification limits according with regulatory guidance. In such a situation, the comparison of different batches is required in order to describe and analyze the variability between them. However, it is unclear how great the variability of the analytical method would be or that in producing the batches. The estimation of the β-expectation tolerance intervals based on the variance components to account for both between-batch and within-batch variability was proposed as a specification limit to control the heterogeneity between batches at the time of manufacture and to verify whether batches meet specification limits. At this point, the variance components were computed by the maximum likelihood method using a linear random model. For this, the protein content, expressed as a percentage of the actual concentration relative to the claim value, and the dimer content (expressed as percentage) were used as critical quality attributes (CQAs) in the monitoring and control process. We used real data from six bevacizumab commercial batches.

Size exclusion chromatography (SEC) with different detection modes was assessed as a means to characterize the type of bevacizumab aggregate that forms under thermal stress, quantitatively monitoring the aggregation kinetics. The combination of SEC with light-scattering (SEC/LS) detection was validated using in-study validation process. This was performed by applying a strategy based on a control chart to monitor the process parameters and by inserting quality control samples in routine runs. The SEC coupled with a differential refractive-index detector (SEC/RI) was validated using a pre-study validation process in accordance with the ICH-Q2 (R1) guidelines and in-study monitoring in accordance with the Analytical Target Profile (ATP) criteria. The total error and β-expectation tolerance interval rules were used to assess method suitability and control the risk of incorrectly accepting unsuitable analytical methods. The aggregation kinetics data were interpreted using a modified Lumry-Eyring model. The true order of the reaction was determined using the initial-rate approach. All the kinetic data show a linear Arrhenius dependence within the studied temperature range. The Arrhenius approach over-predicted the aggregation rate for 5°C, but provides an idea of the aggregation process and amount of aggregate formed. In any case, real-time stability data are necessary to establish the product shelf-life.

In this study, we assessed the usefulness of control charts in combination with the process capability indices, Cpm and Cpk, in the control strategy of an analytical method. The traditional X-chart and moving range chart were used to monitor the analytical method over a 2-year period. The results confirmed that the analytical method is in-control and stable. Different criteria were used to establish the specifications limits (i.e., analyst requirements) for fixed method performance (i.e., method requirements). If the specification limits and control limits are equal in breadth, the method can be considered “capable” (Cpm = 1), but it does not satisfy the minimum method capability requirements proposed by Pearn and Shu (2003). Similar results were obtained using the Cpk index. The method capability was also assessed as a function of method performance for fixed analyst requirements. The results indicate that the method does not meet the requirements of the analytical target approach. A real-example data of a SEC with light-scattering detection method was used as a model whereas previously published data were used to illustrate the applicability of the proposed approach. This article is protected by copyright. All rights reserved

A simple and reproducible UPLC method was developed and validated for the quantitative analysis of finasteride in low-dose drug products. Method validation demonstrated the reliability and consistency of analytical results. Due to the regulatory requirements of pharmaceutical analysis in particular, evaluation of robustness is vital to predict how small variations in operating conditions affect the responses. Response surface methodology as an optimization technique was used to evaluate the robustness. For this, a central composite design was implemented around the nominal conditions. Statistical treatment of the responses (retention factor and drug concentrations expressed as percentage of label claim) showed that methanol content in mobile-phase and flow rate were the most influential factors. In the optimization process, the compromise decision support problem (cDSP) strategy was used. Construction of the robust domain from response-surfaces provided tolerance windows for the factors affecting the effectiveness of the method. The specified limits for the USP uniformity of dosage units assay (98.5–101.5%) and the purely experimental variations based on the repeatability test for center points (nominal conditions repetitions) were used as criteria to establish the tolerance windows, which allowed definition design space (DS) of analytical method. Thus, the acceptance criteria values (AV) proposed by the USP-uniformity of assay only depend on the sampling error. If the variation in the responses corresponded to approximately twice the repeatability standard deviation, individual values for parcentage label claim (%LC) response may lie outside the specified limits; this implies the data are not centered between the specified limits, and that this term plus the sampling error affects the AV value. To avoid this fact, the limits specified by the Uniformity of Dosage Form assay (i.e., 98.5–101.5%) must be taken into consideration to fix the tolerance windows for each factor. All these results were verified by the Monte Carlo simulation. In conclusion, the level of variability for different factors must be calculated for each case, and not arbitrary way, provided a variation is found higher than the repeatability for center points and secondly, the %LC response must lie inside the specified limits i.e., 98.5–101.5%. If not the UPLC method must be re-developed.

An ultra high performance liquid chromatography method was developed and validated for the quantitation of triamcinolone acetonide in an injectable ophthalmic hydrogel to determine the contribution of analytical method error in the content uniformity measurement. During the development phase, the design of experiments/design space strategy was used. For this, the free R-program was used as a commercial software alternative, a fast efficient tool for data analysis. The process capability index was used to find the permitted level of variation for each factor and to define the design space. All these aspects were analyzed and discussed under different experimental conditions by the Monte Carlo simulation method. Second, a pre-study validation procedure was performed in accordance with the International Conference on Harmonization guidelines. The validated method was applied for the determination of uniformity of dosage units and the reasons for variability (inhomogeneity and the analytical method error) were analyzed based on the overall uncertainty.

Stability is a particular problem for biopharmaceutical products because the efficacy of peptides and proteins as therapeutic or diagnostic agents can be affected during preparation, shipping, and storage. A particular formulation may have no immediately apparent effect on physical or chemical stability, and the time required for these studies at ambient temperature can be very lengthy because chemical reactions proceed relatively slowly at low temperatures. Undoubtedly, accelerated and stress testing of stability can provide useful information for future product development. The many methods used to study kinetics in aqueous solution may be experimental or computational. Experimental approaches may be isothermal or non-isothermal. Non-linear and linear regression methods can be used to analyze data from these experimental approaches, and the Monte Carlo method could be useful to obtain information about uncertainties in experimental data. The purpose of this review is to describe and to discuss options for the accelerated study of peptide and protein drugs. These options are not necessarily the same as those used for regulatory testing to set expiration dates. We also review statistical techniques to estimate kinetic parameters (rate constant, activation energy, and pre-exponential factor). Further, we establish the advantages and the limitations of both thermal approaches. We analyze and discuss all aspects using the most recent examples of peptide and protein stability.

A stability-indicating reversed-phase high-performance liquid chromatographic method was developed and validated as per the International Conference on Harmonization (ICH) guidelines to evaluate the reproducibility of batches of synthetic peptides included in a stability program, in particular cholecystokinin (CCK-4) peptide. Both isothermal and nonisothermal approaches were used to determine stability under experimental conditions and the resulting degradation products were identified by liquid chromatography-mass spectrometry (LC-MS). The principal degradation product was the cyclic dimer, although another two products derived from it were also detected, due to the loss of one or two Phe-NH(2) residues. The dimerization follows first-order kinetics, whereas the hydrolytic cleavage implies both consecutive and in-parallel processes. The linear Arrhenius plot indicates that the degradation mechanism and kinetics do not change with temperature or the batch, but the degradation rate does depend on the batch, for example, the shelf-life at 25 degrees C was 2.54 days for batch 3, which is 13-times lower than batch 2. This variability is caused by a change in the synthesis process introduced by the manufacturer. The combination of these two elements: the analytical and stability-evaluating methods provide enough data to establish a stability-indicating profile, as required by the guideline ICH-Q6B for biotechnological/biological products.

Size-exclusion chromatography (SEC) coupled with online laser light-scattering (LS) and refractive index (RI) detection provides an excellent approach to determine the molecular weights (Mw) of proteins by the “two-detector” approach. Mw is determined only at the maximum of a peak, using either peak heights or area ratio from the two detectors. However, proper calibration of the SEC/LS/RI system is critical to obtain high precision. Today, an essential part of any analysis is to evaluate the uncertainty associated with the method. Basically, it is possible to distinguish between factors related to signal nature, precision and those due to signal processing. Given the signal of interest is the peak height or area ratio from two detectors, the signal ratio uncertainty was calculated using the random propagation of error formula. In this case, the effect of signal correlation was evaluated to avoid the uncertainty overestimation. In the second case, the sources of uncertainty affecting analytical measurement were estimated with the information from the precision assessment. For this, two designs with two-factor fully nested were followed for each method. Finally, the contributions from various uncertainty sources related with calibration are also analysed in detail. There are in fact only three main sources of measurement uncertainty: intermediate precision, calibration and repeatability. Of these, method precision is always the greatest, regardless of approach. For all proteins and peptides studied, the Mw calculated using both methods are close to the theoretical results, independently of the design, but the contributions of individual terms to combined uncertainty depend on both the design and method used. For example, the combined uncertainty varied between 223 and 813.2 Da for carbonic anhydrase, although higher values were found for human insulin and ovalbumin dimer. Other considerations that can have a significant impact on the results are discussed. The reproducibility of the two methods versus that based on ASTRA software used as reference method was performed using the concordance correlation coefficient. The methods’ reproducibility depends on the permitted losses in precision and accuracy.

The purpose of this research was to study the thermal stability of a human insulin pharmaceutical preparation using non-isothermal conditions and comparison with classical isothermal experiments. The isothermal studies were performed in the temperature range 20-60 degrees C, whereas non-isothermal stability studies were performed using a linear increasing temperature program, heating rate 0.25 degrees C per hour and temperature interval 30-70 degrees C. Under isothermal conditions, an apparent first-order degradation process was observed at all temperatures. The linear Arrhenius plot suggested that the insulin degradation mechanism was the same within the studied temperature range, with quite large uncertainties due to the small number of degrees of freedom based only on the scatter in the plot, giving an estimated shelf-life at 25 degrees C of 199.1 days. In non-isothermal conditions, the integral approach was used to estimate the activation parameters. It provides results in good agreement with those of the traditional method, but with the advantage that the uncertainty in the final result directly reflects the goodness of fit of the experimental data, since it takes into account the scatter in the original data. The estimated shelf-life in non-isothermal conditions was quite close to the value derived from isothermal data, 191.4 days, although the 95% confidence interval estimated were slightly higher. This is due to the differences in the estimation method and the nature of the experimental errors. The bootstrap technique is also applied to estimating confidence limits for the Arrhenius parameters and shelf-life. This method is very useful when the underlying distribution function of the parameters is unknown. The results obtained indicate that the Arrhenius parameters follow a normal distribution, whereas the shelf-life follows a log-normal distribution. In any case, the results obtained show that there is no difference between the asymptotic and bootstrap confidence intervals.

Protein and peptides production through recombinant techniques has benefited from numerous years of successful research, and is widely accepted as the approach of choice. As a result of these advances, many peptides and proteins are being formulated as biopharmaceuticals; however, to develop these macromolecules into stable formulations still remains a great challenge. Formulation problems in solution arise from the complex native structure of these macromolecules and are often manifested as physical instability, e.g. unfolding, aggregation, and/or precipitation. Understanding these effects is fundamental for research, development, production, and quality control. A variety of different techniques including HPLC, SEC, native gel electrophoresis and other electrophoretic techniques, analytical ultracentrifugation, fluorescence spectroscopy, Fourier transform/IR spectroscopy, mass spectrometry, light scattering, membrane osmometry, and UV spectroscopy have been used to study these processes. The assessment of product quality i.e. identity, content and purity has a major role in the manufacturing process of biopharmacauticals. The biotechnological production process itself usually shows high variability, which introduces high product diversity. Even from production batches, only limited amounts of material may finally be approved for use and made available, requiring sensitive analytical technology. The purpose of the current review is to describe and discuss the latest advances in analytical methodologies used to secure final product quality. © 2007 Bentham Science Publishers Ltd.

Size exclusion chromatography with light scattering detection (SEC-MALLS) was assessed as a means to characterize the type of bevacizumab aggregates that form under mechanical and thermal stress, quantitatively monitoring the aggregation kinetics. The analytical method was monitored and verified during routine use at two levels: (1) the "pre-study" validation shows that the method is specific, linear, accurate, precise, robust and stability indicating; (2) the "in-study" validation was verified by inserting quality control samples and the use of control charts, indicating that the analytical method is in statistical control and stable. The aggregation kinetics data were interpreted using a modified Lumry-Eyring model, but the quality of the fit can be considered poor (R(2)>0.96), especially at higher temperatures. This indicates that the order of the reaction could not be reliably determined, suggesting a different degradation mechanism. The kinetic data set also fit the minimalistic Finke-Watzky (F-W) 2-step model, with an excellent quality of fit (R(2)>0.99), yielding the first quantitative rate constant for the steps of nucleation and growth in bevacizumab aggregation. The bevacizumab pharmaceutical preparation contains (initially) dimers, approximately 1.6% of bevacizumab total concentration, and the effect on aggregation kinetics of seeding was analyzed using the F-W 2-step model assuming [B]0≠0 (for the seeded case). The results suggested that the seeding had no impact on aggregation kinetics. Furthermore, the Arrhenius equation cannot be used to extrapolate the shelf-life since no linear temperature dependence of the rate constant was found within the temperature range. Although the real-time stability data provides the basis for determining the product shelf-life, predictive methodologies such as Vogel-Tammann-Fulcher (VFT) or the Arrhenius approach can be misleading and result in overestimates of the product shelf-life. However, they can be successfully applied to fixing the lower and upper limits of the aggregation rate, i.e. the best and worst-case scenarios regarding the aggregation potential of the product. In conclusion, the present study evaluates the first application of the F-W 2-step model to fitting and interpretation of experimental aggregation data for bevacizumab pharmaceutical preparations, using SEC-MALLS in this context.

Kinetic modelling was applied to predict the stability of cholecystokinin fragment CCK-4 in aqueous solution, which was analyzed by isothermal and nonisothermal methods using a validated stability indicating HPLC method.The isothermal studies were performed in the temperature range 40 to 80 degrees C at pH 12 and ionic strength 0.01 M as constants, whereas nonisothermal stability studies were performed using a linear increasing temperature program, heating rate 0.25 degrees C/h and a temperature interval 40-82 degrees C. The isothermal studies require two-step linear regression to estimate the parameters, resulting in a well-defined confidence interval. Nonisothermal kinetic studies require nonlinear or linear regression by previous transformation of data to estimate the parameters. In this case, the two most popular approaches, derivative and integral, were used and compared.Under isothermal conditions, an apparent first-order degradation process was observed at all temperatures. The linear Arrhenius plot suggested that the CCK-4 degradation mechanism was the same within the studied temperature range, with quite large uncertainties due to the small number of degrees of freedom based only on the scatter in the plot, and giving an estimated shelf life at 25 degrees C of 35.2 days. The derivative approach yields high variability in the Arrhenius parameters, since they are dependent on the number of polynomial terms chosen, so several statistical criteria were applied to select the best model. The integral approach allows activation parameters to be calculated directly from experimental data, and provides results in good agreement with those of the traditional method, but have the advantage that the uncertainty in the final result directly reflects the goodness of fit of the experimental data to the chosen kinetic model. The application of the bootstrap technique to estimating confidence limits for the Arrhenius parameters and shelf life is also illustrated, and shows there is no difference between the asymptotic and bootstrap confidence intervals.Nonisothermal studies give us fast and valuable information about drug stability, although their potential for predicting isothermal behaviour is conditioned by the data analysis method applied.

Recombinant human growth hormone (rhGH), used mainly for the treatment of growth hormone deficiency in children, requires daily subcutaneous injections. The use of controlled release formulations with appropriate rhGH release kinetics reduces the frequency of medication, improving patient compliance and quality of life. Biodegradable implants are a valid alternative, offering the feasibility of a regular release rate after administering a single dose, though it exists the slight disadvantage of a very minor surgical operation. Three laminar implant formulations (F(1), F(2) and F(3)) were produced by different manufacture procedures using solvent-casting techniques with the same copoly(D,L-lactic) glycolic acid (PLGA) polymer (Mw=48 kDa). A correlation in vitro between polymer matrix degradation and drug release rate from these formulations was found and a mathematical model was developed to interpret this. This model was applied to each formulation. The obtained results where explained in terms of manufacture parameters with the aim of elucidate whether drug release only occurs by diffusion or erosion, or by a combination of both mechanisms. Controlling the manufacture method and the resultant changes in polymer structure facilitates a suitable rhGH release profile for different rhGH deficiency treatments.

The proliferation of new peptides and proteins requiring characterisation is a direct result of recent advances in genomics and proteomics, but protein aggregation is particular problem in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage process. To ensure that it meets quality standards, the size, molecular weight and/or molecular weight distribution, and aggregate state must be accurately determined. Traditional analytical methods for determining molecular weight include size-exclusion chromatography (SEC), gel electrophoresis, analytical ultracentrifugation and time-of-flight mass spectrometry. These technologies are time-consuming (some take days), provide data based on relative standards, or cannot characterise very high molecular weight aggregates. Laser light-scattering (LS) detection coupled with SEC system have been used for over a decade to determine the size and molecular weight of bio-molecules such as proteins, peptides, polysaccharides, oligonucleotides, and antibodies, the method of choice being for molar mass determinations and the study of self-association and heterogeneous interaction under native, equilibrium conditions in solution. The purpose of the current review is to describe and discuss the capability of the SEC/LS system to determine absolute molecular weight of proteins and their complexes and the association state of the conjugate, either with itself or with protein receptor/ligands. For this, the "two or three detector" methods, each with its advantages and limitations, can be used to calculate the molecular weight of a simple protein or glycoprotein, and the stoichiometry of their complexes. Also, some alternative techniques for determining the molecular weight are discussed in this review. Applications of all these methodologies are described.

The molecular weight of biopolymers such as peptides or proteins is vital information for understanding their physical/chemical properties. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and size-exclusion chromotography–multi-angle laser light scattering (SEC–MALLS) techniques each with its advantages and limitations were used for molecular weight determination of the SPf66 peptide. The precision of each method was studied using a two-factor fully-nested design with all the analyses performed by the same operator on a single instrument. The overall precision corresponded to the time-different intermediate precision (i.e. time and repeatability conditions). In the case of SEC–MALLS, all potential uncertainty components were carefully evaluated in an Ishikawa diagram, then included and mathematically combined with the uncertainty arising from the accuracy assessment to provide the overall uncertainty. In this case, the refractive-index increment with solute concentration value (dn/dc) provided the most significant contribution to the combined uncertainty. A method for its quantitation is proposed. The accuracy of method B (SEC–MALLS) against reference method A (MALDI-TOF-MS) was assessed using the interval testing hypothesis to limit the risk of unacceptable bias. The results indicate that the bias of B is higher than the limit established at 5%, and is therefore not traceable to A under the studied conditions.

Size-exclusion chromatography (SEC) was used to monitor the time-course of protein degradation induced by high shear rates during the formulation and manufacture of controlled-release pharmaceutical dosage forms. SEC with multi-angle laser light-scattering (MALLS) detection was used to characterize the aggregation products, determining their absolute molecular weight. A stability-indicating method was developed and validated to obtain reliable drug degradation data. The results obtained according to the ICH guidelines confirm that the system and methods proposed are suitable for their intended use. The degradation kinetics are influenced by the type of protein and the effect of the shear rate on their stability. Reversible pseudo-first order degradation kinetics were observed for bovine beta-lactoglobulin, whereas for human (HSA) and bovine serum albumin (BSA), a monomer-dimer transition was observed, independently of the rate of shear. However, trimer formation was also observed for HSA, especially at high shear rates. The kinetic model may thus be described as a two-step process: a monomer-dimer, and dimer-trimer transition.

This paper describes a simple and rapid procedure for the quantitation of the CCK-4 fragment of cholecystokinin by reversed phase high-performance liquid chromatography (RP-HPLC). The eluent for suitable separation on a C-18 column as stationary phase, was a water/acetonitrile (70/30, v/v) mixture with 0.05 trifluoroacetic acid, pH 2; UV detection was set at 280 nm. The method was developed and validated according to the International Conference on Harmonization (ICH) guidelines. The results obtained in the validation process, indicate that the method is specific, linear, accurate (recovery mean = 100.2±3.0%), and reliable (precision=1.21%). Limit of detection is established for 10.9 μg/mL and limit of quantitation for 33.1 μg/mL. In addition, the usefulness of the method, in order to evaluate the stability of the compound, was demonstrated.

The release of two low-molecular weight water-soluble fluoropyrimidines, 5-fluorouracil and 5-fluorouridine, from implants of PLGA films was modulated by varying the area (diameter) and number of layers of film per implant. The aim was to achieve continuous release without burst effect for at least a month. The film implants were prepared by the solvent evaporation technique. Except with 5-fluorouracil films, the in-vitro release profiles were in all cases triphasic, indicating that release proceeds by a combination of diffusion and polymer erosion. The experimental data fit the equation resulting from the sum of two exponentials, one direct and the other inverse. 5-fluorouridine release from simple films presented a relatively minor burst effect (24–28%). In contrast, the delivery of both compounds from sandwich-type implants occurred continuously without a burst effect, and lasted for 17–20 days. During the first phase, both 3- and 5-mm sandwiches released 55% of the dose of 5-fluorouridine, at rate constants of 0.037 ± 0.021 h−1 (n = 3) and 0.009±0.003 h−1 (n = 3), respectively. In the second phase, release was gradual from both simple Alms (k2 = 0.011-0.015 h−1) and sandwiches (k2 = 0.018-0.058 h−1). According to the analysis-of-variance results, neither the area nor type of implant influenced the rate constants significantly. The release profiles of 5-fluorouracil from simple films showed a severe burst effect (64–71%). Release of 5-fluorouracil was gradual only from sandwiches, 5 mm in diameter, showing a lag time unobserved in the 3-mm sandwiches. In the second phase, release was gradual (k2 = 0.014 ± 0.003 h−1) from 3-mm implants. However, the high variability in results for 5-mm implants prevents conclusions being drawn about the model parameters. Therefore, the sandwichtype film implants showed their utility for releasing water-soluble drugs for a prolonged time, without burst effect.

The development and validation of a quantitative size-exclusion chromatography (SEC) method for SPf66 malaria vaccine was achieved. The results show the reliability of the analytical method for the intended use. SPf66 malaria vaccine characterization was performed using both relative techniques such as the conventional SEC and absolute techniques: mass spectrometry and multi-angle laser-light scattering detection. The relative and absolute molecular masses were in the 4600–18 000 Da range. The results clearly indicate the presence of the monomer and dimer species, whereas the third species could be the trimer or tetramer.

A statistical method for testing the equivalence between batches regarding their stability is proposed. This method is based on the statistical linear model making use of a set of dummy variables to code the different batches. The method gives us the point estimates of the slope and zero intercept of one batch, and the differences and the corresponding confidence intervals with the remaining batches. In a second step, zero intercepts and slopes are estimated for all the batches. Stability equivalence assessment is based on the comparison of the confidence intervals for the differences between batches with the maximum difference allowable. The main advantages of this method are the possibility to compare several batches, to disclose the equivalence stability criteria from the statistical hypothesis about the equality between slopes, and the joint estimated of the residual variance whatever the decision to pool or not the data from different batches. This method is illustrated with two data set; the first one, previously published by other authors, involved six batches; the second data set include two batches and arose in a stability study of a commercial human insulin conducted in our laboratory.

The identification of a protein drug and the determination of its purity requires the use of several analytical methods. One identification probe used is the proteolysis of insulin with Staphylococcus aureus protease. This method yields a characteristic pattern of peptide fragments (“fingerprint”) which could be separated by reverse-phase high-performance liquid chromatography (HPLC) with gradient elution and ultraviolet detection. This probe could be used as a stability-indicating method for peptide and proteins, supplementing other traditional methods as reverse-phase (RP) and size-exclusion (SE) HPLC. Proteolysis of commercial human insulin preparations stored under different conditions produces modified fingerprints versus reference standard digested samples. Results vary as a function of the type of preparation and storage conditions since the degradation products detected by SE-HPLC are different.

In this study, we investigated if the size-exclusion chromatography coupled with light-scattering and refractive index detection (SEC/LS/RI) method is fitted for its intended purpose and checked if the analytical method is able to provide enough conforming results. For this, the process capability indices Cp, Cpk, and Cpm were computed. The traditional X-chart and moving range (MR) chart were used by the same analyst to monitor the equipment in the laboratory over a 1-year period. For this, a bovine serum albumin (BSA) sample (0.3 mg mL(-1)) with a nominal Mw of 66.4 kDa was analyzed each working day. The results confirmed that the analytical method is in-control and stable. To determine whether the given process meets the present capability requirement and runs under the desired quality conditions, the Pearn and Shu (2003) method based on the lower confidence bound C on Cpm was used. The estimator Cpm was 1.81, and the lower confidence bound C was 1.40. We therefore conclude that the true value of the method capability Cpm is no less than 1.40 with a 95% level of confidence. This result indicates that the method is satisfactory and no stringent precision control is required. The usefulness of this method was applied in the characterization of bevacizumab commercial pharmaceutical preparations stored under different conditions that lead to aggregation. In this case, the computed Cpm index was 0.98 (0.70, 1.26), which indicates that the method does not comply with the specification limits and needs to be revised. The quality improvement effort should: (1) reduce the uncertainty in the absolute Mw determination; (2) either move the process mean closer to the target value or reduce the process variation, i.e. improve the method accuracy (μ-T) and precision (σ(2)). On this point, the Bayesian posterior distribution of the mean and standard deviation pointed out the need to control the precision but specially accuracy in order to reduce the overall uncertainty of analytical method and thus, the method is capable.

Two commercial human insulin preparations were stored at different conditions in order to study the influence of temperature and shaking on their stability. Deamidation was the predominant degradation process in the case of the suspension, while dimerization prevailed for the solution, mainly when shaking was applied. The formation process of desamido B3 insulin follows first order kinetics; the dimer formation fit better the Prout-Thompkins nucleation model. Results obtained for suspension confirm the suitability of Arrhenius plot for both storage conditions. However, in the case of solution, the Arrhenius relationship was only valid when it was stored without shaking, since with shaking no linearity was observed in the temperature range studied.

The absorption of orally administered polyethylene glycol (PEG) has been used to assess intestinal permeability. We describe a simple HPLC technique to determine the oligomeric profile of PEG excreted in urine. We measured the total (%) PEG excreted in 6 h and the ratio of the four smallest oligomers to the three largest oligomers (expressed as mean percentages). The proposed method differentiates distinct groups of subjects with varying degrees of intestinal permeability detected by intestinal biopsy. The percent of PEG excreted and the oligomer ratio values for healthy subjects were, respectively, 30.1 +/- 3.87 and 0.35 +/- 0.03; for celiac patients on a gluten-free diet, 24.5 +/- 6.65 and 0.45 +/- 0.16; and for celiac patients, 15.0 +/- 5.93 and 1.12 +/- 0.55.

Although the basic science behind current methods for studying biopharmaceutical drug stability has not changed significantly, the techniques available for predicting stability have evolved over the years. This paper therefore describes and discusses various options of data analysis for accelerated degradation studies of peptide and protein drugs based on the Arrhenius equation. Both linear and non-linear regression analyses are also discussed. The results indicate that the simultaneous treatment of all data, as opposed to determining individual rate constants is clearly preferable, combined with the use of the reparameterized Arrhenius equation. The estimated shelf-life at 5 °C varied between 2.2 and 4.0 years in function of the temperature range and procedure used, whereas the precision of the estimated parameter is reflected in the width of the 95% confidence intervals, the classic Arrhenius analysis was maxima. All these results were evaluated by the bootstrap approach.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used in hydrogen exchange studies, exchanging deuteron (H/D) or proton (D/H), to determine the structure and conformational changes of antimalarial Spf66 synthetic peptide in its monomeric and dimeric forms. The accuracy of both analytical methods was assessed along with their suitability to study structural aspects. The results via these two approaches were in agreement, indicating that the dimer presents segments of secondary structure. In this last case, the combination of both methods with enzymatic digestion with pepsin was used in their identification. Although 100% coverage of Spf66 dimer was not observed, the higher levels of deuteration were observed for fragments located in the chain terminal where the structure may be more flexible, while the fragments near the disulfide bonds, which is, in theory, the more rigid region of the molecule, were not detected. This strategy is significantly time saving and allows rapid screening and help to characterize a protein, especially, when no prior structural information is available. However, a single spectrum is not certainly sufficient to obtain structural data; it is just an experimental limitation. Also, changes in peptide structure after storage at different temperatures and time were observed, which lead to a loss in the secondary structure as determined by circular dicroism measurements and an increase in aggregation products, since the trimer and tetramer species were detected by mass spectrometry.

The present study was designed to investigate a new administration model and the antileishmanial activity of a semi-synthetic chalcone, benzylideneacetophenone (trans-chalcone). The antileishmanial activity of this product was first tested in vitro against promastigotes of L. braziliensis, L. tropica, L. infantum and L. amazonensis. An in vivo experiment was carried out using subcutaneous administration of trans-chalcone and implants of synthetic biodegradable polymers, polylactic acid (PLA) and polylactic/glycolic acid (PLGA). This compound showed potent inhibitory effects on the growth of all Leishmania strains examinated. Subcutaneous administration of trans-chalcone at a single dose of 4 mg/kg of body weight reduced lesion development in mice infected with L. amazonensis. A similar inhibition of the lesion growth in mice treated with trans-chalcone and pentamidine was observed. PLA and PGLA implants of trans-chalcone at 4 mg/kg were administered to mice infected with L. amazonensis. PLGA implants induced a highest reduction in the lesion size (31.25%) than PLA implants (10.75%). Treatment in vitro with trans-chalcone at IC50, completely inhibited the pathogenicity of this parasite in vivo. The development of this model provides a new practical technique for delivering drugs and can be useful for experimental leishmaniasis treatment.

Stability studies on the SPf66 antimalarial peptide with different pH and temperature conditions were carried out. The degradation mechanism was elucidated by the size-exclusion chromatography (SEC) technique and the experimental data obtained at 37 degrees C and different pH were fitted to a kinetic degradation model that could explain the loss of its immunogenic capacity. At 5, 25, 37, and 70 degrees C and pH 2, changes were detected in the areas of the different species, although the values obtained could not be fitted to any known degradation kinetics.

The design of a stability study is intended to establish a shelf-life based on testing a limited number of batches of a drug product, which is applicable to all future batches of the drug product manufactured under similar circumstances. In the FDA guidelines for stability testing of new drug products, the matrix design is suggested as the alternative statistical design to the full testing approach. In the last few years, the matrix design has received increasing attention from pharmaceutical companies in order to reduce the cost of stability studies. For this, the full and matrix stability test plans were used on three batches of human insulin pharmaceutical preparation stored under a kinetic mean temperature of 22.7 degrees C for 3 years. The statistical method was suggested by Ruberg and Stegeman (Biometrics, 1991, 47, 1059-1069) and Llabres et al. (Int. J. Pharm. 2000, 204, 61-68), and was used for comparing batches, and the maximum allowable difference in slopes between batches was used as criteria for estimating a common shelf-life. The results obtained show the conservative nature of the matrix approach, although the difference between the matrix and full shelf-life was less than 1 month. The effect of the matrix method on shelf-life was also studied. The results showed that the model for estimating the shelf-life varied as a function of the permutation used, after testing for both slope and intercept poolability at alpha = 0.25. The batches should be pooled for both full testing and matrix design since the critical significance level required to obtain a power of 0.80 was achieved when the maximum allowable difference in slopes between batches was 1%/month, according to Ruberg and Stegeman. In contrast, when the proposed method by Llabres et al. was used, the results varied in function of the maximum allowable difference in slopes.

SPf66 is the first chemically synthesized peptide to elicit a partial protective immune response against malaria. Size-exclusion chromatography (SEC) with multi-angle laser light-scattering (MALLS) detection and hydrogen/deuterium (H/D) exchange monitored by (matrix-assisted laser desorption/ionization) MALDI–TOF (time-of-flight) mass spectrometry (MS) were used to assess the conformation and stability in aqueous solution after storage at different temperatures. Moreover, the feasible conformational changes of this peptide were also measured by circular dichroism (CD)-spectroscopy. The absolute molecular weight of SPf66 monomer and dimer species were 4765 and 8960 Da using SEC with MALLS detection, and 4643 and 9490 Da by MALDI–TOF MS, the discrepancy being between both methods lower than 5.7%, a value quite close to those found in other proteins. The results from H/D exchange monitored by MALDI–TOF MS and CD-spectroscopy show that the SPf66 monomer lacks ordered structure, whereas the SPf66 dimer species presents segments of secondary structure as a determined by CD measurements.

High-performance size-exclusion chromatography (SEC) based on UV-Vis detection is a relative technique for molecular weight determination whereas procedure based on multi-angle laser light scattering (MALLS) is both rapid and absolute. The two methods using recombinant human growth hormone (rHGH) and beta-lactoglobulin samples were compared. A calibration curve for the chromatographic system was generated based on standard proteins and the data were fitted by least squares to a third order polynomial model. The molecular weight from the conventional SEC method for both proteins was higher than the reported values. The molecular weight of rHGH from MALLS was 23.1+/-0.57 and 21.2+/-0.80 kDa using differential refractive index (SEC-MALLS/RI) and UV (SEC-MALLS/UV-Vis) detectors as mass detectors. Both values agree, within experimental error with the molecular weight sequence of rHGH, 22.1 kDa. In contrast, the molecular weight from LS for beta-lactoglobulin was 22.5+/-0.55 kDa by SEC-MALLS/RI and 23.0+/-1.22 kDa by SEC-MALLS/UV-Vis, respectively, values always higher than those supplied by the manufacturer, 18.4 kDa. The reproducibility of the SEC-MALLS/UV-Vis method versus the SEC-MALLS/RI method was performed using the concordance correlation coefficient. The method's reproducibility was accepted by assuming a precision of 98% and a 1% loss in precision.

Two high-performance liquid chromatography methods either in reversed-phase or as size exclusion separation mode were developed and validated for the analysis of insulin and its degradation products in pharmaceutical preparations. The results show the reliability of the analytical methods for the intended used. The static and dynamic light scattering were used to characterize the insulin and its derivatives. The absolute molecular weight of human insulin monomer and dimer were 5800 and 12400 Da respectively whereas its z-average root mean square radius were 21.6+/-0.4 and 40.5+/-0.7 nm, respectively. In contrast, the hydrodynamic diameter varied between 2.69 and 5.50 nm, depending of the association behavior of insulin.