Comparability of a three-dimensional structure in biopharmaceuticals using spectroscopic methods

Area of Science:

• Biochemistry
• Spectroscopy
• Protein Chemistry

Background:

• Protein structure is maintained by interactions, including disulfide bonds, influenced by environmental conditions.
• Free and unoxidized thiols are critical indicators of protein structural integrity.
• Spectroscopic methods offer potential for quantifying these thiols.

Purpose of the Study:

• Validate two spectroscopic methods (DTNB and DLM) for free thiol measurement.
• Assess physicochemical comparability of Rituximab and Etanercept from different manufacturers.
• Evaluate the correlation between thiol quantification techniques and protein structure.

Main Methods:

• Utilized 5,5'-dithionitrobenzoic acid (DTNB) and DyLight Maleimide (DLM) as derivatizing agents.
• Applied spectroscopic techniques to measure free and unoxidized thiols.
• Compared Rituximab and Etanercept products under native, denaturing, and reducing conditions.

Main Results:

• DTNB showed no significant differences between Rituximab products across conditions.
• DTNB indicated comparability for Etanercept under native and denaturing conditions, but revealed discrepancies under reducing conditions.
• DLM supported Rituximab product equality but detected differences in Etanercept, suggesting fewer exposed thiols in Infinitam compared to Enbrel.

Conclusions:

• Both DTNB and DLM are valuable for thiol quantification, but possess distinct capabilities and limitations.
• Methodological choices impact the assessment of protein structural integrity and comparability.
• Further investigation is needed to fully correlate thiol quantification with overall protein structure and function.