Physicochemical Characteristics of Transferon™ Batches

Abstract

Transferon, a biotherapeutic agent that has been used for the past 2 decades for diseases with an inflammatory component, has been approved by regulatory authorities in Mexico (COFEPRIS) for the treatment of patients with herpes infection. The active pharmaceutical ingredient (API) of Transferon is based on polydispersion of peptides that have been extracted from lysed human leukocytes by a dialysis process and a subsequent ultrafiltration step to select molecules below 10 kDa. To physicochemically characterize the drug product, we developed chromatographic methods and an SDS-PAGE approach to analyze the composition and the overall variability of Transferon. Reversed-phase chromatographic profiles of peptide populations demonstrated batch-to-batch consistency from 10 representative batches that harbored 4 primary peaks with a relative standard deviation (RSD) of less than 7%. Aminogram profiles exhibited 17 proteinogenic amino acids and showed that glycine was the most abundant amino acid, with a relative content of approximately 18%. Further, based on their electrophoretic migration, the peptide populations exhibited a molecular mass of about 10 kDa. Finally, we determined the Transferon fingerprint using a mass spectrometry tool. Because each batch was produced from independent pooled buffy coat samples from healthy donors, supplied by a local blood bank, our results support the consistency of the production of Transferon and reveal its peptide identity with regard to its physicochemical attributes.

Figures

Figure 1

Reversed-phase chromatographic profile of Transferon. Comparison between sample matrix (black line) and batch 15A01 of Transferon (blue line). Chromatographic profile exhibits 4 main peaks with k > 1 and absolute retention time of 2.2 min (P1), 4.3 min (P2), 6.4 min (P3), and 8.2 min (P4). Figure S1 shows that this chromatographic profile is consistent between the 10 analyzed Transferon batches. Transferon samples were analyzed using an Acquity™ UPLC™ BEH300 C18 chromatographic column (2.1 mm × 150 mm) and TFA (0.1%)-H2O and TFA (0.1%)-acetonitrile as the mobile phase at 0.4 mL/min using a gradient configuration. The column temperature was maintained at 30°C, and UV detection was performed at 214 nm. Chromatographic profiles were analyzed using Empower™ (ApexTrack method) to obtain the relative area percentage and absolute retention time for each peak. AU: area units.

Figure 2

Amino acid chromatographic profile (aminogram) of Transferon. Comparison between standard mixture of hydrolyzed proteinogenic amino acids (Waters™) (red line) and batch 15A02 of Transferon (blue line). A sample matrix derivatization control is also shown (black line). A total of 17 of the 20 known proteinogenic amino acids were detected using the amino acid standard. The amino acid chromatographic profile also showed three unidentified peaks, which were labeled as U1 (2.25 min), U2 (2.37 min), and U3 (6.80 min). Figure S2 evinces the notion that the aminogram profile is consistent between the 10 Transferon batches. The samples were analyzed using an Acquity C18 column (1.7 µm, 2.1 × 100 mm) with a mixture of acetonitrile-formic acid-water as the mobile phase using a gradient configuration. The column was maintained at 43°C, and UV detection was monitored at 260 nm. AMQ, NH3, and deriv. peaks originate from the reaction of derivatization. AU: area units.

Figure 3

Relative average abundance of proteinogenic amino acids in 10 Transferon batches. Gly (18.30%), Glu (14.49%), and Ala (11.53%) are the most abundant amino acids in Transferon samples, whereas Met (0.95%) and Arg (0.50%) are the least abundant. Total area of the chromatographic peaks of the identified proteinogenic amino acids was considered 100% for calculations of abundance.

Figure 4

Electrophoretic profile of Transferon. (a) Method selectivity. The batch 14E14 of Transferon (85 µg) showed 2 main bands at approximately 10 kDa (line 2). A second 14E14 sample (85 µg) exhibited the same electrophoretic pattern when spiked with 5 µg of equine myoglobin (17 kDa) (line 3). A total of 5 µg of equine myoglobin was used as selectivity control (line 4). (b) Batch reproducibility. The electrophoretic patterns of batches 15A01 (line 2) and 15A02 (line 3) of Transferon (100 µg) are consistent. Figure S3 shows the electrophoretic pattern of the 10 analyzed Transferon batches. Electrophoresis was performed in 16% acrylamide gels using a Tris-Gly system. Bands were detected by silver staining.

Figure 5

Peptide mapping of Transferon batches by MS-TOF. Five typical batches were subjected to spectrometric analysis and their TIC (total ion counting) profile did not exhibit significant differences. Transferon batches: 14E14 (A), 14F16 (B), 14F17 (C), 14G18 (D), and 14G19 (E).