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.

Examination of edge effects with different storage conditions of preplated dimethyl sulfoxide nanospots in ChemLib 1,536- and 3,456-well assay-ready plates.

For ultra-high-throughput screening, 10-30 nl of compound dissolved in 75% dimethyl sulfoxide (DMSO)/25% water (vol/vol) is spotted into 1,536- and 3,456-well ChemLib plates (Aurora Biotechnologies, Carlsbad, CA) and stored appropriately for a short time before screening. Although this practice eliminates the compound plating bottleneck, plated volumes of DMSO slowly evaporate from assay wells if plates are not properly stored in the interim. Since many assays are sensitive to DMSO concentrations, even slight evaporation may cause intra-plate variation and thus decrease assay quality. Using a cytochrome P450 3A4 Vivid Blue assay (Invitrogen, Carlsbad), we investigated the rate, pattern, and quantity of evaporation over a 1-year time frame to identify best practices for long-term (i.e., 6 months or greater) storage of assay-ready compound plates. Our findings regarding evaporation at plate edges indicate that nanospots preplated in ChemLib 1,536- or 3,456-well plates are best stored at -80 degrees C, in a bag, with or without the outer evaporation wells filled or at -20 degrees C, in a bag, with evaporation wells filled.