Improvement of Certolizumab Fab' properties by PASylation technology.

Certolizumab pegol is a Fab' antibody fragment for treatment of rheumatoid arthritis and Crohn's disease which is conjugated to a 40 kDa PEG molecule in order to increase the protein half-life. PEGylation may have disadvantages including immunogenicity, hypersensitivity, vacuolation, decreased binding affinity and biological activity of the protein. To overcome these problems, PASylation has been developed as a new approach. The nucleotide sequence encoding 400 amino acid PAS residues was genetically fused to the corresponding nucleotide sequences of both chains of certolizumab. Then, the bioactivity as well as physicochemical and pharmacokinetic properties of the recombinant PASylated expressed protein was assayed. Circular dichroism spectroscopy demonstrated that the random coil structure of PAS sequences did not change the secondary structure of the PASylated Fab' molecule. It was observed that PASylation influenced the properties of the Fab' molecule by which the hydrodynamic radius and neutralization activity were increased. Also, the antigen binding and binding kinetic parameters improved in comparison to the PEGylated Fab' antibody. Pharmacokinetic studies also showed prolonged terminal half-life and improved pharmacokinetic parameters in PASylated recombinant protein in comparison to the PEGylated and Fab' control molecules. The results reconfirmed the efficiency of PASylation approach as a potential alternative method in increasing the half-life of pharmaceutical proteins.

The Impact of Inadequate Temperature Storage Conditions on Aggregate and Particle Formation in Drugs Containing Tumor Necrosis Factor-Alpha Inhibitors.

PURPOSE: To measure aggregate and particle formation in tumor necrosis factor-alpha (TNF-α) inhibitors etanercept, adalimumab and certolizumab pegol product samples after exposure to freezing temperature conditions similar to storage conditions previously observed in patients' homes. METHODS: TNF-α inhibitors in their original primary and secondary packaging were exposed to 32 freeze-thaw cycles (-10°C for 120min/5°C for 60 min) or continuous low storage temperature (-20°C for 96 h) before thawing at 2-8°C. Non-stressed products were used as controls. The products were analyzed by high pressure size exclusion chromatography (HP-SEC), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), micro-flow imaging (MFI) and second derivative ultraviolet (UV) spectroscopy. RESULTS: Ten out of twenty-one stressed product samples (47.6%) showed increased particle numbers in the submicron and micron size range when compared to controls. For each product, DLS, MFI and NTA detected an increase in particle level in at least one stressed syringe (both continuous freezing and freeze-thaw), whereas HP-SEC and UV spectroscopy showed no differences between stressed and non-stressed products. CONCLUSION: TNF-α inhibitors are relatively resistant to freezing temperatures similar to storage conditions previously observed in patients' homes. However, almost half of the stressed product samples showed formation of particles in the submicron and micron size range.

Molecular Basis for the Neutralization of Tumor Necrosis Factor α by Certolizumab Pegol in the Treatment of Inflammatory Autoimmune Diseases.

Monoclonal antibodies against TNFα, including infliximab, adalimumab, golimumab, and certolizumab pegol, are widely used for the treatment of the inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Recently, the crystal structures of TNFα, in complex with the Fab fragments of infliximab and adalimumab, have revealed the molecular mechanisms of these antibody drugs. Here, we report the crystal structure of TNFα in complex with the Fab fragment of certolizumab pegol to clarify the precise antigen-antibody interactions and the structural basis for the neutralization of TNFα by this therapeutic antibody. The structural analysis and the mutagenesis study revealed that the epitope is limited to a single protomer of the TNFα trimer. Additionally, the DE loop and the GH loop of TNFα play critical roles in the interaction with certolizumab, suggesting that this drug exerts its effects by partially occupying the receptor binding site of TNFα. In addition, a conformational change of the DE loop was induced by certolizumab binding, thereby interrupting the TNFα-receptor interaction. A comprehensive comparison of the interactions of TNFα blockers with TNFα revealed the epitope diversity on the surface of TNFα, providing a better understanding of the molecular mechanism of TNFα blockers. The accumulation of these structural studies can provide a basis for the improvement of therapeutic antibodies against TNFα.