Bioactivity Determination of Native and Variant Forms of Therapeutic Interferons

Abstract

The traditional antiviral assays for the determination of interferon potency are reported to have considerable variability between and within assays. Although several reporter gene assays based on interferon-inducible promoter activities have been reported, data from comprehensive validation studies are lacking and few studies have been conducted to analyze the variant forms of interferons, which could have undesirable clinical implications. Here, a reporter gene assay employing a HEK293 cell line stably transfected with luciferase gene under the control of interferon-stimulated response element promoter was developed and validated. The assay was found to be more sensitive, with a larger detection range than the antiviral assay. Several cytokines tested did not interfere with the test, suggesting the assay possesses a certain degree of selectivity. Moreover, the robustness of the assay was demonstrated by minimal variations in the results generated by different analysts and cell passage number (up to 52 passages). Finally, the method was employed to analyze several interferon variants (interferon-α 2a) and we found that the aggregated form has completely lost its potency; while a modest loss of bioactivity in oxidized interferon was observed (approx. 23%), the deamidated form essentially retained its activity.

Virion-Associated Vpr Alleviates a Postintegration Block to HIV-1 Infection of Dendritic Cells

“…The inability to detect differences in the mRNA expression levels of IFN-␤ in MDDCs infected with the WT and ΔVpr viruses was also mirrored by the absence of differences in protein levels in infected MDDC culture supernatants (data not shown). We used a sensitive bioassay to measure type I IFN production in infected MDDC supernatants and failed to detect any type I IFN production in HIV-1-infected MDDCs over mock-infected controls (data not shown) (70). In contrast, IP-10 was robustly secreted in both Lai-YU2 (WT)-and Lai-YU2/ΔVpr-infected MDDC culture supernatants at day 3 postinfection, although the magnitude of IP-10 induction was donor dependent (Fig.…”

Biosimilars entering the clinic without animal studies

“…For instance, when deamidated and oxidated forms of interferon (IFN)-alfa2b were evaluated in a HEK293 cell line stably transfected with luciferase gene under the control of interferon-stimulated response element promoter, a modest loss of bioactivity in oxidized interferon was observed, but the deamidated form essentially retained its activity. 30 Another example is the contribution of different glycoforms of a mAb to its function since parts of the sugar moiety in the Fc region of IgG molecules contribute to the binding to Fc gamma receptors (FcgR). 31 For example, the binding affinity of a mAb for FcgRIIIa is influenced by the presence or absence of fucose, where a higher degree of afucosylation leads to an increased binding affinity for the receptor.…”

O-Linked Glycosylation Leads to Decreased Thermal Stability of Interferon Alpha 2b as Measured by Two Orthogonal Techniques

“…Samples of IFN α-2b, O-linked glycosylated IFN α-2b and deglycosylated IFN α-2b were prepared as described above and their potency compared using a reporter gene assay as described previously (12). Briefly, this assay utilizes an Interferon Stimulated Response Element (ISRE)-containing promoter coupled to a firefly luciferase gene which is stably transfected in a human kidney cell line (HEK293) (12).…”

“…Briefly, this assay utilizes an Interferon Stimulated Response Element (ISRE)-containing promoter coupled to a firefly luciferase gene which is stably transfected in a human kidney cell line (HEK293) (12).…”

“…A reporter gene assay using a minimal promoter containing Interferon Stimulated Response Element (ISRE) coupled to a firefly luciferase gene, stably transfected in a human kidney cell line (HEK293) (12), was used to assess the potency of the IFN α-2b reference sample, O-linked glycosylated IFN α-2b and deglycosylated IFN α-2b. Results for the assessment are shown in Fig.…”