Long-term stabilization of a new freeze-dried and albumin-free formulation of recombinant human interferon alpha 2b.

In this work, we evaluate the stability of a new freeze-dried and albumin-free formulation of recombinant human IFN alpha 2b (rhIFN-alpha2b) to be used in humans. The freeze-dried, albumin-free formulation was stored at the recommended temperature of 4 degrees C, and under accelerated storage conditions (28 degrees C). The stability of this product was also compared with the stability of a liquid albumin-free formulation of this cytokine. Finally, the stability of the freeze-dried albumin-free formulation was examined after reconstitution and storage at 4 degrees C and room temperature (28 degrees C) for 30 days. Samples were periodically subjected to biological activity assay (antiviral titration), reversed phase high-performance liquid chromatography (RP-HPLC), pyrogens, sterility and enzyme-linked immunosorbent assay (ELISA) testing, abnormal toxicity screening, organoleptic evaluation, and measurement of residual moisture and pH. Accelerated storage (28 degrees C) data for the freeze-dried albumin-free formulation showed biochemical stability of the active ingredient throughout the 6-month study, showing activity between 85 and 125% of its nominal value. RP-HPLC-determined purity showed that rhIFN-alpha2b remained above 95%. Additionally, the formulation was non-pyrogenic, non-toxic, sterile, and organoleptically acceptable. The real-time storage data confirmed the good biochemical long-term (30 months) stability of the freeze-dried formulation of this cytokine. Comparison with the liquid rhIFN-alpha2b albumin-free preparation showed that the freeze-dried albumin-free formulation maintained the stability of the active ingredient better than the liquid preparation. The formulation was also stable after reconstitution and storage at 4 degrees C and 28 degrees C, for 30 days.

Some factors affecting the stability of interferon alpha 2b in solution.

In this paper we evaluated the influence of the protein concentration and a formulation vehicle on the stability of recombinant human Interferon alpha 2b (rhIFN-alpha2b) in solution. The effect of the protein content (from 1 to 100 MIU/ml) at 37 degrees C, showed that higher concentration of this cytokine protected against the loss of bioactivity (antiviral titration) better than the lower concentrations. In contrast, rhIFN-alpha2b at 50 and 100 MIU/ml decreased the SDS/PAGE- and RP-HPLC-determined purity faster than samples at 1 or 10 MIU/ml. According to these results, 10 MIU/ml rhIFN-alpha2b was the best choice to evaluate the influence of a formulation on the stability of this cytokine. Taking this into consideration, we studied the stability of a liquid and albumin-free formulation of this protein at the recommended storage temperature (5+/-3 degrees C) and under accelerated conditions (28+/-2 degrees C). Accelerated storage results showed an acceptable biochemical stability of the active ingredient throughout 2 months. Real-time storage data confirmed the good biochemical stability of this formulation for 30 months.

Influence of packaging material on the liquid stability of interferon-alpha2b.

PURPOSE: In this article we studied the effect of the packaging material on the liquid stability of interferon alpha 2b (rhIFN-alpha2b). METHODS: The compatibility of this cytokine with type I borosilicate glass ampoules was evaluated by ELISA and RP-HPLC, at 4 degrees C and after heat sealing. Additionally, the influence of protein concentration (3 and 10 MIU/ml), buffer species (sodium phosphate, sodium citrate and sodium citrate-phosphate) and additives (polysorbate 80 and EDTA Na(2) x 2H(2)O) were studied in samples with and without contact with chlorobutyl stoppers by RP-HPLC. RESULTS: The compatibility of this cytokine in sodium phosphate buffer, with type I borosilicate glass ampoules showed a significant adsorption at the lowest concentration. This influence was eliminated with a polysorbate 80/benzyl alcohol-based vehicle. The effect of the heat sealing of ampoules on the stability of rhIFN-alpha2b showed two degradation peaks when a volume of 1 ml was dispensed. However, with a lower (0.5 ml) volume, the degradation was not detected. On the other hand, samples in contact with chlorobutyl stoppers increased the apparent degradation rate constant in the range of 6.74 +/- 0.38 to 46.34 +/- 3.11 x 10(3) day-(1). This effect significantly decreased in about 1.2- and 1.1-fold when sodium citrate or sodium citrate-phosphate buffers, respectively, were evaluated. Results from the evaluation of EDTA Na(2) x 2H(2)O or polysorbate 80 showed a similar behavior. These additives reduced the apparent degradation rate constant in the range of 2.01 +/- 0.14 to 25.51 +/- 3.57 x 10(3) day-(1). CONCLUSIONS: The adsorption of the cytokine to type I borosilicate glass ampoules was eliminated with a polysorbate 80/benzyl alcohol-based vehicle, and the deleterious effect of the heat sealing decreased with a lower (0.5 ml) volume. Experimental data indicated that the contact with chlorobutyl stoppers accelerates the degradation of rhIFN-alpha2b. However, protein concentration, buffer species and pharmaceutical excipients can modulate this effect.

Influence of packaging material on the liquid stability of interferon-á2b

Purpose: In this article we studied the effect of the packaging material on the liquid stability of interferon alpha 2b (rhIFN-a2b). Methods: The compatibility of this cytokine with type I borosilicate glass ampoules was evaluated by ELISA and RP-HPLC, at 4ºC and after heat sealing. Additionally, the influence of protein concentration (3 and 10 MIU/ml), buffer species (sodium phosphate, sodium citrate and sodium citrate-phosphate) and additives (polysorbate 80 and EDTA Na2 x 2H2O) were studied in samples with and without contact with chlorobutyl stoppers by RP-HPLC. Results: The compatibility of this cytokine in sodium phosphate buffer, with type I borosilicate glass ampoules showed a significant adsorption at the lowest concentration. This influence was eliminated with a polysorbate 80/benzyl alcohol-based vehicle. The effect of the heat sealing of ampoules on the stability of rhIFN-a2b showed two degradation peaks when a volume of 1 ml was dispensed. However, with a lower (0.5 ml) volume, the degradation was not detected. On the other hand, samples in contact with chlorobutyl stoppers increased the apparent degradation rate constant in the range of 6.74 ± 0.38 to 46.34 ± 3.11 x 103 day-1. This effect significantly decreased in about 1.2- and 1.1-fold when sodium citrate or sodium citrate-phosphate buffers, respectively, were evaluated. Results from the evaluation of EDTA Na2 x 2H2O or polysorbate 80 showed a similar behavior..........(AU)

Objeto: En este artículo se estudió el efecto del material de envasado de líquidos en la estabilidad del interferón alfa 2b (rhIFN-A2B). Métodos: La compatibilidad de este tipo de citoquinas con ampollas de vidrio borosilicato que se evaluó por ELISA y RP-HPLC, a 4 º C y después en caliente. Además, la influencia de la concentración de proteínas (3 y 10 mUI / ml), tampón de especies (fosfato sódico, citrato de sodio y citrato de sodio-fosfato) y aditivos (polisorbato 80 y EDTA Na2 x 2H2O) se estudiaron en las muestras con y sin contacto con clorobutilo tapones por RP-HPLC. Resultados: La compatibilidad de esta citocina en tampón fosfato de sodio, tipo I, con ampollas de vidrio borosilicato mostraron una absorción significativa en la concentración más baja. Esta influencia fue eliminado con un polisorbato 80/benzyl vehículo a base de alcohol. El efecto del calor y el sellado de ampollas en la estabilidad de rhIFN-A2B mostró dos picos de degradación cuando el volumen de 1 ml se dispensaron. Sin embargo, con un menor (0,5 ml) de volumen, la degradación no se detectó. Por otra parte, en contacto con las muestras de clorobutilo tapones aparente aumento de la degradación constante de velocidad en el rango de 6,74 ± 0,38 a 46,34 ± 3,11 x 103 días-1. Este efecto disminuyó significativamente, en alrededor de 1,2 y 1,1 veces al citrato de sodio o citrato de sodio, fosfato de búferes, respectivamente, fueron evaluadas. Los resultados de la evaluación de EDTA Na2 x 2H2O o polisorbato 80 mostraron un comportamiento similar. Estos aditivos de la aparente reducción de la degradación constante de velocidad en el rango de 2,01 ± 0,14 a 25,51 ± 3,57 x 103 días-1......

Stability of Ala 125 recombinant human interleukin-2 in solution.

Herein, we describe the preformulation study of Ala 125- recombinant human interleukin-2 (rhIL-2A(125)) in solution. This modified form of the natural human IL-2 is obtained by the replacement of cysteine with alanine at position 125. The compatibility of this rhIL-2A(125) with type I borosilicate glass vials showed no significant adsorption at liquid-vial interface. The effect of single excipients on the stability of this lymphokine was evaluated through RP-HPLC, SDS-PAGE and biological activity assay. Polysorbate 80 at high concentrations decreased the stability of rhIL-2A(125) in solution. On the other hand, the use of antioxidants (methionine and EDTA Na(2)) diminished the oxidation rate of the active ingredient. Additionally, a group of amino acids (glutamine, alanine, glycine and histidine) stabilized rhIL-2A(125) in different grades, and glycine at 5 mg mL(-1) allowed for the best stability behaviour. Taken together, these preformulation results can be used to design an adequate liquid vehicle for rhIL-2A(125) to be manufactured for human use.

Long-term stabilization of recombinant human interferon alpha 2b in aqueous solution without serum albumin.

The development of parenteral solution dosage forms of interferon alpha 2 (rhIFN-alpha2) without human albumin may significantly diminish the problem of forming highly immunogenic rhIFN-alpha 2b aggregates and the potential risk of blood-transmitted diseases caused by infectious viruses and often living pathogens that may be present in the plasma. With this purpose, we evaluated the compatibility of type I borosilicate glass vials and chlorobutyl stoppers with rhIFN-alpha 2b in an aqueous solution. At the same time, we carried out a targeted formulation screen at 37 degrees C of single or combined (e.g. polysorbate 80, EDTA Na(2), PEG 400) potentially stabilizing excipients. Quantified biochemical results from 12 independent batches of rhIFN-alpha 2b in a polysorbate/benzyl alcohol-based vehicle formulated at pH 7.4 were all found within the limits established by the World Health Organization for this cytokine. Real-time storage data confirmed the excellent biochemical long-term (30 months) stability of rhIFN-alpha 2b in this aqueous solution formulation. Analyses were performed at intervals throughout the time period using reverse-phase high-performance liquid chromatography, a sandwich-type enzyme-linked immunosorbent assay, and antiviral activity as stability-indicating assays. Furthermore, both the physical stability (color, odor, appearance, pH, and absence of particulate material) and the sterility of this formulation were maintained under the proposed shelf conditions.