José B. Fariña

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.

Potential applications of PLGA film-implants in modulating in vitro drugs release

In this work we evaluate poly(lactic/glycolic) acid (PLGA) film-implants as potential biodegradable devices for controlled release of two different drugs: 5-Fluorouridine (5-FUR), a conventional low molecular weight water-soluble compound and SPf66 malaria vaccine, a therapeutic synthetic polypeptide. Three types of devices were prepared by solvent-casting techniques alone or combined with compression method: simple monolithic discs (SMD), multilayer discs with a central monolithic layer (MLDM), and multilayer discs with a central drug-reservoir (MLDR). For the highly water-soluble drug, 5-FUR, in vitro release from SMD showed an initial burst (24% in 2 h) followed by prolonged release over 20 days. In contrast, from a MLDM (two drug-free PLGA discs were added to the SMD) showed an initial lag-time of 12 days followed by a very fast second release phase. Finally, when the load of this system was increased from 3 to 9%, an extended release over 20 days with a low burst effect was obtained. For SPf66, the central reservoir containing the synthetic polypeptide MLDR reduces the possibility of degradation due to peptide contact with polymer solution. When four layers were added, 10 days sustained-release was obtained without any burst effect. With six layers a moderate pulse was obtained, 18-22 days from the beginning of the release. The results show the suitability of the proposed devices to control release and avoid the burst effect with highly water-soluble drugs; as well as modulate in vitro peptide release.

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.

Biodegradable laminar implants for sustained release of recombinant human growth hormone.

Due to its short half-life, renal toxicity and necessity for daily subcutaneous injections, recombinant human growth hormone (rhGH) would best be administered in a controlled release formulation. One approach to this is the use of biodegradable laminar implants based on poly(lactic/glycolic) acid. Two PLGA laminar implant formulations (10% w/w), F(1) and F(2), were designed and statistically compared with a conventional commercial injectable (Norditropin). The following variables were chosen as responses: body weight, organ weights (heart, spleen and thymus), and tibia weights and dimensions. After statistical analysis, the most sensitive responses for detecting differences between formulations were body weight, tibia length and organ weights. Initial in vitro studies of these formulations showed an incomplete gradual release (after 15 days 40% rhGH released from F(1) and 60% from F(2)) and in vivo the best results were obtained with F(1). This formulation showed the best growth curve and the highest values of all the above responses. In conclusion, this type of formulation provides a sustained release of rhGH for at least 15 days and a greater efficacy than the frequent administration of the conventional injectable.

Design of a pediatric oral formulation with a low proportion of hydrochlorothiazide

It is a normal pediatric practice in community and hospital pharmacies to prepare a new drug formulation when no commercial forms of it are available. Any dose or stability control is usually done for these types of compounding formulations due to the effort which means to develop these types of tests in pharmacies. We have studied five different hydrochlorothiazide oral formulations prepared with traditional compounding techniques in pharmacies to treat heart failure and edemas in babies. A Standard Operating Procedure (SOP) was done for every suspension. After the strictly monitoring of the SOP, every suspension was subjected to quality control tests (pH, particle size, viscosity, dose content and stability). There is only one studied formulation that guarantees the correct dose administering and stability after 3 weeks stored at 5 °C and light protected. Both, the percentage of wetting agent and the viscosity of the suspensor vehicle in this formulation make the correct dose administering possible after the formulation is shaken.

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.

Structural properties of biodegradable polyesters and rheological behaviour of their dispersions and films

This paper focuses on the dependence of the rheological properties of PLA-PEG and PLGA dispersions and films on the polymer structural properties, in order to obtain useful information to predict and explain the performance of polyester films as drug-delivery systems. In this study, one PLA-PEG and three PLGA polymers of different molecular mass were synthesized and characterized by NMR, GPC, DSC and TGA–FT-IR. To characterize the viscoelastic behaviour of concentrated solutions in dichloromethane and of the films obtained by a solvent-casting technique, oscillatory shear rheometry was used. The polymer dispersions showed a characteristic Newtonian viscous behaviour, but with different consistency index depending on the nature of the polymer. Freshly prepared, PLGA and PLA-PEG films had elastic modulus (G′) greater than viscous modulus (G″). The decrease in both moduli caused by an increase in temperature from 25 to 37°C was especially marked for the polymers with T g below or around 25°C (PLGA 27 kDa and PLA-PEG 27 kDa). After being immersed in pH 7.4 aqueous solution for one week, PLGA films showed a significant increase in both G′ and G″, due to the promotion of polymer–polymer interactions in a non-solvent medium. In contrast, the PLA-PEG film became softer and more hydrated, due to the amphiphilic character of the polymer. The water taken up by the film acted as a plasticizer and induced the softening of the system. These results suggest that the presence of PEG chains exerts a strong influence on the mechanical properties of polyesters films and, possibly, the performance as coating or matrices of drug-delivery systems.

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.