Reshaping nanobodies for affinity purification on protein a.

Area of Science:

• Biotechnology
• Immunology
• Protein Engineering

Background:

• Nanobodies (Nbs) are small, single-domain antibody fragments with therapeutic and diagnostic potential.
• Current purification methods using His-tags can negatively impact Nb biodistribution.
• Alternative purification strategies are needed for His-tag-free Nbs.

Purpose of the Study:

• To develop and validate an alternative purification method for nanobodies (Nbs) without His-tags.
• To assess the impact of the new purification method on Nb properties and performance.

Main Methods:

• Mutagenesis of non-SpA binding Nbs to enable Protein A (SpA) affinity chromatography.
• Purification of engineered Nbs using SpA affinity chromatography.
• Evaluation of thermostability, antigen affinity, and biodistribution of purified Nb variants.

Main Results:

• Successfully engineered non-SpA binding Nbs for purification via SpA affinity chromatography.
• Mutagenized Nb variants retained their original thermostability and antigen-binding affinity.
• The SpA-based purification method did not negatively affect Nb biodistribution.

Conclusions:

• Protein A affinity chromatography is a viable alternative for purifying His-tag-free nanobodies.
• This method offers a way to improve the biodistribution of Nb-based tracers.
• The developed technique supports the advancement of nanobody applications in diagnostics and therapeutics.