In small molecule drug development, it is often said that the low-hanging fruit has already been harvested. Most structures that are chemically accessible, manufacturable, and druggable have already been considered.
Somewhat analogously, the leading edge in biologics development are proteins that for one reason or other present development or manufacturing challenges. Reasons vary, from difficult transfection or poor expression, to low yields or high losses on purification.
Among the speakers at CHI’s recent “PEGS” conference was Anne London, Ph.D., investigator II and lab head at the Novartis Institutes for BioMedical Research, who spoke on evaluating and streamlining platform approaches to preclinical protein production.
Preclinical protein production is a broad discipline, with expectations and goals differing from project to project. Rapid, efficient production are common goals, but challenges arise with differing maturity of programs (e.g., initial antigen production to late-stage in vivo material) and quality specifications. Dr. London presented case studies reflecting her group’s approach to arriving at a platform capable of adapting to fit production requests, with emphasis on cell-line evaluation and impurity clearance.
“The goals for each production vary based on the protein’s intended use,” she said. Live rabbit studies require a large amount of “clean” protein with very low endotoxin levels. By contrast, endotoxins will not affect proteins used in affinity binding assays, where buffer choice and concentration may be more relevant. “Fully understanding the product’s intended use is critical to guide production to the defined endpoint.”
Preclinical production platforms are “much simpler” than a GMP biologics manufacturing platform because they entail fewer polishing steps and less validation. For example, viral clearance is not required at the preclinical stage, so virus removal and inactivation are not explored. “With the goal of simply making enough protein for the intended use, only steps needed to grow, capture, purify, and deliver the protein are considered at the preclinical stage,” Dr. London explained.
A recurring production theme is low endotoxin material. In one case with the production of a His-tagged protein, endotoxin levels were too high (>1.0 EU/mg) for the intended use after standard purification.
“Guaranteeing product quality below 1.0 EU/mg is difficult, if next to impossible, in a non-GMP setting.” The situation resulted in the evaluation of multiple endotoxin-removing membranes and filters, with the final solution being a detergent extraction. Dr. London reports spending “many hours” troubleshooting this process, but eventually the product met quality specifications.
Project scaleup is another issue facing early-stage protein production. Dr. London’s group encountered an antibody that was evaluated at small scale in multiple transient cell lines before scaling into a Wave bioreactor.
“After picking the highest expressing cell line at the small scale, the culture was scaled up in the Wave with poor results; the expression levels in the flask did not scale linearly into the Wave.” This production required further process development at Wave reactor scale to meet the expression levels seen in shake flasks. “Not every project will scale up well, so time must be budgeted to allow for cell culture process development in these rare cases,” Dr. London warned.