Systematic assessment of process analytical technologies for biologics.

The application of process analytical technology (PAT) for biotherapeutic development and manufacturing has been employed owing to technological, economic, and regulatory advantages across the industry. Typically, chromatographic, spectroscopic, and/or mass spectrometric sensors are integrated into upstream and downstream unit operations in in-line, on-line, or at-line fashion to enable real-time monitoring and control of the process. Despite the widespread utility of PAT technologies at various unit operations of the bioprocess, a holistic business value assessment of PAT has not been well addressed in biologics. Thus, in this study, we evaluated PAT technologies based on predefined criteria for their technological attributes such as enablement of better process understanding, control, and high-throughput capabilities; as well as for business attributes such as simplicity of implementation, lead time, and cost reduction. The study involved an industry-wide survey, where input from subject matter industry experts on various PAT tools were collected, assessed, and ranked. The survey results demonstrated on-line liquid Chromatography (LC), in-line Raman, and gas analysis techniques are of high business value especially at the production bioreactor unit operation of upstream processing. In-line variable path-length UV/VIS measurements (VPE), on-line LC, multiangle light scattering (MALS), and automated sampling are of high business value in Protein A purification and polishing steps of the downstream process. We also provide insights, based on our experience in clinical and commercial manufacturing of biologics, into the development and implementation of some of the PAT tools. The results presented in this study are intended to be helpful for the current practitioners of PAT as well as those new to the field to gauge, prioritize and steer their projects for success.

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.