Detection of PEGylated proteins in polyacrylamide gels by reverse staining with zinc and imidazole salts.

The reverse staining, with imidazole-SDS-zinc, of PEG-linked proteins separated by SDS-PAGE was studied. Using model conjugates (interferon-alpha 2b (IFN-alpha2b) reacted with either a branched-chain (40,000) PEG (PEG2,40) or a linear monomethoxy PEG polymer (Mr of 12,000) and chromatographically purified monoPEG2,40-IFN-alpha2b), conventional small-format analytical gels (<1 mm thick) showed typical detection patterns (i.e., transparent, colorless bands clearly discernible against a zinc imidazolate-generated white gel background), in less than 20 min. Nonreacted (free) PEG was almost undetected, as expected. The reverse-stained PEGylated IFN-alpha2b patterns were qualitatively indistinguishable from those of parallel gels stained with iodine (I2). The LOD was estimated in the low nanogram range (e.g., at about 7 ng for mono- or bi-PEG2,40 IFN-alpha2b per lane on gradient (4-17%) gels). Also, this stain allowed the visualization of Coomassie blue-undetected PEG-IFN bands, and could be restained with I2. PEGylated species of lysozyme, a low-molecular-weight peptide, ovalbumin, and chymotrypsin were used to demonstrate the generality of this stain. We also show (i) how to counteract the adverse effect of some parameters (e.g., gel thickness above 1 mm, long gel length, low (e.g., 4-6%) acrylamide concentration) on the reverse staining process and (ii) that the properties of the reverse-stained PEGylated proteins remain unchanged, as judged by analyzing both the ion exchange chromatography-based positional isomer separation profile and enzyme-linked immunosorbent response of PEG-IFN recovered from gels. Consequently, this technique may be useful for the rapid analysis or the small-scale preparation of PEGylated proteins.

Stability of an extemporaneously prepared recombinant human interferon alfa-2b eye drop formulation.

PURPOSE: The stability of an extemporaneously prepared recombinant human interferon alfa-2b (rh-IFN-alpha2b) eye drop formulation was studied. METHODS: A volume of 3 x 10(6) International Units (IU) of rh-IFN-alpha2b formulated in solution was diluted with 5 mL of a 0.01% benzalkonium chloride solution. The stability of the extemporaneous formulation was evaluated for 30 days at room temperature (5 +/- 3 degrees C) and at 28 +/- 2 degrees C. Solutions were periodically subjected to bioactivity assay (antiviral titration), enzyme-linked immunosorbent assay, preservative-efficacy and sterility testing, organoleptic evaluation, and pH testing. Preservative efficacy was tested against five microorganisms. The organoleptic characteristics were verified by checking for the transparency and absence of suspended solids against light and dark backgrounds. Statistical significance was determined using analysis of variance after a comparison of the homogeneity of variance (Bartlett's test). RESULTS: Results from this evaluation indicated that the formulation was stable for 15 days at 5 +/- 3 C. During this storage period, the biological activity varied between 80 and 125% of the nominal value (0.5 x 10(6) IU/mL). The formulation was sterile and organoleptically acceptable. The pH ranged from 6.7 to 7.3, and the preservative was effective. The formulation was stable for 7 days when stored at 28 +/- 2 degrees C. The formulation remained sterile, colorless, and without suspended solids. The pH range was 6.7-7.3. CONCLUSION: An extemporaneously pre -pared rh-IFN-alpha2b eye drop formulation was stable at 5 +/- 3 degrees C for 15 days and at 28 +/- 2 degrees C for 7 days.

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.

PEGylated interferon-alpha2b: a branched 40K polyethylene glycol derivative.

PURPOSE: The conjugation of interferon-alpha2b (IFN-alpha2b) to a branched-chain (40,000) polyethylene glycol (PEG2,40K) was studied. METHODS: We studied the conjugation of IFN-alpha2b at different pH values (6.5, 7, and 8), using the PEG2,40K reagent in either solution or solid state. MonoPEGylated interferon was isolated by ion-exchange chromatography and characterized using (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) cation exchange high-performance liquid chromatography, (3) bicinchoninic acid protein assay, (4) enzyme-linked immunosorbent assay, (5) cell-based bioassays, (6) thermal stability (at 60 degrees C), (7) tryptic digestion, and (8) pharmacokinetics in rats. RESULTS: PEGylation reaction gave 30-55% PEG2,40K-IFN-alpha2b, 1-10% polyPEGylated interferon, and 35-70% unmodified IFN-alpha2b. Compared to the polyPEGylated IFN-alpha2b species, the pure (96%) monoPEGylated conjugate retained a significantly higher bioactivity (IU/mg): 1.7x10(4)+/-8.5x10(3) vs. 2.8x10(6)+/-1.4x10(6) for antiviral and 1.9x10(4)+/-9.5x10(3) vs. 3.1x10(6)+/-1.6x10(6) for antiproliferative activity. Immunorecognition against IFN was reduced by the PEG2,40K moiety in the conjugate. This monoPEGylated IFN-alpha2b, which migrated as a single band in gel electrophoresis, was found to be a heterogeneous, complex mixture of different positional isomers. PEGylation markedly enhanced both the resistance to tryptic degradation and the thermal stability of IFN-alpha2b. The serum half-life of 40K PEG-IFN was 330-fold longer, while plasma residence time was increased 708 times compared to native IFN. CONCLUSION: The PEG2,40K conjugate of IFN-alpha2b has increased in vitroand in vivo stability as compared to the native cytokine.