L⁴⁴⁵P mutation on heavy chain stabilizes IgG₄ under acidic conditions

Area of Science:

• Biochemistry
• Protein Engineering
• Biopharmaceutical Manufacturing

Background:

• Immunoglobulin G4 (IgG4) antibodies are widely used therapeutics but exhibit lower stability than IgG1 during manufacturing.
• Aggregation and fragmentation are critical challenges impacting IgG4 product quality and yield.
• The heavy chain (HC) C-terminal region of IgG4 is susceptible to acid-induced instability.

Purpose of the Study:

• To investigate the molecular mechanisms underlying IgG4 instability under acidic conditions.
• To identify key residues and conditions that influence IgG4 fragmentation and aggregation.
• To develop strategies for enhancing IgG4 stability during biopharmaceutical manufacturing.

Main Methods:

• Site-directed mutagenesis of key residues (Leu445, Gly446) in the IgG4 heavy chain C-terminal region.
• Comparative stability studies of wild-type and mutant IgG4 antibodies under varying pH conditions (citrate vs. glycine buffers).
• Analysis of fragmentation and aggregation using analytical techniques (details not specified in abstract).

Main Results:

• Mutation of HC C-terminal Leu445 to Pro in IgG4 significantly reduced acid-induced fragmentation and aggregation.
• Mutation of HC C-terminal Pro445 to Leu in IgG1 promoted fragmentation and aggregation.
• Glycine buffer completely inhibited HC C-terminal Gly446 cleavage and subsequent aggregation, unlike citrate buffer.
• Complementarity-determining region (CDR) sequences and target binding influenced HC C-terminal region stability.

Conclusions:

• The IgG4 HC C-terminal region's instability is a critical factor in manufacturing challenges.
• Specific amino acid substitutions (e.g., Leu445 to Pro) and buffer conditions (e.g., glycine) can enhance IgG4 stability.
• Interactions between Fab and CH2-CH3 domains, influenced by CDR sequences and target binding, play a key role in IgG antibody stability.