<p>The paper investigates structure of freeze-dried formulations of a therapeutical monoclonal IgG₁ with non-reducing disaccharides, sucrose and trehalose, as lyoprotectors. Formulations with variable sugar-to-protein ratios are manufactured using different freeze-drying protocols and post-drying temperature treatments. Small-angle X-ray scattering (SAXS) is applied to study packing arrangements of IgG₁ molecules in the crowded solid-state environment. The retention of amorphous structure by the lyoprotectors is confirmed with the simultaneous wide-angle X-ray scattering (WAXS) tests. The new finding is the observation of two IgG₁ SAXS peaks in the crowded environment, with position of one peak (peak A) changing with sugar-to-protein ratio, whereas the second peak (peak B) position is similar in all the formulations tested. The protein center of mass distance, which is calculated from the position of the peak A, increases with the increase in sugar content from 45 Å to 65 Å (sugar-to-protein mass ratio 0.79 to 7.95). Sugar molecules therefore serve as a physical barrier between IgG₁ molecules. While the sugar-to-protein ratio significantly impacts protein separation, other factors, such as disaccharide type, presence of a surfactant, or drying process and post-drying thermal treatment, have minimal effect. The origin of the peak B has not been established yet, with three hypotheses considered. The results highlight applicability of SAXS for quantification of separation distances between protein molecules in freeze-dried formulations, thus improving the fundamental understanding of the stability of protein drugs.</p> Graphical Abstract <p></p>