PROOF-OF-CONCEPT EVALUATION OF THE DIFFUSE REFLECTANCE KINETIC METHOD FOR THE ANALYSIS OF BIOLOGICAL PREPARATIONS IN TURBID MEDIA

INTRODUCTION. The analysis of biological preparations in turbid media is challenging due to residual turbidity, multiple light scattering, and heterogeneous optical properties, which may complicate protein determination in complex adjuvanted formulations. Diffuse reflectance kinetics (DRK), based on monitoring the temporal dynamics of diffusely reflected light from sequential speckle images, offers a potential approach for characterizing turbid samples without prior clarification. AIM. To evaluate, as a proof-of-concept study, the applicability of DRK for protein-related analysis in turbid biological preparations, including concentration-dependent responses, matrix effects, calibration, and repeatability. MATERIALS AND METHODS. Bovine serum albumin (BSA) was analyzed at concentrations of 60, 120, 180, 240, and 360 µg mL⁻¹ in a squalene-containing, TBS-based emulsion as a model of adjuvanted vaccine formulations. Ten DRK descriptors (d1, d2, d3, sd1, sd2, sd3, r1, r2, r3, and R) were calculated from sequential speckle images and evaluated relative to a BSA-free blank matrix using fold-change analysis. The most consistent descriptor (d2) was selected for exploratory linear calibration, and within-day repeatability was assessed at 180 µg mL⁻¹ (n=6). For comparison with DRK, protein concentration was determined using the Pierce BCA Protein Assay Kit in homogeneous antigen-containing samples prior to squalene adjuvant addition. Measurements were performed at a nominal protein concentration of 180 µg mL⁻¹ (90 µg per 0.5 mL dose). RESULTS. The d2 descriptor showed the most consistent monotonic concentration-dependent response, with linear calibration demonstrating a strong correlation with BSA concentration (r = 0.9971; R² = 0.9942). Within-day repeatability was favorable (CV = 1.13%). To further assess the applicability of DRK to VLP preparations, BCA and DRK results were compared, yielding comparable mean concentrations of 179.43 and 179.14 µg mL⁻¹, respectively, with low variability (CV = 0.44% and 1.31%, respectively). CONCLUSION. The findings support the feasibility of the DRK method for protein-related analysis in turbid biological preparations and justify further validation using matrix-matched calibration and multiple recovery levels. The close agreement between the mean values supports the feasibility of DRK for comparative monitoring of protein response in the investigated adjuvanted emulsion system.