Process scale separation of an anti-CD22 immunotoxin charge variant

Area of Science:

• Biopharmaceutical Manufacturing
• Analytical Chemistry
• Protein Chemistry

Background:

• Deamidation is a common post-translational modification in proteins.
• Deamidated variants of immunotoxins can exhibit altered biological activity.
• Controlling impurities is critical for biopharmaceutical product quality.

Purpose of the Study:

• To characterize a deamidated variant of an immunotoxin.
• To develop a process for separating deamidated variants at scale.
• To ensure product quality and biological activity during manufacturing.

Main Methods:

• Analytical ion-exchange high-performance liquid chromatography (HPLC) for variant separation.
• Peptide mapping and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for site of modification identification.
• Cell-based bioassay to assess biological activity.
• Process scale anion-exchange chromatography for impurity removal.

Main Results:

• The site of deamidation was identified in the toxin portion of the immunotoxin.
• Deamidation resulted in reduced biological activity.
• Process scale anion-exchange chromatography effectively reduced the deamidated variant to acceptable levels.
• An analytical HPLC assay served as a surrogate for the cell-based bioassay.

Conclusions:

• Deamidation impacts immunotoxin biological activity and must be controlled during manufacturing.
• Process scale anion-exchange chromatography is effective for controlling deamidated variants.
• Understanding structure-function relationships and utilizing analytical assays are key to successful impurity control.