Alexis Oliva

Development of two high-performance liquid chromatographic methods for the analysis and characterization of insulin and its degradation products in pharmaceutical preparations

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.

Comparative study of protein molecular weights by size-exclusion chromatography and laser-light scattering.

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 methods reproducibility was accepted by assuming a precision of 98% and a 1% loss in precision.

Effect of high shear rate on stability of proteins: kinetic study.

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.

Influence of temperature and shaking on stability of insulin preparations: Degradation kinetics

Abstract 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.

Stability Study of Human Serum Albumin Pharmaceutical Preparations

The influence of temperature on the stability of human serum albumin (HSA) pharmaceutical preparations has been studied by size‐exclusion high‐performance liquid chromatography with multi‐angle laser‐light‐scattering detection and by particle‐size analysis.

In-vitro release of fluoropyrimidines from PLGA film implants.

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.

Chromatographic characterization of synthetic peptides: SPf66 malaria vaccine.

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 perforrmed 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.

Analysis of Peptides and Proteins: Evaluation of Purity, Stability, and Structural Characterization of Insulin

AbstractThe analysis of peptides and proteins is complicated either by their inherent reactivity (as physical change is produced in peptide structure due to aggregation) or by adsorption of sample material onto container surfaces. The characterization of proteins requires the use of several techniques; each probe measures a particular structural or functional feature. Methods for evaluating the purity and stability of peptides and proteins exist as well as various methods for their structural characterization: size, shape, and conformational changes. These are modifications of secondary and tertiary structures produced in the molecule during preparation, manipulation, and storage.

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.

Fingerprint Analysis of Insulin: Application in Stability Studies of Pharmaceutical Preparations

AbstractThe 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.