In order to establish the biosimilarity of a therapeutic drug candidate to the original innovator molecule, multiple batches of both proteins must be characterized. Waters technologies provide the tools to perform a variety of physicochemical analyses both rapidly and robustly. The seamless integration of column chemistries, instrumentation, and informatics provides a complete platform for performing your critical analyses. Find out how we utilized the Biopharmaceutical Platform Solution with UNIFI to perform characterization of infliximab and a candidate biosimilar.

Structural analysis of a biosimilar and its reference product includes:

• Primary structures, such as amino acid sequence
• Higher order structures, including secondary, tertiary, and quaternary structure (including aggregation)
• Enzymatic post-translational modifications, such as glycosylation and phosphorylation
• Other potential variants, such as protein deamidation and oxidation
• Intentional chemical modifications, such as PEGylation sites and characteristics

Intact Mass Analysis

In order to confirm theoretical molecular mass and detect sequence errors or PTMs, an intact mass analysis is often performed first.

• The purified proteins are analyzed by electrospray ionization-MS after a simple desalting step.
• The mass spectra are automatically deconvoluted and mass assignments of each peak are made.
• The mirror plot shows that the candidate biosimilar has one major peak, which corresponds in mass to a major peak in the innovator trace.
• The presence of many peaks in the innovator is the result of variable glycosylation states as well as the presence of terminal lysines

• Removal of glycans via digestion with PNGase-F simplifies the mass spectrum considerably, allowing variations in the polypeptide backbone to be discerned more clearly.
• The deconvoluted mass spectrum of the deglycosylated innovator has three major peaks; the mass differences suggest that the peaks correspond to proteins with a variable number of lysine residues, a common C-terminal variant.
• The candidate biosimilar primarily comprises protein with no extra lysine residues.

• UNIFI can be set up to automatically report on the relative abundance of these variants.
• Looking at the trends across multiple batches of both innovator and biosimilar reveal consistently higher abundance of non-lysine variant in the biosimilar product batches. The biosimilar is actually less heterogeneous than the innovator product.