Mark John Pozzo

Novel approach for optimization of a ‘difficult’ peptide synthesis by utilizing quantitative reaction monitoring assays

Three different synthetic strategies were tested to synthesize EGFRvIII peptide (1), which is a functional variant of the epidermal growth factor receptor, a protein that has been well validated as a target for cancer therapy. The initial synthesis was performed with Applied Biosystem (Carlsbad, CA, USA) 433A peptide synthesizer and it indicated that the last three amino acids coupling and Fmoc removal rates were lower than the rest of the sequence. Purity of the crude peptide was 54.5%. The second synthesis was performed manually utilizing C S Bio (Menlo Park, CA, USA) synthesizer and the Kaiser test for reaction monitoring. Because of non‐optimized reaction conditions and an unexpected by‐product, lower purity crude peptide (40.5%) was obtained. Quantitative assays to monitor reactions were developed and demonstrated in gram scale synthesis with C S Bio synthesizer. The optimized synthetic conditions improved the peptide purity to 68.1%. Copyright

Process development of a FGF21 protein–antibody conjugate

A scalable, viable process was developed for the Fibroblast Growth Factor 21 (FGF21) protein‐antibody conjugate, CVX‐343, an extended half‐life therapeutic for the treatment of metabolic disease. CVX‐343 utilizes the CovX antibody scaffold technology platform that was specifically developed for peptide and protein half‐life extension. CVX‐343 is representative of a growing number of complex novel peptide‐ and protein‐based bioconjugate molecules currently being explored as therapeutic candidates. The complexity of these bioconjugates, assembled using well‐established chemistries, can lead to very difficult production schemes requiring multiple starting materials and a combination of diverse technologies. Key improvements had to be made to the original CVX‐343 Phase 1 manufacturing process in preparation for Phase 3 and commercial manufacturing. A strategy of minimizing FGF21A129C dimerization and stabilizing the FGF21A129C Drug Substance Intermediate (DSI), linker, and activated FGF21 intermediate was pursued. The use of tris(2‐carboxyethyl)phosphine (TCEP) to prevent FGF21A129C dimerization through disulfide formation was eliminated. FGF21A129C dimerization and linker hydrolysis were minimized by formulating and activating FGF21A129C at acidic instead of neutral pH. An activation use test was utilized to guide FGF21A129C pooling in order to minimize misfolds, dimers, and misfolded dimers in the FGF21A129C DSI. After final optimization of reaction conditions, a process was established that reduced the consumption of FGF21A129C by 36% (from 4.7 to 3.0 equivalents) and the consumption of linker by 55% (from 1.4 to 0.95 equivalents for a smaller required amount of FGF21A129C). The overall process time was reduced from ∼5 to ∼3 days. The product distribution improved from containing ∼60% to ∼75% desired bifunctionalized (+2 FGF21) FGF21‐antibody conjugate in the crude conjugation mixture and from ∼80% to ∼85% in the final CVX‐343 Drug Substance (DS), while maintaining the same overall process yield based on antibody scaffold input.