Stability study of a biosimilar of Rituximab after dilution in NaCl 0.9% polyolefin bags, with prolonged temperature excursion to 25°C.

Context
Rituximab is a monoclonal antibody use in many hematological, rheumatoid and autoimmune indications. The manufacturing laboratory claims 30 days stability at 4°C, and only 24 hours at 25°C, after dilution in NaCl 0.9%, which limits its early preparation.

Aims
Study Rituximab’s physical and chemical stability in vials after opening (10 mg/mL), and after dilution (1 and 4 mg/mL) in NaCl 0.9% polyolefin bags, at 4°C for 12 months, and at 25°C for 28 days.

Materials and methods
Bags has been prepared in sterile conditions from 3 different batches. Different validated and orthogonal methods, such as Dynamic Light Scattering (DLS), turbidimetry, ionic chromatography and size exclusion chromatography (SEC), analysis of pH, osmolality and density permits to analyze physical and chemical stability of the biosimilar of Rituximab. Finally, structural stability of the antibody has been analyzed by peptide mapping, infrared (IR) and ultraviolet (UV) spectroscopies, and fluorescence.

Results
No physical or chemical instability has been highlighting, whether it be at 4°C for 12 months or 25°C for 28 days. Whatever the concentrations and the conservation conditions studied, no chromatographic SEC or ionic profiles modification has been seen. It confirms the absence of modification of the initial structure, especially oligomerization, fragmentation or deamidation. DLS showed a monodisperse distribution (pdi = 0.043 ± 0.02), with an average diameter constant over time (12.01 ± 0.52 nm). Moreover, the absence of modification of turbidance confirms the absence of visible and sub-visible aggregate formation (aggregation index < 10). pH, osmolality and density remains constant during the study. Peptide mapping didn’t show any modification of the primary structure of the antibody after 12 months at 4°C and 28 days at 25°C. Neither the UV and IR spectroscopies nor fluorescence show any modification of the aromatics amino-acid environment, confirming the absence of tertiary structure modification of the antibody.

Conclusion
The biosimilar of Rituximab is stable for 12 months at 4°C, diluted at 1 and 4 mg/mL and in vials after opening at 10 mg/mL. Bags can also be stored at 25°C for 28 days. This new stability study allows the early preparation of this biosimilar and it use after a prolonged temperature excursion to 25°C.

Keywords: Rituximab, Biosimilar, Physical stability, Chemical stability.

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