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.

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.

New injectable two-step forming hydrogel for delivery of bioactive substances in tissue regeneration.

A hydrogel based on chitosan, collagen, hydroxypropyl-γ-cyclodextrin and polyethylene glycol was developed and characterized. The incorporation of nano-hydroxyapatite and pre-encapsulated hydrophobic/hydrophilic model drugs diminished the porosity of hydrogel from 81.62 ± 2.25% to 69.98 ± 3.07%. Interactions between components of hydrogel, demonstrated by FTIR spectroscopy and rheology, generated a network that was able to trap bioactive components and delay the burst delivery. The thixotropic behavior of hydrogel provided adaptability to facilitate its implantation in a minimally invasive way. Release profiles from microspheres included or not in hydrogel revealed a two-phase behavior with a burst- and a controlled-release period. The same release rate for microspheres included or not in the hydrogel in the controlled-release period demonstrated that mass transfer process was controlled by internal diffusion. Effective diffusion coefficients, D eff, that describe internal diffusion inside microspheres, and mass transfer coefficients, h, i.e. the contribution of hydrogel to mass transfer, were determined using 'genetic algorithms', obtaining values between 2.64·10-15 and 6.67·10-15 m2/s for D eff and 8.50·10-10 to 3.04·10-9 m/s for h. The proposed model fits experimental data, obtaining an R 2-value ranged between 95.41 and 98.87%. In vitro culture of mesenchymal stem cells in hydrogel showed no manifestations of intolerance or toxicity, observing an intense proliferation of the cells after 7 days, being most of the scaffold surface occupied by living cells.

Structure-Performance Relationships of Temperature-Responsive PLGA-PEG-PLGA Gels for Sustained Release of Bone Morphogenetic Protein-2.

PLGA (poly(lactic-co-glycolic) acid)-PEG (polyethylene glycol)-PLGA synthesis conditions have an impact on the physicochemical features of the copolymer and its usefulness as biomaterial. This study reports on an analysis of the composition and structural properties of PLGA-PEG-PLGA copolymers applying a variety of analytical techniques. Viscoelastic properties and particularly the temperature-responsive behavior of PLGA-PEG-PLGA showed a marked dependence on copolymer structural features. Physicochemical and biological properties, such as bioadhesion, biocompatibility and cell viability, of the raw copolymers and their gels were also evaluated. The most promising copolymer was chosen to formulate the osteoinductive protein bone morphogenetic protein-2 (125I-BMP-2), and the ability of its gels to sustain the release both in vitro and in vivo was monitored in situ using a gamma counter. In vitro diffusion studies were carried out using a bioinspired set-up that included a biorelevant receptor medium. In vivo release tests after implantation in a critical-size calvarial defect model showed an important burst, but then the release fitted well to the square-root kinetics. Importantly, the release rate constants recorded in vitro and in vivo matched each other suggesting close in vitro-in vivo correlation. Overall, the information gathered opens new perspectives in the biomedical application of these temperature-sensitive materials.

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.

Pharmacokinetics analysis of sustained release hGH biodegradable implantable tablets using a mouse model of human ovarian cancer.

This paper presents the pharmacokinetic of human growth hormone (hGH) implantable tablets tested on a human ovarian cancer mouse model. In order to obtain a sustained release device which permits to administer a high dose of the hormone that keeps its integrity and stability, three different formulations of hGH-poly (d,l-lactic-co-glycolic acid) (PLGA) were elaborated by direct compression method varying hormone load, PLGA content and compactation time. In vitro studies showed that drug release was mainly controlled by hormone load. Pharmacokinetic studies were conducted by using immunodeficient female mice. Four days before the insertion of hGH implantable tablets in the peritoneal cavity, every mouse received 5x10(6) human ovarian cancer cells (SKOV3.ip1). Hormone serum levels were monitored through bleeding from eye orbital vessels. The population pharmacokinetic model used was based on the in series tank model and model parameters were estimated using the maximum likelihood method. The null hypothesis test about differences between formulations leads us to the conclusion that the three formulations showed the same kinetic behavior except for the hGH load. The hormone release was extended all over 2 weeks but no increase or decrease in survival time was observed. These results suggest that hGH serum levels do not facilitate tumoral cells proliferation, an expected effect of hGH and this could explain why survival times of mice treated with implantable tablets are not shorter than those treated with the control ones.

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.

Estimation of uncertainty in size-exclusion chromatography with a double detection system (light-scattering and refractive index).

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.

Evaluation of non-isothermal methods in stability studies of human insulin pharmaceutical preparations.

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.

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and hydrogen exchange combined with enzymatic digestion for the structural characterization of antimalaric Spf66 peptide.

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.

Data analysis of kinetic modelling used in drug stability studies: isothermal versus nonisothermal assays.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Applications of multi-angle laser light-scattering detection in the analysis of peptides and proteins.

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.