Bioanalytical Workflow for Qualitative and Quantitative Assessment of Hot-Melt Extruded Lysozyme Formulations

Abstract

Structural and functional integrities of formulated proteins are key characteristics that provide a better understanding of influencing factors and their adjustment during formulation development. Here, the procedures commonly used for protein analysis were applied and optimized to obtain a higher degree of accuracy, reproducibility, and reliability for the analysis of lysozyme extracts from hot-melt extrudates (HME). The extrudates were prepared with polyethylene glycol 20 000. The test lysozyme HMEs were subjected to extraction procedures and analytical methods following the International Council of Harmonization guidelines for testing the active protein ingredient Q 1 A (R2) in its pure and formulated form. Therefore, reversed-phase high-pressure liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization mass spectrometry, and fluorescence-based activity measurements were applied to study lysozyme stability and function after formulation. Long-term accelerated stability studies were performed for the pure and formulated protein. Our findings revealed a high degree of stability for lysozyme toward different temperatures and storage times, confirming that HME is a suitable formulation alternative that preserves lysozyme's properties and stability. The presented methods and workflow are recommended to be exploited for further protein drugs to assess usability and compatibility concerning different pharmaceutical applications.

Figure 1

(a) HPLC chromatogram of 150 μg·mL^–1 lysozyme in phosphate buffer (pH 5.2) using, as the mobile phase, acetonitrile/water /0.1% TFA (each) 20 to 60% for 40 min (b) SDS-PAGE analysis of 1 μg pure lysozyme under nonreducing conditions (lanes 1, 2) and reducing conditions (lanes 3, 4). M is the protein marker covering 3.4–100 kDa. (c) MALDI-TOF mass spectrum of pure lysozyme using 2,5-DHB as the matrix. (d) HPLC chromatogram of extracted H-Lyso/PEG 20 extrudates in phosphate buffer (pH 5.2, ∼50 μM) using the same mobile phase as in (a). (e) SDS-PAGE analysis of 1 μg extracted lysozyme (lanes 1, 2) and H-Lyso/PEG 20 extrudates (lanes 3, 4). M is the protein marker covering 3.4–100 kDa. (f) MALDI-TOF mass spectrum of extracted H-Lyso/PEG 20 extrudates using 2,5-DHB as the matrix.

Scheme 1. Workflow for the Applied Analysis Methods on Hot-Melt Extrudates.

Figure 2

(a) Michaelis–Menten kinetics diagram for lysis of (GlcNAc)₃-MeU by lysozyme in 0.1 M sodium phosphate buffer, pH = 5.2 at 42 °C using GraphPad Prism 7 software (n = 3, K_M = 2.02 × 10^–5 M). (b) % Specific activity of pure unextracted lysozyme samples by turbidimetric (n = 12) and fluorogenic (n = 6) methods. (c) % Specific activity of pure, unextracted (1) considered as 100%; pure, extracted (2); and formulated, extracted (3) lysozyme samples, (n = 6, each) using the fluorogenic activity assay.

Figure 3

MALDI-TOF mass spectra of unextracted H-Lyso/PEG20 sample showed a lysozyme peak around 14 310 Da, PEG 20 000 was observed around 21 500 Da using 2,5-DHB as the matrix.

Figure 4

Content recovery rates (a) and % specific activities (b) of extracted H-Lyso/PEG20 extrudates stored at 4, 25, and 40 °C. Samples were analyzed at the following time points: day 0, week 2, week 4, month 3, and month 6 using the optimized RP-HPLC and the fluorogenic activity assay (n = 3 and # n = 2). Specific activity was calculated as % to that of pure lysozyme stored at −20 °C.

Figure 5

SDS-PAGE gel for 3 months-extracted H-Lyso/PEG20 samples at 4, 25, and 40 °C (lanes 1,2 and 3, respectively), extracted P-Lyso/PEG20 at 4, 25, and 40 °C (lanes 4, 5, and 6, respectively), and extracted P-Lyso samples at 4, 25, and 40 °C, (lanes 7, 8, and 9, respectively) showing a band for lysozyme around 14 kDa, M is the protein marker covering a mass range of 3.4–100 kDa.