Oxidative Damage and Telomere Length as Markers of Lung Cancer Development among Chronic Obstructive Pulmonary Disease (COPD) Smokers.

Lung cancer (LC) constitutes an important cause of death among patients with Chronic Obstructive Pulmonary Disease (COPD). Both diseases may share pathobiological mechanisms related to oxidative damage and cellular senescence. In this study, the potential value of leucocyte telomere length, a hallmark of aging, and 8-OHdG concentrations, indicative of oxidative DNA damage, as risk biomarkers of LC was evaluated in COPD patients three years prior to LC diagnosis. Relative telomere length measured using qPCR and serum levels of 8-OHdG were determined at the baseline in 99 COPD smokers (33 with LC and 66 age-matched COPD without LC as controls). Of these, 21 COPD with LC and 42 controls had the biomarkers measured 3 years before. Single nucleotide variants (SNVs) in TERT, RTEL, and NAF1 genes were also determined. COPD cases were evaluated, which showed greater telomere length (p < 0.001) and increased serum 8-OHdG levels (p = 0.004) three years prior to LC diagnosis compared to the controls. This relationship was confirmed at the time of LC diagnosis. No significant association was found between the studied SNVs in cases vs. controls. In conclusion, this preliminary study shows that longer leucocyte telomere length and increased 8-OHdG serum levels can be useful as early biomarkers of the risk for future lung cancer development among COPD patients.

Opioid prescription patterns in the province of Las Palmas, Canary Islands, Spain (2016-2020): differences between urban and rural areas.

Introduction: The use of opioids has increased markedly in the past decades in European countries, especially for treatment of non-cancer pain including painful chronic musculoskeletal conditions. However, there are some notable differences in the relative levels of use between geographical areas and some distinct, context-specific patterns of weak and strong opioid use. The aim of this work is to describe real world trends in dosage forms and population exposure in the prescription opioid use on isolated geographically area: The Canary Islands of Gran Canaria, Lanzarote and Fuerteventura, Spain. For this, several factors such as living in a rural or urban area, population over 65 years of age, population density or socioeconomic status were analyzed. Methods: Data were extracted from the wholesalers who supply the community pharmacies at the population level. Prescription opioid use was measured as defined daily doses (DDD) per 1,000 inhabitants per day. A model based on covariance analysis with two nested fixed factors and one co-variable was used for contrast analysis at different level. Results: The overall DDD per 1000 inhabitants per day and year variation rate in Spain was very similar to that obtained for Gran Canaria and Fuerteventura (0.967 vs. 1.006), although the levels of dispensation were different (14.75 versus 18.24 for Gran Canaria and 12.7 for Fuerteventura, respectively). Lanzarote is completely different in all issues, where the opioid consumption rate remained stable during the study period, but with a decreasing tendency. The dispensation level of strong opioids varied between islands, from 56.41% for Fuerteventura vs. 17.61% for Gran Canaria, although these values remained stable. Tramadol with acetaminophen and Tramadol in monotherapy were the most consumed forms of the weak opioids, whereas Buprenorphine was the most used strong opioid followed by Fentanyl, although demand for it varied between islands, the transdermal formulations were the most frequent pharmaceutical preparation. Conclusion: The differences in prescription opioid use are most likely explained by the opioid prescribing practices in each island, whereas factors such urbanicity level, population age, population density and status socioeconomic does not help to explain the differences in prescription opioid use across rural and urban areas.

Circulating miR-206 and miR-1246 as Markers in the Early Diagnosis of Lung Cancer in Patients with Chronic Obstructive Pulmonary Disease.

Lung cancer (LC) is the most common cause of cancer death, with 75% of cases being diagnosed in late stages. This study aimed to determine potential miRNAs as biomarkers for the early detection of LC in chronic obstructive pulmonary disease (COPD) cases. Ninety-nine patients were included, with registered clinical and lung function parameters followed for 6 years. miRNAs were determined in 16 serum samples from COPD patients (four with LC and four controls) by next generation sequencing (NGS) at LC diagnosis and 3 years before. The validation by qPCR was performed in 33 COPD-LC patients and 66 controls at the two time points. Over 170 miRNAs (≥10 TPM) were identified; among these, miR-224-5p, miR-206, miR-194-5p, and miR-1246 were significantly dysregulated (p < 0.001) in COPD-LC 3 years before LC diagnosis when compared to the controls. The validation showed that miR-1246 and miR-206 were differentially expressed in COPD patients who developed LC three years before (p = 0.035 and p = 0.028, respectively). The in silico enrichment analysis showed miR-1246 and miR-206 to be linked to gene mediators in various signaling pathways related to cancer. Our study demonstrated that miR-1246 and miR-206 have potential value as non-invasive biomarkers of early LC detection in COPD patients who could benefit from screening programs.

Trends in the Consumption of Antidepressant Drugs before and during the COVID-19 Pandemic in the Canary Islands, Spain: The Case of the Province of Las Palmas.

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.

Drug Shelf Life and Release Limits Estimation Based on Manufacturing Process Capability.

Specification limits are the competence regulatory agencies, whereas the release limit is a manufacturer's internal specification to be applied at the time of batch release to assure that quality attributes will remain within the specification limits until the expiry time. The aim of this work is to propose a method to set the shelf life from drug manufacture process capacity and degradation rate, using a modified version of the proposed method by Allen et al. (1991) Two different data sets were used to do this. The first data set corresponds to analytical method validation to measure the insulin concentration in order to estimate the specification limits, whereas the latter set gathered information on stability data of six batches of human insulin pharmaceutical preparation. In this context, the six batches were divided into two groups: Group 1 (batches 1, 2, and 4) was used to estimate shelf life; Group 2 (batches 3, 5, and 6) was used to test the estimated lower release limit (LRL). The ASTM E2709-12 approach was applied to verify that the future batches fulfill the release criterium. The procedure has been implemented in R-code.

Development and Optimization of a New UPLC-UV/MS Method through DoE and MLR for Detecting Substandard Drug Products to Treat Tuberculosis.

Drug products used for treating tuberculosis are one of the most widely reported medicines to be classified as falsified or substandard in low- and middle-income countries, representing a major hazard to health. The aim of this study was, firstly, to develop an ultra-performance liquid chromatography (UPLC) method which is able to analyze fixed combination tablets with up to four active pharmaceutical ingredients, including isoniazid, pyrazinamide, rifampicin, and ethambutol. Secondly, we aimed to optimize it through the design of experiments and multi-linear regression based on a central composite design and to validate it according to the guidelines of the International Conference on Harmonization. The application of this tools enabled the identification of the influential factors (flow rate, formic acid, and temperature) and their effects on the studied responses (retention factor and percentage for each drug) as part of the quality by design approach. The method proved to be to be linear in the range from 5.0 to 15 µg/mL for isoniazid, pyrazinamide, and rifampicin, being precise (<1%) and accurate (97−101%). In addition, the method validated for ethambutol proved to be linear from 1.4 to 4.2 µg/mL, as well as precise (0.54%) and accurate (97.3%). The method was stability indicated for all the active pharmaceutical ingredients studied and was able to detect two substandard formulations sampled on the African market.

Pitavastatin loaded nanoparticles: A suitable ophthalmic treatment for Acanthamoeba Keratitis inducing cell death and autophagy in Acanthamoeba polyphaga.

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.

Opioid use trends in Spain: the case of the island of La Gomera (2016-2019).

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.

New Quality-Range-Setting Method Based on Between- and Within-Batch Variability for Biosimilarity Assessment.

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.

Limitations of the quality range approach in analytical similarity assessment: Effect of mean shift and relative variability.

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 (<0.5 %) or no differences between the means of the two products, but this percentage decreases by up to 50 % for σT/σR = 1. Effectiveness in detecting the various scenarios was quantified by calculating the probability curves of passing the similarity test, as a function of the two variables for each k value. Alternative methods present the same limitations but with different magnitude in comparison with QR, this being most pronounced in the plausibility-interval QR method.

Application of capability indices and control charts in the analytical method control strategy.

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.

Development of an ultra high performance liquid chromatography method for determining triamcinolone acetonide in hydrogels using the design of experiments/design space strategy in combination with process capability index.

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.

Pre-study and in-study validation of a size-exclusion chromatography method with different detection modes for the analysis of monoclonal antibody aggregates.

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 ß-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.

Fitting bevacizumab aggregation kinetic data with the Finke-Watzky two-step model: Effect of thermal and mechanical stress.

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.

Capability measurement of size-exclusion chromatography with a light-scattering detection method in a stability study of bevacizumab using the process capability indices.

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.

Development and validation of an UPLC method for determination of content uniformity in low-dose solid drugs products using the design space approach.

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 percentage 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 improved methodology for data analysis in accelerated stability studies of peptide drugs: practical considerations.

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.

Anti-tumor effects of adenovirus containing human growth hormone sequences in a mouse model of human ovarian cancer.

Women with ovarian cancer have a low survival rate and develop resistance to chemotherapy, so new approaches to treatment are needed. We unexpectedly found administration of a replication-deficient adenovirus containing human growth hormone sequences (AdXGH) was beneficial in a mouse model of human ovarian cancer. Intraperitoneal injections of AdXGH prolonged median survival from a mean of 31 ± 1.2 to 40 ± 1.4 days in immunodeficient SCID mice given SKOV3.ip1 human ovarian cancer cells in the peritoneal cavity. Adenovirus containing human prolactin or del32-71growth hormone sequences had no effect. Repeated injection of growth hormone or implantation of tablets with sustained growth hormone release did not increase survival. Control mice had overlapping tumors throughout the peritoneal cavity and liver and frequent lung metastases 24 days after tumor cell injection. Mice that received two injections of AdXGH had no lung metastases. Mice that received four injections had no lung or liver metastases and peritoneal fibrosis. They did not survive longer than mice that received two injections, but they had enlarged livers with hepatocellular changes, indicating that a limitation of increasing the dose is liver toxicity.

Application of a validated stability-indicating chromatographic method to evaluate the reproducibility between batches of small peptides in solution.

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.

Solid-state stability studies of cholecystokinin (CCK-4) peptide under nonisothermal conditions using thermal analysis, chromatography and mass spectrometry.

The solid-state stability of cholecystokinin (CCK-4) peptide under nonisothermal conditions was studied by differential scanning calorimetry (DSC), chromatography and mass spectrometry, identifying and schematizing the degradation products. To model the degradation mechanism of the peptide using the combined Kissinger and direct-differential methods, the observed degradation process was characterized by decomposition temperature (T(m)), reacted fraction (alpha(m)), activation energy (E(a)), and pre-exponential factor (A). Results obtained by the two calculation methods were similar. The cleavage reaction on both N- and C-terminal sides of aspartic acid was the principal degradation pathway, although the reaction can occur consecutively and/or in parallel. Therefore to determine the relative importance of the different degradation pathways, a system of differential equations relevant to each degradation reaction was analysed using the R((R)) statistical program. The results obtained show that the consecutive reaction was the less plausible, whereas a slightly better fit was obtained for the reaction with both processes than for the in-parallel reaction. In this situation, the F-test was applied to discriminate between the models, indicating that the simpler model is the most probable. In conclusion, the results demonstrate for the first time that, in solid-state, n-1 cleavage occurs in parallel to n+1 cleavage at aspartic acid residues and not consecutively.