1：Experimental Design and Method Selection:

A custom-made Cross-Flow Filtration (CFF) setup with an on-line measurement loop was developed for Process Analytical Technology (PAT) implementation. The setup included UV/Vis spectroscopy and light scattering (DLS and SLS) for real-time monitoring of protein concentration, tertiary structure, and quaternary structure during VLP reassembly.

2：Sample Selection and Data Sources:

Three chimeric Hepatitis B core Antigen (HBcAg) variants (VLP A, B, and C) provided by BioNTech Protein Therapeutics GmbH were used. Stock solutions were in disassembly buffer (

3：5 M urea, 50 mM Tris, pH 0) and diluted to 1 g/L protein concentration before experiments. List of Experimental Equipment and Materials:

Equipment included a KrosFlo KRIIi CFF unit with mPES hollow fiber membrane module (10 kDa cutoff, 13 cm2 area), Gilson Minipuls 3 peristaltic pump, Zetasizer Nano ZSP photometer with flowcell, Ultimate DAD-3000 Diode Array Detector with flowcell, FR-902 flow restrictor, NI USB-6008 data acquisition device, and various filters and buffers. Materials included HBcAg variants, urea, Tris, potassium phosphate, and ultrapure water.

4：Experimental Procedures and Operational Workflow:

The CFF unit and measurement loop were filled with water, equilibrated, then flushed with disassembly buffer and loaded with protein solution. Diafiltration was initiated at TMPs of

5：25, 5, or 1 bar with reassembly buffer. On-line sensors monitored continuously, and samples were taken every 5 diafiltration volumes for off-line analysis (SEC, DLS, TEM). Data Analysis Methods:

Data was acquired and synchronized using a custom Matlab application. UV/Vis spectra were processed with Savitzky-Golay filters for second derivatives to assess tyrosine and tryptophan solvatization. DLS provided z-average diameters, SLS provided scattered-light intensity. PLS models were calibrated for VLP concentration prediction using cross-validation.