Development and Optimization of a Conjugation Reaction for an efficient PEGylation of full human IgG1 (Anti TNFα) fragments

Monoclonal antibodies are used as clinical and analytical tools for a wide range of applications, including inflammation disease. The basis of this is the high degree of specificity and affinity that IgG presents for its target antigen. Related with this, fragments such as Fab, Fab’2 or Fab’ became a popular tool in applications where the use of full-length IgG could lead to drawbacks, such as immunogenicity.

PEGylation is known as the covalent attachment of polyethylene glycol (PEG) to a variety of compounds and it represents a powerful tool, increasing serum half-live due to the benefits that confer to the protein (reduced proteolysis and renal clearance). However, conjugation between IgG fragments and PEG frequently exhibit reduction or affinity losses, mainly when the antigen binding site is affected.

A wide variety of conjugation reactions are used to obtain PEGylated IgG fragments, but most of these reactions are not studied in detail. The site-specific conjugation of Fab’ has become very successful because it avoids a substantial affinity loss for the antigen but, in general, no effort goes into the design of the conjugate.

The Fab’ fragment of full human IgG1 involved in the inflammatory diseases therapy (AntiTNFα) was modified by adding PEG on a site-specific reaction. Results obtained on the optimization of critical parameters involved in the conjugation reaction, such as, PEG ratio, conjugation time, buffer composition or optimal pH are shown in this work. The characterization of the bioconjugates as well as their final affinity was carried out by SDS-PAGE and ELISA, and further confirmation was achieved by MALDI-TOF.