Validation of a Cell Proliferation Assay to Assess the Potency of a Dialyzable Leukocyte Extract Intended for Batch Release

Abstract

Transferon® is a blood product with immunomodulatory properties constituted by a complex mixture of peptides obtained from a human dialyzable leukocyte extract (DLE). Due to its complex nature, it is necessary to demonstrate batch consistency in its biological activity. Potency is the quantitative measure of biological activity and is also a quality attribute of drugs. Here we developed and validated a proliferation assay using Jurkat cells exposed to azathioprine, which is intended to determine the potency of Transferon® according to international guidelines for pharmaceuticals. The assay showed a linear response (2.5 to 40 µg/mL), coefficients of variation from 0.7 to 13.6% demonstrated that the method is precise, while r2 = 0.97 between the nominal and measured values obtained from dilutional linearity showed that the method is accurate. We also demonstrated that the cell proliferation response was specific for Transferon® and was not induced by its vehicle nor by other peptide complex mixtures (glatiramer acetate or hydrolyzed collagen). The bioassay validated here was used to assess the relative potency of eight released batches of Transferon® with respect to a reference standard, showing consistent results. The collective information from the validation and the assessment of several batches indicate that the bioassay is suitable for the release of Transferon®.

Keywords: Transferon®; biological potency; complex mixture of peptides; development and validation; dialyzable leukocyte extract; quality specifications.

Conflict of interest statement

Carballo-Uicab G, Linares-Trejo JE, Mellado-Sánchez G, López-Morales CA, Estrada-Parra S, Pérez-Tapia SM, and Medina-Rivero E have participated at different stages of the development of Transferon®. Pavón L and Velasco-Velázquez M declare no conflict of interest.

Figures

Figure 1

Effect of Transferon® on the proliferation of Jurkat cells. (A) Comparison of the Transferon® dose–response curve (1–180 µg/mL) using cells exposed (red circles) and not exposed to azathioprine (black squares). (B) Dose–response curve exhibiting a linear behavior in a concentration range from 2.5 to 40 µg/mL Transferon® (blue circles) compared to the response of cells exposed to azathioprine and treated with a vehicle (grey triangles).

Figure 2

Accuracy. (A) Behavior of dilutional linearity at dilution levels of 60% (black squares), 70% (white squares), 100% (white triangles), 130% (black circles), and 140% (white circles) of Transferon®. (B) Relationship between nominal and measured potency at dilution levels from 60 to 140% (r2 = 0.97). O.D.: Optical Density; SD: Standard Deviation.

Figure 3

Specificity. Comparison of the response of Transferon® (blue circles) with Colagenart® (green squares), Copaxone® (white triangles), and the vehicle control (grey triangles).

Figure 4

Consistency of the potency among batches of Transferon®. (A) Dose–response curves of different batches of Transferon®. (B) Relative potency of eight batches of Transferon® with confidence intervals (C.I.) at 95% (n = 3).